Neutrophil Development During Inflammation and Atherosclerosis

PROJECT SUMMARY Neutrophils acutely respond to inflammatory signals in tissues and play a role in cardiovascular disease (CAD). After activation by cholesterol and oxidized low density lipoproteins, neutrophils produce cytokines, degranulate to release myeloperoxidase, azurocidin, and elastase, generate neutrophil extracellular traps (NETs), and trigger lytic cell death. Neutrophils are produced in the bone marrow by stem cell progenitors and, once mature, egress to blood and tissues to fight inflammation. We recently discovered an early committed neutrophil progenitor stem cell (NeP) present in human and mouse bone marrow. Here, we present new data demonstrating the presence of NeP in the peripheral blood of mice and humans with CAD, suggesting that NeP modulate granulopoiesis and inflammation outside of the bone marrow. In this project, we will focus on inflammation via activation of the Nlrp3 inflammasome, as we find that Nlrp3 and ll1b are highly expressed in NeP. Activation of the inflammasome sensor Nlrp3 triggers a robust inflammatory response, including IL-1β production. In Project 1, we hypothesize that activation of the Nlrp3 inflammasome within NeP triggers granulopoiesis and neutrophil heterogeneity which promotes atherosclerosis progression. We will test our hypothesis by studying inflammasome activation in both mice and humans with cardiovascular disease (CAD). In Specific Aim 1 we will test how neutrophil subsets are phenotypically and functionally changed in human CAD patients compared to healthy subjects. These subjects are undergoing medically necessary coronary angiography as part of the ongoing Coronary Assessment in Virginia (CAVA) study, and their extent of atherosclerosis is measured clinically. We will analyze NeP, neutrophil heterogeneity, and the role of the neutrophilic Nlrp3 inflammasome in blood of these subjects and link these findings to clinical parameters. In Specific Aim 2 we will use atherosclerotic mouse models to test how intrinsic action of the Nlrp3 inflammasome in neutrophils impacts granulopoiesis and atherosclerosis. We will use mixed chimeric mouse models with Nlrp3-/-, Gsdmd-/-, and Gsdme-/- mice to assess how inflammasome pathways in NeP regulate granulopoiesis. New neutrophil-specific and macrophage-specific Nlrp3 knock-out mice crossed to atherosclerosis-susceptible, apolipoprotein E-deficient (apoE-/-) mice will be used to directly compare how action of neutrophil vs macrophage Nlrp3 inflammasomes impact neutrophil heterogeneity and atherosclerosis. We will use a new mouse model to assess Nlrp3 action in NeP versus mature neutrophils on atherosclerosis. We will define how Nlrp3 inflammasome activation in neutrophil progenitors impacts granulopoiesis and atherosclerosis, which in turn could lead to new therapeutic approaches targeting Nlrp3 components in neutrophils for atherosclerosis and other inflammatory diseases.