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

描述(申请人提供)：立克次体和康氏立克次体是革兰氏阴性的专性细胞内a-蛋白细菌，已知能在人类中引起落基山斑点热(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细胞的趋向性，导致播散性内皮细胞感染、血管炎症和血管通透性受损。在立克次体侵袭时，激活原本静止的内皮细胞的特征是获得血栓前、促凝血和促炎表型，表现为“立克次体血管炎”。尽管内皮细胞的激活通常涉及多种上游机制的输入，包括Janus Kinase-Signal Transducer and Activator of Transcription(JAK-STAT)蛋白，但这一关键的信号通路在抗病毒宿主防御中与宿主细胞的相互作用中的潜在贡献是完全未知的。已发表的以及我们最近发现的体外和体内初步证据表明：i)感染立克次体的人微血管内皮细胞(ECs)中STAT3的早期和晚期激活；ii)感染细胞的“条件培养液”通过干扰素-b激活新生内皮细胞的STAT1的能力；iii)依赖干扰素-b和STAT1的干扰素刺激基因编码蛋白15 kDa(ISG15)的表达增加；以及iv)干扰素-b、STAT1和ISG15对立克次体的抗菌活性。这些新颖的发现是三个独立的、尽管机械地重叠的具体目标的基础。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激活在感染斑点热立克次体的培养人和小鼠内皮细胞过程中的后果。由于ISG15不仅在天然免疫中发挥重要作用，而且还可以与其他细胞蛋白结合并通过ISG化调节其功能，因此，第二个目的将决定其在宿主防御致病立克次体机制中的作用。具体地说，我们将鉴定和分析感染内皮中ISGylated蛋白的功能，并确定分泌的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，我们将确定干扰素-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。我们将使用细胞生物学、显微镜、分子遗传学和蛋白质组学的当代前沿方法，揭示宿主防御与发病机制的独特见解。由于立克次体病现在正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识别创新的治疗策略，以改变削弱立克次体感染的病程，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.