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Leukocyte Trafficking: Time to Take Time Seriously

Natalia Reglero-Real,1,2,* Loı̈c Rolas,1,2,* and Sussan Nourshargh1,*
1William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London

EC1M 6BQ, UK
2These authors contributed equally

*Correspondence: n.reglero@qmul.ac.uk (N.R.-R.), l.rolas@qmul.ac.uk (L.R.), s.nourshargh@qmul.ac.uk (S.N.)

https://doi.org/10.1016/j.immuni.2019.01.013

Leukocyte trafficking is a key component of steady-state tissue homing and in mounting an inflammatory
response. Two recent publications in Immunity by He et al. (2018) and Adrover et al. (2019) report on the
diurnal regulation of these responses and the associated pathophysiological implications.

Leukocyte migration is a crucial element
of immunity in health and disease. Under
homeostatic conditions, immune cells
shuttle between the vascular system and
tissues and provide immune surveillance.
In response to pathogenic and sterile in-
sults, leukocytes are recruited to inflamed
tissues to eliminate the cause of injury and
contribute to tissue repair (Nourshargh
and Alon, 2014). Leukocyte trafficking is
exquisitely regulated by locally generated
directional cues and factors that induce
vital changes in microvessels to promote
vascular attachment and diapedesis of
leukocytes. These responses occur via a
sequence of well-characterized events,
as described by the leukocyte adhesion
cascade and mediated by adhesive mole-
cules expressed on the cell surface of leu-
kocytes and vascular cells (Nourshargh
and Alon, 2014). Although this established
paradigm is applicable to most peripheral
tissues, recruitment mechanisms may
substantially vary depending on the in-
flammatory stimuli and tissue microenvi-
ronment, including endothelial cell (EC)
and mural cell (pericyte) heterogeneity of
different vascular beds, as well as specific
structural organization, rheological fea-
tures, and local cellular composition of
different organs. Furthermore, it is now
well accepted that a wide variety of addi-
tional parameters, such as age, gender,
diet, gut microbiota, metabolism, and
genetics, can impact the profile and
mechanisms of leukocyte trafficking
(Franceschi et al., 2018). Recent studies
have added circadian rhythm to this list,
demonstrating the importance of diurnal
rhythmicity as a regulator of leukocyte
trafficking (Scheiermann et al., 2013).
Circadian rhythm refers to any biological
process that displays oscillations during
a period of 24 h. Such autonomous fluc-
tuations can be aligned to overlap with the
daily rotation of the Earth by the use of
environmental cues such as light patterns
(Scheiermann et al., 2013). Amongst the
many immune responses that show circa-
dian rhythm, probably the most striking is
the number of blood-circulating leuko-
cytes, which peaks during the night for
humans and during the day for rodents.
In mice, this rhythmic behavior correlates
with increased bone-marrow output at
the beginning of the light phase and
increased leukocyte exit from the circula-
tion during the night (Casanova-Acebes
et al., 2013; Scheiermann et al., 2013).
Through conducting an organism-wide
assessment of pro-migratory molecules,
He et al. (2018) identified distinct mole-
cular migratory codes responsible for
recruitment of different leukocyte subsets
to different organs over the course of the
day. A key finding of this study is that
rhythmic steady-state leukocyte traf-
ficking into tissues (optimal during the
dark phase) was regulated by a molecular
clock in both leukocytes and ECs
(Figure 1). Specifically, lineage-targeted
genetic deletion of Bmal1 (brain and mus-
cle Arnt-like protein-1), a transcription
factor central to circadian clock function,
in different leukocyte subsets or ECs
ablated rhythmic leukocyte recruitment.
This effect was linked to the temporal
expression of pro-migratory molecules
by leukocytes (e.g., integrins and chemo-
kine receptors) and vascular beds (e.g.,
EC adhesion molecules ICAM-1 and
VCAM-1). The functional implications of
these findings were elegantly demon-
strated through chronopharmacological
studies in which optimal efficacy of pro-
migratory blockers was time dependent.
Collectively, these studies shed light
on the molecular basis of time-of-day-
dependent immune-cell trafficking in
different organs.
Adrover et al. (2019) further explored
the rhythmicity of leukocyte recruitment,
focusing on neutrophils. Neutrophils are
the most abundant leukocytes in human
blood, with a relatively short lifespan
(12 h) in the circulation, though this issue
is under exploration in different contexts
(Hellebrekers et al., 2018). Previous
studies have shown that post mobilization
from the bone marrow and during their
circulation time in blood, neutrophils
exhibit a natural phenotypic change that
follows a diurnal regime and ultimately
promotes rhythmic clearance of ‘‘aged
neutrophils’’ from the circulation through
relocation into tissues (e.g., bone marrow)
(Casanova-Acebes et al., 2013). Here, the
term ‘‘aged neutrophils’’ refers to a popu-
lation of CD62Llow CXCR4high mature neu-
trophils that are at the end of their blood
circulation time (Casanova-Acebes et al.,
2013; Zhang et al., 2015). This population
of neutrophils (40% of total blood neu-
trophils) peaks during daylight in mice,
whereas non-aged or ‘‘fresh’’ neutrophils
(CD62Lhigh) are most prevalent in the cir-
culation at night. Most intriguingly, guided
by gene expression analysis, acquisition
of an aged neutrophil phenotype required
a cell-autologous molecular program
whereby BMAL1 regulated the expres-
sion of the chemokine CXCL2, a key
CXCR2 ligand.
In contrast to the pro-aging effects of
CXCR2 activation, the chemokine recep-
tor CXCR4 antagonized the attainment
of neutrophil aging phenotype by blunting
CXCR2 responses (Adrover et al., 2019).
Of note, in addition to neutrophils, the
surface expression of CXCR4 was regu-
lated in a rhythmic manner by numerous
leukocyte subsets (He et al., 2018).

Immunity 50, February 19, 2019 ª 2019 Elsevier Inc. 273


Figure 1. Circadian Rhythm Regulates Leukocyte Trafficking and Functions
At steady state, circadian rhythm regulates the phenotype of leukocytes and ECs and temporal trafficking
of leukocyte into tissues, which coincides with the beginning of the dark phase and a drop in blood
leukocyte numbers. Time-dependent tissue infiltration of aged neutrophils promotes local anti-microbial
efficacy while suppressing susceptibility to vascular damage. ZT, zeitgeber time; EC, endothelial cell; BM,
basement membrane.
Evidence for functional implications of this
expression (He et al., 2018) and the longer
lifespan of most leukocyte subtypes (as
compared to short-lived neutrophils) indi-
cate a broader role for CXCR4 in rhythmic
leukocyte trafficking beyond regulation of
cell aging. Furthermore, because the
ligand for CXCR4 (i.e., CXCL12) is not pro-
duced by neutrophils, neutrophil rhythmic
homing is likely coordinated by extrinsic,
potentially microenvironment-derived cir-
cadian signals, as indicated by He et al.
(2018). In line with this, the unquestion-
able role of vascular mural and perivascu-
lar cells in leukocyte trafficking (Nour-
shargh and Alon, 2014) begs the need
for investigations into how functions of
pericytes, mast cells, and tissue macro-
phages are controlled by time of day.
Both studies show comparable results
in terms of the impact of circadian rhyth-
274 Immunity 50, February 19, 2019
micity on immune cell trafficking. Specif-
ically, during homeostatic conditions,
leukocytes emigrate from blood into tis-
sues during dark phase, leading to a
drop in blood leukocyte numbers (He
et al., 2018). With respect to aged neutro-
phils, through the use of genetically modi-
fied mouse models exhibiting constitutive
or impaired neutrophil aging (i.e., via
deletion of neutrophil Cxcr4 or Bmal1,
respectively), Adrover et al. (2019) show
aging to be essential for neutrophil
clearance into tissues during the dark
phase. Furthermore, leukocytes exhibited
decreased homing to tissues during the
light phase in a model of LPS-driven sys-
temic inflammation (He et al., 2018) and
aged neutrophils (peaking during the light
phase) showed impaired migration into in-
flamed tissues (Adrover et al., 2019). With
respect to the latter, reduced neutrophil
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rolling, stemming from disrupted cortical
actin architecture, was presented as a
mechanism for defective inflammatory
migration of aged neutrophils (Adrover
et al., 2019). Surprisingly, these cells
spontaneously adhered to dermal vascu-
lature under homeostatic conditions,
suggesting expression of activated integ-
rins on aged neutrophils, as previously
described (Zhang et al., 2015). In terms
of breaching venular ECs, because
the cell-autologous CXCL2-CXCR2 axis
drives neutrophil TEM (transendothelial
cell migration) (Girbl et al., 2018), the
pro-migratory activity of neutrophil-
derived CXCL2 could be particularly
relevant in promoting tissue infiltration of
aged neutrophils.
So why does aging instruct a diurnal
switch in the migratory profile of neutro-
phils? Remarkably, evidence is provided
for the ability of aged neutrophils to
exhibit enhanced antimicrobial capability
in a model of Candida albicans infection
by improving fungal clearance in kidneys
(Adrover et al., 2019). At the same time,
clearance of aged neutrophils from the
circulation reduced the occurrence of
intravascular thrombi post ischemia-re-
perfusion injury and protected mice in a
model of acute myocardial infarction.
Thus, it is proposed that tissue-infiltrated
aged neutrophils provide a ‘‘ready-to-
use’’ local immune protective mechanism
while diminishing the risk of detrimental
vascular inflammation. These findings
raise several points. Fundamentally, as
there is at present much interest in func-
tional and phenotypic heterogeneity of
neutrophils in health and disease (Helle-
brekers et al., 2018), it is unclear whether
all neutrophils acquire an ‘‘aging’’ pheno-
type to a similar extent, within the same
timeline, and via comparable mecha-
nisms. Furthermore, the impact of factors
such as chronic inflammation and physio-
logical aging on ‘‘neutrophil aging,’’ most
notably in humans, and their properties
require further explorations. Of note,
although aged neutrophils were protec-
tive against Candida albicans infection
where the kidney is a primary site for
dissemination of the systemic fungus,
decreased survival was observed in a
model of sepsis when conducted during
dark phase, i.e., time of maximal tissue
infiltration of aged neutrophils. Thus, it is
potentially possible that although aged
neutrophils provide protection against
Immunity
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local infections, neutropenia during the
dark phase may render the host suscepti-
ble to systemic infections. However, as
homeostatic clearance of blood neutro-
phils involved intravascular retention of
cells in certain organs (e.g., in lungs) (He
et al., 2018), the relative contribution of
such cellular compartmentalization to
neutrophil-mediated intravascular immu-
nity requires further exploration, as exem-
plified by elegant studies carried out in the
lung microvasculature (Granton et al.,
2018). Finally, contrary to the findings of
Adrover et al. (2019), aged neutrophils
have been reported to rapidly migrate
into sites of inflammation and exhibit
increased phagocytosis (Uhl et al.,
2016), further indicating the need for
greater understanding of the implications
of diurnal immune cell trafficking.
Collectively, these elegant studies
strengthen the case for circadian control
of immune cell functions. In particular,
they provide insight into the physiological
and pathological role of circadian varia-
tions in leukocyte trafficking, though there
remain many unanswered questions,
most notably in humans. Fundamentally,
with greater understanding of this impor-
tant concept, the findings of He et al.
Butyrate Makes Macrophages
‘‘Go Nuclear’’ against Bacterial Pathogens
Lior Lobel1 and Wendy S. Garrett1,*
1Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, 665 Huntington Ave, Boston, MA
02115, USA
*Correspondence: wgarrett@hsph.harvard.edu
https://doi.org/10.1016/j.immuni.2019.01.015
Butyrate is a microbial metabolite with pleiotropic effects. Schulthess et al. (2019) report that butyrate pre-
conditioning of macrophages enhances their anti-bacterial preparedness by inducing anti-microbial proteins
that restrict bacterial growth. This study augments understanding of how microbial metabolites shape host
defense.
Host physiology is profoundly dependent
on the gut microbiota; however, the micro-
bial metabolite signaling underpinning
these transkingdom interactions remain
largely unknown. In this issue of Immunity,
Schulthess et al. (2019) report that short
(2018) and Adrover et al. (2019) endorse
strategies aimed at exploiting time-based
therapeutic interventions for optimal
clinical benefit. Indeed, it is time to take
time seriously in immunity and clinic
practice!
ACKNOWLEDGMENTS
The authors are funded by the Wellcome Trust
(098291/Z/12/Z to S.N.).
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ranging from host metabolism to signal
transduction, and most of our current
knowledge comes from studying patho-
gens. Therefore, findings that reveal direct
mechanisms in which non-pathogenic
commensal bacteria modulate host