Host Cell JAK-STAT Activation and Pathogenesis of Spotted Fever Rickettsioses

描述（由申请人提供）：立克次体立克次体和康氏雷氏杆菌是革兰氏阴性的专性细胞内a-变形菌，已知可引起人类落基山斑疹热（RMSF）和地中海SF。致病性立克次体的一个独特特征是对血管微血管内皮的趋向性，这导致弥散性内皮感染、血管炎症和血管通透性受损。在响应立克次体侵袭时，原本处于静止状态的内皮细胞被激活，其特征是获得血栓原、促凝剂和促炎表型，表现为“立克次体血管炎”。尽管内皮细胞的激活通常涉及多个上游机制的输入，包括Janus激酶-信号转导和转录激活因子（JAK-STAT）蛋白，但这种至关重要的信号通路在立克次体与宿主细胞相互作用中的潜在贡献是完全未知的，它通常涉及抗病毒宿主防御。我们最近发现的已发表的以及初步的体外和体内证据表明：i)感染立克次体的人微血管内皮细胞（ECs）早期STAT3和晚期STAT1激活；ii)来自受感染细胞的“条件培养基”通过IFN-b激活na - ve内皮细胞中的STAT1的能力；iii)干扰素刺激基因编码蛋白15kda （ISG15）依赖于IFN-b和STAT1的表达增加；iv) IFN-b、STAT1和ISG15对立克次体的抑菌活性。这些新发现是三个独立的基础上，虽然机械重叠的具体目标。首先，我们将确定受体介导的和胞浆内的信号机制以及JAK-STAT激活在培养的人和鼠ECs感染斑点热立克次体期间的后果。由于ISG15不仅在先天免疫中发挥重要作用，而且还可以结合其他细胞蛋白并通过isg酰化调节其功能，第二个目标将确定其在宿主防御致病性立克次体机制中的作用。具体来说，我们将鉴定和分析受感染内皮中ISG15基化蛋白的功能，并确定分泌ISG15的细胞因子样活性是否激活自然杀伤细胞介导的宿主防御。最后，我们将定义IFN-b、STAT1和ISG15作为宿主防御的关键决定因素的潜在作用，利用已建立的和新的模拟人类疾病的小鼠感染模型和缺乏IFNAR、STAT1和ISG15作为宿主的小鼠。我们将采用当代最先进的细胞生物学、显微镜、分子遗传学和蛋白质组学方法，揭示宿主防御机制的独特见解。由于立克次体病现在在全球范围内重新出现，全面了解病原体与目标宿主细胞的相互作用将有助于确定创新的治疗策略，以改变使人衰弱的立克次体感染的过程，有利于宿主。

DESCRIPTION (provided by applicant): Rickettsia rickettsii and R. conorii are Gram-negative, obligate intracellular a-proteobacteria known to cause Rocky Mountain spotted fever (RMSF) and Mediterranean SF in humans. A unique trait of pathogenic rickettsiae is the tropism for microvascular endothelium of the blood vessels, which results in disseminated endothelial infection, vascular inflammation, and compromised vascular permeability. Activation of otherwise quiescent endothelium in response to rickettsial invasion is characterized by acquisition of prothrombotic, procoagulant and proinflammatory phenotypes, manifesting as 'rickettsial vasculitis'. Although endothelial activation typically involves input from multiple upstream mechanisms, including Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) proteins, the potential contributions of this critically important signaling pathway, which is typically involved in antiviral host defense, in rickettsial interactios with the host cell are completely unknown. Published as well as preliminary in vitro and in vivo evidence from our recent findings suggests: i) early STAT3 and late STAT1 activation in human microvascular endothelial cells (ECs) infected with SF rickettsiae; ii) ability of 'conditioned medium' from infected cells to activate STAT1 in na¿ve ECs through IFN-b; iii) increased expression of Interferon-Stimulated Gene encoding protein of 15 kDa (ISG15) dependent on IFN-b and STAT1; and iv) anti-bacterial activities of IFN-b, STAT1 and ISG15 against rickettsiae. These novel findings are the basis of three independent albeit mechanistically overlapping specific aims. First, we will identify receptor-mediated and intracytoplasmic signaling mechanisms and consequences of JAK-STAT activation during infection of cultured human and murine ECs with spotted fever rickettsiae. Because ISG15 not only plays an important role in innate immunity, but can also bind to other cellular proteins and modulate their functions via ISGylation, the second aim will determine its role in host defense mechanisms against pathogenic rickettsiae. Specifically, we will identify and analyze the functions of ISGylated proteins in infected endothelium and determine whether cytokine-like activity of secreted ISG15 activates Natural Killer cell-mediated host defense. Finally, we will define the potential roles of IFN-b, STAT1, and ISG15 as critical determinants of host defense exploiting established as well as novel murine models of infection mimicking human disease and mice lacking IFNAR, STAT1, and ISG15 as the host. We will employ contemporary, cutting edge methods of cell biology, microscopy, molecular genetics, and proteomics to reveal unique insights into mechanisms underlying host defense vis-¿-vis pathogenesis. Since rickettsioses are now re-emerging globally, comprehensive understanding of the pathogen interactions with the target host cell will aid in the identification of innovative therapeutic strategies to alter te course of debilitating rickettsial infections in favor of the host.