Project Summary/Abstract Inflammation in the heart causes irreversible cardiac tissue damage. Unlike other organs, a damaged heart cannot regenerate and can lead to serious health problems. The adult heart contains distinct macrophage subsets with different functions. We sought to identify a heart macrophage subset highly specialized in tissue protection. We fully landscaped the immune profile of human pericardial fluid cells using unbiased single cell level analysis techniques, mass cytometry (CyTOF) and single cell RNA sequencing (scRNA-seq). Given the well-characterized phenotype and cutting-edge bioinformatic analysis, we predict that Gata6-expressing macrophages in the pericardial fluid play a heart protective role through tissue repair, efferocytosis, phagocytosis and immune regulation. The goal of this study is to focus on Gata6+ pericardial macrophages for in-depth studies of their role in inflammatory and ischemic heart diseases and to identify their cardioprotective mechanisms. In Aim 1, we will comprehensively characterize the immune phenotype and function of the pericardial effusion cells including Gata6+ macrophages in patients with or without heart diseases. In our preliminary study, we found a decrease of Gata6+ macrophages and an increase of memory T cells in the pericardial fluid of ischemic heart disease and pericarditis patients. We plan to collect 80 pericardial fluid samples from ischemic heart disease, perimyocarditis and pericarditis patients. We will immune profile the human pericardial fluid cells using CyTOF and scRNA-seq. We will also determine if the reduction of Gata6+ pericardial macrophages reflects a more severe disease in these patients by analyzing clinical outcomes. Aim 2 will utilize state-of-the-art macrophage fate-mapping techniques in the Coxsackievirus B3 (CVB3)-induced myocarditis mouse model to determine the origin and fate of GATA6+ pericardial macrophages in naïve and viral conditions. The replenishment of GATA6+ pericardial macrophages by blood circulating monocytes will be tested too. Aim 3 will address the mechanism how Gata6+ pericardial macrophages protect the heart from inflammatory damages using in vivo and in vitro experimental models. Our preliminary study showed that a selective depletion of Gata6+ pericardial macrophages worsened myocarditis in mice. We will profile the transcriptomes expressed by cardiac immune cells and non-hematopoietic stromal cells in Gata6+ macrophage-depleted mice with myocarditis. To prove the protective role of Gata6+ macrophages, we will test their efferocytosis and anti-inflammatory activity using in vitro assays. In addition, we will determine the crosstalk between Gata6+ macrophages and cardiac fibroblasts inhibiting pro-inflammatory monocyte infiltration. This study will reveal (1) a previously unidentified heart protective macrophage subset in the pericardial cavity, (2) the mechanisms of their tissue protective function, and (3) the potential of Gata6+ pericar...