Interplay between Pathogenic and Immune Regulatory Mechanisms in G-alpha-i2 deficient colitis

Abstract: The two major forms of inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC) are gastrointestinal disorders characterized by chronic and relapsing inflammation. Mice deficient in the inhibitory G protein subunit G?i2 spontaneously develop chronic colitis and have been used as a model for IBD, with particular similarities to UC. They have been used in this thesis to study the changes in immune pathology during disease progression; to investigate the role of regulatory T cells (Tregs) in chronic intestinal inflammation; and as a model for testing anti-inflammatory agents. During the progression of the colitis, continuing increases in colon weight/cm and spleen weight, and a gradual decrease in thymus weight were observed. The proteins levels of proinflammatory cytokines/chemokines IL-1?, IL-6, IL-12p40, IL-17, TNF-?, CCL2 and CXCL1 increased during colitis progression and were all significantly increased in mice with moderate and severe colitis in colonic, but not small intestinal tissues. In colon, IFN-? mRNA and IL-27 mRNA were gradually elevated during the course of the colitis, whereas IL-21 mRNA expression was significantly enhanced in mice with severe colitis. Thus, the lack of G?i2 elicits an expansion of gut Th17 responses, possibly as a result of a G?i2–/–-driven epithelial barrier defect; this drives the production of neutrophil-attractant chemokines, resulting in the influx of neutrophils; in turn promoting an adaptive Th1 response. Numbers of lamina propria CD4+FoxP3+ cells were significantly increased in colitis and were dispersed in the tissue, in contrast to non-inflamed colon in which they concentrated within organized lymphoid structures. In vitro data showed that both G?i2–/– and wild-type (WT) splenic Tregs were able to suppress wild type (WT) effector T cells (Teffs) , but that pathogenetic G?i2–/– Teffs could not be suppressed by either type of Tregs. Adoptive transfer experiments in the colitis model showed that neither type of Tregs could prevent disease induced by co-transfer of G?i2–/– Teffs. It is possible that Treg regulatory function is suppressed in the inflamed colonic milieu, and/or that they are unable to overcome the heightened activity of G?i2–/– Teffs. Acute colitis induced by dextran sodium sulphate (DSS) and spontaneous G?i2?/? chronic colitis were used to assess the efficacy of ex vivo anti-inflammatory treatment. The gene profiles reflected the different mechanisms underpinning these two types of colitis. Thus, genes related to pro-inflammatory innate cytokines, chemokines and chemokine receptors were up-regulated in DSS-induced colitis, whereas genes related to T cell markers were preferentially elevated in G?i2?/? colitic tissues. In general, the same panel of genes displayed increased transcription in the in vivo and ex vivo cultured tissues in DSS model. The well-characterised corticosteroid methyl-prednisolone and the proteasome inhibitor MG132, were used to compare the efficacy between in vivo treatment and ex vivo cultures of colon obtained from DSS-induced and G?i2–/– colitic mice. After steroid treatment, IL-1?, IL-6 and iNOS were suppressed in both models, both in in vivo and ex vivo. The anti-inflammatory function of methyl-prednisolone was mostly at the innate level, as shown in DSS-colitis, but MG132 acted effectively on the chronically activated adaptive immune response in the G?i2?/? colitis model. Thus the changes in inflammatory gene expression in the murine ex vivo culture system reflected the in vivo response in the inflamed colonic tissue, suggesting that the murine culture system can be used for validation of future IBD therapies. We conclude that the genetic G?i2 deficiency leads to amplification of gut Th17 responses, possibly mediated by a gut barrier defect, and this leads to a mixed Th17/Th1 effector phenotype in late-stage colitis. The hyper-reactive G?i2?/? effector T cells further amplify the inflammation and increased numbers of memory effector T cells are generated as disease progresses. The lack of appropriate regulation by Tregs further exacerbes the G?i2?/? colitis. Key words: IBD, G?i2?/? colitis, Tregs, Teffs, Th17/Th1, pathogenesis/immunoregulation