Inflammatory response following central nervous system (CNS) injury is a significant, multifaceted process that requires further studies. Microglia and macrophages play key roles in CNS inflammation. However, it is still undefined what activity PD-1 plays in M1 polarization and what mechanisms PD-1 regulates the polarization of microglia and macrophages. This study was design to determine whether PD-1 influences M1 polarization of microglia and macrophages and explore PD-1 regulatory mechanisms in M1 polarization using the model mouse and proteomics analysis. It applied six newborn and sixty 6-8 week-old C57BL/6 mice, sixty PD-1 knockout (KO) mice, sixty PD-L1 KO mice, and fifteen PD-1 over-expressed mice. Our studies have shown that programmed death-1 (PD-1) is upregulated in microglia and macrophages in spinal cord injury (SCI), and polarized stimulation induces the expression of PD-1 in cultured microglia and macrophages. We have also observed that following SCI in a PD-1 KO model mouse, there is an increase in M1 polarization of macrophages. PD-1 and PD-L1 were upregulated with M1 polarization in peritoneal macrophages, Raw 264.7 and cerebral cortex-derived microglia, and M1 polarization was inhibited by PD-1 and PD-L1 through STAT1/NF-κB and Akt/mTOR signaling pathways in bone marrow-derived macrophages of PD-1 KO and PD-L1 KO mice. This study supported that PD-1/PD-L1 inhibits M1 polarization of microglia and macrophages through STAT1/NF-κB and Akt/mTOR signaling pathways. PD-1 regulated M1 polarization could be a key clinical target of novel immune-focused therapeutic strategies for CNS injury and inflammatory response.

Actin filament-associated protein-120kD (AFAP-120) is an alternatively spliced isoform of actin filament-associated protein-110kD (AFAP-110) and contains an additional neuronal insert (NINS) fragment in addition to identical domains to the AFAP-110. Unlike AFAP-110 widely expressed in tissues, AFAP-120 is specifically expressed in the nervous system and plays a role in organizing dynamic actin structures during neuronal differentiation. However, anti-AFAP-120 antibody is still commercially unavailable, and this may hinder the function research for AFAP-120. In this study, we simultaneously used the ABCpred online server and the BepiPred 1.0 server to predict B-cell epitopes in the exclusive NINS sequence of human AFAP-120 protein, and found that a 16aa-peptide sequence was the consensus epitope predicted by both tools. This peptide was chemically synthesized and used as an immunogen to develop polyclonal antibody against AFAP-120 (anti-AFAP-120). The sensitivity and specificity of anti-AFAP-120 were analyzed with immunoblotting, immunoprecipitation, and immunofluorescence assays. Our results indicated that anti-AFAP-120 could react with over-expressed and endogenous human AFAP-120 protein under denatured condition, but not with human AFAP-110 protein. Moreover, native human AFAP-120 protein could also be recognized by the anti-AFAP-120 antibody. These results suggested that the prepared anit-AFAP-120 antibody would be a useful tool for studying the biochemical and biological functions of AFAP-120.