Toxoplasma ROP16I/III ameliorated inflammatory bowel diseases via inducing M2 phenotype of macrophages.
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-rop16 I/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 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 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.
['Xu|Yong-Wei|YW|', 'Xing|Rui-Xin|RX|', 'Zhang|Wen-Hui|WH|', 'Li|Lu|L|', 'Wu|Yi|Y|', 'Hu|Jing|J|', 'Wang|Cong|C|', 'Luo|Qing-Li|QL|', 'Shen|Ji-Long|JL|', 'Chen|Xi|X|']
D000818:Animals D017209:Apoptosis D018938:Caco-2 Cells D018920:Coculture Techniques D016207:Cytokines D015536:Down-Regulation D006706:Homeostasis D006801:Humans D007167:Immunotherapy D007249:Inflammation D015212:Inflammatory Bowel Diseases D007413:Intestinal Mucosa D016086:Lentivirus D008070:Lipopolysaccharides D008264:Macrophages D051379:Mice D010641:Phenotype D011505:Protein-Tyrosine Kinases D015800:Protozoan Proteins D000067996:RAW 264.7 Cells D014122:Toxoplasma
Toxoplasma ROP16I/III Inflammatory bowel disease Caco-2 Alternatively activated macrophages Classically activated macrophages Immunity