New genetic models for C5a receptors

摘要 补体系统不仅是先天性免疫的组成部分，而且还显著地促进免疫应答。 感染相关炎症的威胁性严重程度。危及生命的急性炎症综合征 呼吸窘迫综合征（ARDS）经常由不受控制的细菌性肺炎引起， 持续的公共卫生负担。这些疾病涉及补体系统的激活， 补体过敏毒素C5 a的产生和这些过程的治疗性破坏可以提供 防止组织破坏性炎症。C5 a与其同源受体C5 aR 1和C5 aR 2连接， 由两个相邻的基因编码。C5 aR 1和C5 aR 2在不同组织中的功能作用并不相同。 C5 aR 2的数据存在争议。在我们的初步工作中，我们已经产生了一个鼠标 通过CRISPR/Cas9基因编辑具有两个C5 aR（C5 aR 1/2-/- DKO）的约12.6kb缺失的品系，另一种小鼠 用于报告和条件性缺失C5 aR 2（C5 aR 2LacZ; C5 aR 2flox）的菌株，并且我们可以获得条件性的 C5 aR 1小鼠（C5 aR 1flox）。这些新的工具将有助于检验我们的中心假设， 中性粒细胞上的C5 aR 1与II型肺泡上皮细胞上的C5 aR 2的协同作用决定了 肺损伤和肺炎球菌性肺炎的发展和进展。我们将重点关注三个具体的 目的：（1）在骨髓嵌合体小鼠的基础上，研究人骨髓嵌合体基因的表达和功能。 将研究C5 aR 2在II型肺泡上皮细胞（AECII）中的相关性，包括终点， 肺泡-毛细血管屏障破坏、细胞因子/趋化因子特征和上皮-白细胞串扰 肺损伤和肺炎球菌肺炎。此外，将比较C5 aR 2的存在与 正常人肺组织中的C5 aR 1。(2)我们将描述C5 aRs在Ly 6 G+中性粒细胞中的作用， 肺损伤和肺炎球菌性肺炎，使用C5 aR 1和C5 aR 2的嗜中性粒细胞特异性缺失的小鼠。 为了阐明C5 aR 1对肺侵袭性中性粒细胞亚群及其成熟状态的潜在作用，我们 将使用一种新的基于液滴的高通量方法进行单细胞RNA-Seq研究。(3)我们将 通过比较C5 aRs在炎症反应中的作用，研究C5 aRs的功能协同、冗余和差异， 肺损伤期间C5 aR 1/2-/- DKO至C57 BL/6（Wt）、C5 aR 1-/-和C5 aR 2-/-小鼠的表型， 肺炎球菌性肺炎从上述肺炎实验中分离的中性粒细胞和AECII 将用于在深度测序研究中比较这四种小鼠品系的全转录组 (bulk RNA-Seq），其专注于阐明嗜热粒细胞-AECII相互作用的分子途径，并搜索 新的C5 aR 1/2调节基因。这些调查旨在推进当前的概念，并解决 关于C5 aR的功能性拮抗作用或操作协同作用的持续争论。以后， 这些见解可能有助于推进炎症性疾病治疗干预的概念

Abstract The complement system is not only an integral arm of innate immunity but also significantly contributes to the menacing severity of infection-associated inflammation. The life-threatening inflammatory syndrome of acute respiratory distress syndrome (ARDS) frequently arises from uncontrolled bacterial pneumonia imposing persistent public health burdens. These disorders involve activation of the complement system with excessive generation of the complement anaphylatoxin, C5a, and therapeutic disruption of these processes could provide protection from tissue-destructive inflammation. C5a ligates with its homologous receptors, C5aR1 and C5aR2, encoded by two adjacent genes. The functional roles of C5aR1 and C5aR2 across different tissues are not entirely clear, and the data for C5aR2 is controversial. In our preliminary work, we have generated a mouse strain with a ~12.6 kb deletion of both C5aRs (C5aR1/2-/- DKO) by CRISPR/Cas9 gene editing, another mouse strain for reporting and conditional deletion of C5aR2 (C5aR2LacZ; C5aR2flox) and we have access to conditional C5aR1 mice (C5aR1flox). These novel tools will facilitate the testing of our central hypothesis that a functional cooperation of C5aR1 on neutrophils with C5aR2 on type II alveolar epithelial cells determines the development and progression of lung injury and pneumococcal pneumonia. We will focus on three specific aims: (1) Based on our preliminary findings with bone marrow chimeric mice, the expression and functional relevance of C5aR2 in type II alveolar epithelial cells (AECIIs) will be studied including endpoints such as alveolar-capillary barrier disruption, signatures of cytokines/chemokines and epithelium-leukocyte cross-talk during lung injury and pneumococcal pneumonia. In addition, the presence of C5aR2 will be compared to C5aR1 in normal human lung tissues. (2) We will characterize the roles of C5aRs in Ly6G+ neutrophils during lung injury and pneumococcal pneumonia using mice with neutrophil-specific deletion of C5aR1 and C5aR2. To elucidate potential effects of C5aR1 on subsets of lung invading neutrophils and their maturation states, we will pursue single-cell RNA-Seq studies using a novel droplet-based high-throughput method. (3) We will investigate the functional synergisms, redundancies and diversities of C5aRs by comparing the inflammation phenotypes of C5aR1/2-/- DKO to C57BL/6 (Wt), C5aR1-/- and C5aR2-/- mice during lung injury and pneumococcal pneumonia. Neutrophils and AECIIs isolated from the aforementioned pneumonia experiments will be used for comparing the whole transcriptomes of these four mouse strains in deep sequencing studies (bulk RNA-Seq) focusing on elucidation the molecular pathways of neutrophil-AECIIs interactions and a search for novel C5aR1/2-regulated genes. These investigations will aim to advance the current concepts and resolve ongoing controversies regarding the functional antagonism or operational synergism of the C5aRs. In future, these insights may be helpful for advancing concepts for therapeutic interventions in inflammatory diseases