Intracellular Membrane Attack Complexes and COVID-19 Immune Hyperactivation

细胞内膜攻击​​复合体和 COVID-19 免疫过度激活

• 批准号: 10725199
• 负责人: DANIEL JANE-WIT
• 金额: $ 25.13万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-19 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10725199
• 关键词: 2019-nCoV; Alveolar; Antigens; Binding; Binding Proteins; Biological Assay; Blood capillaries; COVID-19; COVID-19 patient; Cell Death; Cell surface; Cells; Clinical; Complement; Complement Activation; Complement Membrane Attack Complex; Complex; Complication; Cytolysis; Data; Deposition; Diabetes Mellitus; Edema; Endosomes; Endothelial Cells; Family; Functional disorder; GBP1 gene; Homeostasis; Host Defense; Human; Hypertension; Immune; Immunity; In Vitro; Infection; Inflammation; Inflammation Mediators; Inflammatory; Intracellular Membranes; Life; Lung; Mediating; Membrane; Molecular; Mus; Natural Immunity; Orthologous Gene; Outcome; Patient-Focused Outcomes; Patients; Phenotype; Process; Property; Protein Isoforms; Proteins; Proteomics; Role; SARS-CoV-2 antigen; SARS-CoV-2 infection; SARS-CoV-2 spike protein; Sequence Homology; Signal Transduction; Surface; Symptoms; Testing; Tissues; cell type; cytokine; cytokine release syndrome; druggable target; guanylate; improved; in vivo; lung injury; membrane assembly; neoantigens; novel; protein aggregation; protein complex; response; sensor; severe COVID-19; soluble complement C5b-9; stem; tissue injury; vascular inflammation; virtual

PROJECT SUMMARY During severe COVID-19, immune hyperactivation contributes to tissue injury and worsened patient outcomes. Complement (C’) proteins are believed to centrally mediate COVID-19-related immune hyperactivation. C’ are a conserved set of immune proteins involved in host defense that become activated on various cell types in the lung during severe COVID-19. Upon terminal activation, C’ proteins assemble to form pore-like membrane attack complexes (MACs) that insert into target cell surfaces as transmembrane structures. The presence of MACs in pulmonary tissues strongly correlates with immune hyperactivation, but underlying mechanisms are unknown. The immune effects of MACs have been widely attributed to their cytolytic properties. However, widespread MAC deposition occurs on alveolar endothelial cells (ECs) during severe COVID-19 in the absence of significant EC death or vessel rarefaction. Instead, MAC-bound ECs show signs of EC dysfunction with dysregulated NF-B activation and elaboration of pro-inflammatory cytokines. These patient-level observations suggest immune effects of MACs that are separable from their cytolytic properties. In this application we examine a novel role for intracellular MAC proteins as alarmins. We used a SARS-CoV-2-derived antigen, S protein, to induce non-cytolytic MAC assembly on human ECs. Following assembly on ECs, surface-bound MACs became rapidly internalized and transferred to Rab5+ endosomes. The pool of intracellular, but not extracellular or surface-bound MACs, activated NF-B to induce elaboration of inflammatory cytokines. The intraluminal milieu of Rab5+ endosomes caused a MAC protein, C9, to form insoluble aggregates that stimulated aggrephagy, a specialized form of selective macroautophagy, to activate NF-B. Via proteomic profiling of solubilized MAC complexes (Sc5b-9) in COVID-19 sera, we identified guanylate binding protein 4 (GBP4) as a C9-binding protein regulating aggrephagy and NF-B activity. Based on these exciting preliminary data we propose 2 Specific Aims to explore the hypothesis that intracellular MAC proteins act as alarmins to initiate inflammatory signaling. We will consolidate role(s) for GBP4 as an immune sensor by examining binding interactions with C9 using COVID-19 patient sera. Secondly, we will identify mouse orthologs for human GBP4 that mediate C9 sensing and MAC-induced immune hyperactivation in vivo in response to S protein-induced MAC and following infection by replication competent SARS-CoV-2. Our studies introduce a new paradigm for understanding MAC-related immunity and contribute to our long-term aim of understanding C’-induced inflammation. By doing this, druggable targets ameliorating MAC-induced immune complications of severe COVID-19 may emerge.

项目摘要 在严重的COVID-19期间，免疫过度激活导致组织损伤和患者病情恶化 结果。补体（C '）蛋白被认为是中枢介导COVID-19相关的免疫反应。 过度活跃C'是参与宿主防御的一组保守的免疫蛋白， 在严重的COVID-19期间肺部的各种细胞类型。在末端激活后，C'蛋白组装形成 孔样膜攻击复合物（MAC），作为跨膜插入靶细胞表面 结构.肺组织中MAC的存在与免疫过度活化密切相关，但 潜在的机制是未知的。 MAC的免疫效应已被广泛归因于其细胞溶解特性。然而，在这方面， 在严重的COVID-19期间，在没有MAC的情况下，肺泡内皮细胞（EC）上发生广泛的MAC沉积 严重的EC死亡或血管稀疏。相反，MAC结合的EC显示EC功能障碍的迹象， NF-κ B B活化失调和促炎细胞因子的产生。这些病人层面的观察 表明MAC的免疫作用与其细胞溶解特性是分开的。 在这个应用程序中，我们研究了一个新的作用，细胞内MAC蛋白alarmins。我们使用了 SARS-CoV-2衍生抗原S蛋白诱导人EC上的非溶细胞MAC组装。以下 当在EC上组装时，表面结合的MAC迅速内化并转移到Rab 5+内体。 细胞内的MACs，而不是细胞外的或表面结合的MACs，激活NF-κ B B，诱导 炎性细胞因子Rab 5+内体的腔内环境导致MAC蛋白C9形成 不溶性聚集物刺激聚集吞噬，一种选择性巨自噬的特殊形式， NF-κ B B。通过对COVID-19血清中溶解的MAC复合物（Sc 5 b-9）的蛋白质组学分析，我们确定了 鸟苷酸结合蛋白4（GBP 4）作为C9结合蛋白调节聚集吞噬和NF-κ B B活性。 基于这些令人兴奋的初步数据，我们提出了2个具体目标来探索假设， 细胞内MAC蛋白作为警报素启动炎症信号传导。我们将巩固以下方面的作用： 通过使用COVID-19患者血清检查与C9的结合相互作用，将GBP 4作为免疫传感器。 其次，我们将鉴定介导C9传感和MAC诱导的人GBP 4的小鼠直系同源物。 S蛋白诱导MAC和复制感染后体内免疫超活化 SARS-CoV-2病毒。我们的研究为理解MAC相关免疫引入了一种新的范式， 有助于我们理解C '诱导的炎症的长期目标。通过这样做， 可能出现改善严重COVID-19的MAC诱导的免疫并发症。