Necroptosis in alveolar epithelium orchestrates lung ischemia-reperfusion injury: a multi-omics study

Lung ischemia-reperfusion injury (LIRI) is a leading cause of early morbidity and mortality following lung transplantation and other cardiopulmonary procedures. It is characterized by acute sterile inflammation driven by regulated cell death (RCD). While various RCD modalities, including apoptosis, necroptosis, pyroptosis, and ferroptosis, have been implicated in lung injury, their relative contributions and distinct activation patterns in LIRI remain poorly defined.

We employed an integrated multi-omics approach combining transcriptomics and proteomics with histological and functional validations in a murine hilar clamping model of LIRI. Key findings were further corroborated using single-cell RNA sequencing (scRNA-seq) data from human lung transplant recipients. The functional role of necroptosis was validated using pharmacological inhibitors (Nec-1, GSK'872) and Mlkl-deficient (Mlkl^-/-) mice.

LIRI triggered acute, time-dependent lung injury peaking within 24 h of reperfusion. Although transcriptomic profiling suggested broad activation of multiple RCD pathways, proteomic and biochemical analyses revealed a distinct landscape in our experimental setting: markers of apoptosis, pyroptosis, and ferroptosis were either downregulated or showed no significant positive correlation with injury severity and inflammatory peaks. In contrast, the necroptotic pathway emerged as a highly activated modality. Specifically, necroptosis, marked by phosphorylated RIPK1, RIPK3, and MLKL, was localized primarily in alveolar epithelial cells, correlated strongly with cytokine release and histological lung injury, and preceded the inflammatory response. Pharmacological inhibition or genetic ablation of necroptosis significantly attenuated tissue damage and inflammation. This pronounced necroptotic signature appeared distinct from the broad multi-pathway activation observed in lipopolysaccharide (LPS)-induced lung injury. Translational analysis of human scRNA-seq data further confirmed the selective upregulation of necroptosis signatures in alveolar type 2 (AT2) cells following lung transplantation.

Our multi-omics analysis identifies necroptosis, particularly in alveolar epithelial cells, as a critical driver of sterile inflammation and tissue injury in the early phase of LIRI. Targeting alveolar epithelial necroptosis may represent a precise and promising therapeutic strategy for lung transplantation and ischemia-reperfusion-associated pulmonary disorders.