ECDI-fixed donor splenocytes prolong skin allograft survival by promoting M2 macrophage polarization and inducing regulatory T cells

Abstract

Rejection is a common complication of allogeneic tissue transplantation. Fixation of splenocytes (SP) with 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (ECDI) induces immune tolerance in recipients post-transplantation; however, the mechanism underlying this effect remains unclear. Here, we determined the mechanisms of ECDI-fixed donor SP (ECDI-SP) in inducing tolerance in skin allograft transplantation. C57BL/6-recipient mice that received Balb/c full-thickness skin transplants with two infusions of donor-derived ECDI-SP, along with rapamycin showed superior skin allograft survival and lower inflammatory cell infiltration than mice that received rapamycin-only treatment. In ECDI-SP-treated mice, the levels of anti-inflammatory cytokines such as interleukin (IL)-10 in sera were markedly increased, whereas the expression of inflammatory cytokines was significantly suppressed. Splenic macrophages were significantly polarized to the alternative activated macrophage (M2) phenotype, with expansion of CD4+Foxp3+ regulatory T cells (Tregs) in the spleen and draining lymph nodes. Allostimulatory activity of ECDI-SP in vitro and donor-specific ex vivo hyporesponsiveness were observed. C57BL/6 macrophages engulfed allogeneic Balb/c-derived ECDI-SP, polarized to the M2 phenotype, with pronounced cAMP response element-binding (CREB) protein phosphorylation. By facilitating increased IL-10 expression, ECDI-SP induced M2 polarization and Treg production, inhibiting effector T-cell proliferation. Thus, ECDI-SP modulates macrophage M2 polarization by increasing CREB phosphorylation and promoting Treg production to suppress allogeneic skin graft rejection.