PKM2 associated alveolar macrophage remodelling is linked to defective efferocytosis of apoptotic neutrophils in LPS induced acute lung injury

BACKGROUND: Impaired clearance of apoptotic neutrophils by alveolar macrophages (AMs) may sustain inflammation in acute lung injury (ALI). Although PKM2 regulates macrophage inflammatory responses, its relationship with AM efferocytic dysfunction during ALI remains unclear. METHODS: We integrated time-course single-cell transcriptomic reanalysis with primary AM experiments and a murine model of LPS-induced ALI. PKM2 expression and localization and KLF4/MERTK expression were assessed using qPCR, western blotting, immunofluorescence, and flow cytometry. AM efferocytic responses were evaluated using pHrodo-labelled apoptotic neutrophil preparations. TEPP-46 or shikonin was administered 1 h after LPS challenge. UNC2025 was administered before LPS challenge to assess whether MERTK/FLT3 inhibition attenuated the effect of subsequent TEPP-46 treatment on AM efferocytosis. Lung injury was assessed by histology, bronchoalveolar lavage fluid (BALF) protein concentrations, and inflammatory cytokines. RESULTS: Pkm-high AMs exhibited enhanced inflammatory and glycolytic programmes, reduced efferocytosis and pro-resolution programmes, and lower Klf4 and Mertk expression. LPS increased PKM2 abundance and relative nuclear enrichment and reduced KLF4/MERTK expression in experimental AM preparations. These changes were accompanied by reduced AM pHrodo fluorescence and pHrodo-positive cell percentages and increased proportions and absolute numbers of Annexin V-positive BALF neutrophils. TEPP-46 and shikonin reduced PKM2 nuclear enrichment, improved AM efferocytic responses, and decreased dying neutrophil burden, BALF protein and cytokine concentrations, and histological lung injury. UNC2025 attenuated the TEPP-46-induced increases in pHrodo fluorescence and pHrodo-positive AM percentages. CONCLUSIONS: PKM2-associated AM remodelling links inflammatory activation to impaired efferocytic responses in LPS-induced ALI. Pharmacological intervention after LPS challenge improved AM function and attenuated lung injury. These findings support PKM2-associated AM dysfunction as a potentially modifiable component of impaired inflammatory resolution and implicate MERTK-associated signalling in the response to TEPP-46.