Toxoplasma ROP16Ⅰ/Ⅲ ameliorated inflammatory bowel diseases via inducing M2 phenotype of macrophages

摘要

BACKGROUND Inflammatory bowel disease(IBD)is characterized by chronic and non-specific inflammation of the intestinal mucosa and mainly includes ulcerative colitis and Crohn's disease.AIM To explore the beneficial effect of ToxoROP16I/III-induced M2 phynotype macrophages in homeostasis of IBDs through downregulation of M1 inflammatory cells.METHODS RAW264.7 macrophages stimulated by lipopolysaccharide(LPS)(M1 cells)were co-cultured with Caco-2 cells as an inflammatory model of IBD in vitro.The expression of ToxoROP16I/III was observed in RAW264.7 macrophages that were transfected with pEGFP-rop16I/III.The phenotypes of M2 and M1 macrophage cells were assessed by quantitative real-time reverse transcriptase polymerase chain reaction and the expression of tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6,transforming growth factor(TGF)-β1,IL-10,inducible nitric oxide synthase(iNOS),and arginase-1(Arg-1)was detected.The expression of iNOS,Arg-1,signal transducer and activator of transcription 3(Stat3),p-Stat3,Stat6,p-Stat6,programmed death ligand-2(PD-L2),caspase-3,-8,and-9 was analyzed by Western blotting,and Griess assays were performed to detect nitric oxide(NO).TNF-α,IL-1β,IL-6,TGF-β1,and IL-10 expression in the supernatants was detected by enzyme-linked immunosorbent assay,and Caco-2 cell apoptosis was co-culture system.RESULTS M1 cells exhibited significantly increased production of iNOS,NO,TNF-α,IL-1β,and IL-6,while ToxoROP16I/III induced macrophage bias to M2 cells in vitro,showing increased expression of Arg-1,IL-10 and TGF-β1 and elevated production of p-Stat3 and p-Stat6.The mixed M1 and M2 cell culture induced by ToxoROP16I/III exhibited decreased production of NO and iNOS and upregulated expression of Arg-1 and PD-L2.Accordingly,Caco-2 cells became apoptotic,and apoptosis-associated proteins such as caspase-3,-8 and-9 were dampened during co-culture of M1 and M2 cells.Flow cytometry analysis showed that co-culture of M1 cells with Caco-2 cells facilitated the apoptosis of Caco-2 cells,but co-culture of M1 and M2 cells alleviated Caco-2 cell apoptosis.CONCLUSION ToxoROP16I/III-induced M2 macrophages inhibited apoptosis of Caco-2 cells caused by M1 macrophages.This finding may help gain a better understanding of the underlying mechanism and represent a promising therapeutic strategy for IBDs.