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

项目摘要 尽管有数百篇已发表的文章、荟萃分析和综述描述了 关于COVID相关肺结核（CAPA），迄今为止没有任何文献探讨 严重SARS-2感染的个体死于2 °霉菌感染的机制。这项建议 旨在评估新的概念进展，免疫反应靶向细胞内 病毒病原体，如SARS-2，通过释放生物可利用铁，延迟 中性粒细胞（PMN）募集和抗真菌效应物（α FE）表达降低。在目标1 - 3中，我们 探讨肺上皮细胞（PEC）和红细胞中溶解性程序性细胞死亡（PCD） 血细胞溶血增加了包括铁/血红素在内的营养物质的可用性， 依赖于模具生长的加速。同时，SARS-2和抗病毒细胞因子环境减轻PMN 补充和激活导致孢子萌发为大的侵入性菌丝，压倒α FE 在其他免疫活性宿主中实现2 °霉菌感染的机制。在目标1中，我们将量化铁， 血红素、二价阳离子、宿主金属螯合蛋白（MSP）、抗病毒细胞因子和PMN募集 SARS-2 BALs与对照感染组群（IC）中的趋化因子。我们还将使用免疫荧光（IF）， 空间转录组学用于表征FFPE中裂解性PCD、病毒ORF3a介导的细胞裂解和α FE表达 肺组织，并利用同基因突变真菌，以确定关键的宿主和微生物因子介导的霉菌 在BALs中生长。在Aim 2中，我们将利用新型Calu-3敲除（KO）细胞系和1 °正常人细支气管 上皮（NHBE）气液界面细胞培养，以研究下气道环境和直接 SARS-2感染对PEC的影响：裂解性PCD、IFN合成、α FE分泌、金属螯合和反应能力 至2 °真菌刺激。我们还将利用同基因突变真菌，以确定关键途径介导的霉菌生长 以及一种创新的PMN气道迁移模型，以确定I型和III型IFN、铁/血红素 毒性和SARS-2摄取对PMN募集、活化和真菌杀伤的影响。在Aim3中，我们将使用新的 条件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 为了评估SARS-2变异体诱导裂解性PCD的潜力，确定裂解性PCD和IFN信号转导的作用， 在PEC，巨噬细胞（M φ），和中性粒细胞，并评估其对肺病理学，金属释放，中性粒细胞- 募集、孢子活力、真菌生长和感染结果。我们还将解剖关键宿主MSP和真菌 介导霉菌生长的途径，评估M φ和PMN hACE 2对病毒和真菌清除的作用， 确定裂解性PCD的小分子抑制剂、铁螯合剂和铁载体生物合成抑制剂 预防或减轻2 °霉菌感染的发展。我们认为，拟议研究的结果 将揭示新的光的基础生物学介导的CAPA，并可能使发展改善 治疗方案，减轻获得和屈服于这种毁灭性感染的风险。

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.