Transplantation of LPS, IL-4, and TGF-β-induced reparative-biased macrophages promotes early motor functional recovery after spinal cord injury in rats.

OBJECTIVE: Spinal cord injury (SCI) triggers an inflammatory cascade that often culminates in chronic neurological deficits. This study investigates a combined stimulation approach using lipopolysaccharide (LPS), interleukin-4 (IL-4), and transforming growth factor-beta (TGF-β) to generate reparative-biased macrophages and evaluate their early effects after SCI. METHODS: Bone marrow-derived macrophages (BMDMs) from Wistar rats were exposed to LPS, IL-4, and TGF-β for 24 h to generate reparative-biased hybrid macrophages (Mi) in vitro. The anti-inflammatory and pro-regenerative markers of Mi were characterised by qPCR, immunocytochemistry, and proteomic analysis via mass spectrometry. In vivo, rats with a T9/10 clip-contusion-compression SCI received either Mi transplantation or direct inducer administration at the lesion site. Histological outcomes and functional recovery were assessed and compared between injured and uninjured controls over a 14-day period. RESULTS: Following 24 h induction, Mi exhibited significantly elevated mRNA and immunofluorescence expression of Arg1, as well as increased IL-10 mRNA levels, compared to naïve BMDMs in vitro, while iNOS expression remained unchanged. Bioinformatic analysis of proteomic data from Mi revealed enrichment in metabolic activity, immune modulation, and phagocytosis pathways. Fluorescent microsphere phagocytosis assays demonstrated that Mi macrophages retained robust phagocytic activity. In vivo, transplantation of Mi resulted in elevated Arg1 expression and reduced iNOS and IL-1β levels 3 days after SCI, suggesting an anti-inflammatory shift. Immunohistochemical analysis demonstrated significantly reduced lesions and demyelinated areas, as well as increased preservation of motor neurons and oligodendrocytes. Mi transplantation was also associated with reduced apoptosis and improved early functional recovery at 14 days after SCI. CONCLUSION: This study suggests that LPS, IL-4, and TGF-β-induced Mi macrophages display a reparative-biased hybrid profile that modulates inflammation, supports early tissue preservation, and promotes functional recovery after SCI, supporting Mi transplantation as a potential cell-based therapeutic strategy for further investigation. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Transplantation of this reparative-biased hybrid macrophage phenotype may provide a potential therapeutic strategy for the treatment of spinal cord injury.