Dual knockdown of Scn1b and Gsdmd induces an M2-like functional state in macrophages and modulates inflammation and fibrosis in a rat model of acute respiratory distress syndrome.

BACKGROUND: Macrophage-based cell therapy represents a promising approach to the acute respiratory distress syndrome (ARDS) therapy. This study investigates the potential use of adoptive transfer of siRNA-modified macrophages to influence the progression of ARDS. METHODS: Bone marrow-derived macrophages (BMDMs) from male Sprague-Dawley rats were characterized by immunocytochemistry, phagocytosis assays. RNA sequencing was performed following LPS (M1) or IL-4/IL-10 (M2) polarization. Knockdown efficiency, proteomic profiles, metabolic shifts, and effects on mesenchymal stem cells (MSCs) and glioblastoma cells were assessed. Effects of siRNA-dependent knockdown macrophages or M(Scn1b + Gsdmd) were evaluated in the LPS-induced ARDS rat model 24 h and 7 days after macrophage administration. RESULTS: Phagocytic activity was significantly reduced in M2 macrophages. Scn1b and Gsdmd, differentially expressed in M2 vs. M0, were prioritised from RNA sequencing data. Dual knockdown efficiently suppressed both genes, inducing transcriptome changes similar to those observed after IL-4 + IL-10 polarization and shifting metabolism toward oxidative phosphorylation but did not significantly affect phagocytic activity. Conditioned medium from M(Scn1b + Gsdmd) enhanced MSCs proliferation capacity and viability. In ARDS rats, M(Scn1b + Gsdmd) reduced IL-18 and TIMP2 after 24 h. For 7 days, they promoted reparative signatures: increased Vegf and reduced thickness of the interalveolar septa and amount of collagen fiber (according to Mallory's trichrome staining). CONCLUSION: Dual knockdown of Scn1b and Gsdmd leads to the formation of an M2-like macrophage phenotype in vitro. Systemic administration of M(Scn1b + Gsdmd) modulate fibrotic processes in the lungs that develop against the background of intratracheal administration of LPS.