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

Human innate immune response to microbes relies on the coordinated interactions among a variety of cells and secreted factors, with neutrophils as critical first cellular responders. To be effective in restoring health after infection, neutrophils must not only kill and eliminate invading microbes but also resolve inflammation and reestablish homeostasis, which begins with the programmed cell death of neutrophils that have completed their mission. Our studies will provide novel insights into how neutrophils sculpt the inflammatory response, both by regulated their programmed cell death and by release of the proinflammatory cytokine IL-1β, and thereby inspire new treatments for inflammatory diseases.