Activation of Transcription Factor 4 in

Dendritic Cells Controls Th1/Th17 Responses

and Autoimmune Neuroinflammation
This information is current as Indumathi Manoharan, Daniel Swafford, Arulkumaran
of November 14, 2021. Shanmugam, Nikhil Patel, Puttur D. Prasad, Muthusamy
Thangaraju and Santhakumar Manicassamy

Downloaded from http://www.jimmunol.org/ at Red de Bibliotecas del CSIC on November 14, 2021
J Immunol 2021; 207:1428-1436; Prepublished online 4
August 2021;
doi: 10.4049/jimmunol.2100010
http://www.jimmunol.org/content/207/5/1428

Supplementary http://www.jimmunol.org/content/suppl/2021/08/04/jimmunol.210001
Material 0.DCSupplemental
References This article cites 62 articles, 20 of which you can access for free at:
http://www.jimmunol.org/content/207/5/1428.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision
• No Triage! Every submission reviewed by practicing scientists
• Fast Publication! 4 weeks from acceptance to publication
*average

Subscription Information about subscribing to The Journal of Immunology is online at:

http://jimmunol.org/subscription
Permissions Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Email Alerts Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by

The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2021 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
 The Journal of Immunology

Activation of Transcription Factor 4 in Dendritic Cells Controls

Th1/Th17 Responses and Autoimmune Neuroinflammation

Indumathi Manoharan,*,† Daniel Swafford,† Arulkumaran Shanmugam,† Nikhil Patel,‡

Puttur D. Prasad,* Muthusamy Thangaraju,* and Santhakumar Manicassamy*,†,§
Dendritic cells (DCs) are professional APCs that play a crucial role in initiating robust immune responses against invading
pathogens while inducing regulatory responses to the body's tissues and commensal microorganisms. A breakdown of DC-
mediated immunological tolerance leads to chronic inflammation and autoimmune disorders. However, cell-intrinsic molecular

Downloaded from http://www.jimmunol.org/ at Red de Bibliotecas del CSIC on November 14, 2021
regulators that are critical for programming DCs to a regulatory state rather than to an inflammatory state are not known. In this
study, we show that the activation of the TCF4 transcription factor in DCs is critical for controlling the magnitude of
inflammatory responses and limiting neuroinflammation. DC-specific deletion of TCF4 in mice increased Th1/Th17 responses and
exacerbated experimental autoimmune encephalomyelitis pathology. Mechanistically, loss of TCF4 in DCs led to heightened
activation of p38 MAPK and increased levels of proinflammatory cytokines IL-6, IL-23, IL-1b, TNF-a, and IL-12p40. Consistent
with these findings, pharmacological blocking of p38 MAPK activation delayed experimental autoimmune encephalomyelitis onset
and diminished CNS pathology in TCF4DDC mice. Thus, manipulation of the TCF4 pathway in DCs could provide novel
opportunities for regulating chronic inflammation and represents a potential therapeutic approach to control autoimmune
neuroinflammation. The Journal of Immunology, 2021, 207: 1428
1436.

M
ultiple sclerosis (MS) is a chronic autoimmune inflam-
matory condition that leads to multifocal demyelination
in the white matter of the human CNS. Using experi-
mental autoimmune encephalomyelitis (EAE), a mouse model for
MS, studies have shown that dendritic cells (DCs) play a critical
role in the initiation and development of CNS pathology (1
3). DCs
are a specialized subset of APCs that form a critical link between
innate and adaptive immune cells (4, 5). Accumulating evidence
suggests that DCs play a pivotal role in instigating inflammation as
well as suppressing inflammation and restoring immune homeostasis
(4, 5). Recent studies have shown that ablation of DCs in mice
breaks self-tolerance of CD41 T cells and results in spontaneous
fatal autoimmunity (6, 7). Likewise, depletion of DCs in mice
resulted in stronger inflammatory responses with exacerbated EAE
(7, 8). Furthermore, depletion of specific DC subsets in lymphoid or
nonlymphoid tissues during the acute or relapse phase of EAE
results in stronger inflammatory responses and exacerbates the dis-
ease (9
12). Besides, other studies have shown that these cells lose
their regulatory properties, resulting in uncontrolled chronic inflam-
mation (13
15). DCs contribute to CNS pathology through differen-
tiation and activation of naive CD41 T cells to myelin-specific Th1
and Th17 cells (16, 17). Conversely, emerging evidence suggests
that DCs are also critical in resolving inflammation and limiting
immune-mediated pathology in EAE by producing immune regula-
tory factors and driving regulatory T cell (Treg) differentiation and
activation (3, 4, 5). Thus, DCs play a key role in bridging innate
and adaptive immunity. Although DCs are present in low numbers
in the CNS under homeostatic conditions, their numbers increase
drastically during autoimmune inflammation, infection, or trauma
(18). However, cell-intrinsic molecular regulators that are critical for
programming DCs to a regulatory state rather than to an inflamma-
tory state are not known. Thus, understanding these events may pre-
sent promising new targets for therapeutic intervention of various
autoimmune and chronic inflammatory conditions.
Aberrant activation of the wingless/integrated (Wnt) signaling
pathway occurs in several inflammatory diseases, including neurode-
generative and neuroinflammatory diseases (19
21). Many studies
have documented that Wnt ligands are highly expressed in several
chronic inflammatory diseases and autoimmune diseases (22
24).
The T cell factor/lymphoid enhancer
binding factor (TCF/LEF)
family of transcription factors are known to be critical for embryo-
genesis and in the development of hematopoietic stem cells and
immune cells (25). The TCF/LEF family comprises four NFs,
namely TCF1, LEF1, TCF3, and TCF4 (also designated as TCF7,
LEF1, TCF7L1, and TCF7L2) (25). They act as one of the main
downstream mediators of the Wnt/b-catenin signaling pathway (25,
26), and DCs highly express the TCF4 isoform (27, 28). However,
the functional and biological significance of TCF4 in DCs in regu-
lating ongoing inflammation and establishing immune homeostasis
is poorly understood.

*Department of Biochemistry and Molecular Biology, Medical College of
Georgia, Augusta University, Augusta, GA; †Georgia Cancer Center, Medical Col-
lege of Georgia, Augusta University, Augusta, GA; ‡Department of Pathology, Medical
College of Georgia, Augusta University, Augusta, GA; and xDepartment of Medicine,
Medical College of Georgia, Augusta University, Augusta, GA
ORCIDs: 0000-0001-9335-0082 (N.P.); 0000-0002-8455-0186 (P.D.P.); 0000-0002-
2203-3917 (S.M.).
Received for publication January 7, 2021. Accepted for publication June 28, 2021.
This work was supported by the National Institute of Diabetes and Digestive and
Kidney Diseases Awards (DK097271 and DK123360) and Augusta University Awards
(IGPB0003 and ESA00041) (to S.M.).
Address correspondence and reprint requests to Dr. Santhakumar Manicassamy,
Department of Biochemistry and Molecular Biology, Medical College of Georgia,
Augusta University, 1120 15th Street, CN 4153, Augusta, GA 30912. E-mail address:
smanicassamy@augusta.edu
The online version of this article contains supplemental material.
Abbreviations used in this article: DC, dendritic cell; DLN, draining lymph
node; EAE, experimental autoimmune encephalomyelitis; ICS, intracellular
cytokine staining; LRP5/6, low density lipoprotein receptor-related protein 5 and
6; MF, macrophage; MHC II, MHC class II; MOG, myelin oligodendrocyte
glycoprotein; MS, multiple sclerosis; pDC, plasmacytoid DC; pi, post-
immunization; TCF/LEF, T cell factor/lymphoid enhancer
binding factor;
Tr1, type 1 Treg; Treg, regulatory T cell; Wnt, wingless/integrated; WT,
wild-type.
Copyright © 2021 by The American Association of Immunologists, Inc. 0022-1767/21/$37.50

www.jimmunol.org/cgi/doi/10.4049/jimmunol.2100010
The Journal of Immunology
In the current study, we report that during the induction and
effector phase of EAE, TCF4 activation in DCs plays a key role in
regulating the magnitude of inflammatory responses and limiting
collateral damage to the host. Accordingly, our data demonstrate
that the DC-specific deletion of TCF4 in mice results in severe EAE
pathology. This was because of heightened activation of p38 MAPK
and increased expression of proinflammatory cytokines by DCs
lacking TCF4, resulting in increased Th1 and Th17 cell polarization.
In contrast, pharmacological blocking of p38 MAPK activation in
TCF4-deficient DCs markedly reduced the expression of proinflam-
matory cytokines and diminished EAE severity in TCF4DDC mice.
These results reveal a novel mechanism by which the TCF4 in DCs
controls chronic inflammation and limits immune-mediated pathol-
ogy in EAE. Thus, manipulating TCF4 activation in DCs may repre-
sent a convenient therapeutic approach to improve the outcome of
MS and other inflammatory diseases.
Materials and Methods
Mice
C57BL/6, CD11c-cre (29), TCF/LEF-reporter mice (30), and 2D2 myelin
oligodendrocyte glycoprotein (MOG)
specific TCR transgenic mice (31)
were originally obtained from The Jackson Laboratory and bred on site.
TCF4 floxed mice (32) were kindly provided by Dr. Melinda L Angus-Hill
(University of Utah) and were crossbred to CD11c-cre mice to generate
mice lacking TCF4 in DCs (TCF4DDC) (27). The successful cre-mediated
deletion was confirmed by PCR and protein expression analyses. All mice
were housed under specific pathogen-free conditions in the Laboratory Ani-
mal Services of Augusta University. Animal care protocols were approved
by the Institutional Animal Care and Use Committee of Augusta University.
Reagents and Abs
MOG35
55 peptide (MEVGWYRSPFSRVVHLYRNGK) was purchased
from AnaSpec. Abs against mouse CD4 (GK1.5), CD8a (53-6.7), CD45 (30-
F11), Foxp3 (FJK-16s), IL-10 (JES5-16E3), TNF-a (MP6-XT22), CD11c
(N418), CD90.1 (HIS51), IL-17 (TC11-18H10), and IFN-g (XMG1.2) were
purchased from eBioscience and BioLegend. GFP, phospho-p38 MAPK,
p38 MAPK, and TCF4 Abs were obtained from Cell Signaling Technology.
p38 MAPK inhibitor (SB203580) was purchased from Calbiochem.
EAE induction
EAE induction experiments were performed as described in our previous
studies (33, 34). EAE was induced by s.c. immunization in the hind flanks
on day 0 using 100 mg of the MOG35
55 peptide emulsified in CFA contain-
ing 2.5 mg/ml heat-inactivated Mycobacterium tuberculosis (Difco Laborato-
ries). Mice also received 250 ng of pertussis toxin (List Biological
Laboratories) i.p. on days 0 and 2 postimmunization (pi). Disease severity
was assessed on different days pi, according to the following scale: 0, no dis-
ease; 1, flaccid tail; 2, hind limb weakness; 3, hind limb paralysis; 4, fore-
limb weakness; and 5, moribund. In some experiments, EAE-induced
TCF4FL/FL and TCF4DDC were treated with either p38 MAPK inhibitor (5
mg/kg) or vehicle by i.p. injection every 2 d starting on day 3 pi.
CNS leukocyte isolation
Mice were euthanized with CO2 and perfused through the left ventricle with
PBS. The brain and spinal cord were removed from each animal and dis-
sected into small fragments followed by digestion with collagenase type 4 (1
mg/ml) in complete DMEM plus 2% FBS for 30 min at 37 C. Leukocytes
were isolated using 40% Percoll (Sigma-Aldrich) and then were stained for
CD41 T cells expressing Foxp3 and different intracellular cytokines.
1 Spinal cords from EAE-induced mice were removed after perfusion and
CD11c cell isolation
1 fixed using 10% formalin in PBS. Fixed spinal cords were embedded in par-
CD11c DCs were purified from the CNS or draining lymph nodes (DLNs)
and spleen (pooled together) as previously described (33). In brief, the spleen
or brain was cut into small fragments and then digested with collagenase
type 4 (1 mg ml1) in complete DMEM plus 2% FBS for 30 min at 37 C.
Cells were washed twice and enriched for CD11c1 DCs with the CD11c-
specific microbeads from Miltenyi Biotec. The resulting purity of CD11c1
DCs was 95%. Enriched CD11c1 DCs (106 cells/ml) from immunized
mice were cultured ex vivo for 24 h. The supernatants were collected for
cytokine analysis by ELISA, whereas cells were collected for gene expres-
sion analysis by RT-PCR (34, 35).
1429
Flow cytometry
Single-cell suspensions from CNS leukocytes, DLNs, and spleen were sus-
pended in PBS containing 5% FBS. After incubation for 15 min at 4 C with
the blocking Ab 2.4G2 (anti-FcgRIII/I), the cells were stained at 4 C for 30
min with the appropriately labeled Abs. Samples were then washed two
times in PBS containing 5% FBS. The samples were either immediately ana-
lyzed at this point or fixed in PBS containing 2% paraformaldehyde and
stored at 4 C. Intracellular staining for phospho-p38 MAPK-PE and GFP-
FITC was performed using rabbit mAb or with appropriate isotype control in
TBS containing 1% BSA. For intracellular cytokine staining (ICS), single-
cell suspensions from the DLNs or CNS were ex vivo stimulated with PMA/
ionomycin and brefeldin A/monensin for 6 h at 37 C. The cells were then
stained for CD4 and CD8 followed by intracellular staining of IFN-g, IL-
17A, TNF-a, and IL-10. To measure Foxp31 Tregs or phospho-p38 MAPK
or GFP expression, the corresponding Abs were added after permeabilization
and fixation of cells. Flow cytometric analyses were performed using a
FACS LSRII system (BD Biosciences), and the data were analyzed using
Downloaded from http://www.jimmunol.org/ at Red de Bibliotecas del CSIC on November 14, 2021
FlowJo software (Ashland, OR).
Tetramer staining
MOG38
49-IAb tetramers were kindly provided by the National Institutes of
Health Tetramer Core Facility at Emory University. CNS-infiltrating leuko-
cytes were resuspended in PBS containing 5% FBS followed by tetramer
staining for 1 h at 37 C as described in a previous study (34, 36). After 1 h,
cells were stained with anti-CD4 (APC) and anti-CD45 (Alexa Fluor 700)
for 30 min on ice. Cells were washed three times, followed by data acquisi-
tion on the flow cytometer. A nonspecific peptide tetramer was used as a
negative control. The percentage of tetramer-PE
positive cells was deter-
mined in CD4-positive populations based on negative control staining.
2D2 CD41 T cell adoptive transfer
TCF4FL/FL and TCF4DDC recipient mice were reconstituted with 2.5  106
2D2 TCR transgenic CD41 T cells followed by EAE immunization, as
described in our previous study (34). Five days later, DLNs were removed,
and after RBC lysis, in vitro recall responses were assayed by restimulating
DLN cells (2  106/ml) for 6 h with PMA/ionomycin in the presence of bre-
feldin A/monensin for intracellular cytokine detection.
In vitro culture of murine DCs and T cells
In vitro 2D2 CD41 T cell differentiation was performed as previously
described (33, 37). In brief, purified DLNs and splenic CD11c1 DCs (pooled
together) (106 cells/ml) from immunized mice on day 5 were cultured together
with naive 2D2 CD41CD62L1 T cells (105) in 200 ml RPMI-1640 complete
medium in 96-well round-bottom plates in the presence of the MOG35
55 pep-
tide (1 mg/ml). In some experiments, DCs purified from immunized mice
treated with p38 inhibitor or vehicle were cultured with naive 2D2 T cells.
After 96 h, cells were restimulated with PMA/ionomycin for 6 h in the pres-
ence of brefeldin A/monensin for intracellular cytokine detection.
Quantitative real-time PCR
Total RNA was isolated from purified DLNs and spleen (pooled together) or
CNS-infiltrating CD11c1 DCs using the QIAGEN RNeasy Mini Kit, accord-
ing to the manufacturer's protocol (QIAGEN). cDNA was generated using
the superscript First-Strand Synthesis System for RT-PCR and random hex-
amer primers (Invitrogen), according to the manufacturer's protocol. cDNA
was used as a template for quantitative real-time PCR using SYBR Green
Master Mix (Bio-Rad Laboratories) and gene-specific primers, as described
in our previous studies (33, 34). Gene expression across samples was nor-
malized relative to the housekeeping gene GAPDH.
Histopathology
affin and sectioned, after which the sections were stained with H&E to study
leukocyte infiltration and pathology as well as Luxol fast blue stain to dem-
onstrate CNS demyelination (34, 37).
Statistical analysis
Statistical analyses were conducted using Prism (GraphPad). Mean clinical
scores were analyzed using the Mann
Whitney nonparametric t test. The sta-
tistical significance of differences in the means ± SD of cytokines released
by cells of various groups was calculated with the Student t test (one-tailed).
1430 TCF4 DEFICIENCY IN DCs AGGRAVATES THE PATHOGENESIS OF EAE

Results in DC subsets in TCF4DDC mice (Supplemental Fig. 1E). However,

Deletion of TCF4 in DCs exacerbates EAE pathology
To investigate the selective role of TCF4 in DC function during auto-
immune neuroinflammation, we first assessed whether TCF4 is acti-
vated in DCs during autoimmune neuroinflammation using the TCF/
LEF-GFP reporter mice (30). In this reporter mice, GFP reporter gene
is under the control of six copies of a TCF/Lef response element (30).
We immunized TCF-reporter mice with the MOG35
55 peptide plus
CFA and assessed GFP expression in DCs isolated from the spleen dur-
ing the preclinical stage of EAE phase (day 5 pi) and disease phase
(day 14 pi). We observed a marked increase in GFP expression in
splenic DC of TCF/LEF-GFP reporter mice pi during the induction
and disease phase of EAE (Fig. 1A). Likewise, CNS-infiltrating DCs
from the reporter mice showed increased GFP expression during the
clinical stage of EAE (Fig. 1A). Next, we analyzed GFP expression in
TCF4 mRNA levels were comparable in splenic MFs isolated from
WTFL/FL and TCF4DDC mice, demonstrating efficient target gene
deletion in DCs (Supplemental Fig. 1E). Next, we investigated
whether TCF4 deficiency affects DC numbers during the steady
state. We did not observe any significant differences in the fre-
quency and total number of splenic CD11c1MHC II1 DCs between
WTFL/FL and TCF4DDC mice (Supplemental Fig. 1F). Similarly, the
frequencies of DC subsets in the spleen were comparable between
WTFL/FL and TCF4DDC mice (Supplemental Fig. 1G), suggesting
that TCF4 deletion does not affect the development and differentia-
tion of these DC subsets.
Next, we analyzed EAE disease progression and pathology in the
WTFL/FL and TCF4DDC mice. WTFL/FL mice immunized with the
MOG35
55 peptide plus CFA showed onset of neurologic impairment

DC subsets, macrophages (MFs), and T cells. We observed increased
GFP expression in CD81 DCs (CD451MHC class II [MHC II]1
CD11c1CD11bCD8a1), CD11b1 DCs (CD451MHC II1CD11c1
CD11b1CD8a), plasmacytoid DCs (pDCs) (CD451CD11c1B2201
PDCA-11), and MFs (CD451MHC II1CD11cCD11b1CD641F4/
801) (Supplemental Fig. 1A). Of note, we do not detect any reporter
gene expression in CD41 and CD81 T cells during the preclinical
stage of EAE phase (day 5 pi) (Supplemental Fig. 1A). We asked
whether increased GFP expression in DCs is due to changes in the
TCF4 expression during EAE. Enriched splenic and CNS DCs from
the EAE mice showed similar TCF4 mRNA expression levels under
steady-state conditions as well as during the induction and effector
phase of the disease (Supplemental Fig. 1B). These results suggest that
TCF4 is activated in DCs in EAE.
To determine the selective role of TCF4 in DC function in EAE,
we deleted TCF4 specifically in DCs (TCF4DDC) by crossing TCF4
floxed (WTFL/FL) mice to CD11c-cre mice. Subsequently, we con-
firmed the efficient deletion of TCF4 by quantitating the mRNA and
protein expression levels of TCF4 in the splenic DCs isolated from
the WTFL/FL and TCF4DDC mice. As expected, we noted a marked
decrease in the TCF4 mRNA and protein levels in TCF4-deficient
DCs compared with the wild-type (WT) DCs (Supplemental Fig.
1C, 1D). Moreover, TCF4 mRNA levels were markedly decreased
Downloaded from http://www.jimmunol.org/ at Red de Bibliotecas del CSIC on November 14, 2021
occurring around day 14. In contrast, TCF4DDC mice showed an earlier
onset of neurologic impairment and developed a more severe form of
EAE (Fig. 1B). The histopathological analysis of CNS showed a
marked increase in leukocyte infiltration and intense demyelination in
TCF4DDC mice compared with WTFL/FL mice (Fig. 1C, 1D). In line
with these observations, flow cytometric analysis confirmed a signifi-
cant increase in the total number of CD451 cells in the CNS of
TCF4DDC mice. Further characterization of CNS-infiltrating leukocytes
in TCF4DDC mice showed more DCs (CD45hiMHC IIhiCD11c1),
MFs (CD45hiMHC IIhiCD11cCD11b1CD641), monocytes (CD
45hiMHC IICD11cCD11b1Ly6ChiLy6Glow), neutrophils (CD45hi
MHC IICD11cCD11b1Ly6ClowLy6Ghi), and CD41 T cells
(Fig. 1E). Collectively, these data suggest that DC-specific expression
of TCF4 is critical for limiting autoimmune CNS pathology.
TCF4DDC mice display increased infiltration of Th1 and Th17 cells
in the CNS during EAE
CNS-infiltrating CD41 effector T cells contribute to the pathogene-
sis of EAE (16, 17, 38), whereas the CD41 Tregs play a key role in
limiting the disease severity and associated tissue injury. Thus, we
next investigated if increased EAE severity observed in TCF4DDC
mice was due to effector CD41 T cell subsets. There was a signifi-
cant increase in the frequency of the MOG38
49 tetramer
specific

FIGURE 1. Loss of TCF4 in DCs exacerbates EAE. TCF/LEF-GFP reporter mice were immunized with 100 mg of the MOG35
55 peptide in CFA on day 0.
Mice also received 250 ng of pertussis toxin on days 0 and 2 pi. (A) Representative histogram of GFP expression by DCs isolated from the DLNs, spleen (day 5
and 14 pi), and CNS (day 14 pi) of EAE-induced TCF/LEF reporter mice and analyzed by ICS. (B
E) WTFL/FL and TCF4DDC mice were immunized with 100
mg of the MOG35
55 peptide in CFA on day 0. Mice also received 250 ng of pertussis toxin on days 0 and 2 pi. The EAE disease progression was monitored at
various days pi. (B) Mean clinical EAE score in WTFL/FL and TCF4DDC mice. (C and D) H&E and Luxol fast blue staining of spinal cords of WTFL/FL and
TCF4DDC mice to reveal the degree of demyelination and inflammation on day 16 pi. Scale bars (blue), 200 mm. (E) Total number of leukocytes (CD451), DCs
(CD45hiMHC IIhiCD11c1), MFs (CD45hiMHC IIhiCD11cCD11b1CD641), monocytes (CD45hiMHC IICD11cCD11b1Ly6ChiLy6GLow), neutrophils
(CD45hiMHC IICD11cCD11b1Ly6ClowLy6Ghi), and CD41 T cells from the CNS of WTFL/FL and TCF4DDC mice (day 16 pi) analyzed by flow cytometry.
Data are representative of two experiments (n = 4
5 mice per experiment). Error bars show mean values ± SEM. *p < 0.01, **p < 0.001, ***p < 0.0001.
The Journal of Immunology
CD41 T cells in the CNS of TCF4DDC mice when compared with
WT control mice (Fig. 2A, 2B). ICS of CD41 T cells showed a sig-
nificant increase in the frequencies of IFN-g1, IL-171, and TNF-
a1 CD41 T cells in the CNS of TCF4DDC mice (Fig. 2C, 2D). In
contrast, we observed a marked decrease in the frequency of IL-
101CD41 regulatory (type 1 Treg [Tr1]) cells in the CNS of
TCF4DDC mice compared with WT control mice, whereas the fre-
quency of Foxp31CD41 T cells was comparable in both groups
(Fig. 2C, 2D). Consistent with these observations, following ex vivo
MOG stimulation, leukocytes isolated from the CNS of TCF4DDC
mice produced markedly higher levels of IFN-g, IL-17A, and TNF-
a and lower levels of IL-10 compared with the leukocytes isolated
from the CNS of WTFL/FL mice (Fig. 2E). Collectively, these data
suggest that DC-specific expression of TCF4 is critical for limiting
autoimmune CNS pathology, indicating a possible regulatory role
for TCF4 in DCs during ongoing neuroinflammation.
TCF4 programs DCs to induce IL-10 and limit proinflammatory
cytokine expression
These results prompted us to examine whether TCF4 regulates DC
function through the expression of inflammatory and anti-inflamma-
tory cytokines during the preclinical stage and clinical stage of
EAE. We observed significantly increased mRNA levels for IL-6,
TNF-a, IL-1b, IL-23p19, and IL-12p40 and a decreased level of
IL-10 in DCs isolated from the DLNs and spleen of TCF4DDC mice
compared with WT control mice (Fig. 3A). In line with this obser-
vation, TCF4-deficient DCs produced markedly higher levels of IL-
6, IL-1b, TNF-a, IL-23, and IL-12p40 and reduced levels of IL-10
compared with WT DCs (Fig. 3B). In addition to the effector T
cells, DCs infiltrating the CNS contributes to the pathogenesis by
reactivating the primed T cells and functions as effector cells to
cause CNS lesions. Thus, we examined the expression of inflamma-
tory and anti-inflammatory cytokines in CNS-infiltrating DCs. As
shown in Fig. 3C, there was a significant increase in mRNA levels
of IL-6, IL-1b, TNF-a, IL-23p19, and IL-12p40 but decreased lev-
els of IL-10 in TCF4-deficient DCs compared with WT control DCs
infiltrating CNS. Thus, our data demonstrates that in DCs, TCF4 is
FIGURE 2. TCF4 deletion in DCs leads to increased frequencies of Th1 and Th17 cells in the CNS during EAE. (A) Representative FACS plot and
(B) frequencies of the MOG38
49 tetramer
specific CD41 T cells isolated from CNS of WTFL/FL and TCF4DDC mice on day 16 pi. (C) Representative FACS
plots and (D) frequencies of IFN-g1, IL-17A1, TNF-a1, IL-101 (after PMA and ionomycin stimulation), and Foxp31CD41 T cells isolated from the CNS
of WTFL/FL and TCF4DDC EAE mice on day 16 pi. (E) Cytokine concentrations in supernatants obtained after culture of CNS-infiltrating leukocytes stimu-
lated ex vivo with the MOG35
55 peptide for 48 h. Data are representative of two experiments (n = 4
5 mice per experiment). Error bars show mean values ±
SEM. **p < 0.001, ***p < 0.0001.
1431
critical for limiting inflammatory cytokine expression while inducing
IL-10 during the induction and effector phase of EAE.
TCF4 activation in DCs limits Th1 and Th17 differentiation
As DCs dictate the fate of naive CD41 T cells through differential pro-
duction of pro- and anti-inflammatory cytokines (39), we further con-
sidered the functional relevance of DC-specific TCF4-mediated
signaling in naive CD41 T cell differentiation. First, we performed an
ex vivo CD41 T cell differentiation assay by coculturing naive MOG-
specific 2D2 T cells with DCs isolated from the DLNs and spleen of
WTFL/FL and TCFDDC mice challenged with the MOG35
55 peptide
plus CFA. T cells cultured with TCF4-deficient DCs contained signifi-
cantly higher frequencies of Th1 and Th17 cells compared with T cells
cultured with WT DCs (Fig. 4A, 4B). Further, analysis of the culture
supernatant showed that T cells cultured with TCF4-deficient DCs
Downloaded from http://www.jimmunol.org/ at Red de Bibliotecas del CSIC on November 14, 2021
produced markedly higher levels of IFN-g and IL-17A than that of T
cells cultured with WT DCs (Fig. 4C). Therefore, deletion of TCF4 in
DCs augments Th1 and Th17 cell differentiation.
To extend these observations in vivo, we adoptively transferred
naive CD41CD25 T cells from 2D2 mice into WTFL/FL and TCFDDC
mice and then challenged these mice with the MOG35
55 peptide plus
CFA. There was a significant increase in the number of CD412D21 T
cells in the DLNs of TCF4DDC mice when compared with WT mice
on day 5 postchallenge (Fig. 4D). In line with these observations, intra-
cellular cytokine analysis showed a significant increase in donor T cell
differentiation toward Th1 and Th17 cells in TCF4DDC mice compared
with the WT mice (Fig. 4E). Consistent with these observations, donor
T cells isolated from the DLNs and spleen of TCF4DDC mice produced
markedly higher levels of IFN-g and IL-17A compared with the donor
T cells isolated from the WT mice in response to ex vivo MOG33
55
peptide stimulation (Fig. 4F). Therefore, TCF4 signaling in DCs limits
Ag-specific Th1 and Th17 cell differentiation and expansion in vivo.
Next, we asked whether increased inflammatory Th1 and Th17
cell differentiation observed in TCF4DDC mice is due to altered DC
maturation and activation. Characterization of DLN and splenic
DCs from TCF4DDC mice showed no significant difference in the
expression of MHC II, CD40, CD80, and CD86 as compared with
control WT mice on day 5 pi (Fig. 4G). Collectively, our results
1432 TCF4 DEFICIENCY IN DCs AGGRAVATES THE PATHOGENESIS OF EAE

Downloaded from http://www.jimmunol.org/ at Red de Bibliotecas del CSIC on November 14, 2021
FIGURE 3. TCF4 regulates the expression of proinflammatory and anti-inflammatory cytokines in DCs during EAE. (A and C) Quantitative real-time PCR analy-
sis of Il1b, Tnfa , Il6, Il23p19, Il12p40, and Il10 mRNA expression in CD11c1 DCs isolated from the DLNs and spleen or CNS of WTFL/FL and TCF4DDC EAE mice
on day 5 and day 14 pi. (B) Cytokine secretion by CD11c1 DCs isolated from DLNs and spleen of WTFL/FL and TCF4DDC EAE mice (day 5 or 14 pi) after ex vivo cul-
ture for 48 h. Data are representative of two experiments (n > 3 per experiment). Error bars show mean values ± SEM. *p < 0.01, **p < 0.001, ***p < 0.0001.

indicate that TCF4 activation in DCs regulates the differentiation of
naive CD41 T cells into Th1 and Th17 cells through the regulation
of DC-specific cytokine production.
TCF4 limits the expression of inflammatory factors and Th1/Th17
cell differentiation by regulating p38 MAPK activation in DCs
The p38a MAPK signaling pathway is critical for the expression of
inflammatory factors and plays a key role in the pathogenesis of
EAE and other inflammatory diseases (40, 41). Thus, we assessed
the activation status of p38a MAPK in DCs in TCF4DDC and WT
mice during the induction phase and disease phase of EAE. DCs
isolated from the DLNs and spleen of TCF4DDC mice showed a
marked increase in the phosphorylated (active) form of p38a
MAPK compared with DCs isolated from WT control mice both
during the induction and effector phase of EAE (Fig. 5A, 5B). Like-
wise, the activity of p38a MAPK was markedly elevated in CNS-

FIGURE 4. TCF4 activation in DCs limits Th1 and Th17 cell differentiation. (A
C) CD11c1 DCs were isolated from DLNs and spleen on day 5 from
WTFL/FL and TCF4DDC mice immunized with the MOG35
55 peptide plus CFA (MOG) or control mice without immunization (none) and cultured ex vivo with
naive CD41CD62L1 T cells from 2D2 mice. After 5 d, cultured 2D2 cells were stimulated with anti-CD3/CD28 Ab for 48 or 6 h (in the presence of brefeldin
A and monensin). (A) Representative FACS plots and (B) frequencies for IFN-g
and IL-17A
producing CD41 2D2 T cells are shown. (C) Cytokine concentra-
tions in the culture supernatants obtained from differentiated 2D2 cells. (D and E) Naive CD41CD62L1 T cells from 2D2 mice were adoptively transferred into
WTFL/FL and TCF4DDC mice followed by immunization with the MOG35
55 peptide plus CFA (MOG) or control mice without immunization (none). Five days
after the challenge, DLNs cells were stimulated in vitro for 6 h with CD3/CD28 in the presence of brefeldin A and monensin followed by ICS. (D) Total number
of CD41 2D2 cells and (E) cumulative frequencies for IFN-g
and IL-17A
producing CD41 2D2 cells are shown. (F) Cytokine concentrations in supernatants
were obtained after the culture of in vivo differentiated 2D2 cells from DLNs and spleen of WTFL/FL and TCF4DDC mice and stimulated ex vivo with the
MOG35
55 peptide for 48 h. (G) CD11c1 DCs from the DLNs and spleen of WTFL/FL and TCF4DDC mice on day 5 post
EAE induction were analyzed for acti-
vation and maturation. The representative histogram shows the expression of MHC II, CD40, CD80, and CD86 expression on CD11c DCs. Data are representa-
tive of two experiments (n = 4
5 mice per experiment). Error bars show mean values ± SEM. ***p < 0.0001.
The Journal of Immunology 1433

Downloaded from http://www.jimmunol.org/ at Red de Bibliotecas del CSIC on November 14, 2021
FIGURE 5. TCF4 regulates the expression of proinflammatory cytokines and Th1/Th17 cell differentiation through p38 MAPK activity. (A) Representative
histogram of phosphorylated p38 MAPK (p-p38 MAPK) in CD11c1 DCs isolated from the spleen (days 5 and 14 pi) and CNS (day 14 pi) of WTFL/FL
and TCF4DDC EAE mice. (B) Immunoblot analysis of expression of native (p38) and phosphorylated p38 MAPK (phospho-p38 MAPK) in CD11c1 DCs iso-
lated from the spleen of WTFL/FL and TCF4DDC EAE mice (day 5 pi). (C
E) CD11c1 DCs were isolated from the spleen on day 5 from TCF4DDC mice
immunized with the MOG35
55 peptide plus CFA (MOG) and treated daily with either SB203580 or vehicle. (C) Cytokine concentrations in supernatants
were obtained after the culture of CD11c1 DCs isolated from the spleen from TCF4DDC. (D and E) DCs were isolated from TCF4DDC mice and cultured with
naive CD41CD62L1 T cells from 2D2 mice. After 5 d, cultured 2D2 cells were stimulated in vitro for 6 h with CD3/CD28 in the presence or absence of bre-
feldin A and monensin. (D) Frequencies for IFN-g
and IL-17A
producing CD41 2D2 T cells are shown. (E) Cytokine concentrations in the culture superna-
tants obtained from differentiated 2D2 cells stimulated with anti-CD3/CD28 Ab for 48 h. Data are representative of two experiments (n = 3 per experiment).
Error bars show mean values ± SEM. ***p < 0.0001.

infiltrating DCs isolated from TCF4DDC mice as compared with
CNS DCs isolated from WT control mice (Fig. 5A).
Based on these observations, we hypothesized that in EAE, TCF4
regulates the expression of proinflammatory cytokines by controlling
the p38a MAPK activation in DCs. Thus, we examined the effect
of blocking the p38a pathway in TCF4-deficient DCs on the
expression of inflammatory and anti-inflammatory cytokines. To test
this, we immunized TCF4DDC mice with the MOG33
55 peptide
plus CFA (MOG) and treated with either SB203580 or vehicle . As
shown in Fig. 5C, DCs isolated from TCF4DDC mice with
SB203580 produced markedly lower levels of IL-6, TNF-a, IL-23,
and IL-12p40 compared with DCs isolated from mice treated with
vehicle. However, p38 inhibitor treatment had no effect on IL-1b
and IL-10 production by TCF4-deficient DCs (Fig. 5C). These
results indicate that TCF4 limits the expression of proinflammatory
cytokines by regulating the activity of p38a MAPK.
Next, we asked whether blocking p38a MAPK activation in TCF4-
deficient DCs affects Th1/Th17 cell differentiation. For this, we per-
formed ex vivo 2D2 T cell differentiation assay by coculturing naive
2D2 T cells with DCs isolated from TCF4DDC mice immunized with the
MOG35
55 peptide plus CFA (MOG) and treated with either SB203580
or vehicle. T cells cultured with TCF4-deficient DCs obtained from
mice treated with vehicle contained higher frequencies of Th1/Th17
cells compared with T cells cultured with TCF4-deficient DCs obtained
from mice treated with SB203580 (Fig. 5D). Further, analysis of the cul-
ture supernatant showed that SB203580 treatment of TCF4-deficient
DCs markedly reduced IFN-g and IL-17A produced by T cells (Fig.
5E). Based on these results, we conclude that TCF4 activation in DCs
limits Th1/Th17 differentiation by regulating the activity of p38a.
Pharmacological blocking of p38 MAPK activation limits EAE
pathology in TCF4DDC mice
Our data have thus far indicated that p38 MAPK is downstream of
the TCF4 pathway in DCs. So, we explored the possible implication
of pharmacologically blocking the p38 MAPK pathway in TCF4DDC
mice during ongoing neuroinflammation. Thus, we assessed whether
blocking this pathway could ameliorate EAE pathology in TCF4DDC
mice. To study this, we immunized TCF4DDC mice with the
MOG33
55 peptide plus CFA (MOG) and treated with either
SB203580 or vehicle i.p. every 2 d from day 3 pi. The treatment of
TCF4DDC mice with p38 inhibitor markedly delayed EAE onset and
reduced EAE severity compared with vehicle-treated control mice
(Fig. 6A). Consistent with disease severity score, histopathological
analysis of the CNS showed reduced inflammation as marked by
reduced infiltration of leukocytes and diminished demyelination in
the p38 inhibitor
treated group compared with mice in the vehicle-
treated group (Fig. 6B, 6C). Besides, flow cytometric analysis con-
firmed a marked decrease in the total number of leukocytes as well
as decreased number of DCs, MFs, monocytes, neutrophils, and
CD41 T cells in the CNS of TCF4DDC mice treated with SB203580
compared with the vehicle-treated group (Fig. 6D). Further charac-
terization of CNS-infiltrating CD41 T cells showed a significant
reduction in the frequencies of the MOG38
49 tetramer
specific
CD41 T cells, Th1, and Th17 cells upon SB203580 treatment
(Fig. 6E
G) . Consistent with these observations, leukocytes isolated
from the CNS of TCF4DDC mice treated with the p38 inhibitor pro-
duced markedly lower levels of IFN-g and IL-17A compared with
the leukocytes isolated from the CNS of vehicle-treated mice in
response to ex vivo stimulation with the MOG33
55 peptide
(Fig. 6H). Collectively, these results indicate that TCF4 activation in
DCs controls CNS inflammation and EAE disease severity by regu-
lating the p38 MAPK pathway.
Discussion
The current study defines an essential role for the TCF4 transcrip-
tion factor in DCs in controlling CNS inflammation and EAE dis-
ease severity. Accordingly, DC-specific deletion of TCF4 led to an
1434 TCF4 DEFICIENCY IN DCs AGGRAVATES THE PATHOGENESIS OF EAE

Downloaded from http://www.jimmunol.org/ at Red de Bibliotecas del CSIC on November 14, 2021
FIGURE 6. Pharmacological inhibition of p38 MAPK activation diminished EAE pathology in TCF4DDC mice. The progression of EAE disease course in
TCF4DDC mice treated with vehicle alone or SB203580. (A) Mean clinical EAE score in vehicle- (open circle) and SB203580- (closed circle) treated mice.
(B and C) H&E and luxol fast blue (LFB) staining of spinal cords of TCF4DDC mice to reveal the degree of demyelination and inflammation on day 16 pi.
Scale bars (blue), 200 mm. (D) Total number of leukocytes (CD451), DCs (CD45hiMHC IIhiCD11c1), MFs (CD45hiMHC IIhiCD11cCD11b1CD641),
monocytes (CD45hiMHC IICD11cCD11b1Ly6ChiLy6GLow), neutrophils (CD45hiMHC IICD11cCD11b1Ly6ClowLy6Ghi), and CD41 T cells in the
CNS of TCF4DDC EAE mice (day 16 pi) analyzed by flow cytometry. (E) Frequencies of the MOG38
49 tetramer
specific CD41 T cells isolated from CNS
of TCF4DDC EAE mice treated with vehicle alone or SB203580 (day 16 pi). (F) Representative FACS plots and (G) frequencies of IFN-g1 and IL-17A1
CD41 T cells isolated from the CNS of TCF4DDC EAE mice on day 16 pi. (H) Cytokine concentrations in supernatants were obtained after culture of CNS-
infiltrating leukocytes stimulated ex vivo with the MOG35
55 peptide for 72 h. Data are representative of two experiments (n = 4
5 mice per experiment).
Error bars show mean values ± SEM. **p < 0.001, ***p < 0.0001.

increased expression of IL-6, TNF-a, IL-1b, IL-23, and IL-12p40
with diminished production of IL-10 during the induction and effec-
tor phase of EAE. Consequently, the absence of TCF4 signaling in
DCs suppressed Treg responses yet promoted Th1 and Th17 cell
differentiation. Accordingly, mice lacking TCF4 in DCs exhibited a
very severe EAE pathology characterized by intense demyelination
in CNS with increased effector CD41 T cell infiltration in the CNS.
Mechanistically, TCF4 controls the expression of proinflammatory
cytokines and neuroinflammation by regulating p38 activity in DCs.
Finally, pharmacological blocking of p38 MAPK activation in
TCF4-deficient DCs markedly reduced the expression of proinflam-
matory cytokines and delayed EAE onset with diminished neuropa-
thology in TCF4DDC mice. These data indicate an important role for
TCF4 in DCs in controlling CNS inflammation during EAE and
represent a logical target to control chronic inflammatory conditions
such as MS.
Aberrant activation of the Wnt signaling pathway occurs in sev-
eral inflammatory diseases, including neurodegenerative and neuro-
inflammatory diseases (19, 20, 42). Many studies have documented
that Wnt ligands are highly expressed in several chronic inflamma-
tory diseases and autoimmune diseases (22
24). The coreceptors,
low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6),
and coactivator b-catenin are critical for mediating canonical Wnt
signaling in APCs (21, 26, 42). In murine models of intestinal
inflammation and colitis-associated colon cancer, recent studies have
shown that the canonical Wnt pathway in DCs induce T regulatory
response and suppress intestinal inflammation and inflammation-
associated colon cancer. Our previous studies have shown that in
conditional knockout mice that specifically lack LRP5/6 (LRP5/
6DDC) or b-catenin (b-catDDC) in DCs are more susceptible to
inflammatory diseases (28, 43). These mice develop more severe
EAE (34). b-Catenin interacts with several transcription factors,
including TCFs, PPARg (44, 45), Foxo (46
48), VDR (46, 49),
IRF3 (50), and IRF8 (51). Yet, downstream mediators of LRP5/
6
b-catenin signaling in DCs are not known. The present study
shows that TCF4 is one of the key downstream mediators of the LRP5/
6
b-catenin signaling in DCs. As seen in LRP5/6DDC and b-catDDC
mice (34), DC-specific deletion of TCF4 in mice increased Th1/Th17
responses and exacerbated EAE pathology. Furthermore, b-catenin tar-
get genes such as Axin1, Axin 2, and WISP1 are markedly decreased
in DCs isolated from TCF4DDC mice during the induction and effector
phase of the disease (Supplemental Fig. 1H). Many of the LRP5/6- and
b-catenin
controlled cytokines, such as IL-12, IL-6, IL-23, and
TNF-a, are elevated in TCF4DDC mice. These proinflammatory cyto-
kines are also elevated in MS (52
54) and are essential to induce EAE
(55, 56). These observations strongly suggest that TCF4 is a key down-
stream mediator of the LRP5/6
b-catenin signaling in DCs. In mice
and humans, multiple subsets of DCs exist in both lymphoid (CD8a1
DCs, pDCs, and CD11b1 DCs) and nonlymphoid tissues (CD1031
DCs, pDCs, and CD11b1 DCs)2, 3. Depletion of DCs in mice resulted
in stronger inflammatory responses with exacerbated EAE7, 8. Further-
more, depletion of specific DC subsets in lymphoid or nonlymphoid
tissues during the acute or relapsing phase of EAE results in stronger
inflammatory responses and exacerbates the disease9-12. The present
study shows that the TCF4 pathway is active in all three major DC sub-
sets (CD8a1 DCs, CD11b1 DCs, and pDCs). Deletion of TCF4 does
not dramatically alter the frequencies and number of CD8a1 DCs,
CD11b1 DCs, and pDCs. However, further studies are warranted to
understand the role of TCF4 in these subsets in regulating CNS inflam-
mation. Although not analyzed in the current study, it is possible that
TCF4 might modulate EAE by regulating DC migration.
A fine balance between Tregs versus pathological Th1/Th17 cells
underlies disease progression in many inflammatory diseases,
including EAE. Past studies have shown that LRP5/6
b-catenin sig-
naling in APCs limits chronic inflammation through the induction of
Tregs while limiting the differentiation of pathological Th1/Th17
cells (28, 34, 43). Accumulating evidence suggests that Th1/Th17
cells play an important role in the pathogenesis of EAE and MS
(57
59). In contrast, Tregs such as Foxp31 Tregs and IL-
10
producing Tr1 cells play a pivotal role in neuroinflammation
The Journal of Immunology
and EAE disease severity (59). Our studies show that TCF4-defi-
cient DCs are potent in inducing MOG-specific Th1/Th17 cell dif-
ferentiation, at least in part because of increased production of IL-6,
IL-23, and IL-12. Indeed, conditional deletion of TCF4 in DCs
exacerbated EAE severity because of a marked increase in Th1/
Th17 cells and a decrease in IL-10
producing Tr1 cells in the CNS.
A recent study on intestinal inflammation has shown that the b-cate-
nin directly regulates IL-10 production through TCF4 in intestinal
DCs and MFs (28). Furthermore, b-catenin/TCF4 signaling regu-
lates the expression of inflammatory factors through the autocrine
effects of IL-10 on colonic APCs (28). Although not analyzed in
the current study, it is possible that TCF4-mediated signals in DCs
might modulate EAE by inducing Tr1 cell differentiation and regu-
lating the suppressive function of Foxp31 Tregs.
Innate immune receptors, including TLR-mediated signaling in
DCs, play a critical role in the initiation of EAE. These receptors on
DCs sense various danger signals and induce the activation of sev-
eral signaling networks with the secretion of cytokines that drive the
differentiation of naive CD41 and CD81 T cells to pathological
effector T cells or Tregs (39). Activation of most TLRs on DCs
induces secretion of key proinflammatory cytokines that promote
Th1 or Th17 cell differentiation (5, 39). M. tuberculosis, an adjuvant
used for the induction of EAE, activates several TLRs on APCs.
p38 MAPK is one of the key downstream mediators of TLR signal-
ing and is critical for the production of proinflammatory cytokines
upon TLR activation (60). The p38a MAPK signaling pathway
plays a key role in the pathogenesis of EAE and other inflammatory
diseases (40, 61). Moreover, p38 expression and activation in DCs
are critical for the expression of the proinflammatory factors that
drive Th1/Th17 cell differentiation (62). Indeed, DC-specific dele-
tion of p38a in mice increased the expression of the proinflamma-
tory cytokines and Th1/Th17 responses during EAE (62). More
importantly, pharmacological inhibitors of p38 markedly reduced
Th1/Th17 cell differentiation and ameliorated EAE in mice (40).
Our data indicate that TCF4 in DCs limits the expression of proin-
flammatory cytokines and Th1/Th17 differentiation by regulating
the activity of p38a. Accordingly, loss of TCF4 in DCs led to
heightened activation of p38 MAPK and increased levels of proin-
flammatory cytokines IL-6, IL-23, IL-1b, TNFa, and IL-12. Consis-
tent with these observations, pharmacological blocking of p38
MAPK activation in TCF4-deficient DCs markedly reduced the
expression of proinflammatory cytokines and decreased its ability to
drive Th1/Th17 cell differentiation. Besides, the treatment of
TCF4DDC mice with p38 inhibitor markedly delayed EAE onset and
reduced EAE severity. Although not analyzed in the current study,
further studies are warranted to understand whether SB203580 treat-
ment affects the Ag-presenting capacity or the migratory properties
of DCs in TCF4DDC mice. Although our data indicate an important
role for TCF4 in modulating p38 activity in DCs during EAE, it is
not clear whether TCF4 regulates p38 activity directly or indirectly.
A recent study has shown that the b-catenin/TCF4/IL-10 signaling
axis in intestinal APCs regulates IL-6, IL-12, and IL-23 through the
suppression of p38 (28). It is possible that TCF4 could regulate p38
activation indirectly through IL-10. In the current study, we also
show that TCF4 is critical for IL-10 production by DCs during
EAE. However, p38 inhibition in TCF4-deficient DCs failed to
restore the IL-10 production, suggesting that TCF4 regulates IL-10
production independent of p38.
In summary, our study reveals an important role for TCF4 in DCs
in exerting a protective effect on CNS inflammation during EAE by
regulating the expression of key proinflammatory and anti-inflamma-
tory mediators. Furthermore, our study indicates a regulatory role for
the TCF4 during ongoing neuroinflammation, in which activation of
this transcription factor in DCs limits the uncontrolled differentiation
1435
of naive CD41 T cells to Th1 and Th17 cells. Thus, manipulation of
the TCF4 pathway in DCs could provide novel opportunities for regu-
lating chronic inflammation and represents a potential therapeutic
approach to control autoimmune neuroinflammation.
Acknowledgments
We thank Dr. Melinda L Angus-Hill (University of Utah) for kindly provid-
ing TCF4 floxed mice. We also thank Jeanene Pihkala for technical help
with FACS sorting and analysis and Janice Randall for expert technical
assistance with the mice used in this study.
Disclosures
The authors have no financial conflicts of interest.
Downloaded from http://www.jimmunol.org/ at Red de Bibliotecas del CSIC on November 14, 2021
References
1. Terry, R. L., I. Ifergan, and S. D. Miller. 2016. Experimental autoimmune
encephalomyelitis in mice. Methods Mol. Biol. 1304: 145
160.
2. Ganguly, D., S. Haak, V. Sisirak, and B. Reizis. 2013. The role of dendritic cells
in autoimmunity. Nat. Rev. Immunol. 13: 566
577.
3. Steinman, R. M., D. Hawiger, and M. C. Nussenzweig. 2003. Tolerogenic den-
dritic cells. Annu. Rev. Immunol. 21: 685
711.
4. Manicassamy, S., and B. Pulendran. 2011. Dendritic cell control of tolerogenic
responses. Immunol. Rev. 241: 206
227.
5. Pulendran, B., H. Tang, and S. Manicassamy. 2010. Programming dendritic cells
to induce T(H)2 and tolerogenic responses. Nat. Immunol. 11: 647
655.
6. Ohnmacht, C., A. Pullner, S. B. King, I. Drexler, S. Meier, T. Brocker, and D.
Voehringer. 2009. Constitutive ablation of dendritic cells breaks self-tolerance of
CD4 T cells and results in spontaneous fatal autoimmunity. J. Exp. Med. 206:
549
559.
7. Yogev, N., F. Frommer, D. Lukas, K. Kautz-Neu, K. Karram, D. Ielo, E. von Ste-
but, H. C. Probst, M. van den Broek, D. Riethmacher, et al. 2012. Dendritic cells
ameliorate autoimmunity in the CNS by controlling the homeostasis of PD-1
receptor(1) regulatory T cells. Immunity. 37: 264
275.
8. Deshpande, P., I. L. King, and B. M. Segal. 2007. Cutting edge: CNS CD11c1
cells from mice with encephalomyelitis polarize Th17 cells and support
CD251CD41 T cell-mediated immunosuppression, suggesting dual roles in the
disease process. J. Immunol. 178: 6695
6699.
9. Li, H., G. X. Zhang, Y. Chen, H. Xu, D. C. Fitzgerald, Z. Zhao, and A. Rostami.
2008. CD11c1CD11b1 dendritic cells play an important role in intravenous tol-
erance and the suppression of experimental autoimmune encephalomyelitis.
J. Immunol. 181: 2483
2493.
10. Bailey-Bucktrout, S. L., S. C. Caulkins, G. Goings, J. A. Fischer, A. Dzionek,
and S. D. Miller. 2008. Cutting edge: central nervous system plasmacytoid den-
dritic cells regulate the severity of relapsing experimental autoimmune encepha-
lomyelitis. J. Immunol. 180: 6457
6461.
11. Wang, L., Z. Li, B. Ciric, F. Safavi, G. X. Zhang, and A. Rostami. 2016. Selec-
tive depletion of CD11c1 CD11b1 dendritic cells partially abrogates tolerogenic
effects of intravenous MOG in murine EAE. Eur. J. Immunol. 46: 2454
2466.
12. King, I. L., M. A. Kroenke, and B. M. Segal. 12010. GM-CSF-dependent,
CD1031 dermal dendritic cells play a critical role in Th effector cell differentia-
tion after subcutaneous immunization. J. Exp. Med. 207: 953
961.
13. Isaksson, M., B. Ardesj€o, L. R€onnblom, O. K€ampe, H. Lassmann, M. L. Eloranta,
and A. Lobell. 2009. Plasmacytoid DC promote priming of autoimmune Th17
cells and EAE. Eur. J. Immunol. 39: 2925
2935.
14. Duraes, F. V., C. Lippens, K. Steinbach, J. Dubrot, D. Brighouse, N. Bendriss-
Vermare, S. Issazadeh-Navikas, D. Merkler, and S. Hugues. 2016. pDC therapy
induces recovery from EAE by recruiting endogenous pDC to sites of CNS
inflammation. J. Autoimmun. 67: 8
18.
15. Bailey, S. L., P. A. Carpentier, E. J. McMahon, W. S. Begolka, and S. D. Miller.
2006. Innate and adaptive immune responses of the central nervous system. Crit.
Rev. Immunol. 26: 149
188.
16. Bettelli, E., M. Oukka, and V. K. Kuchroo. 2007. T(H)-17 cells in the circle of
immunity and autoimmunity. Nat. Immunol. 8: 345
350.
17. Hirota, K., J. H. Duarte, M. Veldhoen, E. Hornsby, Y. Li, D. J. Cua, H. Ahlfors,
C. Wilhelm, M. Tolaini, U. Menzel, et al. 2011. Fate mapping of IL-17-producing
T cells in inflammatory responses. Nat. Immunol. 12: 255
263.
18. Bailey, S. L., B. Schreiner, E. J. McMahon, and S. D. Miller. 2007. CNS myeloid
DCs presenting endogenous myelin peptides ‘preferentially’ polarize CD41
T(H)-17 cells in relapsing EAE. Nat. Immunol. 8: 172
180.
19. Clevers, H., and R. Nusse. 2012. Wnt/b-catenin signaling and disease. Cell. 149:
1192
1205.
20. Joiner, D. M., J. Ke, Z. Zhong, H. E. Xu, and B. O. Williams. 2013. LRP5 and
LRP6 in development and disease. Trends Endocrinol. Metab. 24: 31
39.
21. Suryawanshi, A., R. K. Tadagavadi, D. Swafford, and S. Manicassamy. 2016.
Modulation of inflammatory responses by Wnt/b-catenin signaling in dendritic
cells: a novel immunotherapy target for autoimmunity and cancer. Front. Immu-
nol. 7: 460.
1436 TCF4 DEFICIENCY IN DCs AGGRAVATES THE PATHOGENESIS OF EAE
22. Rabelo, F. S., L. M. da Mota, R. A. Lima, F. A. Lima, G. B. Barra, J. F. de Car-
valho, and A. A. Amato. 2010. The Wnt signaling pathway and rheumatoid
arthritis. Autoimmun. Rev. 9: 207
210.
23. Hughes, K. R., F. Sablitzky, and Y. R. Mahida. 2011. Expression profiling of
Wnt family of genes in normal and inflammatory bowel disease primary human
intestinal myofibroblasts and normal human colonic crypt epithelial cells.
Inflamm. Bowel Dis. 17: 213
220.
24. Gudjonsson, J. E., A. Johnston, S. W. Stoll, M. B. Riblett, X. Xing, J. J. Kochko-
dan, J. Ding, R. P. Nair, A. Aphale, J. J. Voorhees, and J. T. Elder. 2010. Evi-
dence for altered Wnt signaling in psoriatic skin. J. Invest. Dermatol. 130:
1849
1859.
25. Staal, F. J., and H. C. Clevers. 2005. WNT signalling and haematopoiesis: a
WNT-WNT situation. Nat. Rev. Immunol. 5: 21
30.
26. Suryawanshi, A., M. S. Hussein, P. D. Prasad, and S. Manicassamy. 2020. Wnt
signaling cascade in dendritic cells and regulation of anti-tumor immunity. Front.
Immunol. 11: 122.
27. Hong, Y., I. Manoharan, A. Suryawanshi, T. Majumdar, M. L. Angus-Hill, P. A.
Koni, B. Manicassamy, A. L. Mellor, D. H. Munn, and S. Manicassamy. 2015.
b-catenin promotes regulatory T-cell responses in tumors by inducing vitamin A
metabolism in dendritic cells. Cancer Res. 75: 656
665.
28. Swafford, D., A. Shanmugam, P. Ranganathan, I. Manoharan, M. S. Hussein, N.
Patel, H. Sifuentes, P. A. Koni, P. D. Prasad, M. Thangaraju, and S. Manicass-
amy. 2020. The Wnt-b-catenin-IL-10 signaling axis in intestinal APCs protects
mice from colitis-associated colon cancer in response to gut microbiota. J. Immu-
nol. 205: 2265
2275.
29. Caton, M. L., M. R. Smith-Raska, and B. Reizis. 2007. Notch-RBP-J signaling
controls the homeostasis of CD8- dendritic cells in the spleen. J. Exp. Med. 204:
1653
1664.
30. Ferrer-Vaquer, A., A. Piliszek, G. Tian, R. J. Aho, D. Dufort, and A. K. Hadjan-
tonakis. 2010. A sensitive and bright single-cell resolution live imaging reporter
of Wnt/ß-catenin signaling in the mouse. BMC Dev. Biol. 10: 121.
31. Bettelli, E., M. Pagany, H. L. Weiner, C. Linington, R. A. Sobel, and V. K. Kuch-
roo. 2003. Myelin oligodendrocyte glycoprotein-specific T cell receptor trans-
genic mice develop spontaneous autoimmune optic neuritis. J. Exp. Med. 197:
1073
1081.
32. Angus-Hill, M. L., K. M. Elbert, J. Hidalgo, and M. R. Capecchi. 2011. T-cell
factor 4 functions as a tumor suppressor whose disruption modulates colon cell
proliferation and tumorigenesis. Proc. Natl. Acad. Sci. USA. 108: 4914
4919.
33. Manicassamy, S., R. Ravindran, J. Deng, H. Oluoch, T. L. Denning, S. P. Kasturi,
K. M. Rosenthal, B. D. Evavold, and B. Pulendran. 2009. Toll-like receptor 2-
dependent induction of vitamin A-metabolizing enzymes in dendritic cells pro-
motes T regulatory responses and inhibits autoimmunity. Nat. Med. 15: 401
409.
34. Suryawanshi, A., I. Manoharan, Y. Hong, D. Swafford, T. Majumdar, M. M.
Taketo, B. Manicassamy, P. A. Koni, M. Thangaraju, Z. Sun, et al. 2015. Canoni-
cal wnt signaling in dendritic cells regulates Th1/Th17 responses and suppresses
autoimmune neuroinflammation. J. Immunol. 194: 3295
3304.
35. Manoharan, I., A. Suryawanshi, Y. Hong, P. Ranganathan, A. Shanmugam, S.
Ahmad, D. Swafford, B. Manicassamy, G. Ramesh, P. A. Koni, et al. 2016.
Homeostatic PPARa signaling limits inflammatory responses to commensal
microbiota in the intestine. J. Immunol. 196: 4739
4749.
36. Sabatino, J. J., Jr., J. Huang, C. Zhu, and B. D. Evavold. 2011. High prevalence
of low affinity peptide-MHC II tetramer-negative effectors during polyclonal
CD41 T cell responses. J. Exp. Med. 208: 81
90.
37. Manoharan, I., Y. Hong, A. Suryawanshi, M. L. Angus-Hill, Z. Sun, A. L. Mel-
lor, D. H. Munn, and S. Manicassamy. 2014. TLR2-dependent activation of
b-catenin pathway in dendritic cells induces regulatory responses and attenuates
autoimmune inflammation. J. Immunol. 193: 4203
4213.
38. Ji, Q., and J. Goverman. 2007. Experimental autoimmune encephalomyelitis
mediated by CD81 T cells. Ann. N. Y. Acad. Sci. 1103: 157
166.
39. Manicassamy, S., and B. Pulendran. 2009. Modulation of adaptive immunity
with Toll-like receptors. Semin. Immunol. 21: 185
193.
40. Krementsov, D. N., T. M. Thornton, C. Teuscher, and M. Rincon. 2013. The
emerging role of p38 mitogen-activated protein kinase in multiple sclerosis and
its models. Mol. Cell. Biol. 33: 3728
3734.
41. Kumar, S., J. Boehm, and J. C. Lee. 2003. p38 MAP kinases: key signalling mol-
ecules as therapeutic targets for inflammatory diseases. Nat. Rev. Drug Discov. 2:
717
726.
42. Swafford, D., and S. Manicassamy. 2015. Wnt signaling in dendritic cells: its
role in regulation of immunity and tolerance. Discov. Med. 19: 303
310.
43. Swafford, D., A. Shanmugam, P. Ranganathan, M. S. Hussein, P. A. Koni, P. D.
Prasad, M. Thangaraju, and S. Manicassamy. 2018. Canonical Wnt signaling in
CD11c1 APCs regulates microbiota-induced inflammation and immune cell
homeostasis in the colon. J. Immunol. 200: 3259
3268.
44. Alastalo, T. P., M. Li, V. J. Perez, D. Pham, H. Sawada, J. K. Wang, M. Kosken-
vuo, L. Wang, B. A. Freeman, H. Y. Chang, and M. Rabinovitch. 2011. Disrup-
tion of PPARg/b-catenin-mediated regulation of apelin impairs BMP-induced
mouse and human pulmonary arterial EC survival. J. Clin. Invest. 121:
3735
3746.
45. Liu, J., H. Wang, Y. Zuo, and S. R. Farmer. 2006. Functional interaction between
peroxisome proliferator-activated receptor gamma and beta-catenin. Mol. Cell.
Biol. 26: 5827
5837.
46. Hoogeboom, D., M. A. Essers, P. E. Polderman, E. Voets, L. M. Smits, and
B. M. Burgering. 2008. Interaction of FOXO with beta-catenin inhibits beta-cate-
nin/T cell factor activity. J. Biol. Chem. 283: 9224
9230.
47. Yan, Y., and M. R. Lackner. 2012. FOXO3a and b-catenin co-localization: dou-
Downloaded from http://www.jimmunol.org/ at Red de Bibliotecas del CSIC on November 14, 2021
ble trouble in colon cancer? Nat. Med. 18: 854
856.
48. Kamo, N., B. Ke, R. W. Busuttil, and J. W. Kupiec-Weglinski. 2013. PTEN-
mediated Akt/b-catenin/Foxo1 signaling regulates innate immune responses in
mouse liver ischemia/reperfusion injury. Hepatology. 57: 289
298.
49. Shah, S., M. N. Islam, S. Dakshanamurthy, I. Rizvi, M. Rao, R. Herrell, G.
Zinser, M. Valrance, A. Aranda, D. Moras, et al. 2006. The molecular basis of
vitamin D receptor and beta-catenin crossregulation. Mol. Cell 21: 799
809.
50. Yang, P., H. An, X. Liu, M. Wen, Y. Zheng, Y. Rui, and X. Cao. 2010. The cyto-
solic nucleic acid sensor LRRFIP1 mediates the production of type I interferon
via a beta-catenin-dependent pathway. Nat. Immunol. 11: 487
494.
51. Scheller, M., J. Sch€onheit, K. Zimmermann, U. Leser, F. Rosenbauer, and A.
Leutz. 2013. Cross talk between Wnt/b-catenin and Irf8 in leukemia progression
and drug resistance. J. Exp. Med. 210: 2239
2256.
52. Martino, G., R. Furlan, E. Brambilla, A. Bergami, F. Ruffini, M. Gironi, P. L.
Poliani, L. M. Grimaldi, and G. Comi. 2000. Cytokines and immunity in multiple
sclerosis: the dual signal hypothesis. J. Neuroimmunol. 109: 3
9.
53. Bar-Or, A., E. M. Oliveira, D. E. Anderson, and D. A. Hafler. 1999. Molecular
pathogenesis of multiple sclerosis. J. Neuroimmunol. 100: 252
259.
54. Uccelli, A., E. Pedemonte, E. Narciso, and G. Mancardi. 2003. Biological
markers of the inflammatory phase of multiple sclerosis. Neurol. Sci. 24(0, Suppl
5): S271
S274.
55. Samoilova, E. B., J. L. Horton, B. Hilliard, T. S. Liu, and Y. Chen. 1998. IL-6-
deficient mice are resistant to experimental autoimmune encephalomyelitis: roles
of IL-6 in the activation and differentiation of autoreactive T cells. J. Immunol.
161: 6480
6486.
56. Schiffenbauer, J., W. J. Streit, E. Butfiloski, M. LaBow, C. Edwards III, and
L. L. Moldawer. 2000. The induction of EAE is only partially dependent on TNF
receptor signaling but requires the IL-1 type I receptor. Clin. Immunol. 95:
117
123.
57. Segal, B. M. 2010. Th17 cells in autoimmune demyelinating disease. Semin.
Immunopathol. 32: 71
77.
58. Cua, D. J., J. Sherlock, Y. Chen, C. A. Murphy, B. Joyce, B. Seymour, L. Lucian,
W. To, S. Kwan, T. Churakova, et al. 2003. Interleukin-23 rather than interleu-
kin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature.
421: 744
748.
59. Jones, A., and D. Hawiger. 2017. Peripherally induced regulatory T cells:
recruited protectors of the central nervous system against autoimmune neuroin-
flammation. Front. Immunol. 8: 532.
60. Akira, S., and K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol.
4: 499
511.
61. Ashwell, J. D. 2006. The many paths to p38 mitogen-activated protein kinase
activation in the immune system. Nat. Rev. Immunol. 6: 532
540.
62. Huang, G., Y. Wang, P. Vogel, T. D. Kanneganti, K. Otsu, and H. Chi. 2012. Sig-
naling via the kinase p38a programs dendritic cells to drive TH17 differentiation
and autoimmune inflammation. Nat. Immunol. 13: 152
161.