Regulation of neutrophil extracellular trap formation

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Neutrophils are indispensable components of the innate immune system. A key functional response of these cells is their ability to release so-called neutrophil extracellular traps (NETs) - a meshwork of extruded chromatin filaments decorated with various antimicrobial proteins. NETs entrap (whence the name) microorganisms and promote their killing, both ex vivo and in animal models. As a result, NETs have emerged as a major antimicrobial defense mechanism. Over the last 3 years, we demonstrated the pivotal role of the enzyme, PAD4, for NET generation; conversely, we showed that for most physiological NET inducers, the NADPH oxidase system is dispensable to elicit this response. We also found that NET formation features both immediate-early and late events that are controlled by discrete signaling pathways - a characteristic shared across several classes of physiological neutrophil stimuli. Among the late cellular processes driving NET formation, we recently unveiled the existence of an autocrine/paracrine feedback loop whereby endogenous factors mediate the phenomenon. Despite these advances however, our understanding of the molecular underpinnings of NET formation remains fragmentary. Objectives. The overarching goal of our research program is to shed light on key cellular processes and molecules underlying NET formation, and on the upstream signaling intermediates controlling the phenomenon. In the first project (Aim 1), we will investigate some of the late cellular processes driving NET formation, with a focus (i) on identifying some of the endogenous factors that feed back on neutrophils to trigger NET release, and the signaling pathways controlling their release; and (ii) on processes that affect chromatin extrusion and the formation of long DNA fibers. In the latter instance, we will determine whether filament formation requires macromolecular guides or extracellular scaffolds; and whether chromatin is somehow projected towards nearby cells. In the second project (Aim 2), we will investigate how the phosphoinositol 3-phosphate kinase (PI3K) pathway, which acts belatedly and is central to NET generation, controls this response. In particular, we will explore which PI3K isoforms are involved and whether they are conserved (or differ) among several classes of physiological stimuli; which of the various signaling components downstream of, or associated with, PI3K contribute to NET induction, as well as their inter-relationships; and which late step of NET formation (i.e. endogenous mediator generation, chromatin decondensation, NET extrusion) is affected by relevant signaling molecules identified above. Feasibility. We are well versed in all of the experimental approaches required to carry out the proposed work. Significance. The proposed research program will substantially further our understanding of NET generation, a seemingly unique biological phenomenon, by identifying some of the underlying molecular mechanisms.