Immunoregulatory Defects In Inflammatory Bowel Disease

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Abstract

Project 1: It is now well recognized that mucosal immune responses are controlled by regulatory T cells secreting suppressor cytokines and that many experimental models of mucosal inflammation are due to defects in teh generation of these cells. In the present study we explored the mechanisms of such regulation in a study in which we defined the relation between TGF-beta and IL-10 secretion in the control of the Th1-mediated inflammation occurring in trinitrobenzene sulfonic acid (TNBS)-colitis. In initial studies we showed that feeding of TNP-haptenated colonic protein (HCP) to SJL/J mice induces CD4+ regulatory T cells that transfer protection from induction of TNBS-colitis and that such protection correlates with cells producing TGF-beta, not IL-10. Further studies in which SJL/J mice were fed HCP and then administered either anti-TGF-beta or anti-IL-10 at the time of subsequent TNBS administration per rectum showed that while both antibodies abolished protection, anti-TGF-beta administration prevented TGF-beta secretion but left IL-10 secretion intact whereas anti-IL-10 administration prevented both TGF-beta secretion and IL-10 secretion. Thus, it appeared that the protective effect of IL-10 was an indirect consequence of its effect on TGF-beta secretion. To establish this point further we conducted adoptive transfer studies and showed that anti-IL-10 administration had no effect on induction of TGF-beta-producing T cells in donor mice but did inhibit their subsequent expansion in recipient mice, probably by regulating the magnitude of the Th1 T cell response which would otherwise inhibit the TGF-beta response. These studies thus suggest that TGF-beta production is the primary mechanism of counter-regulation of Th1 T cell-mediated mucosal inflammation and that IL-10 is necessary as a secondary factor that facilitates TGF-beta production. Project 2: Gene therapy utilizing genes encoding regulatory cytokines is an attractive goal for the treatment of autoimmune inflammation because the amount of DNA that is necessary for a therapeutic effect, like the regulatory cytokine it encodes, can be quite modest. In previous studies reported in the last Annual Report, we showed that intranasal delivery of a plasmid encoding active TGF-beta 1 under a CMV promotor could both prevent and treat TNBS-colitis, a Th1-mediated form of mucosal inflammation. More specifically, we showed that the administered plasmid is taken up by T cells and macrophages which traffic to the mucosal surface and which produce both TGF-beta and IL-10. We then showed that the latter cytokines inhibit the Th1 response by blocking both IL-12 synthesis and signaling. Recognizing that TGF-beta may have substantial side effects, in the present study we tested TGF-beta-encoding plasmid under a regulatable pTet-on promotor which only produces TGF-beta1 when the plasmid is co-administered with doxycycline. More particularly, we showed that on intranasally administered doxycycline-regulated plasmid (pTet-on-TGF-beta1) leads to the induction of TGF-beta-producing cells in the mucosal lamina propria that are tightly controlled by the presence or absence of doxycycline. In addition, we showed that such cells can prevent or cure a Th1-mediated mucosal inflammation (TNBS-colitis) in mice co-administered doxycycline. Interestingly, the TGF-beta1 produced by the plasmid-induced cells secondarily evoked a high IL-10 response; thus, the inflammation is down-regulated by two suppressive cytokines. Finally, the pTet-on-TGF-beta1 did not induce fibrosis, but rather blocks fibrosis caused by a known fibrosis-inducing agent, bleomycin. In conclusion, these studies showed that gene therapy of mice by intranasal delivery of a regulatory DNA plasmid encoding TGF-beta can be an effective treatment of TBNS-colitis. Furthermore, such treatment does not cause fibrosis presumably due to the co-secretion of IL-10.