Fig 1: HES-1 was indispensable in naïve CD4+ T cell differentiation into Th22 cells. Naïve CD4+ T cells were transfected with HES-1-siRNA, HES-1 plasmid, or their controls. Subsequently, the transfection cells were cultured in conditions designed to induce Th22 differentiation. a The intracellular IL-17A and IL-22 in transfection CD4+ T cells were assayed by flow cytometry 6 days after the combination of factors induced Th22 cells. b Quantitative analysis of the percentage of IL-22+ IL-17A- cells was performed. c RT-PCR was performed to evaluate the expression of Nicd, Hes-1, Ahr, and Il-22. d Western blot for p-STAT3, STAT3, NICD, Hes-1, AHR, and IL-22 was performed to assess the alteration. e-i The relatively quantified protein bands from western blot were executed with the Quantity One software and data were represented as the means ± S.E.M. of three different experiments. *p < 0.05, ***p < 0.001, compared with the control group; #p < 0.5, ##p < 0.01, for HES-1 siRNA versus M-HES-1siRNA groups; ?p < 0.05, ??p < 0.01, ???p < 0.001, for pCMV6-AC-HES-1 versus vector control groups
Fig 2: NOTCH signaling pathway was involved in differentiation of CD4+ T cells towards Th22 cells. Naïve CD4+ T cells were cultured in conditions designed to induce Th22 differentiation, either 1 µg/mL jagged-1 treatment alone or jagged-1 combined with DAPT (10 µM). a The percentage of Th22 (CD4+IL-22+IFN?-IL-17¯) cells was analyzed by flow cytometry in naïve CD4+ T cells treated with PBS, jagged-1 or jagged-1 plus DAPT. b Percentage quantitation of Th22 cells. c Gene expression of Nicd, Hes-1, Ahr, and Ii-22 was analyzed by RT-PCR. d Representative western blot showing protein levels of p-STAT3, STAT3, NICD, HES-1, AHR, and IL-22 extracted from different groups. e-i Densitometric analysis of p-STAT3, STAT3 NICD, HES-1, AHR, and IL-22 levels was performed with Quantity One software and data were represented as the means ± S.E.M. of three different experiments. **p < 0.01, ***p < 0.001, compared with control group; #p < 0.5, ##p < 0.01, ###p < 0.001, for jagged-1 versus Th22 groups; ??p < 0.01, ???p < 0.001, for DAPT versus jagged-1 groups
Fig 3: IL17A and IL22 production are upregulated in the serum of patients with vitiligo. Serum (A) IL17A and (B) IL22 measured by ELISA in peripheral venous blood samples collected from patients with vitiligo (n=20) and healthy controls (n=20). (C) Representative immunohistochemical and (D) quantitative analyses of skin samples from patients with vitiligo (n=20) and healthy controls (n=10). Scale bar = 100 µm. Correlation between serum (E) IL17A levels and AhR mRNA levels in CD4+ T cells and (F) serum IL22 levels and AhR mRNA levels in CD4+ T cells of patients with vitiligo (n=20). *P 0.05, **P<0.01 and ****P<0.0001. IL, interleukin.
Fig 4: Effect of different concentrations of jagged-1 and DAPT on mediated expression of IL-22. After the naïve CD4+ T cells were pretreated with a combination of factors, different concentrations of jagged-1 (0, 0.1, 0.5, 1, 5 µg/mL) were added to culture medium. a Flow cytometric analysis of the positive IL-22 cell proportion in different doses of jagged-1 treatment. b, c IL-22 proteins were determined by Western blotting. d Naïve CD4+ T cells were treated with the combination of factors, and exposure with or without jagged-1 (1 µg/mL), and different doses of DAPT added (0, 1, 5, 10, 20 µM). Then, the percentages of positive IL-22 cells were assayed by flow cytometry. e, f Following the treatment, expression of IL-22 protein was determined by Western blotting. Data are presented as the mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001 vs. untreated control, ##p < 0.01 vs. jagged-1 (1 µg/mL) without DAPT group
Fig 5: Correlation between serum IL17A and IL22 levels and clinical manifestations of patients with vitiligo. Correlation analyses between (A) serum IL17A levels and disease duration and (B) serum IL17A levels and body surface area in patients with vitiligo (n=20). Correlation analyses between (C) serum IL22 levels and disease duration and (D) serum IL22 levels and body surface area in patients with vitiligo (n=20). IL, interleukin.
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