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

项目摘要 与大多数侵袭性霉菌感染不同，COVID相关性肺曲霉病（CAPA）发生在 具有其他完整免疫系统的个体，提示新的生物学机制介导 易受真菌感染。该提案旨在评估新的概念性进展， 靶向细胞内病毒病原体的免疫应答损害人中性粒细胞的必需性 抗真菌效应子功能需要杀死大的细胞外真菌菌丝。在目标1中，我们将利用 BAL样本和尸检福尔马林固定石蜡包埋（FFPE）肺组织，以表征 人中性粒细胞抗真菌效应物的表达，上皮细胞坏死性凋亡，铁含量和真菌， SARS-CoV-2和CAPA感染期间的呼吸道。在目标2中，我们将使用原发性细支气管 上皮细胞气液界面（ALI）培养物，CRISPR敲除细胞系（MLKL、RIPK 1、RIPK 3）， 细胞死亡途径的化学抑制剂和中性粒细胞：真菌杀伤试验，以确定 SARS-CoV-2介导上皮细胞死亡、铁释放、霉菌生长和人类 中性粒细胞抗真菌效应功能。在目标3中，我们将利用一种新的CAPA感染小鼠模型， 中性粒细胞（MRP 8-Cre）和肺上皮细胞中人ACE 2表达的cre-lox敲低 （CC 10-Cre）沿着与缺铁真菌突变菌株（Δ ftrA、ΔftrA），以评价 SARS-CoV-2感染对肺上皮细胞坏死性凋亡、铁释放、铁载体依赖性真菌 生长和中性粒细胞募集/活化。我们认为，拟议研究的结果将有助于 对介导病毒相关的继发性霉菌感染的基础生物学的新认识， 能够开发改善的治疗方案，从而降低发生CAPA的风险。

Project summary Unlike most invasive mold infections, COVID Associated Pulmonary Aspergillosis (CAPA) occurs in individuals with otherwise intact immune systems suggesting novel biological mechanisms mediating susceptibility to fungal infection. This proposal seeks to evaluate the novel conceptual advancement that immune responses targeting intracellular viral pathogens compromise essential human neutrophil antifungal effector functions required to kill large extracellular fungal hyphae. In Aim 1, we will utilize BAL samples and autopsy formalin-fixed paraffin embedded (FFPE) lung tissues to characterize the expression of human neutrophil antifungal effectors, epithelial cell necroptosis, iron content, and fungi in the respiratory tract during SARS-CoV-2 and CAPA infection. In Aim 2, we will use primary bronchiolar epithelial cell air-liquid interface (ALI) cultures, CRISPR knockout cell lines (MLKL, RIPK1, RIPK3), chemical inhibitors of cell death pathways, and neutrophil:fungal killing assays to determine the mechanism by which SARS-CoV-2 mediates epithelial cell death, iron release, mold growth, and human neutrophil antifungal effector functions. In Aim 3, we will utilize a novel CAPA infection mouse model, cre-lox knockdown of human ACE2 expression in neutrophils (MRP8-Cre) and lung epithelial cells (CC10-Cre) along with iron-deficient fungal mutant strains (ΔsidA, ΔftrA), to evaluate the in vivo impact of SARS-CoV-2 infection on lung epithelial cell necroptosis, iron release, siderophore-dependent fungal growth, and neutrophil recruitment/activation. We believe that the results of the proposed study will shed new light on the fundamental biology mediating viral-associated secondary mold infection which may enable the development of improved therapeutic regimens that mitigate the risk of developing CAPA.