New genetic models for C5a receptors

C5a 受体的新遗传模型

• 批准号: 10433917
• 负责人: Markus Bosmann
• 金额: $ 41.25万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10433917
• 关键词: Acute Respiratory Distress Syndrome; Affinity; Alveolar; Anaphylatoxins; Bacterial Pneumonia; Blood capillaries; Bone Marrow; Breeding; C5AR2 gene; C5a anaphylatoxin receptor; CRISPR/Cas technology; Capillary Permeability; Cells; Chimera organism; Clinical; Complement; Complement 5a; Complement Activation; Complement Receptor; Cre-LoxP; Data; Data Set; Development; Disease; Epithelial; Epithelial Cells; Future; Generations; Genes; Genetic; Genetic Models; Genomics; Histology; Human; Individual; Infection; Inflammation; Inflammation Mediators; Inflammatory; Invaded; Investigation; Knowledge; Leukocytes; Life; Ligands; Ligation; Link; Lung; Methods; Molecular; Mouse Strains; Mus; Natural Immunity; Pathway interactions; Pattern; Peptides; Phenotype; Play; Pneumococcal Pneumonia; Pneumonia; Process; Public Health; Pulmonary Inflammation; RNA analysis; Reporter; Reporting; Role; Scheme; Severities; Structure of parenchyma of lung; System; Techniques; Testing; Therapeutic; Therapeutic Intervention; Tissues; Transgenic Mice; Work; alveolar epithelium; antagonist; arm; base; cell type; chemokine; comparative; complement system; cytokine; deep sequencing; disease phenotype; embryonic stem cell; experimental study; inflammatory lung disease; insight; lung injury; mouse model; neutrophil; novel; programs; receptor; single cell analysis; single-cell RNA sequencing; synergism; targeted treatment; tool; transcriptome; transcriptome sequencing; transcriptomics

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

抽象的 补体系统不仅是先天免疫的一个组成部分，而且对 感染相关炎症的严重程度。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过度激活 补体过敏毒素 C5a 的产生以及这些过程的治疗中断可以提供 防止组织破坏性炎症。 C5a与其同源受体C5aR1和C5aR2连接， 由两个相邻的基因编码。 C5aR1 和 C5aR2 在不同组织中的功能作用并不相同 完全清楚，而 C5aR2 的数据存在争议。在我们的前期工作中，我们生成了一个鼠标 通过 CRISPR/Cas9 基因编辑，两个 C5aR (C5aR1/2-/- DKO) 缺失约 12.6 kb 的菌株，另一只小鼠 用于报告和有条件删除 C5aR2（C5aR2LacZ；C5aR2flox）的菌株，我们可以访问有条件的 C5aR1 小鼠（C5aR1flox）。这些新颖的工具将有助于检验我们的中心假设，即函数 C5aR1 在中性粒细胞上与 C5aR2 在 II 型肺泡上皮细胞上的协同作用决定了 肺损伤和肺炎球菌肺炎的发生和进展。我们将重点关注三项具体工作 目的：（1）根据我们对骨髓嵌合小鼠的初步发现，表达和功能 将研究 C5aR2 在 II 型肺泡上皮细胞 (AECII) 中的相关性，包括以下终点 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期间。此外，C5aR2 的存在将与 正常人肺组织中的 C5aR1。 (2) 我们将描述 Ly6G+ 中性粒细胞中 C5aR 的作用 使用具有中性粒细胞特异性删除 C5aR1 和 C5aR2 的小鼠进行肺损伤和肺炎球菌肺炎。 为了阐明 C5aR1 对肺部侵袭性中性粒细胞亚群及其成熟状态的潜在影响，我们 将使用一种新型的基于液滴的高通量方法进行单细胞 RNA 测序研究。 (3) 我们将 通过比较炎症反应来研究 C5aR 的功能协同性、冗余性和多样性 C5aR1/2-/- DKO 至 C57BL/6 (Wt)、C5aR1-/- 和 C5aR2-/- 小鼠在肺损伤和 肺炎球菌肺炎。从上述肺炎实验中分离出的中性粒细胞和 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）专注于阐明中性粒细胞-AECII 相互作用的分子途径和搜索 用于新的 C5aR1/2 调控基因。这些调查旨在推进当前的概念并解决 关于 C5aR 的功能拮抗或操作协同作用的持续争议。未来， 这些见解可能有助于推进炎症性疾病治疗干预的概念

项目成果

A single-cell lung atlas of complement genes identifies the mesothelium and epithelium as prominent sources of extrahepatic complement proteins.

• DOI: 10.1038/s41385-022-00534-7
• 发表时间: 2022-05
• 期刊: MUCOSAL IMMUNOLOGY
• 影响因子: 8
• 作者: Chaudhary, Neha;Jayaraman, Archana;Reinhardt, Christoph;Campbell, Joshua D.;Bosmann, Markus
• 通讯作者: Bosmann, Markus