Neutrophil Activation Phenotypes in Healthy Subjects and Implications for Bacterial Clearance

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Project Summary/Abstract In work derived from my current K08 (5K08-HL127183, “Biological Significance of Protamine/Heparin Antibodies”), we developed a whole blood assay to measure neutrophil activation/degranulation, as quantified by matrix metalloprotease 9 (MMP9) granule release. In recently published work (Duarte ME, et al. Blood Adv. 2019), we showed that healthy subjects differ in their susceptibility to neutrophil activation by immunecomplexes (ICs), resulting in varying degrees of MMP9 release. With longitudinal testing, we showed that this susceptibility to degranulation represents a fixed phenotype for a given individual: some subjects have neutrophils which always activate readily, while others have neutrophils which are persistently minimally responsive. We have termed this susceptibility to neutrophil degranulation, the “neutrophil activation phenotype”. Because of the focus of my K08, my initial studies were focused on the neutrophil response to a variety of antigen/antibody ICs. However, in new preliminary data, we now show that the neutrophil activation phenotype: 1) is not limited to IC/Fcγ receptor interactions and instead, is more broadly reflective of susceptibility to neutrophil activation by a variety of agonists including the bacterial peptide N-formyl-met-leu-phe (fMLP), lipopolysaccharide (LPS), and bacterial supernatant, 2) is associated with differential sensitivity to PI3K inhibition, a point of convergence for many neutrophil activation pathways, 3) is familial, 4) and is correlated with NET release and ROS generation. Building on this strong preliminary data, we will test the hypothesis that the neutrophil activation phenotype is determined by genetic differences in receptor-mediated signaling responses, resulting in differences in bactericidal activity. In Aim 1, we will determine the cellular and genetic factors which contribute to the neutrophil activation phenotype. Wewill determine if differences in signaling contribute to the neutrophil phenotype. We will examine activation patterns at multiple sites along the PI3K pathway and test the effect of specific kinase inhibitors. In this aim, we will also build on our novel observation that the neutrophil phenotype is familial, and we will perform RNA sequencing of naïve and activated neutrophils from phenotyped subjects to identify genetic determinants of the neutrophil phenotype. In Aim 2, we will determine if the neutrophil activation phenotype has relevance to infectious disease and bacterial clearance. We will use Staphylococcus aureus as a model pathogen to determine if other neutrophil effector functions, beyond degranulation, are associated with the neutrophil activation phenotype. And, we will determine if the phenotype translates into differences in bactericidal activity. In completing the proposed work, we will identify determinants of the heterogeneous neutrophil response in healthy subjects, and we will determine if this variability in neutrophil reactivity results in differential ability to kill bacteria. With the support of this R21, we will generate sufficient preliminary data for a R01 application to determine if the neutrophil activation phenotype contributes to the heterogeneity seen among patients with infection and identify strong predictors of disease outcome.