Effect Of Jagged-1Sognasling On The Differentiation Of Th17Cells And Its Mechanism

Background: Th17cells play an essential role in protection against certainextracellular pathogens, chronic inflammation and autoimmunity diseases. However, themechanism of their differentiation remains unclear. Notch signaling not only regulates T/B celllinesge commitment, but also the differentiation and function of peripheral T cells. Little isknown as to action of it in differentiation of Th17cell subset. Our previous studies suggest thatJagged-1-HES-1signaling can inhibit Th17differentiation. But it has not been reported howJagged-1-Notch signaling influences genes associated with Th17cell differentiation. Obviously,it’s of great scientific significances to explore the change of the genes associated with Th17celldifferentiation, which will lay a foundation for further clarifying theory of Th17celldifferentiation and exploring targets of associated disease therapy.Objective: A soluble Jagged-1/Fc chimera protein (Jagged-1) was directly used to activateJagged-1-Notch signaling to investigate effect of Jagged-1on the differentiation CD4~+T cellsinto Th17cells. Genes most differently expressed in the differentiation of Th17cells werescreened out. Then, a relationship between function of the genes and Th17cell differentiationwas confirmed using different approaches from viouse levels to explore the mechanism ofJagged-1suppressed the skewing of CD4~+T cells toward Th17cells.Methods: CD4~+T cells were isolated from mouse lymph node cells using immuno-magneticbeads-activated cell sorting and treated with Jagged-1. Flow cytometry was performed to analyzeexpressions of IL-17A and IFN-γ in CD4~+T cells. qPCR Array was used to screen differentexpressions of84genes associated with Th17cell differentiation. RT-PCR and qPCR wereemployed to determine alterations of Il17a, Il17f, Il12rb1, Il23a and Rorγt mRNA levels.Western blot was used to detect RORγt, IL-17A and IL-17F protein levels in CD4~+T cells.Immune fluorescence confocal microscopy was adopted to detect changes of IL-17A, IL-17F andRORγt. ELISA was used to determine the level of IL17A and IL-17F from the culturesupernatant of the differently treated CD4~+T cells.Results: Jagged-1suppressed CD4~+T cells to differentiation into CD4~+IL-17+cells. It down-regulated the expressions of19genes and up-regulated the expressions of3genes. Theexpressions of Rorγt, Il17a, Il17f, Il12rb1and Il23a mRNAs were also significantly inhibited by Jagged-1. The expressions of IL-17A, IL-17F and RORγt proteins in CD4~+T cells weredecreased by Jagged-1. Jagged-1further suppressed the secretion of IL-17A and IL-17F in thetreated cells. The changes of IL-17A and IL-17F level were consistent with the RORγtexpression.Conclusions: Jagged-1can inhibit the skewing of CD4~+T cells toward Th17cells, which isassociated with down-regulation of IL-17A, IL-17F and IL-23a expressions via RORγt.