Mechanism of CX3CR1+ macrophage-mediated resolution of eosinophilic allergic lung inflammation

CX3CR1+巨噬细胞介导的嗜酸性变态反应性肺部炎症消退机制 摘要： 最近的研究表明，组织驻留的巨噬细胞不仅参与炎症的启动，而且 还可用于消解和预防局部炎症。精确测定巨噬细胞亚群 致力于解决肺部炎症和了解其功能，我们采用了新的技术 质量细胞术和单细胞RNA-seq(sc-RNA-seq)分析人和小鼠巨噬细胞在 阿龙。我们的支持数据显示，肺泡巨噬细胞(AM)表型多样且高度分化。 应对过敏原挑战的动态反应。基于sc-rna-seq数据，AM可以被聚类为几个 处于稳定状态的组。在这些组中，表达CX3CR1的AM(CX3CR1+AM)的子集是 在表型和基因表达模式方面是独一无二的，与经典的常驻AM相比 CX3CR1均为阴性。在过敏性哮喘患者和哮喘小鼠模型中，我们发现CX3CR1+ 在BAL中，过敏原攻击使AM显著增加。CX3CR1+AM不仅表示 巨噬细胞也有嗜酸性粒细胞标志物，如人类Siglec-8。进一步调查CX3CR1- 记者和EPX-cre(又名EO-cre)报告鼠发现CX3CR1+巨噬细胞以一种 稳定状态和过敏性肺部炎症。CX3CR1+巨噬细胞在小鼠模型中的耗竭 在变态反应性肺中，自发性组织嗜酸性粒细胞处于稳定状态和持续的组织嗜酸性粒细胞增多 发炎。基于这一数据，我们假设新招募的CX3CR1+AM子集促进 清除组织中的嗜酸性粒细胞，促进过敏性肺部炎症的消退。在目标1中，我们将 重点研究CX3CR1+巨噬细胞在变态反应性肺部炎症中的细胞动力学。关于 CX3CR1+介导的嗜酸性粒细胞清除的分子机制，我们研究了潜在的配体 CX3CR1-CX3CL1和CCL26。我们发现CCL26在激活CX3CR1+中起着关键作用 巨噬细胞，而CX3CL1是必不可少的。我们的sc-RNA-seq数据显示，CX3CR1+AM子集是 C1q转录本的唯一来源，C1q是胞吐的关键分子。在体外设置中，CCL26触发 CX3CR1+巨噬细胞以CX3CR1受体介导的方式分泌C1q。此外，C1q和CCL26 过敏性哮喘患者的BAL因过敏原激发而增加。该数据表明CCL26 激活CX3CR1+巨噬细胞，通过分泌C1q促进胞吐作用。在目标2中，我们将研究 CCL26介导的C1q分泌激活CX3CR1+巨噬细胞的详细机制。最后，我们 将把这项研究扩展到使用IRB批准的节段性疾病的翻译人类研究 用一种过敏原进行激发，以评估上述拟议实验的人类相关性。这个 建议的研究是基于我们关于CX3CR1+巨噬细胞在 过敏性肺部炎症的消退。这项研究将有助于更好地理解解决问题的过程 过敏性哮喘。

Title: Mechanism of CX3CR1+ macrophage-mediated resolution of eosinophilic allergic lung inflammation Abstract: Recent studies show that tissue-resident macrophages participate in not only the initiation of inflammation but also in the resolution and prevention of local inflammation. To precisely determine the subsets of macrophages engaged in resolving lung inflammation and gain insight into their functions, we adopted new techniques of mass cytometry and single-cell RNA-seq (sc-RNA-seq) to analyze human and mouse macrophages in the lung. Our supporting data showed that alveolar macrophages (AMs) are phenotypically diverse and highly dynamic in response to allergen challenge. Based on the sc-RNA-seq data, AMs can be clustered into a few groups at a steady status. Among these groups, a subset of CX3CR1-expressing AMs (CX3CR1+ AMs) are unique in terms of their phenotype and patterns of gene expression, compared to classical resident AMs which are CX3CR1 negative. In patients with allergic asthma and a mouse model of asthma, we found that CX3CR1+ AMs are markedly increased in BAL by allergen challenge. The CX3CR1+ AMs express not only the macrophage but also eosinophil markers such as human Siglec-8. Further investigation with the CX3CR1- reporter and Epx-cre (a.k.a. Eo-cre) reporter mice reveals that CX3CR1+ macrophages engulf eosinophils at a steady state and in allergic lung inflammation. Depletion of CX3CR1+ macrophages in mouse models resulted in spontaneous tissue eosinophilia at a steady status and prolonged tissue eosinophilia in allergic lung inflammation. Based on this data, we hypothesized that the newly recruited CX3CR1+ AM subset promote the clearance of tissue eosinophils and facilitates the resolution of allergic lung inflammation. In aim 1, we will focus on the cellular dynamics of CX3CR1+ macrophages in allergic lung inflammation. Regarding the molecular mechanism of CX3CR1+ mediated-eosinophil clearance, we examined the potential ligands for CX3CR1 – CX3CL1 and CCL26. We discovered that CCL26 plays a key role in activating CX3CR1+ macrophages, whereas CX3CL1 is indispensable. Our sc-RNA-seq data revealed that CX3CR1+ AM subset is the sole source of the transcript of C1q - a key molecule for efferocytosis. In in-vitro setting, CCL26 triggers CX3CR1+ macrophages to secrete C1q in a CX3CR1 receptor-mediated manner. Furthermore, C1q and CCL26 are increased in BAL by allergen challenge in patients with allergic asthma. This data suggests CCL26 activates CX3CR1+ macrophages to facilitate efferocytosis via C1q secretion. In aim 2, we will examine the detailed mechanisms of CX3CR1+ macrophage activation through CCL26-mediated C1q secretion. Finally, we will extend the study to translational human research using the IRB-approved protocol for the segmental provocation with an allergen to evaluate the human relevance of the above proposed experiments. The proposed study is based on our strong supporting data on the new roles of CX3CR1+ macrophages in the resolution of allergic lung inflammation. This study will lead to a better understanding of the resolution process of allergic asthma.