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

作　　者：Yong-Wei Xu Rui-Xin Xing Wen-Hui Zhang Lu Li Yi Wu Jing Hu Cong Wang Qing-Li Luo Ji-Long Shen Xi Chen 

机构地区：[1]Department of Gastroenterology,the First Affiliated Hospital of Anhui Medical University,Hefei 230022,Anhui Province,China [2]Department of Pathogen Biology,Provincial Laboratory of Pathogen Biology and Zoonoses Anhui,Anhui Medical University,Hefei 230032,Anhui Province,China

出　　处：《World Journal of Gastroenterology》2019年第45期6634-6652,共19页世界胃肠病学杂志（英文版）

基　　金：Supported by the National Natural Science Foundation of China,No.81471983;the Key Research and Development Plan Project of Anhui Province,Department of Science and Technology 2019,No.201904a07020043;the Key Project of Natural Science Research in the Universities of Anhui Provence,No.KJ2017A202;the Research Fund Project of Anhui Institute of Transforming Medicine,No.2017zhyx04

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 Toxo ROP16Ⅰ/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 Toxo ROP16Ⅰ/Ⅲ was observed in RAW264.7 macrophages that were transfected with p EGFP-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（i NOS）,and arginase-1（Arg-1） was detected.The expression of i NOS,Arg-1,signal transducer and activator of transcription 3（Stat3）,p-Stat3,Stat6,pStat6,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 determined by flow cytometry after mixing M1 cells with M2 cells in a Caco-2 cell co-culture system.RESULTS M1 cells exhibited significantly increased production of i NOS,NO,TNF-α,IL-1β,and IL-6,while Toxo ROP16Ⅰ/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 Toxo ROP16 Ⅰ/Ⅲ exhibited decreased production of NO and i NOS 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-cuBACKGROUND 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

关 键 词：Toxoplasma ROP16Ⅰ/Ⅲ CACO-2 Inflammatory bowel disease IMMUNITY Classically activated macrophages Alternatively activated macrophages 

分 类 号：R73[医药卫生—肿瘤]