We demonstrate conclusively that Il27ra−/− CD45Rbhi T cells are less colitogenic than their WT counterparts in the murine transfer colitis model. Our analysis of the T cell graft after 12 wk of incubation in lymphopenic hosts revealed that a much greater proportion of IL27ra−/− CD45Rbhi T cells assumed a Foxp3+ phenotype, whereas the remaining Foxp3− cells proliferated less and produced much less of the TH1 cytokine IFN-γ. Although there were mild relative increases in Il27ra−/− effector cells producing TH2 and TH17 cytokines, the lower abundance of Il27ra−/− effector cells effectively neutralized this difference, which is consistent with the absence of an increase in neutrophil infiltration into the lamina propria. Therefore, the reduced colitis and weight loss in recipients of Il27ra−/− CD45Rbhi T cells is a consequence of increased Treg conversion, lower IFN-γ production, decreased effector cell proliferation, or a combination of these effects.
Together, the changes in T cell phenotype and abundance form a chicken and egg conundrum: do effector cells proliferate less because there are more Foxp3+
cells present, or is there an increase in Foxp3+
cells because effector cells fail to proliferate and cause inflammation efficiently in the absence of IL-27Ra, which in turn de-represses Treg
conversion? Several of our data points favor the first of these two possibilities: First, IL-27 is known to antagonize TGF-β driven Treg
conversion in vitro (; Neufert et al., 2007
; Huber et al., 2008
), and thus conversion is expected to be de-restrained in Il27ra−/−
T cells in vivo. Second, we observed an increase in absolute numbers, not just relative percentages, of Il27ra−/−
T cells in our OVA-dependent tolerization model (). This difference persisted even in the context of established colitis (), and thus represents a cell intrinsic effect that is independent of the inflammation status of the environment into which the cells are transferred. The OVA oral tolerance model is a much shorter term experiment than the 12-wk colitis study, and is therefore more amenable to mechanistic interpretation. And lastly, although a reduction in IFN-γ production could potentially explain the reduction in colitis, Treg
conversion of IFN-γ−/−
T cells was not enhanced, and they still caused significant wasting disease. Therefore, reduced Treg
conversion is not secondary to reduced production of IFN-γ in the absence of IL-27Ra. In summary, these observations provide strong suggestive evidence that the primary effect of IL-27 in this context is to restrain Treg
We attempted to therapeutically rescue mice with preestablished transfer colitis by introducing Il27ra−/− CD45Rbhi cells, but this experiment was not successful (Fig. S3). Although a trend toward increased Foxp3 expression was still noticed 6 wk after transfer of the Il27ra−/− CD45Rbhi cells into the inflammatory environment, and although the transferred cells clearly produced less IFN-γ (Fig. S3, F and G), we did not detect any changes in weight loss or colon length. This result suggests that although de-repression of Treg conversion through loss of IL-27Ra at the onset of inflammation significantly affects the ultimate outcome, it is not robust enough to overcome preestablished inflammation.
Earlier studies by our group and others showed that IL-27 can induce IL-10 production in T cells and lead to the development of Foxp3−
Tr1 cells (Awasthi et al., 2007
; Fitzgerald et al., 2007
; Stumhofer et al., 2007
; Batten et al., 2008
). Because Tr1 cells have potent immunoregulatory effects (Anderson et al., 2007
; Jankovic et al., 2007
; Trinchieri, 2007
), it has been postulated that this is a mechanism by which IL-27 exerts immune suppression in infectious and autoimmune disease. These earlier observations are at odds with the proinflammatory role of IL-27 described here. However, in transfer colitis, IL-10 production by cells originating from the Foxp3−
graft is minimal (Uhlig et al., 2006
). We found no Il27ra
-dependent difference in IL-10 production by transferred CD45Rbhi
cells, and IL-10 production in general was minimal in this model (unpublished data). Thus, the Foxp3-suppressing effects of IL-27 are not in contradiction with its Tr1-inducing effects; the dichotomy merely reflects the differences between the physiological contexts in which IL-27 stimulation occurs and may explain why the essential function of IL-27 in the regulation of Treg
differentiation has not been noted previously.
It is perhaps appropriate to point out that the majority of in vivo effects assigned to IL-27 have been inferred from the analysis of Il27ra−/−
mice, and only some have been confirmed by studies of Il27p28−/−
mice. This study is no different in that regard. To date, IL-27 is the only confirmed ligand for IL-27Ra. However, IL-35 has been described as an IL-27–related heterodimer with potent immunoregulatory effects (Collison et al., 2007
). IL-35 consists of IL-12p35 and Ebi3, and although its receptor has not been identified, IL-27Ra remains a candidate. Another recently described IL-27–related heterodimer consists of IL-27p28 and cytokine-like factor and appears to bind to IL-27Ra, but conclusive proof that IL-27Ra is required for signaling is currently not available (Crabé et al., 2009
). Thus, it remains possible that IL-27Ra has ligands other than IL-27, which might further contribute to the apparent complexity and dichotomous nature of IL-27 biology in vivo.
In summary, we demonstrate that IL-27 exerts proinflammatory effects in the T cell transfer colitis model. Our experiments demonstrate conclusively that IL-27 acts to suppress induced Treg development in vivo, and thus reveal a hitherto unrecognized proinflammatory mechanism. Collectively, our results suggest that targeting of IL-27 in situations where pathology results from a breakdown in Treg-mediated tolerance may result in significant therapeutic benefit.