Loss of epithelial barrier function is characteristic of inflammatory, infectious, ischemic, and immune mediated intestinal diseases. Synergistic signaling between the TH1 cytokines interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha), which are frequently elevated in these diseases, has been implicated in this barrier dysfunction. In turn, compromised barrier function can allow noxious nominal material to access the lamina propria, stimulate immune cells, and augment IFNgamma and TNFalpha release, culminating in a self-amplifying cycle of epithelial dysfunction. In vivo data show that barrier dysfunction can be reversed by anti-TNF a therapies. We have recently shown that IFN gamma / TNFalpha -induced loss of barrier function is related to increased myosin light chain (MLC) phosphorylation and that both loss of barrier function and increased MLC phosphorylation can be reversed by a novel oligopeptide MLC kinase inhibitor. Despite this, the mechanisms by which IFNgamma / TNFalpha increase MLC phosphorylation and decrease barrier function are not well understood. Characterization of these regulatory mechanisms is important to understanding the pathogenesis of diverse intestinal diseases and may also identify novel targets for therapy of IFNgamma / TNFalpha -driven intestinal disease. This may lead to the development of effective non-immunosuppresive therapies for diseases such as Crohn's disease, enteric infection, ischemia-reperfusion injury, and graft versus host disease. The central hypothesis of this proposal is that IFNgamma and TNFalpha synergize to activate a signaling cascade that results in increased TNF receptor expression, increased MLC kinase expression, increased MLC phosphorylation, tight junction reorganization, and epithelial barrier dysfunction.
The aims of this application are to test this hypothesis by i) determining the role of IFNgamma in enhancing epithelial responsiveness to TNFalpha and the mechanisms by which IFNgamma and TNalpha synergize to increase MLC phosphorylation, ii) defining the effects of IFNgamma and TNFalpha on tight junction protein dynamics using integrated functional, biochemical, and real time imaging approaches, and iii) exploring the effects of IFNgamma and TNFalpha on the regulation of MLC phosphorylation in vivo using knockout mice and pharmacologic agents that prevent IFNgamma / TNFalpha -induced barrier dysfunction in vitro. We expect that these studies will have significant positive effects on human health because they will lead to the development of new understanding of the mechanisms by which barrier function is compromised in disease and will provide the foundation necessary for the development of strategies for enhancement of barrier function as a therapeutic modality.
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