In this study, we demonstrated that helminths living in the duodenum promote TCR-triggered TGF-β production by LPMC isolated from the distal intestine. This study also showed the essential role of TGF-β in regulating intestinal cytokine synthesis, as mice with a T cell-specific TGF-β signaling defect fail to control intestinal Th1 or Th2 cytokine production. Loss of T cell TGF-β signaling abolished helminth-associated priming of immune regulatory mucosal cytokine (TGF-β and IL-10) production. The negligible amount of IL-10 production in LPMC from helminth-infected TGF-βRII DN mice was insufficient to control the robust LP T cells IFN-γ output. Interference with the TGF-β signaling to T cells also abrogated worm-induced regulation of colitis.
Intact TGF-β signaling is required to limit intestinal mucosa IFN-γ production in uninfected mice, as the number of IFN-γ+ LP T cells was increased by 4 to 5-fold and intestinal IFN-γ production by 25-fold in TGF-βRII DN animals compared to WT mice. The IFN-γ signal intensity in cytokine staining Thy1.2+ cells from TGF-βRII DN mice is increased compared to the IFN-γ signal intensity in cytokine staining Thy1.2+ LPMC from their WT counterparts (). The overall IFN-γ cytokine fluorescence intensity by flow cytometry was 2.5-fold increased in Thy1.2+ cells from TGF-βRII DN mice compared to WT animals (). This discrepancy between 4 to 5-fold increase in IFN-γ producing cells and 25-fold increase in IFN-γ output suggests that T cells from the TGF-βRII DN mice release more IFN-γ per cell compared to WT T cells. Similar correlations between cytokine fluorescence intensity by flow cytometry, the spot size by ELISPOT and the amount of cytokine secretion by ELISA have been reported previously [21
]. Helminth colonization did not suppress IFN-γ synthesis in TGF-βRII DN mice, although helminth infection almost completely shut down LP T cell IFN-γ generation from WT animals. Thus, helminthic modulation of intestinal mucosal IFN-γ production requires intact T cell TGF-β signaling.
Regulation of IFN-γ responses could be mediated through several mechanisms. Helminths may induce various Th2 cytokines (e.g. IL-4, IL-13) and other regulatory agents (e.g. IL-10, TGF-β) that may limit or modulate Th1-type activity [23
]. Another possibility is that worms may induce deletion of IFN-γ producing cells from the intestinal mucosa. We found by flow cytometry that IFN-γ-expressing T cells remain in the lamina propria of colonized WT mice. LP T cell IFN-γ production from helminth-infected WT mice could be restored by blocking IL-10 in vitro
]. These data imply that Th1 cell deletion is not the mechanism that prevented TCR-stimulated IFN-γ release in helminth-colonized WT mice.
Strong Th2 cytokine production (IL-4 and IL-5) did not appear to limit mucosal IFN-γ secretion in TGF-βRII DN mice. We found strong Th2 responses in the mucosa of uninfected TGF-βRII DN mice, almost as strong as after helminth colonization in WT mice. T cell TGF-β signaling curtails Th2 cell differentiation presumably by inhibiting T cell GATA3 expression [25
]. In this paper, we demonstrate that T cell TGF-β signaling is essential for limiting either the development or activity of Th2 cells in the normal worm-naïve gut. LPMC IL-4, IL-5 and TGF-β secretion were augmented in helminth-infected WT mice. This suggests that helminthic induction of intestinal IL-4 and IL-5 are not inhibited by the concurrent induction of TGF-β, since abolition of TGF-β-signaling did not result in hyper-expression of IL-4 or IL-5 after worm colonization. It is possible that redundant mechanisms beyond TGF-β signaling limit excessive Th2 responses after helminth colonization.
Helminth-induced Th2 responses can enhance intestinal motility that helps worm elimination [4
]. Although the Th2 cytokine responses were robust in intestinal mucosa of TGF-βRII DN mice, the worm counts at different time points after infection were not significantly altered compared to WT animals. Thus, failure to achieve helminthic regulation of mucosal T cell IFN-γ production could not be attributed to differences in worm numbers between WT and TGF-βRII DN mice.
IL-10 is a master regulator of Th1 cells [26
]. TGF-β is also a strong inhibitor of Th1 differentiation [17
]. In the absence of TGF-β signaling to T cells, LP T cell IFN-γ secretion increases dramatically. LPMC Th1 cytokine production in TGF-βRII DN mice can be regulated by exogenous IL-10, when cells are cultured at lower densities, as seen in WT mice (). T cells are a major source of IL-10 during helminth infection [7
] and helminthic induction of IL-10 by T lymphocytes correlates with down-regulation of T cell IFN-γ output [27
]. As we show here, helminthic induction of intestinal IL-10 requires intact TGF-β signaling to T cells. Thus, T cell TGF-β pathway controls intestinal Th1 responses at multiple levels: TGF-β interferes with Th1 differentiation, thereby curtailing robust Th1 responses and TGF-β signaling to T cells is essential for the induction of intestinal T cell IL-10 production, which is important in suppressing Th1 cell IFN-γ output.
IL-10 producing T cells are also named Tr1 and they can originate from various effector T cell subsets, such as Th1, Th2, Th17 or CD8 T cells [12
]. IL-10 may also originate from FoxP3 positive or FoxP3 negative regulatory T lymphocytes [28
]. TGF-β has been shown to potentiate IL-10 production from Th1, Th17 effector, FoxP3 positive or FoxP3 negative regulatory T cells [15
]. TGF-β may exert its dominant control on LP T cell IL-10 gene expression by activating Smad4 binding to the IL-10 gene promoter [30
]. Helminth colonization results in enhanced TGF-β or Th2 cytokine production, increased T cell FoxP3 gene expression and novel regulatory CD8 T cells in the intestine, while helminths suppress intestinal Th1 and Th17 cytokine output [7
]. Currently it is unclear, which intestinal T lymphocyte subset plays a major role in IL-10 production during worm infection.
Although helminthic regulation of mucosal inflammation or IFN-γ production is in part IL-10-mediated [7
], H. polygyrus
colonization can still regulate IFN-γ production in IL-10 deficient mice [31
]. These results suggest that additional immune regulatory mechanisms besides IL-10 induction are employed by helminths. These mechanisms may include helminthic induction of intestinal TGF-β production. H. polygyrus
stimulates LP T cell TGF-β release in IL-10 deficient animals (unpublished observations) similar to WT mice (). T cell generated TGF-β is an important regulator of intestinal immune balance [34
]. LPMC TGF-β production is diminished in TGF-βRII DN mice. Whether T cell produced TGF-β is important in H. polygyrus
-mediated immune regulation and induction of IL-10 producing T cells remains to be established.
In summary, we demonstrate here that TGF-β signaling to T cells is essential in helminthic mucosal immune regulation. Although helminths regulate mucosal Th1 cytokines and colitis in WT mice [7
], absence of T cell-specific TGF-β signaling prevents helminthic, and in part IL-10-mediated, regulation of IFN-γ secretion. Moreover, absence of T cell-specific TGF-β signaling disallows helminthic regulation of colitis.