Regulation of Paracellular Permeability by IFNgamma and TNFa

描述（由申请人提供）：上皮屏障功能丧失是炎症、感染性、缺血性和免疫介导的肠道疾病的特征。TH1细胞因子干扰素- γ (IFNgamma)和肿瘤坏死因子- α (TNFalpha)之间的协同信号在这些疾病中经常升高，与这种屏障功能障碍有关。反过来，受损的屏障功能可允许有害的名义物质进入固有层，刺激免疫细胞，并增加IFNgamma和TNFalpha的释放，最终导致上皮功能障碍的自我放大循环。体内数据显示，抗tnf - a疗法可以逆转屏障功能障碍。我们最近表明，IFN γ / TNFalpha诱导的屏障功能丧失与肌球蛋白轻链（MLC）磷酸化增加有关，并且屏障功能丧失和MLC磷酸化增加都可以通过一种新型寡肽MLC激酶抑制剂逆转。尽管如此，IFNgamma / TNFalpha增加MLC磷酸化和降低屏障功能的机制尚不清楚。这些调节机制的特征对于理解各种肠道疾病的发病机制非常重要，也可能为IFNgamma / TNFalpha驱动的肠道疾病的治疗找到新的靶点。这可能会导致克罗恩病、肠道感染、缺血再灌注损伤和移植物抗宿主病等疾病的有效非免疫抑制疗法的发展。该建议的中心假设是IFNgamma和TNFalpha协同激活信号级联，导致TNF受体表达增加，MLC激酶表达增加，MLC磷酸化增加，紧密连接重组和上皮屏障功能障碍。本应用程序的目的是通过以下方法来验证这一假设：1)确定IFNgamma在增强上皮细胞对TNFalpha的反应性中的作用，以及IFNgamma和TNalpha协同作用以增加MLC磷酸化的机制；2)使用综合功能、生化和实时成像方法确定IFNgamma和TNFalpha对紧密连接蛋白动力学的影响。iii)利用基因敲除小鼠和药物，探索IFNgamma和TNFalpha对体内MLC磷酸化的调节作用，以防止IFNgamma / TNFalpha诱导的体外屏障功能障碍。我们预计这些研究将对人类健康产生重大的积极影响，因为它们将导致对疾病中屏障功能受损机制的新理解的发展，并将为开发增强屏障功能作为治疗方式的策略提供必要的基础。

DESCRIPTION (provided by applicant): 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.