Broad remodeling of the pulmonary immune landscape occurs during IAPA with specific functional deficits of neutrophil subsets

Abstract

BackgroundInfluenza-associated pulmonary aspergillosis (IAPA) is a severe complication of influenza infection associated with prolonged intensive care unit stay and increased mortality. Although impaired antifungal immunity has been implicated in IAPA pathogenesis, the cell type-specific immune mechanisms driving susceptibility remain incompletely understood. We aimed to characterize the pulmonary immune landscape during IAPA using single-cell transcriptomics and functional neutrophil assays. MethodsMale C57BL/6 mice were assigned to naive control, influenza A/PR/8/34 (H1N1) infection, A. fumigatus (ATCC 42202) infection, or IAPA groups. Lung CD45+ immune cells underwent single-cell RNA sequencing with downstream clustering and CellChat ligand-receptor interaction analysis. Differential gene expression analyses were performed across myeloid, lymphoid, and neutrophil populations. Functional neutrophil responses were evaluated using flow cytometry, myeloperoxidase activity assays, and FLARE (fluorescent Aspergillus reporter) conidia to assess fungal conidia uptake and killing. Cross-species validation was performed using gene set enrichment analysis compared with published human IAPA transcriptomic datasets. ResultsIAPA broadly remodeled the pulmonary immune landscape across myeloid, lymphoid, and neutrophil compartments. Myeloid cells showed coordinated suppression of fungal pattern recognition receptors, lysosomal biogenesis programs, and inflammatory signaling. Lymphoid populations exhibited transcriptional signatures of T cell exhaustion and Th17 suppression. Within the neutrophil compartment, we identified two transcriptionally and functionally distinct populations, conventional and inflammatory neutrophils, with divergent antifungal effector capacities. Inflammatory neutrophils showed selective killing defects, while both subsets exhibited impaired phagocytic uptake during IAPA. Murine transcriptomic findings demonstrated strong concordance with immune dysfunction signatures identified in human IAPA. ConclusionIAPA susceptibility arises from coordinated transcriptional dysfunction spanning innate and adaptive immune compartments. Distinct neutrophil subset dysfunction, impaired fungal recognition pathways, and T-cell exhaustion signatures collectively contribute to defective fungal clearance, providing mechanistic insight into IAPA susceptibility and potential therapeutic targets.