CD11c as a novel target to improve neutrophil effector functions and sepsis outcome

项目概要/摘要 脓毒症仍然与20%至30%的高死亡率相关，每年花费240亿美元， 占美国所有住院总费用的近五分之一。当前脓毒症 管理层是支持的。因此，确定治疗方法是改善 脓毒症的结果。中性粒细胞作为第一道防线消灭微生物的先天免疫细胞。在败血症中， 称为紧急粒细胞生成的过度从头中性粒细胞产生主要通过G-CSF发生 生产然而，未成熟的中性粒细胞也被释放到循环中以满足对中性粒细胞的高需求。 中性粒细胞的数量，但他们有较少的抗菌防御功能，导致更差的主机防御脓毒症 患者G-CSF本身并不触发中性粒细胞的完全成熟。因此，一项旨在实现 增强中性粒细胞成熟对于更好的宿主防御在脓毒症中是关键的。整合素CD 11 c为 被认为是区分脓毒症和全身炎症反应综合征的敏感标志物。我们 先前的研究表明，CD 11 c KO小鼠在盲肠炎诱导的多微生物脓毒症中的存活率较差， 结扎和穿刺（CLP）手术，表明CD 11 c在脓毒症中的关键作用。一直以来， 研究其在体内的功能作用。我们出乎意料地发现，CD 11 c在骨中表达， 骨髓（BM）中性粒细胞（主要是细胞内），其缺乏与BM中性粒细胞减少有关 成熟在一个小鼠模型中重现了紧急粒细胞生成，成熟的中性粒细胞被释放， 与野生型（WT）小鼠相比，CD 11 c KO小鼠中的量显著更少，进一步表明其 中性粒细胞成熟的重要性。我们建立了CD 11 c组成型活性敲入（KI）小鼠， 在稳态条件下和感染期间， 更好的细菌根除和结果。此外，我们的体外中性粒细胞成熟实验， 通过CRISPR/Cas9技术检测的原代鼠CD 11 c KO中性粒细胞或缺乏CD 11 c的HL-60细胞显示， 中性粒细胞成熟较少。基于这些结果，我们假设CD 11 c活性将显著影响 调节稳定状态下中性粒细胞成熟的程度和细胞内在的紧急粒细胞生成 方式我们的初步数据表明，CD 11 c也可以调节中性粒细胞效应子的一个亚群， 与成熟无关的功能。因此，我们将确定CD 11 c在中性粒细胞成熟中的作用， 稳态下的效应器功能和目标1和目标2中的脓毒症。因为IQGAP 1被认为是一个 结合配偶体的CD 11 c，其作用也将进行研究，以阐明如何CD 11 c调节的机制， 中性粒细胞成熟和效应子功能。研究将通过使用人脓毒症受试者和小鼠进行。 模型因为没有CD 11 c小分子激动剂和拮抗剂，我们将筛选它们 目标3。在提案完成后，我们预计我们将巩固CD 11 c将是一个关键的 中性粒细胞成熟和效应子功能的调节剂，用于脓毒症的治疗干预。

Project Summary/ Abstract Sepsis remains to be associated with a high mortality of 20 to 30% with annual cost of $24 billion, accounting for nearly one-fifth of the total aggregate costs in all the hospitalizations in the United States. Current sepsis management is supportive. Therefore, identifying therapeutic approaches is an urgent task to improve the outcome of sepsis. Neutrophils eradicate microbes as the first-line defense innate immune cells. In sepsis, exaggerated de novo neutrophil production called emergency granulopoiesis occurs mainly via G-CSF production. However, immature neutrophils are also released into a circulation to meet a high demand for neutrophil number, but they have less antimicrobial defense functions, leading to worse host defense in septic patients. G-CSF itself does not trigger full neutrophil maturation. Thus, an intervention to attain the enhancement of neutrophil maturation is critical in sepsis for better host defense. Integrin CD11c was considered a sensitive marker to differentiate sepsis from systemic inflammatory response syndrome. We previously showed that CD11c KO mice had worse survival in the polymicrobial sepsis induced by cecal ligation and puncture (CLP) surgery, indicating the critical role of CD11c in sepsis. There has been a paucity of research about its functional role in vivo. We unexpectedly identified that CD11c was expressed in the bone marrow (BM) neutrophils (largely intracellular) and its deficiency was associated with less BM neutrophil maturation. In a mouse model to recapitulate emergency granulopoiesis, mature neutrophils were released in significantly less quantity in CD11c KO mice compared to wild type (WT) mice, further suggesting its importance in neutrophil maturation. We created CD11c constitutively active knock-in (KI) mice, which demonstrated to have more mature neutrophils in the BM in a steady-state condition and during infection with better bacterial eradication and outcomes. In addition, our in vitro neutrophil maturation experiments using primary murine CD11c KO neutrophils or HL-60 cells devoid of CD11c by CRISPR/Cas9 technology showed less neutrophil maturation. Based on these results, we hypothesized that CD11c activity would significantly regulate the degree of neutrophil maturation in a steady state and emergency granulopoiesis in a cell-intrinsic manner. Our preliminary data suggested that CD11c would also regulate a subset of neutrophil effector functions irrelevant of maturation. Thus, we will determine the role of CD11c in neutrophil maturation and effector functions in a steady state and sepsis in Aim 1 and Aim 2. Because IQGAP1 was suggested to be a binding partner for CD11c, its role will also be studied to delineate the mechanism of how CD11c regulates neutrophil maturation and effector functions. Studies will be done by using human sepsis subjects and murine models. Because there are no CD11c small molecule agonists and antagonists available, we will screen them in Aim 3. Upon the completion of the proposal, we expect that we would solidify that CD11c would be a critical regulator of neutrophil maturation and effector functions for a therapeutic intervention in sepsis.