Variability in neutrophil activation

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Project Summary/Abstract I am currently supported by a K08 (5K08-HL127183, awarded 9/4/2015) to study the biological activity of protamine/heparin immune complexes (IC), a newly described immune complication in the cardiopulmonary bypass population. In the course of my work, I developed a whole blood assay to measure IC-mediated neutrophil activation, as measured by matrix metalloprotease 9 granule release. Using this assay, in a recent publication, we report that the neutrophil response to ICs is highly variable among healthy subjects, and this variability represents a fixed response: some individuals have neutrophils which always activate readily in the presence of ICs, while others have neutrophils which are always minimally responsive. We have termed this susceptibility to neutrophil degranulation, the “neutrophil activation phenotype.” In new preliminary data, we now show that the neutrophil activation phenotype: 1) is not associated with Fc receptor variants, 2) is not limited to IC-Fc receptor interactions and is more broadly reflective of susceptibility to neutrophil activation by a variety of receptor- mediated agonists, 3) is determined at the cellular level and is retained with isolated neutrophils, and 4) is correlated with propensity for NET release. Building on this strong new preliminary data, I will test the hypothesis that the neutrophil activation phenotype is determined by differences in receptor-mediated signaling responses and is correlated with differences in procoagulant activity. In this proposal, I will determine the cellular factors which contribute to the neutrophil activation phenotype. In Subaim 1, I will determine if the phenotype is associated with differences in neutrophil effector function beyond degranulation, and I will examine ROS generation, neutrophil adherence, and ability to release proinflammatory lipid and cytokine mediators. In this subaim, I will also quantify low density granulocytes, a subpopulation with enhanced effector functions. In Subaim 2, I will build on preliminary data demonstrating that the response to ICs is highly correlated with the response to fMLP and to LPS, and I will determine if differences in signaling through the PI3K pathway (a point of convergence for neutrophil Fcɣ, G-protein coupled, and Toll-like receptors) contribute to the phenotype. In Subaim 3, I will build on preliminary data demonstrating that “high” subjects have a greater tendency to undergo NETosis, and I will determine if this translates into differences in procoagulant activity. Specifically, I will determine if the phenotype correlates with NET and NET component release, resistance to NET dissolution, and thrombin generation. In completing the proposed work, I will expand on my current K08-supported research objectives and investigate the heterogeneous neutrophil response in healthy subjects. In doing so I will continue to develop new knowledge/skills in immunology and neutrophil biology, learn new laboratory techniques, and foster new collaborative relationships. With the support of this R03, I will generate sufficient preliminary data for a R01 application to identify strong predictors of disease outcome and to identify therapeutic targets in disease. All of these skills will be essential to my long-term success as I transition to an independent investigator.