Th17/Treg balance is regulated by myeloid‐derived suppressor cells in experimental autoimmune myocarditis

Figures

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  Figure 1. The proportion of MDSCs in Control and EAM mice. (A) On Day 21, H&E‐stained heart sections from Control and EAM mice. The EAM clinical scores were measured at each time point (Days 0, 10, 14, 21, and 28) following immunization of the EAM mice with MyHC‐α (n = 5 mice/group). (B) Representative flow cytometric images of CD11b+Gr‐1+ MDSCs in the spleens or blood of Control and EAM mice at different stages of EAM development (d10, d14, d21, and d28). (C) The dynamic percentages of CD11b+Gr‐1+ MDSCs from Control and EAM mice tissues (n = 4–11 mice/group). The percentages of CD11b+Gr‐1+ MDSCs in immunocytes from the heart of Control and EAM mice on Day 21 (n = 4–6 mice/group). (D) Spearman correlation analysis of MDSC percentages and EAM clinical scores at different stages of EAM (n = 25 mice). Values are means ± SEM. Three or more independent experiments have been conducted. ns p > .05, *p < .05 and **p < .01 versus Control group. EAM, experimental autoimmune myocarditis; H&E, hematoxylin and eosin; MDSC, myeloid‐derived suppressor cell.

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  Figure 2. MDSC characterization in EAM mice. (A) MDSC purity as determined by Ly6G+Gr‐1+ magnetic beads. (B) Wright–Giemsa staining of PMN‐MDSCs (Ly6G+ cells) and M‐MDSCs (Gr‐1+ cells) isolated from Control and EAM mice spleens. (C) Representative flow cytometric images of PMN‐MDSCs (CD11b+Ly6GhighLy6Clow cells) and M‐MDSCs (CD11b+Ly6GlowLy6Chigh cells) in the EAM mouse heart. On Day 21, statistical analysis revealed the percentages of MDSC phenotypes in the blood, spleens, and hearts of Control and EAM mice (n = 4–5 mice/group). (D) The expression of functional and chemotactic markers (IFN‐γR, IL‐4Rα, iNOS2, CCR2, and CCR7) on splenic CD11b+Gr‐1+ MDSCs from Control and EAM mice. At least three independent experiments have been carried out. *p < .05 and **p < .01 versus the Control group. EAM, experimental autoimmune myocarditis; MDSC, myeloid‐derived suppressor cell; PMN, polymorphonuclear.

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  Figure 3. Adoptive transfer (AT) and MDSC depletion improved EAM progression. (A) A flow chart of mice in the AT and Gem groups. On Days 0 and 7, AT was carried out via intravenous injection of sorted MDSCs (5 × 106 cells). We depleted MDSCs by administering gemcitabine intraperitoneally every 3 days beginning on Day 7 after the second immunization. (B) Flow cytometry was used to determine the proportion of CD11b+Gr‐1+MDSC in mouse spleen (n = 5 mice/group). (C) H&E‐stained cardiac tissue damage in mice was assessed, and the EAM clinical score and the HW/BW ratio in mice were statistically analyzed (n = 4–15 mice/group). (D) Flow cytometry was used to analyze the ratio of Th17 cells and Tregs in the mice spleens of each group (n = 4–7 mice/group). Three or more independent experiments have been conducted. *p < .05 and **p < .01 versus Control group, # p < .05 and ## p < .01 versus EAM group. EAM, experimental autoimmune myocarditis; H&E, hematoxylin and eosin; MDSC, myeloid‐derived suppressor cell.

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  Figure 4. In gemcitabine‐treated EAM mice, MDSC transfer played a minor role in the pathogenesis of the late stage of EAM. (A) The flow chart of mice in the Gem and Gem(p)+adoptive transfer (AT) treated groups. After a week of gemcitabine treatment, adoptive transfer of MDSCs (5 × 106 cells) from EAM mice was performed on Day 14 to observe the accumulation of peripheral MDSCs in later stage. (B) Flow cytometry was used to determine the percentages of CD11b+Gr‐1+ MDSCs mouse spleen (n = 5 mice/group). (C) H&E‐stained cardiac tissue damage in mice was assessed, and the EAM clinical score and the HW/BW ratio in mice were statistically analyzed (n = 6–7 mice/group). (D) The ratio of Th17 cells and Tregs in CD4+ cells in the spleens of each group was analyzed by flow cytometer (n = 4–8 mice/group). Three or more independent experiments have been conducted. *p < .05 and **p < .01 versus Con group, # p < .05 and ## p < .01 versus EAM group. EAM, experimental autoimmune myocarditis; H&E, hematoxylin and eosin; MDSC, myeloid‐derived suppressor cell.

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  Figure 5. MDSCs induce the differentiation of Th17 cells under Th17‐polarizing conditions in vitro. For 96 h, sorted MDSCs were cocultured with CD4+ T cells from mice in the Control group. (A) Flow cytometry was used to determine the percentages of CD4+ cells. (B, C) Co‐cultures with MDSCs increased the percentages of IL‐17+ cells in CD4+ cells (B) and CD4+IL‐17+ cells (C). * p < .05, ** p < .01. MDSC, myeloid‐derived suppressor cell.

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  Figure 6. MDSCs inhibit Treg differentiation in vitro under Treg‐polarizing conditions. Sorted MDSCs were cocultured for 96 h with CD4+ T cells from Control mice inTreg‐polarizing conditions. (A–C) Co‐cultures with MDSCs, the percentages of CD4+ cells (A) were increased, whereas the percentages of Foxp3+ cells in CD4+ cells (B) and CD4+Foxp3+ cells (C) were both decreased. At less three or more independent experiments have been carried out. *p < .05, **p < .01 and ns denotes nonsignificant. MDSC, myeloid‐derived suppressor cell.