A novel mechanism of immunosuppression in sepsis: Depletion of monocytes and macrophages

脓毒症是一种危及生命的疾病，在美国每年影响超过100万患者。 越来越多的证据表明，免疫抑制是脓毒症死亡率的主要驱动力。 巨噬细胞在机体对病原体的免疫应答中起重要作用。先前的临床研究表明， 脓毒症患者的外周单核细胞通过细胞凋亡被耗尽。最近的体外研究表明， 细菌组分、鞭毛蛋白、III型分泌系统的杆状蛋白（T3 SS）或LPS诱导 通过激活炎性体途径引起巨噬细胞的焦亡。在本提案中，我们提供 有令人信服的证据表明，外周血单核细胞和组织巨噬细胞都是由于细胞凋亡而耗尽的， 小鼠脓毒症模型，包括盲肠结扎穿孔（CLP）模型。我们发现静脉注射 鞭毛蛋白或杆状蛋白的注射诱导组织中外周单核细胞和巨噬细胞的消耗。 我们进一步证明，小鼠中这些细胞的耗竭损害了免疫应答，并增加了免疫应答。 随后用E.杆菌重要的是，我们的数据表明， 从热性凋亡单核细胞和巨噬细胞中释放出的毒素触发弥散性血管内凝血（DIC）。所以我们 研究结果确定单核细胞/巨噬细胞耗竭是免疫抑制和DIC的新机制， 败血症本申请的目的是描述单核细胞/巨噬细胞的潜在机制， 在脓毒症中，其消耗及其对免疫抑制的贡献。目的1是阐明 脓毒症时单核细胞和巨噬细胞的死亡。目前的假设是炎性小体 活化和随后的焦亡在脓毒症期间单核细胞/巨噬细胞消耗中起关键作用。 半胱天冬酶-1、半胱天冬酶-11、半胱天冬酶-1/11双重或GSDMD缺陷的小鼠模型（全身和全身） 巨噬细胞特异性）将用于阐明炎性小体激活的详细机制， 单核细胞/巨噬细胞耗竭中的焦亡。目的2是确定杆蛋白和 鞭毛蛋白诱导导致单核细胞/巨噬细胞耗竭的细胞凋亡。我们最近发现了一种细胞内 EprJ的结合伴侣，巨噬细胞中的酰基辅酶A脱氢酶家族成员9型（ACAD 9）。我们将 使用不同的方法来测试假设，ACAD 9依赖性途径有助于 巨噬细胞耗竭目的3是确定单核细胞/巨噬细胞耗竭对 脓毒症期间的免疫抑制。我们将使用脓毒症模型的组合来研究 单核细胞/巨噬细胞耗竭在脓毒症期间的免疫抑制中的作用。脓毒症患者外周血单核细胞耗竭 患者也将接受调查。完成拟议的研究将揭示一种新的分子机制 革兰氏阴性菌引起的免疫抑制。这些发现将大大推动我们的 了解脓毒症的发病机制，并确定这种致命疾病的新药物靶点。

Sepsis is a life-threatening condition that affects more than 1 million patients a year in the United States. Growing evidence indicates that immunosuppression is a major driving force for mortality in sepsis. Macrophages play essential roles in immune response to pathogens. Previous clinical studies have shown that peripheral monocytes are depleted in septic patients through apoptosis. Recent in vitro studies revealed that bacterial components, flagellin, the rod protein of the type III secretion system (T3SS), or LPS induce pyroptosis of macrophages through activation of inflammasome pathways. In this proposal, we provide convincing evidence that both peripheral monocytes and tissue macrophages are depleted due to pyroptosis in mouse sepsis models including the cecal ligation and puncture (CLP) model. We show that intravenous injection of flagellin or the rod proteins induced depletion of peripheral monocytes and macrophages in tissues. We further demonstrate that depletion of these cells in mice impaired immune response and increased mortality rate by subsequent challenged with E. coli. Importantly, our data indicate that tissue factor released from pyroptotic monocytes and macrophages triggers disseminated intravascular coagulation (DIC). Thus, our findings identified monocyte/macrophage depletion as a novel mechanism of immunosuppression and DIC in sepsis. The goal of this application is to delineate the underlying mechanisms of monocyte/macrophage depletion and its contribution to immunosuppression in sepsis. Aim 1 is to delineate the mechanisms of pyroptotic monocyte and macrophage death during sepsis. The working hypothesis is that inflammasome activation and subsequent pyroptosis play a critical role in monocyte/macrophage depletion during sepsis. Mouse models deficient in caspase-1, caspase-11, caspase-1/11 double, or GSDMD (whole-body and macrophage-specific) will be used to elucidate the detailed mechanism of inflammasome activation and pyroptosis in monocyte/macrophage depletion. Aim 2 is to identify the mechanism by which rod protein and flagellin induce pyroptosis leading to monocyte/macrophage depletion. We recently identified an intracellular binding partner of EprJ, the acyl-CoA dehydrogenase family member type 9 (ACAD9) in macrophages. We will use different approaches to test the hypothesis that the ACAD9-dependent pathway contributes to macrophage depletion. Aim 3 is to identify the contribution of monocyte/macrophage depletion to immunosuppression during sepsis. We will use a combination of sepsis models to investigate the role of monocyte/macrophage depletion in immunosuppression during sepsis. Peripheral monocyte depletion in septic patients will also be investigated. Completion of the proposed studies will reveal a novel molecular mechanism of immunosuppression induced by Gram-negative bacteria. Such findings will significantly advance our understanding about the pathogenesis of sepsis and identify new drug targets for this deadly disease.