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微生物和非微生物刺激是如何引发炎症的，并从治疗的角度控制这一过程。由于Toll样受体(TLR)的功能似乎是炎症的核心，TLR4的功能受细胞外基质(ECM)的调节，本项目的总体目标是确定局部环境是如何控制炎症的，以及这种控制如何被打破而引发全身炎症反应综合征(SIRS)。我们的中心假设是，炎症刺激在健康组织中被完整的ECM抑制，而疾病中ECM的降解促进或触发局部炎症，并潜在地引发SIRS。我们计划通过两个特定的目标来验证这一中心假说：1)在体外鉴定完整ECM的TLR4抑制成分(S)；2)在3种SIRS(内毒素血症、蓝蛋白诱导的急性胰腺炎、对乙酰氨基酚诱导的酸性肝炎)小鼠模型中，建立完整的ECM的体内作用及其降解作用。我们推测，完整ECM中的硫酸乙酰肝素蛋白多糖直接连接TLR4单体，从而阻止受体二聚化和TLR4激动剂的激活。此外，我们假设体内早期的SIRS事件(例如，血清细胞因子升高)是由与ECM接触很少的循环细胞介导的。这些细胞激活降解组织细胞外基质的酶。这种ECM的破坏释放了在实质细胞上表达的TLR4，这些细胞的激活然后导致晚期SIRS事件(例如，远端器官功能障碍)。我们计划通过使用TLR4阳性和TLR4阴性的HEK293细胞、ECM板和我们实验室生产的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大学获得的或我们实验室培育的TLR4阳性和TLR4阴性的C57 NF-kB EGFP报告小鼠中产生几种不同的嵌合TLR4小鼠来实现第二个特定目的。这些嵌合小鼠将接受感染和非感染的SIRS模型，并将测量早期和晚期的SIRS事件。这些研究将为之前在体外观察到的TLR4提供一个机制解释，并为几个临床相关疾病模型中的系统性炎症提供一个通用的模型。这些结果也将为SIRS提供潜在的新的治疗和诊断靶点。 与公共卫生相关：这些研究将描述炎症是如何从肝脏和胰腺传播的。这些数据可能适用于许多不同的疾病，并给出了炎症的一般模型。这一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.