The Role Of Regulatory T Cells In The Treg/Th17Imbalance On The Patients With Systemic Sclerosis

Background:Immune imbalance between Th17and regulatory T cells (Treg) is a characteristic of systemic sclerosis (SSc). The functional heterogeneity and differentiation dynamics can be clearly shown by separating Treg cells into three subsets based on the expression of FoxP3and CD45RA.Objective:To investigate subsets levels of Treg from the patients with naive SSc and assess their roles in the immune balance between Treg and Thl7.Methods:Peripheral blood from31patients with SSc and33health controls were collected. Peripheral blood mononuclear cells (PBMCs) were prepared and analyzed for the expression of CD4, CD25, CD45RA, CTLA-4, CD69, CD279, GITR, FoxP3and IL-17with flow cytometry. Measuring Treg suppressive capacity against proliferation of CD4+CD25-T cells in coculture experiments by a CFSE based. The expression of FoxP3, CTLA-4, IL-17A and RORC mRNA were studied with real-time PCR.Results:(1) The frequency of CD4+CD25+FoxP3+Treg cells were significantly elevated in patients with SSc (3.62+1.14) compared with controls (1.97+0.75, P<0.001); the expression of CTLA-4were lower in Treg cells of SSc patients compared with control(P=0.034), but no differenece of CD69, PD-land GITR between two groups; the expression of CTLA-4and FoxP3mRNA were lower in the patients with SSc compared with control(P<0.05, respectively), the immunosuppressive capacity of Treg cells were diminished in the patients with SSc (P=0.034).(2) In the patients with SSc, the frequency of CD45RA-FoxP3high activated Treg cells (aTreg, FrⅡ) were lower than control (0.25+0.16vs0.66+0.41among CD4+P<0.001;12.42±5.23vs30.01±1.74among Treg, P<0.001), CD45RA-FoxP3lo non-inhibitory T cell (FrⅢ) were higher than control(6.23±2.29vs2.90±0.91among CD4+, P<0.001;73.71±9.62vs57.96±9.90among Treg, P<0.001), There were no significant difference in two groups with CD45RA+FoxP3lo resting Treg cells (rTreg, FrⅠ),which were relatively lower in Treg cells (1.87±0.94vs1.63±0.97among CD4+, P=0.320;23.19±10.60vs29.63+11.77among Treg, P=0.025); in health control, the immunosuppressive capacity of FrⅡ cells were greater than FrI cells (P=0.025); in the patients with SSc, there were no difference of the immunosuppressive capacity between FrII cells and FrI cells (P=0.974), but their immunosuppressive capacity were both lower than health control(P=0.011,P<0.001); FrⅢ cells had no immunosuppressive capacity neither in the patients with SSc nor with control.(3) In the patients with SSc, the expression of CTLA-4mRNA in FrⅡ and FrⅢ cells were decreased significantly (P<0.001, respectively), but not in FrI cells; in control, the expression of CTLA-4mRNA in FrII cells were higher than FrI, FrⅢ and CD4+CD25-T cells (P<0.001, respectively), but in the patients with SSc, there were no difference of CTLA-4mRNA expression between FrII and FrI cells.(4) The expression of IL-17were higher in FrⅢ cells than FrI and FrⅡ cells significantly in both groups (P<0.001, respectively), but there were no difference of FrⅢ between these two groups; Th17cells were increased in the patients with SSc (P<0.001); in the patients with SSc, CD25+FoxP3+IL17+cells among CD4+cells were increased(0.075±0.032vs0.049+0.027, P=0.029), but no difference of CD25+FoxP3+IL17-with control; the expression of IL-17A and RORC mRNA were increased in the FrⅢ cells of SSc patients than control (P<0.001, respectively).Conclusion:Decreased aTreg with functional deficiency and increased CD45RA-FoxP3lo T cells could explain increased Treg with dysfunction in the patients with SSc. Increased Treg cells were not the real regulatory T cells, but non-suppressive CD45RA-oxP3lo cells, which is the main provider for FOXP3+IL17+cells without immunosuppressive capacity. Background:non-suppressive CD45RA-FoxP3lo cells increasing and immune imbalance between Thl7and regulatory T cells (Treg) are the major characteristic of systemic sclerosis (SSc). Tacrolimus (FK506) is a widely used T cell targeted immunosuppressive drug, known as a calcineurin inhibitor. The immunosuppressive function of FK506depends on the inhibition of T cell activation and proliferation.Objective:To estimate the effects of FK506on the proliferation of Treg and its subsets in the patients with naive SSc. To assess the modificated capacity of FK506in the abnormal Treg and immune imbalance between Treg and Th17.Methods:PBMC of5patients with SSc and6health control were cultured with Ong/ml,0.1ng/ml,1ng/ml and10ng/ml FK506for72hours, analyzed for the expression of CD4, CD25, CD45RA, FoxP3and IL-17using flow cytometry. CFSE labeled PBMC and CD4+cells were cultured with Ong/ml,0.1ng/ml,1ng/ml and10ng/ml FK506for96hours, stained the CD25,FoxP3and CD45RA antibodies, analyzed the proliferation by flow cytometry. Measure the expression of IL-17A, IL-22, IL-6and IL-1β by FlowCytoMix.Results:(1) CD4+CD25+FoxP3+Treg cells were decreased in the patients with SSc by the effect of FK506, but there was no obvious effect of FK506on CD4+CD25+FoxP3+Treg cells in the control.(2) FrI cells were decreased both in the patients with SSc and control by the effect of FK506, but there were on effect on the frequency of FrI in Treg; there were no obvious variation of FrII cells by FK506, which increased the frequency of FrII in Treg relatively; there were significantly decreased of FrⅡ both in the patients with SSc and control induced by FK506.(3) The levels of IL-17A, IL-22, IL-6and IL-1β in the serum of SSc patients were higher than control(P<0.05, respectively), there were obvious decreased of Th17cells and FoxP3+IL-17+cells by presenting FK506with decreased IL-17A and IL-1β in culture supernatant.(4) The proliferation capacity of CD4+cells were inhibited by FK506, with the higher effect on the FoxP3-cells than FoxP3+cells; there were no impact on the proliferation of FrI and FrII cells by FK506in the patients with SSc, but the proliferation of FrⅢ cells were significantly decreased by presenting FK506.Conclusion:Decreased aTreg with functional deficiency and increased CD45RA-FoxP3loT cells could explain increased Treg with dysfunction and imbalance between Treg and Thl7cells in the patients with SSc. FK506could reduced the level of CD45RA-FoxP3lo T cell and augment aTreg relatively through inhibiting the proliferation of CD45RA-FoxP3lo T cell, which could modify the abnormal Treg and immune imbalance between Treg and Th17cells in the patients with SSc.