Substance P promotes epidural fibrosis via induction of type 2 macrophages

[Image: see text] In response to spinal surgery, neurons secrete a large amount of substance P into the epidural area. Substance P is involved in macrophage differentiation and fibrotic disease. However, the specific roles and mechanisms of substance P in epidural fibrosis remain unclear. In this study, we established a mouse model of L1–L3 laminectomy and found that dorsal root ganglion neurons and the macrophages infiltrating into the wound area released sphingolipids. In vitro experiments revealed that type 1 macrophages secreted substance P, which promoted differentiation of type 1 macrophages towards a type 2 phenotype. High-throughput mRNA-seq analysis revealed that the sphingolipid metabolic pathway may be involved in the regulation of type 2 macrophages by substance P. Specifically, sphingomyelin synthase 2, a component of the sphingolipid metabolic pathway, promoted M2 differentiation in substance P-treated macrophages, while treating the macrophages with LY93, a sphingomyelin synthase 2 inhibitor, suppressed M2 differentiation. In addition, substance P promoted the formation of neutrophil extracellular traps, which further boosted M2 differentiation. Blocking substance P with the neurokinin receptor 1 inhibitor RP67580 decreased the number of M2 macrophages in the wound area after spinal surgery and alleviated epidural fibrosis, as evidenced by decreased fibronectin, α-smooth muscle actin, and collagen I in the scar tissue. These results demonstrated that substance P promotes M2 macrophage differentiation in epidural fibrosis via sphingomyelin synthase 2 and neutrophil extracellular traps. These findings provide a novel strategy for the treatment of epidural fibrosis.