Multi-omics profiling of acute <i>Pseudomonas aeruginosa</i> pneumonia unmasks conventional NK cell depletion and stage-specific therapeutic targets

<i>Pseudomonas aeruginosa</i> (PA) is a key pathogen in hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP), challenging clinical medicine. This study aims to elucidate the characteristics of the host’s innate immune response following inhalational PA infection. We developed a mouse model by aerosolized intratracheal inoculation with PA and conducted a comprehensive analysis at the protein, cellular, and gene expression levels. Protein analysis revealed a substantial increase in inflammatory proteins in the bronchoalveolar lavage fluid and serum, indicating a robust inflammatory response in the lungs and systemic circulation. Cellular investigations showed an increase in neutrophils, monocytes, and alveolar macrophages during infection, whereas NK cells showed a marked reduction from 5.88% pre-infection to 2.41% at 24 h (<i>p</i> = 0.0102) and 1.55% by 48 h (<i>p</i> = 0.0023). To assess gene expression changes, RNA-sequencing technology was employed to map the temporal shifts in the transcriptional profile of the host lung post-infection. We analysed the expression patterns and dynamic transcriptional characteristics of differentially expressed genes (DEGs), describing the inflammation progression. Importantly, Through the analysis of single-cell RNA sequencing (scRNA-seq) datasets in public repositories, we observed the reduction in conventional natural killer (cNK) cells, rather than tissue-resident natural killer (trNK) cells in the early stages of PA infection. Sequential scRNA-seq analysis resolved NK-subset heterogeneity, revealing that cNK dominance (77.8% of total NK cells) under homeostasis collapsed to 9.2% within 24 h post-infection. Our findings establish cNK attrition as the earliest immune checkpoint failure in PA pneumonia and provide proof-of-concept for cNK-targeted immunotherapies to counteract lethal pulmonary infections. Keywords: <i>Pseudomonas aeruginosa</i>, aerosolized intratracheal inoculation, conventional NK cells, innate immunity, RNA-sequencing