Fig 2: IGFBP5 maintained the balance of mucosal Th17/Treg in the inflamed colon. The expression of (A) RORγt and (C) FOXP3 in colon tissue were detected by immunohistochemistry (4× and 20×), and the quantitative analysis of RORγt and FOXP3 positive cells were shown respectively, in (B) and (D). (E) The concentrations of TNF-α、; IL-β and IFN-γ in the serum of mice in each group were determined by ELISA (n=5). The experiment was repeated three times independently. One-way ANOVA was used to determine statistical significance. The data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: IGFBP5 ameliorated DSS-induced colitis in mice. (A) Daily body weight during the experiment. (B) Disease activity index (DAI) every day during the experiment. (C) Representative photo of the colon in each group and (D) Quantitative analysis of colon length in each group (n=5). (E) Hematoxylin and eosin (H&E) staining of colon sections were examined after the mice were sacrificed (4× and 20×). (F) Histological damage index (HAI) was determined. One-way ANOVA was used to determine statistical significance. The data are presented as the mean ± SEM (n=5). The experiment was repeated three times independently. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: IGFBP5 increased the percentage of Treg cells and decreased the percentage of Th17 cells. Flow cytometry and RT-qPCR were used to detect the effect of IGFBP5 on T cell subsets in vitro. (A, C) Flow cytometry showed that the percentage of CD4+ IL-17A+ Th17 cells in the CD3/CD28 activated T cells decreased after the treatment of recombinant IGFBP5. (B, D) Flow cytometry showed that the percentage of CD4+ CD25+ Treg cells in the CD3/CD28 activated T cells increased after IGFBP5 treatment. (E, G) Flow cytometry showed that the percentage of CD4+ IFN-γ+ Th1 cells in the CD3/CD28 activated T cells decreased slightly after the treatment of recombinant IGFBP5. (F, H) Flow cytometry showed that the percentage of CD4+ IL-4+ Th2 cells in the CD3/CD28 activated T cells increased slightly after IGFBP5 treatment. (I, J) RT-qPCR showed that the expression of RORγt was significantly reduced and the expression of Foxp3 was markedly increased in IGFBP5 group. (K, L) RT-qPCR showed that the expression of T-bet was slightly reduced and there was little effect on the expression of Gata3 after the treatment of IGFBP5. (M-Q) The relative mRNA levels of Ki-67 and activation-related CD25, CD44, CD69 and IL-2. Student’s t-test was used to determine statistical significance. The data are presented as the mean ± SEM (n=3). The experiment was repeated three times independently. *p < 0.05, **p < 0.01; ns, not significant.

Fig 5: IGFBP5 maintained the balance of Th17/Treg cells in the mesenteric lymph nodes of mice. Cells were isolated from the mesenteric lymph nodes of mice with DSS-induced colitis and stained with fluorochrome-conjugated antibodies against CD4, CD25 and IL-17A. (A) The number of Th17 were expressed as percentages of the CD4+ IL-17A+ cell population. (B) Statistical analysis of Th17. (C) The number of Tregs were expressed as percentages of the CD4+ CD25+ cell population. (D) Statistical analysis of Tregs. The experiment was repeated three times independently. One-way ANOVA was used to determine statistical significance. The data are presented as the mean ± SEM (n=5). *p < 0.05, **p < 0.01, ***p < 0.001.