Determinants of human neutrophil fate after phagocytosis

人类对微生物的先天免疫反应依赖于多种微生物之间的协调相互作用 细胞和分泌因子，以多形核白细胞(PMN)为主 响应者。中性粒细胞作为吞噬细胞，以膜结合的方式吞噬猎物 吞噬小体，其中来自NADPH氧化酶的氧化剂和来自颗粒的蛋白质协同作用，产生有毒的 促进吞噬微生物死亡和降解的环境。在某些情况下，一个重要的 部分摄取的微生物在PMN内存活。吞噬小体内存活微生物的持久性不是 不仅为持续感染提供了一种机制，而且还可以通过以下方式调节局部炎症基调 改变PMN的程序性细胞死亡，促进PMN释放促炎细胞因子。 干扰吞噬诱导的中性粒细胞凋亡(PICD)，或通过延迟凋亡，如所见 淋球菌(NGC)或参与一种新的坏死性细胞死亡途径，如金黄色葡萄球菌 (SA)，阻碍炎症反应的解决，并导致宿主衍生的危险信号的释放， 促进炎症和继发性组织损伤。因此，有效的先天免疫反应不需要 不仅是入侵微生物的死亡和降解，而且还包括炎症的消退和重建 动态平衡。这一提议的首要假设是，PMN未能经历凋亡 细胞死亡破坏了炎症反应的解决阶段，反而放大了疾病。 因为活化的人中性粒细胞(HPMN)及其分泌产物可以在体内塑造炎性基调 组织，我们建议使用NGC和SA-这两种人类病原体生存在PMN内，改变吞噬作用- 诱导细胞命运路径并引发深刻的局部炎症变化--作为探索机制的工具 决定吞噬细胞的命运并及时消退炎症。我们将追求两个具体目标： 目的1：确定人类中性粒细胞命运的调控机制 。 A.确定不同的信号通路引导hPMN走向生存还是程序化 细胞死亡(细胞凋亡与原发坏死)。 B.测定hPMN中核糖体(细胞内复合体)的组成和活性 吞噬作用 C.确定增殖细胞核抗原(增殖细胞核抗原)在吞噬hPMN的命运中的作用 目的2：探讨巨噬细胞吞噬过程中hPMN分泌IL-1β的机制 A.确定炎性小体在人多形核白细胞吞噬过程中产生IL-1β中的作用 B.确定丝氨酸蛋白酶在产生IL-1β中的重要性 C.确定受体相互作用蛋白-3(RIPK-3)在hPMN IL-1β分泌中的作用

Human innate immune response to microbes depends on the coordinated interactions among a variety of cells and secreted factors, with polymorphonuclear leukocytes (PMN) typically prominent among the first responders. In their capacity as phagocytic cells, PMN sequester ingested prey in membrane-bound phagosomes, where oxidants from the NADPH oxidase and proteins from granules synergize to create a toxic environment that promotes death and degradation of the engulfed microbe. In certain settings, a significant fraction of ingested microbes survives within PMN. The persistence of viable microbes within phagosomes not only provides a mechanism for sustained infection but also can modulate the local inflammatory tone by altering the programmed cell death of PMN and promoting PMN release of proinflammatory cytokines. Interference with phagocytosis-induced apoptosis of PMN (PICD), either by delaying apoptosis, as seen with N. gonorrhoeae (Ngc) or engaging a novel necrotic cell death pathway, as seen with Staphylococcus aureus (SA), thwarts resolution of the inflammatory response and causes release of host-derived danger signals that promote inflammation and secondary tissue damage. Thus, effective innate immune response requires not only death and degradation of invading microbes but also resolution of inflammation and reestablishment of homeostasis. The overarching hypothesis of this proposal is that the failure of PMN to undergo apoptotic cell death derails the resolution phase of the inflammatory response and instead amplifies disease. Because activated human PMN (hPMN) and their secreted products can sculpt the inflammatory tone in tissue, we propose to use Ngc and SA -- both human pathogens that survive within PMN, alter phagocytosis- induced cell fate pathways and elicit profound inflammatory local changes – as tools to probe mechanisms that dictate phagocyte fate and timely resolution of inflammation. We will pursue two Specific Aims: Aim 1: To determine the mechanisms that regulate human PMN fate . A. Determine the signaling pathways that differentially direct hPMN towards survival vs programmed cell death (apoptosis vs primary necrosis). B. Determine the composition and activities of the ripoptosome (intracellular complexes) in hPMN after phagocytosis C. Determine the role of Proliferating Cell Nuclear Antigen (PCNA) in the fate of phagocytosing hPMN Aim 2: To determine the mechanisms underlying hPMN secretion of IL-1β during phagocytosis A. Identify the role of inflammasomes in IL-1β production by hPMN during phagocytosis B. Determine the importance of serine proteases in generation of IL-1β C. Determine the role of Receptor-interacting kinase-3 (RIPK-3) in hPMN IL-1β secretion