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The Myeloid Differentiation Factor 88
(MyD88) Is Required for CD4 + T Cell
Effector Function in a Murine Model of
Inflammatory Bowel Disease
Masayuki Fukata, Keith Breglio, Anli Chen, Arunan S.
Vamadevan, Tyralee Goo, David Hsu, Daisy Conduah,
Ruliang Xu and Maria T. Abreu
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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 © 2008 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2008; 180:1886-1894; ;
doi: 10.4049/jimmunol.180.3.1886
http://www.jimmunol.org/content/180/3/1886
The Journal of Immunology
The Myeloid Differentiation Factor 88 (MyD88) Is Required
for CD4ⴙ T Cell Effector Function in a Murine Model of
Inflammatory Bowel Disease1
Masayuki Fukata,* Keith Breglio,* Anli Chen,* Arunan S. Vamadevan,* Tyralee Goo,*
David Hsu,* Daisy Conduah,* Ruliang Xu,† and Maria T. Abreu2*
I
nflammatory bowel disease (IBD)3 is characterized by
chronic intestinal inflammation in the absence of a recognized bacterial pathogen. Advances in human genome research have revealed that several candidate genes in IBD are
linked to innate immunity (1– 6). In contrast, abnormal T cell responses to luminal commensal bacteria are crucial to the pathogenesis of IBD (7–9). The link between genetic defects in innate
immunity and sustained activation of T cells remains unclear.
Recognition of commensal bacteria largely depends on TLRs in the
intestinal mucosa (10). TLRs are pattern recognition receptors expressed by immune and nonimmune cells in the intestinal mucosa that
signal in response to pathogen-associated molecular patterns
(PAMPs) expressed by microbes (11). The main purpose of TLR
signaling is to provide a rapid response against pathogens to protect
the host. Eleven TLRs have been identified and individual or pairs of
*Inflammatory Bowel Disease Center, Division of Gastroenterology, Department of
Medicine, and †Department of Pathology, Mount Sinai School of Medicine, New
York, NY 10029
Received for publication August 7, 2007. Accepted for publication November
27, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AI052266 (to
M.T.A.) and DK069594 (to M.T.A.), and a Uehara Memorial Foundation Research
Fellowship (to M.F.).
2
Address correspondence and reprint requests to Dr. Maria T. Abreu, Mount Sinai
School of Medicine, 1 Gustave Levy Place, Box 1069, New York, NY 10029. E-mail
address: [email protected]
3
Abbreviations used in this paper: IBD, inflammatory bowel disease; PAMP, pathogen-associated molecular pattern; Treg, regulatory T cell; DC, dendritic cell; LP,
lamina propria; MLN, mesenteric lymph node.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
www.jimmunol.org
TLRs recognize distinct PAMPs. Most TLRs, with the exception of
TLR3, signal via the adapter molecule MyD88 to the IL-1 receptor
associated kinase and TNF receptor-associated factor 6 to TGF-␤activated kinase 1, leading to the nuclear translocation of NF-␬B and
activation of mitogen-activated protein kinase (12). Previous studies
have demonstrated that MyD88-deficient mice develop profound defects in Ag-specific T cell responses, suggesting that TLR signaling
plays a role in the development of adaptive immune responses (13,
14), but the effect of MyD88 was thought to be mediated by APCs.
Mucosal immune homeostasis requires a balanced interplay between effector and regulatory T cells (Tregs). Naive T cells differentiate into effector cells that are divided into three distinctive
types, Th1, Th2, and Th17 cells, depending on the type of cytokines secreted. The fate of T cell differentiation into Th1, Th2, or
Th17 type T cells or Tregs is largely regulated by the interaction
between naive T cells and dendritic cells (DCs) (15). The recognition of PAMPs by TLRs on DCs promotes stimulation of Ag
presentation, up-regulation of costimulatory molecules, and secretion of cytokines, which in turn leads to the induction of T cell
differentiation, proliferation, and survival of Ag specific CD4⫹ T
cells (11).
Although most TLR functions have been attributed to their role
in DC activation, CD4⫹ T cells also express TLRs and may regulate functional responses of CD4⫹ T cells in an APC-independent
manner (16 –21). For example, TLR2 is a potent costimulatory
receptor found on CD4⫹ T cells, which may increase proliferation
and IFN-␥ secretion following TCR stimulation (22, 23). TLR3
signals have been suggested to prolong CD4⫹ T cell survival (17).
TLR5 and TLR7 have been shown to enhance TCR stimulation in
memory CD4⫹ T cells (16). TLR4 signaling on T cells mediates
adherence to fibronectin and expression of suppressor of cytokine
signaling 3 (24). TLR9-mediated CD4⫹ T cell activation has been
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Abnormal T cell responses to commensal bacteria are involved in the pathogenesis of inflammatory bowel disease. MyD88 is an
essential signal transducer for TLRs in response to the microflora. We hypothesized that TLR signaling via MyD88 was important
for effector T cell responses in the intestine. TLR expression on murine T cells was examined by flow cytometry. CD4ⴙCD45Rbhigh
T cells and/or CD4ⴙCD45RblowCD25ⴙ regulatory T cells were isolated and adoptively transferred to RAG1ⴚ/ⴚ mice. Colitis was
assessed by changes in body weight and histology score. Cytokine production was assessed by ELISA. In vitro proliferation of T
cells was assessed by [3H]thymidine assay. In vivo proliferation of T cells was assessed by BrdU and CFSE labeling.
CD4ⴙCD45Rbhigh T cells expressed TLR2, TLR4, TLR9, and TLR3, and TLR ligands could act as costimulatory molecules.
MyD88ⴚ/ⴚ CD4ⴙ T cells showed decreased proliferation compared with WT CD4ⴙ T cells both in vivo and in vitro.
CD4ⴙCD45Rbhigh T cells from MyD88ⴚ/ⴚ mice did not induce wasting disease when transferred into RAG1ⴚ/ⴚ recipients. Lamina
propria CD4ⴙ T cell expression of IL-2 and IL-17 and colonic expression of IL-6 and IL-23 were significantly lower in mice
receiving MyD88ⴚ/ⴚ cells than mice receiving WT cells. In vitro, MyD88ⴚ/ⴚ T cells were blunted in their ability to secrete IL-17
but not IFN-␥. Absence of MyD88 in CD4ⴙCD45Rbhigh cells results in defective T cell function, especially Th17 differentiation.
These results suggest a role for TLR signaling by T cells in the development of inflammatory bowel disease. The Journal of
Immunology, 2008, 180: 1886 –1894.
The Journal of Immunology
Materials and Methods
Mouse transfer colitis
MyD88⫺/⫺ mice were purchased from Oriental BioService and were backcrossed to C57BL/6 mice ⬎10 times. C57BL/6 mice and RAG1⫺/⫺ mice
(C57BL/6 background) were obtained from The Jackson Laboratory. Sixto ten-wk-old gender-matched mice were used in this study. Mice were
kept in specific-pathogen free conditions and fed by free access to a standard diet and water. All experiments were performed according to Mount
Sinai School of Medicine Animal Experimental Ethics committee
guidelines.
Spleen and mesenteric lymph node (MLN) were taken from WT
(C57BL/6) mice and MyD88⫺/⫺ mice. Single cell suspensions from the
spleen and MLN were prepared to isolate CD4⫹ T cells using MACS
magnetic separation system (Miltenyi Biotec). Enriched CD4⫹ T cells
were labeled with FITC-conjugated anti-CD45Rb (BD Biosciences), and
allophycocyanin-conjugated CD25 (Biolegend). CD4⫹CD45Rbhigh and
CD4⫹CD45Rblow CD25⫹ cell fractions were sorted with a FACS Vantage cell sorter (BD Biosciences). The purity of cell separation was over
98% as measured by flow cytometry. Sorted cells were resuspended in
sterile PBS; 5 ⫻ 105 CD4⫹CD45Rbhigh cells were injected i.p. into each
RAG1⫺/⫺ mouse. Additional mice were injected with WT
CD4⫹CD45Rbhigh cells (5 ⫻ 105) and CD4⫹CD45Rblow CD25⫹ cells
(5 ⫻ 105) isolated from either WT or MyD88⫺/⫺ mice (34, 35).
Mice were monitored weekly for body weight, stools, and the gross
appearance of wasting disease. Mice were euthanized 9 wks after T cell
transfer. Cecum and proximal and distal halves of the colon were removed
and fixed in 10% buffered formalin, paraffin-embedded, sectioned, and
stained with H&E. Histological assessment was performed by a pathologist
blinded to the treatment. Histologic score was a combined score of crypt
damage (0 – 4), acute inflammatory cell infiltrate (0 – 4), edema (0 – 4), ero-
sion/ulceration (0 – 4), chronic inflammatory cell infiltrate (0 –2), epithelial
regeneration (0 –3), and crypt distortion/branching (0 –3).
Isolation of lamina propria (LP) cells
Colons were removed, washed in PBS with 5% penicillin/streptomycin, cut
into small pieces, and incubated with Ca2⫹ and Mg2⫹ free HBSS containing 0.002 mol/L EDTA for 12 min with gentle agitation to remove epithelial cells. Tissue pieces were then incubated while shaking in complete
medium containing 5% FCS, 0.02 mol/L HEPES, L-glutamine, 5% penicillin and streptomycin with 1 mg/ml collagenase, 1.5 mg/ml dispase in
RPMI 1640 at 37°C for 60 min. The supernatant was centrifuged and the
pellet was washed two times with RPMI 1640 containing 5% penicillin/
streptomycin. LP cells were isolated by lymphocyte separation medium
(Mediatech) density gradient centrifugation (800 ⫻ g for 20 min) and collected at the interface. DCs were isolated from LP cells by positive selection using the anti-CD11c MACS beads (Miltenyi Biotec). Using the DC
isolation flow through, CD4⫹ cells were further isolated by negative selection with the CD4 MACS system (Miltenyi Biotec).
Real time PCR
Total RNA was isolated from the colonic tissues using RNA Bee (Tel-Test)
according to the manufacturer’s instructions. A total of 1 ␮g RNA was
used as the template for single strand cDNA synthesis utilizing the Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer’s instructions. Quantitative real-time PCR was performed for IL23p19, IL-6, and ␤-actin. The primers and probes used in this study are as
follows: (5⬘33⬘ direction), for mouse IL-23p19: sense primer, C TGT
GCC TAG GAG TAG CAG TC, anti-sense primer, A GTC TCT TCA
TCC TCT TCT TCT CT, and probe, C TCA GTG CCA GCA GCT CTC
TCG GA; mouse IL-6 primers and probe were designed by Applied Biosystems (Mn00446190_m1); for mouse ␤-actin: sense primer, ATG ACC
CAG ATC ATG TTT G, anti-sense primer, TAC GAC CAG AGG CAT
ACA, and probe, CGT AGC CAT CCA GGC TGT GC. All TaqMan
probes and primers were designed using Beacon Designer 3.0 (Premier
Biosoft International). The cDNA was amplified using TaqMan universal
PCR Master Mix (Roche) on the ABI Prism 7900HT sequence detection
system (Applied Biosystems), programmed for 95°C for 10 min and then
40 cycles of 95°C for 15 s and 60°C for 1 min. The amplification results
were analyzed using SDS 2.2.1 software (Applied Biosystems), and the
genes of interest were normalized to the corresponding ␤-actin results.
Western blot analysis
Whole cell lysates were prepared from LP CD4⫹ T cells using a lysis buffer
containing 50 mM Tris-HCl, 50 mM NaF, 1% Triton X-100, 2 mM EDTA,
and 100 mM NaCl with a proteinase inhibitor mixture (Calbiochem). Protein concentration was determined by the Bradford method using Bio-Rad
Protein Assay Dye and SmartSpec 3000 (Bio-Rad). Twenty-five micrograms of the lysates were subjected to 10% SDS-PAGE and transferred to
Immobilon-P membranes (Millipore). The membrane was blocked in 5%
skim milk and was immunoblotted with the primary Abs for 1 h, followed
by HRP-conjugated goat anti-rabbit IgG, or Streptavidin-HRP (Zymed
Laboratories) corresponding to the primary Abs used. The membrane
was exposed on x-ray film using an enhanced chemiluminescent substrate SuperSignal West Pico Trial Kit (Pierce). Abs specific for
phoshpo (Ser 727)-STAT3 and phospho (Tyr 705)-STAT3 and STAT3
were purchased from Cell Signaling Technology.
Immunohistochemistry
Paraffin-embedded colon tissue samples (n ⫽ 5 each for mice that received
WT T cells or mice that received MyD88⫺/⫺ T cells) were double stained
with CD4 and phospho-STAT3. Ag retrieval by microwave was performed
after deparaffinization. After blocking the nonspecific binding with 5%
skim milk, sections were incubated with rabbit anti-phospho (Ser 727)STAT3 Ab (1/100, Cell Signaling Technologies) overnight at 4°C. After
washing in PBS, sections were incubated with tetramethylrhodamine isothiocyanate-conjugated goat anti-rabbit IgG (1/200, Sigma-Aldrich) for 1 h
at room temperature. Sections were then reincubated with 5% skim milk
and stained with FITC-conjugated anti-mouse CD4 Ab (1/100, BD Pharmingen, BD Biosciences) overnight at 4°C. Sections were mounted with
SlowFade Gold antifade reagent with 4⬘,6-diamidino-2-phenylindole (Invitrogen Life Technologies) after rinsing with PBS. Double stained tissue
slides were examined using a Leica TCS-SP (UV) confocal microscope.
In vitro T cell differentiation
The method for in vitro T cell differentiation was adapted from previous
studies (36, 37). In brief, CD4⫹CD45Rbhigh T cells isolated from
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shown to be MyD88 dependent (25). Recently, IL-4 receptor associated kinase, the most proximal downstream signaling molecule
after MyD88, has been shown to be essential in eliciting CD4⫹ T
cell activation (26). Finally, TLRs modulate Treg proliferation and
their ability to suppress T cell activation (27–29). These data suggest a significant role for MyD88 in CD4⫹ T cell activation.
Commensal bacteria are required to initiate chronic intestinal
inflammation in most animal models of IBD (30). The
CD4⫹CD45RBhigh cell adoptive transfer model of colitis is one of
the animal models of IBD that most closely reflects human IBD
with respect to gene expression profiles (31). In this model of
colitis, CD4⫹CD45RBhigh T cells are activated in response to
commensal bacteria to initiate chronic inflammation in the absence of Tregs (32). This T cell population has also been suggested to be important in the pathogenesis of human IBD (33).
Therefore, we hypothesized that TLR signaling via MyD88 is
important for the development of colitis in the murine
CD4⫹CD45Rbhigh T cell adoptive transfer model.
In the current study, we first determined the expression of TLRs
on CD4⫹CD45Rbhigh T cells. The finding that CD4⫹CD45Rbhigh
T cells express significant levels of TLRs led us to test their role
in the development of colitis. We examined the effects of MyD88
deficiency on CD4⫹ T cell activation in vivo and in vitro using
MyD88 knockout (MyD88⫺/⫺) mice as donor mice. In the adoptive transfer model of colitis, CD4⫹CD45Rbhigh T cells from
MyD88⫺/⫺ mice did not induce wasting disease. MyD88⫺/⫺ T
cells also showed defective proliferation in vitro and in vivo. The
secretion of IL-2 and IL-17 was significantly reduced in CD4⫹ T
cells from mice receiving MyD88⫺/⫺ T cells compared with mice
receiving WT T cells. In vitro, TLR ligands could act as costimulatory molecules in the context of anti-CD3 stimulation. In addition, Tregs from MyD88⫺/⫺ mice showed a decreased ability to
suppress T cell proliferation in vivo and in vitro. These results
suggest that MyD88 signaling by CD4⫹ T cells is required for
expansion and differentiation in the intestinal compartment. Our
results provide a link between an innate immune defect and the
sustained activation of T cells in the pathogenesis of IBD.
1887
1888
MyD88 REGULATES ADAPTIVE IMMUNITY IN CHRONIC COLITIS
FIGURE 1. TLR expression on
CD4⫹CD45Rbhigh CD4⫹ T cells.
Expression of TLRs on CD4⫹
CD45Rbhigh CD4⫹ T cells. Splenic
CD4⫹CD45Rbhigh CD4⫹ T cells
were examined for TLR2, TLR3,
TLR4, TLR5, and TLR9 expression
by flow cytometry (black peaks are
CD4⫹CD45Rbhigh CD4⫹ T cells of
WT
mice,
gray
peaks
are
CD4⫹CD45Rbhigh CD4⫹ T cells
from individual knock-out mice, and
white peaks are RAW 264.7 cells).
The isotype control Ab is used for
TLR5 staining.
ELISA
To measure cytokine production, DCs and CD4⫹ T cells were isolated
from the LP. CD4⫹ T cells (1 ⫻ 105) were cocultured with LP-DCs (5 ⫻
103) isolated from the same mouse in RPMI 1640 supplemented with 10%
FCS, 5% penicillin/streptomycin in 96-well anti-CD3 precoated (1 ␮g/ml)
flat-bottom plates (BD Biosciences) for 48 h. IFN-␥, IL-2, IL-10, and IL17A concentrations in the supernatant were determined by ELISA (R&D
Systems) according to the manufacturer’s instructions.
Flow cytometry
CD4⫹CD45Rbhigh T cells were isolated from spleens. RAW 264.7 cells
were used as a positive control for TLR staining. Spleen cells from knockout mice (TLR4⫺/⫺, TLR2⫺/⫺, TLR3⫺/⫺, and TLR9⫺/⫺) or isotype
matched Ab (mouse IgG2a␬ for TLR5) were used as negative controls.
Cells were stained with biotinylated anti-mouse TLRs (TLR2, TLR3,
TLR4, and TLR9) (eBioscience) or anti-mouse TLR5 (IMGENEX, San
Diego, CA) for 20 min at 4°C, followed by streptavidin-FITC or FITCconjugated goat anti-mouse IgG (Zymed Laboratories), respectively. Cells
were then fixed and permeabilized with the eBioscience Fixation/Permeabilization kit (eBioscience), and restained with the same Abs for 20 min
at 4°C to obtain intracellular staining. Cells were washed in PBS and analyzed by FACSCalibur (BD Biosciences) and CellQuest software (BD
Biosciences).
Cell proliferation assays
Mice were injected with 120 mg/kg of BrdU (Sigma-Aldrich) i.p. 90 min
before sacrificing. Paraffin sections of colon and MLN were incubated with
5% skim milk and stained with FITC-conjugated anti-BrdU (BD Pharmingen), followed by rabbit anti-mouse CD4 and tetramethylrhodamine isothiocyanate-conjugated anti-rabbit IgG. Proliferating CD4⫹ T cells were
determined by fluorescence microscopy.
In vitro cell proliferation was analyzed in freshly isolated CD4⫹ T cells
following 3 days of stimulation in anti-CD3 precoated 96-well plates (BD
Biosciences) with 1 ␮g/ml anti-CD28. Single cell suspensions of splenocytes were separated into CD4⫹CD25⫺ and CD4⫹CD25⫹ subsets using
negative selection with the anti-CD4 MACS system followed by positive
selection with an anti-CD25 MACS column (Miltenyi Biotec). In certain
experiments, we used TLR ligands, Pam3CSK4 (1 ␮g/ml; Invivogen), poly
I:C (90 ␮g/ml; Invivogen), LPS (1 ␮g/ml; Eschericha coli 0111: B4, Invivogen), and CpG-ODN (1 ␮g/ml; Invivogen) as an alternative to antiCD28. [3H]Thymidine was added during the last 18 h of culture. To assess
Treg suppressor activity, freshly isolated CD4⫹CD25⫹ Treg cells were
cocultured with CD4⫹CD25⫺ T cells at a ratio of 0.125:1 (0.125 ⫻ 105 vs
1 ⫻ 105 per well). [3H]Thymidine incorporation was analyzed with a beta
scintillation counter.
For CFSE labeling, CD4⫹ T cells were incubated in 5 ␮M CFSE (Molecular Probes, Invitrogen Life Technologies) in PBS at 37°C for 15 min.
CFSE-labeled cells (5 ⫻ 105) were injected i.p. into RAG1⫺/⫺ mice. After
7 days, CD4⫹ T cells were isolated from spleen, MLN, and the colon as
described above. Cell division was analyzed by flow cytometry detecting
CFSE fluorescence.
Statistical analysis
Results were expressed as mean ⫾ SD. Data were analyzed by Student’s
t test using the statistics package within Microsoft Excel. The p values were
considered significant when ⬍0.05.
Results
CD4⫹CD45Rbhigh T cells express TLRs
Several studies have previously demonstrated the expression of
TLRs on T cells (16 –21). However, most of the previous work
only examined expression at the mRNA level. We examined the
expression of TLR protein in CD4⫹CD45Rbhigh T cells isolated
from spleens of WT mice by flow cytometry (Fig. 1). We focused on TLR2, TLR4, TLR5, and TLR9 because of their relevance to luminal bacterial Ags. We also examined the expression of TLR3 because of viral pathogens. Although TLR
expression was lower than that seen in the mouse macrophage
cell line RAW 264.7 cells, CD4⫹CD45Rbhigh T cells expressed
significant levels of TLR2, TLR4, TLR9, and TLR3. TLR5 was
barely detectable.
MyD88 signaling modulates T cell proliferation
We next addressed whether TLR signaling contributes to T cell
function using MyD88⫺/⫺ T cells. To examine T cell activation,
CD4⫹CD25- T cells were cultured for 3 days with anti-CD3 and
anti-CD28 and proliferation was measured by [3H]thymidine incorporation. MyD88⫺/⫺ T cells showed 50% less proliferation
than WT T cells (Fig. 2A). In addition, we found that exogenous
TLR ligands (TLR2, TLR4, and TLR9 ligands) could act as a
costimulus to anti-CD3 activation in WT CD4⫹CD25⫺ T cells but
not in MyD88⫺/⫺ CD4⫹CD25⫺ T cells (Fig. 2B). By contrast, The
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MyD88⫺/⫺ or WT mice spleen were cultured in RPMI 1640 supplemented
with 10% FCS, 5% Pen/Strep at 37°C in 5% CO2. Cells were primed for
5 days with plate-bound anti-CD3 (2 ␮g/ml; BD Pharmingen), anti-CD28
(2 ␮g/ml; BD Pharmingen), and anti-IL-4 (4 ␮g/ml; 11B11 L, Santa Cruz
Biotechnology). The following cytokines were added for 5 days as indicated. For Th1 differentiation, IL-12 (4 ng/ml; PeproTech) was added. For
Th17 differentiation, IL-6 (100 ng/ml; PeproTech) plus TGF-␤ (5 ng/ml;
PeproTech), or IL-23 (10 ng/ml; R&D Systems) were added. Cells were
then washed and stimulated for 24 h with plate-bound anti-CD3 (1 ␮g/ml;
BD Biosciences) at a density of 1 ⫻ 106 cells/ml. Cell-free supernatants
were analyzed simultaneously for IL-17 or IFN-␥ production using specific
ELISA kits (R&D Systems).
The Journal of Immunology
TLR3 ligand caused similar degrees of proliferation between WT
T cells vs MyD88⫺/⫺ T cells, consistent with being an
MyD88-independent TLR.
Given the decrease in proliferation in MyD88⫺/⫺ T cell with
TCR activation, we investigated whether there were differences in
total T cell numbers between WT mice and MyD88⫺/⫺ mice. We
isolated CD4⫹ cells from spleens and LP of WT or MyD88⫺/⫺
mice and found similar numbers of CD4⫹ T cells, CD8⫹ T cells,
B cells (CD19), Gr-1 positive cells, or CD4⫹CD25⫹ Tregs. Thus,
in the MyD88⫺/⫺ mouse, other compensatory mechanisms result
in normal numbers of cells even in the LP.
To examine the function of MyD88⫺/⫺ Tregs, WT
CD4⫹CD25⫺ T cells were cocultured with either WT or
MyD88⫺/⫺ CD4⫹CD25⫹ Tregs. MyD88⫺/⫺ Tregs showed a decreased ability to suppress the proliferation of allogeneic T cells
challenged with anti-CD3 and anti-CD28 (Fig. 2C). Thus, in
MyD88⫺/⫺, mice there are no apparent defects in T cell numbers
in the periphery or the intestine under homeostatic conditions, but
in vitro functional responses are perturbed.
FIGURE 3. Defective function of MyD88⫺/⫺ naive T cells and Tregs in
the T cell transfer colitis model. A, MyD88⫺/⫺ CD4⫹CD45Rbhigh CD4⫹ T
cells do not cause weight loss in RAG1⫺/⫺ mice after transfer.
CD4⫹CD45Rbhigh T cells isolated from MyD88⫺/⫺ (n ⫽ 12) and WT (n ⫽
8) mice spleen were injected i.p. into RAG1⫺/⫺ mice. Mice were weighed
weekly. The graph shows percent body weight change. The data represent
the average (⫾SD) of five independent experiments (ⴱ, p ⬍ 0.05). B, Histology of the colon of RAG1⫺/⫺ mice nine weeks after transfer of naive T
cells. The colons in mice receiving WT T cells (WT hi) show a severe
inflammatory cell infiltrate, crypt loss, and distortion. Mice receiving
MyD88⫺/⫺ T cells (MyD88⫺/⫺ hi) have less inflammation compared with
the mice receiving WT T cells. C, Tregs from MyD88⫺/⫺ mice do not
protect from colitis induced by WT CD4⫹CD45Rbhigh CD4⫹ T cell transfer in RAG1⫺/⫺ mice. CD4⫹CD45Rbhigh T cells isolated from WT mice
were cotransferred with either WT Tregs or MyD88⫺/⫺ Tregs (n ⫽ 6 each)
into RAG1⫺/⫺ mice. The graph shows weekly percent body weight change.
The data represent the average (⫾SD) of three independent experiments
(ⴱ, p ⬍ 0.05). D, Histology of the colon of RAG1⫺/⫺ mice 9 wk after
cotransfer of Tregs with WT naive T cells. WT Tregs but not MyD88⫺/⫺
Tregs protect against naive T cell induced colitis.
MyD88⫺/⫺ CD4⫹CD45Rbhigh T cells are defective in their
ability to induce colitis in RAG1⫺/⫺ mice
To clarify the role of TLR signaling in CD4⫹CD45Rbhigh T cells
in vivo, we adoptively transferred CD4⫹CD45Rbhigh T cells isolated from MyD88⫺/⫺ or WT spleen into RAG1⫺/⫺ mice. After 9
wk, mice transferred with WT T cells demonstrated significant loss
of body weight as previously described (34). However, mice receiving MyD88⫺/⫺ CD4⫹CD45Rbhigh T cells showed significantly less weight loss (Fig. 3, A and B). Histological examination
of the colon showed that mice receiving MyD88⫺/⫺ T cells had
significantly decreased severity of colitis compared with that of
WT T cell transferred mice (colitis score: 11.1 ⫾ 2.9 vs 3.6 ⫾ 3.7,
p ⬍ 0.001, max score: 20). Importantly, MyD88⫺/⫺ lymphocytes
did home to the intestine and other compartments but did not induce colitis. Indeed, MyD88⫺/⫺ T cells in peripheral blood expressed the mucosal homing marker ␣4␤7 to a similar extent compared with WT T cells (data not shown).
Defective Treg function in TLR2⫺/⫺ mice has been previously
reported (38). We therefore examined MyD88⫺/⫺ Treg function in
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FIGURE 2. Absence of MyD88 signaling results in defective CD4⫹ T
cell function in vitro. A, MyD88⫺/⫺ T cells have decreased proliferation.
Proliferation of CD4⫹CD25⫺ T cells in the presence of anti-CD3 and
anti-CD28 mAbs, measured by [3H]thymidine incorporation after 3 days in
culture. Values indicate average counts (⫾SD) of triplicate wells for three
individual experiments (ⴱ, p ⬍ 0.05). B, T cell proliferation by exogenous TLR ligands. CD4⫹CD25⫺ T cells were stimulated with antiCD3 in the presence or absence of TLR ligands (without anti-CD28).
Cell proliferation was measured by [3H]thymidine incorporation after 3
days in culture. Values indicate average counts (⫾SD) of triplicate
wells for three individual experiments (ⴱ, p ⬍ 0.05). C, MyD88⫺/⫺
Tregs have decreased suppressor function. CD4⫹CD25⫹ Treg cells
were cocultured with CD4⫹CD25⫺ T cells at a ratio of 0.125: 1
(0.125 ⫻ 105 vs 1 ⫻ 105 per well) for 3 days with stimulation by
anti-CD3 and anti-CD28 mAbs. Values indicate average counts (⫾SD)
in triplicate wells in three individual experiments (ⴱ, p ⬍ 0.05).
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the colitis transfer model. WT CD4⫹CD45Rbhigh T cells were cotransferred with either WT Tregs or MyD88⫺/⫺ Tregs, and the
severity of the colitis was compared after 9 wk. Tregs from
MyD88⫺/⫺ mice did not protect against colitis induced by WT
CD4⫹CD45Rbhigh T cells (Fig. 3, C and D). Histological severity
of the colitis revealed a marginal suppressive effect of the
MyD88⫺/⫺ Tregs, but the severity score was significantly higher
than in mice cotransferred with WT Tregs (colitis score:
MyD88⫺/⫺ Tregs 7.7 ⫾ 3.9 vs WT Tregs 3.6 ⫾ 3.1, p ⬍ 0.01,
max: 20). These results indicate that CD4⫹CD45Rbhigh T cells
from MyD88⫺/⫺ mice exhibit an impaired ability to induce
chronic colitis in the adoptive transfer model. In addition, MyD88dependent TLR signaling is required for optimal Treg function
in vivo.
CD4⫹CD45Rbhigh T cells from MyD88⫺/⫺ mice exhibit
decreased proliferation in MLNs and the LP when transferred
into RAG1⫺/⫺ mice
Secretion of IL-2 and IL-17 is significantly lower in colonic
CD4⫹ T cells from RAG1⫺/⫺ mice receiving MyD88⫺/⫺ T cells
than in mice receiving WT T cells
Given the results suggesting proliferative defects in MyD88⫺/⫺
CD4⫹CD45Rbhigh T cells, we next addressed whether cytokine
expression and T cell polarization were altered in MyD88⫺/⫺ T
cells in the colitis model. CD4⫹ T cells isolated from the LP in
mice receiving MyD88⫺/⫺ T cells were cocultured with LP DCs,
isolated from the same mouse, and stimulated with anti-CD3 for
48 h. Supernatants were analyzed for cytokine production by
ELISA. There were no differences in the production of IFN-␥ or
IL-10 between MyD88⫺/⫺ CD4⫹ T cells and WT CD4⫹ T cells
(Fig. 5). By contrast, the production of IL-2 and IL-17 was significantly lower in MyD88⫺/⫺ CD4⫹ T cells than in WT CD4⫹ T
cells (Fig. 5). These results suggest that MyD88⫺/⫺ T cells are
specifically defective in Th17 polarization. In addition, impaired
expression of IL-2 may be part of the reason for defective T cell
proliferation observed in MyD88⫺/⫺ CD4⫹ T cells.
FIGURE 4. Decreased proliferation of MyD88⫺/⫺ T cells after transfer into RAG1⫺/⫺ mice. A, The number of CD4⫹ T cells in the LP of
RAG1⫺/⫺ mice 9 wks after naive T cell transfer. The data represent the
mean (⫾SD) of three mice each (ⴱ, p ⬍ 0.05). B, BrdU labeling of
proliferating cells in the LP and MLN taken from RAG1⫺/⫺ mice receiving WT or MyD88⫺/⫺ naive T cells. Representative pictures show
BrdU positive cells (green) and CD4⫹ cells (red), appearing yellow.
RAG1⫺/⫺ mice receiving WT T cells have more proliferating cells in
both the LP and MLN than mice receiving MyD88⫺/⫺ T cells. C, Decreased MyD88⫺/⫺ CD4⫹ T cell proliferation in MLN of RAG1⫺/⫺
mice observed 7 days after transfer. CD4⫹ T cells isolated from
MyD88⫺/⫺ and WT mice spleen were labeled with CFSE and injected
i.p. into RAG1⫺/⫺ mice. Division of CFSE labeled cells was analyzed
by flow cytometry. White peaks show the initial CFSE fluorescence
(before injection). WT CD4⫹ T cells show more dividing cells than
MyD88⫺/⫺ CD4⫹ T cells in the MLN, represented by the broader
shoulder of dividing cells in the WT MLN panel (upper left panel).
Mucosal IL-6 and IL-23 expression are decreased in RAG1⫺/⫺
mice receiving MyD88⫺/⫺ T cells
T cell polarization is regulated by cytokines present during the
initial stage of TCR ligation (39). Th17 polarization is specifically
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Given the in vitro findings that MyD88⫺/⫺ T cells have a decreased ability to proliferate in response to TCR stimulation yet
have normal numbers of T cells in situ, we next addressed whether
this decreased ability to proliferate also occurred when T cells
were transferred to a TLR sufficient host, namely the RAG1⫺/⫺
mice. First, we compared the total number of CD4⫹ T cells isolated from LP between MyD88⫺/⫺ T cell transferred mice and WT
T cells transferred mice (Fig. 4A). RAG1⫺/⫺ mice receiving
MyD88⫺/⫺ T cells had significantly fewer LP CD4⫹ T cells than
mice receiving WT T cells.
To confirm whether this difference in T cell number was due to
defective proliferation of MyD88⫺/⫺ T cells in vivo, we examined
local T cell proliferation by BrdU labeling at 9 wk after T cell
transfer (Fig. 4B). Double staining of the colon and MLN with
anti-CD4 and anti-BrdU showed less proliferation of CD4⫹ T
cells in mice receiving MyD88⫺/⫺ CD4⫹CD45Rbhigh T cells
than those receiving WT CD4⫹CD45Rbhigh T cells. We next
examined the dynamic proliferative ability of the CD4⫹ T cells
using CFSE labeling. CD4⫹ T cells from WT mice and
MyD88⫺/⫺ mice were labeled with CFSE and transferred to
RAG1⫺/⫺ mice. Cell division was analyzed by flow cytometry on
the seventh day after transfer (Fig. 4C). Transferred T cells from
MyD88⫺/⫺ mice showed fewer cell divisions in MLN than T cells
from WT mice. These results suggest that MyD88⫺/⫺
CD4⫹CD45Rbhigh T cells have defective proliferation in vivo.
The Journal of Immunology
mediated by DC-derived cytokines IL-23 and IL-6 under the influence of TGF-␤ (40). To clarify whether defective Th17 differentiation of MyD88⫺/⫺ T cells is associated with a decrease in
these upstream cytokines, we examined the expression of IL23p19 and IL-6 in the colonic mucosa using real time PCR. Compared with mice receiving WT T cells, mice that received
MyD88⫺/⫺ T cells had significantly decreased mRNA expression
of both IL-23 and IL-6 (Fig. 6). Expression of IL-1␤ and IL-18
mRNA in the colonic mucosa was slightly decreased in mice receiving MyD88⫺/⫺ T cells but did not reach statistical significance
(data not shown). These results suggest that MyD88 function in
CD4⫹CD45Rbhigh T cells is required to prime LP APCs to produce these cytokines.
MyD88 is required for the induction of Th17 differentiation
in vitro
Thus far, we found that differentiation of MyD88
CD4⫹CD45Rbhigh T cells into Th17 cells was defective in vivo
and that expression of IL-6 and IL-23 were also decreased in vivo.
We then wished to examine whether the defect in Th17 differen-
FIGURE 6. Decreased mucosal expression of IL-6 and IL-23 mRNA in
RAG1⫺/⫺ mice receiving MyD88⫺/⫺ T cells. TaqMan real-time PCR demonstrates up-regulation of mucosal IL-6 and IL-23 expression in the colon
of mice receiving WT T cells but not in mice receiving MyD88⫺/⫺ T cells
9 weeks after transfer (n ⫽ 7 each). Values indicate average relative values
(⫾SD) (ⴱ, p ⬍ 0.05).
FIGURE 7. MyD88 is required for induction of Th17 differentiation
in vitro. A, Defective in vitro Th17 differentiation in MyD88⫺/⫺
CD4⫹CD45Rbhigh T cells. MyD88⫺/⫺ CD4⫹CD45Rbhigh T cells or WT T
cells were stimulated as described in Materials and Methods in the presence of IL-12, IL-6 plus TGF-␤ or IL-23 and IL-17 production measured
(ⴱ, p ⬍ 0.05). B, MyD88 is not required Th1 differentiation. MyD88⫺/⫺
CD4⫹CD45Rbhigh T cells or WT T cells were stimulated as described in
Materials and Methods in the presence of IL-12 and IFN-␥ measured (NS;
not significant).
tiation could be overcome with exogenous administration of cytokines in vitro. Polarization of T cells was performed as previously described (36, 37). WT and MyD88⫺/⫺ CD4⫹CD45Rbhigh T
cells primed with IL-12 and treated with anti-IL-4 expressed equal
amounts of the Th1 cytokine IFN-␥ (Fig. 7B). In contrast, Th17
differentiation was very different between MyD88⫺/⫺
CD4⫹CD45Rbhigh T cells and WT T cells. Whereas priming with
IL-6 plus TGF-␤, or IL-23-induced IL-17 expression in WT
CD4⫹CD45Rbhigh T cells, MyD88⫺/⫺ CD4⫹CD45Rbhigh T cells
expressed significantly less IL-17 (Fig. 7A). These results suggest
that MyD88⫺/⫺ CD4⫹CD45Rbhigh T cells have an inherent defect
in Th17 but not Th1 polarization.
STAT3 phosphorylation in mucosal CD4⫹ T cells is decreased
in MyD88⫺/⫺ T cell transferred mice
STAT3 is active in the colon in IBD (41, 42), and plays a pivotal
role in the pathogenesis of colitis following adoptive T cell transfer
(43). In addition, activation of STAT3 in response to IL-6 and
IL-23 is required for Th17 development by CD4⫹ T cells (15, 44,
45). IL-17 further induces the activation of STAT3, leading to a
positive feedback loop to sustain inflammation (46). We analyzed
the phosphorylation state of STAT3 in LP CD4⫹ T cells of both
WT and MyD88⫺/⫺ T cell transferred RAG1⫺/⫺ mice (Fig. 8A).
Activation of STAT3 is regulated by phosphorylation of Tyr705
and Ser727. Consistent with the altered expression of IL-17,
STAT3 phosphorylation was greater in the LP CD4⫹ T cells from
mice receiving WT T cells than the cells from mice receiving
MyD88⫺/⫺ T cells.
We also examined the localization of phosphorylated STAT3
(Ser727) in colonic tissues of those mice by immunohistochemistry (Fig. 8B). Most of the LP CD4⫹ T cells were positive for
phospho-STAT3 in mice receiving WT T cells but very few phospho-STAT3 positive cells were found in the mice receiving
MyD88⫺/⫺ T cells. Cytoplasmic as well as nuclear localization of
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FIGURE 5. Decreased IL-2 and IL-17 production by MyD88⫺/⫺ T cells
in LP after transfer into RAG1⫺/⫺ mice. CD4⫹ T cells isolated from the LP
in mice receiving MyD88⫺/⫺ T cells and mice receiving WT T cells were
cocultured with LP DCs isolated from the same mouse, and stimulated with
anti-CD3 for 48 h. IFN-␥, IL-10, IL-2, and IL-17 in the supernatant were
analyzed by ELISA. Values indicate average concentrations (⫾SD) of duplicate wells in three individual experiments (ⴱ, p ⬍ 0.05).
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phospho-STAT3 was observed in CD4⫹ T cells in mice receiving
WT T cells but no nuclear localization was seen in mice receiving
MyD88⫺/⫺ T cells. These results suggest that the observed defects
of MyD88⫺/⫺ T cells in the development of the colitis might be
due to decreased STAT3 activation.
Discussion
Many aspects of the interface between innate and adaptive immunity
remain unexplored. It is particularly interesting to ponder how adaptive immune responses are regulated in the intestine given its coexistence with the microflora. We show that CD4⫹CD45Rbhigh T cells
from MyD88⫺/⫺ mice exhibit multiple defects in establishing a
pathogenic adaptive response to host signals and the luminal microenvironment during chronic colitis. Among the defects we identified,
MyD88⫺/⫺ T cells are unable to be activated and to differentiate into
Th17 effector cells, thereby preventing expansion and sustained inflammation. MyD88⫺/⫺ CD4⫹ T cells show decreased production of
IL-2 and a reduction in proliferation in response to TCR stimulation.
Tregs from MyD88⫺/⫺ mice were also defective in protecting against
colitis induced by WT CD4⫹CD45Rbhigh T cells. Therefore, signaling via MyD88 on CD4⫹CD45Rbhigh T cells appears to be required
for the proliferation and Th17 differentiation of T cells. MyD88 signaling is also required for Treg-mediated suppression of colitogenic T
cells. These results suggest a significant role for TLR signaling by T
cells in the development of IBD.
The adoptive transfer model of colitis provides an ideal mechanism
by which to test the hypothesis that TLR signaling by T cells is important for their function. Extensive previous work in this model has
highlighted the dependence of luminal bacteria in the development of
colitis (35, 47). In particular, Powrie et al., (34) has shown that intestinal bacteria are necessary in the recipient mouse to develop colitis.
CD4⫹CD45Rbhigh T cells from germ-free mice can still induce wasting disease as long as bacteria are present in the recipient mouse. Our
study has taken advantage of MyD88⫺/⫺ mice with the notion that
these mice are effectively unable to signal through TLRs. A previous
study has found that crossing MyD88⫺/⫺ mice to IL-10⫺/⫺ mice
protects against the development of colitis (48). But in that study, all
the cells of the mouse are null for both IL-10 and MyD88 including
APCs and T cells. By contrast, our study allows us to dissect the role
of MyD88-dependent signaling within the T cell compartment. Given
what was known about T cell development in MyD88⫺/⫺ mice, we
had initially hypothesized that the transfer of CD4⫹CD45Rbhigh
MyD88⫺/⫺ T cells into a TLR-positive environment, namely the
RAG1⫺/⫺, would be sufficient to induce colitis. We were surprised by
the degree to which MyD88⫺/⫺ CD4⫹CD45Rbhigh T cells were unable to cause colitis.
We described significant expression of TLR2, TLR3, TLR4, and
TLR9 protein on murine CD4⫹CD45Rbhigh T cells. There are several
conflicting reports in terms of TLR expression in murine T cells (17,
49, 50). Marsland et al. demonstrated TLR4, TLR7, and TLR9
mRNA expression, whereas Sobek et al. reported TLR1, TLR2, and
TLR6 mRNA expression. Another study described expression of
TLR2, TLR3, TLR4, TLR5, and TLR9 mRNA (17), but we could not
detect TLR5 protein expression. Although the discrepancy may be
due to the differences between mRNA and protein expression, others
have shown the absence of TLR5 mRNA in CD4⫹CD45Rbhigh T
cells consistent with our finding of absent TLR5 protein (19). Interestingly, it has been reported that TLR4 and TLR2 mRNA expression
become undetectable while TLR3 and TLR9 mRNA are up-regulated
in naive T cells (CD44lowCD25⫺CD4⫹) after stimulation with antiCD3 and anti-CD28 (17). Therefore, TLR expression may vary with
T cell activation state and highlights the developmental role of TLRs
on T cells.
Our results demonstrate that there is impaired activation of
MyD88⫺/⫺ CD4⫹CD45Rbhigh T cells after adoptive transfer into
RAG1⫺/⫺ mice. Although this population is meant to represent a
population of activation naive cells, clearly this may be a heterogeneous population (51). Nevertheless, the CD45Rb surface Ag permits
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FIGURE 8. STAT3 activation in mucosal CD4⫹ T
cells. A, Western blot analysis of serine and tyrosine
phosphorylation of STAT3 in CD4⫹ T cells isolated
from the LP. RAG1⫺/⫺ mice receiving MyD88⫺/⫺ T
cells and the mice receiving WT T cells were examined
for STAT3 activation 9 wk after transfer. Mice receiving
MyD88⫺/⫺ T cells show less phosphorylation of STAT3
in CD4 T cells. B, Immunofluorescent staining of
CD4⫹ mucosal cells (green) and phospho-STAT3
(Ser727) (red), 4⬘,6⬘-diamidino-2-phenylindole (blue)
identifies nuclei. The top panel shows colonic mucosa of
RAG1⫺/⫺ mice receiving WT T cells and the bottom
panel shows the mucosa of mice receiving MyD88⫺/⫺ T
cells. Cytoplasmic localization of phospho-STAT3 is
represented by yellow and nuclear localization of phospho-STAT3 is represented by pink.
The Journal of Immunology
involved in IL-17 production, and conversely, IL-17 activates
STAT3 (15, 44 – 46). Therefore, the sequence of events leading
to defective STAT3 activation and IL-17 production may underlie the inability of MyD88⫺/⫺ CD4⫹CD45Rbhigh T cells to
induce chronic colitis in RAG1⫺/⫺ mice.
In this study, we illustrate an important role of MyD88 in the
establishment of pathogenic adaptive immunity. In the absence of
MyD88 signaling, CD4⫹CD45Rbhigh T cells can home to the intestine but do not acquire the phenotype of colitogenic T cells
characterized by IL-17 production, STAT3 activation, and aberrant, nonhomeostatic levels of T cell proliferation. Therefore, traditional innate immune signaling may have a greater role in adaptive immunity than originally recognized. Our results enhance the
understanding of the link between innate immune defects and abnormal T cell activation in response to luminal commensal bacteria
in IBD. Inhibition of MyD88 in select T cell compartments may be
useful in the treatment of IBD.
Disclosures
The authors have no financial conflict of interest.
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us to identify a subpopulation of cells that has been well characterized
in this animal model. Several possible mechanisms may underlie the
defect in MyD88⫺/⫺ T cells. APCs express TLRs normally in the
RAG1⫺/⫺ mice. The primary defects in T cell function seen must
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In vivo, APCs expressing CD28 also could not compensate for the
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The second possibility accounting for defective T cell function
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(13, 14, 54). Because MyD88⫺/⫺ T cells express similar amounts
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in the absence of MyD88-dependent signaling (55, 56). By contrast, expression of IL-17 was decreased in MyD88⫺/⫺ T cells,
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whether they receive WT T cells or MyD88⫺/⫺ T cells. STAT3 is
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27.
MyD88 REGULATES ADAPTIVE IMMUNITY IN CHRONIC COLITIS