The results from several studies have indicated that T cell–derived IL-10 is important for the prevention of colitis9,10,12,23
. However, here we did not identify a major role for Treg
cell–derived IL-10 in the transfer model of colitis. The reasons for our discordant findings could reflect aspects of the endogenous flora in different colonies and/or the intensity of the pathogenesis. Regardless of the source of the discrepancy, our results have demonstrated that in some circumstances, Treg
cells can prevent colitis by means other than IL-10 secretion. Most notably, we made the unexpected observation that IL-10 produced by cells other than T lymphocytes was required for Treg
cell function, despite the ability of the donor Treg
cells to secrete IL-10. Treg
cells transferred into Il10−/−Rag1−/−
recipient mice expanded in number in vivo
and homed to various tissues, including the intestine. However, these cells failed to maintain Foxp3 expression and suppressive activity in the absence of IL-10 signaling.
Despite rigorous sorting of Treg cells on the basis of Foxp3 expression, it was possible that a small population (<1%) of contaminating activated effector T cells outgrew the transferred Foxp3+ Treg cells. However, the results from an experiment in which we deliberately transferred congenic activated memory cells together with Treg cells demonstrated that these cells did not outgrow the Treg cells in Il10−/−Rag1−/− recipients. Therefore, loss of Foxp3 expression is the most plausible explanation for the ineffectiveness of Treg cells in the absence of IL-10 signaling.
Studies have indicated that the phenotype and function of Treg
cells can be unstable. For example, a minority of Treg
cells lose Foxp3 expression in vitro
when IL-6 is added24
. Also, antibody ligation of T cell immunoglobulin mucin 1 causes loss of Foxp3 mRNA expression and Treg
cell function in vitro25
. The effect of engagement of T cell immunoglobulin mucin 1 on Foxp3+
cells by its ligand Tim-4 in vivo
remains to be analyzed. In addition, a sub-population of CD25−
cells has a tendency to lose Foxp3 expression26
. However, we excluded that subset from our analysis, as our Treg
cell populations were selected for high expression of CD25. Moreover, transferred Treg
cells can generate follicular helper T cells in the Peyer’s patches under the influence of CD40 expression by B lymphocytes27
. A study using a Foxp3 reporter lineage marker system has shown that there is some spontaneous loss of Foxp3 expression in vivo
, which is enhanced in nonobese diabetic mice with autoimmune disease28
. As in our experiments, these formerly Treg
cells secrete proinflammatory cytokines.
Many cell types, including mast cells, epithelial cells and dendritic cells, synthesize IL-10 (refs. 29–32
). Our data have indicated that a population of CD11b+
myeloid cells in the LPL constitutively produced IL-10 and that a phenotypically similar population of IL-10-producing cells was greater in abundance in the MLNs early after T cell transfer. Many of the IL-10-producing myeloid cells were probably macrophages, given their expression of F4/80, although we did not exclude the possibility that other cell types were involved. Our findings are in agreement with the results of a study showing that IL-10 from lamina propria macrophages is important for the induction of Foxp3 expression33
. However, that investigation focused on cells from the small intestine rather than the large intestine and studied induction rather than the maintenance of Foxp3 expression. Despite that, the concept is emerging that intestinal macrophages are important for supporting natural and induced Treg
Why is Treg cell–derived IL-10 not sufficient for the maintenance of Foxp3 expression in mice developing colitis? We suggest that the requirement for host cell–derived IL-10 is a matter of kinetics. At early time points, few Treg cells were producing IL-10, although at 6 weeks after transfer, Treg cells in the intestine were the main IL-10 producers. Our data therefore suggest that Treg cell–derived IL-10 cannot sustain Foxp3 expression because it is induced relatively late.
The outcome of the transfer of Il10rb−/−
cells suggested that the IL-10 produced by cells in the Rag1−/−
hosts acted in part directly on the Treg
cells to maintain Foxp3 expression, although these findings do not exclude the possibility that IL-10 serves additional functions by acting on other cell types that might influence Treg
cell function. A caveat to our conclusion is that IL-10Rβ can participate in the signals delivered by several other cytokines34
. However, the concordance with the results obtained from transfer to Il10−/−Rag1−/−
recipients suggests that the ineffectiveness of the Il10rb−/−
cells was due to the absence of Treg
cell IL-10 signaling.
Our data have indicated that colitis in the Rag1−/− recipients also contributed to the loss of Treg cell Foxp3 expression. At relatively early times after transfer, when inflammation was less severe, the loss of Foxp3 was less pronounced and more localized to the intestine. However, Foxp3+ T lymphocytes developed in essentially normal numbers in Il10rb−/− mice. The Il10rb−/− mice we used were mainly on the colitis-resistant C57BL/6 background and were only 8–10 weeks old, and therefore they did not have signs of colitis (data not shown). The results from the transfer of mixed populations of Treg cells into recipients that developed colitis indicated that colitis alone could not explain the loss of Foxp3. Furthermore, Foxp3 expression was not much lower in Il10rb−/− Treg cells in recipients of mixed Treg cell populations that did not develop colitis. We conclude, therefore, that the combined effects of the absence of IL-10 signaling and the inflammatory milieu in mice with severe colitis were responsible for loss of Foxp3 expression and Treg cell function.