Project 1:? ? To investigate the immunopathogenesis of inflammation-associated fibrosis we analyzed the recurrent colitis and late-developing fibrosis occurring in BALB/c mice administered weekly doses of intra-rectal trinitrobenzene sulfonic acid (TNBS). We showed first that in this model an initial Th1 response involving IL-12p70 and IFN-g subsides after three weeks only to evolve into a IL-23/IL-25 response beginning after 4-5 weeks. This evolution is followed by gradually increasing production of IL-17 and cytokines ordinarily seen in a Th2 response, particularly IL-13, that reaches a plateau at 8-9 weeks. In vitro stimulation studies suggest that this IL-13 production is dependent on IL-23 and IL-25, but not on IL-12p70. We then show that IL-13 production results in the induction of a novel IL-13 receptor formerly thought to function only as a decoy receptor, IL-13Ra2, and this receptor is critical to the production of TGF-b1 and the onset of fibrosis. Thus, if IL-13 is blocked by soluble IL-13Ra2-Fc, TGF-b1 is not produced and/or fibrosis does not occur. These studies show that in recurrent TNBS colitis, fibrosis is dependent on the development of an IL-13 response that acts through a novel cell-surface-expressed IL-13 receptor to induce TGF-b1. A similar mechanism may obtain in certain forms of human IBD. ? ? ? Project 2:? ? To explore the function of Stat4 in Th1 mucosal inflammation we studied mice with reduced (but not absent) Stat4 expression due to the presence of a doxycycline-regulated Stat4 anti-sense transgene or intra-rectal administration of a Stat4 anti-sense plasmid. Initially we showed that SJL/J mice treated with doxycycline and expressing the transgene do not develop TNBS-colitis, a Th1 mucosal inflammation. This seemed due to a cell traffic problem since these mice exhibited accumulation of potential effector cells in the spleen (rather than in the intestine) that displayed decreased Th1-chemokine receptor expression. To further examine this possibility we transferred spleen cells from mice administered TNBS with or without doxycyline to recipients given low doses of TNBS; we found that transfer of cells from mice administered doxycycline did not result in colitis whereas those administered doxycline did, even though both recipient post-transfer spleens contained potential Th1 effector T cells. Finally, we showed that mice administered a plasmid expressing the Stat4 anti-sense construct also displayed decreased TNBS-colitis and reduced chemokine receptor expression. These studies demonstrate that Stat4 down-regulation leads to failure of effector cell traffic to the lamina propria and such down-regulation is a viable approach to the treatment of Crohn?s disease.
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