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，中性粒细胞引起的炎症反应对于心肌梗死的发生至关重要。 清除细胞碎片和组织修复;但如果不加管制，它可能会带来比实际更长期的伤害。 效益我们知道中性粒细胞计数升高是心肌梗死患者心力衰竭和死亡的强预测因子， 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。这是由于 由于缺乏对中性粒细胞功能及其与其他促炎和抗炎信号传导的相互作用的了解， 心肌梗死后心脏中的成分。我们最近报道，浸润性中性粒细胞的初始波放大了 通过部署关键损伤相关分子模式（DAMP）， S100A8/A9。在这里，我们提供了有希望的发现，表明虽然S100 A8/A9参与煽动， 在炎症中，它们也可能在炎症的解决中具有潜在的作用。我们在全球范围内 在MI后第3天获得的心脏白细胞的转录组学分析（RNA seq），并且发现在MI后第3天获得的心脏白细胞的转录组学分析（RNA seq）中， 通过MafB和吞噬作用上调A类清道夫受体（SR），如Msr 1和Marco 受体（Mertk，CD 36通过Nr 4a 1），分别对DAMP和死细胞的清除至关重要。我们 进一步表明S100 A8/A9可以通过接合Ly 6Chi上的toll样受体（TLR 4）来上调SR和Mertk， 单核细胞，这是第二种浸润梗死心脏的细胞类型。这些新发现支持了 嗜中性粒细胞衍生的S100 A8/A9通过使单核细胞参与炎症的消退， 有效清除DAMP，以及促进单核细胞成熟为巨噬细胞， MI后红细胞增多和炎症最终消退。我们将使用两个具体目标来测试这一假设： 目的1，我们将明确S100 A8/A9和Ly 6Chi单核细胞之间相互作用的机制， DAMP的初始清除率。我们将特别关注表征时间依赖性表型和 单核细胞与中性粒细胞或S100 A8/A9相互作用后的转录组学变化 流式细胞术（CyTOF）和单细胞RNA测序。然后，我们将破译TLR 4-MafB-SR的作用， 包括S100 A8/A9在内的DAMP的内吞摄取中的信号级联， 炎症在目标2中，我们将定义S100 A8/A9在协调细胞周期的转变中的旁分泌功能。 Ly 6Chi至Ly 6Clo单核细胞/巨噬细胞用于最终清除死细胞和解决炎症。 在这里，我们将使用谱系追踪研究来绘制Ly 6Chi向Ly 6Clo单核细胞的转化，并研究Ly 6Chi在单核细胞中的作用。 的Nr 4a 1，和中性粒细胞或S100 A8/A9对这种转换的影响。对于每种策略， DAMP、死亡细胞、心脏重塑和功能将作为终点进行研究。我们的研究结果 将产生新的见解的机制，其中嗜热链球菌衍生的S100 A8/A9诱导促分辨信号， 并且还揭示了抑制与MI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.