A dialogue between neutrophils and monocytes for effective resolution of inflammation following acute myocardial injury

项目摘要 心肌梗死(MI)后，以中性粒细胞为先导的炎症反应对 清除细胞碎片和组织修复；但如果不加以监管，它可能会带来比实际更长期的伤害 利益。我们知道中性粒细胞计数升高是心肌梗塞患者心力衰竭和死亡的强烈预测因子，但 旨在抑制中性粒细胞功能的策略在改善结果方面并不成功。这是到期的 由于缺乏对中性粒细胞功能及其与其他促炎和抗炎信号的相互作用的了解 心肌梗死后心脏的成分。我们最近报道，渗透的中性粒细胞的第一波放大了 心肌梗死后炎症反应通过部署关键的损伤相关分子模式(DAMP)，如 S100A8/A9。在这里，我们提供了有希望的发现来表明，当S100A8/A9参与煽动 除了炎症，它们还可能在消解炎症方面发挥潜在作用。我们在全球演出 心肌梗死后第3天获得的心脏白细胞的转录分析(RNA Seq)，并发现一个稳健的 MafB上调Msr1和Marco等A类清道夫受体(SR)和吞噬功能 受体(MerTK、CD36通过Nr4a1)分别对湿气和死亡细胞的清除起关键作用。我们 进一步证明S100A8/A9可通过激活Ly6chi上的Toll样受体(TLR4)上调SRS和MerTK 单核细胞，第二顺位细胞类型，渗透到梗塞的心脏。这些新奇的发现支持这一假设 中性粒细胞来源的S100A8/A9通过与单核细胞接触来协调炎症的消退 有效清除湿气，促进单核细胞成熟为巨噬细胞，增强 心肌梗死后的胞吐和炎症的最终消退。我们将使用两个特定目标来检验这一假设： 目的1，我们将确定S100A8/A9与Ly6chi单核细胞相互作用的机制 最初对湿气的清除。我们将特别集中于表征依赖于时间的表型和 单核细胞与中性粒细胞或S100A8/A9相互作用后转录水平的变化 细胞计数(CyTOF)和单细胞RNA测序。然后我们将破译TLR4-MafB-SR的作用 包括S100A8/A9在内的DAMP内吞过程中的信号级联 发炎。在目标2中，我们将定义S100A8/A9在协调 Ly6chi至Ly6Clo单核/巨噬细胞，用于最终清除死亡细胞和消解炎症。 在这里，我们将使用谱系追踪研究来映射Ly6chi到Ly6Clo单核细胞的转换，并研究其作用 以及中性粒细胞或S100A8/A9对这种转化的影响。对于每一种策略， 湿气、死细胞、心脏重塑和功能将作为终点进行研究。我们的研究成果 将对中性粒细胞衍生的S100A8/A9诱导前解析信号的机制有新的见解。 也揭开了抑制与心肌梗死相关的炎症的新靶点。

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 which neutrophil-derived S100A8/A9 induce pro-resolving signals, and also unravel novel targets to suppress inflammation associated with MI.