Treg Cells In The Pathogenesis Of Ankylosing Spondylitis

ObjectiveAnkylosing spondylitis(AS)is a kind of chronic immune inflammatory disease that mainly involves the spine,sacroiliac joint and peripheral joints,and the underlying pathogenesis of AS is not clear now.In recent years,studies have shown that CD4+ T lymphocyte subsets,such as regulatory T cells(Treg cells),T helper cells 17(Th17 cells)and T-helper cells 1(Th1 cells)may be involved in the pathogenesis of AS.CD4+CD25+FoxP3+ Treg cells are important inhibitory T cell and play a central role in maintaining immune tolerance and immune homeostasis,which can inhibit other immune cells in vivo and in vitro.The animals will develop severe autoimmune diseases if their Treg cells develop abnormally.Previous literatures show that Treg cells are involved in a variety of autoimmune diseases,such as multiple sclerosis(MS)and type 1 diabetes(T1D).However,the relationships between the CD4+ T cell subsets,especially Treg cells and the pathogenesis of AS is not clear.In order to investigate whether and how CD4+ T cell subsets,especially Treg cells participate in the pathogenesis of AS,we conducted the study.MethodsThe peripheral blood mononuclear cells(PBMCs)were separated from the peripheral blood of active AS patients and stable AS patients.The SYTOX Green-CD4+CD25-CD45RA+ na?ve T cells(Tn cells)and SYTOX Green-CD4+CD25high Treg cells were sorted from PBMCs.The first part detected the proliferation and apoptosis of Tn cells,and the ability to differentiate into CD4+ T cell subsets Th17 cells.For the detection of proliferation,freshly sorted Tn cells were labelled with 5,6-carboxyfluorescein succinimidyl ester(CFSE)and cultured in the presence of anti-CD3/CD28 beads.On day 5,Tn cell proliferation was determined based on CFSE fluorescence measurements by flow cytometry.For the detection of apoptosis,the sorted Tn cells were cultured in the presence of anti-CD3/CD28 beads for 24 or 72 hours.Then,the cells were collected and stained with Annexin Ⅴ and propidium iodide(PI).After being washed the samples were processed with a BD FACS Calibur for further analyses.For the detection of the ability to differentiate into Th17 cells,the IL-23 R expression of the PBMCs was detected.At the same time,the sorted Tn cells were activated with anti-CD3/CD28 beads and cultured under the following Th17 skewing conditions: recombinant human IL-6(rhIL-6),recombinant human TGF-β1(rhTGF-β1),recombinant human IL-1β(rhIL-1β)and recombinant human IL-23(rhIL-23).The percentages of CD4+ IL-17a+ Th17 cells and CD4+ IFN-γ+ Th1 cells were assessed on day 3 and day 7.The second part detected the proportions of CD4+ T cell subsets Treg cells,Th17/Th1 cells in PBMCs and the corresponding mean fluorescence intensity(MFI).After the PBMCs were stained with appropriate Flow antibody,the samples were processed by flow cytometry.The third part detected the suppression of Tn cells proliferation by Treg cells.Freshly sorted Tn cells were labelled with CFSE and cultured with or without sorted Treg cells in the presence of anti-CD3/CD28 beads.On day 5,Tn cell proliferation was determined based on CFSE fluorescence measurements by flow cytometry.In addition,on day 5,the supernatant was collected and stored at-80°C until enzyme-linked immunosorbent assay(ELISA)analysis.The concentrations of IL-10,transforming growth factor-β(TGF-β),Granzyme B and IL-2 in the supernatants were assessed.The fourth part detected the IL-2 signal pathway of Treg cells.For the detection of the IL-2 expression in Tn cells,the sorted Tn cells were cultured with or without sorted Treg cells in the presence of anti-CD3/CD28 beads.On day 5,the cells were collected and total RNA was extracted,after which the RNA was reverse transcribed to cDNA and the expression levels of the IL-2 gene in Tn cells and ten genes in Treg cells were detected by real-time quantitative polymerase chain reaction(qPCR).For the phosphorylation of signal transducer and activator of transcription 5(STAT5)in sorted Treg cells,sorted Tregs were cultured in the presence of IL-2 for different time and the phosphorylation of STAT5 were detected.The fifth part detected the methylation status of Cp G islands in the conserved noncoding sequence(CNS)2 region of Fox P3 gene in Treg cells.DNA was extracted from the sorted Treg cells and the CpG islands methylation status of the CNS2 region in FOXP3 gene were checked via bisulfite sequencing.Calculations were performed using GraphPad Prism software version 7.6.1.p values less than 0.05 were considered significant.Results1.No differences in the proliferation,apoptosis and Th17 cell differentiation of the Tn cells between patients with active AS and healthy controls.2.No differences in the proportions of Treg cells and Th17/Th1 cells in PBMCs of patients with stable AS or active AS and healthy controls.However,the FoxP3 MFI of Treg cells were significantly decreased in patients with active AS,which suggested that Treg cells were defective.3.Treg cells from patients with active AS can’t inhibit the proliferation of Tn cells effectively,which indicated that Treg cells in patients with active AS have defects in the suppressive function.4.Patients with active AS had higher IL-2 levels in the supernatant of T cell(Treg and Tn cells)co-cultures.In the same time,the Tn cells from patients with active AS had the same IL-2 mRNA expression levels as those from healthy controls and the Tn cells from patients with active AS had greater IL-2 usage.All the data indicated that patients with active AS harbour Treg cells that are defective in using IL-2,thereby causing them to use less IL-2.Further detection showd that the patients with active AS exhibited higher STAT5 expression in their Treg cells.Although the percentage of Tregs expressing phosphorylated STAT5 did not increase in these patients,the increased STAT5 expression in Tregs seemed to be compensatory.These results indicate that patients with active AS exhibit inadequate or unstable STAT5 phosphorylation in their Treg cells.This finding also confirmed that patients with active AS have abnormal IL-2 signalling in their Treg cells,which can affect Treg cell function.5.The Treg cells from patients with active AS had higher methylated CpG islands in CNS2 range in FOXP3 gene.ConclusionIn this study,we first investigated whether CD4+ T cell subsets,such as Treg cells,are involved in the pathogenesis of AS.Firstly we detected the proliferation,apoptosis,and the ability to differentiate into Th17 cells of Tn cells.But we did not found abnormity.We further assessed the proportions of Treg cells and Th17/Th1 cells in PBMCs and the corresponding MFI in patients with AS,and we firstly found a decrease in FoxP3 MFI in Treg cells of patients with active AS,which suggested the dysfunction of the Treg cells.So we next detected the suppression of Tn cells proliferation by Treg cells and found that the Treg cells from patients with active AS did not inhibit the proliferation of Tn cells effectively,which suggested a defect in the suppressive function of Treg cells from patients with active AS.Because of the results above suggested that Treg cells of patients with active AS exhibit suppressive defects.We then studied the mechanisms underlying the Treg cell dysfunction.On one side,we detected the IL-2 signalling of the Treg cells,which includes IL-2 usage and the downstream IL-2 pathway.We found that Treg cells of patients with active AS had defects in using IL-2 and hence used less IL-2.In the same time,there is inadequate or unstable STAT5 phosphorylation in the Treg cells from patients with active AS,and the STAT5 is the downstream molecule in the IL-2 pathway.On the other side,we detected the methylation status of CpG islands in CNS2 region of FOXP3 gene in Treg cells and found that the Treg cells of patients with active AS have higher levels of CpG methylation in CNS2.The abnormal IL-2 signalling and CNS2 hypermethylation further induces the intrinsic dysfunction of Treg cells in patients with active AS.The new cure for autoimmune diseases and inflammatory diseases through restoring the normal function of Treg cells are developed.Now,it has been made some achievement in many diseases,such as the systemic lupus erythematosus(SLE),MS,psoriasis and ulcerative colitis(UC).Thus,our finding has opened up a new line of investigation into a possible cure for AS through restoring the normal function of Treg cells.