The Impact of SARS-CoV-2 Immune Dysregulation on Antifungal Immunity

项目摘要 尽管已发表了数百篇文章、荟萃分析和综述，描述了该病的临床综合征 COVID相关性肺曲霉菌病(CAPA)，到目前为止还没有任何出版物探讨 严重SARS-2感染者死于2°霉菌感染的机制。这项建议 旨在评估免疫反应针对细胞内的新概念进展 病毒病原体，如SARS-2，通过延迟释放生物有效铁促进2°霉菌感染 中性粒细胞(PMN)募集，抗真菌效应物(αFE)表达减少。在目标1-3中，我们 肺上皮细胞(PEC)和RED细胞裂解程序性死亡(PCD)假说的探讨 血细胞溶血增加了包括铁/血红素在内的营养物质的利用率，铁/血红素驱动铁载体- 依赖于霉菌生长的加速。同时，SARS-2和抗病毒细胞因子环境可减轻PMN 重新招募和激活导致孢子萌发成大的侵袭性菌丝，从而压倒αFE 使2°霉菌在其他具有免疫能力的宿主中感染的机制。在Aim1中，我们将对铁进行量化， 血红素、二价阳离子、宿主金属隔离蛋白(MSP)、抗病毒细胞因子和PMN-Recruiting SARS-2感染队列中的趋化因子与对照感染队列(IC)的比较。我们还将使用免疫荧光(IF)和 用空间转录组学方法研究裂解PCD、病毒ORF3a介导的细胞裂解和αFE在FFPE中的表达 肺组织，并利用同基因突变真菌鉴定关键宿主和微生物因子中介霉菌 BALS的增长。在AIM2中，我们将利用新的Calu-3基因敲除(KO)细胞系和1°正常人细支气管壁 上皮(NHBE)气液界面细胞培养对下呼吸道环境的影响及直接 非典-2感染对肺内皮细胞的影响：裂解细胞死亡、干扰素合成、αFE分泌、金属固存和应答能力 至2°真菌刺激。我们还将利用同基因突变真菌来确定调节霉菌生长的关键途径。 和一个创新的PMN气道迁移模型，以确定I型和III型IFN、铁/血红素的影响 毒性，以及SARS-2对PMN募集、激活和真菌杀灭的影响。在Aim3中，我们将利用小说 条件性KO小鼠、荧光病毒和真菌报告菌株、IF、流式细胞术和单细胞测序 评估SARS-2变异体诱导裂解性PCD的可能性，明确裂解性PCD和干扰素信号转导的作用 在PECs、巨噬细胞(Mφ)和中性粒细胞中，并评价它们对肺病理、金属释放、中性粒细胞- 招募、孢子存活、真菌生长和感染结果。我们还将剖析关键宿主MSP和真菌 介导霉菌生长的途径，评估Mφ和PMNhACE2在病毒和真菌清除中的作用 确定裂解PCD的小分子抑制剂、铁络合剂和铁载体生物合成抑制剂 防止或减轻2°霉菌感染的发展。我们相信拟议研究的结果 将为CAPA介导的基础生物学提供新的线索，并可能使改良的 减少感染和死于这种毁灭性感染的风险的治疗方案。

Project Summary Despite hundreds of published articles, meta-analyses, and reviews describing the clinical syndrome of COVID-Associated Pulmonary Aspergillosis (CAPA), there have been zero publications to date exploring the mechanism by which individuals with severe SARS-2 infection succumb to 2° mold infection. This proposal seeks to evaluate the novel conceptual advancement that immune responses targeting intracellular viral pathogens, like SARS-2, promote 2° mold infection via release of bioavailable iron, delayed neutrophil (PMN) recruitment, and decreased antifungal effector (αFE) expression. In Aims 1-3, we explore the hypotheses that lytic programmed cell death (PCD) in pulmonary epithelial cells (PEC) and red blood cell hemolysis increase the availability of nutrients including iron/heme which drive siderophore- dependent acceleration in mold growth. Concurrently, SARS-2 and the antiviral cytokine milieu mitigate PMN recruitment and activation resulting in spore germination into large invasive hyphae that overwhelm αFE mechanisms enabling 2° mold infection in an otherwise immunocompetent host. In Aim1, we will quantify iron, heme, divalent cations, host metal sequestration proteins (MSP), antiviral cytokines, and PMN-recruiting chemokines in SARS-2 BALs vs control infection cohorts (IC). We will also use immunofluorescence (IF) and spatial transcriptomics to characterize lytic PCD, viral ORF3a-mediated cell lysis, and αFE expression in FFPE lung tissues and utilize isogenic mutant fungi to identify critical host and microbial factors mediating mold growth in BALs. In Aim2, we will utilize novel Calu-3 knockout (KO) cell lines and 1°normal human bronchiole epithelial (NHBE) air liquid interface cell cultures to study the impact of the lower airway milieu and direct SARS-2 infection on PEC: lytic PCD, IFN synthesis, αFE secretion, metal sequestration, and ability to respond to 2° fungal stimuli. We will also utilize isogenic mutant fungi to identify critical pathways mediating mold growth and an innovative PMN airway transmigration model to determine the impact of Type I and III IFNs, iron/heme toxicity, and SARS-2 uptake on PMN recruitment, activation, and fungal killing. In Aim3, we will utilize novel conditional KO mice, fluorescent viral and fungal reporter strains, IF, flow cytometry, and single cell sequencing to assess the potential of SARS-2 variants to induce lytic PCD, define the role of lytic PCD and IFN signaling in PECs, macrophages (Mφ), and PMNs, and evaluate their impact on lung pathology, metal release, PMN- recruitment, spore viability, fungal growth, and infection outcome. We will also dissect key host MSP and fungal pathways mediating mold growth, evaluate the role of Mφ and PMN hACE2 on viral and fungal clearance and determine if small molecule inhibitors of lytic PCD, iron chelators, and siderophore biosynthesis inhibitors prevent or mitigate the development of 2° mold infection. We believe that the results of the proposed study will shed new light on the fundamental biology mediating CAPA and may enable the development of improved therapeutic regimens that mitigate the risk of acquiring and succumbing to this devastating infection.