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.

项目总结 在严重的新冠肺炎期间，免疫过度激活会导致组织损伤和病情恶化 结果。补体(C‘)蛋白被认为是新冠肺炎相关免疫的中枢调节因子 过度活跃。C‘是参与宿主防御的一组保守的免疫蛋白，它们在 重症新冠肺炎时肺内有多种细胞类型。在末端激活时，C‘蛋白质组装形成 以跨膜形式插入靶细胞表面的孔状膜攻击复合体(MACs) 结构。肺组织中MACs的存在与免疫过度激活密切相关，但 潜在的机制尚不清楚。 MACs的免疫作用被广泛归因于它们的细胞溶解特性。然而， 严重新冠肺炎时，肺泡内皮细胞广泛沉积。 严重的EC死亡或血管稀疏。相反，与MAC绑定的EC显示出EC功能障碍的迹象 失调的NF-B激活和促炎细胞因子的产生。这些患者层面的观察 提示Mac的免疫效果可与其细胞溶解特性相分离。 在这个应用中，我们研究了细胞内MAC蛋白作为警报蛋白的一个新角色。我们使用了一个 SARS-CoV-2衍生抗原S蛋白诱导人内皮细胞非细胞溶解MAC组装。跟随 在ECs上组装，表面结合的MACs迅速内化并转移到Rab5+内吞体内。 细胞内而不是细胞外或表面结合的MACs池激活了NF-B以诱导精加工 炎性细胞因子。Rab5+内小体的腔内环境导致了MAC蛋白C9的形成 刺激吞噬的不溶性聚集体，这是选择性巨噬的一种特殊形式，以激活 通过对新冠肺炎血清中溶解的复合体(Sc5b-9)的蛋白质组学分析，我们鉴定了 鸟苷结合蛋白4(Gbp4)是一种C9结合蛋白，调节吞噬功能和核因子-B活性。 基于这些令人兴奋的初步数据，我们提出了两个具体目标来探索以下假设 细胞内的MAC蛋白充当启动炎症信号的警报器。我们将巩固(S)的角色 通过使用新冠肺炎患者血清检测与C9的结合相互作用，将GBP4作为免疫传感器。 其次，我们将确定介导C9感觉和MAC诱导的人GBP4的小鼠同源基因 S蛋白诱导的MAC和复制感染后的体内免疫激活 有能力的SARS-CoV-2。我们的研究介绍了一种理解MAC相关免疫和 有助于我们理解C‘引起的炎症的长期目标。通过这样做，可下药的靶点 重症新冠肺炎可能出现改善MAC诱导的免疫并发症。