Spinal cord injury elicits an inflammatory response that plays an important role during the pathological response process by activating the resident microglia and infiltrating macrophages.Macrophages and microglia can be polarized into either the classically activated proinflammatory M1 phenotype or the alternatively activated anti-inflammatory M2 phenotype.M1 macrophages and microglia secrete proinflammatory cytokines and upregulate inducible nitric oxide synthase and reactive oxygen species that are neurotoxic,thereby preventing axonal regeneration.In contrast,M2 macrophages and microglia release trophic factors (e.g.insulin-like growth factor,brain-derived neurotrophic factor,and nerve growth factor) and anti-inflammatory cytokines (e.g.interleukin-4,interleukin-10,and transforming growth factor-3) that are neuroprotective and promote axonal regeneration.The mechanisms underlying the modulation of macrophages and microglia polarization are not very clear.In this study,we demonstrated that programmed cell death protein 1 (PD-1) was inducibly expressed in macrophages and microglia under polarizing stimulation,and PD-1 deficiency promoted macrophages and microglia susceptibility to M1 polarization with high expression of inducible nitric oxide synthase and proinflammatory cytokines when stimulated with lipopolysaccharide (LPS) + interferon-y (IFN-v) in vitro.In addition,PD-1 deficiency reduced M2 polarization by decreasing arginase 1 (Arg1) and CD206 expression on macrophages and microglia,as well as anti-inflammatory cytokine release after stimulation with interleukin-4 in vitro.PD-1 deficiency induced M1 polarization by increasing signal transducers and activators of transcription 1 (STAT1) phosphorylation or suppressed M2 polarization by reducing STAT6 phosphorylation in vitro.In in vivo experiments,we also showed that PD-1 deficiency delayed switching of macrophages and microglia from M1 to M2 phenotype at the injured site and reduced locomotor recovery after spinal cord injury.PD-1 deficient mice showed enhanced M1 response through the activation of several transcription factors,including activators of transcription 1 and nuclear factor-kappa B.We also found remarkable differences in the polarized macrophages and microglial phagocytosis during in vitro studies.M2 macrophages derived from wild type mice displayed higher phagocyte ability than M1 macrophages.However,in the microglial cells isolated from wild type mice,the M2 phenotype displayed a lower phagocytic ability than did the M1 cells.PD-1 deficiency caused divergence in phagocytosis ability of macrophages and microglia.In general,PD-1 deficiency decreased the phagocytic ability of macrophages,while maintaining the characteristic disparities between M1 and M2 phenotypes similar to those observed in wild type mice.Interestingly,PD-1 deficiency increased the phagocytic ability of microglia in general without altering the characteristic disparities between M1 and M2 phenotypes in wild type mice.These results indicate that the PD-1 signaling pathway plays an important role in the regulation of macrophages and microglia polarization,and when compromised,these cells polarize towards the M1 phenotype.Our results provide new insights into the modulation mechanisms of macrophage and microglial polarization and could aid in developing new therapies for spinal cord injury via the regulation of macrophage and microglial polarization through PD-1 signaling.