Defects in junctional adhesion molecule-A and Intestinal Permeability: Identification of Novel Mechanisms Driving Non-Alcoholic Steatohepatitis

项目摘要 非酒精性脂肪性肝病(NAFLD)对越来越多的美国人构成了主要的健康风险。 非酒精性脂肪性肝炎(NASH)是一种更具侵袭性的肝脏病变，与NAFLD一起出现的是肝脏 代谢综合征(METS)的表现，胰岛素抵抗和炎症被认为是 关键驱动因素。这一建议利用了紧密连接(TJ)中肠道通透性缺陷的小鼠。 由于靶向删除连接粘连而发展的肠上皮的 分子A(果酱-A)。初步数据，从JAM-A全球基因敲除(JAM-A-/-)和VillinCreJAM- AFL/FL小鼠集落饲喂肥胖性饮食(HFCD)，表现出强烈的脂肪性肝炎和早期肝脏 8周后星状细胞(H、S、C)激活。这些数据提出了一些关键的悬而未决的问题 关于与肠道通透性相关的分子功能障碍以及与 纳什进展。提出的模型将提供一个独特的机会来阐明分子和细胞 肠道屏障功能受损，脂多糖(LPS)显著易位的机制， 体内测定，有利于NASH的发生发展。因此，中央 这一应用的假说是西方饮食诱导的肠道生物失调与受损的屏障功能协同作用 VillinCreJAM-AFL/FL小鼠促进内毒素转位到肝脏，促进由此产生的炎症和纤维化 在纳什进程中。核心假说将通过以下综合的具体目标进行检验。目标1： 证明肠道内毒素和肝脏先天免疫激活之间的直接联系，从而驱动NASH 肠通透性改变的发生和发展。我们假设肝脏 肠道内毒素刺激的TLR激活促进HFCD喂养的VillinCreJAM-AFL/FL小鼠的肝脏炎症。我们会 通过确定一种可以结合内毒素，西维拉姆， 减轻HFCD喂养的VillinCreJAM-AFL/FL小鼠的肝脏炎症和纤维化。目标2：测试 NASH的发展依赖于肠道通透性增加之间的协同关系 在HFCD喂养的VilllinCreJAM-AFL/FL小鼠和HFCD诱导的促炎肠道微生物群中：WE 假设西方饮食诱导的肠道微生物失调会导致黏膜炎症，从而进一步 破坏肠道TJ分子组装，严重损害肠道屏障功能。目标3：核实 HFCD喂养的VilllinCreJAM-AFL/FL小鼠因自然杀伤细胞受损而加速纤维化 (NK)细胞功能，促进肝星状细胞(HSC)存活。我们假设HFCD介导的 NK细胞耗竭或功能受损可促进VillinCreJAM-AFL/FL小鼠HSC存活。给定 初步数据、提出的假设和目标，这一独特的NASH模型将提供机械性的见解 这将很有可能成为NASH潜在治疗的临床适用性。

PROJECT ABSTRACT Non-alcoholic fatty liver disease (NAFLD) poses a major health risk to a growing sector of Americans. Non-alcoholic steatohepatitis (NASH) - a more aggressive liver lesion - along with NAFLD are the hepatic manifestations of metabolic syndrome (MetS), for which insulin resistance and inflammation are thought to be key drivers. This proposal takes advantage of mice with an intestinal permeability defect in tight junctions (TJs) of the intestinal epithelium that develops as a consequence of a targeted deletion of the junctional adhesion molecule A (JAM-A). Preliminary data, obtained from both JAM-A global knockout (JAM-A-/-) and VillinCreJAM- AFL/FL mouse colonies fed an obesogenic diet (HFCD), demonstrate robust steatohepatitis and early hepatic stellate cell ( H S C ) activation after eight weeks. These data raise critical unanswered questions regarding the molecular dysfunction associated with intestinal permeability and direct association with NASH progression. The proposed model will provide a unique opportunity to elucidate the molecular and cellular mechanisms responsible for impaired intestinal barrier function, significant lipopolysaccharide (LPS) translocation, as measured in vivo, that are conducive to development and progression of NASH. Therefore, the central hypothesis of this application is western diet-induced gut dysbiosis synergizes with impaired barrier function of VillinCreJAM-AFL/FL mice to facilitate translocation of LPS to the liver, promoting inflammation and fibrosis resulting in NASH progression. The central hypothesis will be tested by the following integrated specific aims. AIM 1: Demonstrate a direct link between gut LPS and hepatic innate immune activation that drives NASH development and progression in the setting of altered intestinal permeability. We hypothesize that hepatic TLR activation driven by gut LPS drives hepatic inflammation in HFCD-fed VillinCreJAM-AFL/FL mice. We will substantiate our hypothesis by determining whether treatment with an agent that can bind LPS, sevelamer, attenuates hepatic inflammation and fibrosis in HFCD-fed VillinCreJAM-AFL/FL mice. AIM 2: To test whether NASH development is dependent on a synergistic relationship between increased intestinal permeability in HFCD-fed VilllinCreJAM-AFL/FL mice and a HFCD-induced pro-inflammatory gut microbiota: We hypothesize that Western diet-induced gut microbial dysbiosis results in mucosal inflammation that further disrupts intestinal TJ molecular assembly and severely compromises gut barrier function. AIM 3: To verify that HFCD-fed VilllinCreJAM-AFL/FL mice have accelerated fibrosis as a consequence of impaired natural killer (NK) cell function that promotes hepatic stellate cell (HSC) survival. We hypothesize that HFCD-mediated depletion or functional impairment of NK cells promotes HSC survival in VillinCreJAM-AFL/FL mice. Given the preliminary data, proposed hypothesis and aims, this unique model of NASH will provide mechanistic insights that will have a high likelihood of clinical applicability for potential treatment for NASH.