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. 2015 Feb 4:5:8234.
doi: 10.1038/srep08234.

Jagged-1 signaling suppresses the IL-6 and TGF-β treatment-induced Th17 cell differentiation via the reduction of RORγt/IL-17A/IL-17F/IL-23a/IL-12rb1

Yuan Wang  1 , Feiyue Xing  1 , Siqi Ye  2 , Jia Xiao  2 , Jingfang Di  2 , Shan Zeng  2 , Jing Liu  3
Affiliations

Jagged-1 signaling suppresses the IL-6 and TGF-β treatment-induced Th17 cell differentiation via the reduction of RORγt/IL-17A/IL-17F/IL-23a/IL-12rb1

Yuan Wang et al. Sci Rep. .

Abstract

Jagged-1 signaling has recently been reported to be involved in the Th17 cell differentiation. However, little is known about its mechanisms. Soluble Jagged-1 was used to activate the Jagged-1-Notch signaling to interfere with the IL-6 and TGF-β-induced Th17 cell skewing. Genes relevant to the autoimmunity or inflammation were screened for the first time in this system by qPCR array for the differential expressions. The 18 genes out of 84, including Clec7a, Il12b, Il12rb1, Il12rb2, Csf3, Il15, Il17a, Il17f, Il17rc, Il17rd, Il17re, Il23a, Myd88, Socs1, Stat4, Stat5a, Sykb and Tbx21, were downregulated, but only Cxcl2, Cxcl12 and Mmp3 were upregulated. The expressions of the genes, Rorγt, Il17a, Il17f, Il12rb1 and Il23a, induced by simultaneous IL-6 and TGF-β treatment were significantly suppressed by Jagged-1, followed by the reduction of RORγt, IL-17A, and IL-17F. Consistent with the attenuation of RORγt, and the reduced production and secretion of IL-17A and IL-17F in the cell supernatant and the in situ stained cells, the number of CD4(+)IL-17(+) cells was also diminished. It is concluded that the Jagged-1-Notch signaling can suppress the IL-6 and TGF-β treatment-induced Th17 cell skewing through the attenuation of RORγt and, hence by, the down-regulation of IL-17A, IL-17F, IL-23a, and IL-12rb1.

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Figures

Figure 1
Figure 1. The inhibition of CD4+ T cells towards Th17 cell differentiation by Jagged-1 signaling.
The isolated CD4+ T cells were treated by Jagged-1 and quantitatively analyzed by the flow cytometry, 72 h after the treatment. (A) The levels of intracellular IL-17 in the treated CD4+ T cells. (B) Intracellular IL-17 and IFN-γ production in the treated CD4+ T cells. (C) Percentage of CD4+IL-17+ T cells. (D) Proportion of IL-17+ cells in the lower right quadrant in the FACS plots. Three independent experiments were repeated. **p < 0.01, vs. the corresponding control.
Figure 2
Figure 2. Differential expressions of the genes relevant to the Th17 cell differentiation by Jagged-1 signaling.
(A) The heat map provides a graphical representation of expression fold between the Jagged-1-treated CD4+ T cells (Jagged-1 group) and the untreated CD4+ T cells (control group). Red indicates the upregulated genes and green, the downregulated genes. (B) The both groups are overlaid onto the qPCR Array plate layout with genes' name and their location. (C) The relative expressional levels for each gene in the Jagged-1 group and the control group are plotted against each other in the scatter plot. Red shows the upregulated genes and green, the downregulated genes. (D) Up/downregulated genes in CD4+ T cells from the Jagged-1 group are compared with the control group. Fold-change values greater than two hint the positive regulation, and fold-change values less than two, the negative regulation. Two independent experiments were repeated.
Figure 3
Figure 3. The decreased expressions of Il17a, Il17f, Il12rb1 and Il23a genes in the Jagged-1-treated CD4+ T cells.
(A–D) The expressions of Il17a, Il17f, Il12rb1 and Il23a mRNA were evaluated by RT-PCR, and the PCR products were separated by electrophoresis on the 1.5% agarose gel. (E–H) the relative quantification of Il17a, Il17f, Il12rb1 and Il23a mRNA expressions was performed using the 2−ΔΔCt method by qPCR. The results are representative of the three independent experiments. *p < 0.05, **p < 0.01 vs. the corresponding control.
Figure 4
Figure 4. The downregulation of IL-17A, IL-17F and the RORγt expressions in the Jagged-1-treated CD4+ T cells.
The isolated CD4+ T cells were treated with Jagged-1 for 72 h. (A–C) The relative quantification of Il-17a, Il-17f and Rorγt mRNA expressions was detected using 2−ΔΔCt method by qPCR. (D–F) The levels of IL-17A, IL-17F and RORγt proteins were determined by Western Blot. (G and H) The levels of IL-17A and IL-17F secreted by the treated CD4+ T cells were measured by ELISA. The results are representative of the three independent experiments. *p < 0.05, **p < 0.01.
Figure 5
Figure 5. The in situ localization and distribution of IL-17A and IL-17F in the CD4+ T cells treated with Jagged-1.
The isolated CD4+ T cells were treated with Jagged-1 for 72 h. The cells were fixed, permeablized and stained with monoclonal antibodies specific for IL-17A (green) and IL-17F (red). The nuclei were stained with DAPI (blue). All the cells were observed at magnification ×200. Three independent experiments were repeated. *p < 0.05, vs. the corresponding control.
Figure 6
Figure 6. The in situ expressional relationship of both IL-17A and IL-17F to RORγt in the CD4+ T cells treated with Jagged-1.
The isolated CD4+ T cells were treated with Jagged-1 for 72 h. The cells were fixed, permeablized and stained with monoclonal antibodies specific for IL-17A (green), IL-17F (red) and RORγt (blue). All the cells were observed at magnification ×200. Three independent experiments were repeated. *p < 0.05, vs. the corresponding control.
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