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 that are decorated with histones, as well as with proteins originating from the cytoplasm or intracellular granules. NETs were first described a decade ago, as structures that entrap (whence the name) various microorganisms and can thus participate in their killing. NETs have since been detected in a variety of inflammatory contexts, and were shown to help in preventing the dissemination of microorganisms in animals models. As a result, it has become a widely held view that NETs must represent a major defense mechanism against infection. Despite this, the molecular mechanisms underlying NET formation, as well as the upstream signaling pathways, remain poorly understood. In the last decade, we published several studies documenting the signaling machinery of neutrophils and how it controls functional responses such as cytokine generation and delayed apoptosis. More recently, we began exploring how discrete signaling molecules also govern NETosis (i.e. NET generation) and in doing so, have observed that there are probably more steps involved in this process than previously appreciated. Objectives. The general goal of our research program is to shed light on the cellular processes and molecular mechanisms that control NETosis. In the first project (Aim 1), we will focus on cellular processes that affect chromatin extrusion and the formation of long DNA fibers. This will involve observing the entire NETosis phenomenon as it unfolds by live cell imaging, and identifying processes that only affect extrusion or filament formation. We will also determine whether filaments result from physical traction by moving cells; whether filament formation requires macromolecular guides or extracellular scaffolds; and whether chromatin might somehow be projected towards nearby cells. In the second project (Aim 2), we will investigate how discrete signaling components participate in controlling NETosis (and at which steps), as well as their inter-relationships; and whether NETosis can occur in the absence of reactive oxygen species production, which are deemed essential for this response. Feasibility. We have expertise in all of the experimental approaches that are required to carry out the proposed work. Significance. The proposed research program will substantially further our understanding of NETosis, a seemingly unique biological phenomenon, by identifying its discrete phases. These investigations will also shed light on various aspects of how NETosis is regulated, by identifying some of the underlying molecular mechanisms.