Spontaneous amelioration of inflammation (often accompanied by fibrosis) is a well-known, but poorly understood outcome of many chronic inflammatory processes, including that occurring in Crohn's disease. We studied this phenomenon using the chronic TNBS-colitis model developed initially by Lawrance and his colleagues. As shown here and previously, the colonic inflammation in this model begins as an acute and severe inflammation dominated by the production of IL-12p70 and IFN-γ. It then morphs into a less intense but on-going inflammation characterized by IL-23, IL-25 and IL-17 production. This set of cytokines then supports the development of IL-13 and TGF-beta1 responses that play a major role in the development of a fibrotic program that is quite distinct from the underlying inflammatory program. In the present study, in which the chronic colitis was observed over a longer period of time than previously, it was found that the IL-23 and IL-17 responses peaked on day 49 and then underwent a precipitous decline, so that at day 91 they had returned to near background levels. This drop occurred during a resolution phase of the colitis during which inflammation subsided and was presumably the cause of such subsidence. On the other hand, the IL-13 responses were much more persistent and never dropped to baseline levels. This corresponded to the fact that resolution of the inflammation was dependent on the continued secretion of IL-13, as evidenced by the fact that maneuvers that blocked such secretion led to continued colitis. It was therefore evident that IL-13 was driving a program that ameliorated inflammation. In searching for a mechanism to explain how IL-13 signaling could be leading to modulation of the disease we considered the possibility that IL-13 could be affecting the function of GSK-3beta, a previously well studied serine/threonine kinase central to glucose/glycogen metabolism and insulin activity that also has been shown to regulate the reciprocal production of pro-inflammatory cytokines (e.g., IL-12p70) and anti-inflammatory cytokines (e.g., IL-10) in antigen-presenting cells. A key observation made in this study that in fact connected IL-13 to GSK-3beta and resolution of colitis was our demonstration that GSK-3beta phosphorylation status reflected the pattern of inflammation in chronic TNBS-colitis and that IL-13 signaling via STAT6 could result in GSK-3beta phosphorylation. In addition, we showed that in vivo blockade of IL-13 signaling by sIL-13Ralpha2-Fc as well as by STAT6 decoy oligonucleotides or STAT6-deficiency not only blocked resolution of the inflammation, but also led to facilitation of NF-kappaB signaling. Finally, and perhaps most importantly, we showed that administration of two separate and specific inhibitors of GSK-3beta activation (which maintained GSK-3beta in a phosphorylated state and thus mimicked the effect of IL-13 signaling) throughout the late phase of TNBS-colitis led to resolution of the colitis in the face of a block in IL-13 signaling. Similarly, late stage in vivo down-regulation of GSK-3beta by administration of a GSK-3beta-specific siRNA also had the effect of causing resolution of colitis. In studies of the proximal mechanism of inflammation resolution we focused on the role of the anti-inflammatory cytokine, IL-10. It has been shown in prior studies that IL-13-induced GSK-3βphosphorylation leads to increased IL-10 production and, indeed, high level IL-10 production occurs throughout the resolution phase of chronic TNBS-colitis. Such IL-10 could be tied to upstream IL-13 effects in the present study by showing that blockade of IL-13 signaling by sIL-13Ralpha2-Fc administration inhibited IL-10 production. In summary, the data presented in this study demonstrates that IL-13 induced during the course of chronic TNBS-colitis is the central factor in the spontaneous resolution of this colitis. The key mechanism underlying this IL-13 effect is the inactivation (phosphorylation) of GSK-3beta which in turn leads to blockade of TLR-induced inflammation;in addition such inactivation leads to the up-regulation of IL-10, an anti-inflammatory factor.
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