The Myeloid Differentiation Factor 88 (MyD88) Is Required for CD4⁺ T Cell Effector Function in a Murine Model of Inflammatory Bowel Disease

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. Six- to 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⁺CD45Rb^high and CD4⁺CD45Rb^low 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⁺CD45Rb^high cells were injected i.p. into each RAG1^−/− mouse. Additional mice were injected with WT CD4⁺CD45Rb^high cells (5 × 10⁵) and CD4⁺CD45Rb^low 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), erosion/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 μg 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 IL-23p19, IL-6, and β-actin. The primers and probes used in this study are as follows: (5′→3′ 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 β-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 β-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⁺CD45Rb^high T cells isolated from 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 μg/ml; BD Pharmingen), anti-CD28 (2 μg/ml; BD Pharmingen), and anti-IL-4 (4 μg/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-β (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 μg/ml; BD Biosciences) at a density of 1 × 10⁶ cells/ml. Cell-free supernatants were analyzed simultaneously for IL-17 or IFN-γ production using specific ELISA kits (R&D Systems).

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 μg/ml) flat-bottom plates (BD Biosciences) for 48 h. IFN-γ, IL-2, IL-10, and IL-17A concentrations in the supernatant were determined by ELISA (R&D Systems) according to the manufacturer’s instructions.

Flow cytometry

CD4⁺CD45Rb^high T cells were isolated from spleens. RAW 264.7 cells were used as a positive control for TLR staining. Spleen cells from knock-out mice (TLR4^−/−, TLR2^−/−, TLR3^−/−, and TLR9^−/−) or isotype matched Ab (mouse IgG2aκ 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 FITC-conjugated 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 μg/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 μg/ml; Invivogen), poly I:C (90 μg/ml; Invivogen), LPS (1 μg/ml; Eschericha coli 0111: B4, Invivogen), and CpG-ODN (1 μg/ml; Invivogen) as an alternative to anti-CD28. [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 μM 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.

CD4⁺CD45Rb^high T cells express TLRs

Several studies have previously demonstrated the expression of TLRs on T cells (16, 17, 18, 19, 20, 21). However, most of the previous work only examined expression at the mRNA level. We examined the expression of TLR protein in CD4⁺CD45Rb^high 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⁺CD45Rb^high T cells expressed significant levels of TLR2, TLR4, TLR9, and TLR3. TLR5 was barely detectable.

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FIGURE 1.

TLR expression on CD4⁺CD45Rb^high CD4⁺ T cells. Expression of TLRs on CD4⁺CD45Rb^high CD4⁺ T cells. Splenic CD4⁺CD45Rb^high CD4⁺ T cells were examined for TLR2, TLR3, TLR4, TLR5, and TLR9 expression by flow cytometry (black peaks are CD4⁺CD45Rb^high CD4⁺ T cells of WT mice, gray peaks are CD4⁺CD45Rb^high 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.

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. 2⇓A). 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. 2⇓B). By contrast, The TLR3 ligand caused similar degrees of proliferation between WT T cells vs MyD88^−/− T cells, consistent with being an MyD88-independent TLR.

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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 (∗, p < 0.05). B, T cell proliferation by exogenous TLR ligands. CD4⁺CD25⁻ T cells were stimulated with anti-CD3 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 (∗, 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 (∗, p < 0.05).

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. 2⇑C). 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.

MyD88^−/− CD4⁺CD45Rb^high T cells are defective in their ability to induce colitis in RAG1^−/− mice

To clarify the role of TLR signaling in CD4⁺CD45Rb^high T cells in vivo, we adoptively transferred CD4⁺CD45Rb^high 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⁺CD45Rb^high 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 α4β7 to a similar extent compared with WT T cells (data not shown).

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FIGURE 3.

Defective function of MyD88^−/− naive T cells and Tregs in the T cell transfer colitis model. A, MyD88^−/− CD4⁺CD45Rb^high CD4⁺ T cells do not cause weight loss in RAG1^−/− mice after transfer. CD4⁺CD45Rb^high 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 (∗, 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⁺CD45Rb^high CD4⁺ T cell transfer in RAG1^−/− mice. CD4⁺CD45Rb^high 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 (∗, 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.

Defective Treg function in TLR2^−/− mice has been previously reported (38). We therefore examined MyD88^−/− Treg function in the colitis transfer model. WT CD4⁺CD45Rb^high 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⁺CD45Rb^high 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⁺CD45Rb^high T cells from MyD88^−/− mice exhibit an impaired ability to induce chronic colitis in the adoptive transfer model. In addition, MyD88-dependent TLR signaling is required for optimal Treg function in vivo.

CD4⁺CD45Rb^high T cells from MyD88^−/− mice exhibit decreased proliferation in MLNs and the LP when transferred into RAG1^−/− mice

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. 4⇓A). RAG1^−/− mice receiving MyD88^−/− T cells had significantly fewer LP CD4⁺ T cells than mice receiving WT T cells.

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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 (∗, 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).

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. 4⇑B). 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⁺CD45Rb^high T cells than those receiving WT CD4⁺CD45Rb^high 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. 4⇑C). Transferred T cells from MyD88^−/− mice showed fewer cell divisions in MLN than T cells from WT mice. These results suggest that MyD88^−/− CD4⁺CD45Rb^high T cells have defective proliferation in vivo.

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⁺CD45Rb^high 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-γ 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.

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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-γ, 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 (∗, p < 0.05).

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 mediated by DC-derived cytokines IL-23 and IL-6 under the influence of TGF-β (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 IL-23p19 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-1β 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⁺CD45Rb^high T cells is required to prime LP APCs to produce these cytokines.

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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) (∗, p < 0.05).

MyD88 is required for the induction of Th17 differentiation in vitro

Thus far, we found that differentiation of MyD88 CD4⁺CD45Rb^high 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 differentiation 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⁺CD45Rb^high T cells primed with IL-12 and treated with anti-IL-4 expressed equal amounts of the Th1 cytokine IFN-γ (Fig. 7⇓B). In contrast, Th17 differentiation was very different between MyD88^−/− CD4⁺CD45Rb^high T cells and WT T cells. Whereas priming with IL-6 plus TGF-β, or IL-23-induced IL-17 expression in WT CD4⁺CD45Rb^high T cells, MyD88^−/− CD4⁺CD45Rb^high T cells expressed significantly less IL-17 (Fig. 7⇓A). These results suggest that MyD88^−/− CD4⁺CD45Rb^high T cells have an inherent defect in Th17 but not Th1 polarization.

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FIGURE 7.

MyD88 is required for induction of Th17 differentiation in vitro. A, Defective in vitro Th17 differentiation in MyD88^−/− CD4⁺CD45Rb^high T cells. MyD88^−/− CD4⁺CD45Rb^high T cells or WT T cells were stimulated as described in Materials and Methods in the presence of IL-12, IL-6 plus TGF-β or IL-23 and IL-17 production measured (∗, p < 0.05). B, MyD88 is not required Th1 differentiation. MyD88^−/− CD4⁺CD45Rb^high T cells or WT T cells were stimulated as described in Materials and Methods in the presence of IL-12 and IFN-γ measured (NS; not significant).

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. 8⇓A). 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.

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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.

We also examined the localization of phosphorylated STAT3 (Ser727) in colonic tissues of those mice by immunohistochemistry (Fig. 8⇑B). 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 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.