The myeloid differentiation factor 88 (MyD88) is required for CD4+ T cell effector function in a murine model of inflammatory bowel disease - PubMed (original) (raw)

The myeloid differentiation factor 88 (MyD88) is required for CD4+ T cell effector function in a murine model of inflammatory bowel disease

Masayuki Fukata et al. J Immunol. 2008.

Abstract

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. CD4(+)CD45Rb(high) T cells and/or CD4(+)CD45Rb(low)CD25(+) regulatory T cells were isolated and adoptively transferred to RAG1(-/-) 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 [(3)H]thymidine assay. In vivo proliferation of T cells was assessed by BrdU and CFSE labeling. CD4(+)CD45Rb(high) T cells expressed TLR2, TLR4, TLR9, and TLR3, and TLR ligands could act as costimulatory molecules. MyD88(-/-) CD4(+) T cells showed decreased proliferation compared with WT CD4(+) T cells both in vivo and in vitro. CD4(+)CD45Rb(high) T cells from MyD88(-/-) mice did not induce wasting disease when transferred into RAG1(-/-) recipients. Lamina propria CD4(+) T cell expression of IL-2 and IL-17 and colonic expression of IL-6 and IL-23 were significantly lower in mice receiving MyD88(-/-) cells than mice receiving WT cells. In vitro, MyD88(-/-) T cells were blunted in their ability to secrete IL-17 but not IFN-gamma. Absence of MyD88 in CD4(+)CD45Rb(high) 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.

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Figures

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.

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 three 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 three 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 co-cultured with CD4+CD25− T cells at a ratio of 0.125 : 1 (0.125×105 vs. 1×105 per well) for three 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).

Figure 3. Defective function of MyD88−/− naïve 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 (*P<0.05). B. Histology of the colon of RAG1−/− mice nine weeks after transfer of naïve 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 to 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 co-transferred 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 nine weeks after co-transfer of Tregs with WT naïve T cells. WT Tregs but not MyD88−/− Tregs protect against naïve T cell induced colitis.

Figure 4. Decreased proliferation of MyD88−/− T cells after transfer into RAG1−/− mice

A. The number of CD4+ T cells in the lamina propria of RAG1−/− mice nine weeks after naïve T cell transfer. The data represent the mean (±SD) of three mice each (*P<0.05). B. BrdU labeling of proliferating cells in the lamina propria and MLN taken from RAG1−/− mice receiving WT or MyD88−/− naïve 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 lamina propria and MLN than mice receiving MyD88−/− T cells. C. Decreased MyD88−/− CD4+ T cell proliferation in MLN of RAG1−/− mice observed seven 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).

Figure 5. Decreased IL-2 and IL-17 production by MyD88−/− T cells in lamina propria after transfer into RAG1−/− mice

CD4+ T cells isolated from the lamina propria in mice receiving MyD88−/− T cells and mice receiving WT T cells were co-cultured with lamina propria DCs isolated from the same mouse, and stimulated with anti-CD3 for 48 hours. 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).

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 nine weeks after transfer (n=7 each). Values indicate average relative values (±SD) (*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 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+CD45Rbhigh T cells or WT T cells were stimulated as described in Methods in the presence of IL-12 and IFN-γ measured (NS; not significant).

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 lamina propria. RAG1−/− mice receiving MyD88−/− T cells and the mice receiving WT T cells were examined for STAT3 activation nine weeks 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), DAPI (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.

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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 - PubMed (original) (raw)