Inflammatory Response and SARS Pathogenesis: An in vitro Model

简介(申请人提供)：严重急性呼吸系统综合症(SARS)，由一种名为SARS-CoV的新型冠状病毒引起，是一种高度传染性和危及生命的呼吸系统疾病，以肺部为主要病理目标。SARS的确切发病机制尚不清楚。在SARS患者中，肺泡上皮细胞胞浆内可见典型的冠状病毒样颗粒，提示上皮细胞不仅对SARS冠状病毒具有容许性，而且在SARS的病理生物学中起重要作用。重要的是，我们已经证明了人支气管上皮CALU-3细胞对SARS-CoV感染的反应是通过产生几种NFkappa B介导的细胞因子，包括IL-6和IL-8。由于SARS被认为是一种病毒性肺炎，很可能是由不受控制的、往往是过度的先天炎症反应引起的，因此迫切需要充分了解这些由SARS冠状病毒感染的肺上皮细胞分泌的炎性因子如何与巨噬细胞和树突状细胞(DC)相互作用，这两个细胞成分是先天免疫系统的两个关键细胞成分，从而诱导一连串的炎症反应，最终影响感染的结果。基于这种高度病理相关性的SARS-CoV感染的Calu-3细胞模型产生的强大的初步数据，我们在这一应用中假设，感染SARS-CoV的高分化Calu-3细胞释放的细胞因子将改变巨噬细胞和DC的内在功能，导致炎症反应加剧。我们还假设，SARS冠状病毒感染的肺上皮细胞最初炎症分泌的减弱将减轻随后的巨噬细胞和/或DC介导的炎症反应。将追求两个具体目标：(1)确定人肺上皮细胞在宿主对SARS-CoV的反应中的作用(S)。基于初步数据的工作假设是，感染SARS冠状病毒的人肺上皮细胞分泌的先天炎症介质是巨噬细胞和DC的强大调节器，导致全身炎症反应的级联(细胞因子风暴？)。(2)鉴定SARS冠状病毒诱导的人肺上皮细胞先天信号通路。需要检验的假设是，SARS-CoV通过TLR-3、TLR-8和/或RIG-I介导的途径激活CALU-3细胞，导致分泌依赖于NFkappa B的炎症因子。这些研究完成后，将深入了解SARS的发病机制，并为SARS的治疗和预防措施提供细胞靶点。SARS是一种传染性很高的人类呼吸道疾病，通常是致命的。SARS的发病机制尚不清楚，也没有有效的治疗措施。该项目与公共卫生相关，因为它不仅侧重于确定SARS发病的细胞和分子基础，而且还致力于确定关键的炎性“生物标志物”，这将有助于对未来SARS爆发的诊断和治疗干预。

DESCRIPTION (provided by applicant): Severe acute respiratory syndrome (SARS), caused by a novel coronavirus known as SARS-CoV, is a highly contagious and life-threatening respiratory disease, with the lungs as its main pathological target. The exact mechanism of SARS pathogenesis remains unknown. In SARS patients, extensive epithelial disruption is seen with typical coronavirus-like particles in the cytoplasm of pneumocytes, indicating that epithelial cells are not only permissive to SARS-CoV but also important in the pathobiology of SARS. Importantly, we have shown that human bronchial epithelial Calu-3 cells responded to SARS-CoV infection by producing several NFkappa B-mediated cytokines, including IL-6 and IL-8. Because SARS is considered a viral pneumonia, likely caused by unregulated and often excess innate inflammatory responses, a full understanding is urgently needed as to how these inflammatory factors secreted by SARS-CoV-infected lung epithelial cells would interact with macrophages and dendritic cells (DC), two of the key cellular elements of the innate immune system, with regard to the induction of a cascade of inflammatory responses that ultimately influence the outcome of infection. Based on the strong preliminary data generated with this highly pathologically relevant Calu-3 cell model of SARS-CoV infection, we hypothesize in this application that cytokines released from highly differentiated Calu-3 cells that were infected by SARS-CoV will alter intrinsic functions of both macrophages and DC, resulting in exacerbated inflammatory responses. We also hypothesize that attenuation of the initial inflammatory secretion by SARS-CoV-infected lung epithelial cells will alleviate the subsequent macrophage and/or DC-mediated inflammatory responses. Two specific aims will be pursued: (1) Define the role(s) of human lung epithelial cells in the host responses to SARS-CoV. The working hypothesis, based on preliminary data, is that innate inflammatory mediators secreted by SARS-CoV-infected human lung epithelial cells are potent modulators of macrophages and DC, leading to cascades of systemic inflammatory responses (a cytokine storm?). (2) Identify SARS-CoV-induced innate signaling pathways in human lung epithelial cells. The hypothesis to be tested is that SARS-CoV activates Calu-3 cells through TLR-3-, TLR-8, and/or RIG-I-mediated pathways, resulting in secretion of NFkappa B-dependent inflammatory factors. Upon completion, these studies will provide insight into SARS pathogenesis and cellular targets for therapeutic and preventive measures against SARS. SARS is a highly contagious and often fatal human respiratory disease. The pathogenesis of SARS remains unknown and effective therapeutic measurements are not available. This project is of relevance to public health, because it focuses not only on determining the cellular and molecular basis for SARS pathogenesis, but it also on identifying key inflammatory "biomarkers" that will be useful for diagnostic and therapeutic interventions against future outbreaks of SARS.