Project Summary After myocardial infarction (MI), the ensuing inflammatory response spearheaded by neutrophils is essential for removal of cellular debris and tissue repair; but if unregulated, it may confer more long-term harm than actual benefit. We know elevated neutrophil count is a strong predictor of heart failure and death in MI patients, yet strategies aimed at suppressing neutrophil function have not been successful in improving outcome. This is due to the lack of knowledge of neutrophil function and its cross-talk with other pro- and anti-inflammatory signaling components in the heart post-MI. We recently reported that the initial wave of infiltrating neutrophils amplify the inflammatory response post-MI by deploying key damage associated molecular patterns (DAMPs) such as S100A8/A9. Here, we provide promising findings to show that while S100A8/A9 participates in instigating inflammation, they may also have a potential role in the resolution of inflammation. We performed global transcriptomic analysis (RNA seq) of cardiac leukocytes obtained at day 3 post-MI, and found a robust upregulation of class A scavenger receptors (SRs) such as Msr1 and Marco via MafB, and phagocytosis receptors (Mertk, CD36 via Nr4a1) that are crucial for the clearance of DAMPs and dead cells, respectively. We further show that S100A8/A9 could upregulate SRs and Mertk by engaging the toll-like-receptor (TLR4) on Ly6Chi monocytes, the second-in-line cell type to infiltrate the infarcted heart. These novel findings support the premise that neutrophil-derived S100A8/A9 orchestrate the resolution of inflammation by engaging monocytes for effective clearance of DAMPs, as well as facilitating the maturation of monocytes into macrophages to enhance efferocytosis and final resolution of inflammation after MI. We will test this hypothesis using two specific aims: In Aim 1, we will define the mechanisms underlying the interaction between S100A8/A9 and Ly6Chi monocytes for the initial clearance of DAMPs. We will specifically focus on characterizing the time-dependent phenotypic and transcriptomic changes in monocytes following their interaction with neutrophils or S100A8/A9 using mass cytometry (CyTOF) and single-cell RNA sequencing. We will then decipher the role of the TLR4-MafB-SR signalling cascade in the endocytic uptake of DAMPs including S100A8/A9 during the initial phase of inflammation. In Aim 2, we will define the paracrine function of S100A8/A9 in orchestrating the transition of Ly6Chi to Ly6Clo monocytes/ macrophages for the final clearance of dead cells and the resolution of inflammation. Here, we will use lineage-tracing studies to map the conversion of Ly6Chi to Ly6Clo monocytes and study the role of Nr4a1, and the impact of neutrophils or S100A8/A9 on such conversion. For each strategy, the clearance of DAMPs, dead cells, cardiac remodeling and function will be studied as endpoints. The outcome of our studies will yield new insights into the mechanisms by w...