Single-cell transcriptomics unveils alveolar microenvironment remodeling in sepsis-induced ARDS

Abstract

Acute respiratory distress syndrome (ARDS) is a life-threatening disorder with high mortality, predominantly triggered by sepsis. Sepsis-associated ARDS involves not only focal lung injury but also systemic immune dysregulation. Despite advances in supportive care, effective therapies targeting the underlying mechanisms of sepsis-associated ARDS remain limited, and a comprehensive single-cell landscape of the alveolar immune microenvironment remains poorly defined. In this study, single-cell RNA sequencing was performed on bronchoalveolar lavage fluid (BALF) samples from five patients with sepsis-induced ARDS within 24 h of intubation, one in-house healthy donor (HD), and four HD from public databases. Unsupervised clustering identified five major cell populations, with neutrophils being the most abundant. Subpopulation analyses revealed significant transcriptional heterogeneity in macrophages, neutrophils, lymphocytes, and epithelial cells between the ARDS and HD groups. Flow cytometry further validated the cellular proportions in BALF, revealing that BALF neutrophil proportions were positively correlated with SOFA score, C‑reactive protein, procalcitonin, lactate, and multiple organ‑injury markers (ALT, AST, BUN, CREA), and negatively correlated with PaO₂/FiO₂ ratio. Cell-cell communication analysis showed weak, homeostatic intercellular interactions in HD. In contrast, ARDS patients displayed intense, dynamic crosstalk between immune and epithelial cells. We propose that sepsis‑induced ARDS is characterized by functional state remodeling, manifested by the depletion of protective subsets including lipid‑metabolic homeostatic macrophages (Mac1), resting‑like NK/T cells (NK/T1), ciliated epithelial cells (Epi4) and interferon‑responsive neutrophils (Neu3), the expansion of pathological pro‑inflammatory subsets (Mac2, NK/T3, Epi2, Neu1), and rewiring of intercellular signaling: attenuation of homeostatic reparative pathways alongside hyper‑activated CXCL/CCL chemokine signaling cascades, which collectively contribute to alveolar barrier disruption and sustained pulmonary inflammation. This single-cell atlas highlights disease-associated cellular subsets and candidate molecular mediators ( CCL3 , CCL4 , and CXCL2 ) for future mechanistic investigation and evaluation of their diagnostic or therapeutic relevance.