ABSTRACT
Objective To evaluate the effect of Notch 1 signaling pathway on the differentiation and function of Th17 cells in peripheral blood of patients with psoriasis.Methods Peripheral blood samples were obtained from 35 patients with psoriasis and 32 healthy controls.Flow cytometry was performed to determine the proportion of Th17 cells in CD4+ T cells in peripheral blood mononuclear cells (PBMCs),real-time RT-PCR to measure the mRNA expression of retinoic acid receptor-related orphan receptor γt (RORγt),interleukin (IL)-17,Notch 1 and hairy-and-enhancer-of-split-1 (Hes-1),and enzymelinked immunosorbent assay (ELISA) to detect levels of IL-17 in the serum and culture supernatant of PBMCs stimulated with phorbol ester,calcium ionophore and brefeldin A.The correlation of Notch 1 mRNA expression with psoriasis area and severity index (PASI) was analyzed,so were its correlation with the proportion of Th17 cells,mRNA expression of RORγt,and mRNA and protein expression of IL-17.PBMCs isolated from the patients with psoriasis were divided into 5 groups to be treated with γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester (DAPT) at different concentrations of 0(control group),2.5,5.0,10.0 and 20.0μmol/L,respectively,so as to evaluate the effects of blocking the Notch1 signaling pathway by DAPT on the proportion of Th17 cells in PBMCs,levels of RORγt and IL-17.Results Compared with the healthy controls,patients with psoriasis showed a significant increase in the proportion of Th17 cells in CD4+ T cells in PBMCs (2.863％ ± 0.969％ vs.0.604％ ± 0.124％,P ＜ 0.01),mRNA expression of RORγt (5.255 ± 0.998 vs.1.530-± 0.485,P ＜ 0.01),Notch1 (6.743 ± 1.756 vs.1.731 ± 0.456,P ＜ 0.01),Hes-1 (6.384 ± 1.665 vs.1.627 ± 0.485,P ＜ 0.01) and IL-17 (6.944 ± 1.626 vs.1.698 ± 0.329,P ＜ 0.01),and serum level of IL-17 ([36.444 ± 5.936] ng/L vs.[11.762 ± 2.260] ng/L,P ＜ 0.01).Among the patients with psoriasis,the mRNA expression of Notch1 was positively correlated with PASI scores,proportion of Th17 cells,mRNA expression of RORγt and IL-17,and serum level of IL-17 (r =0.584,0.544,0.518,0.549 and 0.511,respectively,all P ＜ 0.05).There were significant differences in the proportion of Th17 cells,mRNA expression of RORγt and IL-17,and level of IL-17 in the culture supematant among the control group,2.5-,5.0-,10.0-and 20.0-μmol/L DAPT groups (F =79.527,82.239,78.086 and 80.558,respectively,all P ＜ 0.01).The above indices were significantly lower in the 2.5-,5.0-,10.0-and 20.0-μmol/L DAPT groups than in the control group (all P ＜ 0.05),and decreased along with the increase of DAPT concentrations.Conclusion Notch1 signaling pathway can promote the differentiation of Th17 cells and the expression of RORγt,IL-17 and Hes-1 in the peripheral blood of patients with psoriasis.
ABSTRACT
Objective:To determine the effect of Notch1 signaling pathway on the differentiation and function of Th17 cells in murine psoriasis model.Methods: BALB/c mice were randomly divided into psoriasis model group and control group.Murine psoriasis model was established by topical 5% imiquimod application in combination with intraperitoneal injection of α-2b interferon.The CD4+ T lymphocytes were isolated by magnetic activated cell sorter (MACS).Flow cytometric analysis (FCM) was performed to detect the percentage of Th17 cells.Real-time RT-PCR was employed to measure the mRNA levels of RORγt,IL-17A,Notch1 and Hes-1.The CD4+ T lymphocytes were then divided into γ-secretase inhibitor DAPT groups and control group,and the expression differences of Notch1 signaling molecule and its target gene Hes-1 mRNA levels,Th17 cell percentage,RORγt and IL-17A mRNA levels,and IL-17A concentrations in cell-free supernatant were detected.Results: The expression levels of Th17 cell percentage and RORγt,IL-17A,Notch1 and Hes-1 mRNA in CD4+ T lymphocytes of murine psoriasis model were significantly higher than control mouse[(2.97±0.86)% vs.(0.65±0.11)%,t=15.083;(5.75±0.61) vs.(1.57±0.43),t=21.630;(7.83±0.97) vs.(1.63±0.31),t=25.348;(7.10±1.37) vs.(1.47±0.34),t=17.386;(7.30±1.15) vs.(1.67±0.48),t=18.840,respectively,all P<0.01].Compared with control group,Th17 cell percentage,mRNA expression levels of Notch1,Hes-1,RORγt and IL-17A,and IL-17A concentrations in cell-free supernatant from cultured CD4+ T lymphocytes of murine psoriasis model were dramatically decreased in DAPT treated groups in a dose-dependent way (F=74.368,89.719,126.572,94.558,124.323 and 123.231 respectively,all P<0.01).Conclusion: Notch1 signaling pathway can regulate the differentiation and function of Th17 cells in murine psoriasis model,and may have potential value for the target immunotherapy of psoriasis.