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

Figure 1 Stable transfection of RAW264. 7 cells with LV-rop16_I/III recombinant lentivirus. A: Fluorescence microscopy was used to observe the expression of green fluorescent protein in macrophages, Lv-Mφ and Lv-rop16_I/III-Mφ cells stably transfected with recombinant lentivirus. B: Macrophages, Lv-Mφ, and Lv-rop16_I/III-Mφ stably-transfected cells were analyzed by Western blotting. C: Statistical analysis of protein expression in Lv-rop16_I/III-Mφ cells relative to non-transfected macrophages and mock Lv-Mφ by Western blotting. ^aP < 0.001 vs Mφ. Mφ: Macrophages; LV-Mφ: Lentivirus transfer into macrophages; LV-rop16_I/III-Mφ: Lentivirus-rop16_I/III transfer into macrophages.

Figure 2 Lipopolysaccharide polarized to M1 cells. Protein was extracted from M1 cells at different time intervals over 24 h. iNOS expression was significantly increased at 6 h. iNOS: Inducible nitric oxide synthase.

Figure 3 The proinflammatory cytokines produced by M1, and mixed M1 and M2 cells. A-D: The mRNA expression levels of interleukin (IL)-6, IL-1β, tumour necrosis factor (TNF)-α, and iNOS were significantly increased in M1 cells by quantitative real-time reverse transcriptase polymerase chain reaction. Levels were normalized to those of β-actin. E-G: E-G: Expression of IL-6, IL-1β, and TNF-α was remarkably increased in the M1 cells in comparison with the control when examined by ELISA. D: The relative mRNA expression of iNOS was also increased in the M1 cell group. H: The nitric oxide (NO) was upregulated relative to normal macrophages as measured by the Griess method. The production of proinflammatory cytokines was markedly decreased in mixed M1 and M2 cells compared with M1 cells. The NO was measured by the Griess method. ^aP < 0.001 vs Mφ; ^bP < 0.001 vs lipopolysaccharide + Mφ. iNOS: Inducible nitric oxide synthase; NO: Nitric oxide; IL: Interleukin; LPS: Lipopolysaccharide; LV- Mφ: Lentivirus transfer into macrophages; LV-rop16_I/III- Mφ: Lentivirus-rop16_I/III transfer into macrophages.

Figure 4 Western blotting analysis for the detection of M1 and M2 cell signatures. A-C: LV-rop16_I/III polarized macrophages to M2 cells via the phosphorylation of Stat3 and Stat6. The expression of p-Stat3 and p-Stat6 was significantly elevated in M2 cells relative to normal macrophages. D and E: The expression of PD-L2 (33 kDa) was obviously increased in M2 cells while Lv-Mφ was significantly increased in M1 cells relative to macrophages. F-H: The expression of Arg-1 was obviously increased in M2 cells while iNOS was significantly increased in M1 cells relative to macrophages. ^aP < 0.01 vs Lv-Mφ; ^bP < 0.001 vs Lv-Mφ; ^cP < 0.001 vs Mφ. Stat3: Signal transducer and activator of transcription 3; Stat6: Signal transducer and activator of transcription 6; p-Stat3: Phosphorylate signal transducer and activator of transcription 3; p-Stat6: Phosphorylate signal transducer and activator of transcription 6; iNOS: Inducible nitric oxide synthase; PD-L2: Programmed death ligand-2; Arg-1: Arginase-1; Mφ: Macrophages; LPS: Lipopolysaccharide; LV-Mφ: Lentivirus transfer into macrophages; LV-rop16_I/III-Mφ: Lentivirus-rop16_I/III transfer into macrophages.

Figure 5 Cytokine expression was detected in M2 eclls and mixed M1 and M2 cells. A-C: The relative mRNA expression of interleukin (IL)-10, transforming growth factor (TGF)-β1 and Arg-1 in M2 cells was markedly upregulated in Lv-rop16_I/III-Mφ cells relative to LV-Mφ cells, while the production of cytokines was increased in mixed M1 and M2 cells relative to M1 cells. D and E: The culture supernatants were collected and analyzed by ELISA for IL-10 and TGF-β1, consistent with the result of relative mRNA. ^aP < 0.05 vs lipopolysaccharide (LPS) + Mφ; ^bP < 0.001 vs LPS + Mφ; ^cP < 0.01 vs Lv-Mφ, ^dP < 0.001 vs Lv-Mφ. Mφ: Macrophages; LV-Mφ: Lentivirus transfer into macrophages; LV-rop16_I/III-Mφ: Lentivirus-rop16_I/III transfer into macrophages.

Figure 6 Caco-2 cell apoptosis was restrained by M1 cells mixed with M2 cells. Lipopolysaccharide (LPS)-induced macrophages with the M1-like phenotype were co-cultured with Caco-2 cells. A-D: Western blotting indicated that the expression of caspase-3, caspase-8, and caspase-9 was significantly increased in M1 cells compared to normal macrophages. M1 cells and M2 cells were co-cultured with Caco-2 cells, the expression levels of apoptosis-associated proteins were significantly reduced compared to the M1 cell group. The above proteins were detected by Western blotting, and the data were analyzed by grey values. ^aP < 0.01 vs Mφ; ^bP < 0.01 vs LPS + Mφ; ^cP < 0.001 vs Mφ, ^dP < 0.001 vs LPS + Mφ. Mφ: Macrophages; LV-Mφ: Lentivirus transfer into macrophages; LV-rop16_I/III-Mφ: Lentivirus-rop16_I/III transfer into macrophages; LPS: Lipopolysaccharide.

Figure 7 M1 cells mixed with M2 cells lead to reduction of Caco-2 cell apoptosis in co-culture. A: M1 cells co-cultured with Caco-2 cells for 24 h induced higher Caco-2 cell apoptosis relative to normal macrophages, as detected by FCM. A and B: M1 cells mixed with M2 cells in the co-culture system resulted in a notable decrease in Caco-2 cell apoptosis M1 cells. ^aP < 0.001 vs Mφ; ^bP < 0.001 vs lipopolysaccharide + Mφ. Mφ: Macrophages; LPS: Lipopolysaccharide; LV- Mφ: Lentivirus transfer into macrophages; LV-rop16_I/III - Mφ: Lentivirus-rop16_I/III transfer into macrophages; LPS: Lipopolysaccharide.

Figure 8 M2 cells reduced iNOS expression in M1 inflammatory macrophages. LV-rop16_I/III-induced M2-like macrophages restricted the production of iNOS in M1 cells. A and B: The iNOS level was downregulated in mixed M1 and M2 cells relative to that in M1 cells. A, C-E: Arg-1 and PD-L2 expression was higher in M2 cells than in Lv-Mφ cells. The above proteins were detected by Western blotting, and the data were analyzed by grey values. ^aP < 0.05 vs lipopolysaccharide (LPS) + Mφ; ^bP < 0.01 vs Mφ; ^cP < 0.001 vs Lv-Mφ, ^dP < 0.001 vs LPS + Mφ. iNOS: Inducible nitric oxide synthase; PD-L2: Programmed death ligand-2; Arg-1: Arginase-1; Mφ: Macrophages; LPS: Lipopolysaccharide; LV-Mφ: Lentivirus transfer into macrophages; LV-rop16_I/III-Mφ: Lentivirus-rop16_I/III transfer into macrophages.