Integrated single-cell and spatial transcriptomic atlas of multi-etiology acute lung injury reveals etiology-dependent neutrophil fate decisions and a prognostic neutrophil-monocyte/macrophage interaction signature.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a life-threatening syndrome with heterogeneous etiologies and no effective pharmacological therapy. Although neutrophils are central mediators of lung injury, their functional diversity and interplay with monocyte/macrophage (Mo/MΦ) populations across etiologies remain poorly defined. Here, we integrated single-cell RNA-sequencing profiles from 180,031 murine lung cells across seven ALI models spanning infectious, sterile, and extrapulmonary insults to construct a multi-etiology neutrophil atlas. Augur identified neutrophils as the most transcriptionally perturbed immune population. Gene-set variation analysis revealed etiology-specific programs, including NF-κB/IL-6-driven inflammation in infection/sepsis, dual interferon responses in influenza, tissue-remodeling and stress-adaptive programs in bleomycin injury, and ROS-dominated injury with metabolic reprogramming after radiation. Spatial transcriptomics further mapped the spatiotemporal dynamics of neutrophil reprogramming during influenza-induced lung injury. High-resolution re-clustering resolved 13 neutrophil subtypes organized into four macro-states, and pseudotime analysis uncovered an etiology-dependent bifurcation from a bone marrow-like immature origin toward either an IFN/inflammasome-associated effector branch or an NF-κB-driven inflammatory branch, with sepsis and bacterial infection retaining neutrophils near the immature origin. In parallel, we defined 12 Mo/MΦ subtypes and derived a 26-gene Neutrophil-Monocyte/Macrophage Interaction Index with prognostic value for sepsis mortality. Importantly, immunobiological validation showed that conditioned media from LPS-, P. aeruginosa- and CLP-derived neutrophils contained elevated THBS1 and activated NF-κB-MAPK-NLRP3 signaling in macrophages, whereas blockade of THBS1-CD36 with CD36 peptide P(93-110) attenuated inflammatory activation, promoted reparative polarization, and reduced circulating neutrophils in vivo. Together, these findings define an etiology-resolved atlas of ALI and identify THBS1-CD36 as a mechanistically relevant therapeutic target in infection- and endotoxin-driven ALI.