CMPK2 promotes M1 macrophage polarization in sepsis-induced acute lung injury via NLRP3/NF-κB signalling

Sepsis-induced acute lung injury (SALI) is a life-threatening complication characterized by uncontrolled inflammation and alveolar macrophage (AM) activation, and contributes significantly to sepsis-related mortality. Cytidine/uridine monophosphate kinase 2 (CMPK2) has emerged as a potential regulator of inflammatory responses, but its role in SALI remains poorly understood. This study aimed to elucidate the molecular mechanisms by which CMPK2 modulates SALI, focusing on its impact on AM polarization and inflammatory signalling pathways, to identify novel therapeutic targets for improving clinical outcomes.

The biological impact of CMPK2 on SALI was assessed by evaluating the survival rate and histological appearance of lung tissue from a cecal ligation and puncture (CLP)-induced sepsis mouse model. The concentrations of inflammatory factors and oxidative stress indicators were subsequently assessed via enzyme-linked immunosorbent assay (ELISA) and biochemical kits. Flow cytometry and real-time quantitative PCR were utilized to evaluate the polarization types of AMs. RNA sequencing was conducted on AMs from wild-type and CMPK2-deficient mice to explore potential molecular mechanisms involved. Transmission electron microscopy (TEM) was used to examine the mitochondrial ultrastructure in lipopolysaccharide (LPS)-stimulated macrophages. The subcellular localization of CMPK2 in RAW264.7 macrophages was mapped via high-resolution confocal microscopy. Dual-luciferase reporter assays and coimmunoprecipitation (co-IP) were used to investigate the interaction of CMPK2 with NF-κB signalling components.

CMPK2 was significantly upregulated in the lung tissue of CLP-induced septic mice, which correlated with increased systemic and pulmonary inflammation, as evidenced by elevated IL-1β, IL-6, and TNF-α levels and a higher lung wet-dry (W-D) ratio. RNA sequencing revealed 2843 DEGs in CLP versus sham mice, with enrichment in Th1/Th2 cell differentiation, NF-κB, NOD-like receptor, and B-cell receptor signalling pathways. CMPK2 deficiency significantly improved survival; reduced lung edema, inflammatory cell infiltration, and microvascular leakage; and decreased cytokine levels in the BALF. TEM revealed that CMPK2 knockdown in LPS-stimulated macrophages mitigated mitochondrial edema and cristae disruption, restoring the mitochondrial membrane potential and ATP levels. Flow cytometry and RT-qPCR confirmed reduced M1 macrophage polarization in CMPK2^-/-^ mice, with decreased expression of M1-specific markers. Mechanistically, CMPK2 interacts with IKKα/β via its C-terminal domain, enhancing NF-κB phosphorylation and NLRP3 inflammasome activation, with the C-terminal domain being essential for this pro-inflammatory activity, whereas deletion of the N-terminal mitochondrial targeting sequence did not abolish CMPK2-mediated NF-κB activation. Inhibition of NF-κB with BAY11-7082 reversed CMPK2-mediated inflammatory effects. Confocal microscopy revealed that LPS stimulation induced CMPK2 translocation from the cytoplasm to the nucleus, suggesting that LPS plays a dynamic role in inflammatory signalling.

CMPK2 exacerbates SALI, at least in part, by promoting M1-skewed alveolar macrophage responses and enhancing NF-κB/NLRP3-associated inflammatory signalling. Mechanistically, CMPK2 associates with IKKα/β through its C-terminal domain, whereas the canonical N-terminal mitochondrial targeting sequence is not strictly required for its pro-inflammatory activity in this context. These findings highlight CMPK2 as a novel therapeutic target for alleviating SALI, offering potential for precision interventions to improve the clinical management of sepsis-related lung injury.