Dengue Infected Endothelial Cells Enhance Immune Cell Activation

描述（由申请人提供）：登革热（DF）、登革出血热（DHF）和登革休克综合征（DSS）是由4种血清型登革病毒（DV）引起的疾病。DHF/DSS中发现的水肿和出血性疾病是由血管渗漏引起的，这些严重的表现被预先存在的DV抗体和随后的第二种DV血清型感染所增强。因此，免疫增强的疾病过程有助于DHF/DSS病例中内皮细胞（EC）通透性增加。DV感染人外周血白细胞、树突状细胞和内皮细胞，DV增强血管通透性的机制刚刚开始被确定。DV感染免疫细胞的能力导致了许多关于外周血淋巴细胞（PBL）感染及其趋化因子应答的研究。两者对于病毒发病机理都是明显重要的，并且容易在来自人或DV感染的鼠疾病模型的血液中进行研究。虽然EC更难以在体内研究，但它们形成血管系统的主要流体屏障，并且最终水肿或出血性疾病是由改变内皮的屏障功能引起的。DV感染患者和AG 129小鼠模型的尸检研究表明，肝、肺和脾中的EC被感染。DV感染EC的能力为感染提供了改变毛细血管通透性、复制和诱导EC趋化因子应答的手段，所述EC趋化因子应答激活免疫细胞并将其募集到内皮。然而，DV感染的EC对免疫增强和血管通透性的贡献尚未被考虑到DV疾病过程中。DV感染培养物中的EC，然而先前的体外研究仅在2-10%感染的EC上进行。相反，我们最近对DV感染的EC的研究是通过同步感染>80%的原代人EC并监测病毒和细胞应答来进行的。我们发现DV有效地感染原代人EC，感染后1天病毒滴度快速增加（约105/ml）。我们的研究结果表明，DV感染的EC有助于病毒血症，病毒传播和DV抗原在EC上的呈递，使其成为抗体和免疫细胞的靶点。我们对EC对DV感染的反应的分析揭示了趋化因子的高水平诱导，所述趋化因子指导免疫细胞的募集和活化。注射后24小时，在同步感染的EC中，细胞因子CXCL 10、CXCL 11、IL-7、RANTES和BAFF分别被诱导337、45、128、84和119倍CXCL 10/11和RANTES分泌募集并激活白细胞和T细胞，而IL-7刺激B、T和NK细胞增殖和T细胞成熟。BAFF是一种B和T细胞活化因子，在活化的EC表面释放或表达。DV感染还诱导备解素（P因子，34倍），其激活旁路途径（AP）补体系统，产生C3 a/C5 a：趋化性过敏毒素，其触发局部炎症、肥大细胞脱粒和血管通透性。重要的是，高水平的C3 a存在于重症登革热患者中，并与严重疾病、血浆泄漏和休克相关。这些新发现的EC反应表明，EC不仅是DV感染的靶点，而且释放增强免疫细胞趋化性和感染内皮细胞渗透性的因子。总的来说，这些发现表明DV感染的EC有助于病毒血症、增强的免疫应答和血管通透性，这些是DHF和DSS的基本组分。我们建议定义EC对DV感染的反应，通过激活和招募免疫细胞到内皮来增强免疫反应。 公共卫生相关性：登革热病毒由蚊子携带，每年感染5000万至1亿人，引起血管渗漏疾病，每年有50万至100万人患水肿和出血性疾病，死亡率为5-30%。登革热导致免疫增强的疾病过程，其中第二次感染从不同的血清型更严重。DV感染免疫细胞以及毛细血管内皮细胞，调节水肿和出血。我们已经发现DV感染的内皮细胞引起增强免疫介导的应答的应答，所述免疫介导的应答有助于血管渗漏并有助于增加病毒产生和传播。我们在这项研究中解决了内皮细胞的这一过程。

DESCRIPTION (provided by applicant): Dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are diseases caused by 4 serotypes of dengue virus (DV). Edema and hemorrhagic disease found in DHF/DSS result from vascular leakage, and these severe manifestations are enhanced by preexisting DV antibodies and subsequent infection by a second DV serotype. Thus an immune enhanced disease process contributes to increased endothelial cell (EC) permeability in DHF/DSS cases. DVs infect peripheral human leukocytes, dendritic cells and endothelial cells, and mechanisms by which DVs enhance vascular permeability are just beginning to be define. The ability of DVs to infect immune cells has resulted in many studies on infection of peripheral blood lymphocytes (PBLs) and their chemokine responses. Both are clearly important to viral pathogenesis and are readily studied in blood from humans or DV infected murine disease models. Although ECs are more difficult to study in vivo, they form the primary fluid barrier of the vasculature, and ultimately edema or hemorrhagic disease result from altering barrier functions of the endothelium. Postmortem studies of DV-infected patients and the AG129 mouse model demonstrate that ECs in the liver, lung, and spleen are infected. The ability of DVs to infect ECs provides a means for infection to alter capillary permeability, replicate and induce EC chemokine responses that activate and recruit immune cells to the endothelium. However, the contribution of DV infected ECs to immune enhancement and vascular permeability has yet to be factored into the DV disease process. DV infects ECs in culture, however prior in vitro studies were performed on ECs that were only 2-10% infected. In contrast, our recent studies of DV infected ECs were performed by synchronously infecting >80% of primary human ECs and monitoring viral and cellular responses. We found that DV productively infects primary human ECs, with a rapid increase in viral titer (~105/ml) 1 day p.i. Our findings suggest that DV infected ECs contribute to viremia, viral dissemination and the presentation of DV antigens on ECs that make them targets of antibodies and immune cells. Our analysis of EC responses to DV infection revealed the high level induction of chemokines that direct immune cell recruitment and activation. Cytokines CXCL10, CXCL11, IL-7, RANTES and BAFF are induced 337, 45, 128, 84 and 119 fold, respectively, in synchronously infected ECs by 24 hrs p.i. CXCL10/11 and RANTES secretion recruit and activate leukocytes and T-cells while IL-7 stimulates B, T and NK cell proliferation and T cell maturation. BAFF is a B and T cell activating factor that is released or expressed on the surface of activated ECs. DV infection also induced properdin (Factor P, 34-fold), which activates the alternative pathway (AP) complement system producing C3a/C5a: chemotactic anaphylatoxins which trigger localized inflammation, mast cell degranulation and vascular permeability. Importantly, high levels of C3a are present in severely ill dengue patients and associated with severe disease, plasma leakage and shock. These newly discovered EC responses indicate that ECs are not only targets of DV infection, but release factors that potentiate immune cell chemotaxis and permeability of the infected endothelium. Collectively these findings suggest that DV infected ECs contribute to viremia, enhanced immune responses and vascular permeability which are fundamental components of DHF and DSS. We propose to define EC responses to DV infection that enhance immune responses by activating and recruiting immune cells to the endothelium. PUBLIC HEALTH RELEVANCE: Dengue virus is carried by mosquitos and infects 50-100 million people/ year, causing a vascular leak disorder responsible for edema and hemorrhagic disease in 500,000-1,000,000/ year with a 5-30% mortality rate. Dengue results in an immune enhanced disease process whereby the 2nd infection is more severe from a distinct serotype. DV infects immune as well as endothelial cells that line capillaries and regulate edema and bleeding. We have found that DV infected endothelial cells elicit responses that enhance immune mediated responses that contribute to vascular leakage and contribute to increased virus production and spread. We address this process in endothelial cells in this study.