Introduction: Human neutrophils are a major barrier to graft survival in xenotransplantation, particularly in the early stages of the innate immune response. In our previous studies, we reported that CD47 and CD31 suppress neutrophil activation. On the other hand, recent studies on anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) have shown that Plexin-B2 acts as a ligand for neutrophil semaphorin 4D (SEMA4D) and suppresses neutrophil activation. Semaphorins are a group of approximately 30 molecules with a wide range of functions discovered since 1992, including "immune semaphorins," which regulate the immune system from macrophages to T cells. However, the inhibitory effect of Plexin-B2 in xenotransplantation remains unclear. In this study, we compared the inhibitory effects of Plexin-B2 expression in swine endothelial cells (SECs) on neutrophil-mediated cytotoxicity, reactive oxygen species (ROS) production, and neutrophil extracellular trap (NET) formation with those of CD47 and CD31.
Methods: The cDNAs for Plexin-B2, CD47, and CD31 were cloned into the expression vector pCXN2 (chicken β-actin promoter + CMV enhancer) and transfected into SECs to generate SEC/Plexin-B2, SEC/CD47, and SEC/CD31 cell lines, respectively. Expression was confirmed by flow cytometry. Human neutrophils were isolated from healthy volunteers, and the inhibitory functions of these molecules were assessed by cytotoxicity assays using 10 or 50 nanomolar concentrations of phorbol 12-myristate 13-acetate (PMA). In addition, ROS production and NET formation in neutrophils were evaluated using flow cytometry and SYTOX Green staining, respectively. Statistical significance was determined using Student’s t-test.
Results: First, the expression of each molecule on SECs was confirmed by flow cytometry. The cytotoxicity of SEC/Plexin-B2 was significantly reduced compared to wild-type cells with 10 nM PMA stimulation (P = 0.0145) and 50 nM PMA stimulation (P = 0.00053) for five hours. In contrast, the suppression of cytotoxicity was not clearly observed in SEC/CD47, but it was significantly reduced in SEC/CD31 compared to wild-type cells (P = 0.0213 at 10 nM, P = 0.0056 at 50 nM).
Furthermore, Plexin-B2 expression significantly attenuated ROS production (P = 0.019) and NET formation (P = 0.0063) compared to wild-type cells, confirming its inhibitory effect on neutrophil activation. For SEC/CD47, the ROS assay did not show clear suppression but indicated significant downregulation of NET formation compared to wild-type cells (P = 0.0025).
Conclusion: These findings suggest that endothelial expression of Plexin-B2 may provide a novel strategy to suppress neutrophil-induced xenograft injury and improve graft survival by targeting neutrophil–endothelial cell interactions in xenotransplantation.