Regulation of Toll-like Receptor 4 in Acute Inflammatory Disease

描述（由申请人提供）：我们实验室的长期目标是发现炎症是如何由微生物和非微生物刺激触发的，并在治疗上控制该过程。由于Toll样受体（TLR）功能似乎是炎症的核心，而TLR 4功能受细胞外基质（ECM）调节，因此本项目的总体目标是确定局部环境如何控制炎症以及如何破坏这种控制以启动全身炎症反应综合征（SIRS）。我们的中心假设是，健康组织中的炎症刺激被完整的ECM抑制，而疾病中的ECM降解促进或触发局部炎症，并可能导致SIRS。我们计划通过两个特定的目的来测试该中心假设：1）在体外鉴定完整ECM的TLR 4抑制组分; 2）在3种SIRS（内毒素血症、蛙皮素诱导的急性胰腺炎、对乙酰氨基酚诱导的急性肝炎）的鼠模型中建立完整ECM及其降解的体内作用。我们假设完整ECM中的硫酸乙酰肝素蛋白聚糖直接束缚TLR 4单体，从而阻止受体二聚化和TLR 4激动剂的激活。此外，我们假设体内早期SIRS事件（例如，血清细胞因子的升高）由几乎没有ECM接触的循环细胞介导。这些细胞激活降解组织ECM的酶。这种ECM分解释放在实质细胞上表达的TLR 4，其活化然后导致晚期SIRS事件（例如，远端器官功能障碍）。我们计划通过使用TLR 4阳性和TLR 4阴性HEK 293细胞、ECM板和我们实验室生产或商业获得的基质降解剂来实现第一个目标。我们计划通过分别从北卡罗来纳州大学获得的TLR 4阳性和TLR 4阴性的C57 NF-kB EGFP报告基因小鼠或我们实验室产生的小鼠产生几种不同的嵌合TLR 4小鼠来实现第二个具体目的。这些嵌合小鼠将经历SIRS的感染性和非感染性模型，并且将测量早期和晚期SIRS事件。这些研究将为先前的体外TLR 4观察结果提供机制解释，并为几种临床相关疾病模型中的全身性炎症提供可推广的模型。这些结果也将为SIRS提供潜在的新的治疗和诊断靶点。 公共卫生相关性：这些研究将描述炎症如何从肝脏和胰腺传播。这些数据可以应用于许多不同的疾病，并给出炎症的一般模型。这种模型将为治疗炎症提供新的药物靶点。

DESCRIPTION (provided by applicant): Our laboratory's long-term goal is to discover how inflammation is triggered by microbial and non-microbial stimuli and to control that process therapeutically. Since Toll-like receptor (TLR) function appears central to inflammation and TLR4 function is regulated by extracellular matrix (ECM), the overall objective of this project is to determine how local environment controls inflammation and how this control may be breached to initiate the systemic inflammatory response syndrome (SIRS). Our central hypothesis is that inflammatory stimuli are suppressed in healthy tissue by intact ECM while ECM degradation in disease facilitates or triggers local inflammation and, potentially, SIRS. We plan to test this central hypothesis through two specific aims: 1) Identify TLR4 inhibiting component(s) of intact ECM in vitro; 2) Establish the in vivo role of intact ECM and its degradation in 3 murine models of SIRS (endotoxemia, cerulein-induced acute pancreatitis, acetaminophen-induced acijte hepatitis). We hypothesize that heparan sulfate proteoglycans in intact ECM directly tether TLR4 monomers and thus prevent receptor dimerization and activation by TLR4 agonists. Moreover, we hypothesize that early SIRS events in vivo (e.g., elevation of serum cytokines) are mediated by circulating cells that have little ECM contact. These cells activate enzymes that degrade tissue ECM. This ECM breakdown releases TLR4 expressed on parenchymal cells whose activation then leads to late SIRS events (e.g., distal organ dysfunction). We plan to achieve the first aim by using TLR4 positive and TLR4 negative HEK293 cells, ECM plates, and matrix-degrading agents generated in our laboratory or obtained commercially. We plan to achieve the second specific aim by generating several different chimeric TLR4 mice from TLR4 positive and TLR4 negative C57 NF-kB EGFP reporter mice obtained from University of North Carolina or generated in our laboratory respectively. These chimeric mice will undergo infectious and non-infectious models of SIRS and both early and late SIRS events will be measured. These studies will provide a mechanistic explanation for previous in vitro TLR4 observations and a generalizable model for systemic inflammation in several clinically relevant disease models. These results will also provide potential new therapeutic and diagnostic targets for SIRS. PUBLIC HEALTH RELEVANCE: These studies will describe how inflammation spreads from the liver and pancreas. This data could apply to many different diseases and give a general model for inflammation. This model would give new drug targets to treat inflammation.