Effect Of IL-35 On Modulating Macrophage Phenotypes

Background: Macrophage plays an important role in all three stages of wound healing by converting from the inflammatory M1 phenotype to anti-inflammatory M2 phenotype in response to microenvironmental changes in the tissue.IL-35 is a novel anti-inflammatory cytokine belonging to the IL-12 family of cytokines which share the IL-12p35 and EBI3 of IL-27 subunits.IL-35 is produced mainly by regulatory T cells and B lymphocytes,which are cells often associated with inflammation and chronic wounds.In this study,we hypothesized that IL-35 produced in wound healing tissue would act on macrophage to switch macrophages from the M1 to M2 phenotype by suppressing pro-inflammatory cytokines production.Materials and Methods: THP-1 cells were cultured with GM-CSF and M-CSF for three days to drive M1 to M2 macrophage differentiation.M1 and M2 phenotypes were examined by stimulating the THP-1 cell with IFNγ and LPS.In some experiments,conditioned media from cultures of IL-35 expressing CHO cells and control CHO cells were added into the cultures.Pro-inflammatory cytokines,IL-23 and TNF-α,as well as anti-inflammatory cytokine IL-27 were detected by ELISA in THP-1 cell culture media with or without stimulation.Cytokines and cell surface receptors including IFNγreceptors and IL-35 receptors(IL-12Rb2 and gp130)gene expression were examined using Real-time PCR.Results:1.THP-1 showed clear M1 and M2 macrophage phenotype in culture with GM-CSF for M1 and M-CSF for M2 macrophages.2.GM-CSF priming of THP-1 is critical for cell responding to IFNγ stimulation for pro-inflammatory cytokine IL-23 and TNF production.3.GM-CSF induced IFNγ receptors to respond IFNγ stimulation.IL-35 suppressed IFNγ stimulated IL-23 and TNF-α production.Conclusions: IL-35 is a potent immune suppressing cytokine which converts M1 inflammatory macrophages to M2 anti-inflammatory macrophages by reducing IFNγinduced IL-23 and TNF-α production in M1 macrophages.