Neutrophil-Mediated Bacterial Dissemination and Host Immunopathogenesis in a Murine Scrub Typhus Model

摘要 恙虫病是一种危及生命的人畜共患传染病，由恙虫病东方体生物体传播， 被恙螨的幼虫感染世界上大约三分之一的人口面临感染的危险。但 细菌从皮肤传播的机制和先天免疫细胞不能控制局部的原因 感染是未知的;在体内感染期间追踪细菌在技术上具有挑战性。该项目将 使用我们新建立的皮内（i.d.）感染小鼠模型和荧光标记细菌以确定 细菌传播的机制。我们的初步小鼠数据显示， 中性粒细胞中CFSE标记东方体体外至少12小时。我们还发现中性粒细胞是 在小鼠皮肤中对失活东方体应答的浸润细胞群。鉴于中性粒细胞减少 通过抗体促进小鼠在致死感染（i. v.感染模型）中对抗急性感染的存活，我们 我想测试东方菌可以劫持和利用中性粒细胞作为特洛伊木马的假设， 宿主杀菌活性，允许细菌从最初的皮肤感染部位传播到内脏器官。 特异性目的1将检查皮肤中骨髓细胞募集的动力学和独特的细菌负荷。 使用多色流式细胞术检测细胞群体。我们还将评估东方体感染对 中性粒细胞杀菌活性，包括活性氧生成，中性粒细胞胞外陷阱 形成和颗粒/细胞因子产生。具体目标2将定义中性粒细胞- 介导的东方体传播和免疫发病机制。我们将跟踪CFSE标记的Orientia， 在i.d.接种。蜂窝 还将检查反向迁移的标志。进一步证实中性粒细胞的致病作用 在疾病进展期间，我们将使用选择性和有效的拮抗剂调节中性粒细胞迁移。 这项工作将为东方体感染的先天免疫决定因素提供新的见解， 我们的长期目标是为这种和其他载体开发有前途的治疗策略， 通过共同的病原体传播途径传播的疾病。

Abstract Scrub typhus is a life-threatening zoonosis caused by Orientia tsutsugamushi organisms that are transmitted by the larvae of trombiculid mites. About one third of the world’s population is at risk of infection. However, the mechanism of bacterial dissemination from the skin and the reason that innate immune cells fail to control local infection are unknown; it is technically challenging to track the bacteria during infection in vivo. This project will use our newly established intradermal (i.d.) infection mouse model and fluorescence-labeled bacteria to define the mechanisms of bacterial dissemination. Our preliminary mouse data revealed containment and survival of CFSE-labeled Orientia in neutrophils for at least 12 hours in vitro. We also found neutrophils as the major infiltrating cell population responding to inactivated Orientia in the mouse skin. Given that neutrophil depletion via antibodies promoted mouse survival against acute infection in a lethal infection (i.v. infection model), we want to test the hypothesis that Orientia bacteria can hijack and utilize neutrophils as Trojan horses against host bactericidal activities, allowing bacterial dissemination from the initial skin infection sites to visceral organs. Specific Aim 1 will examine the dynamics of myeloid cell recruitment in the skin and the bacterial load in unique cell populations using multi-color flow cytometry. We will also evaluate effects of Orientia infection on neutrophil bactericidal activity, including reactive oxygen species generation, neutrophil extracellular trap formation, and granules/cytokine production both in vitro and in vivo. Specific Aim 2 will define neutrophil- mediated Orientia dissemination and immunopathogenesis. We will track CFSE-labeled Orientia among various organs (e.g. draining lymph nodes, bone marrow, lung, and spleen) after i.d. inoculation. Cellular markers of reverse migration will also be examined. To further validate the pathogenic role of neutrophils during disease progression, we will modulate neutrophils migration using selective and potent antagonists. This work will provide new insights into the innate immune determinants of Orientia infection and will lay the groundwork for our long-term goal of developing promising therapeutic strategies for this and other vector- borne diseases that share a common route of pathogen dissemination.