Role of T cell TGF-beta signaling in intestinal cytokine responses and helminthic immune modulation

Abstract

Colonization with helminthic parasites induces mucosal regulatory cytokines, like IL-10 or TGF-beta, that are important in suppressing colitis. Helminths induce mucosal T cell IL-10 secretion and regulate lamina propria mononuclear cell (LPMC) Th1 cytokine generation in an IL-10-dependent manner in WT mice. Helminths also stimulate mucosal TGF-beta release. As TGF-beta exerts major regulatory effects on T lymphocytes, we investigated the role of T lymphocyte TGF-beta signaling in helminthic modulation of intestinal immunity. T cell TGF-beta signaling is interrupted in TGF-beta receptor II dominant negative (TGF-betaRII DN) mice by T-cell-specific over-expression of a TGF-betaRII DN. We studied LPMC responses in WT and TGF-betaRII DN mice that were uninfected or colonized with the nematode, Heligmosomoides polygyrus. Our results indicate an essential role of T cell TGF-beta signaling in limiting mucosal Th1 and Th2 responses. Furthermore, we demonstrate that helminthic induction of intestinal T cell IL-10 secretion requires intact T cell TGF-beta-signaling pathway. Helminths fail to curtail robust, dysregulated intestinal Th1 cytokine production and chronic colitis in TGF-betaRII DN mice. Thus, T cell TGF-beta signaling is essential for helminthic stimulation of mucosal IL-10 production, helminthic modulation of intestinal IFN-gamma generation and H. polygyrus-mediated suppression of chronic colitis.

Figure 1

TCR-triggered TGF-β production in LPMC from uninfected control or H. polygyrus-infected WT and TGF-βRII DN mice. LPMC from uninfected or helminth-infected WT or TGF-βRII DN mice were cultured in vitro alone (Cells) or with anti-CD3/CD28 mAb (Cells + αCD3/28) for 48 h in TGF-β culture medium. Supernatants then were analyzed for TGF-β by ELISA. Data show mean ± SD from three independent experiments with each experiment containing multiple determinations. (WT: unstimulated LPMC Uninfected vs. Infected, p>0.05, NS; anti-CD3/CD28 stimulated Uninfected vs. Infected, **p<0.01).

Figure 2

Helminths cannot modulate the Th1 response and only weakly promote the Th2 response in LPMC from TGF-βRII DN mice. LPMC from uninfected or helminth-infected WT or TGF-βRII DN mice were stimulated in vitro with anti-CD3/CD28 mAb for 48 mAb. Supernatants were harvested and analyzed for IFN-γ, IL-4 and IL-5 by ELISA. Data show mean ± SD from at least three independent experiments with each experiment containing multiple determinations. (IFN-γ: WT Uninfected vs. Infected, ***p<0.001; TGF-βRII DN Uninfected vs. Infected, p>0.05, NS. IL-4: WT Uninfected vs. Infected, ***p<0.001; TGF-βRII DN Uninfected vs. Infected, *p<0.05. IL-5: WT Uninfected vs. Infected, ***p<0.001; TGF-βRII DN Uninfected vs. Infected, *p<0.05).

Figure 3

Helminth-associated changes in cytokine producing LP T cells from WT and TGF-βRII DN mice. Representative dot plots of LPMC from (A) uninfected WT, (B) helminth-infected WT, (C) uninfected TGF-βRII DN or (D) helminth-infected TGF-βRII DN mice that were stimulated overnight in vitro with anti-CD3/28. Brefeldin A was added to culture supernatants for the last 12 hours. Subsequently, cells were harvested and stained for Thy1.2, IFN-γ and IL-4 for FACS analysis. (E) Data show mean ± SD of the percentage IFNγ (upper panel) or IL-4 (lower panel) producing Thy1.2 positive LP cells from four (WT) and three (TGFβ RII DN) independent experiments. (IFN-γ: WT Uninfected vs. Infected, p>0.05, NS; TGF-βRII DN Uninfected vs. Infected, p>0.05, NS. IFN-γ: WT Uninfected vs. TGFβ RII DN Uninfected, *p<0.05; WT Infected vs. TGFβ RII DN Infected, *p<0.05. IL-4: WT Uninfected vs. Infected, *p<0.05; TGF-βRII DN Uninfected vs. Infected, p<0.05; WT Uninfected vs. TGFβ RII DN Uninfected *p<0.05; WT Infected vs. TGFβ RII DN Infected, p>0.05, NS).

Figure 4

Helminthic induction of IL-10 in LPMC is dependent on T cell TGF-β signaling. LPMC from uninfected or helminth-infected WT or TGF-βR DN mice were stimulated in vitro with anti-CD3/CD28 mAb for 48 hr. Supernatants were harvested and analyzed for IL-10 by ELISA. Data show mean ± SD from three independent experiments with each experiment containing multiple determinations. (WT Uninfected vs. Infected, ***p<0.001; TGF-βRII DN Uninfected vs. Infected, p>0.05, NS).

Figure 5

Effect of exogenous IL-10 on LPMC IFN-γ production from WT and TGF-βRII DN mice. LPMC from uninfected WT (A and C) or TGF-βRII DN (A and B) mice were stimulated in vitro with anti-CD3/CD28 (white bars) for 48 hr, in indicated cell numbers per well, with parallel cultures also containing 20 ng/ml (gray bars) or 100 ng/ml recombinant IL-10 (black bars). Supernatants were harvested and analyzed for IFN-γ by ELISA. Data show mean ± SD of (A) IFNγ secretion (two independent experiments with each experiment containing multiple determinations) or (B-C) the percentile ratio of LPMC IFNγ secretion in each condition to IFN-γ output of anti-CD3/28-stimulated culture (three independent experiments with each experiment containing multiple determinations). ((A) WT Uninfected vs. Infected, ***p<0.001; TGF-βRII DN Uninfected vs. Infected, p>0.05, NS). (B-C) (*p<0.05 for IFN-γ secretion between IL-10 containing and IL-10 naïve cultures in incubations with the corresponding cell numbers).

Figure 6

The reduction in worm population in WT and TGF-βRII DN mice is similar. The number of duodenal helminths was counted in 6 WT and 6 TGF-βRII DN mice at each time point, 2, 4 and 6 weeks after worm colonization. Data from 18 WT and 18 TGF-βRII DN mice in 3 different time points were accumulated in 3 independent experiments. Data show mean ± SD at each time point. (2 wk: WT vs. TGF-βRII DN, p>0.05; 4 wk: WT vs. TGF-βRII DN, p>0.05; 6 wk: WT vs. TGF-βRII DN, p>0.05, NS).

Figure 7

H. polygyrus colonization does not reverse spontaneous colitis in TGF-βRII DN mice. (A) Representative example of dissected colons from 12-14 wk old WT, uninfected control TGF-βRII DN and H. polygyrus-infected TGF-βRII DN mouse. (B-D) Representative example of colonic histology of 12-14 wk old WT (B), uninfected control TGF-βRII DN (C) and H. polygyrus-infected TGF-βRII DN (D) mouse (4x magnified). (E) Colonic tissues of uninfected or helminth-infected TGF-βRII DN mice were analyzed by histopathology. Data show mean ± SD of histologic colitis scores from 10 uninfected and 13 H. polygyrus-infected mice in 3 independent experiments (TGF-βRII DN Uninfected vs. Infected, p>0.05, NS).