Pathogenic Mechanisms of Vascular Dysfunction in Scrub Typhus

森林斑疹伤寒是一种由东方体引起的危及生命的疾病，东方体是一种内毒素阴性细菌， 在内皮细胞和吞噬细胞中优先复制。虽然每年有100万人感染， 由于世界上约有三分之一的人口面临感染风险，缺乏有效的感染控制战略。 关于疾病发病机制和免疫失调的信息有限。为了应对这些挑战，我们 已经开发出模仿人类丛林斑疹伤寒某些关键病理特征的小鼠模型。我们发现 内源性损伤相关分子模式(DAMP)分子，如HMGB1和 髓过氧化物酶(MPO)是与血管生成素2相关的分子功能障碍的关键调节因子 (ANG2)、Tie2和CD41血小板。我们还发现了中性粒细胞持续激活的有害影响。 在血小板减少和血管损伤中的作用，以及Ang2和miR-200b作为独特的生物标志物 血管功能障碍。本研究的目的是明确血管功能障碍的发病机制。 以及利用钩端螺旋体EC靶向模型治疗严重恙虫病的方法 感染。我们的中心假设是，感染引发的湿气释放加剧了O。 恙虫病通过改变中性粒细胞/血小板的激活而导致的血管损伤；旨在 保持血管完整性或吞噬细胞功能有助于诱导对急性组织的平衡免疫 损伤和严重的丛林斑疹伤寒。这一假设将在三个具有凝聚力的具体目标中得到检验。目标1将 检测hMGB1和肿瘤坏死因子介导的人皮肤致敏和加重的机制 东方体感染中的内皮细胞。我们将使用人微血管内皮细胞(HMEC)来测试 东方体触发HMGB1、肿瘤坏死因子、Ang2和miR-200b及其靶基因/蛋白的释放，如果 针对这些机制的治疗方法可以减轻EC损伤。目标2将检查通过哪些机制 中性粒细胞和HMGB1在小鼠感染期间导致血管和血小板功能障碍。我们将设置 CXCR2-/-或MPO-/-小鼠在不同感染阶段的致死性和亚致死性感染。同样，删除 RAGE(一种HMGB1受体，RAGE-/-小鼠)或抗RAGE抗体将揭示HMGB1/RAGE的特定作用 组织浸润性白细胞、血小板和血管功能的信号转导。目标3将测试抗Ang2和 他汀类药物通过调节血管功能和吞噬细胞激活促进宿主存活 老鼠。小鼠将接受抗Ang2抗体、重组Ang1或他汀类药物(单独或联合使用)治疗。 在致命性和亚致死性感染之前或期间。我们将检查细菌传播，中性粒细胞/血小板 激活，以及Tie2和标志性细胞因子/DAMPS的水平。这项以机理为重点的研究充分利用了 关于几个研究团队之间的协同作用。血管特异性生物标记物的发现，新的调控 它们对东方病免疫反应的影响是及时的、创新的和非常重要的。

Scrub typhus is a life-threatening disease caused by Orientia tsutsugamushi, a LPS-negative bacterium that replicates preferentially in endothelial cells (EC) and phagocytes. While one million people are infected yearly, with about one-third of world population at risk of infection, effective strategies for infection control are lacking. Information on disease pathogenesis and immune dysregulation is limited. To address these challenges, we have developed mouse models that mimic certain key pathological features of human scrub typhus. We found that endogenous damage-associated molecular pattern (DAMP) molecules such as HMGB1 and myeloperoxidase (MPO) are key regulators responsible for molecular dysfunctions involving angiopoietin 2 (Ang2), Tie2, and CD41+ platelets. We also discovered the detrimental effect of sustained neutrophil activation in thrombocytopenia and vascular injury, and the potential of Ang2 and miR-200b as unique biomarkers for vascular dysfunction. The objective of this study is to define pathogenic mechanisms of vascular dysfunction and therapeutic modalities for severe scrub typhus by utilizing the EC-targeting model of O. tsutsugamushi infection. Our central hypothesis is that the infection-triggered release of DAMPs exacerbates O. tsutsugamushi-induced vascular damage by altering neutrophil/platelet activation; interventions aimed at preserving vascular integrity or phagocyte function help to elicit a balanced immunity against acute tissue damage and severe scrub typhus. This hypothesis will be tested in three cohesive Specific Aims. Aim 1 will examine the mechanisms of HMGB1- and TNF-mediated sensitization and exacerbation of human endothelium in Orientia infection. We will use human microvascular endothelial cells (HMEC) to test whether Orientia triggers the release of HMGB1, TNF, Ang2, and miR-200b and their target genes/proteins, and if therapeutics targeting these mechanisms mitigate EC injury. Aim 2 will examine mechanisms by which neutrophils and HMGB1 contribute to vascular and platelet dysfunction during infection in mice. We will set up lethal and sublethal infections in CXCR2-/- or MPO-/- mice at different infection stages. Likewise, the deletion of RAGE (a HMGB1 receptor, RAGE-/- mice) or anti-RAGE antibody will reveal specific roles of HMGB1/RAGE signaling in tissue-infiltrated leukocytes, platelet and vascular function. Aim 3 will test whether anti-Ang2 and statin-based therapeutics promote host survival via modulating vascular function and phagocyte activation in mice. Mice will be treated with anti-Ang2 antibody, recombinant Ang1, or statins (alone or in combination) before or during lethal and sublethal infection. We will examine bacterial dissemination, neutrophil/platelet activation, and the levels of Tie2 and signature cytokines/DAMPs. This mechanism-focused study capitalizes on the synergism among several research teams. Discovery of vascular-specific biomarkers, novel regulatory pathways, and their impact on immune responses to Orientia is timely, innovative and highly significant.