Mincle and STING Activation in Proinflammatory Responses to Orientia Infection

项目总结/摘要 恙虫病是一种由恙虫病东方体（一种LPS阴性细菌）引起的危及生命的疾病。这 细菌是高度传染性的，优先在吞噬细胞和内皮细胞中复制，留下三分之一的 世界人口面临感染风险。丛林斑疹伤寒疫苗尚未问世;关于宿主免疫的信息 认识和发病机制有限。我们最近开发了O.恙虫 卡普菌株感染，模仿人类患者的严重感染，已经揭示了过量的1型，但 缺乏2型，免疫反应导致多个器官的急性血管损伤。使用NanoString 通过这项技术，我们检测了炎症组织（肺、脾、脑）中的1,200多个免疫基因。我们发现了一个 Mincle/Clec 4 e（一种C型凝集素受体）和STING（一种DNA 传感器）在小鼠急性/致死性感染期间和感染的原代吞噬细胞培养物中。底层 假设在O.恙虫病感染的单核细胞对于 效应T细胞引发和失调的Mincle/STING活化可导致过度的1型炎症 和急性组织损伤目标1将测试持续的细菌复制是否可以启动Mincle和STING 激活并触发其下游信号通路。小鼠和人的原代巨噬细胞和 单核细胞将用于在各种条件下的体外感染， Mincle或STING（通过siRNA、药理学抑制剂或基因敲除）。目标2将决定是否 树突状细胞中的Mincle/STING活化可以有效地引发/活化T效应细胞，并且如果Mincle/STING活化， 导致东方体感染期间T细胞反应失调。来自Mincle-/-和STING-/-的树突状细胞 将感染C57 BL/6 J小鼠，并检查体外和体内致敏幼稚CD 4 T细胞;敲除小鼠 将接受非致命或致命的东方体感染。先天性和适应性免疫反应谱 将在单细胞和分子水平上进行评价，并与细菌学/病理学水平相结合 问题研究虽然来自某些人类病原体或受损自身组分的Mincle和STING配体是 据报道，Mincle或STING在Orientia感染期间的作用从未被探索过。本研究将 揭示，第一次，这些先天传感器如何共同促进宿主对专性 细胞内细菌，这是研究不足，但有公共卫生的影响。可行性是由我们的 研究专业知识和BSL 3设施。发现初始免疫激活/功能障碍的本质将有助于 揭示了恙虫病预后的标志性宿主特征和药理学治疗的潜在途径 干预这项及时的研究将对其他细胞内病原体产生广泛的影响。

Project Summary/Abstract Scrub typhus is a life-threatening disease caused by Orientia tsutsugamushi, an LPS-negative bacterium. This bacterium is highly infectious, replicating preferentially in phagocytes and endothelial cells, leaving one third of world population at risk of infection. Scrub typhus vaccines are not yet available; information on host immune recognition and disease pathogenesis is limited. Our recently developed mouse models of O. tsutsugamushi Karp strain infection, which mimic severe infection in human patients, have revealed that excessive type 1, but deficient type 2, immune responses result in acute vascular damage in multiple organs. Using the NanoString technology, we examined more than 1,200 immune genes in inflamed tissues (lung, spleen, brain). We found a significant/selective increase in transcripts of Mincle/Clec4e (a C-type lectin receptor) and STING (a DNA sensor) during acute/lethal infection in mice and in infected primary phagocyte cultures. The underlying hypothesis is that Mincle/STING pathway activation in O. tsutsugamushi-infected monocytes are critical for effector T cell priming, and dysregulated Mincle/STING activation can lead to excessive type 1 inflammation and acute tissue damage. Aim 1 will test whether sustained bacterial replication can initiate Mincle and STING activation and trigger their downstream signaling pathways. Both mouse and human primary macrophages and monocytes will be used for in vitro infection under various conditions with normal or attenuated expression of Mincle or STING (via siRNA, pharmacological inhibitors, or genetic knockouts). Aim 2 will determine whether Mincle/STING activation in dendritic cells can effectively prime/activate T effector cells and if Mincle/STING contribute to dysregulated T-cell responses during Orientia infection. Dendritic cells from Mincle-/- and STING-/- C57BL/6J mice will be infected and examined for priming naïve CD4 T cells in vitro and in vivo; knockout mice will receive a non-lethal or lethal Orientia infection. The spectrum of innate and adaptive immune responses will be evaluated at the single-cell and molecular levels and integrated with those from bacteriologic/pathologic studies. While Mincle and STING ligands from certain human pathogens or damaged self-components are reported, the roles of Mincle or STING during Orientia infection have never been explored. This study will reveal, for the first time, how these innate sensors collectively contribute to host responses to an obligate intracellular bacterium, which is poorly studied but has public health impact. The feasibility is justified by our research expertise and BSL3 facilities. Discovery of the nature of initial immune activation/dysfunction will help reveal signature host profiles for scrub typhus prognosis and potential avenues for pharmacological intervention. This timely study will have broad implications for other intracellular pathogens.