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SELECTED ARTICLES APRIL 2015 www.jem.org

INNATE IMMUNITY
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 Welcome
I n n a t e I m mu n i t y
The Journal of Experimental Medicine now prints topic-specific mini collections to showcase a handful
of our recent publications. In this installment, we highlight papers focusing on two innate cell subsets,
dendritic cells and macrophages in health and disease.
Immunotherapy has proven successful in many types of cancer treatment, but has also been
associated with dangerous inflammatory responses in some patients. Our collection begins with
an insight by Merghoub and Wolchok discussing the findings of Mirsoian et al. who show that
lethal inflammation in response to anti-CD40 and IL-2 immunotherapy is triggered by increased
inflammatory macrophages in the accumulated visceral fat of aged mice. Similarly, young obese
mice succumbed to treatment while older mice on diets were protected.The study highlights factors
to be considered with use of immunotherapeutics.
In the gut, the immune system must balance recognition of infectious pathogens with minimal responses to commensal
bacteria. An Insight by Giorgio Trinchieri describes these immune challenges and the findings from Longman et al. showing that
in both mice following Citrobacter rodentium infection and in patients with colitis, CX3CR1+ mononuclear phagocytes (MNPs)
are potent producers of IL-23 and IL-1b. The production of these cytokines by MNPs efficiently induces IL-22 production by
group 3 innate lymphoid cells to promote barrier integrity and mucosal protection.
Lambrecht and Guilliams examine genetic fate mapping of myeloid cells that distinguishes macrophages derived from
embryonic precursors vs. circulating monocytes. They highlight the contribution to macrophage biology from Molowi et al.
reporting that cardiac macrophages are initially of embryonic origin, but are progressively replaced by monocyte-derived cells as
mice age. They speculate about how manipulation of different macrophage populations could be used in therapeutics.
Myelin destruction in multiple sclerosis (MS) is mediated by inflammatory macrophages, but the origin of these cells has been
unclear. An Insight from Michael Heneka discusses findings from Yamasaki et al., who use a mouse model of MS to distinguish
tissue-resident microglial cells from infiltrating monocytes. Using double chemokine reporter mice, the authors find that
monocyte-derived macrophages initiate myelin destruction, mainly in the nodes of Ranvier, while microglia-derived macrophages are
involved with clearing debris.
Two papers from the Liu and Nussenzweig groups track the differentiation of human progenitor cells into dendritic cells
(DCs). They show that a granulocyte/monocyte/DC progenitor gives rise to a monocyte-DC progenitor that in turn generates
both monocytes and a common DC progenitor. The common DC progenitor produces the three major subsets of human DCs,
such as CD1c+ cDCs, CD141+ cDCs and pDCs. They go on to identify the immediate precursor of CD1c+ and CD141+ DCs
in the circulation of healthy donors. These precursor cells (hpre-cDC) were detectable in cord blood, bone marrow, blood, and
peripheral lymphoid organs. An Insight by Frederic Geissmann describes the novel in vitro culture system used in the studies to
parse out the development of human DCs as well as a discussion of the developmental similarities between mouse and human DCs.
Together these studies characterize the many roles and functions of innate immune cells. We hope you enjoy this
complimentary copy of our Innate Immunity collection. We invite you to explore additional collections at www.jem.org
and to follow JEM on Facebook, Google+, and Twitter.

Selected Articles April 2015

Immunotherapy and the belly of the beast

Taha Merghoub and Jedd D. Wolchok

Adiposity induces lethal cytokine storm after systemic administration of stimulatory immunotherapy
regimens in aged mice
Annie Mirsoian, Myriam N. Bouchlaka, Gail D. Sckisel, Mingyi Chen, Chien-Chun Steven Pai, Emanuel Maverakis,
Richard G. Spencer, Kenneth W. Fishbein, Sana Siddiqui, Arta M. Monjazeb, Bronwen Martin, Stuart Maudsley,
Charles Hesdorffer, Luigi Ferrucci, Dan L. Longo, Bruce R. Blazar, Robert H. Wiltrout, Dennis D. Taub,
and William J. Murphy
Critical role for CX3CR1+ mononuclear phagocytes in intestinal homeostasis
Giorgio Trinchieri

CX3CR1+ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22
Randy S. Longman, Gretchen E. Diehl,1 Daniel A. Victorio, Jun R. Huh,1 Carolina Galan, Emily R. Miraldi,
Arun Swaminath, Richard Bonneau, Ellen J. Scherl, and Dan R. Littman

Monocytes find a new place to dwell in the niche of heartbreak hotel

Bart Lambrecht and Martin Guilliams

Progressive replacement of embryo-derived cardiac macrophages with age

Kaaweh Molawi, Yochai Wolf, Prashanth K. Kandalla, Jeremy Favret, Nora Hagemeyer, Kathrin Frenzel,
Alexander R. Pinto, Kay Klapproth, Sandrine Henri, Bernard Malissen, Hans-Reimer Rodewald, Nadia A. Rosenthal,
Marc Bajenoff, Marco Prinz, Steffen Jung, and Michael H. Sieweke

Macrophages derived from infiltrating monocytes mediate autoimmune myelin destruction

Michael T. Heneka

Differential roles of microglia and monocytes in the inflamed central nervous system
Ryo Yamasaki, Haiyan Lu, Oleg Butovsky, Nobuhiko Ohno, Anna M. Rietsch, Ron Cialic, Pauline M. Wu,
Camille E. Doykan, Jessica Lin, Anne C. Cotleur, Grahame Kidd, Musab M. Zorlu, Nathan Sun, Weiwei Hu,
LiPing Liu, Jar-Chi Lee, Sarah E. Taylor, Lindsey Uehlein, Debra Dixon, Jinyu Gu, Crina M. Floruta, Min Zhu,
Israel F. Charo, Howard L. Weiner, and Richard M. Ransohoff

The real thing: How to make human DC subsets

Frederic Geissmann

Restricted dendritic cell and monocyte progenitors in human cord blood and bone marrow
Jaeyop Lee, Galle Breton, Thiago Yukio Kikuchi Oliveira, Yu Jerry Zhou, Arafat Aljoufi, Sarah Puhr, Mark J. Cameron,
Rafick-Pierre Skaly, Michel C. Nussenzweig, and Kang Liu

Circulating precursors of human CD1c+ and CD141+ dendritic cells

Galle Breton, Jaeyop Lee, Yu Jerry Zhou, Joseph J. Schreiber, Tibor Keler, Sarah Puhr, Niroshana Anandasabapathy,
Sarah Schlesinger, Marina Caskey, Kang Liu, and Michel C. Nussenzweig
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INSIGHTS

Immunotherapy and the belly of the beast

Immunotherapy has emerged as an effective means to restore immune recognition of cancer.
Numerous forms of immunotherapy are currently being explored clinically. The most common are
vaccines (dendritic cell, viral, and whole tumor cell based), adoptive T cell therapy and immune
checkpoint blockade. The recent successes in melanoma, renal cancer, and lung cancer have gener-
ated renewed optimism for treatment of multiple cancer types that were believed not amenable to
immune-based therapies. However, immune-related events such as colitis, dermatitis, and, less
frequently, endocrinopathy and pneumonitis have been reported and can be a challenge in the
clinical use of these approaches. Cytokine release syndrome has been reported in the case of CAR Insight from Taha Merghoub (left)
(chimeric antigen receptor) T cell therapy and treatment with agonist antibodies. In this issue, and Jedd Wolchok (right)
Mirsoian et al. provide evidence that adiposity in aged mice induces a lethal cytokine storm follow-
ing systemic administration of stimulatory immunotherapy consisting of anti-CD40 agonist antibody with IL-2.
This is an elegant follow up to a previous study by the same authors in which they showed that systemic immunotherapy adminis-
tration in aged mice resulted in the induction of a rapid and lethal cytokine storm. In the current paper, they find that it is the fat that
accumulates during aging, and mainly the visceral fat, that is associated with toxicity. This results in increased levels of proinflammatory
M1 macrophages within the peritoneal cavity and visceral adi-
pose tissues, leading to heightened production of TNF. In or-
der to confirm that it is indeed the fat that is associated with
lethal effects, the authors repeated their studies in young mice
with genetic (ob/ob) or diet-induced obesity (DIO). While
young obese mice also displayed severe toxicity to the therapy,
it was much more severe in older mice suggesting that age is
also a significant factor. Reciprocally, calorie-restricted aged
mice had lower visceral body fat content and reduced cytokine
levels, with increased survival following immunotherapy.
Obese mice were also protected by macrophage depletion or
TNF blockade. These data demonstrate the need to consider
age and body fat content as variables in preclinical assessment
of therapeutics and when modeling diseases such as cancer.
Since many cancer patients fall into the elderly category,
this study brings up a critical point that aged mice respond dif-
ferently to immunotherapy than younger mice. This also
highlights an important issue when considering potential tox-
icities associated with any therapy, as most animal studies are
performed in young, healthy mice. Another interesting point
is the potential impact of body mass index and adipose accu-
mulation in predicting side effects when investigating and uti-
lizing immunotherapies. This is a very important and impactful
The Murphy group previously showed that immunotherapy with anti-CD40 finding for the field, and further investigations in tumor bear-
and IL-2 leads to a productive immune response in young mice (A) but ing mice with commonly used therapies are warranted. If the
lethal cytokine storm in older mice (B). They now find that the visceral fat findings are confirmed in other therapeutic settings, the inclu-
that accumulates in aged mice (or young obese mice) is the primary trigger
sion of supportive measures, such as TNF blockade, should
of inflammation after immunotherapy, resulting in increased inflammatory
M1 macrophages and toxic cytokine storm (B). Immunotherapy-induced
continue to be considered for early management of immune-
lethality was prevented by blocking TNF or depleting macrophages related toxicities to improve clinical outcomes.
(B). Whether the success of immunotherapy in the treatment of tumor- Mirsoian, A., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/
bearing mice will also be impaired by obesity remains to be determined. jem.20140116.

Taha Merghoub and Jedd D. Wolchok, Memorial Sloan Kettering Cancer Center: merghout@mskcc.org and wolchokj@mskcc.org

2 INSIGHTS | The Journal of Experimental Medicine

 Article

Adiposity induces lethal cytokine storm

after systemic administration of stimulatory
immunotherapy regimens in aged mice
Annie Mirsoian,1* Myriam N. Bouchlaka,1* Gail D. Sckisel,1 Mingyi Chen,2
Chien-Chun Steven Pai,1 Emanuel Maverakis,1 Richard G. Spencer,5
Kenneth W. Fishbein,5 Sana Siddiqui,5 Arta M. Monjazeb,3
Bronwen Martin,5 Stuart Maudsley,5 Charles Hesdorffer,5 Luigi Ferrucci,5
Dan L. Longo,5 Bruce R. Blazar,6 Robert H. Wiltrout,7 Dennis D. Taub,4,8**
and William J. Murphy4**
1Department of Dermatology, 2Department of Pathology and Laboratory Medicine, 3Department of Radiation Oncology,
and 4Department of Dermatology and Internal Medicine, University of California, Davis, Sacramento, CA 95817
5National Institute on Aging-Intramural Research Program, National Institutes of Health, Biomedical Research Center,

Baltimore, MD 21224
6Division of Blood and Marrow Transplantation, Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455
7National Cancer Institute, Frederick, MD 21702
8Hematology and Immunology Translational Research Center, VA Medical Center, Washington, DC 20422

Aging is a contributing factor in cancer occurrence. We recently demonstrated that sys-

temic immunotherapy (IT) administration in aged, but not young, mice resulted in induction
of rapid and lethal cytokine storm. We found that aging was accompanied by increases in
visceral fat similar to that seen in young obese (ob/ob or diet-induced obese [DIO]) mice.
Yet, the effects of aging and obesity on inflammatory responses to immunotherapeutics are
not well defined. We determine the effects of adiposity on systemic IT tolerance in aged
compared with young obese mice. Both young ob/ob- and DIO-generated proinflammatory
cytokine levels and organ pathologies are comparable to those in aged ad libitum mice
after IT, culminating in lethality. Young obese mice exhibited greater ratios of M1/M2
macrophages within the peritoneal and visceral adipose tissues and higher percentages of
TNF+ macrophages in response to CD40/IL-2 as compared with young lean mice. Macro-
phage depletion or TNF blockade in conjunction with CD40/IL-2 prevented cytokine
storms in young obese mice and protected from lethality. Calorie-restricted aged mice
contain less visceral fat and displayed reduced cytokine levels, protection from organ
pathology, and protection from lethality upon CD40/IL-2 administration. Our data dem-
onstrate that adiposity is a critical factor in the age-associated pathological responses to
systemic anti-cancer IT.

CORRESPONDENCE
William J. Murphy:
wmjmurphy@ucdavis.edu
Abbreviations used: AL, ad
libitum; ALT, alanine amino
transferase; CR, calorie restricted;
DIO, diet-induced obese; HD,
high dose; IT, immunotherapy;
LD, low dose; MRI, magnetic
resonance imaging.
The use of immunotherapy (IT) in cancer has
recently resulted in impressive responses.Yet, the
usages of IT regimens, especially those involving
cytokine therapies, have also resulted in the in
duction of severe systemic toxicities often not pre
viously characterized in their preclinical animal
studies.Because such toxicities often require inten
sive care management of patients, the causes for
*A. Mirsoian and M.N. Bouchlaka contributed equally to
this paper.
**D.D. Taub and W.J. Murphy contributed equally to
this paper.
the discrepancies in observations between clin
ical and preclinical toxicity merit further study.
Cancer has been considered a disease of aging,
with persons over 65 accounting for over 60%
of newly diagnosed malignancies (Balducci and
Ershler, 2005). Consequences of aging include
gradual decreases in immunological function,
thymic involution leading to decreased naive
T cell output, restriction within T cell repertoire
2014 Mirsoian et al. This article is distributed under the terms of an Attribution
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the publication date (see http://www.rupress.org/terms). After six months it is
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J. Exp. Med. 2014 Vol. 211 No. 12 23732383 2373
www.jem.org/cgi/doi/10.1084/jem.20140116
diversity, increases in genomic instability, and increases in cel
lular senescence (Campisi, 2013). However, despite the ma
jority of patients being of this demographic, the overwhelming
majority of preclinical studies are performed in young inbred
mice.We recently demonstrated that treatment of young mice
with a combination IT regimen consisting of agonistic mono
clonal antibody to CD40 (CD40) given in conjunction with
IL-2 resulted in synergistic anti-tumor effects and was well
tolerated (Murphy et al., 2003).Yet, administration of the same
regimen in an aged cohort resulted in 100% lethality within
two days of treatment (Bouchlaka et al., 2013). Importantly, the
mortality in the aged was perpetuated by an aberrant cytokine
storm mirroring a systemic inflammatory response syndrome
(SIRS), which resulted in multi-organ damage (Bouchlaka et al.,
2013). These systemic toxicities were closely representative of
those previously noted clinically with FDA-approved immu
nostimulatory IT therapeutics such as high-dose IL-2 and
IFN- (Lee and Margolin, 2011).
Aging is associated with a gradual decline in immunolog
ical function but is also accompanied by the coincidence of
a systemic, chronic, and low-grade proinflammatory response
termed inflammaging that leads to increased systemic cyto
kine levels, namely IL-1, IL-6, and TNF (Franceschi, 2007).
Emerging data suggests that inflammaging may be influenced
by age-associated changes in body mass composition such as
decreased lean muscle mass, increased adiposity, and increases
in overall body mass index (BMI; Franceschi et al., 2000).
Whether adiposity plays a role in the generation of systemic
toxicities or whether it is primarily an age-associated occur
rence remains unknown.
Like aging, hallmark to the obese microenvironment is
the development of a low-grade chronic meta-inflammatory
state. Excess adiposity is associated with increased infiltration
of macrophages and proinflammatory mediators such as den
dritic cells, NK cells, and T cells into fat depots that ultimately
results in environmental remodeling. Consequences include
increased circulating levels of C-reactive protein (CRP), IL-6,
leptin, and TNF (Ronti et al., 2006;Vzquez-Vela et al., 2008).
Importantly, the immunomodulatory effects of increased adi
pose tissue extends through the ability for both adipocytes and
the stromal vascular fraction to express a variety of immunologi
cally important surface receptors, such as the leptin receptor,
IL-6 receptor, and both p55 and p75 TNF receptors (Ailhaud,
2000). Systemic consequences of increased adiposity are also
seen at distance sites through premature induction of aging-
associated changes such as thymic involution, which leads to
the restriction of naive T cell output and ultimately restriction
of the T cell repertoire (Yang et al., 2009).
Collectively, the field of IT has made great progress in the
last decade toward the development of therapeutic candidates
against cancer.Yet, these candidates have been met in the clinic
with the induction of severe, often limiting, systemic toxici
ties that hinder their usage. Additionally, given the rise of
obesity within society, as well as the estimation that cancer is
a disease primarily of the aged, using lean and young models
may not accurately reflect patient outcomes. Therefore, this
2374
study seeks to establish the consequences of increased adipose
tissue accumulation throughout aging upon IT-induced tox
icities. Here, we find that adiposity results in a skewing toward
increased levels of proinflammatory M1 macrophages within
the peritoneal cavity and visceral adipose tissues that ultimately
result in heightened production of proinflammatory cytokines,
namely TNF, during strong immune stimulation with IT.
Importantly, aged ad libitum (AL)fed mice and young obese
mice succumb to TNF-mediated pathological responses culmi
nating in rapid lethality, which are prevented through caloric
restriction in the aged or through either macrophage depletion
or TNF blockade.
RESULTS
IT administration in the aged results in rapid lethal cytokine
storm and is associated with increased accumulation
of visceral adipose tissue
The combination IT regimen consisting of CD40 and IL-2
(CD40/IL-2) has previously shown synergistic anti-tumor
effects resulting in the regression of metastatic tumors in
young (<6 mo) mouse models (Murphy et al., 2003). Re
cently, we demonstrated that administration of the therapeu
tic dose (high dose [HD]) of CD40/IL-2 into aged mice
(15 mo) resulted in a heightened cytokine storm that prompted
multi-organ damage and resulted in 100% lethality by day 2
of treatment (Bouchlaka et al., 2013). To address whether the
increased lethality was specific to this therapeutic dosage in
the aged, we administered CD40/IL-2 at a lower dose (LD),
less than half, into aged AL-fed mice. Consistent with our
previous findings, treatment of aged mice with LD IT re
sulted in 100% mortality by day 2 of treatment (Fig. 1 A) and
demonstrated increased serum levels of proinflammatory cy
tokines TNF, IL-6, and IFN-, indicative of a cytokine storm
(Fig. 1 B). In addition, to ensure that the lethality observed in
the aged mice was not IT regimenspecific, we administered
an IT combination of CpG and IL-2 (CpG/IL-2) into young
WT and aged AL mice. Confirming our previous findings,
aged mice treated with CpG/IL-2 therapy incurred a similar
cytokine storm that culminated in rapid lethality by day 3
(Fig. 1, CE), in contrast to young mice which exhibited lower
levels of cytokines and demonstrated complete treatment tol
erance (Fig. 1, CE).
Normal aging is inherently associated with changes in body
mass compositionspecifically with decreases in muscle mass
and increases in intra-abdominal visceral adiposity (Harris,
2002). Equally, within our animal studies an observable differ
ence between aged and young mice is noted in overall size, in
particular increased waist circumference (Fig. 1 F). On average,
aged mice weigh twice as much as young (Fig. 1 G).To deter
mine whether the difference in size is due to increases in fat
deposits, we sought to quantify differences in body mass and
the presence of visceral adiposity. Young (<6 mo) and aged
(15 mo) C57BL/6 mice were analyzed through magnetic
resonance imaging (MRI) to quantify the presence of visceral
adiposity. Aged mice exhibited markedly increased body mass
indices and total visceral fat volumes in comparison with young
Obesity induces immunostimulatory therapy toxicity | Mirsoian et al.

mice (Fig. 1, G and I).These results indicate that aged mice are
not only heavier and larger in size but that these size differences
are attributed to increases in adipose tissue accumulation.
Adipose tissue is widely published to be a highly active
metabolic organ with immune modulatory capabilities. To
address whether in our aged AL-fed mice, who were allowed
to freely access food, the increased adiposity noted was con
tributory toward the development of IT-induced toxicities in
the aged, we aimed to determine the effects of IT administra
tion into an aged calorie-restricted (CR) cohort. CR mice are
placed on a food restriction diet beginning at 14 wk of age
and maintained throughout life. To ensure that age-matched
CR aged mice contained a decreased accumulation of body
fat in comparison with aged AL mice, we quantified total body
weight and visceral fat through MRI (Fig. 1, H and I). CR
mice weighed significantly less than AL aged mice and exhib
ited a decreased presence of visceral adiposity to levels that
JEM Vol. 211, No. 12
Ar ticle
Figure 1. Aged AL mice show increased
proinflammatory cytokines, rapid lethality
to LD IT, and display increased fat volume
ratios in comparison to aged CR mice.
(A) Ad libitumfed C57BL/6 mice were treated
with LD CD40/IL-2 or rIgG/PBS and survival
was monitored, n = 58 mice/group. (B) Serum
cytokines TNF, IL-6, and IFN- were measured
day 2 by CBA after the start of CD40/IL-2 or
rIgG/PBS therapy in aged AL mice, n = 35
mice/group. (C) Young AL (3 mo) and aged AL
(18 mo) mice were treated with CpG/IL-2 IT
and survival was monitored, n = 5 mice/group.
(D and E) Serum cytokine levels of TNF (D) and
IL-6 (E) were measured by CBA on day 2 after
the start of therapy, n = 5 mice/group.
(F) Representative image of aged and young
C57BL/6 mice showing size and waist circum-
ference. (G) Total body weight (left graph) and
visceral fat weight (right graph), was mea-
sured for young (2 mo) and aged AL mice
(20 mo), n = 5 mice/group. (H) Total body
weight (left graph) or visceral fat (right graph)
was measured in naive 15-mo-old C57BL/6
mice on an AL or CR diet, n = 34 mice/group.
(I) Visceral fat in young (4 mo), middle-aged
(9 mo), and aged (15 mo) mice on either an AL
(left) or CR (right) diet was evaluated by MRI.
WS is indicative of water suppression, where
fat is bright. FS is indicative of fat suppression,
fat is dark/black. Data in all panels are repre-
sentative of one of at least five experiments
with similar results. MRI scans are representa-
tive of one of at least three mice imaged per
group with similar results. Survival analysis
was plotted according to the Kaplan-Meier
method, and statistical differences were deter-
mined with the log-rank test. Bar graph (mean
value SEM) statistics were performed using
either one-way or two-way ANOVA with
Bonferroni post-hoc tests. ***, P < 0.001; **,
P < 0.01; *, P < 0.05.
were similar to young mice (Fig. 1, GI). MRI scans of young
(4 mo), middle-aged (9 mo), and aged (15 mo) mice that were
either AL-fed or CR throughout life showed significant dif
ferences in adipose accumulation over time (Fig. 1 I). We found
that with increasing age, AL-fed mice have a proportional in
crease in body fat content, whereas mice placed on a CR diet
lack increased fat accumulation regardless of age. Aged CR
mice show no significant difference in fat volume ratios to
their middle-age and young counterparts, and remain similar
to young AL mice (Fig. 1 I).
Caloric restriction in aged mice results in decreased
accumulation of adiposity and protection
from IT-induced toxicity
To examine the effect of adiposity and aging on the develop
ment of systemic toxicities after IT, aged AL, aged CR, and
young AL mice were administered IT or a control therapy
2375
Figure 2. Calorie restriction in the aged confers protection by decreasing cytokine levels and IT-associated organ pathology, thereby
allowing increased survival during IT. (AC) Aged AL and age-matched aged CR mice (18 mo) were treated with HD CD40/IL-2 or rIgG/PBS; at day 2
of IT, mice were sacrificed for histological analysis of livers (A) and intestines (C). Serum was collected on day 2 and assessed for ALT (B) levels, n = 3 mice/
group. Representative H&E images of liver and intestines (gut) for aged AL and aged CR groups. Bars, 200 m. Asterisks represent steatosis and arrows
denote areas of severe immune infiltration and/or necrosis. (DF) Serum was analyzed day 2 after treatment for TNF (D), IL-6 (E), and IFN- (F). (G) Aged
(18 mo) C57BL/6 mice on AL or CR diet were treated with either LD or HD of CD40/IL-2 and survival was monitored. Control groups received rIgG/PBS.
All data panels are representative of one of four independent experiments with similar results for all panels. Survival analysis was plotted according to
the Kaplan-Meier method and statistical differences were determined by using the log-rank test. Bar graph (mean value SEM) statistics were performed
using one-way ANOVA with Bonferroni post-hoc test. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01.

(rIgG/PBS). IT resulted in increased lymphocytic infiltrates
to the liver of aged CR and aged AL mice, in contrast to age-
matched control-treated mice (Fig. 2, AC). To assess whether
IT resulted in organ damage, livers and intestines were col
lected on day 2 of treatment, the day of mortality in aged AL
mice, and assessed for pathological changes. Importantly, livers
and intestines from aged CR mice demonstrated significantly
less inflammation and less necrosis in comparison with aged
AL mice (Fig. 2, A and C). Serum analysis of alanine amino
transferase (ALT) enzyme levels, a correlate of liver damage,
showed a significant reduction in ALT levels in aged CR mice
in comparison with aged AL mice (Fig. 2 B).Additionally, serum
proinflammatory cytokine analysis of TNF (Fig. 2 D), IL-6
(Fig. 2 E), and IFN- (Fig. 2 F) resulted in all cytokines being
significantly decreased in aged CR mice in comparison with
the aged AL mice. These decreases in cytokine values were to
levels that were either lower-than or as-low-as those in young
AL mice that received IT (Fig. 2, DF). To determine if these
lower cytokine values during IT therapy would confer protec
tion against IT-induced mortality, aged AL mice and aged CR
mice were administered either LD IT or HD IT and monitored
for mortality. Independent of treatment dose, aged AL mice all
succumb to mortality on day 2 of treatment (Fig. 2 G). How
ever, 100% of aged CR mice placed on the HD amount of
IT demonstrated complete protection from CD40/IL-2
mediated lethality (Fig. 2 G). Collectively, these data suggest
that increased body fat plays a critical role in the induction of
systemic toxicities by IT. Increased adiposity played a critical
role in the age-associated cytokine storm and subsequent organ
pathologies after IT and calorie restriction diminished the de
velopment of such toxicities conferring a protective effect that
allowed for increased survival in the aged.
Increased adipose tissue accumulation through obesity, in the
absence of age, results in IT-induced lethal cytokine storm
To further dissect the role of adipose tissue in the IT-associated
inflammatory responses seen in aged mice, we next sought to
determine the impact of adiposity, in the absence of age, as a
possible cofactor.Young ob/ob mice (B6.Cg-Lepob/ob) are leptin-
deficient and therefore start exhibiting obesity by 3 wk of age.
In comparison to age-matched young WT (2 mo) control mice
placed on an AL diet (young AL), ob/ob mice have significantly
higher body masses and weigh, on average, 2.5 greater
(Fig. 3 A). MRI analysis of body fat content demonstrated

2376 Obesity induces immunostimulatory therapy toxicity | Mirsoian et al.


that young ob/ob mice have higher total body fat content that
is similar in accumulation to the aged mice, being deposited
largely in the intra-abdominal cavity, in comparison with young
AL mice (Fig. 3 B).
To determine if adiposity could solely induce toxic con
sequences in aged AL, young ob/ob and age-matched young
AL mice were treated with HD IT and at day 2 of treatment
analyzed for the presence of immune-mediated organ patholog
ical changes within the liver and intestines (Fig. 3, C and D).
IT resulted in mild inflammatory infiltrates in young AL mice
but resulted in moderate to severe inflammation in the ob/ob
mice, which was comparable to the aged within the intestines
(Fig. 3 D).Yet, analysis of IT-induced liver pathology was hin
dered by the presence of extensive steatosis in ob/ob mice, re
sulting in a paradoxical interference in determining the intensity
of immune-mediated inflammatory liver damage (Fig. 3 C).
IT resulted in both the intestines and liver displaying the most
immune-mediated inflammatory damage within the ob/ob that
JEM Vol. 211, No. 12
Ar ticle
Figure 3. Increased adiposity indepen-
dently of age results in systemic release of
proinflammatory cytokines and organ pa-
thology after IT in young ob/ob (obese)
and aged AL mice. (A) Body weights of naive
young (2 mo) WT and young ob/ob C57BL/6
mice, n = 6 mice/group. Representative pic-
tures of young-AL WT mice and obese ob/ob
naive mice are shown. (B) Total fat content in
naive young WT (2 mo), young ob/ob (2 mo),
and aged (18 mo) C57BL/6 mice was visualized
by MRI. Fat is bright. (C and D) Young AL (2 mo),
young ob/ob (2 mo), and aged AL (18 mo) mice
were treated with HD CD40/IL-2 and liver
(C) and gut (D) were stained with H&E and
scored for histopathology on day 2. Bars,
200 m. Arrows indicate areas of steatosis
and/or immune cell infiltration and asterisks
indicate patchy necrosis and/or fibrosis.
(EG) Young AL (2 mo), young ob/ob (2 mo),
aged AL (18 mo), and aged CR (18 mo) were
treated with LD CD40/IL-2 and serum was
collected day 2 of treatment and analyzed by
CBA for levels of: TNF (E), IL-6 (F), and IFN-
(G), n = 3 mice/group. (H and I) Young AL (2 mo),
young ob/ob (2 mo), aged AL (18 mo), and
aged CR (18 mo) were treated with LD CD40/
IL-2 (H) or CpG/IL-2 (I) or rIgG/PBS control and
survival was monitored. (J) Serum levels of TNF
were measured on day 2 of CpG/IL-2 therapy
by CBA. Data in all panels are representative of
one of at least three experiments with similar
results. MRI scans are representative of at
least three mice imaged per group with similar
results. Survival data were plotted using the
Kaplan-Meier method and statistical differ-
ences were determined with the log-rank test.
Bar graph (mean value SEM) statistics per-
formed using Students t test or one-way
ANOVA with Bonferroni post-hoc tests. ***,
P < 0.001; **, P < 0.01; *, P < 0.05.
was comparable to the aged AL mice and significantly greater
than the young AL (Fig. 3, C and D).
Because HD IT resulted in significant changes within the
obese ob/ob mice, we next administered the lower dose (LD)
of IT to young ob/ob, young age-matched AL mice, aged AL,
and aged CR mice. Administration of LD IT still resulted in
significant increases in TNF and IL-6 proinflammatory cyto
kine levels within the young ob/ob that were not significantly
different to the aged AL mice (Fig. 3, E and F). Both ob/ob and
aged AL were significantly greater than both the young AL
and aged CR mice, where CR in the aged led to significant
down-regulation in both TNF and IL-6 cytokine production
(Fig. 3, E and F). Although the young ob/ob and the aged AL
mice displayed increased levels of IFN-, these levels were not
significant among the groups (Fig. 3 G). Consistent with the
increase in proinflammatory cytokines at day 2 of IT treatment,
100% of young ob/ob mice succumbed to IT-induced lethality
within 4 days of treatment in contrast to 100% survival in the
2377
Figure 4. IT toxicity is TNF-dependent through M1 macrophage accumulation. (AD) Young (2 mo) WT and young (2 mo) ob/ob mice were
treated with either control rIgG/PBS or LD CD40/IL-2. Visceral adipose tissue and peritoneal lavages were collected on day 2 and assessed for the pres-
ence of macrophages identified as CD45+CD19F4/80+CD11b+. Ratio of M1/M2 macrophages (A), and total numbers (B) and percentages (C) of TNF-
producing macrophages. Macrophages identified as M1 macrophages were characterized as CD206; M2 macrophages were gated as CD206+. (EH)
Young AL (2 mo) and young ob/ob (2 mo) were treated with either control or macrophage depleting clodronate liposomes before and during LD CD40/IL-2
and survival was monitored; n = 5 mice/group. (F and G) On day 2, visceral adipose tissues and peritoneal lavages were collected and assessed for the
percentages TNF+ macrophages (F and G) as characterized in experiments in AD and serum TNF levels (H), n = 4 mice/group. (IL) Young AL (2 mo) and
young ob/ob (2 mo) mice were treated with either control hIgG/PBS or subcutaneous injection of 1.5 mg/0.1 ml etanercept before and during LD CD40/
IL-2 and survival was monitored (I), n = 5 mice/group. (J and K) On day 2, visceral adipose tissue and peritoneal lavages were collected and assessed by
flow cytometry for the percentages of TNF+ macrophages (J and K) and serum TNF levels (L); n = 4 mice/group. Data in all panels are representative of one
of at least three experiments with similar results. Survival data were plotted using the Kaplan-Meier method and statistical differences were determined
with the log-rank test. Bar graph (mean value SEM) statistics performed using one-way ANOVA with Bonferroni post-hoc tests. **, P < 0.01; *, P < 0.05.

young WT cohort (Fig. 3 H). 100% of aged AL mice yielded
to lethality from the IT by day 2 of treatment, whereas 100%
of aged CR mice survived the entire regimen (Fig. 3 H).
The pathological and lethal response observed within the
young ob/ob mice was not CD40/IL-2 IT administration
specific. Young AL and age-matched young ob/ob mice were
administered CpG in combination with HD IL-2 and, like
wise, resulted in lethality of 75% of young ob/ob mice by day 5
of treatment but resulted in complete tolerance by young
AL mice (Fig. 3 I). CpG/IL-2 therapy led to significantly in
creased levels of serum proinflammatory cytokine TNF in an
analogous trend as CD40/IL-2 administration on day 2 of
therapy (Fig. 3 J).
This data further supports that increased adiposity plays a
crucial role in the induction of systemic toxicities after IT.
These data are suggestive that not only does an aging environ
ment result in lethal toxicities but toxicity may also be depen
dent on the increase in fat accumulation as the young ob/ob
mice succumb to cytokine storm induction and mortality after
IT, similar to that observed in the aged mice.
IT toxicity is TNF- and M1 macrophagedependent within
the peritoneum and visceral adipose tissues
A hallmark of obesity is the increasing infiltration of macro
phages into adipose tissues (ATMs) through the secretion of
MCP-1 by adipocytes. Phenotypically, these macrophages are

2378 Obesity induces immunostimulatory therapy toxicity | Mirsoian et al.


predominately classically activated M1 cells that also express
proinflammatory mediators IL-1, TNF, and IL-6 (Lumeng
et al., 2007; Fujisaka et al., 2009). As such, we next sought to
determine if increases in M1 macrophages within the perito
neal cavity and visceral adipose tissues could account for the
pathological increases in systemic TNF during IT administra
tion.Young AL and age-matched young ob/ob mice were ad
ministered LD CD40/IL-2. At day 2 of treatment, visceral
adipose tissues and peritoneal lavages were phenotypically as
sessed by flow cytometry for the presence of M1 (CD206)
macrophages, M2 (CD206+) macrophages, and percentages
of TNF-expressing macrophages. Consistently, young ob/ob
mice demonstrated significant increases in M1 macrophage
proportions during IT administration, whereas young AL mice
were able to maintain consistent M1-to-M2 ratios (Fig. 4 A).
Additionally, both the visceral adipose tissues and peritoneal
lavages of ob/ob mice experienced significant increases in per
centages of TNF-expressing macrophages as opposed to young
AL mice (Fig. 4, BD). Significant changes were not observed
in the spleen of either young ob/ob or AL mice (unpublished
data).This data, consistent with obesity literature, suggests that
increases in adipose tissue accumulation within the visceral
cavity may impact regulation of M1 and M2 macrophage ra
tios that ultimately may affect TNF responses to immune stim
ulation with immunotherapeutic regimens.
Given the increases in M1 macrophages within the obese
mice and dysregulation observed with IT, young ob/ob and
young AL mice were treated with LD CD40/IL-2 in conjunc
tion with either liposomal clodronate for macrophage deple
tion (Fig. 4, EH) or etanercept (Enbrel) for TNF blockade
JEM Vol. 211, No. 12
Ar ticle
Figure 5. DIO succumb to cytokine storm,
organ pathology, and lethality similar to aged
AL mice after IT. Young C57BL/6 mice were
placed on a 60% fat diet starting at 3 wk of age
to induce obesity (young DIO). Lean age-matched
controls (young lean) were fed a 10% fat diet
beginning at 3 wk of age. Young mice were ana-
lyzed at 5 mo of age, and aged AL mice were
analyzed at 18 mo of age, n = 6 mice/group. Av-
erage body weight (A), representative pictures (B),
and total fat content as measured by MRI (C) are
shown for each group. (DF) Young lean (5 mo),
young DIO (5 mo), and aged AL (18 mo) mice
were treated with HD CD40/IL-2 or rIgG/PBS.
Serum was collected day 2 after the start of
treatment and analyzed by CBA for levels of TNF
(C), IL-6 (D), and IFN- (E) and survival was ana-
lyzed (F). Data in AC are representative of one of
at least three experiments with similar results and
data in DG are representative of one of at least
four experiments with similar results. Survival
data were plotted using the Kaplan-Meier method
and statistical differences were determined with
the log-rank test. Bar graph (mean value SEM)
statistics performed using one-way ANOVA with
Bonferroni post-hoc tests. ****, P < 0.0001;
***, P < 0.001; **, P < 0.01; *, P < 0.05.
(Fig. 4, IL). Both macrophage depletion and co-treatment
with etanercept during LD CD40/IL-2 resulted in complete
protection of young ob/ob mice from lethality (Fig. 4, E and I).
As expected, treatment with liposomal clodronate led to signif
icantly decreased levels of TNF-producing macrophages within
both the visceral fat and peritoneal lavages (Fig. 4, F and G),
which significantly reduced systemic circulating serum levels
of proinflammatory cytokine TNF (Fig. 4 H). Additionally,
co-administration of LD CD40/IL-2 with etanercept did
not reduce the percentages of TNF-expressing macrophages
within the visceral fat or peritoneal cavity (Fig. 4, J and K) but
mediated complete protection through an overall reduction
in systemic TNF levels (Fig. 4 L).
Together, these data indicate that proinflammatory M1
macrophages are the cellular mediator in the induction of IT
toxicity, particularly through increased presence within the peri
toneal and visceral adipose tissues. Importantly, these effects are
TNF-dependent, as blocking TNF levels either through etan
ercept administration or macrophage depletion resulted in com
plete protection of young ob/ob mice from lethality.
Diet-induced obese (DIO) mice succumb to cytokine
storm, organ pathology, and lethality similar to ob/ob
and aged AL mice after IT
ob/ob mice are congenic for the spontaneous mutation in the
OB gene, resulting in leptin hormone deficiency. To extend
our findings, we generated young DIO mice. Mice were placed
on a purified 60% fat diet to induce obesity beginning at 3 wk
of age and were determined to be obese when weighing >40 g
(DIO). Age-matched counterparts were placed on a matching
2379
purified diet that was 10% fat and served as lean controls. In
creases in body mass were validated in DIO mice before IT
administration (Fig. 5, AC). DIO mice displayed increases in
overall body weight and size (Fig. 5, A and B). MRI analysis
of body fat content showed increased total body fat accumu
lation, largely as increased visceral fat, in young DIO mice that
was similar to the aged AL mice (Fig. 5 C).
Confirming our previous findings, DIO mice treated with
HD CD40/IL-2 displayed increased serum TNF, IL-6, and
IFN- levels compared with age-matched young mice on a
10% fat diet after IT administration (Fig. 5, DF).The increases
in proinflammatory cytokines in DIO mice were lower, yet not
significantly so, than aged mice after IT (Fig. 4, CE). Young
DIO mice started succumbing to IT lethality at day 3 of treat
ment (>60%), and <40% were still alive by day 4 (Fig. 5 G).
Together, these results suggest that adipose tissue is a criti
cal component to the development of systemic cytokine storm
and lethality after IT.Yet, due to the observation that treatment
of DIO mice did not result in 100% of mice succumbing to
lethality, aging itself may also likely contribute to the overall
heightened inflammatory responses incurred by IT.
DISCUSSION
After initiation of strong immune stimulation with IT, we
show that both young obese and aged AL mice demonstrate
increased levels of adiposity, which led to aberrant increases in
both TNF and IL-6 levels that ultimately resulted in multi-
organ damage. Similar to aging, obesity has been shown to be
immunomodulatory, where the cross talk between adipocytes
and macrophages is thought to lead toward the development
and perpetuation of a meta-inflammatory state through con
tinual NF-B activation, which is currently hypothesized to
be responsible for the induction of metabolic diseases. A study
conducted by Spaulding et al. (1997) examined the effects of
age and calorie restriction on the production of TNF and IL-6
in resting mice and demonstrated that serum levels of both
cytokines were significantly higher in aged mice in compari
son to young, and yet aged mice subjected to long-term calo
rie restriction resulted in TNF and IL-6 serum levels that
were comparable to the young mice.These findings lend sup
port to the protective effects seen within our studies in the
aged CR cohort administered IT.
Importantly, the data presented here demonstrate the need
for using age and body fat content as variables in preclinical
assessment of therapeutics and modeling diseases, such as can
cer. However, the vast majority of inbred mouse studies are
housed in specific pathogen-free colonies where normal aging
may result in higher body fat content and yet less immune
challenge from pathogens making them more susceptible to
proinflammatory responses and toxicities after immune thera
pies. It will therefore be important that future studies ascer
tain responses in mice that have been exposed to pathogens
and immune challenges as they age.
Furthermore, our data demonstrate that an obese phenotype
resulted in lethal consequences that were ameliorated through
calorie restriction in the aged. Also, differences in lethality
2380
were observed between young obese and aged AL mice, where
aged mice die by day 2 but young obese mice die at day 4 of
treatment. Therefore, although our data strongly demonstrates
that adiposity plays a central role in the induction of toxicities,
aging is also a likely key factor. Therefore, our findings high
light the importance in considering both the status of aging
and obesity of patients receiving systemic administration of stim
ulatory IT regimens. It will be important to conduct studies
dissociating the consequences of aging upon increased cytokine
responsiveness, possibly through either investigating epigene
tic modifications induced by the meta-inflammatory and/or
inflammaging states associated with increased adipose accumu
lation or whether aging is accompanied by changes in expres
sion levels of various receptors that allow for a quicker response.
The development of IT regimens continues on a positive
trajectory with increasing successes within a variety of can
cers. Active IT has taken a greater comprehensive form focus
ing on activating humoral and cell-mediated immunity, and
these strategies have included antigen-specific vaccines (i.e.,
MART-1 vaccines), DC-based vaccines (i.e., Sipuleucel-T),
inhibitors of checkpoint blockade (i.e., CTLA-4 and PD-1),
and systemic stimulatory regimens through cytokine based
therapies and application of agonistic antibodies (i.e., CD40,
IFN-, and IL-2). Our data presented herein demonstrates
that both young obese and aged AL mice succumb to rapid
lethality during systemic administration of stimulatory thera
pies. Our data strongly suggests that excess adiposity may be a
prognostic factor in determining patient outcome and treat
ment tolerance. However, additional studies need to be con
ducted examining the effects of adiposity on the development
of adverse reactions within other classes of IT regimens, such
as checkpoint inhibitors.
Clinically, we hypothesize that taking into account patient
BMI, adipose accumulation, and age may better modulate treat
ment type choices as well as the inclusion of supportive thera
peutics such as TNF blockade in conjunction with anti-cancer
therapies that, overall, may increase patient survival and lead
to greater anti-cancer responses. Follow-up investigations in
tumor-bearing models merit further study.
MATERIALS AND METHODS
Animals and diets. Female young (25 mo old), middle-aged (912 mo old),
and aged (1522 mo old) C57BL/6 mice were purchased from Charles River
or from the animal production area at the National Cancer Institute. Young
female B6.Cg-Lepob/ob and young age-matched female WT control C57BL/6
mice were purchased from The Jackson Laboratory at 812 wk of age.
DIO obese and control lean mice were generated from WT C57BL/6
mice purchased at 3 wk of age from Charles River or from the animal produc
tion area at the National Cancer Institute. Mice were placed on an open-source
purified diet consisting of either 60% high-fat (D12492) or 10% low-fat
(D12450J) constitution (Research Diets, Inc.). Age-matched mice were placed
on either a high-fat or low-fat diet beginning at 3 wk of age for 1620 wk
and weighed weekly. Mice were deemed obese upon reaching a minimum of
40 g of weight.
For studies comparing aged AL and CR dietary effects, both female aged
CR and age-matched female AL-littermates (1522 mo old) were purchased
from the CR rodent colony at the National Institutes on Aging, National In
stitutes of Health (NIH). In brief, mice were either raised through free access,
AL, feeding on the NIH31 regular chow, or placed on a limited daily feeding
Obesity induces immunostimulatory therapy toxicity | Mirsoian et al.

Table 1. Grading of acute liver damage
Global assessment
0: None
I: Minimal/indeterminate Portal inflammatory infiltrate is in a minority of the portal triads, which is minimal
II: Mild Inflammatory infiltrate in 50% of the triads, which is generally mild, and confined within the portal spaces
III: Moderate Inflammatory infiltrate, expanding most or all of the triads, and associated with mild interface and lobular activity
IV: Severe As above for moderate, with spillover into periportal areas and moderate to severe perivenular inflammation that
extends into the hepatic parenchyma and is associated with perivenular hepatocyte necrosis
schedule with the NIH31-fortified chow. CR is initiated at 14 wk of age at
10% restriction, increased to 25% restriction at 15 wk, and to 40% restriction
at 16 wk of age where it is maintained throughout the life of the animal. In
formation on survival curves, food intake curves, and body weights of the
NIA strains has been previously published (Turturro et al., 1999).
All experimental mice were housed in the Animal Facilities at the Uni
versity of California, Davis under specific pathogen-free (SPF) conditions.
For the comparison in fat volume ratios, young, middle-aged, and aged AL
and CR mice were ordered and sent to the National Institute on Aging for
MRI analysis and also housed under SPF conditions. All of the animal proto
cols were approved by the Institutional Animal Care and Use Committees of
both institutions. Body weights were also measured for several mice at differ
ent ages and on dietary regimens. Mice were euthanized by CO2 asphyxia
tion and the visceral fat pads (mesenteric, gonadal, and renal) were collected
and weighed together for each mouse to determine the visceral fat weight.
Reagents. The agonistic antimouse CD40 antibody (clone FGK115B3)
was generated via ascites production in our laboratory as previously described
(Murphy et al., 2003; Bouchlaka et al., 2013). The endotoxin level of the
CD40 was <1 endotoxin unit/mg of antibody as determined by a quantita
tive chromogenic limulus amebocyte lysate kit (QCL-1000; Bio Whittaker).
Recombinant human IL-2 (rhIL-2; TECIN Teceleukin) was provided by the
National Cancer Institute (NCI). Murine CpG ODN 1826 was purchased
from InvivoGen. All were injected i.p.
Schedule of CD40/IL-2 and CpG/IL-2 IT treatments. Mice were
treated with agonist CD40 antibody and rhIL2 as previously described
(Murphy et al., 2003; Bouchlaka et al., 2013). CD40 or CpG was adminis
tered daily for a total of 5 consecutive days (days: 0, 1, 2, 3, and 4) and IL-2
was administered twice a day for a total of 4 d (days: 1, 4, 8, and 12). Control
mice received rat IgG (rIgG; Jackson ImmunoResearch Laboratories, Inc.)
and PBS (Cellgro). Survival was monitored daily. An HD or LD of CD40/
IL-2, or HD CpG/IL-2, was administered as follows on the same scheduling
pattern as previously described (Bouchlaka et al., 2013): HD CD40/IL-2,
mice received 80 g of agonist CD40 and 106 IU of IL-2 in 0.2 ml PBS i.p.;
control mice received 80 g rIgG in PBS; LD CD40/IL-2, mice received
40 g of agonist CD40 and 4 105 IU of IL-2 in 0.2 ml PBS i.p; control serum samples and standards were incubated for 1 h at 25C with a bead
mice received 40 g rIgG in PBS; CpG/HD IL-2, mice received 100 g CpG
ODN 1826 (InvivoGen) and 106 IU of IL-2 in 0.2 ml PBS i.p.
Macrophage depletion and TNF blockade with etanercept (Enbrel).
In some experiments, mice were in vivo depleted of macrophages using lipo
somal clodronate or control-loaded liposomes (Encapsula NanoScience) be
fore and during IT administration, as previously described (Bouchlaka et al.,
2013). For experiments where mice were euthanized by day 2 of treatment,
0.2 ml per dose was injected i.p. of either clodronate or control liposomes on
days 2 and 0. For all survival experiments, clodronate or control liposomes
were administered on days 2, 0, 2, and 4.
TNF blockade was preformed through in vivo subcutaneous injection
of 1.5 mg/0.1 ml etanercept (Enbrel) on days 1 and 1 for experiments where
mice were euthanized on day 2 of IT therapy. For all survival experiments,
etanercept was administered on days 1, 1, 3, 7, and 9.
JEM Vol. 211, No. 12
Ar ticle
Criteria
No portal inflammation
Murine macrophage studies. On day 2 of treatment, murine macrophages
were isolated from either the peritoneal cavity or visceral adipose tissue. For
peritoneal lavages, 4 ml of cold PBS was injected into the peritoneal cavity
of each mouse and then aspirated. Lavages were spun down at 1,200 rpm for
5 min and the resulting pellet was resuspended in RF10c media and plated
for flow cytometry staining.
The stromal vascular fraction of visceral adipose tissues was collected day
2 of treatment. Adipose tissues from the visceral cavity (gonadal) were col
lected and then incubated for 60 min in a digestion solution consisting of
2 mg/ml type IV collagenase (Sigma-Aldrich) dissolved fresh in a 2% BSA in
PBS solution. After incubation samples were centrifuged at 1,200 rpm for
5 min for layer separation of the stromal vascular fraction from adipocytes.
The resulting stromal vascular fraction was then removed, resuspended into
a single cell suspension, counted, and plated for flow cytometry staining.
Flow cytometry analysis of macrophages. Single cell suspensions from
the peritoneal lavages and visceral adipose tissues were plated in RF10c media
with the addition of GolgiPlug (containing Brefeldin A) and GolgiStop (con
taining Monensin) Protein Transport Inhibitors (BD) according to manufac
turers protocols. Plates were incubated for 9 h at 37C and 5% CO2.
After incubation, cells were washed with PBS and stained for flow analy
sis. Single cell suspensions were labeled with Fc block (purified antimouse
CD16/32; BD) for 10 min, followed by labeling with antibodies for 20 min
at 4C. Cells were washed twice with a staining buffer consisting of 5% FBS
(Gemini Bio-products) in DPBS (Corning). For intracellular staining, the
Cytofix/Cytoperm kit (BD) was used per manufacturers protocols. After
staining, cells were analyzed using a custom configured Fortessa cytometer,
and by using FACSDiva software (BD). Data were analyzed using FlowJo
software vX (Tree Star). Antibodies used to distinguish macrophages were:
PB-conjugated antimouse CD45, BV711-conjugated antimouse CD11b,
BV605- and PE-conjugated antimouse CD206, BV785-conjugated anti
mouse CD19, APC-conjugated antimouse F4/80, and PE-Cy7conjugated
antimouse TNF (BioLegend).
Serum cytokine bead array. Serum levels of TNF, IFN-, and IL-6 were
quantified by multiplex measurement using the Cytometric Bead Array
(CBA; BD) kit as described previously (Bouchlaka et al., 2013). In brief,
mixture containing bound antibodies to TNF, IFN-, and IL-6 according to
manufacturers instructions. Samples and standards were resuspended in wash
buffer and analyzed by flow cytometry. Data were acquired on a custom-
configured Fortessa cell analyzer using FACSDiva software (BD) and analyzed
using FlowJo software (Tree Star). Each sample was analyzed in triplicate.
Upon analysis of raw data, the mean fluorescent intensities (MFIs) of each
bead cluster were quantified.Where indicated in the figures, protein concen
trations were extrapolated relative to a standard curve created by serial dilu
tion of the mouse positive control cytokine.
Colorimetric liver enzyme assay. Serum ALT was quantified using the
ID Labs ALT Enzymatic Assay kit (ID Labs Biotechnology Inc.) as previously
described (Bouchlaka et al., 2013). Colorimetric determination of ALT levels
was performed by reading the absorbance of each well at 340 nm on a plate
reader (VERSAmax turntable plate reader).The concentration of ALT (U/liter)
2381
Table 2. Grading of acute intestinal damage
Global assessment Criteria
0: None No inflammation
I: Minimal/indeterminate Scattered infrequent inflammatory infiltrate in a minority of crypts, which is minimal
II: Mild Inflammatory infiltrate in 50% of the crypts, which is generally mild, and confined within the mucosa epithelium with
mild architectural distortion
III: Moderate Inflammatory infiltrate involving most of the crypts, and associated with architectural distortion (villous blunting and
focal mucosal atrophy/erosion) and luminal inflammatory exudate
IV: Severe Extensive inflammatory infiltrate (large lymphoid aggregates) with expanding into the lamina/muscular propria, and
associated with prominent mucosal ulceration/denuding, muscular wall necrosis, and/or perforation

in each sample was then directly determined from the change in absorbance
within 5 min time. Dilutions of the Pyruvate Control, included in the kit,
were used to construct a standard curve to calibrate the assay. Every serum
sample was assayed in triplicate.
Histopathology and grading/scoring. Liver and whole intestines were
collected on day 2 of IT, flushed, and fixed in 10% paraformaldehyde, embed
ded in paraffin, cut into 5-m sections, and stained with hematoxylin and
eosin (H&E). All tissues were prepared and stained at Histology Consultation
Services, Inc. (Everson, WA). Images were captured with a microscope (BX4;
Olympus) equipped with a Q-color3 camera and 10 numerical aperture
objective lens. Magnification for each captured image is specified in the figure
legends. Grading of histopathological inflammation was performed using a
scale from 0 to 4 in a blind fashion by a board-certified pathologist (M. Chen)
at the UC Davis Medical Centers Department of Pathology and Laboratory
Medicine. Specifically, the grading score for liver and gastrointestinal tract
followed our previously published grading method (Bouchlaka et al., 2013),
summarized in Tables 1 and 2.
normal body temperature using warm air. Mice were inserted into the scan
ner in a head-first prone orientation. Spin-echo T1-weighted images were
obtained through the mouse body (neck-to-base of the tail) using a 72-mm
linear volume coil. Scan sequence parameters were the following: TR 1,000 ms,
TE 15 ms, and 2 averages. The field of view was 7.7 3.85 2.0 cm, with a
matrix size of 256 128 40. The corresponding voxel size was 0.3 0.3
0.5 mm. Images were acquired using ParaVision 5.0 software. After acquisi
tion, images were transferred into Invenon Research Workplace 4.0 software
(Siemens Preclinical) allowing fat to be displayed with high intensity (white).
Statistical analysis. Statistical analyses were performed using Prism soft
ware (GraphPad Software Inc.). Data were expressed as mean SEM. For
analysis of three or more groups, a nonparametric ANOVA test was per
formed with a Bonferroni post-hoc test. Analysis of differences between two
normally distributed test groups was performed using the Students t test.
Non-parametric groups were analyzed with the Mann-Whitney test.Welchs
correction was applied to datasets with significant differences in variance be
fore Students t test.

We thank Monja Metcalf and Weihong Ma for technical help.

MRI. To quantify abdominal fat, MR images were acquired in collaboration
with the National Institute on Aging. Images were acquired using a Biospec 7
Tesla 30-cm MRI scanner (Bruker Biospin) with a 72-mm diameter transmit-
receive birdcage coil. In each experiment, two mice were inserted into the
scanner, side by side, in feet-first prone orientation immediately after sacrifice.
During scanning, the mice were cooled with a stream of cold air from a vortex
tube (Exair, Inc.) to minimize decomposition. Two different pulse sequences
were used: a heavily T1-weighted fast spin echo (RARE) sequence yielding
bright-fat (water-suppressed [WS]) images and a fat-suppressed proton density
weighted fast gradient echo (FLASH) sequence yielding fat-suppressed (FS)
images. 3D datasets were acquired in axial orientation with a field of view of
72 36 80 mm (left-right anterior-posterior head-foot) and matrix size
256 128 128.The voxel size (spatial resolution) was 281 281 625 m.
The spectral bandwidth in both sequences was 100 kHz (391 Hz/pixel) and
two averages were acquired for improved signal-to-noise ratio. For the RARE
sequence, the RARE factor (i.e., number of spin echoes per shot or number
of k-space lines per segment) was 8, the repetition time (TR) was 125 ms, the
actual echo time (TE) was 11.4 ms, and the effective echo time (TEeff) was
46.3 ms. Each RARE scan took 8 min and 32 s to acquire. For the FLASH
sequence, the repetition time was 25 ms and the echo time was 3.2 ms. The
excitation pulse had a flip angle of 30. Each FLASH scan took 13 min 39 s to
acquire. Image processing was performed in ImageJ 1.45 (NIH). The statisti
cal analyses were performed using one-way ANOVA and the Newman-Keuls
post-hoc test. P < 0.05 was considered to be significant.
To visualize fat distribution and content imaging of young ob/ob, DIO,
and lean age-matched controls were conducted in collaboration with the
Center for Molecular and Genomic Imaging (CMGI) Facility at the Univer
sity of California, Davis. Mice were anesthetized before imaging using 2%
isoflurane in an induction chamber and then maintained on 12% isoflurane
throughout imaging via a nose cone fitted for anesthetic inhalation. Mice
were imaged using a BioSpec 70/30 7T (Bruker Biospin) horizontal bore
system designed specifically for small animal imaging. Animals were kept at
This work has been supported by NIH grants CA0905572 and AG034874 and
by the Intramural Research Program of the National Institute on Aging, NIH.
The authors declare no competing financial interests.
Author contributions: A. Mirsoian, M.N. Bouchlaka, D.D. Taub, and W.J. Murphy
designed the research; A. Mirsoian, M.N. Bouchlaka, G.D. Sckisel, M. Chen, C.C.S. Pai,
A.M. Monjazeb, and D.D. Taub performed the research; A. Mirsoian, M.N. Bouchlaka,
D.D. Taub, and W.J. Murphy analyzed the data; M. Chen analyzed and scored all
histology data; E. Maverakis provided etanercept (Enbrel) for TNF blockade studies;
R.G. Spencer, K.W. Fishbein, and S. Siddiqui performed and analyzed MRI studies;
B. Martin, C. Hesdorffer, L. Ferrucci, and S. Maudsley provided aged mice and assisted
with data interpretation; A. Mirsoian, M.N. Bouchlaka, D.D. Taub, and W.J. Murphy
wrote the paper; and G.D. Sckisel, D.L. Longo, B.R. Blazar, and R.H. Wiltrout helped with
the editing of the paper. All authors read and approved the manuscript.
Submitted: 18 January 2014
Accepted: 6 October 2014
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2383
INSIGHTS

Cr itical role for CX 3 CR1 + mononuclear phagocytes in

intestinal homeostasis
A key challenge at the intestinal barrier is to minimize responses to commensal bacteria, which can lead to
inflammatory bowel disease (IBD) in genetically predisposed individuals, but retain the ability to recognize
and control the growth of infectious pathogens. Group 3 innate lymphoid cells (ILC3) help maintain intes-
tinal homeostasis by producing the cytokine IL-22, which promotes mucosal healing and maintains barrier
integrity. Microbial signals trigger production of IL-23 and IL-1b, which stimulate ILC3s to produce IL-22,
leading to the induction of antibacterial peptides and epithelial cell regeneration.
But the identity of the cell type producing IL-23 in response to microbial signals is unclear and has
Insight from been the subject of much debate; resident mononuclear phagocytes, inflammatory monocytes, and
Giorgio Trinchieri
conventional dendritic cells have all been implicated. In this issue, Longman et al. provide compelling
evidence, both in mouse following Clostridium rodentium infection and in patients with colitis, that
CX3CR1+ mononuclear phagocytes (MNPs) are the most potent producers of IL-23 and IL-1b and are very efficient in
inducing IL-22 production by ILC3.
The authors demonstrated the importance of microbial stimulation in IL-22 induction
in patients with surgical diversion of the fecal streamIL-22 production by ILC3 was lower in
the sites unexposed to the gut microbiota compared with exposed sites. Microbial TLR4 and
TLR9 agonists were particularly efficient at inducing IL-23 and IL-1b production by CX3CR1+
MNPs. The authors also discovered that a gene significantly associated with both ulcerative
colitis and Crohns disease in GWAS, TNF-like ligand 1A (TL1A or TNFSF15), is overexpressed
in mouse CX3CR1+ MNPs and synergizes with IL-23 and IL-1b to induce IL-22 production
in both human and mouse ILC3.
The study does not completely exclude the role of other cell types in the production of IL-23 and
induction of IL-22 production but in the conditions studied (mouse infection with C. rodentium and
human colitis), CX3CR1+ MNPs were crucial in mediating this mucosal protective loop. In basal
conditions or in other types of inflammatory or preneoplastic conditions, and under stimulation by
different microbial TLR agonists (e.g., flagellin), it is quite possible that other cell types, including
conventional dendritic cells, may play an important role, as suggested by published studies. However, Confocal immunofluorescence
the study by Longman et al. greatly contributes to our understanding of the mechanisms of human image of mouse colon shows
IBD and reassures us that when mouse data are carefully combined with experimental human studies juxtaposition (white arrows)
and GWAS, they are powerful in providing mechanistic evidence that explains human pathology. of CX3CR1+ MNPs (green) and
Longman, R.S., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/jem.20140678. RORgt+ ILC3 (red).

Giorgio Trinchieri, National Cancer Institute, National Institutes of Health: trinchig@mail.nih.gov

2 INSIGHTS | The Journal of Experimental Medicine

 Article

CX3CR1+ mononuclear phagocytes support

colitis-associated innate lymphoid cell
production of IL-22
Randy S. Longman,1,3 Gretchen E. Diehl,1 Daniel A. Victorio,1,3
Jun R. Huh,1 Carolina Galan,1 Emily R. Miraldi,1,5,6 Arun Swaminath,4
Richard Bonneau,5,6 Ellen J. Scherl,3,4 and Dan R. Littman1,2
1The Kimmel Center for Biology and Medicine of the Skirball Institute and 2Howard Hughes Medical Institute, New York
University School of Medicine, New York, NY 10016
3The Jill Roberts Center for IBD, Department of Medicine, Weill-Cornell Medical College, New York, NY 10021
4Division of Digestive and Liver Diseases, Department of Medicine, Columbia University Medical Center, New York, NY 10032
5Center for Genomics and Systems Biology, Department of Biology; and 6Courant Institute of Mathematical Sciences,

Computer Science Department, New York University, New York, NY10003

Interleukin (IL)-22producing group 3 innate lymphoid cells (ILC3) promote mucosal heal-
ing and maintain barrier integrity, but how microbial signals are integrated to regulate
mucosal protection offered by these cells remains unclear. Here, we show that in vivo
depletion of CX3CR1+ mononuclear phagocytes (MNPs) resulted in more severe colitis and
death after infection with Citrobacter rodentium. This phenotype was rescued by exogenous
IL-22, which was endogenously produced by ILC3 in close spatial proximity to CX3CR1+
MNPs that were dependent on MyD88 signaling. CX3CR1+ MNPs from both mouse and
human tissue produced more IL-23 and IL-1 than conventional CD103+ dendritic cells
(cDCs) and were more efficient than cDCs in supporting IL-22 production in ILC3 in vitro
and in vivo. Further, colonic ILC3 from patients with mild to moderate ulcerative colitis or
Crohns disease had increased IL-22 production. IBD-associated SNP gene set analysis
revealed enrichment for genes selectively expressed in human intestinal MNPs. The product
of one of these, TL1A, potently enhanced IL-23 and IL-1-induced production of IL-22
and GM-CSF by ILC3. Collectively, these results reveal a critical role for CX3CR1+ mono-
nuclear phagocytes in integrating microbial signals to regulate colonic ILC3 function in IBD.

CORRESPONDENCE
Randy S. Longman:
ral2006@med.cornell.edu
OR
Dan R. Littman:
dan.littman@med.nyu.edu
Abbreviations used: CD,
Crohns disease; DT, diphtheria
toxin; DTR, DT receptor; IBD,
inflammatory bowel disease;
ILC3, group 3 innate lymphoid
cell; MAMP, microbe-associated
molecular pattern; MNP,
mononuclear phagocyte;
PAMP, pathogen-associated
molecular pattern; UC, ulcer-
ative colitis.
Inflammatory bowel disease (IBD) has been de-
fined as a dysregulated cellular immune response
to environmental triggers in genetically predis-
posed individuals. Although the initial discovery
linking single-nucleotide polymorphisms in the
IL23R locus with susceptibility to IBD (Duerr
et al., 2006) was consistent with a role for IL-23
responsive T cells, more recent evidence supports
the importance of IL-23responsive innate lym-
phoid cells (ILC) in maintaining epithelial ho-
meostasis (Sonnenberg and Artis, 2012). These
RORt-dependent ILCs (now named group 3
ILCs, or ILC3 (Spits et al., 2013)) were initially
R.S. Longman and G.E. Diehl contributed equally
to this paper.
J.R. Huhs present address is University of Massachusetts
Medical School, Worcester, MA 01605.
A. Swaminaths present address is Lennox Hill Hospital,
New York, NY 10075.
characterized in mouse models of colitis as pre-
dominant producers of IL-22 (Satoh-Takayama
et al., 2008), an IL-10 family member that sig-
nals via STAT3 to regulate mucosal healing, a
critical clinical endpoint in IBD (Pickert et al.,
2009; Hanash et al., 2012). In light of their ro-
bust production of IL-22 and close proximity
to the intestinal epithelial layer (Cella et al., 2009),
ILC3 have been proposed to play an important
role in mucosal healing and maintenance of
barrier integrity, and understanding how they
are induced to produce IL-22 has great poten-
tial for therapeutic benefit.
2014 Longman et al. This article is distributed under the terms of an Attribution
NoncommercialShare AlikeNo Mirror Sites license for the first six months
after the publication date (see http://www.rupress.org/terms). After six months
it is available under a Creative Commons License (AttributionNoncommercial
Share Alike 3.0 Unported license, as described at http://creativecommons.org/
licenses/by-nc-sa/3.0/).

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J. Exp. Med. 2014 Vol. 211 No. 8 1571-1583 1571
www.jem.org/cgi/doi/10.1084/jem.20140678
 Mononuclear phagocytes (MNPs) are sentinels of the intes-
tinal lamina propria, capable of responding to microbial prod-
ucts, and play a crucial role in orchestrating intestinal lymphocyte
homeostasis. MNPs can be subdivided based on their expression
of CD103 or CX3CR1, and each group has been ascribed criti-
cal functions in maintaining intestinal homeostasis (Bogunovic
et al., 2009; Merad et al., 2013). CD103+ cells, which can be
further subdivided based on the expression of CD11b, differen-
tiate from a common DC precursor and are thought to be
the conventional, migratory myeloid DCs (Varol et al., 2010).
CD103+ CD11b DCs require Irf8, Id2, and Batf3 for their de-
velopment and are thought to play a critical role in cross-priming
virus- and tumor-specific CTLs (Hildner et al., 2008; Merad
et al., 2013). Loss of these cells, however, does not alter intestinal
T cell homeostasis or lead to spontaneous inflammation (Edelson
et al., 2010). CD103+CD11b+ DCs, in contrast, require Notch2
signaling, produce IL-23 in response to flagellin-induced TLR5
activation, resulting in IL-22 production by ILC3, and have ad-
ditionally been proposed to support Th17 polarization (Lewis
et al., 2011; Kinnebrew et al., 2012). These cells can produce
retinoic acid, which promotes the expression of the gut-homing
receptor CCR9 and synergizes with TGF to induce regulatory
T cells (Sun et al., 2007). One recent study suggests that Notch2-
dependent CD103+ CD11b+ DCs regulate protection from
C. rodentiuminduced colitis (Satpathy et al., 2013). However,
specific depletion of CD103+ CD11b+ intestinal DCs revealed
that these cells are not the MNP subset required for protection
against C. rodentium or IL-22 production (Welty et al., 2013).
In contrast to CD103+ cDCs, CX3CR1+ MNPs differenti-
ate from monocyte precursors (Varol et al., 2010). Although
these cells were previously thought to be tissue-resident and to
promote local Treg differentiation (Hadis et al., 2011), recent data
from our group showed that they can up-regulate CCR7 and
migrate to secondary lymphoid organs, suggesting a broader role
in orchestrating immunity (Diehl et al., 2013). Notably, we ob-
served that interaction with microbiota limits the migration of
these cells to mesenteric LNs (MLNs; Diehl et al., 2013), and an
increase in CX3CR1+ cells has been described in the lamina
propria during mouse (Zigmond et al., 2012) and human colitis
(Kamada et al., 2008). A recent study reported that fractalkine
receptor (CX3CR1) expression supports innate celldependent
clearance of C. rodentium infection (Manta et al., 2013), but a
functional role for CX3CR1+ MNPs in regulating colitis-
associated ILC3 remains unclear.To evaluate this question, we
employed novel mouse models to enable selective depletion of
CX3CR1+ MNPs in vivo. Our results reveal a critical role for
CX3CR1+ MNPs from both mouse and human tissue in sup-
porting IL-22 induction in ILC3 in vitro and in vivo. Moreover,
we identify the ability of TL1A produced by MNPs to potently
enhance IL-23 and IL-1induced production of IL-22 and
GM-CSF by ILC3.
RESULTS
CX3CR1+ cells protect against C. rodentiuminduced colitis
To investigate the role of the expanded population of CX3CR1+
cells in the intestinal lamina propria during colitis, we generated
1572
a mouse with the diphtheria toxin receptor (DTR) cDNA in-
serted into the Cx3cr1 locus (Diehl et al., 2013). Analysis of
colonic lamina propria mononuclear cells (LPMCs) after infec-
tion of DT-treated mice revealed a reduction in the percentage
of CD11c+ MHCII+ LPMCs (Fig. 1 A), which reflected a pref
erential loss of the CX3CR1+ CD11b+ CD14+ fraction of MNPs
(Fig. 1 B; Tamoutounour et al., 2012), as well as CX3CR1+
monocytes in Cx3cr1DTR/+ mice compared with control mice.
CD103+ CD11b+ cDCs were not depleted (Fig. 1 C).To induce
colitis, mice were infected with C. rodentium, a mouse model
for infectious colitis (Zheng et al., 2008; Sonnenberg et al., 2011;
Qiu et al., 2012). DT-treated infected Cx3cr1DTR/+ mice, but
not uninfected or control infected mice, lost more weight
(Fig. 1 D), displayed more severe intestinal pathology (Fig. 1 E),
and ultimately succumbed to infection (Fig. 1 F). Infected
Cx3cr1DTR/+ mice also had increased bacterial burden in the
spleen, consistent with the loss of barrier integrity (Fig. 1 G).
To examine potential involvement of signaling pathways
for receptors of pathogen- or microbe-associated molecular
patterns (PAMPs or MAMPs) in mediating this phenotype, mice
with a conditional deletion of MyD88 in CD11c-expressing
MNPs (CD11c-Cre/Myd88fl/fl) were infected with C. roden-
tium. Infection of CD11c-Cre/Myd88fl/fl mice, but not litter-
mate controls, was lethal by 15 d after infection (Fig. 2 A),
implicating PAMP/MAMP signaling as having a critical role
in barrier protection mediated by CD11c-expressing MNPs.
The C. rodentium colitis model depends on IL-22 for protec-
tion (Zheng et al., 2008). Thus, to test if exogenous IL-22
could rescue the susceptibility phenotype described above,
CD11c-Cre/Myd88fl/fl (Fig. 2 A) and DT-treated CX3CR1-
DTR (Fig. 2 B) mice were hydrodynamically injected with a
plasmid encoding IL-22 (Qiu et al., 2012). The exogenous
IL-22 rescued both lines of mice from colitis-induced death.
Colonic CX3CR1+ MNPs regulate ILC3 production of IL-22
High-dose infection with C. rodentium is controlled by ILC3,
which represents the large majority of LPMCs producing IL-22
(Sonnenberg et al., 2011). At day 7 after infection, both the
percentage and absolute number of IL-22+ colonic, lineage,
CD90hi, and RORt+ ILCs (Fig. S1 shows gating strategy)
from mice depleted for CX3CR1+ cells were reduced in com-
parison to ILCs from mice with intact CX3CR1+ cells (Fig. 2,
CE). Depletion of CX3CR1+ cells did not affect the abso-
lute number of ILC3 (Fig. 2 F). Although T cells can also con-
tribute to IL-22 production in low-dose C. rodentium infection
(Basu et al., 2012), no statistically significant difference in the
total IL-22+ (or IL-17+) T cells was noted in mice depleted
for CX3CR1+ cells (Fig. 2 G). To assess the ability of colonic
CX3CR1+ MNPs to interact with ILC3 within the colonic tis-
sue, Cx3cr1GFP/+ mice were used to visualize CX3CR1+ MNPs
in situ ( Jung et al., 2000). Consistent with the ability of these
cells to regulate ILC3 function, confocal microscopy revealed
the spatial proximity of RORt+ ILC3 cells with CX3CR1+
MNPs in the colonic lamina propria (Fig. 2 H).
To evaluate the ability of intestinal CX3CR1+ cells and
cDCs to support ILC3 activation, CX3CR1+ (GFP+) cells and
CX3CR1+ MNPs regulate ILC3 | Longman et al.

CD103+ CD11b+ DCs were sorted from the lamina propria
of Cx3cr1GFP/+ mice (Fig. 3 A) and co-cultured with intestinal
ILCs. TLR-stimulated CX3CR1+ cells were markedly more
efficient than CD103+ CD11b+ DCs in supporting IL-22 pro-
duction (Fig. 3, B and C). CX3CR1+ cells in the LP include
Ly6Chi monocytes and Ly6Clo MHCIIhi MNPs (Fig. 3 D;
Zigmond et al., 2012; Diehl et al., 2013).To evaluate the role of
these distinct CX3CR1+ populations in supporting ILC3 func-
tion, we sorted CX3CR1+ monocytes and CX3CR1+ MNPs
and co-cultured them with ILCs in the presence of TLR stim-
uli.TLR-stimulated MNPs were much more potent inducers of
IL-22 than monocytes (Fig. 3, E and F). In an effort to confirm
the functional potential of MNPs compared with monocytes
in vivo, we selectively ablated CD11c-expressing CX3CR1+
MNPs. CD11c-cre mice were bred to mice engineered to
JEM Vol. 211, No. 8
Ar ticle
Figure 1. Intestinal CX3CR1+ cells pro-
tect mice from C. rodentium-induced
colitis. (A and B) Depletion efficiency in the
Cx3cr1DTR/GFP mice was assessed by flow
cytometry of small intestinal lamina propria
cells Cx3crDTR/GFP or littermate control mice
after administration of DT to both groups
daily for 2 d. (A) Surface staining for CD11b
versus CX3CR1-GFP. (B) MHCII+ CD11c+ cells
were assessed for expression of CX3CR1,
CD103, and CD14. Results are representative
of five independent experiments with a mini-
mum of three animals per group. (C) Total
number of MHCII+ CD11c+CD103+ (left) and
MHCII+ CD11c+CX3CR1+ cells (right) per intes-
tine as determined by flow cytometry analy-
sis. n.s., P > 0.05; **, P 0.01. Two-tailed
Students t test. Error bars represent the SEM.
Results are representative of five independent
experiments with a minimum of 3 animals per
group. (D) Weight of DT-treated littermate WT
(control) mice or Cx3cr1DTR/+ mice following
infection with C. rodentium (n = 7 mice/
group). DT was administered at days 2, 1,
and 0 and every other day after infection.
Data are representative of two independent
experiments. (E) Representative colonic histol-
ogy from littermate control mice or Cx3cr1DTR/+
mice (analyzed in D) infected with C. rodentium
at day 7 after infection. <, areas of lympho-
cyte infiltration; *, areas of epithelial erosion.
Bar, 100 m. (F) Survival curves of DT-treated
Cx3cr1DTR/+ and littermate control mice in-
fected with C. rodentium or uninfected (n = 7
mice/group). Data are representative of three
independent experiments. Animals were
treated with DT as in D. (G) Bacterial CFUs of
spleens from littermate WT (control, n = 5)
mice or Cx3cr1DTR/+ mice (n = 5) infected with
C. rodentium and treated with DT as above.
*, P 0.05. Two-tailed Students t test. Error
bars represent the SEM. One of two represen-
tative experiments is shown.
express DTR only upon cre-mediated deletion of a LoxP-Stop
cassette inserted into the Cx3cr1 locus (Diehl et al., 2013). In-
jection of DT resulted in a selective loss of Ly6Clo MHCIIhi
MNPs, which express both intermediate and high levels
of CX3CR1-GFP, and spared the Ly6Chi monocytes, which
express intermediate levels of CX3CR1 (Fig. 4 A; Diehl
et al., 2013). Similar to results with CX3CR1DTR/+ mice, in
which both monocytes and MNPs were ablated, depletion of
CX3CR1+ CD11c+ cells led to reduction in colitis-induced
ILC3 production of IL-22 (Fig. 4 B).
Because both IL-23 and IL-1 regulate ILC3, we wished
to determine the contribution of these cytokines to the ob-
served regulation of IL-22 production by MNPs. LPS and CpG
stimulation induced markedly increased IL-23 and IL-1 pro-
duction by CX3CR1+ MNPs compared with CD103+ CD11b+
1573

DCs in vitro (Fig. 4, C and D). To evaluate the role of MNP-
derived IL-23 and IL-1, co-cultures were performed with in-
testinal CD11c+ cells derived from WT or Il23p19/ mice and
ILCs from WT or Il1r/ mice. IL-23deficient MNPs and
IL1R-deficient ILCs yielded significantly reduced production
of IL-22 (Fig. 4 E), consistent with the importance of MNP-
derived IL-1 and IL-23 in supporting IL-22 production.
These data reveal a mechanistic role for IL-23 and IL-1 pro-
duced by colonic MNPs in supporting colitis-associated ILC3
secretion of IL-22.
Human intestinal ILC3 production of IL-22
is regulated by microbial stimulation of MNPs
To evaluate the regulation of intestinal ILC3 in humans with
IBD, we prepared LPMCs from descending colon biopsies of
patients with endoscopically mild to moderate Crohns disease
1574
Figure 2. CX3CR1+ cells support colonic ILC3 pro-
duction of IL-22. (A) Survival curves of C. rodentium
infected Myd88f/f littermate controls (n = 10, open circle)
as compared with CD11c-cre/Myd88f/f mice without
(n = 13, filled circle) or with (n = 5, open triangle) exog-
enous hydrodynamic delivery of an IL-22producing
plasmid. Results are a composite of two independent
experiments. (B) Survival curves of DT-treated Cx3cr1DTR/+
mice infected with C. rodentium after hydrodynamic
delivery of a plasmid expressing IL-22 (n = 8) or control
vector (n = 9). DT was administered at days 2, 1, and 0
and every other day after infection. Results are a com-
posite of three independent experiments. (CE) Percent-
age (C and D) and total number (E) of colonic Lin
CD90.2+ ILCs producing IL-22 from DT-treated Cx-
3cr1DTR/+ (n = 10) and littermate control mice (n = 9) 7 d
after C. rodentium infection and from uninfected mice
(NT; n = 3). Results are a composite of two independent
experiments. DT was administered at days 2, 1, and 0
and every other day after infection. Intracellular IL-22 was
assayed by flow cytometry after 4-h culture. A represen-
tative flow cytometry plot from each group is shown
in C. **, P 0.01. One way ANOVA with Bonferronis
correction. Error bars represent the SEM. (F) Total
number of ILC3 per colon in Cx3cr1DTR/+ (n = 9) or control
(n = 8) mice administered DT. Error bars represent the
SEM. Results are one of three representative experiments.
(G) Total number of IL-17+ or IL-22+ colonic CD4+ T cells
from DT-treated Cx3cr1DTR/+ (n = 10) and littermate con-
trol mice (n = 9) 7 d after C. rodentium infection and
from uninfected mice (n = 3). Intracellular IL-22 and IL-17
was assayed by flow cytometry after 4-h culture.
*, P 0.05. One way ANOVA with Bonferronis correction.
Error bars represent the SEM. Results are a composite of
two independent experiments. (H) Confocal immuno-
fluorescence of colonic samples from Cx3Cr1GFP/+ mice
stained for CD3 and RORt. Bar, 10 m. White arrows
indicate sites of MNP and ILC3 juxtaposition.
(CD, n = 8) or ulcerative colitis (UC, n = 6;Table S1) as well as
age-matched non-IBD control patients undergoing routine
screening colonoscopy (n = 8). Analysis of intracellular cytokine
production revealed significantly increased IL-22 production
in the CD3 fraction of colonic LPMCs from sites of colonic
inflammation in both CD and UC compared with the non-IBD
controls (Fig. 5 A and Fig. S2). In contrast, IL-22 production
by T cells was not significantly different between the groups.
Further characterization of the nonT cells producing IL-22
revealed that a large fraction of these cells expressed c-Kit and
CD56, markers of ILCs (Cella et al., 2009; Fig. 5 B). Consis-
tent with their being ILC3, these cells were lineage negative
(Lin; Fig. S3 A); expressed RORt (Fig. 5 C); were CD45int
CD127+ (Fig. 5 D); expressed CD161, NKp44, and CCR6
(Fig. 5 E), which are phenotypic surface markers of ILC3; and
produced IL-22 in response to IL-23 stimulation (Fig. 5 F). As
CX3CR1+ MNPs regulate ILC3 | Longman et al.

Myd88 deficiency abrogated ILC3 production of IL-22, we
hypothesized that signals from the microbiota could induce
ILC3 to produce IL-22. To investigate this in human tissue, we
evaluated three patients who had a surgical diversion of the
fecal stream (i.e., a diverting ostomy) as part of their therapy
for IBD. Endoscopic biopsies were taken from a site proximal
to the diversion (afferent limb), where the mucosa was exposed
to intestinal microbiota in the fecal stream, and from mucosa
distal to the diversion (efferent limb), that was unexposed to
the fecal stream. ILCs were present at both mucosal locations,
but not in PBMCs from the same donor (Fig. S3 B). In all
three donors, ILCs from tissue exposed to bacteria in the fecal
stream produced substantially more IL-22 compared with
ILCs from unexposed tissue (postdiversion; Fig. 5 G).
JEM Vol. 211, No. 8
Ar ticle
Figure 3. TLR-stimulated CX3CR1+ MNPs
are stronger inducers of ILC3 production
of IL-22 than CD103+ CD11b+ DCs and
monocytes. (AC) CD103 or CX3CR1+ MHCII+
CD11c+ CD11b+ cells were isolated from the
lamina propria of CX3CR1GFP/+ mice (sort strat-
egy shown in A and co-cultured with Lin
RORt-GFP+ ILCs with or without the indi-
cated bacterial TLR ligands or IL-23. IL-22 was
assessed by intracellular staining of CD90.2+
ILCs after 18 h. A representative flow cytom-
etry plot is shown in B. (C) Percent IL-22+ ILCs
is shown from seven independent experi-
ments. **, P 0.01; *, P 0.05. One way
ANOVA with Bonferronis correction. Error
bars represent SEM. (DF) Ly6C+ MHCIIlo
(monocytes) and Ly6C MHCIIhi (MNPs) were
isolated from CX3CR1+ CD11b+ lamina propria
cells (sort strategy is shown in D and co-
cultured with intestinal ILCs with LPS or IL-23
as indicated. Intracellular cytokine staining for
IL-22 is shown after 18 h (E). Supernatants
were harvested after 18 h and IL-22 produc-
tion quantitated by ELISA (F). Results are rep-
resentative of two independent experiments
performed in triplicate. ***, P 0.001. One-
way ANOVA with Bonferroni correction. Error
bars represent the SEM.
Parallel subpopulations of CD11c+ MNPs present in the
mouse intestine similarly exist in the human intestine (Fig. 6 A;
Merad et al., 2013). To evaluate whether these distinct sub-
populations of MNPs from human intestinal tissue functioned
similarly, we examined the phenotypic properties of CD103+
DCs and CD14+ MNPs (which express CX3CR1; Kamada
et al., 2008) within the CD11c+ MHCII+ fraction of LPMCs
(Fig. 6 B). In contrast to CD103+ DCs, CD14+ MNPs ex-
pressed CD64 as well as higher levels of CD86. Consistent with
the phenotypic characterization of these subsets, transcriptional
analysis of these populations by RNA-seq revealed higher
levels of CLEC9A, XCR1, and CD207 expression in the
CD103+ cells, whereas MERTK, STAB1, and CX3CR1 were
higher in the CD14+ cells (Fig. 6 C). We tested the potential
1575
Figure 4. CX3CR1+ MNP-derived IL-23 and IL-1 activate ILC3 to produce IL-22. (A) Phenotype analysis of colonic LPMCs from Cx3cr1STOP-DTR/GFP
mice with or without CD11c-cre after DT injection for 2 d. (top) Selective depletion of CX3CR1hi MNPs. (bottom) Expression of Ly6C and MHCII on
CX3CR1hi and CX3CR1int populations. (B) Expression of IL-22 in Lin CD90.2+ colonic ILCs from Cx3cr1STOP-DTR/+ (Stop-DTR) or CD11c-Cre x Cx3cr1STOP-DTR/+
(Cre-DTR) mice at 7 d after C. rodentium infection. DT was administered at days 2, 1, and 0 and every other day postinfection. One representative
intracellular cytokine flow cytometry plot is shown on the left and a composite graph (n = 6/group) on the right. *, P 0.05, two-tailed Students t test.
Error bars represent the SEM. Results are a composite of two independent experiments. (C) Supernatants from APC-ILC co-cultures (Fig. 3, B and C) were
harvested after 18 h and assayed for IL-23 by ELISA. Results are averages of three independent experiments and the SEM is shown. (D) CX3CR1+ MNPs or
CD103+ CD11b+ DCs were sorted and incubated with media or CpG for 18 h and supernatants were assayed for IL-1 by ELISA. Results are the mean of
two independent experiments performed in duplicate and the SEM is shown. *, P 0.05; **, P 0.01. (E) Lin CD90.2hi ILCs from WT or Il1r/ mice were
co-cultured with sorted intestinal MNPs from WT or Il23p19/ mice, with or without CpG, as indicated. IL-22 production by the ILCs was assessed after
18 h by ELISA. Data are combined from three independent experiments performed in duplicate. *, P 0.05; ***, P 0.001. One-way ANOVA with Bonferroni
correction. Error bars represent the SEM.

of these subsets to induce IL-22 production by co-culturing
TLR-stimulated CD14+ MNPs and CD103+ DCs from human
intestinal resections with intestinal ILCs. Intracellular cyto-
kine staining at 18 h revealed that the CD14+ MNP were more
effective than the CD103+ DCs at stimulating IL-22 production
by ILCs (Fig. 6 D). Neither cell population induced signifi-
cant IL-17 or IFN- production by ILCs (Fig. 6, D and E).
Consistent with the importance of IL-23 and IL-1 in
the mouse co-culture experiments, a higher level of IL23A
expression was observed by RNA-seq in CD14+ MNPs com-
pared with CD103+ DCs (Fig. 6 C). Stimulation of these human
MNP subsets with LPS or flagellin revealed increased IL-23p19
mRNA and IL-1 protein produced by the CD14+ cells com-
pared with the CD103+ cells (Fig. 6 F). In accord with this
finding, in co-cultures of human intestinal ILC3 and CD14+
MNPs, IL-23 and IL-1 antibody blockade blunted IL-22
production (Fig. 6 G).These data reveal a mechanistic role for
the expanded population of CD14+ MNPs (Kamada et al.,
2008) in supporting colitis-associated IL-22 production by
ILC3, through their secretion of IL-23 and IL-1.
CX3CR1+ MNP-derived TL1A synergizes
with IL-23 and IL-1 to induce IL-22
We hypothesized that MNP-derived factors in addition to
IL-23 and IL-1 would contribute to the regulation of ILC3
function. The RNA-seq data from CD14+ human MNPs re-
vealed a significant enrichment for genes associated with IBD in
GWAS studies, suggesting that IBD-associated pathways are im
portant in these cells (Fig. 7 A and Table S2). Notably, we iden
tified TNF-like ligand 1A (TL1A, also designated TNFSF15)
as a significant contributor to the IBD GWAS-derived gene
set enrichment. Evaluation of Tnfsf15 transcript in sorted
mouse colonic APC subsets by qPCR confirmed higher ex-
pression in CX3CR1+ MNPs (Fig. 7 B). DR3/TNFRSF25,

1576 CX3CR1+ MNPs regulate ILC3 | Longman et al.

 Ar ticle

Figure 5. Increased ILC3 production of IL-22 in mild to moderate IBD correlates with presence of fecal stream. (A) LPMCs isolated from de-
scending colon biopsies from patients with endoscopically mild to moderate Crohns disease (n = 8, gray) or ulcerative colitis (n = 6, black; Table S1),
as well as age-matched non-IBD control patients undergoing routine screening colonoscopy (n = 8, white), were stimulated ex vivo with PMA/ionomycin
and evaluated by intracellular cytokine staining for expression of IL-17 and IL-22. The percentage of CD3+ or CD3 fraction expressing IL-17 or IL-22 is
indicated. *, P 0.05, two-tailed Students t test. Black bars represent the geometric mean. (B) Expression of c-Kit and CD56 in electronically gated CD3
IL-22+ (black lines) and CD3 IL-22 (gray) LPMCs. (C) Expression of RORt by c-Kit+CD56+ LPMCs. Lin cells (CD14/CD19/CD3/CD11b/CD11c/TCR;
Fig. S3 A) were stained with antibodies to surface markers c-Kit and CD56 and to intracellular RORt. Lin CD56+ c-Kit+ ILC3 (black line) were compared
with Lin CD56+ c-Kit NK cells (gray) for RORt expression. (D) Surface staining of Lin c-Kit+ ILCs for the indicated markers (black line) compared with
isotype control (gray) and all live LPMCs (dotted line) (E) LPMCs stained for intracellular IL-22 after stimulation with IL-23 for 3 h (solid line) or with con-
trol media (dotted line). Cells shown were gated on Lin CD56+ c-Kit+. The isotype control is in gray. (F) CD11c+ MHCII+ human colonic APCs were elec-
tronically gated for expression of CD103 and CD14. One of three donors is shown. (G) Lamina propria cells from biopsy samples of tissue exposed
(prediversion) or not exposed to the fecal stream (post-diversion) were cultured for 3 h and ILC3 production of IL-22 was assessed by flow cytometry.
(left) Result from one representative donor. (right) Percentage of IL-22+ ILCs in afferent (Pre) and efferent (Post) limbs of three diverted patients.
**, P 0.01, two-tailed Students t test. Black bars represent the geometric mean.

the receptor for TL1A, is expressed both on T cells and ILC
subsets, with the highest expression on ILC2 and ILC3 (Meylan
et al., 2014). Ex vivo stimulation with mouse or human re-
combinant TL1A significantly enhanced the ability of IL-23
and IL-1 to induce IL-22 by both mouse (Fig. 7 C and Fig. S4)
and human (Fig. 7 D) intestinal ILC3, respectively. TL1A and
IL-1 also cooperated in inducing GM-CSF production by
mouse ILC3 (Fig. 7 E). To assess the specificity of TL1A for
DR3/TNFRSF25 on ILCs and its functional role in enhancing
CX3CR1+ support of ILC3 activation, DR3 expression was
specifically knocked down using siRNA nucleofection of sorted
mouse intestinal ILC3. Efficiency of knock-down was con-
firmed by surface staining for DR3, comparing with a scram-
bled control siRNA (Fig. 7 F). Compared with the scramble
control, cells targeted with siRNA for Tnfrsf25 had signifi-
cantly reduced enhancement of IL-22 production upon treat-
ment with recombinant TL1A (Fig. 7, G and H).The targeted
ILCs also produced reduced amounts of IL-22 upon co-culture
with LPS-stimulated CX3CR1+ MNPs. These data reveal a
potent ability of TL1A to enhance IL-22 production via DR3/
TNFRSF25 on ILC3 in both mouse and human.
DISCUSSION
Mononuclear phagocytes are spatially and functionally poised
to integrate microbial signals from the luminal microbiota
(Niess et al., 2005; Varol et al., 2010). Although MNPs were
previously thought to remain in the tissue, we recently showed
that CX3CR1+ MNPs can migrate to draining lymph nodes
and initiate immune responses under conditions of dysbiosis
(Diehl et al., 2013). In the context of inflammation, however,
these CX3CR1+ MNPs expand within the lamina propria dur-
ing chemical (Zigmond et al., 2012) or infectious colitis and
in IBD patients (Kamada et al., 2008), and their function has
remained obscure. Conventional DCs (cDCs), rather than the
MNPs, have been postulated to regulate intestinal Th17 cell
differentiation in response to microbiota (Denning et al., 2011;

JEM Vol. 211, No. 8 1577

Figure 6. Human ILC3 production of IL-22 is supported by IL-23 and IL-1 produced by TLR-stimulated CD14+ and CX3CR1+ MNPs.
(AC) HLA-DR+ CD11c+ cells from intestinal resection tissue were sorted into CD103+ DCs and CD14+ MNPs subpopulations and transcriptional profiles
were assessed by RNA-seq. (A) Sorting strategy. (B) Each subset was examined for expression of the indicated cell surface markers. Isotype controls are
shown in gray. One of three donors is shown. (C) Heatmap of relative expression of relevant MNP-related genes. Values represent the mean of two inde-
pendent donors, and an asterisk denotes individual genes differentially expressed at an FDR = 0.01. (D and E) Induction of IL-22 in human ILCs in co-
culture with CD14+ MNPs or CD103+ DCs in the presence of media alone, LPS, or flagellin, as indicated. c-Kit+ cells were examined for intracellular IL-22
production after 18-h culture. Data are representative of five independent experiments. (F) CD14+ MNPs or CD103+ DCs sorted from human intestine (as
in A) were stimulated with the indicated TLR ligands for 18 h and qPCR or cytometric bead array analysis were used to quantitate IL-23p19 and IL-1,
respectively. Results are averaged from three independent donors, and technical replicates were performed in duplicate or triplicate, respectively. *, P 0.05.
N.D., not detected. Two-tailed Students t test. Error bars represent the SEM. (G) Sorted human intestinal CD14+ MNPs and ILCs were left unstimulated or
were co-cultured in the presence of LPS with or without neutralizing antibodies against IL-1 and IL-23. IL-22 ELISA was performed after 18h. Results
are averaged from two independent donors performed in duplicate. *, P 0.05. Two-tailed Students t test. Error bars represent the SEM.

Lewis et al., 2011; Persson et al., 2013; Schlitzer et al., 2013;
Goto et al., 2014), but there has been conflicting evidence as
to which myeloid cell populations regulate IL-22 produc-
tion by ILC3 cells (Kinnebrew et al., 2012; Manta et al., 2013;
Satpathy et al., 2013).
Our data reveal that colonic CX3CR1+ MNPs regulate
ILC3 production of IL-22 and likely play a critical role in pro-
moting mucosal healing during colitis. The requirement for
CX3CR1+ MNPs in regulating colitis-induced ILC3 shown
here appears to contrast with recent work suggesting that
Notch2-dependent CD103+ CD11b+ cDCs are required for
the control of C. rodentiuminduced colitis (Satpathy et al., 2013).
These disparate results may reflect a coordinated contribution
of multiple myeloid subsets in the immune response to C. ro-
dentium (Schreiber et al., 2013), and our data do not exclude a
contribution of other myeloid subsets. Alternatively, Notch2
may affect cellular subpopulations in addition to CD103+
CD11b+ cDCs. This latter hypothesis is supported by the re-
port that an alternate selective depletion strategy for CD103+
CD11b+ cDCs, by expression of DT under the human Lan-
gerin promoter (Welty et al., 2013), did not result in increased
susceptibility to C. rodentium. Some of the conflicting data
may additionally reflect differential roles of inflammatory mono-
cytes and MNPs in colitis. Inflammatory monocytes may ex-
acerbate pathology, as antibody-mediated depletion of Ly6Chi
monocytes during DSS treatment improved pathology in DSS

1578 CX3CR1+ MNPs regulate ILC3 | Longman et al.

 Ar ticle

Figure 7. CX3CR1+ MNP-derived TL1A synergizes with IL-23 and IL-1 to induce IL-22 in intestinal ILC3. (A) Gene-set enrichment analysis
was used to determine whether the indicated disease-related SNP were differentially expressed between CD14+ MNPs and CD103+ DCs. Significance was
estimated using the hypergeometric cumulative distribution, with a raw p-value cutoff of 0.05 for differential expression. Data were averaged from two
independent donors. (B) B cells (CD3 CD19+), CX3CR1+ MNPs, CD103+ DCs, and Ly6C+ monocytes were sorted from the intestinal lamina propria of
Cx3cr1GFP/+ and quantitative PCR for TL1A was performed. Relative quantitation was performed by Ct normalized to GAPDH expression. Data are from
two biological replicates performed with two technical replicates. *, P 0.05. Two-tailed Students t test. (CE) Sorted intestinal ILCs from mice (C and E)
or cultured human intestinal ILCs (D) were stimulated with media alone, IL-1, or IL-23 with or without TL1A as indicated for 18 h. Brefeldin was added
to the cultures 4 h before intracellular cytokine staining for IL-22 (C and D) or GM-CSF (E). Data are representative of six independent experiments.
(FH) Sorted intestinal ILCs were transfected with siRNA targeting Tnfrsf25 or a scramble control. (F) Knockdown efficiency was assessed after 24 h by
flow cytometry comparing scramble control (solid line) with Tnfrsf25 siRNA. One of two representative experiments is shown. ILCs were then cultured
with media alone (-) or IL-23 and TL1A or co-cultured with CX3CR1+ MNPs with or without LPS as indicated for an additional 18 h. (G) IL-22 production
was measured by intracellular flow cytometry. Brefeldin was added to the cultures 4 h before intracellular cytokine staining. Data are representative
of two independent experiments. (H) IL-22 secretion by samples from G were assessed by ELISA, performed in duplicate, before addition of Brefeldin.
*, P 0.05; **, P 0.01. Two-tailed Students t test. Error bars represent SEM.

colitis (Zigmond et al., 2012). Selective depletion of such
monocytes may hence explain the intermediate results seen
in the CCR2-deficient mice exposed to C. rodentium (Satpathy
et al., 2013). In addition to supporting ILC3, MNPs may also
directly promote epithelial barrier repair (Wynn et al., 2013).
Although our data suggest a critical role for CX3CR1+ MNPs
in regulating production of IL-22 by ILC3 during acute coli-
tis, it will be important to examine the role for these myeloid
subsets during chronic colitis and their impact on another major
clinical endpoint in IBDtumorigenesis (Huber et al., 2012;
Kirchberger et al., 2013).
Our results also highlight a beneficial role for microbial sig-
nals in promoting intestinal homeostasis and driving ILC3 pro-
duction of IL-22 through TLR/MyD88-dependent induction
of IL-23 and IL-1. At steady state, commensal-dependent sig-
naling may negatively regulate ILC production of IL-22 via in-
testinal epithelial cell production of IL-25 (Sawa et al., 2011),
but we and others (Satoh-Takayama et al., 2008) find that
microbes support colitis-associated ILC production of IL-22,
which promotes mucosal healing. Although intravenous de-
livery of flagellin can induce CD103+ CD11b+ cDCs to produce
IL-23 that regulates ILC3 in the small intestine (Kinnebrew
et al., 2012), we and others (Kamada et al., 2008) found that
in vivo colonic inflammation and other bacterial-derived signals
induced more robust production of IL-23 and IL-1 by MNPs,
suggesting that the type of stimulation and/or colonic inflam-
mation may confer specificity. Similar to our results in mice,
we found marked reductions in IL-22 production by intestinal

JEM Vol. 211, No. 8 1579

ILC3 from human tissue distal to a surgical diversion of the
fecal stream.These findings may offer clinically relevant mech-
anistic insight into diverted IBD patients with persistent in-
flammatory disease or even non-IBD patients with de novo
mucosal inflammation after diversion (e.g., diversion colitis;
Harig et al., 1989). Although some evidence supports a role
for luminal replacement of short chain fatty acids in diversion
colitis (Harig et al., 1989;Vernia et al., 1995), further work is
required to determine if particular metabolites or intestinal
microbes may regulate ILC3 function to promote healing.
In light of their robust production of IL-22 and close
proximity to the intestinal epithelial layer (Cella et al., 2009),
ILC3 have been proposed to play an important role in muco-
sal healing and maintenance of barrier integrity, but their char-
acterization in IBD patients has been limited. ILCs producing
IL-17 or IFN- (classified as ILC1) have also been described in
mouse models of colitis (Buonocore et al., 2010; Klose et al.,
2013) and human IBD (Geremia et al., 2011; Bernink et al.,
2013), supporting a potentially important role for ILCs in IBD
pathogenesis. Here, we observed a specific increase in IL-22
production by ILCs from colonic biopsies of patients with mild
to moderate CD and UC. Despite characteristically distinct
phenotypes, both CD and UC share genetic susceptibility risk
alleles within the IL-23 pathway (Duerr et al., 2006), which
may underlie a common role for ILCs during colonic inflam-
mation in both diseases. In contrast to previous studies, we did
not observe any significant IL-17 production by CD3 cells
from patients with CD (Geremia et al., 2011) or a reduction
in ILC3 in CD patients (Bernink et al., 2013). This difference
may reflect clinical differences in the patient cohorts and
samplescolonoscopic biopsies in our cohort were taken from
ambulatory patients with mild to moderate colonic CD and UC,
in contrast to surgical resection of ileal tissue from medically
refractory patients in the other studies. As such, mild to mod-
erate colitis may be the ideal human model to evaluate ILC3-
producing IL-22 promoting mucosal healing. The association
of clinical phenotype with ILC phenotype and the potential
plasticity of these ILCs in colitis remain to be evaluated.
Consistent with our results in mouse models, CX3CR1+
MNPs from human intestinal tissue were more potent than
CD103+ DCs in supporting human intestinal ILC3 produc-
tion of IL-22. In addition to the increased production of IL-23
and IL-1 by the CX3CR1+ subset described by us and oth-
ers (Kamada et al., 2008), RNA seq analysis of these subsets
revealed enrichment for transcripts from genes having IBD-
associated SNPs, notably TL1A/TNFSF15. Polymorphisms
in TNFSF15, the gene that encodes TL1A, are strongly asso-
ciated with IBD, and expression of both TL1A and its recep-
tors DR3 and DcDR3 is elevated in IBD patients (Kugathasan
et al., 2008). TL1A has been shown to enhance Th17 differen-
tiation and effector function (Pappu et al., 2008; Kamada et al.,
2010); promote Treg expansion and ameliorate allergic asthma
(Schreiber et al., 2010); and promote IL-13 production by
ILC2s (Meylan et al., 2014). Interestingly, microbial stimula-
tion and Fc-receptor engagement induces TL1A/TNFSF15
in monocytes and monocyte-derived mononuclear phagocytes
1580
(Shih et al., 2009).The data shown here reveal a potent ability
of TL1A to enhance ILC3 production of IL-22 and suggest
that the increased expression of TL1A by colonic CX3CR1+
MNPs enables their selective ability to potently support ILC3.
Furthermore, the ability of TL1A to induce GM-CSF pro-
duction by ILC3 is consistent with recent data suggesting
that, even at steady state, CSF1R-dependent MNPs support
ILC3 production of GM-CSF that, in turn, regulates oral tol-
erance (Mortha et al., 2014). Collectively, these data support a
broader role for colonic CX3CR1+ MNPs in regulating mu-
cosal homeostasis.
Mucosal ILC3 play a crucial role in barrier homeostasis,
mucosal healing, and oral tolerance. CX3CR1+ MNPs are aptly
poised to integrate microbial signals to regulate ILC3 and
may serve as important targets for therapeutic manipulation.
Further understanding of the contributions of discrete commen-
sal bacterial species and of the mechanistic interactions be-
tween CX3CR1+ MNPs and ILC3 may provide novel strategies
to promote intestinal healing. In light of the specific and po-
tent regulation of ILC3 by colonic CX3CR1+ MNPs during
colitis, diagnostics that correlate susceptible genotypes (Hueber
et al., 2012) with clinical disease immunophenotype will pro-
vide insight into therapeutic strategies to manipulate colonic
MNPs in the clinical management of IBD.
MATERIALS AND METHODS
Antibodies and flow cytometry. Staining of human cells was performed
with c-Kit-e450 (104D2), CD56-PECy5.5 (CMSSB), HLA-DR-PE (LN3),
CD11c-FITC (3.9), CD3-e780 (UCHT1), CD19-PerCP5.5 (HIB19), CD103
PECy7 (B-Ly7), CD14 PE (61D3), CD86-PE (IT2.2), CD123 PE (6H6),
CD11b PE (CBRM1/5), and RORt (AFKJS-9) obtained from eBiosci-
ence; CD161-FITC, CD83-PE, CD11c PE (555392), CD127 FITC (M21),
CCR6-biotin (11A9) were purchased from BD; BDCA-1-APC were ob-
tained from Miltenyi Biotec; CD45-APC (4505) and CD64 (6404) were ob-
tained from Invitrogen; NKp44-APC (p44-8.1) was purchased from R&D
Systems; and BDCA-3 APC (M80) was purchased from BioLegend. Intra
cellular human cytokine staining was performed with IL-22 PE (IC7821P;
R&D Systems), IL-17A-FITC (eBio64CAP17; eBioscience), or IFN- (45.
B3; eBioscience). Staining of mouse cells was performed with CD90.2-e450
(532.1), CD3e (145-2C11), NKp46-e710 (29A1.4), RORt-PE (B2D),
CD11c-PECy7 (N418), MHCII A700 (M5/114.15.2), CD103 APC (2E7),
CD14 PerCP5.5 (Sa2-8), CD11b e780 (M1/70), Ly6C PerCP5.5 (AL-21),
and F4/80 PE (BM8) were from eBioscience. DR3-PE (4C12) was obtained
from BioLegend. Intracellular mouse cytokine staining was performed with
IL-22 APC (IL22JOP), IFN- PECy7 (XMG1.2), IL17A FITC (eBio17B7),
all from eBioscience. Intracellular cytokine staining was performed according
to the manufacturers protocol (Cytofix/Cytoperm buffer set; BD). Intra
nuclear staining for RORt was performed according to manufacturers protocol
(Intracellular Fixation and Permeabilization kit; eBioscience). Flow cytome-
try and analysis were performed with a LSR II (BD) and FlowJo software
(Tree Star). Dead cells were excluded using the Live/Dead fixable aqua dead
cell stain kit (Invitrogen).
Mice. C57BL/6, Myd88flox, CD11c-cre (Caton et al., 2007), and Il1r/ mice
were purchased from The Jackson Laboratory. Myd88-deficient mice were
obtained from S. Akira (Osaka University, Osaka, Japan; Adachi et al., 1998)
and Il23p19/ mice were obtained from D. Cua (Merck Research Labora-
tories, Palo Alto, CA; Cua et al., 2003). RORc(t)GFP/+ (Eberl et al., 2004),
Cx3cr1GFP/+ (Jung et al., 2000) and inducible CX3CR1-DTR mice (Diehl et al.,
2013), were previously described. The latter mice were subsequently bred to
a germline-expressing cre mouse to excise the transcriptional stop sequence
CX3CR1+ MNPs regulate ILC3 | Longman et al.

and produce the Cx3cr1DTR/+ mice. All mice were kept in specific pathogen
free (SPF) conditions at the animal facility of the Skirball Institute. Mouse
experiments were performed with at least three mice per group and multiple
experiments were combined to assess statistically significant differences as
noted. Littermates of the same genotype were randomly assigned to experi-
mental groups. Animals were used between 8 and 16 wk of age. Males and
females were used in approximately equal ratios. All animal experiments were
performed in accordance with approved protocols for the NYU Institutional
Animal Care and Usage Committee.
Preparation of LPMCs. Endoscopic biopsies were obtained under IRB-
approved protocols at Weill Cornell Medical College (1103011578 and Co-
lumbia University Medical Center (AAAE5448) including patients >18 yr of
age and able to give informed consent. IBD sample was defined based on en-
doscopic inflammation with history of ulcerative colitis or Crohns disease.
Endoscopic score (mild, moderate, or severe) was based on Mayo Endoscopic
subscore or SES-CD (1, 2, 3, respectively) at the site of biopsy (Pineton de
Chambrun et al., 2010). All endoscopic biopsies were taken from the sigmoid
or descending colon to reduce sampling variation. Study sample sizes for
human biopsies were based on preliminary data and powered to achieve sta-
tistically significant differences in the production of IL-22 or IL-17. Surgical
resections were obtained under an IRB-approved protocol at New York Uni-
versity Langone Medical Center. Mouse and human intestines were washed
in PBS and 1 mM DTT twice with 30 mM EDTA, and then digested in col-
lagenase 8 (Sigma-Aldrich) and DNase-containing media with 10% fetal bo-
vine serum. Digested material was passed through a cell strainer and separated
on a discontinuous 40%/80% Percoll gradient. LPMCs were cultured ex vivo
in the presence of GolgiPlug (BD) for 4 h or stimulated with phorbol my-
ristate acetate (PMA; 20 ng/ml) and ionomycin (1 g/ml) or IL-23 (40 ng/ml;
eBioscience) in the presence of GolgiPlug (BD) for 4 h before staining.
Intestinal ILC cultures. Surgical resections were obtained from the NYU
Biorepository (Rachel Brody). Lin c-Kit+ CD45int ILCs were sorted and
cultured in tissue culture media (RPMI 1640; Invitrogen) supplemented with
10% (vol/vol) heat-inactivated FBS (HyClone), 50 U penicillin-streptomycin
(Invitrogen), 2 mM glutamine, and 50 M -mercaptoethanol), supplemented
with IL-7 (50 ng/ml; PeproTech) and IL-2 (1,000 U/ml; PeproTech) for 810 d
before stimulation. Lin, CD90.2+, RORt-GFP mouse ILC3 were sorted
from LPMCs and resuspended in RPMI-based tissue culture media for stim-
ulation directly ex vivo. Human and mouse ILCs were stimulated with human
or mouse IL-23 (eBioscience; 40 ng/ml), IL-1 (eBioscience; 10 ng/ml), or
TL1A (R&D Systems; 200 ng/ml) as indicated, respectively. After 18 h, super-
natants were harvested for IL-22 ELISA (eBioscience) and Golgi Plug (BD)
was added to cells for 4 h for subsequent intracellular cytokine staining.
Co-culture assay. ILCs and APC populations were sorted on a FACSAria
and co-cultured with 5 103 and 2.5 103 cells, respectively, in 96-well
round-bottom plates in tissue culture media.TLR stimulation was performed
with 1 g/ml LPS (Escherichia coli; Sigma-Aldrich), 1 M CpG 1668 (mouse),
1 M CpG 2216 (human), or 1 g/ml flagellin (Salmonella Typhi; InvivoGen).
Cultures were incubated for 18 h. Supernatants were harvested for ELISA
and remaining cells were incubated with Golgi Plug (BD) for 4 h and sub
sequently analyzed by flow cytometry.
siRNA transfection. Sorted intestinal ILCs were cultured overnight in IL-7
(20 ng/ml) and SCF (20 ng/ml). After 24 h, 4 105 ILCs were transfected
using AMAXA T cell nucleofection protocol. 300 pmol of Tnfrsf25 siRNA
pool or scramble control (Thermo Fisher Scientific) was used per transfec-
tion. Cells were rested overnight and harvested at 24 h for experimental use.
Knockdown efficiency was assessed at 24 h by DR3 surface staining.
Colitis models. C. rodentium DBS100 (ATCC 51459; American Type Culture
Collection) was harvested at log phase growth and 1010 CFU were delivered by
gavage in PBS. 200 ng of DT was administered i.p. as indicated for depletion.
Plasmid DNA expressing IL-22 or control plasmid (Qiu et al., 2012) were
JEM Vol. 211, No. 8
Ar ticle
delivered i.v. at 5 g DNA/mouse diluted in TransIT-EE Hydrodynamic Deliv-
ery Solution (Mirus) at 0.1 ml/g body weight. Immune cell functional analysis
and histology were performed at day 7 after exposure. Spleens were harvested on
day 21, homogenized, and plated at serial dilutions to determine CFU/spleen.
RNA-seq processing and gene set enrichment analysis. Sequence reads
were mapped to the human genome (version hg19) by Tophat (version 2.0.6),
using Bowtie2 (version 2.0.2) and Samtools (version 0.1.18). Reads are depos-
ited at Bioproject PRJNA219394. Reads mapped per transcript served as input
to DESeq (version 1.12.0), an R package that calculates differential gene ex-
pression. To improve detection of APC lineage-dependent gene-expression
changes and overcome donor-dependent variability, we used the following
strategy: independently for each donor, differential gene expression, comparing
CD103+ to CD14+ human myeloid cells, was estimated using the negative bi-
nomial distribution (nbinomTest), and then results from biological replicates
were combined using Fishers method. Several gene set enrichment techniques
(e.g., hypergeometric test and area under precision-recall curve) were used to
test whether specific gene sets (Table S2) were significantly differentially regu-
lated between the two lineages.The GWAS gene sets are derived from disease-
associated SNPs from the NHGRI GWAS Catalog. False-discovery rates
(FDRs) were calculated using the Benjamini-Hochberg procedure. The en-
richment analyses were implemented in Matlab R2013a (8.1.0.604).
qPCR. RNA from primary intestinal APCs stimulated as indicated was pre-
pared with TRIzol (Invitrogen). RNA was reverse transcribed into cDNA
(SuperScript III; Invitrogen) and QPCR was performed with a Roche Light-
Cycler with SYBR Green Supermix (Bio-Rad Laboratories), 20 pmol forward
and reverse primers, and 0.1 g of cDNA from 5-TGTTCCCCATATC-
CAGTGTGG-3 and 5-CTGGAGGCTGCGAAGGATTT-3 for human
IL23p19, 5-ATGCTTCGGGCCATAACAGA-3 and 5-TGAAGGC-
CATCCCTAGGTCA-3 for mouse TL1A; 5-ACCACAGTCCATG
CCATCAC-3 and 5-TCCACCACCCTGTTGCTGTA-3 for human
GAPDH; and 5-AATGTGTCCGTCGTGGATCT-3 and 5-CATCGA
AGGTGGAAGAGTGG-3 for mouse GAPDH. The thermocycling pro-
gram was 40 cycles at 95C for 15 s, 60C for 30 s, and 72C for 30 s, with
an initial cycle of 95C for 2 min. Relative levels of target gene were deter-
mined by using the delta Ct value compared with delta Ct (GAPDH).
Immunofluorescence. Intestinal tissue was Swiss-rolled before fixing for 4 h
in 4% paraformaldehyde. Tissue was incubated overnight in 30% sucrose be-
fore freezing in OCT. Tissue was cut into 5-M sections. Tissue was blocked
in PBS-XG (0.1% Triton X-100, 10% goat serum) before incubating over-
night in primary antibody in PBS-XG. Tissue was washed and then incu-
bated with secondary antibody for 1 h before DAPI staining. The following
primary antibodies are from eBioscience: antihuman/mouse RORt (clone
AFKJS-9) and antimouse CD3e (clone 145-2C11). Secondary antibodies
are from Jackson ImmunoResearch Laboratories (Cy3-AffiniPure goat antirat
IgG and goat antiArmenian hamster). Tissue was imaged using an LSM 710
confocal (Carl Zeiss) and images were processed using ImageJ.
Online supplemental material. Fig. S1 shows the gating strategy for co-
lonic ILC3. Fig. S2 shows increased production of IL-22 in ILCs from pa-
tients with IBD. Fig. S3 shows Lin gating and surface phenotype for human
intestinal ILC3. Fig. S4 shows that TL1A enhances IL-23 and IL-1 induc-
tion of IL-22 by intestinal ILC3s. Online supplemental material is available at
http://www.jem.org/cgi/content/full/jem.20140678/DC1.
We thank Rachel Brody (NYU Biorepository), Fatiha Chabouni, Priyanka Patel, Ryan
Warren and members of The Roberts Center for IBD for help with sample collection
as well as the patients that participated in this study. We would like to thank
Michael Cammer for assistance with microscopy.
This work was supported by the American Gastroenterological Association
Research Foundation (R.S. Longman), National Institutes of Health K08 DK099381
(R.S. Longman), American Cancer Society (G.E. Diehl), NIH T32 CA009161 (G.E. Diehl),
K99DK091508 (J.R. Huh), and the Howard Hughes Medical Institute (D.R. Littman).
The authors declare no competing financial interests.
1581
Author contributions: R.S. Longman and G.E. Diehl designed and performed the
experiments. R.S. Longman, G.E. Diehl and D.R. Littman planned experiments and
wrote the manuscript with input from the co-authors. J.R. Huh, C. Galan, and
D.A. Victorio helped plan and perform experiments. E.R. Miraldi and R. Bonneau
performed data analysis. A. Swaminath and E.J. Scherl helped with sample collection
and experimental design.
Submitted: 9 April 2014
Accepted: 18 June 2014
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1583
INSIGHTS

Monocytes find a new place to dwell in the niche of

heartbreak hotel
Monocytes and macrophages belong to the mononuclear phagocyte system (MPS). The con-

Lambrecht Photo courtesy of

cept of the MPS is based on the idea that circulating monocytes represent the immediate
precursors of tissue macrophages. Recent genetic fate mapping of myeloid cells caused a con-
ceptual frameshift, as it was found that many macrophages are derived from embryonic pre-
cursors that seed the tissues before birth and then self-maintain without any significant

www.vib.be
contribution from circulating monocytes. In this new view, monocytes merely represent an
emergency squad that can be rapidly recruited to sites of inflammation to provide a transient
Insight from Bart Lambrecht (left)
supplement of inflammatory macrophages to the local tissue-resident macrophage pool of and Martin Guilliams (right)
embryonic origin.
In this issue, Molawi et al. challenge this new concept by reporting that cardiac macrophages are initially of embryonic
origin but are progressively replaced by monocyte-derived cells. These newcomer macrophages lack the capacity to self-renew
and are slowly but constantly replaced by circulating monocytes. In this regard, the heart resembles the skin and intestine.
There, macrophages are also mainly derived from circulating monocytes. Because the skin and the intestine are barrier sites with
an important influence of the microbiome, a continuous low-grade inflammation could explain continuous recruitment of
circulating monocytes. Molawi et al. report that cardiac macrophages of embryonic origin gradually lose the capacity to self-
renew with age and hypothesize that this allows circulating monocytes to occupy the heart niche and join the cardiac macrophage
pool. Why embryonic macrophages would lose their self-renewal capacity in the heart but not in the liver, the lung, or the
spleen remains to be determined. Another unexplored hypothesis is that continuous microtrauma induced by cardiac contraction
is the driver of monocyte influx to the sterile heart.
Tissue-resident macrophages are astonishingly diverse in terms of gene expression and are adapted to perform specific functions
in their tissue of residence. The unique functions of cardiac macrophages remain largely unknown. Molawi et al. describe
four subsets of cardiac macrophages based on the expression of CX3CR1 and MHC class II (see figure). Embryonic macrophages
preferentially give rise to MHC class IIlow cells, while most monocyte-derived macrophages express high levels of MHC class II.
This implies a different capacity to
interact with lymphocytes and distinct
functional adaptations for embryonic
macrophages. Macrophages play an im-
portant role in wound healing, and many
cardiovascular diseases are associated with
poor or exaggerated tissue repair. It will
therefore be interesting to investigate
whether embryonic macrophages are
better at tissue repair compared with
monocyte-derived macrophages, and
whether the presence of the embryonic
pool explains the superior regeneration
capacity of the heart at a young age.
Manipulation of macrophage self-renewal
might yield important therapeutic appli-
Cardiac macrophages derived from embryonic progenitors gradually lose their capacity to cations to increase macrophage popula-
self-renew and are continually replaced in adulthood by macrophages derived from circulating tions involved in tissue homeostasis and
monocytes, even in the absence of inflammation. repair, while avoiding the recruitment of
macrophages that fuel inflammation.
With these exciting prospects and new concepts, macrophage biology is going through a revival period, and this study
certainly contributes to that. Given the breadth of tissue-specific macrophage heterogeneity, both in terms of functional specialization
and cellular origin, we have many years ahead until we fully understand the cell that initiated immunology research more than
a century ago.
Molawi, K., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/jem.20140639.

Bart Lambrecht and Martin Guilliams, VIB Inflammation Research Center, UGent: bart.lambrecht@irc.vib-Ugent.be and martin.guilliams@irc.ugent.be
 Brief Definitive Report

Progressive replacement of embryo-derived

cardiac macrophages with age
Kaaweh Molawi,1,2,3,4 Yochai Wolf,5 Prashanth K. Kandalla,1,2,3
Jeremy Favret,1,2,3 Nora Hagemeyer,6 Kathrin Frenzel,6 Alexander R. Pinto,8
Kay Klapproth,9 Sandrine Henri,1,2,3 Bernard Malissen,1,2,3
Hans-Reimer Rodewald,9 Nadia A. Rosenthal,8,10 Marc Bajenoff,1,2,3
Marco Prinz,6,7 Steffen Jung,5 and Michael H. Sieweke1,2,3,4
1Centre dImmunologie de Marseille-Luminy (CIML), Aix-Marseille Universit, UM2, 13288 Marseille, France
2Institut National de la Sant et de la Recherche Mdicale (INSERM), U1104, 13288 Marseille, France
3Centre National de la Recherche Scientifique (CNRS), UMR7280, 13288 Marseille, France
4Max-Delbrck-Centrum fr Molekulare Medizin (MDC), Robert-Rssle-Strasse 10, 13125 Berlin, Germany
5Department of Immunology, The Weizmann Institute of Science, 7610001 Rehovot, Israel
6Institute of Neuropathology and 7BIOSS Centre for Biological Signaling Studies, University of Freiburg, 79106 Freiburg, Germany
8Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton 3800, Victoria, Australia
9Division of Cellular Immunology, German Cancer Research Center (DKFZ), D-69120 Heidelberg, Germany
10National Heart and Lung Institute, Imperial College London, London SW7 2AZ, England, UK

Cardiac macrophages (cM) are critical for early postnatal heart regeneration and fibrotic
repair in the adult heart, but their origins and cellular dynamics during postnatal develop-
ment have not been well characterized. Tissue macrophages can be derived from embryonic
progenitors or from monocytes during inflammation. We report that within the first weeks
after birth, the embryo-derived population of resident CX3CR1+ cM diversifies into
MHCII+ and MHCII cells. Genetic fate mapping demonstrated that cM derived from
CX3CR1+ embryonic progenitors persisted into adulthood but the initially high contribution
to resident cM declined after birth. Consistent with this, the early significant prolifera-
tion rate of resident cM decreased with age upon diversification into subpopulations.
Bone marrow (BM) reconstitution experiments showed monocyte-dependent quantitative
replacement of all cM populations. Furthermore, parabiotic mice and BM chimeras of
nonirradiated recipient mice revealed a slow but significant donor contribution to cM.
Together, our observations indicate that in the heart, embryo-derived cM show declining
self-renewal with age and are progressively substituted by monocyte-derived macrophages,
even in the absence of inflammation.

CORRESPONDENCE
Michael H. Sieweke:
sieweke@ciml.univ-mrs.fr
Abbreviations used: cM,
cardiac macrophages; HSC,
hematopoietic stem cell;
YS, yolk sac.
Cardiovascular disease represents a leading
cause of death in the developed world, and
many pathologies result from insufficient repair
of cardiac injury. Mammalian wound healing
mechanisms in the adult heart involve scar for-
mation, but for a short time window after birth
the neonatal heart maintains full regeneration
capacity of cardiac tissue (Porrello et al., 2011),
a process which requires macrophages (Aurora
et al., 2014). Studies of cardiac repair after myo-
cardial infarction in the adult have highlighted
the critical role of infiltrating monocytes and
monocyte-derived macrophages for the healing
K. Molawi and Y. Wolf contributed equally to this paper.
Steffen Jung and M.H. Sieweke contributed equally to this paper.
process (Nahrendorf et al., 2007; Swirski et al.,
2009). The focus of these studies has been on
monocyte-derived cells, consistent with the tra-
ditional view that macrophages are part of the
mononuclear phagocyte system and monocyte-
derived (van Furth and Cohn, 1968; Geissmann
et al., 2010). Recent evidence, however, sug-
gests that monocyte contribution to macro-
phages only represents an emergency pathway,
as many tissue macrophage populations get
seeded in the developing embryo and can self-
maintain without major monocyte contribution
2014 Molawi et al. This article is distributed under the terms of an Attribution
NoncommercialShare AlikeNo Mirror Sites license for the first six months
after the publication date (see http://www.rupress.org/terms). After six months
it is available under a Creative Commons License (AttributionNoncommercial
Share Alike 3.0 Unported license, as described at http://creativecommons.org/
licenses/by-nc-sa/3.0/).

The Rockefeller University Press $30.00

J. Exp. Med. 2014 Vol. 211 No. 11 2151-2158 2151
www.jem.org/cgi/doi/10.1084/jem.20140639
Figure 1. cM develop into 4 subpopulations after birth. (A and B) Cytometry analysis of cM from adult CX3CR1GFP/+ mice. (A) FACS profiles of
cM populations, pregated on living single CD11b+ cells with low CD11c and Ly6C expression. (B) Mean of total and subpopulations of cM in absolute
numbers (left) or as percentage of total (right) as determined by bead-normalized flow cytometry. Error bars represent SEM (n = 4). (C and D) Cytometry
analysis (C) and mean percentage (D) of cM subpopulations from CX3CR1GFP/+ mice of indicated age. Error bars represent SEM (n = 34). (E) MHCII and
MHCII+ cM from adult CX3CR1cre:R26-yfp mice (black line) were analyzed for YFP expression and compared with Cre littermate controls (gray area;
n = 34). Data in all panels are representative of at least two independent experiments.

(Chorro et al., 2009; Ginhoux et al., 2010; Hoeffel et al.,
2012; Schulz et al., 2012; Hashimoto et al., 2013;Yona et al.,
2013). Macrophages can massively expand in the tissue by
local proliferation in response to challenge (Jenkins et al.,
2011; Hashimoto et al., 2013; Sieweke and Allen, 2013) and
can extensively self-renew without loss of differentiated func-
tion in culture upon inactivation of MafB and cMaf tran-
scription factors (Aziz et al., 2009). This has led to the
proposition that tissue macrophages may have a long-term
self-renewal capacity akin to that of stem cells (Sieweke and
Allen, 2013).
Examples of tissue macrophages that are independent
from monocytes are microglia in the brain and epidermal
Langerhans cells (Ajami et al., 2007; Chorro et al., 2009;
Ginhoux et al., 2010; Hoeffel et al., 2012). In contrast, intesti-
nal or dermal macrophages have a high turnover rate and are
constantly replaced from Ly6C+ blood monocytes (Zigmond
et al., 2012; Tamoutounour et al., 2013). Thus, origin and
turnover of tissue macrophages are highly tissue specific and
need to be assessed individually for different organ systems
(Sieweke and Allen, 2013).
Resident macrophages can be found in all tissues, where
they fulfill a variety of tissue-specific functions contributing
to homeostasis, development, and regeneration (Davies et al.,
2013), making them prominent candidates for therapeutic in-
tervention.This holds in particular for the heart, where tissue-
resident cardiac macrophages (cM) have been described as
CX3CR1+ cells that can be found throughout the myocar-
dium (Pinto et al., 2012). A recent study identified several
cM populations, including short-lived monocyte-derived
cells that resemble monocyte-derived DCs as well as tissue-
resident cM, which were suggested to be primarily of embry-
onic origin and to self-maintain under homeostatic conditions
(Epelman et al., 2014).

2152 Replacement of embryonic cardiac macrophages with age | Molawi et al.


Here, we examined the origin and turnover of tissue-
resident cM during postnatal development. Using genetic
lineage tracing, parabiotic mice, and unconditioned BM chi-
meras, we demonstrate that embryo-derived cM are gradu-
ally replaced by monocyte-derived macrophages with age and
in the absence of inflammation or injury. This replacement is
mirrored by dynamic changes in the resident cM population
composition and decreasing cM self-renewal over time.
RESULTS AND DISCUSSION
We focused on the resident cM population and applied a
rigorous flow cytometry gating strategy to exclude potential
infiltrating leukocytes (Fig. S1, AC). Tissue-resident macro-
phages were identified as positive for the core macrophage
signature markers CD14, CD64, and MerTK (Gautier et al.,
2012; Tamoutounour et al., 2013) and the classical macro-
phage marker F4/80 (Fig. 1 A). The chemokine receptor
CX3CR1 is widely expressed in the mononuclear phagocyte
system (Jung et al., 2000) and cM have been reported to be
CX3CR1-positive (Pinto et al., 2012). Therefore, we refined
our analysis of the global cM population by testing CX3CR1
expression in CX3CR1GFP/+ knock-in mice (Jung et al.,
2000). Additionally, we included MHCII in our analysis,
which can be differentially expressed on tissue macrophages
JEM Vol. 211, No. 11
Br ief Definitive Repor t
Figure 2. Decreased contribution of embryo-
derived macrophages to cM with age.
(A) CX3CR1creER:R26-yfp embryos were treated with
TAM on E9 or E13 and analyzed on the day of delivery
(P0) or 6 wk after delivery (P42). (B) Percentage of
microglia-normalized YFP+ cM and representative
cytometry plots pregated on total cM at P0 and
P42 after E13 labeling. Bars show median (n = 913).
***, P 0.005, Mann-Whitney test. Data were pooled
from two independent experiments. (C) Representa-
tive cytometry plot showing YFP+ within total cM
at P42 and percentage of microglia-normalized YFP+
cM at P0 and P42 after E9 labeling. Bars show
median (n = 7) *, P 0.05. Data were pooled from
three independent experiments. (D) Representative
cytometry plots showing YFP+ cells within MHCII
and MHCII+ cM at P0 and P42 after E13 labeling
(n = 913). (E) Quantification of YFP+ cells within
MHCII and MHCII+ cM at P42 after E13 labeling.
Bars show the median (n = 913). ***, P 0.005,
Mann-Whitney test. Data were pooled from two
independent experiments. (F) Adult TAM-treated
CX3CR1creER:R26-yfp mice were analyzed for YFP+
cM and microglia at 1 and 4 wk after treatment.
Percentage of microglia-normalized YFP+ cM is
shown as median with extreme samples as error
bars (n = 3). Representative cytometry plots
show YFP+ within total cM. Unless otherwise
indicated data in all panels are representative of
two independent experiments.
(Tamoutounour et al., 2013). We found that in 8-wk-old
mice, the majority of cM was CX3CR1+ (80%) and
MHCII+ (70%), allowing the delineation of four distinct
cM subpopulations (Fig. 1, A and B). Additional populations
of CD11c+MHCII+ or Ly6C+ cells, referred to as cM
by others (Epelman et al., 2014), were excluded from our
analysis (Fig. S1 B), as data from other tissues suggest that
these are monocytes and monocyte-derived dendritic cells
(Tamoutounour et al., 2013). Consistent with this, these cells
have been shown to be short-lived and monocyte-derived
(Epelman et al., 2014).
To investigate the development of the four cM popula-
tions, we analyzed CX3CR1 and MHCII expression in cM
from newborn to 30-wk-old mice (Fig. 1, C and D). We ob-
served that almost all embryo-derived cM present at birth
were CX3CR1+MHCII. This homogenous cM compart-
ment diversified with age into four subpopulations with a pro-
gressive increase of MHCII+ cM and a decrease of CX3CR1+
cM. Importantly, genetic fate mapping analysis using
CX3CR1cre mice crossed to R26-yfp reporter mice (CX3CR1cre:
R26-yfp; Yona et al., 2013) revealed that all adult cM sub-
populations must have developed from a CX3CR1+ stage
(Fig. 1 E). These results suggested two not mutually exclusive
possibilities for cM subpopulation development: persistence
2153
Figure 3. Decreased proliferation rate of embryo-derived cM with age. (A) cM subpopulations of adult CX3CR1GFP/+ mice were analyzed for
BrdU incorporation and expression of Ki67 by flow cytometry four hours after BrdU injection (i.p.). Bars show median (n = 1213). *, P 0.05; **, P 0.01;
***, P 0.005; Wilcoxon test. (B and C) Total (B) or CX3CR1+MHCII (C) cM of CX3CR1GFP/+ mice of indicated age were analyzed for BrdU incorporation
and Ki67 expression by flow cytometry 4 h after BrdU injection (i.p.). Bars show median (n = 415). ***, P 0.005, Mann-Whitney test. Data in all panels
were pooled from 23 independent experiments.

and diversification of embryo-derived cM as suggested else-
where (Epelman et al., 2014), or replacement of embryo-
derived cM by adult monocyte-derived macrophages.
To address these alternatives, we first analyzed the persis-
tence of embryo-derived cM by genetic lineage tracing using
CX3CR1-driven tamoxifen (TAM)-inducible Cre recom-
binase (CreERT2) and R26-yfp reporter mice (CX3CR1creER:
R26-yfp; Fig. 2 A; Yona et al., 2013). Tamoxifen-induced
pulse labeling at E9 permits us to identify cells derived from
yolk sac (YS) CX3CR1+ macrophages before the onset of
definitive hematopoiesis. Labeling at E13 cannot distinguish
YS- and hematopoietic stem cell (HSC)derived macro-
phages, but should increase cM labeling because the heart
is colonized by CX3CR1+ macrophages at this time (Epelman
et al., 2014). cM labeling was normalized to YFP+ mi-
croglia, which are YS-derived, remain CX3CR1+ throughout
development, self-maintain without contribution of HSC-
derived cells (Ginhoux et al., 2010; Kierdorf et al., 2013),
and therefore represent an internal control for maximal
CX3CR1 labeling efficiency. E13 labeling revealed that rela-
tive cM labeling declined from 35% in newborn mice
to 18% in 6-wk-old mice (Fig. 2 B), indicating a loss of
embryo-derived cM with age. E9 labeling showed that
embryo-derived cM were at least partially of YS origin and
similarly declined from 14% in neonates to 4% in 6-wk-
old mice (Fig. 2 C). Interestingly, all embryo-derived YFP+
cM were MHCII at birth but had partially differentiated
into MHCII+ cM after 6 wk (Fig. 2 D). Although this
demonstrated that both populations can develop from cells of
embryonic origin, the relative contribution of embryo-derived
YFP+ cM to MHCII+ cM was significantly lower than to
MHCII cM, (Fig. 2 E).
We further assessed the turnover of CX3CR1+ cM in
the adult heart by pulse labeling adult CX3CR1creER:R26-yfp
(Fig. 2 F). 1 wk after treatment, labeling in cM corresponded
to 60% of microglia labeling but drastically decreased to
20% after 4 wk. By comparison, microglia labeling was
nearly complete after 1 wk and remained unchanged there-
after (Goldmann et al., 2013). Collectively, our lineage tracing
experiments argue that a significant proportion of macro-
phages established in early embryonic development is still
present at birth but is gradually lost with age.
The relatively fast turnover of labeled tissue-resident cM
suggested that local self-renewal was insufficient to maintain
the resident cM pool. We therefore analyzed the prolifera-
tive activity of cM subpopulations in adult mice by BrdU
incorporation after a 4-h pulse labeling period and by Ki67
staining (Fig. 3 A). The overall number of proliferating mac-
rophages in adult hearts was low, particularly in MHCII+
populations, but the proliferative rate of CX3CR1+MHCII
cM significantly exceeded that of all other subpopulations.
Interestingly, this population was also the only cM subset
present at birth but declined with age at the expense of the other
cM populations (Fig. 1, C and D). We therefore analyzed the
evolution of cM proliferation with age. Both BrdU incor-
poration and Ki67 staining showed a high proliferative rate
in newborn CX3CR1+MHCII cM, which gradually de-
creased by 810-fold both in total cM (Fig. 3 B) and in the
CX3CR1+MHCII cM population (Fig. 3 C). Together,
these data suggest that both a successive loss of the cell popu-
lation with the highest proliferative rate (CX3CR1+MHCII
cM) and a strong decrease of the proliferation rate collectively
result in progressively decreasing self-renewal of the tissue-
resident cM pool.

2154 Replacement of embryonic cardiac macrophages with age | Molawi et al.

 Br ief Definitive Repor t

Figure 4. Monocytes contribute to four cM subpopulations in adult mice. (A) WT mice were lethally irradiated and reconstituted with BM from
Ubow:CX3CR1GFP/+ mice. cM were analyzed for contribution of dTomato+ cells at indicated time points after reconstitution and graft-derived cells were
analyzed for CX3CR1 and MHCII expression. Data are presented as mean percentage SEM (n = 37) and derived from two independent experiments.
(B) Mixed BM chimeras were generated by reconstituting lethally irradiated WT mice (CD45.1/CD45.2) with BM from CCR2/:CX3CR1GFP/+ (CD45.2) and
WT CX3CR1GFP/+ (CD45.1) mice. cM, circulating CD11b+CD115+ monocytes (Ly6C+ and Ly6C) and B220+ B cells were analyzed for WT (CD45.1) and
CCR2/ (CD45.2) contribution. Host- and graft-derived cM were analyzed for the ratio of MHCII+/MHCII cM. Bars show median (n = 4). *, P 0.05,
Mann-Whitney test. (C) Parabiosis was established between adult CCR2/ (CD45.2) and WT (CD45.1) mice. After 10 wk, CCR2/ mice were analyzed for
contribution of CD45.1+ non-host cells to cM (total, MHCII+, and MHCII), circulating monocytes (Ly6C+ and Ly6C), and B cells. For CD45.1 host and
CD45.1+ non-host cM, the ratio of MHCII+/MHCII cM was calculated and compared. Bars show median (n = 4). **, P 0.01, Mann-Whitney test.
(D) BM chimeras were generated by transfer of LT-HSC isolated from panYFP mice into nonirradiated Rag2/c/KitW/Wv mice. cM (total, MHCII+, and
MHCII) and neutrophils (Gr1hi and SSChi) were analyzed for contribution of grafted YFP+ cells 8 wk after transplantation. Bars show median (n = 6).
**, P 0.01, Mann-Whitney test. Data in all panels are representative of two independent experiments.

We next addressed the question of how the age-dependent
loss of embryo-derived macrophages is compensated. To test
the ability of monocytes to contribute to cM populations,
we generated BM chimeras using WT hosts and BM from
transgenic mice expressing dTomato from a ubiquitously ex-
pressed human Ubiquitin-C promoter (Ubow mice; Ghigo
et al., 2013) and GFP from the CX3CR1 locus that makes it
possible to monitor grafted cells in all four cM populations.
Graft-derived dTomato+ cM contributed to all four cM
populations from 4 wk after transplantation and almost com-
pletely replaced host cM populations within 8 wk (Fig. 4 A).
However, a small radio-resistant population (10%) persisted
for 36 wk without monocyte contribution. To further analyze
whether cM replacement was monocyte-dependent, we
generated mixed chimeras with BM from WT (CD45.1)
and CCR2/ (CD45.2) CX3CR1GFP/+ mice in CD45.1/
CD45.2 double-positive WT hosts (Fig. 4 B). CCR2/ mice
have reduced numbers of Ly6C+ monocytes in the blood
(Serbina and Pamer, 2006) and can therefore be used to ana-
lyze the contribution of circulating monocytes and CCR2-
dependent progenitors to tissue macrophage populations (Yona
et al., 2013; Tamoutounour et al., 2013). Blood analysis indeed

JEM Vol. 211, No. 11 2155

confirmed that the vast majority of monocytes but not B cells
were of WT origin. Graft-derived cM were almost com-
pletely derived from WT cells (Fig. 4 B). Collectively these
two experiments established that HSC-derived CCR2-
dependent cells, most likely Ly6C+ monocytes, have the capac-
ity to differentiate into all cM subsets. Residual host-derived
cM included both MHCII+ and MHCII cM but showed
a lower proportion of MHCII+ cells than grafted monocyte-
derived cM (Fig. 4 B).
To examine monocyte contribution to cM populations
under homeostatic conditions, we generated parabiotic cou-
ples of WT (CD45.1) and CCR2/ (CD45.2) mice and
analyzed non-host contributions after 10 wk of parabiosis in
CCR2/ partners (Fig. 4 C). Circulating B cells were equally
distributed between the partner mice. In the CCR2-deficient
host Ly6C+ monocytes had reached 70% non-host chime-
rism. About 16% of total cM in CCR2-deficient mice were
of non-host origin. Given a 70% chimerism in Ly6C+ mono-
cytes, the likely cells of origin, and the possibility that de-
pending on lifetime and exchange rate cM may not be fully
equilibrated after 10 wk, these observations indicated a signif-
icant monocyte contribution to tissue-resident cM. Again,
non-host contribution to MHCII+ cM (30%) was signifi-
cantly higher compared with MHCII cM (5%) as high-
lighted by MHCII+/MHCII cM ratios for non-host and
host-derived cells (Fig. 4 C).
As a complimentary experimental approach that does not
depend on myeloablative conditioning, we used Rag2/c/
KitW/Wv mice, which are universal HSC recipients that accept
grafts without prior irradiation (Waskow et al., 2009), and
generated BM chimeras by transplantation of HSC from
panYFP mice (Luche et al., 2013). More than 90% of neutrophils
were YFP+ after 8 wk, demonstrating efficient reconstitution
(Fig. 4 D). Analysis of cM revealed 7% YFP+ cells in the
total population. Again, donor cells showed a higher contribu-
tion to MHCII+ (8% YFP+) than to MHCII cM (4%
YFP+) (Fig. 4 D). In the same mice, no donor contribution
to microglia cells was observed (K. Klapproth and H.R.
Rodewald, personal communication). Together, these experi-
ments clearly demonstrated a slow but significant replacement
of tissue-resident cM by infiltrating monocyte-derived mac-
rophages, which preferentially contributed to MHCII+ cM.
In the present study, we have analyzed homeostasis, origin,
and turnover of resident macrophages in the heart during
postnatal development. We have shown that the cM com-
partment is undergoing dynamic changes with age. Embryo-
derived macrophages present in newborn mice are all CX3CR1+
MHCII and at least partially YS-derived. After birth, the
cM compartment diversifies into four subpopulations
defined by MHCII and CX3CR1 expression. Our results
show that this diversification is due both to differentiation
of persisting embryo-derived CX3CR1+MHCII cM and
infiltrating monocyte-derived macrophages, which both can
contribute to all four cM subpopulations. However, mono-
cytes preferentially give rise to MHCII+ cM, whereas embryo-
derived or radio-resistant cM had a higher proportion of
2156
MHCII cells, explaining the relative increase of MHCII+ cM
and the reduction of the CX3CR1+MHCII cM subpopu-
lation with age.The dynamic change of the cM subpopulation
distribution therefore appears to reflect the gradual replace-
ment of embryo-derived cM by monocyte-derived macro
phages. The increasing contribution of monocyte-derived
macrophages to the cM pool with age may be explained by
the decreasing self-renewal of embryo-derived cM. Although
CX3CR1+MHCII cM that contain the majority of em-
bryo-derived macrophages have a higher proliferative rate than
the other subpopulations, overall cM self-renewal is decreas-
ing with age and is insufficient to sustain the cM pool with-
out input from infiltrating monocytes. It will be interesting to
determine whether the loss of self-renewal capacity in cM is
permanent or might be reactivated. The observed mechanism
of cM homeostasis differs from other macrophage popula-
tions such as microglia, Langerhans cells, or alveolar macro-
phages, which in the absence of tissue damage or inflammation
appear to self-maintain in tissues without replacement from
circulating cells. It is also distinct from the high turnover rate of
dermal tissue macrophages or intestinal lamina propria macro-
phages that are constantly replenished from circulating mono-
cytes (Zigmond et al., 2012; Tamoutounour et al., 2013). In
contrast to what was proposed elsewhere (Epelman et al., 2014),
our results also show that inflammation or injury are not neces-
sarily required for replacement of embryo-derived cardiac
tissue macrophages by monocytes. It will be interesting to
determine whether dynamic age-dependent changes in resi-
dent tissue macrophage populations also occur in other tissues
and whether they may be important for the differences in re-
pair mechanisms and regenerative capacity observed between
young and old individuals.
MATERIALS AND METHODS
Mice. CD45.1 and CD45.2 C57BL/6 mice were obtained from Charles
River. All transgenic mice used in this study have a C57BL/6 background
(>7 backcrosses). CX3CR1GFP/+ mice (Jung et al., 2000), CCR2/, Rosa-26-
yfp, CX3CR1cre (JAX Stock No. 25524 B6.C-Cx3cr1<tm1.1(cre)Jung>/J), and
CX3CR1creER mice (JAX Stock No. 20940 B6J.129-Cx3cr1<tm1.1(cre/
ERT2)Jung>/J; Yona et al., 2013), Ubow mice (Ghigo et al., 2013), Rag2/c/
KitW/Wv mice (Waskow et al., 2009), and panYFP mice (Luche et al., 2013)
were described elsewhere. Experiments were performed on 610-wk-old
mice, if not stated otherwise. Experiments involving CX3CR1cre and
CX3CR1creER mice included littermate controls. For other experiments
C57BL/6 mice served as controls. All mouse experiments were performed
under specific pathogen-free conditions. Animals were handled according
to protocols approved by the Weizmann Institute Animal Care Committee
(Israel), the Federal Ministry for Nature, Environment and Consumers Pro-
tection of the state of Baden-Wrttemberg (Germany) or the animal ethics
committee of Marseille (France) and were performed in accordance to the
respective international, national, and institutional regulations.
Parabiosis. 9-wk-old C57BL/6 CD45.1 mice were sutured together with
9-wk-old B6 CCR2/ CD45.2 and subsequently kept under Bactrim for
10 wk before analysis.
BM chimeras. 710-wk-old host animals were lethally irradiated and were
reconstituted with donor BM by i.v. injection of minimum 106 BM cells.
Donor BM was isolated from femurs and tibias of donor mice, filtered through
a 70-m mesh and resuspended in PBS for i.v. injection. Rag2/c/KitW/Wv
Replacement of embryonic cardiac macrophages with age | Molawi et al.

mice were reconstituted with long-term (LT)HSC from panYFP BM
without prior conditioning as previously described (Waskow et al., 2009).
LT-HSCs were defined as lineage-negative (B220, CD3e, CD4,
CD8a, CD11b, CD19, Gr1, Ter119, NK1.1), Kit+, Sca1+, CD48, Fondation pour la Recherche Mdicale (DEQ. 20071210559 and DEQ.
and CD150+.
Tamoxifen treatment. Tamoxifen (TAM; Sigma-Aldrich) was dissolved in
100% ethanol to get a 1 g/ml solution and was 10-fold diluted in corn oil
(Sigma-Aldrich) to get the 100 mg/ml final solution for oral or i.p. adminis-
tration. To induce Cre-mediated gene recombination in 5- to 7-wk-old
CX3CR1creER mice, 5 mg TAM was administrated orally for 5 consecutive
days (25 mg total). For Cre induction in the embryo, 200 l of 20 mg/ml
TAM and 10 mg/ml Progesterone dissolved in corn oil was injected i.p. into
pregnant females at 9 or 13 days post coitum (dpc).
BrdU pulsing. BrdU was purchased from Sigma-Aldrich. Mice were in-
jected with 0.1 mg BrdU/g body weight. Newborn mice were injected sub-
cutaneously, older mice intraperitoneally. cM isolated from BrdU injected
mice were analyzed 4 h after injection for BrdU incorporation using the
BrdU Flow kit (BD).
Flow cytometry. Cells were acquired on FACSCanto, LSRII, and LSRFor
tessa systems (BD) and analyzed with FlowJo software (Tree Star). The fol-
lowing antibodies were used for staining cells: anti-CD11b (clone M1/70;
eBioscience), anti-CD11c (clone N418; eBioscience), anti-CD14 (clone
Sa2-8; eBioscience), anti-F4/80 (clone BM8; eBioscience), anti-MHCII
(clone M5/114.15.2; eBioscience), anti-CD45.1 (clone A20; eBioscience),
anti-CD45.2 (clone 104; eBioscience), anti-B220 (clone RA3-6B2; eBiosci-
ence), anti-CD115 (clone AFS98; eBioscience), anti-CD117 (clone 2B8;
eBioscience), anti-Sca1 (clone D7; eBioscience), anti-CD48 (clone JM48-1;
eBioscience), anti-Gr1 (clone RB6-8C5; BioLegend), anti-CD64 (clone
X54-5/7.1; BioLegend), anti-Ly6G (clone 1A8; BioLegend), anti-CD150
(clone TC15-12F12.2; BioLegend), anti-Ly6C (clone AL21; BD), anti-
Ki67 (clone B56; BD), anti-CD4 (clone H129.19; BD), anti-CD8 (clone
536.7; BD), anti-CD19 (clone ID3; BD), Ter119 (clone Ter119; BD),
NK1.1 (clone PK136; BD), anti-F4/80, anti-MerTK (clone BAF591; R&D
Systems), anti-BrdU (clone MoBU-1; Life Technologies). Before staining,
cells were preincubated with Fc receptor blocking antibody (clone 2.4 G2;
BD). LIVE/DEAD Fixable Aqua Dead Cell Stain kit (Life Technologies)
was used to stain dead cells. Absolute cell numbers were determined by
using Flow-Count Fluorosperes (Beckman Coulter) according to the manu
facturers instructions.
Preparation of tissue samples. Peripheral blood leukocytes were isolated
by density separation using Lympholite-M solution (Cedarlane) according to
manufacturers instructions. For preparing single cell suspension from heart
and brain, the mice were perfused with PBS before collecting the tissue. For
cM, the heart was macerated and incubated with 1 mg/ml collagenase-2
(Worthington Biochemical Corporation) and 0.15 mg/ml DNase1 (Sigma-
Aldrich) at 37C for 30 min during constant agitation. Resulting cell suspen-
sion was filtered through a 70-m mesh and erythrocytes were removed by
ACK lysis. For microglia isolation a gradient of 70, 37, 30% Percoll was used.
The brain tissue was homogenized and incubated with HBSS solution con-
taining 2% BSA (Sigma-Aldrich), 1 mg/ml collagenase d (Roche), and
0.15 mg/ml DNase1, filtered through a 70-m mesh, and resuspended in
70% Percoll, before density centrifugation (800 g for 30 min at 20C with
low acceleration and no brake).
Online supplemental material. Fig. S1 depicts the full gating strategy and
population characterization of cM. Online supplemental material is avail-
able at http://www.jem.org/cgi/content/full/jem.20140639/DC1.
We thank Dr. Sandrine Sarrazin for critical reading of the manuscript and help
with statistical analysis, L. Razafindramanana for animal handling, and M. Barad,
A. Zouine, and S. Bigot for cytometry support.
JEM Vol. 211, No. 11
Br ief Definitive Repor t
K. Molawi was supported by an HFSP long-term fellowship. The work
was supported by grants to M.H. Sieweke from Aviesan ITMO IHP exploratory
Project A12194AS and CNRS PICS program No. 5730. M.H. Sieweke is a
20110421320) and INSERM-Helmholtz group leader. The work was further
supported by the Israel Science Foundation (ISF), the Deutsche Forschungs
gemeinschaft (DFG) Research Unit (FOR) 1336 (S. Jung and M. Prinz), and
DFG-SFB 938-project L (H.-R. Rodewald and K. Klapproth).
The authors declare no competing financial interests.
Submitted: 6 April 2014
Accepted: 19 August 2014
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Replacement of embryonic cardiac macrophages with age | Molawi et al.
INSIGHTS

M acrophages der ived from infiltrating monocytes mediate

<doi>10.84/jem2nsight1<do>ajem.218insght</ad>uMicelT.Hnka</>AF1UiverstyofBn</AF1>crmihael.nk@ub-ode</cr>haInsigt</doce> piNws</doct>

autoimmune myelin destruction

Macrophages mediate myelin destruction in multiple sclerosis (MS), but the origin of these cells (whether de-
< ID >j e m .2 1 8i n s gh t f j p e < / ID>

rived from tissue-resident microglial cells or infiltrating monocytes) has been widely debated. Now, Yamasaki
and colleagues distinguish these cells in a mouse model of MS and show that monocyte-derived macrophages
(MDMs) mediate myelin destruction, whereas microglia-derived macrophages (MiDMs) clear up the debris.
Previous attempts to decipher the nature and role of cells involved in autoimmune demyelination have
proven challenging. Although ontogenetically distinct, it has not been possible to distinguish macrophages
derived from tissue-resident or -infiltrating cells based on morphological features (by light microscopy) or
surface phenotype. Previous attempts to address this problem include parabiosis and bone marrow trans-
Insight from
plantation after irradiation, both strategies with substantial technical problems and limitations. Michael Heneka
Yamasaki et al. studied double chemokine receptor (CCR2-RFP+; < I D > j em . 2 1 8i ns g h t f p < / I D >

CX3CR1-GFP+) mice in the experimental autoimmune encephalo-

Nodes of Ranvier represent a prime
site of attack for MDMs at the onset
of EAE. This 3D reconstruction of SBF-
SEM images shows a monocyte-derived
macrophage encircling the node of
Ranvier, as shown by the two primary
processes (white and black arrows).
myelitis (EAE) mouse model of MS. Inflammatory lesions were filled with both MDMs and
MiDMs. Confocal immunohistochemistry, serial block-face scanning electron microscopy
(SBF-SEM), and subsequent 3D reconstruction revealed that myelin destruction was initiated
by MDMs, often at the nodes of Ranvier, whereas MiDMs were not detected at this site.
Disruption of MDM infiltration by CCR2 deficiency completely abolished the presence of
macrophages at the nodes of Ranvier. Gene expression profiling of both cell types at disease
onset revealed substantial differences, which correlated well with the observations obtained
by SBF-SEM. MDMs expressed genes attributable to effector functions, including those
involved in phagocytosis and cell clearance. In contrast, MiMD gene expression patterns at
disease onset were characteristic of a repressed metabolic state.
This paper sets a new standard for further studies in the field. For the first time,
MDMs and MiDMs have been clearly differentiated and their morphological relation-
ship to axoglial structures has been analyzed. The finding that MDMs rather than
MiDMs initiate myelin destruction at disease onset should enable this cell population to
be targeted more effectively in future. The next stage is to verify these findings in human
tissue. Future research should also assess further time points over the entire disease
course, in particular to exclude that MiDMs do not join MDMs at the node of Ranvier
at later stages of disease. A precise distinction between local and infiltrating cell popula-
tions may also contribute to a better understanding of pathogenesis in other CNS disor-
ders such as stroke and brain trauma and will hopefully lead to the development of new
therapeutic strategies.
Yamasaki, R., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/jem.20132477.

Michael T. Heneka, University of Bonn: michael.heneka@ukb.uni-bonn.de

 Article

Differential roles of microglia and monocytes

in the inflamed central nervous system
Ryo Yamasaki,1 Haiyan Lu,1 Oleg Butovsky,5 Nobuhiko Ohno,2
Anna M. Rietsch,1 Ron Cialic,5 Pauline M. Wu,2 Camille E. Doykan,2
Jessica Lin,1,6 Anne C. Cotleur,1 Grahame Kidd,2 Musab M. Zorlu,1,7
Nathan Sun,8 Weiwei Hu,2,9 LiPing Liu,1 Jar-Chi Lee,3 Sarah E. Taylor,10
Lindsey Uehlein,1,6 Debra Dixon,1,11 Jinyu Gu,1 Crina M. Floruta,1,12 Min Zhu,1
Israel F. Charo,13 Howard L. Weiner,5 and Richard M. Ransohoff 1,4,11
1Neuroinflammation Research Center and 2Department of Neurosciences, Lerner Research Institute; 3Department
of Quantitative Health Sciences; and 4Mellen Center for Multiple Sclerosis Treatment and Research, Neurological Institute,
Cleveland Clinic, Cleveland, OH 44106
5Center for Neurological Diseases, Brigham and Womens Hospital, Harvard Institutes of Medicine, Boston, MA 02115
6Ohio State University College of Medicine, Columbus, OH 43210
7Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey
8Vanderbilt University, Nashville, TN 37235
9Department of Pharmacology, School of Basic Medical Sciences, Zhejiang University, Hangzhou, 310058 Zhejiang, China
10Case Western Reserve University, School of Medicine, Cleveland, OH 44106
11Cleveland Clinic Lerner College of Medicine, Cleveland, OH 44106
12Baylor University, Waco, TX 77030
13Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, San Francisco, CA 94158

In the human disorder multiple sclerosis (MS) and in the model experimental autoimmune
encephalomyelitis (EAE), macrophages predominate in demyelinated areas and their num-
bers correlate to tissue damage. Macrophages may be derived from infiltrating monocytes
or resident microglia, yet are indistinguishable by light microscopy and surface phenotype.
It is axiomatic that T cellmediated macrophage activation is critical for inflammatory
demyelination in EAE, yet the precise details by which tissue injury takes place remain
poorly understood. In the present study, we addressed the cellular basis of autoimmune
demyelination by discriminating microglial versus monocyte origins of effector macro-
phages. Using serial block-face scanning electron microscopy (SBF-SEM), we show that
monocyte-derived macrophages associate with nodes of Ranvier and initiate demyelination,
whereas microglia appear to clear debris. Gene expression profiles confirm that monocyte-
derived macrophages are highly phagocytic and inflammatory, whereas those arising from
microglia demonstrate an unexpected signature of globally suppressed cellular metabolism
at disease onset. Distinguishing tissue-resident macrophages from infiltrating monocytes
will point toward new strategies to treat disease and promote repair in diverse inflamma-
tory pathologies in varied organs.

CORRESPONDENCE
Richard M. Ransohoff:
ransohr@ccf.org
Abbreviations used: CNS,
central nervous system; EAE,
experimental autoimmune
encephalomyelitis; MDM,
monocyte-derived macrophage;
MiDM, microglia-derived mac-
rophage; MS, multiple sclerosis;
SBF-SEM, serial block-face
scanning electron microscopy.
Blood-derived monocytes and resident microglia
can both give rise to macrophages in the cen-
tral nervous system (CNS). In tissue sections,
macrophages derived from these two distinct
precursors are indistinguishable at the light
microscopic level both morphologically and by
surface markers. Using flow cytometry, microglia-
and monocyte-derived macrophages can be
isolated separately from CNS tissue lysates and
expression profiling suggests distinct functional
R. Yamasaki, H. Lu, and O. Butovsky contributed equally to
this paper.
capacities (Gautier et al., 2012; Chiu et al., 2013;
Butovsky et al., 2014).
Microglia and monocytes are ontogeneti-
cally distinct: microglia derive from yolk-sac pro-
genitors during embryogenesis (Ginhoux et al.,
2010; Schulz et al., 2012), whereas monocytes
continuously differentiate throughout postnatal
life from bone marrow hematopoietic stem cells
2014 Yamasaki et al. This article is distributed under the terms of an Attribution
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J. Exp. Med. 2014 Vol. 211 No. 8 1533-1549 1533
www.jem.org/cgi/doi/10.1084/jem.20132477
(HSCs), which require the transcription factor Myb. Microg-
lial precursors are Myb independent, and microglia self-renew
independently of bone marrow HSCs (Gomez Perdiguero
et al., 2013). Distinct developmental origin and renewal mech-
anisms imply that monocyte-derived macrophages (MDMs)
and microglia-derived macrophages (MiDMs) might exert dif-
ferent functions in pathological processes. Microglia represent
one instance of tissue-resident macrophages, which reside in all
organs. Studying the CNS as compared with other organs
may carry advantages for distinguishing tissue-resident my-
eloid cells from infiltrating monocytes during disease, as there
is virtually no background trafficking of monocytes in the
CNS parenchyma of healthy animals.
In EAE, which models inflammatory aspects of MS
(Williams et al., 1994; Ransohoff, 2012), macrophages dominate
the inflammatory infiltrates and their numbers correlate to
EAE severity (Huitinga et al., 1990, 1993; Ajami et al., 2011).
However the cellular mechanisms by which macrophages
promote disease progression are uncertain. Whether MiDMs
or MDMs are functionally distinct and whether the two cell
types differentially initiate demyelination or promote repair
(Steinman et al., 2002) also remains elusive (Bauer et al., 1995).
In MS autopsy tissues, prominent macrophage accumulation
correlates with active demyelination (Ferguson et al., 1997;
Trapp et al., 1998). Based on kinetics of cell accumulation and
differential marker expression, its estimated that 3050% of
activated macrophages in active MS lesions derive from mi-
croglia (Brck et al., 1995; Trebst et al., 2001). Therefore, dif-
ferential functions of MDMs and MiDMs are relevant for
human demyelinating disease.
To date, no research techniques have permitted distinction
between monocytes and microglia in CNS tissue without ir-
radiation chimerism or parabiosis, techniques that confound
interpretation or impose practical limitations (Ajami et al.,
2007, 2011; Ransohoff, 2007). When F4/80+ macrophages
were isolated from CNS and analyzed by flow cytometry
using cells from double-heterozygous Ccr2rfp::Cx3cr1gfp mice
with EAE, GFP was expressed by CD45dim/Ly6C microglia,
whereas RFP was restricted to CD45high/Ly6C+ monocytes
(Saederup et al., 2010; Mizutani et al., 2012). These findings
suggested an approach to clarifying distinct roles of MDMs
and MiDMs in EAE based on differential expression of GFP
and RFP reporters. Here, we use that strategy to extend pre-
vious findings and address the hypothesis that MDMs and
MiDMs exert different functions in neuroinflammation. We
detected detailed ultrastructural characterization of MDMs
and MiDMs at EAE onset.
Unexpectedly, this approach provided insight into the cel-
lular basis for autoimmune demyelination, which has remained
obscure despite >80 yr of study in the EAE model. Here we
provide evidence that MDMs initiate demyelination, often at
nodes of Ranvier. In contrast, phagocytic microglia appear rela-
tively inert at disease onset. Results from expression profiling
provided insight into mechanisms and signaling pathways un-
derlying the disparate effector properties of MDMs and MiDMs
in EAE.The distinct functions of tissue-resident myeloid cells
as compared with infiltrating macrophages broadly underlie
disease pathogenesis in manifold circumstances and also hold
promise for innovative treatment strategies.
RESULTS
In the CNS of mice of EAE, MDMs and MiDMs
exhibit different accumulation kinetics
The histological strategy in this study is shown in Table 1. At
onset of EAE, two pools of CD11b+ mononuclear phagocytic
cells (putative red MDMs and green MiDMs) predominated
in spinal cord (Fig. 1 A), indicating that fluorochrome mark-
ers could be distinguished at this time point. Using cells iso-
lated from Ccr2rfp/+::Cx3cr1gfp/+ spinal cords at disease onset,
flow cytometry demonstrated distinct expression of RFP and

Table 1. Histology analysis strategy

Method Purpose Finding
Confocal analysis of 0.2 mm optical To distinguish MDMs (RFP+)from MDMs and MiDMs can be distinguished by cell volume and primary
sections (n = 104 cells). MiDMs (GFP+). processes.
SBF-SEM inspection in 0.2 mm sections To detect MDMs and MiDMs in SBF- Using criteria detected in the previous step, it is possible to distinguish
from 14 lesions, 7 mice at EAE onset. SEM using cell volume and process MDMs and MiDMs in SBF-SEM images.
criteria.
SBF-SEM inspection of ultrastructure To detect ultrastructural characteristics MDMs and MiDMs show characteristic ultrastructural differences
of MDMs and MiDMs. of MDMs and MiDMs. regarding their mitochondria, nuclei, osmiophilic granules and
microvilli.
Quantification of relation of MDMs To determine relationship of MDMs Most (55/75; 73%) axoglial units are contacted in limited fashion by
(n = 169) and MiDMs (n = 86) to and MiDMs to axoglial units and MDMs and MiDMs. If one cell type is present (20/75 cells), its nearly
axoglial units (n = 29 intact axons, characterize presence of myelin debris. always (18/20 segments) MDMs.
46 demyelinated axons).
Reconstruction of 3D shape of four To detect relationship of MDMs with In all, 49 MDMs interacting with axoglial units in absence of nearby
representative MDMs at axoglial axoglial units at EAE onset. MiDMs, 2-3 MDMs were attached to each (n = 18) axoglial unit.
units. MDMs have close relationship with nodes of Ranvier (7 MDMs/75
axoglial units). 3D reconstructions showed four representative MDMs
at axoglial units: show one carrying out active demyelination, three at
nodes of Ranvier.
1534 Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.
 Ar ticle

GFP by F4/80+/CD45high MDMs and F4/80+/CD45dim
MiDMs, respectively (Fig. 1 B). Enumeration of cells recovered
from cell sorting using F4/80+RFP+ as MDMs gate and F4/
80+GFP+ as MiDMs gate indicated that MDMs and MiDMs
showed equal numbers at disease onset when explosive MDM
accumulation occurred. MiDM expansion began at peak
(Fig. 1 B). At recovery, MiDMs were found near preonset
numbers as MDM frequency continued to decline, which is
compatible with previous studies (Ajami et al., 2011). There-
fore, there were unequal numbers of MiDMs/MDMs before
and after disease onset (Fig. 1 B). Morphological analyses and
definitions of relations between myeloid cells and axoglial
units were conducted at disease onset so that equal numbers
of MDMs and MiDMs could be assayed and early events in
the demyelinating disorder could be explored.
Morphological features distinguish
MDMs and MiDMs at EAE onset
Immunofluorescence staining for RFP and GFP in spinal cord
at EAE onset showed that red MDMs exhibited elongated or
spindle shape, whereas green MiDMs showed a process-bearing
morphology (Fig. 1 C). Quantification in 3D reconstructions
from 0.2-m confocal z-stack images showed that MiDMs
exhibited much larger size than MDMs along with multiple
primary processes, which were sparse in MDMs (Fig. 1 D).
Several 3D shape parameters also discriminated between MDMs

Figure 1. MDMs and MiDMs exhibit different time courses of accumulation in the CNS of mice with EAE and morphological characteristics
can distinguish them. (A) Immunohistochemistry shows expression of CD11b for red RFP+ MDMs and green GFP+ MiDMs in the spinal cords of
Cx3cr1gfp/+::Ccr2rfp/+ mice at EAE onset. Bars: 25 m. We studied 6 mice at EAE onset from 3 EAE inductions. In each EAE induction, 810 mice were used and
2 mice were selected from each induction. (B) Flow cytometric analysis of CCR2-RFP+ and CX3CR1-GFP+ populations in cells gated for F4/80 expression
(top); CD45 expression of F4/80+RFP+ MDMs and F4/80+GFP+ MiDMs populations (middle); and MDMs and MiDMs numbers at EAE onset, peak, and recovery
(bottom). We studied 3 mice for naive groups; 12 for onset; 15 for peak; 13 for recovery from 5 EAE inductions. For each induction, 810 mice were used
and 23 mice were selected at each time point (onset, peak, and recovery) for analysis. (C) Confocal microscopy assessment of myeloid cell morphology in
lumbar spinal cord from mice at EAE onset. We studied 54 MDMs and 51 MiDMs of 5 EAE onset mice from 3 EAE inductions for (CE); 2 sections/mouse;
46 cells/section; 812 cells/mouse. In each EAE induction, 810 mice were induced and 12 EAE onset mice were selected from each experiment. Bars,
25 m. (D) Cell volumes of 500 m3; surface areas of 1,000 m2; primary process numbers 3 or 5; solidity3D of 0.4; and Formfactor3D of 0.3 discrimi-
nate between MDMs and MiDMs. (E) Model plot of cell volume against primary process number to distinguish MDMs (red symbols and pink area) from
MiDMs (green symbols and green area).

JEM Vol. 211, No. 8 1535

and MiDMs (Fig. 1 D). We observed scant overlap of several
values between MDMs and MiDMs (Fig. 1 D), and entirely
nonoverlapping distributions for cell volume and primary
processes (Fig. 1 E).
MDMs and MiDMs exhibit differentiating
ultrastructural characteristics at EAE onset
We used confocal microscopy in 0.2-m optical sections to
correlate structural features of MDMs and MiDMs with RFP
or GFP fluorescence, as a bridge to characterizing cells in 0.2 m
SBF-SEM images (Table 1). Using this approach (Fig. 1 E),
MDMs and MiDMs were identified by estimating volume
and counting primary processes. Volume estimations came
from multiplying the midcell area by the number of sections in
which the cell was identified. In electromagnetic (EM) images,
quantitative analysis also demonstrated differentiating ultrastruc-
tural characteristics for mitochondria, nuclei, cytoplasmic os-
miophilic granules and microvilli (unpublished data). MDMs
had shorter, thicker mitochondria than MiDMs (unpublished
data). Total mitochondrial numbers and volumes were equal
in MDMs and MiDMs (unpublished data). MDMs had bi-
lobulated or irregular nuclei, whereas MiDMs had round nu-
clei (unpublished data). MDMs, but not MiDMs, frequently
contained osmiophilic granules and microvilli (unpublished
data). Collectively, these ultrastructural features provided con-
firmatory ultrastructural characteristics to distinguish MDMs
from MiDMs.
MDMs initiated demyelination at EAE onset
Results from confocal and EM analysis yielded a secure basis
for examining the relationships of MDMs and MiDMs to ax-
oglial units at EAE onset (n = 7 mice; 14 lesions) using serial
block-face scanning electron microscopy (SBF-SEM), as pre-
sented diagrammatically (Table 1). We quantified contacts
made by MDMs (n = 169) and MiDMs (n = 86) with axoglial
units (n = 75; Fig. 2), and observed that most (55/75; 73%) of
all segments (both intact and demyelinated) contacted both
MDMs and MiDMs (Fig. 2). Where only one myeloid cell
type was present (20/75; 27%), nearly all axoglial units made
contacts to MDMs (Fig. 2). In particular, 8/29 intact and 10/46

Figure 2. SBF-SEM shows MDMs initiating demyelination at EAE onset. Quantitation of MiDMs and MDMs interacting with axoglial units in SBF-
SEM images of CNS at EAE onset. Intact (69%) and demyelinated (76%) segments interacted with MDMs and MiDMs. Red and pink, MDMs; green and
light green, MiDMs; yellow, both MDMs and MiDMs. We studied 29 intact axon segments, 46 demyelinated axon segments, 86 MiDMs, and 169 MDMs in
14 lesions of 7 EAE onset mice from 3 EAE inductions as follows: 810 mice were immunized at each experiment and 23 onset mice were selected from
each induction.

1536 Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.


demyelinated axoglial units were contacted solely by MDMs.
We found 23 MDMs attached to each of the 18/20 (90%)
axoglial units where only MDMs were present (Fig. 2). More
than half of all analyzed MDM and MiDM cells (n = 255
total) contained myelin debris, regardless of whether axon
segments were intact or demyelinated (Fig. 2). Of the MDMs
found in sole contact with axoglial units, virtually all (>90%)
MDMs contained myelin when found in sole contact with a
demyelinated axon (Fig. 2). These findings motivated evalua-
tion of relationships of MDMs to axoglial units by 3D recon-
struction of SBF-SEM image stacks.
MDMs frequently exhibited morphological characteristics
suggesting an involvement in active demyelination. Reconstruc-
tion of one representative image stack shows MDMs with
large intracellular myelin inclusions tightly encircling a partially
demyelinated axon (Fig. 3 A). The myelin peeled away from
the axon remained in continuity with a large myelin inclusion
JEM Vol. 211, No. 8
Ar ticle
inside the MDMs (Fig. 3 A). Remaining myelin was undergoing
vesicular breakdown (Fig. 3 A). In contrast, a nearby MiDM
encompassed a large fragment of myelin debris (Fig. 3 B) and
contacted the nearby MDMs (Fig. 3 B), but made minimal
connection to the axoglial unit (Fig. 3 B). In our SBF-SEM
data, only MDMs seemed to be implicated in active damage
to myelin. These observations suggested that MDMs initiated
demyelination at the onset of EAE.
MDMs surrounded apposed and invaded
nodes of Ranvier at EAE onset
We analyzed axoglial units to examine the nature of contacts
with myeloid cells. Unexpectedly, 7/75 (9%) of axoglial units
demonstrated MDMs attached to nodes of Ranvier. In each
case, the contact between MDMs and node appeared to be
pathogenic. One representative monocyte surrounded a node
of Ranvier with two microvilli interposed between myelin
Figure 3. SBF-SEM shows example of
MDM-initiating demyelination at EAE
onset. (A) Representative MDMs encircles the
axoglial unit. A myelin ovoid within an intra-
cellular phagolysosome shows physical conti-
nuity with myelin remaining attached to an
axoglial unit which is undergoing active de-
myelination. In serial images, disrupted myelin
shows continuity from outside to inside the
MDM. (B) Rotated view from B demonstrating
MDM-extensive attachment to axoglial unit
and MiDM nearby with limited attachment to
axon. A, axon; m, myelin; c, cytosol; n, nucleus.
Red, MDM cytosol; green, MiDM cytosol;
yellow: nuclei; blue, myelin and myelin debris;
gray, axoplasm; red line, MDM plasma mem-
brane. We studied 14 lesions from 7 EAE on-
set mice from 3 EAE inductions as follows:
810 mice were immunized at each experi-
ment and 23 EAE onset mice were selected
from each induction. Bar, 2 m.
1537
and axolemma near the paranode complex (Fig. 4 A). The ax-
oglial unit appeared otherwise healthy and no myelin debris
was found in the MDM cytosol. This observation suggested
that initial MDMaxoglial contacts might occur at nodes of
Ranvier. Further, we detected an intratubal (Stoll et al., 1989)
MDMs with myelin debris interposed between compact my-
elin and axolemma near a node of Ranvier (Fig. 4 B). Addi-
tionally we identified an MDM apposed to a node of Ranvier
and actively phagocytizing myelin (Fig. 4 C). At this node,
paranode loops were disrupted and surrounded by MDM cy-
tosol (Fig. 4 C), indicating likely involvement in damaging my-
elin near the node. No MiDMs contacted nodes of Ranvier.
Nodal pathology without demyelination
at EAE onset in Ccr2rfp/rfp::Cx3cr1gfp/+ mice
We interpreted our ultrastructural findings to indicate that
MDMs recognized altered nodal structure and initiated demy-
elination at EAE onset. CCR2 is essential for monocyte recruit-
ment to CNS tissues during immune-mediated inflammation
(Fife et al., 2000; Izikson et al., 2000; Savarin et al., 2010). To
1538
address the role of MDMs in demyelination at EAE onset, we
investigated clinical characteristics in relation to node pathology
and demyelination in Ccr2rfp/rfp::Cx3cr1gfp/+ mice in which
MDMs were virtually absent from inflamed EAE tissues and
replaced in large part by neutrophils (Saederup et al., 2010).We
observed equivalent magnitude of weight loss in Ccr2rfp/+::
Cx3cr1gfp/+ and Ccr2rfp/rfp::Cx3cr1gfp/+ mice at preonset and
onset stages of EAE, showing that CCR2 deficiency did not
affect systemic inflammation in this model (Fig. 5 A). There was
a moderate delay in disease onset (Fig. 5 B) and slight reduction
in EAE onset severity (Fig. 5 A) in Ccr2rfp/rfp::Cx3cr1gfp/+ mice.
SBF-SEM was used to evaluate nodal pathology, myeloid
cell relations to axoglial units and demyelination at and before
EAE onset. In three distinct tissues from individual Ccr2rfp/+
::Cx3cr1gfp/+ mice with EAE preonset, we found five MDMs
attached to disrupted nodes of Ranvier. In an equivalent sam-
ple of EAE tissues from three Ccr2rfp/rfp::Cx3cr1gfp/+ mice, only
one MDM was found in contact with a node of Ranvier, despite
the presence of disrupted nodes in proximity to neutrophils.
One representative MDM from Ccr2rfp/+::Cx3cr1gfp/+ tissue
Figure 4. MDMs surrounded, apposed,
and invaded nodes of Ranvier at EAE
onset. (A) SBF-SEM images and 3D reconstruc-
tion of SBF-SEM images of MDMs with a node
of Ranvier. White and black arrow: microvil-
lus. (B) SBF-SEM images and 3D construction
of intratubal MDMs with demyelinated axon
and node of Ranvier. (C) SBF-SEM images and
3D reconstruction of an MDM with intracel-
lular myelin debris apposed to a node of Ran-
vier. Red, MDM cytosol; yellow, nucleus; blue,
myelin; gray, axoplasm. M, myelin; c, cytosol.
red line, MDM plasma membrane. We studied
14 lesions from 7 EAE onset mice collected as
follows: 810 mice were immunized at each
induction and 23 EAE onset mice were
selected from each immunization. Bar, 2 m.
Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.
 Ar ticle

Figure 5. Nodal pathology without demyelination at EAE onset in Ccr2rfp/rfp::Cx3cr1gfp/+ mice. (A) Magnitude of weight loss in Ccr2rfp/+::Cx3cr1gfp/+ and
Ccr2rfp/rfp::Cx3cr1gfp/+ mice at preonset and onset stages of EAE. Clinical score in Ccr2rfp/+::Cx3cr1gfp/+ and Ccr2rfp/rfp::Cx3cr1gfp/+ mice at EAE onset stage. (B) Days
at disease preonset and onset stages of EAE. We studied 28 Ccr2rfp/+::Cx3cr1gfp/+ mice and 26 Ccr2rfp/rfp::Cx3cr1gfp/+ mice for A and B. Data were collected from
12 EAE inductions in Ccr2rfp/+::Cx3cr1gfp/+ mice and 19 EAE inductions in Ccr2rfp/rfp::Cx3cr1gfp/+ mice as follows: 810 mice were immunized at each induction and
13 EAE recovery mice were selected from each immunization. **, P < 0.01; ***, P < 0.001. (C) SBF-SEM imaging of MDMs with nodes of Ranvier phagocytosis in
Ccr2rfp/+::Cx3cr1gfp/+ mice at EAE preonset. Pink, MDM cytosol; red arrow, myelin inclusion of MDM connecting to the node of Ranvier. We studied 3 tissues from 3
Ccr2rfp/+::Cx3cr1gfp/+ EAE mice at preonset stage from 3 EAE inductions: 810 mice were immunized at each experiment and one EAE preonset mouse was selected
from each induction. Bar, 2 m. (D) SBF-SEM of disrupted nodes (black arrows) in preonset spinal cord tissues of Ccr2rfp/rfp::Cx3cr1gfp/+ mice. Bar, 2 m. (E) SBF-
SEM of neutrophil is with myelin phagocytosis from internode at preonset stage of Ccr2rfp/rfp::Cx3cr1gfp/+ mouse. Blue, neutrophil cytosol. For DE, we studied
three tissues from three Ccr2rfp/rfp::Cx3cr1gfp/+ EAE mice at preonset stage from 3 EAE inductions: 810 mice were immunized at each experiment and one EAE
preonset mouse was selected from each induction. Bar: 2 m. (F) Histochemical staining and with aurohalophosphate complexes (black gold staining) and quanti-
fication of demyelinated area of Ccr2rfp/+::Cx3cr1gfp/+ mice and Ccr2rfp/+::Cx3cr1gfp/+ mice. We studied 5 naive Ccr2rfp/+::Cx3cr1gfp/+ mice, 5 naive Ccr2rfp/rfp
::Cx3cr1gfp/+ mice, 5 onset Ccr2rfp/+::Cx3cr1gfp/+ mice, and 5 onset Ccr2rfp/rfp::Cx3cr1gfp/+ mice from 3 EAE inductions as follows: 810 mice were immunized at each
experiment and 12 onset mice were selected from each induction. **, P < 0.01. Bar: 250 m.

JEM Vol. 211, No. 8 1539

Figure 6. Inflammatory signature in MiDMs versus MDMs in the CNS of Cx3cr1gfp/+::Ccr2rfp/+ mice with EAE. (A) Quantitative nCounter expres-
sion profiling of 179 inflammation related genes was performed in CNS-derived GFP+ microglia and RFP+ recruited monocytes from naive and EAE mice
at onset, peak and recovery stages. Each row of the heat map represents an individual gene and each column an individual group from pool of 5 mice at
each time point. The relative abundance of transcripts is indicated by a color (red, higher; green, median; blue, low). For AH, we studied five mice in each
time point (onset, peak, recovery) from 3 EAE inductions; 810 mice were immunized in each induction. (B) Heat map of differentially expressed microglia

1540 Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.


having concave nucleus (Fig. 5 C, left) had multiple intracellu-
lar myelin inclusions, one of which (Fig. 5 C, left middle) was
physically connected to a myelin sheath (Fig. 5 C, right mid-
dle) at a paranode (Fig. 5 C, right), indicating active ongoing
demyelination at a node of Ranvier. By distinct contrast, EAE
onset tissues of Ccr2rfp/rfp::Cx3cr1gfp/+ mice were characterized
by nodal pathology often without cellular infiltrates (Fig. 5 D).
In one instance, we detected a neutrophil abstracting myelin
from the myelin internode (Fig. 5, left and right) despite a
nearby disrupted node (Fig. 5, left) in tissues from a Ccr2rfp/rfp::
Cx3cr1gfp/+ mouse. Importantly, there was no evidence for
neutrophil recognition of disrupted nodes of Ranvier. We in-
terpreted these observations to suggest that MDMs specifically
recognized nodal components to initiate demyelination, and that
absence of MDMs at disrupted nodes of Ccr2rfp/rfp::Cx3cr1gfp/+
mice with EAE was caused by the virtual absence of infiltrat-
ing monocytes (Saederup et al., 2010).
To quantify the outcome of these ultrastructural differences,
we monitored demyelination using histochemical staining with
aurohalophosphate complexes at disease onset in Ccr2rfp/rfp::
Cx3cr1gfp/+ and Ccr2rfp/+::Cx3cr1gfp/+ mice. Demyelination was
significantly reduced at EAE onset in CCR2-deficient mice
(Fig. 5 F), indicating the importance of MDM recognition of
disrupted nodes for efficient inflammatory demyelination. Fur-
thermore, as nodal pathology was equivalent in Ccr2rfp/rfp::
Cx3cr1gfp/+ (Fig. 5 D) and Ccr2rfp/+::Cx3cr1gfp/+ mice at the
preonset stage of EAE, the results suggested that inflammatory
nodal disruption could be reversible if MDMs were prevented
from initiating demyelination at those sites.
Expression profiling demonstrates differential
MiDMs and MDMs gene expression
across the time course of an EAE attack
We reasoned that different phenotypes (Fig. 1) and effector
properties (Figs. 24) of MDMs and MiDMs should be re-
flected in distinct gene expression profiles in the dynamic CNS
microenvironment during EAE. To address this hypothesis,
nCounter digital multiplexed gene expression analysis (Kulkarni,
2011) was performed using directly ex vivo naive microglia
and splenic F4/80+ macrophages (here termed monocytes and
considered similar to microglia by expression profiling; Gautier
et al., 2012), as well as flow-sorted MiDMs or MDMs across the
time course of an EAE attack. Microglia and MiDMs clus-
tered together during unsupervised hierarchical clustering, as
did monocytes and MDMs (Fig. 6, A and B). In both MiDMs
and MDMs, naive and recovery-stage expression profiles were
more alike than were onset and peak-stage profiles (Fig. 6,
A and B) suggesting a return to homeostasis at EAE recovery.
and monocyte genes. (C) Enriched monocyte genes as compared with resident microglia. Bars represent fold changes of gene expression across naive and
all disease stages versus resident microglia. (D) Enriched microglia genes as compared with recruited monocytes. Bars represent fold changes of gene
expression across naive and all disease stages versus recruited monocytes. (EH) K-means clustering of inflammation genes in resident microglia and
recruited monocytes. K-means clustering was used to generate 5 disease stagerelated clusters in MiDMs. Heat map (E) and expression profile (F) of in-
flammation genes in MiDMs are shown by generated clusters. MDM expression matrix overlaid on microglial based clusters shows (G) heat map and
(H) expression profile.
JEM Vol. 211, No. 8
Ar ticle
We noted a subset of genes that were expressed in microglia
and highly regulated in MiDMs during EAE, but not ex-
pressed at all in monocytes or MDMs (Fig. 6, A and B). Con-
versely, a subset of MDM-enriched genes were dynamically
regulated in monocytes and MDMs but not in microglia
(Fig. 6, A and B). MDM-enriched genes were sharply up-
regulated from naive monocytes to onset and peak-stage
MDMs (Fig. 6 B), descending toward naive levels during
recovery (Fig. 6 B). In contrast, MiDM-enriched genes
were strongly expressed in naive cells, almost uniformly si-
lenced at onset, and began a return toward naive levels at peak
and recovery (Fig. 6 B). Comparing MDM-enriched genes
with MiDM-enriched genes showed that MDMs were
more likely to express effector functions, including secreted
factors and surface molecules (18/28; 64.3% of MDM-
enriched genes encoded effector functions; Fig. 6 C: and
Table S1, purple genes). In contrast, only 18/48 (37.5%) of
MiDM-enriched genes encoded effector functions (Fig. 6 D,
Table S1, purple genes). These observations indicated that
MiDMs and MDMs exhibited markedly distinct expression
profiles during EAE.
Differential expression of macrophage
effector functions by MiDMs and MDMs
Our ultrastructural analysis of myeloid cells in EAE focused
on myeloid cell relationships to tissue elements. Expression
profiling also addressed the cytokine and growth factor output
of MiDMs and MDMs, potentially providing insight into disease
pathogenesis.We used k-means clustering to discriminate five
distinct patterns of MiDM gene expression during the course
of EAE (Fig. 6, E and F). The red, blue and green groups in-
creased in MiDMs at onset, peak, and recovery, respectively.
Red group genes involved several surface molecules. Green
group genes, up-regulated at onset and transiently further
up-regulated at peak, were comprised mainly of complement-
system elements (C3aR1; C4a, C1qa, C1qa, C3, and Cfb);
mononuclear cellspecific chemokines (CCl2, 3, 4, 5, 7, and
CXCL9); proliferation related genes ( fos, jun, myc, and CSF1);
and acute inflammationrelated genes (IL1a, IL1b, TNF,
CEBP, STAT1). Cell growthrelated genes expressed at this
time point correlated to reported patterns of microglial pro-
liferation during EAE (Ajami et al., 2011). Blue group genes
up-regulated at recovery included heterogeneous cytokines
(IFN-, IFN-, TGFB3, IL2, IL3, IL4, IL12, IL12,
PDGFA, CSF2, and CXCL2). Both yellow group and golden
group genes were strongly expressed in naive microglia, re-
duced drastically at onset, and either returned to preEAE lev-
els during recovery (yellow) or failed to do so (golden).These
1541
genes included a large spectrum of intracellular signaling com-
ponents from the MAP-kinase pathways, as well as TGF and
receptor, both of which are implicated in the nave microglial
phenotype (Butovsky et al., 2014).
These five gene groups were also analyzed for MDM ex-
pression patterns during EAE (Fig. 6, G and H). None of the
gene groups showed coordinate regulation patterns in MDMs,
as were observed in MiDMs (Fig. 6 H). This observation un-
derscored disparate responses of MiDMs and MDMs to the
inflammatory CNS microenvironment of EAE, despite their
being present in close proximity (Fig. 1 A).
Expression patterns at EAE onset
in relation to MiDM and MDM function
To determine whether gene expression patterns could be in-
formative for understanding the relationships of cells to ax-
oglial elements in tissues at EAE onset, we interrogated naive
versus onset MDM and MiDM gene expression related to
cellular functions (Fig. 7). MiDMs showed highly significant
up-regulation of functions associated with cell movement, che-
moattraction, and migration (Fig. 7 B). In the Ingenuity IPA
database, the terms cell movement, chemoattraction, and mi-
gration indicated production of chemokines such as CCL2,
CCL3, CCL4, CCL5, and CCL7, which are up-regulated at
onset and further increased at peak (Fig. 6, E and F, green
group and genes). In other respects, MiDMs exhibited a re-
pressed metabolic and activation phenotype by comparison
to naive microglia (Fig. 7 B) including proliferation, RNA
metabolism, cytoskeletal organization, microtubule dynamics,
extension of processes, phagocytosis and generation of reac-
tive oxygen species.
MDMs showed up-regulation of functions associated to
macrophages, including phagocytosis, calcium signaling, pro-
duction of prostanoids, adhesion, autophagy, and cell clearance
(Fig. 7 B).This pathway analysis corresponded well to effector
properties displayed by MDMs in our SBF-SEM analysis
(Figs. 24). No functions were reported to be down-regulated
in MDMs at EAE onset as compared with naive monocytes.
A comprehensive listing (Table S1) of all genes regulated
by at least twofold in MiDMs or MDMs as compared with
expression levels in naive mice affirmed and extended these
interpretations. At EAE onset when SBF-SEM analyses were
performed, MiDMs predominantly suppressed the distinctive
gene expression pattern which correlates to their unique phe-
notype (Chiu et al., 2013), reflected by the observation that
MiDMs down-regulated far more genes than were up-regulated
(Table S1). In contrast, MDMs up-regulated far more genes
than did MiDMs and up-regulated more transcripts than were
down-regulated. Additionally, the extent of gene up-regulation
in MDMs exceeded that seen in MiDMs.
MiDM and MDM gene expression kinetics reflected
return toward homeostasis in recovery stage
These expression profiles showed consonant changes for the
vast majority of genes analyzed: if a gene was up-regulated at
any time point, then its expression level showed an increase at
1542
other time points as well. However, a substantial minority of
genes both for MDMs and MiDMs showed some dissonant
time points, at which a previously down-regulated gene might
show up-regulation (unpublished data). We show this subset of
recovered genes in Fig. 8. In virtually every case (Fig. 8, AD),
these dissonant compensatory changes took place during recov-
ery and almost always showed an increase in a gene that had
been down-regulated during onset and peak. Both MiDMs
(Fig. 8 B) and MDMs (Fig. 8 D) demonstrated this pattern of
gene-expression kinetics.
Convergent and divergent responses to upstream
regulatory signaling by MiDMs and MDMs
Translation of observations made using expression profiles can
be enabled through identification of upstream regulators. We
used Ingenuity IPA software to identify putative upstream reg-
ulators of the gene expression alterations demonstrated by
MiDMs and MDMs at disease onset. Putative regulatory ele-
ments were then grouped in signaling modules and subjected
to pathway analysis. Cell motility pathways were clearly differ-
ent in MiDMs and MDMs (unpublished data). Core elements
such as RhoA (Xu et al., 2009) were regulated divergently and
associated signaling components were predicted to be enhanced
in MDMs but depressed in MiDMs, consistent with our pheno-
typic characterization using SBF-SEM. Both HIF-1 (Fig. 9 A)
and TNF pathways (not depicted) were also differentially reg-
ulated in MiDMs and MDMs. By contrast, type I IFN pathway
(Fig. 9 B) was regulated virtually identically in MiDMs and
MDMs. Collectively, these data suggest that HIF-1 and TNF
signaling may partly drive pathogenic properties of MDMs.
Additionally, these data indicated that the separate ontogeny
of microglia and monocytes will lead, probably by epigenetic
influences, to divergent responses to some but not all environ-
mental stimuli, with phenotypic consequences according to
the CNS microenvironment.
DISCUSSION
In this study, we developed a novel strategy to discriminate
MDMs from MiDMs. We used SBF-SEM to address the de-
tailed relationships of MiDM and MDM to axoglial units in
the spinal cords of mice at EAE onset and expression profiling
to examine potential mechanisms. Selection of the EAE disease
model ensured that both recruited monocytes and resident mi-
croglia were exposed to the same intensely inflammatory en-
vironment to increase the likelihood that ambient conditions
could activate these two myeloid cell types toward a convergent
inflammatory phenotype. Instead, we found strikingly diver-
gent relationships of MDMs and MiDMs to axoglial units, by
quantitative and qualitative ultrastructural analysis. Results
from expression profiling supported this interpretation by show-
ing that MiDM metabolism was severely down-regulated,
whereas expression profiles of MDMs reflected the activated
phagocytic phenotype observed through SBF-SEM.
Several salient new observations emerged from these ex-
periments. First, we showed that MDMs initiate demyelination
at EAE onset, as MDMs were the overwhelmingly dominant
Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.
 Ar ticle

cells found in isolation attached to axoglial units and demon-
strated destructive interactions with myelinated axons in 3D
reconstructions. Second, MDMs were unexpectedly observed
at nodes of Ranvier in 9% of axoglial units and showed remark-
ably invasive behavior, including extension of microvilli (Fig. 4 A)
or localization of cell soma (Fig. 4 B) between axolemma and
myelin sheath. Our observed frequency of MDMnodal inter-
action represents a minimum estimate as MDMs found at hemi-
nodes adjacent to a demyelinated segment (Fig. 3 B) were not
scored. Comparison of Ccr2rfp/rfp::Cx3cr1gfp/+ and Ccr2rfp/+::
Cx3cr1gfp/+ mice at and before EAE onset emphasized the
importance of MDMs for this mechanism of demyelination. In
particular, neutrophils in inflamed CNS of Ccr2rfp/rfp::Cx3cr1gfp/+
mice did not recognize disrupted nodes. These observations
are clinically pertinent: our detection of MDMs at nodes of
Ranvier is consistent with recent reports of nodal pathology
in clinical demyelinated tissues (Fu et al., 2011; Desmazires
et al., 2012).The present observations extend this concept and
provide a cellular basis for nodal pathology at the earliest stages
of demyelination. Given the presence of potential phagocytic
signals at nodes (antibodies to paranodal proteins such as contac-
tin and neurofascins; Meinl et al., 2011); complement-derived

Figure 7. Affected functions in MiDMs and MDMs at EAE onset. nCounter inflammatory gene expression data were uploaded to IPA. Genes with
fold change (EAE onset vs. Naive) 1.5 or 1.5 were included in downstream effects analysis. (A) MDMs up-regulated functions, sorted by activation
z-score. (B) MiDMs up-regulated (left) and down-regulated (right) functions, sorted by activation z score. The bias term indicates imbalanced numbers of
up- and down-regulated genes associated with a distal function requiring significance at the P < 0.01 level. We studied pooled samples from 5 mice in
each time point (onset, peak, recovery) from 3 EAE inductions; 810 mice were immunized in each induction.

JEM Vol. 211, No. 8 1543

Figure 8. Restoration of affected inflammatory genes in resident microglia and recruited monocytes at recovery stage. For each gene, fold
change of all different disease stages versus naive state were calculated. Genes that contained at least one fold change >2 or < 0.5 and average fold
change for peak and onset >2 or <0.5 were presented. (A) Microglial up-regulated; (B) Microglial down-regulated; (C) monocyte up-regulated; (D) monocyte
down-regulated. We studied pooled samples from five mice in each time point (onset, peak, recovery) from three EAE inductions; 810 mice were immu-
nized in each induction. FC, fold change.

1544 Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.

 Ar ticle

Figure 9. Function networks in MiDMs and MDMs. nCounter inflammatory gene expression data were uploaded to IPA. Genes with fold change
(EAE onset vs. Naive) 1.5 or 1.5 were included in upstream regulators analysis. Predicted upstream regulators were manually curated to form func-
tional clusters. Clusters were uploaded to IPA using Z scores as reference value for each gene. Networks were generated for each cluster consisting of
uploaded genes and additional predicted molecules. (A) Typical example of functions with dissimilar activation pattern in MiDMs and MDMs: HIF1A.
(B) Function with similar activation pattern in MiDMs and MDMs: Type I IFN. Red object denotes positive (>2) z score and green object denotes negative

JEM Vol. 211, No. 8 1545

opsonins (Nauta et al., 2004); and stress-induced eat-me sig-
nals (Hochreiter-Hufford and Ravichandran, 2013), it may be
feasible to identify a direct molecular pathway for initiating de-
myelination in this model. Third, we characterized a molecu-
lar signature for resident microglia at EAE onset. Grouping of
regulated genes into functional categories demonstrated a
remarkable down-regulation of microglial metabolism at the
nuclear, cytoplasmic and cytoskeletal levels.
In our initial experiments we found that the presence of
myelin debris at the peak of EAE did not discriminate MDMs
from MiDMs. We considered that SBF-SEM would exhibit
advantages for spatial resolution (Denk and Horstmann, 2004)
required for characterizing relationships of myeloid cells to
axoglial units during the inflammatory demyelinating process
at EAE onset.To take advantage of this technique we developed
methods based on cell volume and process number (Fig. 1 E),
to distinguish MDMs from MiDMs in 0.2-m confocal optical
sections, and translated this approach directly to SBF-SEM
image sets at 0.2-m intervals.We also noted differential nuclear
morphology, mitochondrial shape, and osmiophilic granule
content between MDMs and MiDMs. These characteristics of
MDMs and MiDMs may not be universally present in other
pathological circumstances but demonstrate an approach
to ultrastructural distinction of myeloid cell populations in
tissue sections.
Gene expression profiling across the time course of EAE
yielded intriguing kinetics as analyzed by k-means clustering.
Five patterns were observed. Red group genes (increased at
onset) comprised the smallest number and involved several sur-
face molecules: CCR1, CCR7, CXCR2, and CD40. Of these,
CCR7 and CD40 have been reported on activated microglia,
including those observed in MS tissue sections (Kiviskk et al.,
2004; Serafini et al., 2006). GAPDH was up-regulated in
MiDMs at onset. Although often regarded as a housekeeping
gene, GAPDH is found in complexes that limit the translation
of inflammatory gene transcripts in activated mouse macro-
phages (Mukhopadhyay et al., 2009; Arif et al., 2012). As pre-
viously reported (Chiu et al., 2013), MiDM gene expression
during the course of EAE did not correspond to the M1/M2
pattern of peripheral macrophage responses to infection or tis-
sue injury. Microglial morphological transformation can be
relatively uniform regardless of the inflammatory process that
provokes it. Despite this apparent uniformity, gene expression
by morphologically identical microglia can differ drastically
contingent on context (Perry et al., 2007).
Unsupervised hierarchical clustering provided insight into
gene expression patterns of MDMs and MiDMs. Naive and
recovery patterns were similar for both cell types. At disease
onset, microglia showed drastic down-regulation of the expres-
sion profile observed in cells from healthy brain. Brisk mi-
croglial proliferation (Ajami et al., 2011) may have accelerated
(<2) z-score. Orange object denotes predicted activation of the network object. Blue object denotes predicted inhibition of the network object. Predicted
relationships (connecting lines): orange, leads to activation; blue, leads to inhibition; yellow, finding inconsistent with state of downstream molecule;
gray, effect not predicted.
1546
a gradual return toward a homeostatic expression profile, as
suggested by MiDM up-regulation of fos, jun, myc, and CSF1
(Wei et al., 2010) at disease onset. By striking contrast, MDMs
up-regulated a large suite of inflammation-associated genes at
EAE onset, with subsequent regression to the expression phe-
notype of circulating monocytes.
Blood monocytes and resident microglia were exposed to
the same inflammatory environment. However, their preEAE
states were extremely distinct, with monocytes being gener-
ated from a bone marrow progenitor within weeks of entry
into CNS, whereas microglia originated during early embryo-
genesis and had inhabited a serum-free unique environment
from midgestation. In a recent study, we characterized resident
microglia by profiling mRNA, miRNA, and protein in com-
parison with infiltrated brain macrophages, nonmicroglial resi-
dent brain cells, and peripheral macrophages (Butovsky et al.,
2014). The detailed profiling after separating cells via CD45dim
status showed distinct mRNA, miRNA, and protein expres-
sion by microglia as compared with infiltrating monocytes or
neuroepithelial brain cells (Butovsky et al., 2014). The study
described transcription factors and miRNAs characteristic of
microglia in healthy brain but not in peripheral monocytes.
These findings partially explain a divergent response of these
two cell types to the same stimuli (Butovsky et al., 2014).
The strength of the study is that the dual reporter system
is sufficient to accurately distinguish monocyte versus mi-
croglial cells and thus to address the general concept that
monocytes and microglia can exert differential functions in
a CNS disease process. At the onset of EAE, the time point
at which our imaging studies were focused, we are able to
make an unequivocal distinction between resident microglia
(CX3CR1gfp) and infiltrating monocytes (CCR2rfp). Two em-
pirical observations underline this discrimination: microglia
are uniformly CX3CR1+ from early embryonic time points
through adulthood (Cardona et al., 2006; Ginhoux et al., 2010;
Schulz et al., 2012), and CCR2+Ly6C+ cells constitute the vast
majority of infiltrating monocytes at EAE onset (Saederup
et al., 2010; Mizutani et al., 2012).
There were unavoidable limitations of our research; spe-
cifically, to address how monocytes and microglia respond to
a shared microenvironment, we focused on a single, patho-
genically relevant time point: onset of EAE. For this reason, it
was beyond the scope of our study to decipher the phenotypic
fate of infiltrated monocytes. In peripheral models of inflam-
mation, Ly6Chi/CCR2rfp monocytes down-regulate the re-
porter over time and show phenotypic evolution. Furthermore,
our conclusions should not be generalized beyond the present
disease paradigm: in other models, such as spinal cord contusion,
the inflammatory infiltrate includes Ly6Clow/CX3CR1gfp
monocytes, which are highly pathogenic (Donnelly et al.,
2011). Our findings carry biological and medical significance
Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.

by demonstrating and characterizing differential responses of
infiltrating monocytes and resident microglia in a relevant dis-
ease model at a prespecified time point, at which point patho-
genic events are taking place.Therefore, we focused our analysis
on the day of EAE onset rather than subsequent events to
challenge our overall hypothesis that infiltrating monocytes
versus resident microglia respond very differently to acute in-
flammatory stimuli.
Activated myeloid cells are the proximate effectors of a
bewildering array of acute and chronic disorders (Wynn et al.,
2013). The technical and conceptual approach taken in this
study may be applicable to other tissues and disease processes.
In many pathological conditions, tissues harbor a mixed pop-
ulation of activated resident and recruited monocytes. The
therapeutic strategy will differ conclusively based on the spe-
cific effector properties of each cell type and the stage of dis-
ease. In particular, if monocytes are pathogenic, then their
trafficking should be blocked using a peripherally active agent.
The optimal application of agents that regulate leukocyte mi-
gration and intracellular signaling will be promoted by de-
tailed examination of each individual myeloid population.
MATERIALS AND METHODS
Mice. C57BL/6 mice were obtained from the National Cancer Institute.
Ccr2rfp/+::Cx3cr1gfp/+ mice were generated by crossbreeding Ccr2rfp/rfp::C57BL/6
mice (Saederup et al., 2010) with Cx3cr1gfp/gfp::C57BL/6 mice ( Jung et al.,
2000). Ccr2rfp/rfp::Cx3cr1gfp/gfp mice were generated by breeding Ccr2rfp/+
::Cx3cr1gfp/+ mice. Ccr2rfp/rfp::Cx3cr1gfp/+ mice were generated by crossbreed-
ing Ccr2rfp/rfp::C57BL/6 mice with Ccr2rfp/rfp::Cx3cr1gfp/gfp mice. Animal ex-
periments were performed according to the protocols approved by the
Institutional Animal Care and Use Committee at the Cleveland Clinic fol-
lowing the National Institutes of Health guidelines for animal care.
EAE induction and clinical evaluation. EAE was induced in Ccr2rfp/+
::Cx3cr1gfp/+ mice and Ccr2rfp/rfp::Cx3cr1gfp/+ mice of 2428 wk of age using
myelin-oligodendrocyte-glycoprotein peptide 3555 (MOG) as previously
described (Huang et al., 2006). All mice were weighed and graded daily for
clinical stages as previously reported (Saederup et al., 2010).We defined clinical
stage of EAE as follows: pre-onset was the day sudden weight loss for 810%
occurred; onset was the day EAE signs appeared; peak was the second day score
didnt increase after sustained daily worsening; and recovery was the second
day score didnt decrease after a period of sustained daily improvement.
To address our research questions, we integrated flow cytometry, immuno
histochemistry with quantitative morphometry, cell sorting for expression
profiling, and serial block-face scanning electronic microscopy. In all, we per-
formed 12 EAE immunizations in Ccr2rfp/+::Cx3cr1gfp/+ mice and 19 immu-
nizations in Ccr2rfp/rfp::Cx3cr1gfp/+ mice for this project, with 810 mice in
each immunization. We selected EAE mice at onset, peak or recovery de-
pending on the specific studies underway at that time, with the majority of
mice coming from the onset stage of EAE. Each experiment incorporated
samples from at least three separate immunizations. Details of mouse numbers
and how they were selected for each experiment were included in the figure
legends as requested.
Cell isolation and flow cytometry. Brains and spinal cords were removed
and homogenized. Mononuclear cells were separated with a 30%/70% Per-
coll (GE Healthcare) gradient as previously reported (Pino and Cardona,
2011). Single-cell suspensions from CNS were stained with antiF4/80-APC
(BM8; eBioscience) and antiCD45-PerCP (30-F11; BioLegend). Cells were
either analyzed on a LSR-II (BD) or sorted on a FACSAria II (BD) running
Diva6. Data were analyzed with FlowJo software (Tree Star).
JEM Vol. 211, No. 8
Ar ticle
Histological and immunohistochemical analysis. Spinal columns were
removed after mice were perfused with 4% paraformaldehyde (PFA). For im-
munofluorescence assay, free floating sections of the lumbar spinal cord were
prepared as previously described (Huang et al., 2006). For immunofluores-
cence assay, sections were blocked with 10% normal serum for 2 h and stained
with primary antibodies at 4C for 2448 h. After washing with PBS-T (PBS
with 0.1% Triton X-100; Sigma-Aldrich) three times, the sections were incu-
bated with secondary antibodies at room temperature for 2 h and mounted
in ProLong Gold antifade reagent (Invitrogen). Antibodies used include rat
anti-CD11b (BD), mouse anti-GFP (Abcam), rabbit anti-RFP (Abcam), Alexa
Fluor 488 goat antimouse IgG (Invitrogen), Alexa Fluor 594 goat antirabbit
IgG (Invitrogen), and Alexa Fluor 647 goat antirat IgG (Invitrogen). Nuclei
were labeled by DAPI. Images were collected by confocal laser-scanning mi-
croscope (SP5; Leica).
Quantitative 3D morphology. Quantitative 3D morphology of MDMs
and MiDMs was analyzed in confocal images from spinal cord of mice at
EAE onset. Free floating sections of the lumbar spinal cord were stained with
RFP for MDMs, GFP for MiDMs, and DAPI for nuclei. Stack images were
taken at 0.2-m step size along the z-direction with a 63 objective (numer-
ical aperture [NA] = 1.4) and zoom factor 2. A square (1,024 1,024 pixels)
corresponding to 123 123 m2 was used for the analysis. Cells were 3D re-
constructed by ImageJ software and all analyses were performed using ImageJ
with 3D Convex Hull plugin. The parameters analyzed include voxel (volu-
metric pixel), convex voxel, volume, convex volume, surface, and convex sur-
face area. Other calculated parameters were: Solidity3D = volume/convex
volume; Convexity3D = convex surface area/surface area; Formfactor3D =
3 36 volume 2 surface area 3 . The number of primary processes was esti-
mated visually. We included 5 mice, 54 MDMs; 51 MiDMs in this assay with
2 sections/mouse, 46 cells/section and 812 cells/mouse. Those mice came
from three EAE inductions.
SBF-SEM. Spinal cords were removed after mice were perfusion-fixed
using 4% PFA with 1% glutaraldehyde. Lumbar spinal cord sections were
made on a vibratome (Leica). Sections were stained with 0.4% OsO4, uranyl
acetate and lead aspartate, then embedded in epon resin (Electronic Micros-
copy Sciences). SBF-SEM images were acquired using a Sigma VP SEM
(Carl Zeiss) with 3View (Gatan). Serial image stacks of images at 100-nm
steps were obtained by sectioning 48 48 20 m3 tissue blocks (length
width depth) at a resolution of 8192 8192 pixels. Image stacks were pro-
cessed for 3D reconstruction by TrakEM2 in FIJI software (National Institutes
of Health). Alternating sections from the same stacked images were chosen to
make stacks for 3D reconstructions which matched the 0.2-m step size used
for acquiring confocal stacked images. In SBF-SEM images, we discriminated
MDMs and MiDMs using the volume/primary processes model (Fig. 1 E)
generated from analyzing confocal images. Quantifications of myeloid-cell
spatial relationships to axoglial units, including myelin incorporation, were
done in SBF-SEM images.
Quantification of nuclei and mitochondria. Characterizations of nu-
clear shapes were conducted in SBF-SEM images. Nuclei were categorized
as follows: round, round shape and smooth surface with ratio of length/
width 1.5; elongated, elongated or oval shape with length/width >1.5, and
may have small indentations; Bilobulated: two connected lobes with single
intervening large indentation; Irregular: complicated shape with corrugated
surface, and may have multiple and variable sizable indentations. Blinded ob-
servers (n = 3) scoring the nuclear morphology from SBF-SEM images in-
cluded a research student, a research fellow and a neuroscientist. Observers
were trained on the same nuclear examples in each category and practiced
using 20 nuclei comprising all shapes before scoring the nuclei. Kappa test
showed good pairwise agreement rates among observers (>0.8) and the data
from the neuroscientist are used. Quantifications were done in 3 individual
mice from 3 EAE inductions including 2835 cells from two separate lesions
from each mouse in the assay.
1547
 Mitochondria of MDMs and MiDMs at EAE onset were reconstructed
from SBF-SEM images to 3D images using TrakEM2. 5 MDM and 5 MiDM
cells from 3 separate mice at EAE onset (total 10 cells) were included in the
assay.Those mice came from 3 EAE inductions. Mitochondria were quantified
for length, cross-sectional area, volume and ratio of length/cross-sectional
area using Fiji software.
Quantification of demyelination. Black-gold staining was performed ac-
cording to a protocol described previously (Liu et al., 2010). In brief, 5 free
floating lumbar spinal cord sections were stained in 0.2% black-gold solution
at 65C water bath for 10 min. After staining with black-gold, sections were
pictured by 3-CCD video camera interfaced with an Image-Pro Plus Analy-
sis System (Version 4.1.0.0; MediaCybernetics) and analyzed with ImageJ
software. Demyelinated areas are those void of black-gold staining. Mean
percentage of demyelinated areas in white matter were calculated. We in-
cluded 5 mice from 3 EAE inductions in this assay.
Statistical analysis of cellular elements. Statistical analyses were per-
formed using SAS (SAS Institute Inc.), PRISM (GraphPad Software) and
SPSS 17.5 (SPSS Inc.). Flow cytometry data were analyzed by two-way
ANOVA test and Wilcoxon matched-pairs signed rank test. Nuclear shape
quantifications were compared by paired Students t tests and logistic regres-
sion. Mitochondrial quantifications were compared by Mann-Whitney U
test and linear mixed model. Quantitative relationships of myeloid cells to
axoglial units were compared using logistic regression with generalized esti-
mating equations (GEE). Clinical characteristics of EAE mice were analyzed
using two-way ANOVA test with Bonferroni post test. Percentage of demye-
lination was compared by Students t test. Data were shown as mean SEM
or median (the first quartilethe third quartile) and P < 0.05 was considered
statistically significant.
Gene expression. Mononuclear cells were prepared from brains and spinal
cords as described previously. Cells were sorted on a BD FACSAria II by gat-
ing on F4/80+GFP+ for MiDMs and F4/80+RFP+ for MDMs. RNA was
isolated from FACS-sorted cells mixed from three mice from six EAE induc-
tions per data point in TRIzol Reagent (Ambion) according to manufactur-
ers protocol. RNA samples were analyzed by nCounter gene expression
analysis and quantified with the nCounter Digital Analyzer (NanoString
Technologies). Expressions of 179 genes were analyzed using nCounter GX
Mouse Inflammation kit.
Nanostring data normalization. Normalization was conducted with
nSolver Analysis Software1.1. Data were normalized using positive and nega-
tive controls and housekeeping genes probes. Background level was calcu-
lated for each sample as mean of negative control probes + (x2 SD). Calculated
background was subtracted from each gene expression value. In cases where
the calculated value was <1, values were set to 1.
Hierarchical and k-means clustering analysis. Hierarchical cluster analy
sis was performed using Pearson correlation for distance measure algorithm
to identify samples with similar patterns of gene expression. MiDM samples
expression data were used in k-means clustering using Pearson correlation
for distance measures (Multi Experiment Viewer v. 4.8).
IPA (Ingenuity) analysis. Data were analyzed using IPA (Ingenuity Sys-
tems). Differentially expressed genes (EAE onset MiDMs versus naive mi-
croglia and EAE onset MDMs versus naive splenic monocytes) were used in
downstream effects and upstream regulators analyses. Uploaded dataset for
analysis were filtered using cutoff definition of 1.5-fold change. Level of con-
fidence for analysis was set to high-predicted and experimentally observed.
Terms used in IPA analyses. The p-value is a measure of the likelihood
that the association between a set of genes in the uploaded dataset and a re-
lated function or upstream regulator is due to random association.The smaller
the p-value, the less likely it is that the association is random and the more
significant the association. In general, P < 0.05 indicate a statistically significant,
1548
nonrandom association. The p value of overlap is calculated by the Fishers
Exact Test.
The activation z-score is a value calculated by the IPA z-score algorithm.
The z-score predicts the direction of change for a function or the activation
state of the upstream regulator using the uploaded gene expression pattern
(upstream to the function and downstream to an upstream regulator). An ab-
solute z-score of 2 is considered significant. A function is increased/upstream
regulator is activated if the z-score is 2. A function is decreased/upstream
regulator is inhibited if the z-score -2.
The bias term is the product of the dataset bias and the bias of target
molecules involved in a particular function annotation or upstream regulator
activity. A biased dataset is one where there is more up- than down-regulated
genes or vice versa. The dataset bias is constant for any given analysis and the
function/upstream regulator bias is unique for each upstream regulator/function.
When the absolute value of this term is 0.25 or higher, then that function/up-
stream regulators prediction is considered to be biased and the Fishers exact
p-value must be 0.01 or lower for the analysis to be considered significant.
We thank Dr. Bruce D. Trapp for invaluable suggestions. We thank Flow core
in Cleveland Clinic Foundation for the flow cytometry experiments. We thank
Aishwarya Yenepalli for help with quantification.
This research was supported by grants from the US National Institutes of
Health, the Charles A. Dana Foundation, the National Multiple Sclerosis Society, and
the Williams Family Fund for MS Research, as well as a Postdoctoral Fellowship
from National Multiple Sclerosis Society (to N. Ohno).
The authors have no competing financial interests.
Submitted: 28 November 2013
Accepted: 9 June 2014
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INSIGHTS

The real thing: How to make human DC subsets

The developmental relationship between monocytes, dendritic cells (DCs), and macrophages has been
well defined in mice, but human DC development is less well understood and has been hampered by
the lack of a suitable culture system. Now, two papers published in this issue describe a novel in vitro
culture system for human DC progenitors and the use of this system to elucidate the pathway of human
DC development.
The extent to which mouse studies are useful to understand the human immune system is debatable.
Insight from One expectsand observesimportant similarities, but there are also differences in the way the building
Frederic blocks of the immune system are assembled in different species of vertebrates that have evolved in different
Geissmann contexts and milieus, and with different lifespans. Laboratory mice, maintained as inbred strains under
selected housing conditions, are amendable to controlled genetic studies. However, genetic and environmental
variations are difficult to control in human studies, and experimental limitations make it difficult to assess whether "mouse"
immunology "works" in humans.
The two studies in this issue, by Lee et al. and Breton et al., represent an important advance in the DC field. The work
describes, at the population and single cell level, a hierarchy of human myeloid precursors that closely parallel the hierarchy in
the mouse bone marrow and blood. The authors used a combination of technically impressive qualitative and quantitative
approaches, including in vitro differentiation of human hematopoietic precursors cultured on a stromal cell line with defined
sets of cytokines, in vivo "cultures" of human precursors in immunodeficient NOD-scid g mice, and observational studies in
humans. This allowed them to map out the development of monocytes, conventional DCs (cDCs), and plasmacytoid DCs
(pDCs), and the sequential relationship between the different precursor populations. They showed that a granulocyte-
monocyte-DC progenitor (hGMDP) develops into a monocyte-DC progenitor (hMDP), which itself differentiates into mono-
cytes and into a common DC progenitor (hCDP) that produces the three major human DC subsets (CD1c+ cDCs, CD141+ cDCs,
and pDCs). Furthermore, they report the identification of an immediate DC precursor (hpre-cDC) that originates from the
hCDP, circulates in the human blood,
and increases in response to plasma levels
of the cytokine FLT3.
These results are a significant advance
in the field. They illustrate the similari-
ties between mice and humans regarding
the development of monocytes, cDCs,
and pDCs, and provide a guideand
experimental systemsto further in-
terrogate the genetic and molecular
control of human monocyte/DC devel-
opment and their functions in disease.
It is possible to imagine that DC-based
therapy may benefit from knowledge Schematic of the developmental relationship between human monocytes, cDCs, and pDCs.
of the "real thing."
Breton, G., et al. 2015. J. Exp. Med. http://dx.doi.org/10.1084/jem.20141441.
Lee, J., et al. 2015. J. Exp. Med. http://dx.doi.org/10.1084/jem.20141442.

Frederic Geissmann, Centre for Molecular and Cellular Biology of Inflammation, Kings College London: frederic.geissmann@kcl.ac.uk

CORRESPONDENCE
Kang Liu:
kl2529@columbia.edu
OR
Michel C. Nussenzweig:
nussen@rockefeller.edu
Abbreviations used: cDC, con-
ventional DC; CDP, common
dendritic progenitor; CLP,
common lymphoid progenitor;
CML, chronic myelogenous
leukemia; CMP, common
myeloid progenitor; GMP,
granulocyte-macrophage
progenitor; hGMDP, human
granulocyte-monocyte-DC
progenitor; hMDP, human
monocyte-dendritic progenitor;
HSC, hematopoietic stem cell;
HSPC, hematopoietic stem
and progenitor cell; LMPP,
lymphoid-primed multipotent
progenitor; MDP, macrophage/
dendritic progenitor; MLP,
multilymphoid progenitor;
MPP, multipotent progenitor;
NSG, NOD-scid-IL2Rgnull;
pDC, plasmacytoid DC; SCF,
stem cell factor.
The Rockefeller University Press $30.00
J. Exp. Med. 2015 Vol. 212 No. 3 385399
www.jem.org/cgi/doi/10.1084/jem.20141442
Ar ticle
Restricted dendritic cell and monocyte
progenitors in human cord blood
and bone marrow
Jaeyop Lee,1* Galle Breton,2* Thiago Yukio Kikuchi Oliveira,2
Yu Jerry Zhou,1 Arafat Aljoufi,1 Sarah Puhr,1 Mark J. Cameron,4
Rafick-Pierre Skaly,4 Michel C. Nussenzweig,2,3** and Kang Liu1**
1Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY 10032
2Laboratoryof Molecular Immunology and 3Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065
4Case Western Reserve University, Cleveland, OH 44106
In mice, two restricted dendritic cell (DC) progenitors, macrophage/dendritic progenitors
(MDPs) and common dendritic progenitors (CDPs), demonstrate increasing commitment to
the DC lineage, as they sequentially lose granulocyte and monocyte potential, respectively.
Identifying these progenitors has enabled us to understand the role of DCs and monocytes in
immunity and tolerance in mice. In humans, however, restricted monocyte and DC progenitors
remain unknown. Progress in studying human DC development has been hampered by lack
of an in vitro culture system that recapitulates in vivo DC hematopoiesis. Here we report a
culture system that supports development of CD34+ hematopoietic stem cell progenitors into
the three major human DC subsets, monocytes, granulocytes, and NK and B cells. Using this
culture system, we defined the pathway for human DC development and revealed the sequential
origin of human DCs from increasingly restricted progenitors: a human granulocyte-monocyte-DC
progenitor (hGMDP) that develops into a human monocyte-dendritic progenitor (hMDP),
which in turn develops into monocytes, and a human CDP (hCDP) that is restricted to produce
the three major DC subsets. The phenotype of the DC progenitors partially overlaps with
granulocyte-macrophage progenitors (GMPs). These progenitors reside in human cord
blood and bone marrow but not in the blood or lymphoid tissues.
DCs, monocytes, and macrophages are closely In the mouse, DC differentiation is depen-
related cell types whose interrelationship were dent on a hematopoietin, Flt3L, whose recep-
long debated and only recently elucidated in tor, Flt3 (CD135), is expressed throughout DC
the mouse (Geissmann et al., 2010; Merad et al., development (McKenna et al., 2000; Karsunky
2013). In mice, DCs and monocytes arise from et al., 2003; Waskow et al., 2008). In contrast,
a macrophage/dendritic progenitor (MDP; other hematopoietin receptors such as monocyte
Fogg et al., 2006), which produces monocytes, colony-stimulating factor receptor (M-CSFR
and a common dendritic progenitor (CDP) or CD115) and granulocyte macrophage colony-
that is restricted to the DC fate (Shortman and stimulating factor receptor (GM-CSFR or
Naik, 2007; Liu et al., 2009; Geissmann et al., CD116) are restricted to hematopoietic pro-
2010; Merad et al., 2013). The CDP produces genitors of DCs but not expressed on all ma-
preplasmacytoid DCs (pDCs) and pre ture DCs (Kingston et al., 2009).
conventional DCs (cDCs), the latter of which DC development in the human is far less
leaves the BM and circulates in the blood be- well understood than in the mouse. Human
fore entering tissues and developing into the monocytes can be induced to differentiate into
different DCs subsets (Naik et al., 2006, 2007; potent antigen-presenting cells with some
Onai et al., 2007b, 2013; Ginhoux et al., 2009;
Liu et al., 2009; Onai et al., 2013). 2015 Lee et al. This article is distributed under the terms of an Attribution
NoncommercialShare AlikeNo Mirror Sites license for the first six months after
*J. Lee and G. Breton contributed equally to this paper. the publication date (see http://www.rupress.org/terms). After six months it is
available under a Creative Commons License (AttributionNoncommercialShare
**K. Liu and M.C. Nussenzweig contributed equally to Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/
this paper. by-nc-sa/3.0/).
385

phenotypic features of DCs after in vitro culture with cock-
tails of cytokines (Sallusto and Lanzavecchia, 1994). However,
these monocyte-derived DCs are more closely related to ac-
tivated monocytes than to cDCs (Naik et al., 2006; Xu et al.,
2007; Cheong et al., 2010; Crozat et al., 2010). Progress in
defining the human DC lineage has been hampered, in part,
by a paucity of reliable markers to distinguish these cells from
monocytes, limited access to human tissues, the relatively
small number of circulating DCs in blood, and the lack of
386
Figure 1. Stromal culture system for DCs
and other leukocytes. (a) Flow cytometry
plots show DCs obtained from peripheral
blood (PBMC) and DCs obtained from
cultures of cord blood CD34+ cells with
Flt3L+SCF+GM-CSF+IL-4 (FSG-4) or mouse
BM stromal cells (MS5)+Flt3L or MS5+FSG.
pDCs, CD1c+ cDCs, and CD141+ cDCs are
shaded green, blue, and red, respectively. Pie
charts indicate the relative representation of
each DC subset in each group. Plots are repre-
sentative of two Flt3L+SCF+GM-CSF+IL-4 or
more than three MS5+Flt3L and MS5+FSG
experiments. (b) Graphs show output of the
indicated cells derived from CD34+ cells in
MS5 with Flt3L (F) versus FSG. Error bars indi-
cate SEM. (c) Flow cytometry plots show
phenotype of cells developing from 2,000
CD34+ HSPCs purified from human cord blood
and cultured in MS5+FSG for 14 d. Represen-
tative culture of five different donors. Pie
chart shows CD45+ cell composition.
a robust tissue culture system for the in vitro development of
all DC subsets (Poulin et al., 2010; Ziegler-Heitbrock et al.,
2010; Proietto et al., 2012).
Here we report a stromal cell culture system that supports
the development of CD34+ hematopoietic stem cell (HSC)
progenitors into the three major subsets of human DCs,
monocytes, granulocytes, and NK and B cells. Using this cul-
ture system, we have been able to define the sequential origin
of human DCs from a human granulocyte-monocyte-DC
Progenitor of human dendritic cells | Lee et al.

progenitor (hGMDP), which develops into a more restricted
human monocyte-dendritic progenitor (hMDP), which pro-
duces monocytes, and a human CDP (hCDP), which is re-
stricted to produce the three major subsets of DCs.
RESULTS
Human DC subsets develop in stromal cellcontaining
cultures in vitro
CD34+ hematopoietic stem and progenitor cells (HSPCs) cul-
tured in the presence of cytokines produce CD1c/BDCA1+
and CD141/BDCA3+ cDCs but fail to produce pDCs
(CD303/BDCA2+; Fig. 1 a; Poulin et al., 2010). Stromal cells
have been used to facilitate differentiation of pDCs (Spits et al.,
2000; Chicha et al., 2004; Olivier et al., 2006), but their ability
to support differentiation of all DC subsets as well as other
hematopoietic lineages has not been evaluated. In an attempt
to develop a method that would support development of all
three major types of DCs, we used a combination of mouse
BM stromal cells (MS5; Itoh et al., 1989) and defined human
cytokines. The combination of MS5 and Flt3L was sufficient
to support development of cord blood CD34+ HSPCs into
multiple cell types, including the three DC subsets, in pro-
portions similar to those found in peripheral blood (Fig. 1 a).
Addition of human stem cell factor (SCF) and human GM-
CSF (MS5+FSG, herein) increased the overall yield of DCs
(Fig. 1, a and b). MS5+FSG cultures produced granulocytes
(CD66b+), monocytes (CD14+CD16), NK cells (CD56+),
B cells (CD19+), pDCs, and both subsets of human cDCs
from human CD34+ cells, as determined by expression of cell
surface markers by flow cytometry (Fig. 1 c).
To further characterize the culture-derived DCs and
monocytes and compare them with primary cells from blood,
we performed whole transcriptome expression analysis on all
four subsets (Fig. S1). Using sparse hierarchical clustering, we
showed that all four cell types clustered separately from each
other and that cultured and primary monocytes clustered to-
gether, as did pDCs and cDCs (Fig. 2 a, Fig. S2, and Table S5).
We performed two separate analyses: monocytes versus pDCs
(Fig. 2 b) and CD141+ cDCs versus CD1c+ cDCs (Fig. 2 c).
Sparse hierarchical clustering showed that culture-derived
pDCs and monocytes are closely related to their in vivo coun
terparts (Fig. 2 b and Table S1), as are culture-derived CD1c+
and CD141+ cDCs to theirs (Fig. 2 c and Table S2).
A closer look at the expression of genes that constitute the
molecular signature of each subset (e.g., transcription factors
and surface receptors; Robbins et al., 2008; Crozat et al., 2010;
Schmidl et al., 2014) again indicates strong similarity between
cultured-derived cells and their in vivo counterparts (Fig. 2 d
and Table S3). For example, IRF8, BATF3, Zbtb46, and FLT3
were similarly and selectively expressed in cultured and pri-
mary CD141+ cDCs; ETS2, ID2, Zbtb46, and FLT3 in cul-
tured and primary CD1c+ cDCs; FOS, CD14, and CSF1R in
cultured and primary monocytes; and IRF7,TCF4, SPIB, and
IL3RA in cultured and primary pDCs (Fig. 2 d).
Within the cDC population, primary and cultured CD141+
cDCs clustered together, whereas primary and cultured
JEM Vol. 212, No. 3
Ar ticle
CD1c+ cDCs did not, which suggests that blood- and culture-
derived cells are not identical (Fig. 2 a and Fig. S2). Notably,
all of the culture-derived CD1c+ and CD141+ cDCs differed
from primary bloodderived counterparts in a similar man-
ner in that they were enriched for expression of genes that
mediate cell division, as determined by Gene Set Enrichment
Analysis (GSEA; Fig. 2 e, Fig. S2, and Table S4).This alteration
in gene expression is likely the result of increased prolifera-
tion in the cultures caused by high levels of Flt3L. Consistent
with this idea, primary peripheral bloodderived CD141+
cDCs phenocopy the culture-derived cells by acquiring
CD1c expression when they are placed into the MS5+FSG
(Fig. 2 f) or skin culture (Haniffa et al., 2012), and peripheral
blood CD141+ cDCs also coexpress CD1c in individuals that
are treated with Flt3L (see accompanying manuscript Breton
et al. in this issue). Finally, both culture-derived and primary
CD1c+ cDCs purified from peripheral blood acquire CD14
expression in culture (Fig. 2 f ).
Gene array data were confirmed by flow cytometry using
selected markers. Similar to primary pDCs, culture-derived
pDCs express high levels of CD123 and CD45RA and low
levels of HLA-DR but differ from mature cDCs in that they
do not express CD11c or CD86 (Fig. 3 a). In contrast, culture-
derived CD1c+ and CD141+ cDCs resemble their primary
peripheral bloodderived counterparts in their differential
expression of CX3CR1 and CD172a and in expressing high
levels of CD11c, HLA-DR, and CD86 but not CD83 or
CD80 (MacDonald et al., 2002; Lindstedt et al., 2005; Mittag
et al., 2011). Of note, CD1a and DC-SIGN, which are ex-
pressed by monocyte-derived DCs and absent on primary
CD1c+ cDCs (Chang et al., 2000), are not expressed on
culture-derived CD1c+ or CD141+ cDCs (Fig. 3 a). In addition,
culture-derived CD141+ cDCs express CLEC9a (DNGR1;
Fig. 3 a), a marker specifically expressed on primary CD141+
cDCs (Poulin et al., 2010, 2012).
To examine the functional properties of cultured-derived
cDCs, we measured their responses to TLR ligands. Like their
primary bloodderived counterparts, only the culture-
derived pDCs produced IFN- in response to CpG (Fig. 3 b;
Ito et al., 2005; Liu, 2005). Similarly, culture-derived and pri-
mary CD141+ cDCs produced the highest amount of IFN-
and IL-12 in response to Poly(I:C) (Fig. 3 b; Kadowaki et al.,
2001; Poulin et al., 2010). We conclude that MS5+FSG sup-
ports differentiation of multiple hematopoietic lineages from
their progenitors, including human pDCs and CD1c+ and
CD141+ cDCs.
DC-restricted progenitors
Several different, early, CD34+ hematopoietic progenitors pu-
rified from human cord blood or BM are reported to give rise
to DCs (Chicha et al., 2004; Ishikawa et al., 2007; Doulatov
et al., 2010; Kohn et al., 2012). These include common lym-
phoid progenitors (CLPs; Galy et al., 1995; Chicha et al., 2004;
Ishikawa et al., 2007), common myeloid progenitors (CMPs;
Akashi et al., 2000; Manz et al., 2002; Chicha et al., 2004;
Ishikawa et al., 2007), granulocyte-macrophage progenitors
387
Figure 2. Culture-derived DCs resemble primary DCs. (ae) Transcriptional profiling of pDCs, monocytes, and CD1c+ and CD141+ cDCs purified
from primary peripheral blood (blood; six healthy individuals) or from culture of CD34+ cells in MS5+Flt3L for 14 d (culture; four cord blood donors) as
in Fig. S1. (a) Hierarchical clustering dendrogram of cultured versus primary pDCs, monocytes, and CD1c+ and CD141+ cDCs. This dendrogram was gen-
erated using the top 611 differentially expressed genes selected by unsupervised clustering (sparse hierarchical clustering using all genes; Table S5).
(b) Heat map showing the sparse hierarchical clustering of mRNAs expressed by primary and culture-derived pDCs and monocytes. This analysis showed
that a minimal number of 78 genes is enough to distinguish one cell type from another. The normalized expression values for the top 78 differentially
expressed genes (Table S1) are displayed. (c) Heat map showing the sparse hierarchical clustering of mRNAs expressed by primary and culture-derived

388 Progenitor of human dendritic cells | Lee et al.

 Ar ticle

Figure 3. Culture-derived cells resemble

their ex vivo counterparts in phenotype
and function. (a) Histograms show cell sur-
face markers of human monocytes and DCs
isolated from blood (top rows) and MS5+FSG
cultures (bottom rows). (b) Cord blood CD34+-
derived DC subsets were cultured for 14 d,
purified by FACs, and exposed to the indicated
TLR stimuli. Graphs indicate concentration of
IFN- and IL-12p70 in the supernatant mea-
sured by ELISA after 48 h from three indepen-
dent experiments. n (number of donors) = 3.
Error bars indicate SEM.

(GMPs; Manz et al., 2002; Chicha et al., 2004; Ishikawa et al.,
2007; Doulatov et al., 2010), a myeloid DC progenitor (Olweus
et al., 1997), multilymphoid progenitors (MLPs; Doulatov et al.,
2010), and lymphoid-primed multipotent progenitors (LMPPs;
Kohn et al., 2012). However, none of these cell types is re-
stricted to the monocyte or DC lineage (Chicha et al., 2004;
Ishikawa et al., 2007; Doulatov et al., 2010; Kohn et al., 2012).
In mice, monocyte- and DC-restricted progenitors can be
identified by differential expression of CD117 (cKit), CD135
(Flt3), CX3CR1, CD115 (M-CSFR), and Ly6C (Fogg et al.,
2006; Naik et al., 2007; Onai et al., 2007a, 2013; Hettinger
et al., 2013; Merad et al., 2013). In humans, all myeloid and
lymphoid progenitors express CD34, CD117, and CD135
(Doulatov et al., 2010). Thus, additional markers are required
to further purify human monocyte and DC progenitors. To
attempt to distinguish human monocyte and DC progenitors
from earlier and less restricted precursors, we combined two
separate sets of antibodies: one set that can separate human
CD34+ hematopoietic progenitor cells into six populations
with distinct lineage potential (Fig. 4 a; Doulatov et al., 2010)
and the other specific against receptors for Flt3L (CD135),
GM-CSF (CD116), M-CSF (CD115), and IL-3 (CD123),
which are differentially expressed by monocytes and DCs
(Breton et al., 2015).
We found that among the six populations of cord blood
CD34+ cells, namely HSCs/multipotent progenitors (MPPs),
MLPs, megakaryocytic and erythroid progenitors, B/NK,
CMPs, and GMPs, only GMPs (CD34+CD38hiCD135+CD
45RA+CD10) exhibit heterogeneity in CD115+, CD116+,
and CD123hi expression (Fig. 4 b) and contain cells with DC
progenitor activity (Olweus et al., 1997; Chicha et al., 2004;
Doulatov et al., 2010). To determine whether GMP can be
further fractionated into monocyte- and DC-restricted pro-
genitors, we separated them into five populations on the basis
of CD115, CD116, and CD123 expression (Fig. 5 a) and cul-
tured 200 cells from each purified population in MS5+FSG
cultures for 7 d. We expected that DC progenitors show po-
tential to produce all three DC subsets. CD123hiCD115,

CD1c+ and CD141+ cDCs. This analysis showed that a minimal number of 80 genes is enough to distinguish one cell type from another. The normalized
expression values for the top 80 differentially expressed genes (Table S2) are displayed. (d) Heat map showing the hierarchical clustering of mRNAs for
selected genes (Table S3) expressed by primary and culture-derived pDCs, monocytes, and CD1c+ and CD141+ cDCs. (e) Top 50 enriched KEGG metabolic
pathways (Table S4) for genes shared by both subsets of cultured cDCs but not primary cDCs according to GSEA analysis. (f) Phenotype change of blood
CD141+ and CD1c+ cDCs in culture. Blood CD141+ and CD1c+ cDCs were purified and cultured for 7 d in MS5+FSG. Flow cytometry plots of gated CD45+
cells show cell surface markers of output cells.

JEM Vol. 212, No. 3 389

Figure 4. Fractionation of cord blood progenitors based on cytokine receptor expression. (a and b) Flow cytometry plots show exhaustive sepa-
ration of CD34+ cord blood cells into six populations, HSCs/MPPs, MLPs, B and NK progenitors (B/NK), CMPs, megakaryocytic and erythroid progenitors
(MEP), and GMPs (a), and expression of CD115, CD116, CD135, and CD123 on each of the cord blood CD34+ populations in a (b).

CD123intCD116CD115+, and CD123intCD116CD115 CD123intCD116CD115+ cells produced pDCs, cDCs, and

cells gave rise to pDCs and CD1c+ and CD141+ cDCs
(Fig. 5 b). Although CD123intCD116+CD115 or CD123int
CD116+CD115+ cells gave rise to CD1c+ cDCs, they failed
to produce pDCs or CD141+ cDCs. These two populations
were therefore excluded as CDPs (Fig. 5 b).
CD123hiCD115 cells were restricted to DCs and pro-
duced no monocytes or granulocytes, reminiscent of the
CDPs in the mouse (Naik et al., 2007; Onai et al., 2007b),
and we will refer to them as hCDPs. These cells constitute
0.41% (range of 0.030.87%) of cord blood CD34+ cells.
CD14+CD1c monocytes (Patterson et al., 2005; Granelli-
Piperno et al., 2006), but produced few, if any, granulocytes
and are therefore the human equivalent of the mouse MDP
(Fogg et al., 2006) and will be referred to as hMDPs. In cord
blood samples, the hMDPs constitute 0.56% (range of 0.18
0.70%) of CD34+ cells and can be identified using the CD34+
CD45RA+CD123intCD115+ phenotype because of the neg-
ligible frequency of CD123intCD116+CD115+ (0.04% of
CD34+ cells). In contrast, CD123intCD116CD115 cells
produced DCs, monocytes, and granulocytes and will be

390 Progenitor of human dendritic cells | Lee et al.


referred to as hGMDPs (Fig. 5 b), which constitute 10.08%
(range of 6.1513.2%) of cord blood CD34+ cells. As might
be expected, the less differentiated hGMDPs and hMDPs
have a higher proliferative potential in vitro than hCDPs
(Fig. 5 c). All of these DC progenitors express HLA-DR but
not CD11c (Fig. 5 d). Morphologically, hGMDPs, hMDPs,
JEM Vol. 212, No. 3
Ar ticle
Figure 5. Characterization of cord blood
progenitors. (a) Flow cytometry plots show
gating of cord blood CD34+CD38hiCD10CD45
RA+CD123int/hi GMP cells (Doulatov
et al., 2010) and further separation into
five separate populations based on CD123,
CD115, and CD116 expression: CD123hiCD115
(CD123hi), CD123intCD115+CD116
(CD115+), CD123intCD115+CD116+ (DP),
CD123intCD115CD116+ (CD116+), and
CD123intCD115CD116 (DN). (b) Differen-
tiation potential of 200 purified cells from
each of the five populations indicated in a in
MS5+FSG culture harvested after 7 d. Flow
cytometry plots show CD45+CD56CD19
cells. (c) Graph indicates output/input ratio
of total number of CD45+ cells obtained from
each of the five populations sorted in a.
Bars and error bars are means and SEM,
respectively, from three independent experi-
ments. (d) Histograms show expression of
indicated markers on hGMDPs, hMDPs, and
hCDPs. (e) Morphology of purified cord blood
hGMDPs, hMDPs, and hCDPs by Giemsa
staining of cytospin preparations. Bars, 10 m.
(f) Graph indicates the differentiation poten-
tial of hCDPs, hMDPs, hGMDPs, and CMPs in
methylcellulose colony formation assays
in vitro (as in Materials and methods). Colonies
were enumerated at 14 d after culture. BFU-E,
burst-forming unit erythroid; GEMM, granu-
locyte, erythrocyte, macrophage, megakaryo-
cyte; GM, granulocyte and macrophage; G,
granulocyte; M, macrophage. Bars are means
and error bars are SEM from three independent
experiments. (g) Cross-phenotyping hCDPs,
hMDPs, and hGMDPs with DC-associated pro-
genitors. hCDPs, hMDPs, and hGMDPs were
identified by flow cytometry and overlaid
with previously identified progenitors (gray).
CLPs were gated as CD45+Lin(CD3/19/56/
14)CD34+CD10+CD45RA+ (Galy et al., 1995;
Ishikawa et al., 2007), LMPPs as CD45+
LinCD34+CD10CD62L+CD45RA+ (Kohn
et al., 2012), CMPs as CD45+LinCD34+CD38+
CD10CD45RACD123+, GMPs as CD45+Lin
CD34+CD38+CD10CD45RA+CD123+/hi (Chicha
et al., 2004; Doulatov et al., 2010), MLPs as
CD45+LinCD34+CD38CD45RA+ (Doulatov
et al., 2010), and myeloid DC progenitors as
CD45+LinCD34+CD123hi (Olweus et al., 1997).
n (number of donors) = 3.
and hCDPs were very similar and showed a high nucleus/
cytoplasm ratio as well as multilobulated nuclei (Fig. 5 e).
In view of the more limited outgrowth of granulocytes
from the MS5+FSG cultures, we examined the DC pro-
genitors potential to produce monocytes and granulocytes
in CFU assays. In agreement with the MS5+FSG cultures,
391

Figure 6. Developmental potential of single progenitor cells.
(a) Graph shows the percentage of positive wells obtained from culturing
single hCDP, hMDP, hGMDP, CD123intCD115CD116+ (CD116+), and
CD123intCD115+ CD116+ (DP) cells in MS5+FSG culture. Clonal effi-
ciency calculated based on the number of positive wells is indicated.
Data are pooled from three independent experiments. (bd) Bar graphs
summarize the cellular output of all positive single cell cultures of
hCDPs (b), hMDPs (c), and hGMDPs (d) from three independent experi-
ments (n = 3 donors). The number of wells per category is noted on
top of each bar. DC, pDC and/or cDC; G, granulocyte; L, lymphocyte;
M, monocyte.
hCDPs produced rare CFU, hMDPs produced mostly mono-
cytes but no granulocytes, and hGMDPs produced both
monocytes and granulocytes but no erythrocytes (Fig. 5 f ).
To define the relationship between hCDPs, hMDPs, and
hGMDPs and previously reported DC progenitors, we per-
formed cross-phenotyping experiments.We found that, GMPs
include hCDPs, hMDPs, and hGMDPs; myeloid DC pro-
genitors overlap with hCDPs; LMPPs partially overlap with
hCDPs, hMDPs, and hGMDPs, whereas CLPs, MLPs, and
CMPs do not overlap with hCDPs, hMDPs, and hGMDPs
(Fig. 5 g). We conclude that populations containing hGMDPs,
hMDPs, and hCDPs can be isolated from human cord blood
by using cytokine receptor expression to distinguish them
from less committed leukocyte precursors.
Single cell assays
To examine the developmental potential of individual cells in
the hGMDP, hMDP, and hCDP populations, we purified and
cultured single cells from each population. The relative clonal
392
efficiency, as measured by their ability to give rise to CD45+
cells, was 22% for hCDPs (89/408 wells), 80% for hMDPs
(152/191 wells), and 58% for hGMDPs (103/179 wells; Fig. 6 a).
In contrast, CD123intCD116+CD115 or CD115+ cells
showed lower clonal efficiency, 11% (13/122 wells) and 10%
(9/95 wells), respectively (Fig. 6 a). All positive wells were fur-
ther evaluated for production of specific cell lineages, and clonal
potential was categorized as multilineage or unilineage. The
multilineage clones yielded combinations of granulocytes,
monocytes, and DCs (G+M+DC), monocytes and DCs
(M+DC), granulocytes and monocytes (G+M), myeloid
(G/M/DC), and lymphoid (B or NK cells; G/M/DC+L), and
the unilineage ones produced only lymphoid cells (B or NK, L),
granulocytes (G), monocytes (M), or DCs (Fig. S3). Of the 89
positive wells produced by hCDPs, 84 contained pDCs and/or
cDCs and no monocytes or granulocytes (Fig. 6 b and Fig. S4).
Importantly, two clones produced both pDCs and cDCs (Fig. S4),
formally proving the existence of a clonal progenitor that pos-
sesses both pDC and cDC potential. Of the 152 positive
wells obtained from hMDPs, 31% contained only monocytes,
41% only DCs, 13% contained both monocytes and DCs, and
rare clones produced granulocytes (3.9%) or lymphocytes
(1.3%; Fig. 6 c and Figs. S5 and S6). Thus, hMDPs show little
granulocyte or lymphocyte potential and are restricted primar-
ily to monocytes and DCs. Among the 103 positive wells pro-
duced by hGMDPs, 22% contained only granulocytes, 4% only
monocytes, 20% only DCs, 17% contained monocytes and
DCs, 6% showed granulocytes, monocytes, and DCs, and 14%
produced lymphoid cells alone or in combination with my-
eloid cells (Fig. 6 d and Figs. S7 and S8). Thus, hGMDPs
contain clonal progenitors that can produce granulocytes,
monocytes, and DCs, but this population is heterogeneous and
also contains cells with the potential to give rise to lympho-
cytes (Fig. 6 d and Figs. S7 and S8). The residual lymphocyte
potential in this population may result in part from contamina-
tion of MLPs, which are difficult to separate from hGMDPs
using CD38 (Doulatov et al., 2010).
Progenitorprogeny relationships
of hGMDPs, hMDPs, and hCDPs
The sequential loss of differentiation potential to granulocytes,
and then to monocytes at the hMDP and hCDP stages, sug-
gests that a differentiation hierarchy exists between hGMDPs,
hMDPs, and hCDPs. To determine whether, in fact, these
progenitors are related in this way, we initially performed
in vivo transfer experiments. Purified hGMDPs were transferred
into the bone cavity of NOD-scid-IL2Rgnull (NSG) mice
(Material and methods; Kalscheuer et al., 2012). 7 d after
transfer, both hMDPs and hCDPs were detected among the
donor-derived cells in the BM, indicating that hMDPs and
hCDPs descend from hGMDPs (Fig. 7 a).
To further refine this developmental hierarchy, we exam-
ined the development of purified hGMDPs, hMDPs, and
hCDPs in tissue culture over time using flow cytometry.
Whereas hGMDPs and hMDPs retained CD34 expression
for at least 4 d, hCDPs down-regulated CD34 within 2 d,
Progenitor of human dendritic cells | Lee et al.
 Ar ticle

Figure 7. Relationship of hGMDPs to hMDPs and to hCDPs. (a) 10,000 hGMDPs were purified from cord blood and adoptively transferred into the
bone cavity of the preconditioned NSG mice (Materials and methods). 7 d later, BM cells were analyzed. Flow cytometry plots show phenotype of BM cells
from NSG mice receiving PBS or hGMDPs (n = 3 mice). (b) 200400 hGMDP, hMDP, and hCDP cells were purified and cultured in MS5+FSG. Cultures were
analyzed on days 1, 4, and 8. Flow cytometry plots gated on live CD45+Lin(CD3/19/56/14)DC(CD1c/141/303)CD45RA+ cells show cell surface markers
and frequency of hCDPs, hMDPs, and hGMDPs. Results are representative of three independent experiments.

indicating that hCDPs are the most differentiated among the
three groups. Further phenotypic analysis of the CD34+ cells
revealed that hGMDPs sequentially produced hMDPs and
then hCDPs and that hMDPs produced hCDPs (Fig. 7 b). In
addition, both hGMDPs and hMDPs maintained CD34 ex-
pression after 4 d in culture (Fig. 7 b), suggesting they may
have the ability to self-renew. In contrast, hCDPs rapidly lost
CD34 and produced all three subsets of DCs, but did not
produce hMDPs or hGMDPs (Fig. 7 b). We conclude that
hGMDPs gave rise to hMDPs, which produced monocytes
and hCDPs, which are finally restricted to pDCs and cDCs.
Distribution in adult hematopoietic organs
To determine whether hGMDPs, hMDPs, and hCDPs
also participate in hematopoiesis in the adult, we examined
human BM samples, peripheral blood, and tonsils. Adult
BM contained hGMDPs (mean of 5.22%, range of 1.76
7.40% of CD34+ cells), hMDPs (mean of 0.56%, range of
0.081.94%), and hCDPs (mean of 1.98%, range of 0.05
7.29%; Fig. 8 a). When these populations were purified and
cultured in MS5+FSG, they showed differentiation poten-
tial similar to those exhibited by their counterparts in cord
blood (Fig. 8 b). In contrast, hGMPs, hMDPs, and hCDPs
were undetectable in the peripheral blood or tonsils (Fig. 8 a),
indicating that in the steady-state, these progenitors are
retained in the BM. Of note, hGMDPs from BM produced
more monocytes (P < 0.05) and less CD1c+ cDCs (P <
0.01) than their counterparts from cord blood, whereas BM
hCDPs produced less pDCs than their cord blood counter-
parts (P < 0.05; Fig. 8 c). Moreover, CD141+ cDCs derived
from BM hCDPs did not fully up-regulate CLEC9a expres-
sion (Fig. 8 b).

JEM Vol. 212, No. 3 393


DISCUSSION
We have delineated the developmental pathway that leads to
production of human DCs and enumerated these cells in cord
blood and BM. This pathway involves sequential loss of devel-
opmental potential from a progenitor that can produce granulo-
cytes, monocytes, and DCs (hGMDPs), to one that produces
monocytes and DCs (hMDPs), to a committed DC progenitor
that produces cDCs and pDCs (hCDPs). Others have shown
that human GMPs, CLPs, and LMPPs can produce human DCs
(Chicha et al., 2004; Ishikawa et al., 2007; Kohn et al., 2012).
Our findings are not inconsistent with these observations be-
cause GMPs and LMPPs are heterogeneous groups of cells that
contain subpopulations with the cell surface features of DC pro-
genitors, as revealed by cross-phenotyping (Fig. 5 g).
394
Figure 8. Distribution of hGMDPs, hMDPs,
and hCDPs in adult hematopoietic organs.
(a) Representative flow cytometry plots of
gated CD45+Lin(CD3/19/56/14)CD34+ cells
show hGMDPs, hMDPs, and hCDPs in human
BM (n = 4), peripheral blood (PBMC; n = 4), and
tonsils (n = 4). (b) hGMDPs, hMDPs, and hCDPs
were purified from human BM, and 2,500 pro-
genitors were cultured in MS5+FSG for 7 d.
Flow cytometry plots of gated live CD45+ cells
show phenotype of output cells, including
granulocytes (brown), CD141+ cDCs (red),
CD1c+ cDCs (blue), monocytes (orange), and
pDCs (green). Data represent three independent
experiments. (c) Graph shows output/input cell
ratio in percentage of the indicated cells de-
rived from BM (n = 3) or cord blood (CB; n = 3)
cultures of hGMDPs, hMDPs, and hCDPs in
MS5+FSG for 7 d. Statistical significance was
determined using unpaired Students t test.
*, P < 0.05; **, P < 0.001.
Identification of restricted monocyte and DC progenitors
in human BM facilitates understanding of diseases involving
aberrant DC hematopoiesis. For example, patients harboring
GATA2 mutations develop a newly identified form of pri-
mary immunodeficiency characterized by recurrent infec-
tions and disseminated BCG infection after vaccination (Vinh
et al., 2010). These patients lack B cells, NK cells, mono-
cytes, cDCs, and pDCs in the blood, but have a normal gran-
ulocyte compartment (Bigley et al., 2011). Analysis of their
CD34+ progenitor compartment revealed an absence of
CD45RA+ cells, including MLPs and GMPs (Bigley et al.,
2011), which contain hCDPs, hMDPs, and hGMDPs. The
normal granulocyte development in these patients suggests
that acquiring CD45RA marks a crucial developmental step
Progenitor of human dendritic cells | Lee et al.

for monocytes and DCs, but not for granulocytes. Addition-
ally, mutations of IRF8 cause monocyte and DC deficiency
without affecting granulocytes (Hambleton et al., 2011), im-
plying an IRF8-dependent developmental block in the tran-
sition from hGMDPs to hMDPs.
A second example involves chronic myelogenous leuke-
mia (CML). CML is caused by BCR-Abl translocation and is
a myeloproliferative disease of granulocytes. CML patients
show a dramatic decrease of blood pDCs and cDCs but rela-
tively normal numbers of monocytes (Boissel et al., 2004).
This is accompanied by a relative deficiency in CD34+CD123hi
hCDPs but only slightly reduced CD123intCD45RA+ cells,
which include hMDPs and hGMDPs (Diaz-Blanco et al.,
2007), suggesting a block in the transition from hMDPs to
hCDPs. The ability to purify human DC progenitors and fol-
low their differentiation after transfer into NSG mice (Fig. 7 a;
Doulatov et al., 2010) should facilitate the study of human
DC differentiation in vivo.
Defining human DC progenitors required that we de-
velop an efficient tissue culture method that supports devel-
opment of all three major human DC subtypes: CD1c+
cDCs, CD141+ cDCs, and pDCs. Although others have
shown that human CD141+ cDCs could be obtained by ex-
panding HSPCs in vitro and then subculturing them in SCF,
Flt3L, GM-CSF, and IL-4 (Poulin et al., 2010) and that stro-
mal cells could facilitate pDC development (Spits et al., 2000;
Chicha et al., 2004; Olivier et al., 2006), defining DC pro-
genitors required a culture system that would produce all
three types of DCs and also support development of mono-
cytes, granulocytes, and lymphoid cells. Addition of stromal
cells to the cocktail of Flt3L, SCF, and GM-CSF is sufficient
to support efficient development of CD34+ human HSPCs
to lymphoid and myeloid cells, including B cells, NK cells,
granulocytes, and all three DC subsets without pre-expansion
of stem cells in vitro. Importantly, the DC subsets derived
from the stromal cell cultures closely resemble primary CD1c+
cDCs, CD141+ cDCs, and pDCs obtained from the blood of
normal donors as determined by gene expression, surface
phenotype, and cytokine production.
Flt3L, M-CSF, GM-CSF, and IL-3 exert distinct func-
tions on development and homeostasis of monocytes, cDCs,
and pDCs (Schmid et al., 2010; Merad et al., 2013). Our
experiments show that human CD34+ progenitors are het-
erogeneous for expression of these cytokine receptors, but
they are all contained in the GMP fraction in cord blood.
GMP can be divided into five populations based on CD115,
CD116, and CD123 expression. Three of these populations
show potential to produce granulocytes, monocytes, and
DCs: hGMDPs, hMDPs, and hCDPs. In healthy individu-
als, these hGMDPs, hMDPs, and hCDPs are found in cord
blood and in the BM. They do not circulate and are not
found in peripheral lymphoid organs. Most of CD141+
cDCs derived from BM progenitors, particularly those de-
rived from hCDPs, did not up-regulate CLEC9a. This may
result from subtle differences between progenitors from adult
BM and cord blood.
JEM Vol. 212, No. 3
Ar ticle
Although the cell surface markers that define DC precur-
sors are not entirely conserved between mouse and human,
there is significant overlap, especially in cytokine receptor
expression. Importantly, using cytokine receptor expression
is crucial for purification of lineage-restricted progenitors.
For instance, the originally discovered mouse MDPs
(LinCX3CR1-GFP+cKit+) lack granulocyte potential (Fogg
et al., 2006). However, a subpopulation with 13-fold higher
clonal efficiency to produce DC-monocyte/macrophage
(Sathe et al., 2014) can be purified using the CD115+CD135+
phenotype from the original MDP population. Moreover,
the sequential loss of granulocyte and monocyte potential in
humans parallels DC differentiation in the mouse. For exam-
ple, in both species, MDP is a stage marked with loss of gran-
ulocyte potential and expression of CD115 (Fogg et al., 2006;
Waskow et al., 2008), the receptor which enables monocyte
and macrophage development (Witmer-Pack et al., 1993;
Greter et al., 2012). This degree of conservation is an indica-
tion of the relative importance of this pathway during verte-
brate evolution.
MATERIALS AND METHODS
Cell samples. Human umbilical cord blood and leukophoretic peripheral
blood (buffy coat) were purchased from the New York Blood Center.
Human BM was obtained from total hip arthroplasty by J. Schreiber at Hos-
pital for Special Surgery (New York). Tonsils were obtained from routine
tonsillectomies performed at the Babies and Childrens Hospital of Columbia-
Presbyterian Medical Center. Informed consent was obtained from the
patients, and/or samples were exempt from informed consent being residual
material after diagnosis and fully de-identified. All samples were collected
according to protocols approved by the Institutional Review Board at Co-
lumbia University Medical Center (CUMC) and The Rockefeller Univer-
sity. The specimens were kept on ice immediately after surgical removal.
Tonsil samples were minced, treated with 400 U/ml collagenase (Roche) at
37C for 20 min, and proceeded to cell isolation. BM samples were pre-
served in solution containing 1,000 U/ml heparin (National Drug Code
#63323-540-11) and digested in RPMI containing 20 mg/ml collagenase
IV (Sigma-Aldrich) for 15 min at 37C. After density centrifugation using
Ficoll-Hypaque (GE Healthcare), aliquots of mononuclear BM cells were
frozen and stored in liquid nitrogen for future analysis.
Cell isolation and flow cytometry. Fresh mononuclear cells were iso-
lated by density centrifugation using Ficoll-Hypaque (GE Healthcare). Sam-
ples from cord blood, peripheral blood, BM, and tonsil were incubated with
fluorescent-labeled antibodies for direct analysis on the LSRII flow cytome-
ters (BD) or further purification by fluorescence-activated cell sorting on the
Influx or FACSAria (BD), both using HeNe and argon lasers. Sorted popu-
lation showed >95% purity.
For purification of differentiated DCs and monocytes from peripheral
blood and culture, cells were stained with LIVE/DEAD (Life Technologies),
CD45 (HI30, Alexa Fluor 700 [AF700]; BioLegend), CD66b (G10F5,
PerCP-Cy5.5; BioLegend), CD56 (B159, Pacific Blue; BD), CD19 (HIB19,
APC-Cy7; BioLegend), CD14 (TuK4, Qdot-655; Invitrogen), CLEC9a
(8F9, PE; BioLegend), CD1c (L161, PE-Cy7; BioLegend), CD303 (201A,
FITC; BioLegend), CD123 (6H6, Brilliant Violet [BV] 510; BioLegend),
and CD141 (AD5-14H12, APC; Miltenyi Biotec) for 40 min on ice. Alter-
natively, we used CD335 (9E2, BV421; BioLegend), CD11c (3.9, A700;
eBioscience), CD3 (S4.1, PE Texas Red; Invitrogen), and CD19 (SJ25-C1,
PE Texas Red; Invitrogen).
For surface marker analysis, CD11c (3.9; BioLegend), HLA-DR (G46-6;
BD), CD80 (2D10, Biotin; BioLegend), CD83 (HB15e; BioLegend), CD86
(IT2.2; BioLegend), and DC-SIGN/CD209 (DCN46; BD) were used in
395
PerCP-Cy5.5, and CD123 (9F5; BD), CX3CR1 (2A9-1; BioLegend),
SIRP/CD172 (SE5A5; BioLegend), CD45RA (HI100; eBioscience),
or CD1a (HI149; BD) was used in PE.
For single progenitor lineage potential and developmental hierarchy rela-
tionship experiments, CD34+ cells were first enriched from cord blood using
the CD34 MicroBead kit and LS MACS magnetic columns (Miltenyi Bio-
tec). Enriched CD34+ cells (4095% purity) were incubated with antibodies
against CD3 (OKT3, BV650; BioLegend), CD19 (HIB19, BV650; BioLegend),
CD56 (HCD56, BV650; BioLegend), CD14 (Qdot-655), CD34 (581,
AF700; BioLegend), CD38 (HIT2, BV421; BioLegend), CD45RA (HI100,
BV510; BioLegend), CD123 (9F5, PE; BD), CD10 (HI10a, PE-Cy7; Bio-
Legend), CD116 (4H1, FITC; BioLegend), and CD115 (9-4D2-1E4, APC;
BioLegend). From CD34+ HSPCs, hGMDPs were sorted as Lin(CD14/
19)CD34+CD38hiCD45RA+CD123intCD10CD115CD116, hMDPs
as LinCD34+CD38hiCD45RA+CD123intCD10CD115+CD116, and
hCDPs as LinCD34+CD38hiCD45RA+CD123hiCD115. For surface
phenotype analysis, CD45 (HI30, APC-Cy7; eBioscience), CD123 (6H6,
BV421; BioLegend), CD135 (4G8, PE; BD), and CD62L (DREG-56, PB;
BD) were used alternatively.
For progenitor-progeny experiments, cells from either culture or NSG
BM were stained for LIVE/DEAD, CD45 (AF700), CD14 (Qdot-655),
CD3 (OKT3, BV650; BioLegend), CD19 (HIB19, BV650; BioLegend), CD56
(HCD56, BV650; BioLegend), CD1c (L161, Biotin; BioLegend), CD141 (M80,
PE-Cy7; BioLegend), CD34 (581, APC-Cy7; BioLegend), CD123 (9F5, PE;
BD), CD45RA (BV510), CD303 (FITC), and CD115 (APC) for 40 min on
ice. Secondary staining was performed with PerCP-Cy5.5conjugated strep-
tavidin on ice for 40 min.
Morphological analysis. Purified hGMDPs, hMDPs, and hCDPs were ana-
lyzed by Giemsa staining of cytospin preparations. As few as 5 104 cells were
cytospun for 5 min at 800 rpm on a glass slide and then stained with the He-
macolor stain kit (Harleco) as recommended by the manufacturer. Slides were
then imaged on an Axioplan 2 microscope (Carl Zeiss) at 100 magnification.
Cell culture. For cord blood and BM stromal culture, MS5 cells, provided
by B. Reizis (Columbia University) and originally obtained from Leibniz-
Institut DSMZ, were maintained and passed in complete -MEM medium
(Invitrogen) with 10% FCS and penicillin/streptomycin (Invitrogen). After
3 h of 10 g/ml mitomycin C (Sigma-Aldrich) treatment and wash, 3.75
104 MS5/ml was seeded per well in 96-well plates or 1.5 105 cells per well
in 24-well plates 24 h before culturing hematopoietic cells. Bulk CD34+
cells or purified progenitor populations were seeded in medium containing
100 ng/ml Flt3L (Celldex Therapeutics), 20 ng/ml SCF (PeproTech), and
10 ng/ml GM-CSF (PeproTech). Cells were harvested between days 1 and
14 for flow cytometry analysis.
Stroma cellfree cultures were performed according to previously re-
ported methods (Poulin et al., 2010). In brief, 5 104 purified bulk CD34+
cells were cultured in 1 ml StemSpan medium (STEMCELL Technologies)
with penicillin/streptomycin and 100 ng/ml SCF, 100 ng/ml Flt3L, 20 ng/ml
IL-3, and 20 ng/ml IL-6 for expansion. After 2 wk of culture with one me-
dium change at day 7, cells were washed and replated at 6.25 104 cells/ml in
RPMI 1640 with glutamine, penicillin/streptomycin, 2-betamercaptoethanol
(all from Invitrogen), and 10% FCS in the presence of 20 ng/ml SCF, 20 ng/ml
GM-CSF, 20 ng/ml IL-4, and 100 ng/ml Flt3L (Flt3L from Celldex Thera-
peutics, all other cytokines from PeproTech) to induce differentiation of
CD1c+ and CD141+ cDCs. Cells were cultured for 2 wk with one medium
change at day 7, before flow cytometry analysis. Absolute CD45+ cell numbers
from cultures were calculated relative to a well with a known number of
CD45+ cells (i.e., 10,000 cells). CFU assay was performed using MethoCult
(H4434; STEMCELL Technologies), containing SCF, GM-CSF, IL-3, and
EPO. CFU-cells (CFU-Cs) were counted after 14 d of culture.
Functional analysis of DC function. DC subsets were sorted from culture,
and 40,000 cells/100 l were plated in complete RPMI medium containing
10% FCS and penicillin/streptomycin. Cells were stimulated with 1 M CpG
396
ODN2216 (InvivoGen), 50 g/ml Poly(I:C) (InvivoGen), or 10 g/ml LPS
(Sigma-Aldrich). 48 h later, supernatant was collected, and IFN- and IL-
12p70 were analyzed using ELISA (Mabtech and eBioscience, respectively).
In vivo transplantation into NSG mice. NOD.Cg-Prkdcscid-IL2rgtmlWjl/
Sz (NSG) mice were developed at the Jackson Laboratory. All experiments
were performed according to the guidelines of the institutional animal care
and use committee at CUMC. NSG mice were injected intraperitoneally
with busulfan (30 g/g of body weight; Sigma-Aldrich) to ablate the endog-
enous hematopoietic system. Human progenitors purified from cord
blood were resuspended in 10 l PBS and injected into the bone cavity.
Mice were injected intraperitoneally for five consecutive days with 10 g
Flt3L (provided by T. Keller from Celldex Therapeutics), starting at 1 d
after transfer. 7 d after transplantation, BM was harvested from recipient
mice and analyzed for human CD45+ cells.
Clonal analysis of progenitors. Progenitors were first sorted as separate
populations, which were then individually sorted as single cells directly into
96-well plates containing mitomycin Ctreated stromal cells. The sorting effi-
ciency was determined using CFSE-labeled cells, resulting in 9.5% empty
wells, 90.5% wells containing one cell, and 0% containing more than one cell.
Each well was harvested and stained with LIVE/DEAD, CD45, CD66b,
CLEC9a, CD14, CD1c, CD303, CD141, CD19, and CD56. Positive clones
were determined by the detection of at least 10 human CD45+ cells using flow
cytometry. We defined each positive clones lineage output potential by posi-
tively scoring when more than seven cells were found for each cell type.
Array analysis of RNA expression. RNA was extracted using RNeasy
Plus procedures (QIAGEN) according to the manufacturers protocol. Total
RNA was checked for quantity and quality using a NanoDrop 2000c spec-
trophotometer (Thermo Fisher Scientific) and Experion automated electro-
phoresis system (Bio-Rad Laboratories). Only RNA samples with a 28S/18S
ratio >1.5 were processed for array analysis. 500 ng of total RNA was ampli-
fied using the Illumina Total-Prep RNA amplification kits (Applied Biosys-
tems), as recommended by the manufacturer. 750 ng of the biotinylated cRNA
was hybridized onto HumanHT-12_V4 Expression BeadChips (Illumina) at
58C for 20 h and quantified using an iScan System and GenomeStudio
software (both Illumina).
For analysis, signature transcripts were selected and clustered using the
sparse hierarchical clustering tool and visualized with the HeatMap Viewer
of the GenePattern genomic analysis platform (Reich et al., 2006). Accord-
ing to Witten and Tibshirani (2010), standard hierarchical clustering clusters
observations using all of the genes, whereas sparse hierarchical clustering will
adaptively choose a subset of the genes to use in the clustering. The goal is
to identify a small set of genes that is relevant to the clustering and identify a
tighter and less noisy clustering of the observations using only the relevant
genes. Each gene will be given a nonnegative weight, and depending on the
tuning parameter used, many of the genes will have zero weights. If a genes
weight is zero, then it is not involved in the clustering. The weights of the
genes can be used to rank the genes in terms of importance to the clustering
(the larger the weight, the more important the gene).
For sparse hierarchical clustering, data were log scaled and standardized
to have mean zero and standard deviation one. The sum of gene weights was
selected via a permutation approach, and genes and samples were clustered
by centroid-based method. Selected genes shown in Fig. 2 d were also clus-
tered by the hierarchical clustering method. GSEA (Subramanian et al.,
2005) was conducted using expression profiles from culture and primary
bloodderived CD1c+ and CD141+ cDCs and KEGG metabolic pathways
gene sets. Microarray data are available in the National Center for Biotech-
nology Information GEO DataSets under accession no. GSE65128.
Online supplemental material. Fig. S1 shows the gating strategy for iden-
tification and purification of monocytes, pDCs, CD1c+ cDCs, and CD141+
cDCs. Fig. S2 shows transcriptional profiling of all primary versus cultured
monocyte and DC subsets. Fig. S3 shows lineage potential analysis of single
Progenitor of human dendritic cells | Lee et al.

progenitor cultures. Figs. S4S8 show hCDP (Fig. S4), hMDP (Figs. S5 and
S6), and hGMDP (Figs. S7 and S8) single cell lineage potential. Tables S1
S5 are included in a separate Excel file. Table S1 shows the top 78 regulated
genes in cultured and primary pDCs and monocytes. Table S2 shows the
top 80 regulated genes in cultured and primary CD1c+ cDCs and CD141+
cDCs. Table S3 shows comparison of selected gene expression for pDCs,
monocytes, CD1c+ cDCs, and CD141+ cDCs. Table S4 lists all metabolic
pathways enriched in cultured cDCs when compared with primary cDCs.
Table S5 shows the top 611 regulated genes in primary or cultured pDCs,
monocytes, CD1c+ cDCs, and CD141+ cDCs. Online supplemental material
is available at http://www.jem.org/cgi/content/full/jem.20141442/DC1.
This work was inspired by Ralph M. Steinman.
We thank Klara Velinzon (Flow Cytometry Core Facility, Laboratory of Molecular
Immunology, The Rockefeller University) for technical support with polychromatic
flow cytometry sorting and Dr. Chiara Borsotti (Columbia Center for Translational
Immunology, Columbia University Medical Center) for teaching intrabone cavity
injection. We thank Peter Wilkinson (Case Western Reserve University) and
Stephanie Richards (Collaborative Genomics Center, Vaccine and Gene Therapy
Institute of Florida) for their input in microarray analysis. We thank Heidi Schreiber
(Laboratory of Molecular Immunology, The Rockefeller University) for help with
human BM protocol and Joseph Schreiber for providing human BM specimen. We
thank Tibor Keller for giving us Flt3L.
Research reported in this publication was supported by the Empire State Stem
Cell Fund through New York State Department of Health Contract #C029562
(to K. Liu), Helmsley Foundation (to K. Liu), National Institutes of Health (NIH) grants
AI101251 (to K. Liu) and NS084776 (to S. Puhr), Iris and Junming Le Foundation
(to G. Breton), NIH grant 1U19AI111825-01 (to M.C. Nussenzweig), Clinical and
Translational Science Awards, The Rockefeller University Center for Clinical and
Translational Science (RUCCTS) grant no. UL1RR024143 from the National Center
for Research Resources, NIH, and NIH grant AI13013. The RUCCTS is supported, in
part, by a Clinical and Translational Science Award (CTSA) and the National Center
for Advancing Translational Sciences (NCATS), part of the NIH. M.C. Nussenzweig is
a Howard Hughes Medical Institute investigator.
The authors declare no competing financial interests.
Submitted: 30 July 2014
Accepted: 28 January 2015
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 Article

Circulating precursors of human CD1c+

and CD141+ dendritic cells
Galle Breton,2* Jaeyop Lee,1* Yu Jerry Zhou,1 Joseph J. Schreiber,5 Tibor Keler,6
Sarah Puhr,1 Niroshana Anandasabapathy,2,4 Sarah Schlesinger,2
Marina Caskey,2 Kang Liu,1** and Michel C. Nussenzweig2,3**
1Columbia University Medical Center, Department of Microbiology and Immunology, New York, NY 10032
2Laboratory of Molecular Immunology, 3Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065
4Department of Dermatology Brigham and Womens Hospital, Boston, MA 02115
5Hospital for Special Surgery, New York, NY 10021
6Celldex Therapeutics, Hampton, NJ 08827

Two subsets of conventional dendritic cells (cDCs) with distinct cell surface markers and
functions exist in mouse and human. The two subsets of cDCs are specialized antigen-
presenting cells that initiate T cell immunity and tolerance. In the mouse, a migratory cDC
precursor (pre-CDC) originates from defined progenitors in the bone marrow (BM). Small
numbers of short-lived pre-CDCs travel through the blood and replace cDCs in the peripheral
organs, maintaining homeostasis of the highly dynamic cDC pool. However, the identity and
distribution of the immediate precursor to human cDCs has not been defined. Using a tissue
culture system that supports the development of human DCs, we identify a migratory precur-
sor (hpre-CDC) that exists in human cord blood, BM, blood, and peripheral lymphoid organs.
hpre-CDCs differ from premonocytes that are restricted to the BM. In contrast to earlier
progenitors with greater developmental potential, the hpre-CDC is restricted to producing
CD1c+ and CD141+ Clec9a+ cDCs. Studies in human volunteers demonstrate that hpre-CDCs
are a dynamic population that increases in response to levels of circulating Flt3L.

CORRESPONDENCE
Kang Liu:
kl2529@columbia.edu
OR
and Michel C. Nussenzweig:
nussen@rockefeller.edu
Abbreviation used: cDCs, con-
ventional DCs; CDP, common
dendritic progenitor; pre-CDC,
cDC precursor; MS5+FSG,
MS5 stromal cells with Flt3L,
SCF, and GM-CSF cytokines.
Conventional DCs (cDCs) induce immunity
or tolerance by capturing, processing, and pre-
senting antigen to T lymphocytes (Banchereau
and Steinman, 1998). In the mouse, cDCs are
short-lived cells, whose homeostasis in lymphoid
and nonlymphoid tissues is critically dependent
on continual replenishment from circulating
pre-CDC (Liu et al., 2007; Liu and Nussenzweig,
2010). Murine pre-CDCs are BM-derived cells
that are present in very small numbers in the
blood but increase in response to Flt3L injec-
tion (Liu et al., 2007, 2009). pre-CDCs have a
very short dwell time in the blood, 65% of these
cells leave the circulation within 1 min after leav-
ing the BM (Liu et al., 2007, 2009). Upon leav-
ing the circulation, pre-CDCs seed tissues where
they differentiate to cDCs, which divide further
under the control of Flt3L (Liu et al., 2007, 2009).
Thus, in addition to the BM and blood, mouse
pre-CDCs are also found in peripheral lym-
phoid organs and nonlymphoid tissues (Naik
*G. Breton and J. Lee contributed equally to this paper.
**K. Liu and M. Nussenzweig contributed equally to
this paper.
et al., 2006; Bogunovic et al., 2009; Ginhoux
et al., 2009; Liu et al., 2009;Varol et al., 2009).
Mouse cDCs can be divided into two major
subsets, CD11b+ DCs and CD8+/CD103+ DCs
that differ in their microanatomic localization,
cell surface antigen expression, antigen-processing
activity, and ability to contribute to immune re-
sponses to specific pathogens (Merad et al., 2013;
Murphy, 2013). Despite these important differ-
ences, both CD11b+ and CD8+/CD103+ cDC
subsets of mouse DCs are derived from the same
immediate precursor (pre-CDC) that expresses
CD135 (Flt3), the receptor for Flt3L, a cyto-
kine that is critical to DC development in vivo
(McKenna et al., 2000; Waskow et al., 2008).
Similar to the mouse, humans have two major
subsets of cDCs. CD141 (BDCA3)+Clec9a+ DCs
(CD141+ cDC herein) appear to be the human
counterpart of mouse CD8+/CD103+ DCs,
expressing XCR1, Clec9a, IRF8, and TLR3
2015 Breton et al. This article is distributed under the terms of an Attribution
NoncommercialShare AlikeNo Mirror Sites license for the first six months after
the publication date (see http://www.rupress.org/terms). After six months it is
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J. Exp. Med. 2015 Vol. 212 No. 3 401413 401
www.jem.org/cgi/doi/10.1084/jem.20141441
and producing IL-12 (Robbins et al., 2008; Bachem et al.,
2010; Crozat et al., 2010; Jongbloed et al., 2010; Poulin et al.,
2010; Haniffa et al., 2012). CD1c (BDCA1)+ cDCs appear to be
more closely related to mouse CD11b+ DCs, expressing IRF4,
inducing Th17 differentiation upon A. fumigatus challenge,
and imprinting intraepithelial homing of T cells (Robbins
et al., 2008; Crozat et al., 2010; Schlitzer et al., 2013;Yu et al.,
2013). In the mouse, the superior ability of CD8+/CD103+
DCs to cross-present exogenous antigens to CD8+ T cells is
attributed to both differential antigen uptake (Kamphorst et al.,
2010) and to increased expression of proteins and enzymes
that facilitate MHC class I presentation (Dudziak et al., 2007).
Human CD141+ cDCs are more efficient than CD1c+ cDCs
in cross-presentation (Bachem et al., 2010; Crozat et al., 2010;
Jongbloed et al., 2010; Poulin et al., 2010), but this difference
appears to result from differences in antigen uptake and cyto-
kine activation rather than a specialized cell-intrinsic program
( Segura et al., 2012; Cohn et al., 2013; Nizzoli et al., 2013).
Both CD1c+ cDCs and CD141+ cDCs are present in
human blood and peripheral tissues. Each subset in the blood
resembles its tissue counterpart in gene expression but appears
less differentiated (Haniffa et al., 2012; Segura et al., 2012;
Schlitzer et al., 2013). These observations are consistent with
the idea that less differentiated human cDCs travel through the
blood to replenish the cDC pool in the peripheral tissues
(Collin et al., 2011; Segura et al., 2012; Haniffa et al., 2013).
Others have postulated the existence of a less differentiated cir-
culating DC progenitor based on absence of CD11c, expres-
sion of CD123, and response to Flt3L (ODoherty et al., 1994;
Pulendran et al., 2000), but the progenitor potential of these
putative precursors that produced large amounts of IFN- was
never tested directly and they appear to correspond at least in
part to plasmacytoid DCs (Grouard et al., 1997; Siegal et al.,
1999). Thus, whether there is an immediate circulating pre-
cursor restricted to human immature and mature CD1c+ and
CD141+ cDCs is not known.
Here, we report the existence of a migratory pre-CDC in
humans (hpre-CDC) that develops from committed DC pro-
genitors (hCDPs) in the BM (Lee et al., 2015) and is the im-
mediate precursor of both CD1c+ and CD141+ cDCs, but
not pDCs or monocytes. hpre-CDCs are present in BM, cord,
and peripheral blood, as well as peripheral lymphoid tissues.
In studies of human volunteers, Flt3L injection induces ex-
pansion of hpre-CDCs in the circulation. Thus, human cDC
precursors constitute a dynamic circulating population whose
homeostasis is regulated by Flt3L, a cytokine that is responsive
to inflammatory and infectious agents.
RESULTS
Identification of an immediate precursor to human cDCs
To define cell surface markers that might be expressed by cir-
culating hpre-CDCs, we examined DCs and monocytes for
expression of cytokine receptors that are found on BM DC
progenitors (Lee et al., 2015). Whereas CD116 (GM-CSF re-
ceptor) is expressed on differentiated monocytes and DCs,
CD135 (Flt3L receptor) is restricted to DCs, CD115 (M-CSF
402
receptor) is expressed only on monocytes, and CD117 (SCF
receptor) is found on the surface of CD141+ cDCs and most
CD1c+ cDCs, but not on pDCs or monocytes (Fig. 1).
CD45RA, a marker expressed by several different hematopo-
etic cell progenitors including granulocyte monocyte pro-
genitors (GMP) and common lymphoid progenitors (CLP;
Chicha et al., 2004; Doulatov et al., 2010), is highly expressed
only on pDCs (Fig. 1). Finally, CD34 is expressed on early
DC progenitors (Chicha et al., 2004; Doulatov et al., 2010;
Lee et al., 2015), but not on fully differentiated DCs or mono-
cytes, suggesting that loss of CD34 would precede cDC de-
velopment (Fig. 1). Collectively, the data suggests that if a
hpre-CDC exists in human blood it may be found in the CD
34CD115CD116+CD117+CD135+ fraction.
To try to identify hpre-CDCs in cord blood, we ex-
cluded terminally differentiated lymphoid cells (CD19+CD3+
CD56+), granulocytes (CD66b+), monocytes (CD14+), pDCs
(CD303/BDCA2+), and cDCs (CD1c+ and CD141+). The
remaining CD45+ cells were subdivided into 4 groups by ex-
pression of CD34, CD117, and CD135, as follows: CD34+
CD117+ (CD34+), CD34CD117 (CD117), CD34
CD117+CD135 (CD135), and CD34CD117+CD135+
(CD135+; Fig. 2 a). Pools of 1,000 purified cells were cultured
on MS5 stromal cells with Flt3L, SCF, and GM-CSF cytokines
(MS5+FSG herein) under conditions that support DC devel-
opment in vitro (Lee et al., 2015). Only CD34+CD117+ and
CD34CD117+CD135+ cells produced identifiable progeny
(Fig. 2 b). As expected, CD34+CD117+ cells, which should
contain the least committed progenitors because they retain
CD34 expression, developed into monocytes, granulocytes,
pDCs, CD1c+ and CD141+ cDCs, and B cells, indicating
that they are indeed multipotent (Fig. 2 b and not depicted).
In contrast, CD34CD117+CD135+ cells were restricted
to CD1c+ and CD141+ cDCs and monocytes, suggesting
that this pool of cells contains the immediate precursor to
human cDCs and monocytes (Fig. 2 b). To further purify
the hpre-CDC, we fractionated the CD34CD117+CD135+
pool based on expression of CD116, CD115, and CD45RA
(Chicha et al., 2004; Doulatov et al., 2010) and assayed
their potential in MS5+FSG cultures (Fig. 2 c). Whereas
CD116+CD45RA+CD115+ cells produced primarily CD14+
CD1c monocytes, a small number of CD1c+CD14+ cDCs
(Patterson et al., 2005; Granelli-Piperno et al., 2006), and no
CD141+ cDCs; CD116+CD45RA+CD115 cells yielded
primarily CD1c+ and CD141+ cDCs and only rare mono-
cytes and pDCs (Fig. 2 d). These results were reproducible
in multiple different human donors (Fig. 2 e). Therefore,
CD116+CD45RA+CD115 cells show the restricted de-
velopmental potential expected of hpreDCs and CD116+
CD45RA+CD115+ cells of premonocytes. hpre-CDCs ex-
press high HLA-DR, an intermediate level of CD123, and a
low level of CD11c (Fig. 2 f). Morphologically, hpre-CDCs
are mononuclear cells that present a distinctive formation of
multilobulated nucleus and a few veiled processes but do not
possess the membrane extensions typically associated with
cDCs (Fig. 2 g).
Circulating precursors of human dendritic cells | Breton et al.

hpre-CDCs in adult blood and lymphoid organs
In the mouse, pre-CDCs are found in circulation and in tis-
sues. To determine the physiological distribution of hpre-
CDCs in adult humans, we examined BM, peripheral blood,
and tonsils using the same cocktail of surface markers used to
JEM Vol. 212, No. 3
Ar ticle
Figure 1. Expression of cytokine receptors on
DCs and monocytes. Flow cytometry plots show
expression of CD117, CD135, CD115, CD123, CD45RA,
CD34, and CD116 on gated CD3CD19CD56 cells
(gray), differentiated monocytes (CD14+ orange),
pDCs (CD303+ green), CD1c+ cDCs (blue), and CD141+
cDCs (red) from peripheral blood. Table summarizes
results of flow cytometry.
analyze cord blood. LinCD34CD117+CD135+ cells in
the BM resembled those in the cord blood and contained
CD116+CD115 hpre-CDC-like cells and CD116+CD115+
premonocyte-like cells (Fig. 3 a). These hpre-CDC-like (Lin
CD34CD117+CD135+CD116+CD115) cells were also
403
Figure 2. Screening of populations in the cord blood for committed cDC lineage potential. (a) Flow cytometry plots show gating of CD45+CD3
CD19CD56CD14CD66bCD1cCD141CD303 cells in human cord blood can be divided into four populations based on CD117, CD34, and CD135:
CD34+CD117+ (CD34+), CD34CD117 (CD117), CD34CD117+CD135 (CD135), and CD34CD117+CD135+ (CD135+). Numbers indicate the frequency
of respective gates. (b) Differentiation potential of 1,000 purified cells from each of 4 populations indicated in (a) in MS5+FSG cultures for 7 d. Flow cy-
tometry plots show gated live human CD45+ cells. (c) Flow cytometry plots show CD135+ cells indicated as in (a) can be further separated into 4 populations

404 Circulating precursors of human dendritic cells | Breton et al.

 Ar ticle

detected in the peripheral blood and the tonsil. In contrast,
the premonocyte-like (LinCD34CD117+CD135+CD116
+CD115+) cells are lacking in the peripheral blood and tonsil
(Fig. 3 a). To determine the developmental potential of the
hpre-CDC-like cells in different tissues, we purified them
and cultured them under conditions that support DC devel-
opment. Although occasional hpre-CDCs remained undiffer-
entiated, all cultures, irrespective of hpre-CDC origin from
cord blood, BM, blood, or tonsils, predominantly produced
CD1c+ and CD141+ cDCs and few, if any, CD14+CD1c
monocytes (Fig. 3 b). CD1c+ cDCs produced in our culture sys-
tem can express some CD14, but gene array data show that these
CD1c+ CD14+ cells are cDCs and not monocytes (Lee et al.,
2015). In contrast, the CD115+CD116+ premonocytes in the
BM, which are absent from the blood and tonsils, produced
primarily monocytes and no CD141+ cDCs (Fig. 3 c). Cells
showing the hpre-CDC phenotype constitute 0.008% (range,
0.0010.016%) of the CD45+ cells in cord blood, 0.117%
(range, 0.0560.188%) in the BM, 0.001% (range of 0.001
0.002%) in the peripheral blood, and 0.001% (range, 0.001
0.002%) in the tonsil (Fig. 3 a). Thus, whereas hpre-CDCs
originate in the BM and travel through the blood to seed lym-
phoid tissues, the monocyte precursor is retained in the BM.
Proliferative capacity and clonal potential of hpre-CDC
To examine the proliferative potential of hpre-CDC, we pu-
rified them from cord or peripheral blood and compared
their developmental potential to CD34+ hematopoietic stem
and progenitor cells (HSPCs) purified from cord blood in
MS5+FSG culture (Lee et al., 2015). Whereas CD34+ pro-
genitors expanded 156-fold during the 7-d culture period,
hpre-CDCs from cord blood or peripheral blood expanded
only 8- and 6-fold, respectively (Fig. 4 a). The relatively low
level of hpre-CDC expansion could result from low prolifer-
ative capacity or poor clonal efficiency. To determine their
proliferative capacity, we loaded purified hpre-CDCs or
CD34+ HSPCs with CFSE and documented division by dye
dilution at days 2 and 7 of culture. On day 2, HSPCs re-
mained CD34+ and produced neither CD1c+ nor CD141+
cDCs, whereas hpre-CDCs differentiated into CD1c+ and
CD141+ cDCs (Fig. 4, b and c). On day 7, both CD34+ HSPC
and hpre-CDC cultures contained CD1c+ and CD141+ cDCs
(Fig. 4 b). CD34+ HSPCs divided up to 9 times in 7 d, whereas
hpre-CDCs underwent a maximum of 4 divisions (Fig. 4 c).
Comparison of CFSE dilution revealed that hpre-CDC-
derived CD1c+ cDCs and CD141+ cDCs underwent at least
one more division than hpre-CDCs in the culture (Fig. 4 c), in-
dicating that cDCs are also capable of cell division. Consistent
with this notion, purified blood CD1c+ and CD141+ cDCs also
divided 34 times in culture (Fig. 4 c).Thus, hpre-CDCs have
a more limited proliferative capacity than CD34+ HSPCs
in vitro. Nevertheless, a single hpre-CDC may be able to give
rise to as many as 256 cDCs.
To determine the clonal efficiency of hpre-CDCs, we
compared them to CD34+ HSPCs and progenitors in limit-
ing dilution assays. Whereas 1 in 4.57 CD34+ cells produced
CD45+ cells (i.e., granulocytes, monocytes, DCs, or lym-
phoid cells), 1 in 7.84 and 1 in 6.55 hpre-CDCs from cord
blood or peripheral blood, respectively, produced CD45+
cells during the culture period (Fig. 5 a). This is in line with
our observation that some hpre-CDCs remained undifferen-
tiated in our cultures (Fig. 3 b). Although CD34+ HSPCs had
higher clonal potential, only 12 out of 43 (28%) were able to
produce cDCs (Fig. 5 c and Fig. S1), and both cord blood
and peripheral blood hpre-CDCs generated mainly cDCs
(Fig. 5 c, Fig. S2, and Fig. S3). Individual hpre-CDCs pro-
duced either CD1c+ cDCs or CD141+ cDCs or both (Fig. 5 b).
Of 81 cells assayed, 88% of all productive clones of cord
blood hpre-CDCs produced cDCs; 32% yielded only
CD141+ cDCs, 50% only to CD1c+ cDCs, and 6% produced
both CD1c+ and CD141+ cDCs (Fig. 5 c and Fig. S2). Of
105 cells assayed, 90% of hpre-CDCs from the PBMC pro-
duced only cDCs; 11% gave rise to CD141+ cDCs only, 65%
only to CD1c+ cDCs, and 14% produced both CD1c+ and
CD141+ cDCs (Fig. 5 c and Fig. S3). This demonstrates that
hpre-CDCs purified from peripheral or cord blood have
clonal efficiencies and differentiation potential similar to CD1c+
and CD141+ cDCs (Fig. 5 c). Only a very small fraction of
hpre-CDCs produced monocytes alone or monocytes and
DCs (Fig. 5 c; 11.5 and 10.5% from cord blood and periph-
eral blood, respectively). Thus, the hpre-CDC population in
peripheral and cord blood contains single cells with potential
to differentiate into both major subsets of human cDCs.
Progenitors of hpre-CDCs
We have identified a common DC progenitor in BM and
cord blood that produces pDCs and cDCs (hCDP; Lee et al.,
2015). To determine whether there exists a progenitorprogeny
relationship between the hCDP and hpre-CDC (Fig. 6 a),
we purified hCDPs from cord blood (Fig. 6 b) and tested
whether they differentiate into hpre-CDCs in MS5+FSG
culture. hCDPs down-regulated CD34 as early as 1 d after
culture and diverged into two populations: one group of cells
up-regulated CD303 and assumed a pDC phenotype, the other
group was CD303 but expressed CD45RA, CD117, and
CD116 resembling hpre-CDCs. The number of hpre-CDCs

based on CD116, CD115, and CD45RA: CD135+CD116 (CD116), CD135+CD116+CD45RA(CD45RA), CD135+CD116+CD45RA+CD115+ (CD115+), and
CD135+CD116+CD45RA+ CD115 (CD115). (d and e) Differentiation potential of 100 purified cells from each of 4 populations indicated in (c) in
MS5+FSG cultures for 7 d. (d) Flow cytometry plots of CD45+ cells gated as in (c), showing expression of CD141, CLEC9a, CD1c, and CD19. (e) Graphs
show mean output of pDC, CD1c+ cDC, CD141+ cDC and monocytes from each population from three independent experiments. (f) Histograms show ex-
pression of CD11c, HLA-DR, CD123, CD135, CD117, CD45RA, CD116, and CD115 on indicated cell-type. (g) Morphology of purified cord blood hpre-CDCs
by Giemsa staining of cytospin preparations (100). Dotted lines indicate cropping out of white space between cells. Bar, 10 m.

JEM Vol. 212, No. 3 405


Figure 3. hpre-CDCs in human lymphoid organs and blood.
(a) Representative flow cytometry plots of gated Lin(CD3/19/56/14/66b)
DC(CD1c/141/303)CD34 cells show gating strategy and composition
of hpre-CDCs (SSCloCD117+CD116+CD135+ CD45RA+CD115, red) and
premonocytes (SSCloCD117+CD116+CD135+ CD45RA+CD115+, green) in
human cord blood (CB), BM, peripheral blood (PB), and tonsil. Numbers
indicate frequency of cells of parent gate (CB, n = 5; BM, n = 4, PB, n = 5;
Tonsil, n = 3; n, number of donors). (b) Differentiation potential of puri-
fied hpre-CDCs indicated in (a) in MS5+FSG cultures for 5 d. Flow cytom-
etry plots of gated live human CD45+ cells show culture output of CD141+
cDC (red) and CD1c+ cDC (blue). Representative results of four (CB), three
406
peaked at day 2, and this was followed by increase of CD1c+
cDCs and CD141+ cDCs (Fig. 6 c). We conclude that hCDPs
lose CD34 expression and give rise to hpre-CDCs and pDCs
in culture.
Flt3L injection induces expansion
of hpre-CDCs in human blood
Flt3L stimulates DC hematopoiesis (Karsunky et al., 2003)
and is an acute phase reactant that can be elevated in infection
in both mice and humans, accounting for DC expansion in
patients with malaria infection and in volunteers injected
with Flt3L (Maraskovsky et al., 2000; Pulendran et al., 2000;
Guermonprez et al., 2013). In humans, Flt3L injection is associ-
ated with a rapid increase in circulating DCs and in CD11c
CD123+ cells that produce large amounts of IFN- when
cultured with influenza virus (Pulendran et al., 2000). The
latter were initially thought to be DC progenitors but were
subsequently shown to be pDCs (Siegal et al., 1999; Liu, 2005).
To determine whether Flt3L mobilizes DC progenitors
into the blood, we made use of samples from three volunteers
subcutaneously injected with 25 g/kg of Flt3L for 10 con-
secutive days. 14-color flow cytometry was used to assess the
number of cDCs, pDCs, and hpre-CDCs (Fig. S4). The
number of circulating cDCs increased on day 5 and peaked
on day 14 after the initial injection (Fig. 7, a and b). The de-
gree of cDC expansion varied with a 32234-fold increase in
CD1c+ cDCs, a 40171-fold increase in CD141+ cDCs, and
1622-fold increase in pDCs in the blood (Fig. 7 b). Similar
to CD141+ cDCs obtained from MS5+FSG culture (Lee
et al., 2015), Flt3L-induced CD141+ cDCs express variable
levels of CD1c, and all DC subtypes in Flt3L-injected indi-
viduals down-regulated expression of the Flt3L receptor,
CD135 (Fig. 7 c; Waskow et al., 2008). Thus, as others have
found, both subtypes of cDCs and pDCs increase in the blood
in response to Flt3L injection (Pulendran et al., 2000).
Like the more differentiated DCs, hpre-CDC also in-
creased in the blood of the same patients, starting on day 5 but
peaking earlier, on days 1112 after injection (Fig. 7, a and b).
Flt3L-expanded hpre-CDCs were similar to their uninduced
counterparts and cord blood derived hpre-CDCs in producing
CD1c+ and CD141+ cDCs in MS5+FSG culture (Fig. 7 d).
In contrast, the immediate precursors of hpre-CDCs, hCDPs,
and more distant progenitors such as hGMDPs or hMDPs
were undetectable in the peripheral blood of individuals in-
jected with Flt3L at any time point examined (Fig. S4). We
conclude that Flt3L expands human DCs and their progenitors
and recruits hpre-CDCs, but not earlier DC progenitors, into
the circulation.
(BM), three (PB), and two (tonsil) independent experiments are shown.
(c) Differentiation potential of purified premonocytes from cord blood
(CB) and BM in MS5+FSG cultures for 5 d. FACs plots show phenotype
of gated live human CD45+ culture-derived cells including monocytes
(orange) and CD1c+ cDCs (blue). Representative of three independent
experiments are shown.
Circulating precursors of human dendritic cells | Breton et al.
 Ar ticle

Figure 4. Proliferative capacity of hpre-CDCs. (a) Expansion of purified hpre-CDCs or CD34+ HSPCs from peripheral blood (PB) or cord blood (CB) in
MS5+FSG cultures for 7 d. Graph shows the mean fold change of live human CD45+ cells from 100 input cells from three independent experiments. *, P <
0.005, unpaired two-tailed Students t test. (b and c) CD34+ HSPCs and hpre-CDCs were purified from CB, labeled with CFSE, and cultured in MS5+FSG for
2 or 7 d; proliferation was assessed by flow cytometry. FACs plots show (b) gated CD45+ culture-derived cells, including CD34+ cells (purple), CD34CD1c
CD141 cells (orange), CD141+ cDCs (red), and CD1c+ cDCs (blue) and (c) their CFSE dilution. Dotted lines mark last division by hpre-CDCs. Plots are repre-
sentative of three independent experiments.

Other myeloid populations, such as monocytes and gran-
ulocytes also expand in response to Flt3L, starting on day 5
and peaking on day 14 after the initial injection (Fig. 7,
e and f). Interestingly, comparison of the fold change at day
14 indicates that Flt3L preferentially increases the number
of human cDCs and their progenitors rather than the other
myeloid subsets, i.e., pDCs, monocytes, and granulocytes in
blood (Fig. 7 g).
DISCUSSION
DCs turn over in tissues and must be replaced continuously to
maintain homoeostasis (Liu et al., 2007; Liu and Nussenzweig,
2010). In the mouse, lymphoid and nonlymphoid tissue
resident DCs are continually replenished by pre-CDCs,
which are produced in the BM, enter the circulation and then
emigrate into tissues to differentiate into both major subsets
of cDCs (Naik et al., 2006; Bogunovic et al., 2009; Ginhoux
et al., 2009; Liu et al., 2009; Varol et al., 2009). Defining
the mouse pre-CDC was essential to establishing that DCs
represent a unique cell lineage because pre-CDCs do not
produce monocytes, or pDCs; their differentiation potential
is restricted to cDCs.
Human DC development has been difficult to delineate,
in part because of limited access to human tissues and limited
tissue culture methods. Earlier human trials showed increased
numbers of DCs in the blood after Flt3L injection, and sug-
gested that a CD11cCD123+ IFN- producing cell might
be a DC progenitor (Pulendran et al., 2000). However, pro-
genitor potential was never tested directly and it is likely that
those cells represent pDCs, which are CD123+ and induced
by Flt3L injection (Fig. 7 a). More recently, circulating cDCs
were shown to be less mature than their tissue-derived coun-
terparts and it has been proposed that these less mature cells
are the precursors of tissue cDCs (Collin et al., 2011; Segura
et al., 2012; Ginhoux and Schlitzer, 2014). Our experiments
identify a human pre-CDC that resides in the BM, peripheral
blood, and peripheral lymphoid organs that is the direct pro-
genitor of both major subsets of human cDCs. Whether the
hpre-CDC differentiates into cDCs in the blood or tissues or
both has yet to be determined.
The cell surface markers used to define mouse DCs and
their progenitors are not entirely conserved by their human
counterparts. For example, lack of cell surface MHCII expres-
sion, one of the key features that distinguishes mouse pre-CDCs

JEM Vol. 212, No. 3 407


from fully differentiated DCs, cannot be used to define human
DC progenitors because MHCII is expressed on the surface
of most early human hematopoietic cells. Similarly, CD11c used
as marker to identify pre-CDCs in the mouse is expressed
at low levels on hpre-CDCs (Fig. 2 f) and should not be used
to identify this cell in humans. However, both human and
mouse pre-CDCs express CD135. This receptor marks the
entire DC pathway in both species from the earliest BM pre-
cursor to the fully differentiated cDCs in tissues (Chicha et al.,
2004; Fogg et al., 2006; Naik et al., 2007; Onai et al., 2007;
Waskow et al., 2008; Doulatov et al., 2010; Lee et al., 2015).
In the mouse, DC progenitors can be distinguished from
other cells based on increasingly restricted expression of cyto-
kine receptors (Karsunky et al., 2003; Onai et al., 2007; Waskow
et al., 2008; Liu et al., 2009). We applied this principle to
identify the hpre-CDC and made use of differential expres-
sion of CD116 (GM-CSF receptor), CD135 (Flt3L recep-
tor), CD115 (M-CSF receptor), CD117 (SCF receptor), and
CD45RA (marker for DC progenitors) on monocytes, cDCs,
and pDCs to uncover the hpre-CDC, which is LinCD34
CD117+CD135+CD115CD116+CD45RA+. In contrast,
otherwise identical cells that are CD115+ appear to be the
immediate precursors of monocytes, resembling the mouse
common monocyte progenitor (cMOP), in its differentiation
potential as well as in its absence in the circulation (Hettinger
et al., 2013).
hpre-CDCs enter the circulation and emigrate into tissues
as incompletely differentiated progenitors. In the mouse, this
feature of the cDC lineage enables pre-CDC differentiation
408
Figure 5. Clonal efficiency and potential
of hpre-CDC. (a) Limiting dilution outgrowth
assay show clonal efficiency of CD34+ HSPCs
(n = 3) or hpre-CDCs from peripheral (PB, n = 4)
and cord blood (CB, n = 3) in MS5+FSG cul-
tures for 5 d (for hpre-CDCs) or 14 d (for
CD34+ HSPCs; Materials and methods). **, P <
0.05, pairwise test by ELDA. (b) Representative
flow cytometry plots of gated CD45+ cells
derived from single hpre-CDC clones indi-
cated in (a) show output of CD1c+ cDCs (blue)
and CD141+ cDCs (red). (c) Graphs summarize
the lineage output of single clones from CD34+
HSPCS, and hpre-CDCs from cord (CB) or pe-
ripheral blood (PB). G, granulocyte; M, mono-
cyte; L, lymphocytes.
into specialized subsets in different tissues. We speculate that
similar tissue specific heterogeneity in cDC subtypes will also
be found in human. In contrast, the closely related human
monocyte progenitor is restricted to the BM (Fig. 3). Similar
to monocytes, pDCs enter the circulation as fully differenti-
ated effector cells.
Like all cDCs and their early progenitors, hpre-CDCs
express CD135, the receptor for Flt3L. Our experiments
show that like more mature cDCs (Maraskovsky et al., 1997;
Pulendran et al., 2000), hpre-CDCs are mobilized into the
blood by Flt3L injection. However, the immediate precursor
of hpre-CDCs, hCDPs, are not. Flt3L levels in serum increase
in response to DC depletion in mice and are also increased in
humans that lack cDCs and other myeloid cells due to GATA2
mutation (Dickinson et al., 2014). In addition, levels of this
hematopoietin are also increased in the serum of humans in-
fected with Plasmodium falciparum, and in mice infected with
Plasmodium chaubodii or Plasmodium yoelli or cytomegalovirus
(Eidenschenk et al., 2010; Guermonprez et al., 2013). More-
over, in humans infected with P. falciparum, increased serum
Flt3L is associated with an increase in the number of circulat-
ing cDCs. Thus, cDC levels in humans are regulated in part by
hpre-CDC recruitment from the BM in response to Flt3L.
In both humans and mice, pre-CDCs constitute a very
small population of cells in the peripheral blood (0.001 and
0.02%, respectively). Although present in only small num-
bers, the flux of pre-CDCs through the blood stream is remark-
ably high due to their short residence time in circulation. In the
mouse, 65% of pre-CDCs are cleared from the circulation
Circulating precursors of human dendritic cells | Breton et al.
 Ar ticle

Figure 6. hpre-CDCs descend from hCDPs. (a) Flow cytometry plots show comparison of hCDP (purple) and hpre-CDC (orange) in cord blood. (b) Flow
cytometry plots of gated CD45+Lin(CD3/19/56/14)CD34+ show phenotype and purity of magnetically enriched cord blood (CB) CD34+ cells (presorting,
upper) and sorted cells (post-sorting, bottom). hCDPs were gated as CD34+CD38hiCD45RA+CD10CD123hi. (c) Differentiation kinetics of hCDPs purified
from CB in MS5+FSG cultures for 1, 2, or 4 d. FACs plots show culture output of live human CD45+ cells, including CD34+ cells (purple), pDC (green),
CD1c+ cDC (blue), CD141+ cDCs (red), and hpre-CDCs (orange). Representative of four independent experiments are shown. Graphs summarize composi-
tion of indicated populations among total hCD45+ cells. Bars are mean values from four independent experiments, and error bars are SEM.

within 1 min after entering the blood, indicating a t1/2 < 1 min
(Liu et al., 2007, 2009). Thus, a cell with a half-life in circu
lation of 24 h would have to be present in circulation at a
1,440-fold higher concentration than the pre-CDC to achieve
the same overall flux. Similar behavior has been demonstrated
for HSCs in the circulation (Wright et al., 2001). Like their
mouse counterparts, hpre-CDCs have limited expansion po-
tential (Fig. 4 a; Liu et al., 2009), but their immediate prog-
eny, CD1c+ and CD141+ cDCs, can proliferate to further
expand the DC pool in the periphery (Fig. 4 c; Kabashima
et al., 2005; Liu et al., 2009; KC et al., 2014). We speculate that
this highly dynamic pool of specialized antigen-presenting
cells allows rapid adaptation to acute antigenic challenges.
MATERIALS AND METHODS
Cell samples. Human umbilical cord blood and leukophoretic peripheral
blood (buffy coat) were purchased from New York Blood Center. Human
BM was obtained from total hip arthroplasty at Hospital for Special Surgery
(New York, NY). Tonsils were obtained from routine tonsillectomies per-
formed at the Babies and Childrens Hospital of Columbia-Presbyterian
Medical Center (New York, NY). Informed consent was obtained from the
patients and/or samples were exempt from informed consent being residual
material after diagnosis and fully de-identified. All samples were collected
according to protocols approved by the Institutional Review Board at Co-
lumbia University Medical Center and The Rockefeller University (New
York, NY). The specimens were kept on ice immediately after surgical re-
moval. Tonsil samples were minced, treated with 400 U/ml collagenase
(Roche) at 37C for 20 min, and put into cell isolation. BM samples were
preserved in solution containing 1000 U/ml heparin (National Drug Code

JEM Vol. 212, No. 3 409


#63323-540-11) until digestion. The samples were then digested in RPMI
containing 20 mg/ml collagenase IV (#C-5138; Sigma-Aldrich) for 15 min
at 37C. After density centrifugation using Ficoll-Hypaque (GE Healthcare),
aliquots of mononuclear BM cells were frozen and stored in liquid nitrogen
for future analysis.
Cell isolation and flow cytometry. Fresh mononuclear cells were isolated
by density centrifugation using Ficoll-Hypaque (GE Healthcare). Samples
410
Figure 7. Flt3L administration increases
circulating DC subsets and hpre-CDCs in
humans. PBMCs from Flt3L-treated volun-
teers (n = 3; 25 g/kg for 10 consecutive
days) were analyzed by flow cytometry over a
21-d period to assess the expansion of DC
subsets (CD1c+ cDCs [blue], CD141+ cDCs
[red], and pDCs [green]; hpre-CDCs (gray);
monocytes (orange); and granulocytes
(brown). (a) Representative flow cytometry
dot plots show DC subsets and hpre-CDCs in
blood (gating strategy in Fig. S4). (b) Graphs
show the kinetics of cell number of cDC sub-
sets, pDCs, and hpre-CDCs over 21 d in
3 Flt3L-treated volunteers. The absolute num-
bers per milliliter of blood were obtained by
multiplying the number of cells (obtained by
flow cytometry) by the total number of
PBMCs per milliliter of blood. (c) Representa-
tive histograms show CD135 expression on
CD141+ cDCs, CD1c+ cDCs, pDCs and hpre-
CDCs over 21 d in one Flt3L-treated volunteer.
(d) Differentiation potential of purified hpre-
CDCs from blood of Flt3L-injected volunteers
in MS5+FSG cultures after 7 d. Flow cytom-
etry plots of gated CD45+ cells from culture
show output of CD141+ cDCs (red), CD1c+
cDCs (blue), and lack of pDCs (green gate).
(e) Representative flow cytometry dot plots
show CD14+ monocytes and CD66b+ granulo-
cytes in blood (gating strategy in Fig. S4).
(f) Graphs show the kinetics of cell number of
monocytes and granulocytes over 21 d in
3 Flt3L-treated volunteers. The absolute num-
bers per milliliter of blood were obtained by
multiplying the number of cells (obtained by
flow cytometry) by the total number of
PBMCs per milliliter of blood. (g) Graph show-
ing the mean fold change increase from the
three patients (d1 vs. d14) of hpre-CDCs
(gray), CD1c+ cDCs (blue), CD141+ cDCs (red),
pDCs (green), monocytes (orange), and granu-
locytes (brown) per milliliter of blood. Error
bars are SDs.
from cord blood, peripheral blood, BM, and tonsil were incubated with
fluorescent-labeled antibodies for direct analysis on the BDLSR II flow
cytometers (BD) or further purification by fluorescence-activated cell sort-
ing on the BD FACSAria or Influx, both using HeNe and argon lasers.
For isolation of rare hpre-CDCs from cord blood and peripheral blood,
an enrichment step was performed before sorting. In brief, mononuclear cells
were incubated with antibodies against CD135 (4G8; PE; BD) and CD117
(A3C6E2; Biotin; BioLegend) for 40 min at 4C. After washing, antibody
Circulating precursors of human dendritic cells | Breton et al.

against PE (PE001; Biotin; BioLegend) was added and incubated for another
10 min at 4C. After washing, CD117+ and CD135+ cells were positively se-
lected using anti-biotin MicroBeads and LS MACS magnetic columns (Mil
tenyi Biotec). For sorting hpre-CDCs, enriched cells (from CB or PB) or total
mononuclear cells (from CB, PB, BM, or tonsils) were stained for CD14
(TuK4; Qdot-655; Invitrogen), CD3 (OKT3; Brilliant Violet [BV] 650;
BioLegend), CD19 (HIB19; BV650; BioLegend), CD56 (HCD56; BV650;
BioLegend), CD66b (G10F5; PerCP-Cy5.5; BioLegend), CD303 (201A;
PerCP-Cy5.5; BioLegend), CD1c (L161; APC-Cy7; BioLegend), CD141
(M80; PE-Cy7; BioLegend), CD34 (581; Alexa Fluor 700; BioLegend),
CD117 (104D2; BV421; BioLegend), CD135 (4G8; PE; BD), CD45RA
(HI100; BV510; BioLegend), CD116 (4H1; FITC; BioLegend), and CD115
(9-4D2-1E4; APC; BioLegend) for 40 min on ice. hpre-CDCs were isolated
as Lin(CD3/19/56/14)Granulocyte(CD66b)pDC(CD303)cDC(CD1c/
CD141)CD34CD117+CD135+SSClo CD116+CD115CD45RA+.
In addition to these antibodies, for surface marker phenotype charac-
terization of fully differentiated cells, CD45 (HI30; Alexa Fluor 700; Bio-
Legend), CD34 (581; APC-Cy7; BioLegend), CD3 (UCHT1; Biotin; BD),
CD19 (HIB19; Biotin; BD), CD56 (B159; Biotin; BD), CD115 (12-3A3-
1B10; PE; eBioscience), CD123 (9F5; PE; BD) and CD45RA (120458-42;
eBioscience; PE) were used alternatively, followed by a secondary stain with
streptavidin-conjugated PerCP-Cy5.5 for 40 min on ice.
Cultured cells were harvested and stained with LIVE/DEAD (Life
Technologies), CD45 (AF700), CD56 (B159; Pacific Blue; BD), CD66b
(G10F5; PerCP-Cy5.5; BioLegend), CD19 (HIB19; APC-Cy7; BioLeg-
end), CLEC9a (8F9; PE; BioLegend), CD14 (Qdot-655), CD1c (L161; PE-
Cy7; BioLegend), CD303 (201A; FITC; BioLegend), CD123 (6H6; BV510;
BioLegend), CD141 (AD5-14H12; APC; Miltenyi Biotec) for 40 min on
ice and analyzed for lineage potential by flow cytometry. Absolute cell counts
have been calculated relative to a well containing a known number of CD45+
cells (i.e., 10,000 cells), which were added on the day of harvest.
For CFSE assays, cultured cells were stained with LIVE/DEAD (Life
Technologies), CD45 (AF700), CD34 (APC-Cy7), CLEC9a (PE), CD14
(Qdot-655), CD1c (PE-Cy7), and CD141 (APC) for 40 min on ice.
For progenitorprogeny relationship experiments, CD34+ cells were first
enriched from cord blood using CD34 MicroBead kit and LS MACS magnetic
columns (Miltenyi Biotec). Enriched CD34+ cells (4095% purity) were then
incubated with Lin (CD3/19/56/14; BV650), CD34 (AF700), CD38 (HIT2;
BV421; BioLegend), CD10 (HI10a; PE-Cy7; BioLegend), CD45RA (BV510),
CD123 (PE), CD116 (FITC), and CD115 (APC). hCDPs were sorted as Lin
CD34+CD38hiCD10CD45RA+CD123hiCD115. Cultured cells were har-
vested at specific time points and stained with LIVE/DEAD, CD45 (AF700),
CD14 (Qdot-655), CD3 (BV650), CD19 (BV650), CD56 (BV650), CD303
(FITC), CD1c (PE-Cy7), CD141 (APC), CD34 (APC-Cy7), CD117 (BV421),
CD123 (PE), CD45RA (BV510), and CD116 (FITC) for 40 min on ice.
Morphological analysis. Purified hpreDCs were analyzed by Giemsa stain-
ing of cytospin preparations. As few as 5 104 cells were cytospun for 5 min
at 800 rpm on a glass slide and then stained with the Hemacolor stain kit as
recommended by the manufacturer (HARLECO). Slides were then imaged
on an Axioplan 2 microscope at 100 magnification (Carl Zeiss).
Cell culture. MS5 BM stromal cells were maintained and passed in com-
plete -MEM medium (Invitrogen) with 10% FCS and penicillin/strepto-
mycin (Invitrogen). 24 h before hpre-CDC culture, MS5 stromal cells were
treated with 10 g/ml of mitomycin C (Sigma-Aldrich) for 3 h, washed, and
reseeded at 3.75 104 MS5 cells per well in 96-well plates. Purified popula-
tions were seeded in medium containing 100 ng of Flt3L/ml (Celldex),
20 ng/ml SCF (PeproTech), and 10 ng/ml GM-CSF (PeproTech). Cells
were harvested between day 1 and 14 for flow cytometry analysis.
To determine cellular divisions in culture, input populations were la-
beled for 15 min with 5 M CFSE (Molecular Probes) at 37C.
Limiting dilution assay. For limiting dilution experiments, cells were
sorted directly into 96-well plates containing MS5+FSG at 1, 2, 4, 8, or 16
JEM Vol. 212, No. 3
Ar ticle
cells per well for CD34+ HSPCs, and at 1, 5 or 10 cells per well for hpre-
CDCs. The percentage of detection failure for cDCs was calculated as 100%
(1-k/n), where k is the number of positive wells and n the total number of wells
combined from three or four independent experiments (CB and PB, respec-
tively). We classified each well as positive if more than 5 cells were detected for
cDCs or other lineages among live human CD45+ cells.Then, for every posi-
tive clone, we scored positive for cDCs or other lineages based on their re-
spective gates to determine the clonal lineage potential. The frequency of
DC-producing precursors was determined by Loi de Poisson using the Ex-
treme Limiting Dilution Analysis software provided by the Walter and Eliza Hall
Institute of Medical Research Bioinformatics (Parkville,Victoria, Australia.
Flt3L injection in volunteers. 3 healthy volunteers received a 25 g/kg/
day subcutaneous injection of CDX-301 (a clinical formulation of recombi-
nant human Flt3L; Celldex) for 10 d. Blood samples were collected before
and at 5, 8, 10, 11, 12, 14, and 21 d after initial administration. PBMCs were
isolated from heparinized blood using Ficoll-Hypaque and stored in liquid
nitrogen for further analysis.
PBMCs were stained with CD116 (4H1; FITC; BioLegend), CD135
(4G8; PE; BD), CD66b (G10F5; PerCP-Cy5.5; BioLegend), CD335 (9E2,
PerCP-Cy5.5, BioLegend), CD303 (201A; PerCP Cy5.5; BioLegend),
CD141 (M80; PE-Cy7; BioLegend), CD117 (104D2; BV421; BioLegend),
CD123 (6H6; BV510; BioLegend), CD20 (2H7; BV605; BioLegend), CD3
(OKT3; BV605; BioLegend), CD45RA (MEM-56; Qdot 655; Life Tech-
nologies), LIVE/DEAD Fixable Blue Dead Cell Stain (Life Technologies),
CD14 (Tk4; Qdot 800; Life Technologies), CD115 APC (9-4D2-1E4;
APC; BioLegend), CD34 (581; Alexa Fluor 700; BioLegend), CD1c (L161;
APC-Cy7; BioLegend). For surface staining, titrated antibodies were added
to 2 million cells in 50 l PBS for 20 min at 4C.
Washed cells were fixed in 2% formaldehyde and stored at 4C until
analysis, which was performed using an LSR II flow cytometer (BD). The
whole sample was acquired and analysis was performed using Flow Jo 9.1
software (Tree Star). hpre-CDCs were isolated as Lin(CD3/20/335/14)
Granulocyte(CD66b)DC(CD303CD1c/CD141)CD34CD117+CD135+
CD116+ CD115CD45RA+CD123+.
Online supplemental material. Fig. S1 shows clonal output of cord blood
CD34+ HSPCs. Fig. S2 shows clonal output of cord blood hpre-CDCs. Fig. S3
shows clonal output of peripheral blood hpre-CDCs. Fig. S4 shows 14-color
gating strategy for identification of human hpre-CDCs in blood and that
hCDPs, hMDPs, and hGMDPs are undetectable in the blood before and after
Flt3L administration in healthy volunteers. Online supplemental material is
available at http://www.jem.org/cgi/content/full/jem.20141441/DC1.
This work was inspired by Dr. Ralph M. Steinman. We thank Klara Velinzon (Flow
Cytometry Core Facility, Laboratory of Molecular Immunology, The Rockefeller
University) for technical support with polychromatic flow cytometry sorting. We thank
Heidi Schreiber for help with human BM protocol (Laboratory of Molecular Immunology,
The Rockefeller University). We acknowledge the assistance of Arlene Hurley and the
clinical team at the Rockefeller University Hospital. We also thank James Pring, Popi
Sarma and Christine Trumpfheller for the management of the Flt3L patient samples
(Laboratory of Cellular Physiology and Immunology, The Rockefeller University).
Research reported in this publication was supported by the Empire State Stem
Cell Fund through New York State Department of Health Contract #C029562
(to K.Liu), Helmsley Foundation (to K. Liu), National Institute of Allergy and Infectious
Diseases of National Institutes of Health under award numbers of AI101251 (to K. Liu)
and U19AI111825 (to M.C. Nussenzweig), National Institute of Neurological Disorder
and Stroke of NIH under award number of NS084776 (to S. Puhr), Iris and Junming Le
Foundation (to G.Breton), the Dermatology Foundation (to N. Anandasabapathy),
National Institute of Arthritis and Musculoskeletal and Skin Diseases AR063461-
01A1 (to N. Anandasabapathy), and CTSA, RUCCTS grant no. UL1RR024143 from
the National Center for Research Resources, NIH, and by NIH grant AI13013. The
Rockefeller University Center for Clinical and Translational Science is supported, in
part, by a Clinical and Translational Science Award (CTSA), and the National Center for
Advancing Translational Sciences (NCATS), part of the NIH. The content is solely the
responsibility of the authors and does not necessarily represent the official views of
411
the National Institutes of Health or the Empire State Stem Cell Board, the NY State
Department of Health or the State of New York or any other applicable federal
funding agency. M.C. Nussenzweig is an HHMI Investigator.
T. Keler works for Celldex Therapeutics. The authors declare no additional
financial interests.
Submitted: 30 July 2014
Accepted: 30 January 2015
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