Molecular and cellular mechanism regulating innate immunity and inflammation during pattern recognition receptor activation and respiratory virus infection

先天免疫构成促炎反应，启动炎症反应进行适应性免疫反应 清除病毒所需的。促炎(和炎症)反应需要“严格”调控 在病毒感染期间，因为过度的炎症会导致炎症性疾病。激活 模式识别受体(PRRs)是触发先天免疫和炎症的关键 感染人类呼吸道合胞病毒(RSV)和甲型流感病毒(IAV)。因此，重要的是要 研究转导最佳先天免疫的潜在细胞/分子机制 PRR激活时的炎症(和促炎)反应。我们出人意料地发现了一部小说 PRR激活后最佳促炎反应所需的信号网络。我们的 初步结果表明，氧固醇(一种生物活性脂质)25-羟基胆固醇(25HC)起着 在PRRs和PRRs激活后放大和加强促炎反应中的重要作用 在病毒(RSV、IAV)感染期间。令人惊讶的是，我们观察到25HC与细胞表面整合素和 进一步的初步研究提出了一种可能的机制。从机制上讲，我们假设25HC释放 来自PRR激活(和病毒感染)的细胞作为细胞外可溶性介质激活25HC 整合素(51，V3整合素)-粘着斑激酶-最佳促炎作用的核因子B途径 回应。因此，我们可以预见，细胞外25HC作为一个“连接物”(通过自分泌/旁分泌机制)发挥作用 在PRR通路和整合素通路之间架起桥梁，以实现最佳的促炎反应。我们假设-a) 胞外25HC连接PrR途径和整合素途径；b)PrR-25HC-整合素-FAK-NFB信号 网络放大促炎反应和炎症；以及c)25HC-整合素-FAK信号 在RSV和IAV感染过程中，途径导致过度炎症，从而导致发展 肺炎和毛细支气管炎等加重的呼吸道疾病。在目前的提案中，我们选择了一个 膜结合的PRR(即Toll样受体3或TLR3)和胞质PRR(即NOD2)以及两个 临床重要呼吸道病毒25HC-整合素-FAK-NFB的作用 促进最佳和最大促炎反应的途径。在AIM-1中，我们将研究相互作用 与整合素结合可激活整合素-FAK-NF-B通路 巨噬细胞的炎症反应。在AIM-2中，我们将研究25HC-整合素-FAK-NFB的作用 PRR(TLR3，NOD2)诱导巨噬细胞最佳促炎反应的途径 激活和病毒(RSV、IAV)感染。最后，在AIM-3中，我们将利用基因敲除小鼠来研究生理学 25HC-整合素-FAK通路在PRR(NOD2，TLR3)激活和病毒(RSV，IAV)感染中的活体作用 意义-我们目前的建议对开发抗RSV、IAV和 其他激活PRR的病原体。

Innate immunity constitutes pro-inflammatory response to initiate inflammation for adaptive immune response required for virus clearance. Pro-inflammatory (and inflammation) response need to be “tightly” regulated during virus infection since exaggerated inflammation contributes to inflammatory diseases. Activation of Pattern Recognition Receptors (PRRs) is critical for triggering innate immunity and inflammation during infection with human respiratory syncytial virus (RSV) and influenza A virus (IAV). Therefore, it is important to study the underlying cellular/molecular mechanism involved in transducing optimal innate immunity and inflammatory (and pro-inflammatory) response upon PRR activation. We have surprisingly identified a novel signaling network that is required for optimal pro-inflammatory response following PRR activation. Our preliminary result suggested that an oxysterol (a bioactive lipid) 25-hydroxycholesterol (25HC) play an important role in magnifying and intensifying pro-inflammatory response following activation of PRRs and during virus (RSV, IAV) infection. Surprisingly we observed interaction of 25HC with cell surface integrins and further preliminary studies suggested a possible mechanism. Mechanistically, we postulate that 25HC released from PRR activated (and virus infected) cells act as an extracellular soluble mediator to activate 25HC--- integrin (51, V3 integrins)---FAK (focal adhesion kinase)---NFB pathway for optimal pro-inflammatory response. Thus, we envision that extracellular 25HC serve as a “linker” (via autocrine/paracine mechanism) to bridge PRR pathway with integrin pathway for optimal pro-inflammatory response. We hypothesize that - a) extracellular 25HC links PRR pathway with integrin pathway; b) PRR---25HC---integrin---FAK---NFB signaling network magnifies pro-inflammatory response and inflammation; and c) 25HC--- integrin---FAK signaling pathway contributes to exaggerated inflammation during RSV and IAV infection, thus leading to development of exacerbated airway diseases like pneumonia and bronchiolitis. In the current proposal we have selected a membrane bound PRR (i.e. toll-like receptor 3 or TLR3) and a cytosolic PRR (i.e. Nod2) along with two clinically important respiratory viruses (RSV, IAV) to elucidate the role of 25HC---integrin---FAK---NFB pathway in promoting optimal and maximal pro-inflammatory response. In aim-1 we will investigate interaction of 25HC with integrins leading to activation of integrin---FAK---NFB pathway and subsequent pro- inflammatory response in macrophages. In aim-2 we will examine the role of 25HC---integrin---FAK---NFB pathway in triggering optimal pro-inflammatory response in macrophages following PRR (TLR3, Nod2) activation and virus (RSV, IAV) infection. Finally, in aim-3 we will utilize knockout mice to study physiological in vivo role of 25HC---integrin---FAK pathway during PRR (Nod2, TLR3) activation and virus (RSV, IAV) infection. Significance –Our current proposal has wide implication in development of therapeutics against RSV, IAV and other pathogens that activate PRRs.