The Role Of Tang Cells In The Pathogenesis Of Systemic Sclerosis

ObjectiveSystemic sclerosis(SSc)is an autoimmune disease characterized by fibrogenesis of multiple organs and systems,with an unknown etiology and pathogenesis.Early diagnosis is difficult,disease-related damage is often irreversible,and lacking specific treatment.SSc is a rare clinical orphan disease,but its mortality rate is higher than that of any other rheumatic disease,and the diagnosis and treatment are challenging.Pulmonary arterial hypertension(PAH)is defined as an organ-based complication,with poor prognosis and high mortality rate.To seek biomarkers predicting SSc associated PAH is crucial to improving prognosis.The study found that T cells and their subgroup cells,in the early stages of the SSc,were localized before the formation of vascular disease and endothelial cell damage,and may participate in triggering and promoting pathological processes,resulting in vascular disease or fibrosis.The imbalance and function of the T cell subgroups are also closely related to the development of SSc.The angiogenic T cells as a new type of the T cell subgroup which is involved in the repair of angiogenesis and endothelial cells is becoming more and more concerned,but the level and function of Tang cells in SSc are unclear.In addition,multiple cytokines participate in the pathogenesis of SSc.Interleukin-6(IL-6)as a preinflammatory cytokines takes an important role on SSc fibrosis and collagen deposition.Some clinical studies with small sample have shown that the treatment of the SSc for the IL-6 receptor is good for the treatment of SSc,however,it has not yet been reported in the related to Tang cells in SSc.In this study,we have explored the changes of quantity,percentage and function of Tang cells in SSc patients,the effects of the IL-6R antibody on the Tang cells and mouse model,which lay a foundation to further exploring the role of Tang subsets in SSc pathogenesis and targeting specific Tang cell in SSc treatment.MethodsAccording to the 2010 ACR/EULAR SSc Criteria,30 SSc patients who were not treated or had stopped glucocorticoids and immunosuppressive agents for at least 3months were enrolled from department of clinical immunology,First Affiliated Hospital of Air Force Medical University.And 15 gender and age matched healthy controls(HC)were enrolled at the same time.The first part detected the proportion and absolute number of Tang cells,the proportion of CD4~+Tang and CD8~+Tang cells in PB of SSc patients by flow cytometry(FCM)as well as these in SSc subgroups of different Clinical characteristics.Correlation analysis was performed between the percentage of Tang cells or their subsets and course of disease,CRP and MRSS scores.In the second part,FCM was used to analyze these following indicators:(1)the percentages of Treg,Th1,Th17 and CD4~+CD161~+T cells in PB of SSc patients;expression of Granzyme B,perforin,IFN-γand TNF-αon CD8~+Tang cells;Expression of CD39,CD73,TGF-β,IL-10 and CTLA-4 on Tang cells;the number of Tang cells of PBMC in SSc with IL-6 or IL-6R antibodies.(2)Luminex technology was used to detect the levels of cytokines,IL-6,IL-9,TGF-β,IFN-γ,IL-17,VEGF,VCAM,TNF-α.(3)Immunomagnetic beads were used to isolate and purify Naive T cells(Tn)and Treg cells from PB,The inhibitory function of Treg cells were detected by in vitro co-culture suppression assay.(4)Effector T(Teff)cells,Treg cells,Tang and CD3~+CD31~+CXCR4~-cells were separated from the PB of HC and SSc patients.The inhibitory function of Tang cells was detected by in vitro co-culture suppression assay.In the third part,BALB/c mice were used to construct a bleomycin-induced scleroderma(BIS)mouse model.After treatment with anti-IL-6 receptor antibodies,The contents of Hydroxyproline and collagen were measured,and pathological sections were stained with CD31,CXCR4 andα-SMA.At the same time,Tang and Treg cells were detected by FCM in PB and spleen of BIS.The data was statistically described and analyzed using SPSS17.0 and Graph Pad Prism 6.0 software,p<0.05 was considered statistically significant.Results1.The number of Tang cells in PB of SSc patients was significantly higher than that of HC group(p=0.0211),and the proportion of Tang cells was higher than that of HC group(p=0.028).The proportion of CD8~+Tang cells in SSc group was significantly higher than that in the HC group(p=0.0029).The proportion of CD8~+Tang cells in SSc-PAH was significantly higher than that of HC and SSC-non-PAH patients(p<0.001,p=0.0114).The number of Tang cells and the proportion of CD8~+Tang cells were significantly higher in SSc patients with nucleolus type than in HC(p=0.002,p=0.0221)2.The proportion of Treg cells in SSc patients was lower than that of HC(p=0.0256),and the proportion of CD4~+CD161~+cells in SSc patients was higher than that of HC (p=0.0289).The proportion of Th17 cells in lc SSc is higher than that of HC(p=0.0138).IL-6 levels in Plasma of SSc patients were higher than those in HC(p=0.0157),while IL-9,IFN-γ,IL-18,IL-10,TNF-αand IL-17A levels were not significantly different from those of HC.The number of Tang cells were increased or decreased stimulating with IL-6 or IL-6R antibody in PBMC of SSc patients,respectively.3.In SSc patients,the levels of vascular endothelial growth factor(VEGF)and vascular cell adhesion molecule(VCAM)were higher than those of HC(p=0.0068,p=0.0180).Correlation analysis showed that the proportions of Tang cells and CD8~+Tang cells in VEGF level in SSc patients were positively correlated(r=0.4037,p=0.0269).r=0.3924,p=0.0320).VCAM level was positively correlated with the percentage of CD8~+Tang cells(r=0.4501,p=0.0126).4.The proliferation of Tn cells in SSc patients is not significantly changed compared with HC.Treg cells in SSc patients could not effectively inhibit the proliferation of Tn cells(p=0.082),while HC could(p=0.0058),In vitro co-culture inhibition experiment,Tang cells sorted from PB of SSc patients inhibited Teff cells proliferation,Tang combined with Treg cells co-culture group inhibited Teff cells proliferation more strongly,while CD3~+CD31~+CXCR4~-cells inhibition not.Further percentages of CD73 and CD39 on Tang cell in the PB of SSc patients were higher than in HC.5.The percentages of Granzyme B,Perforin,IFN-γ,and TNF-αin the PB of SSc patients were not significantly different from those in the HC group(all p>0.05).6.The average thickness of BIS skin was significantly thicker than of control groups,and the difference was statistically significant(p=0.0061).The hydroxyproline level and collagen content of BIS skin are significantly higher than that of the PBS control groups(p=0.0028,p=0.0005),HE stain showed that the thickness of the dermal layer increased and the expression of CD31 andα-SMA increased,the percentages of Treg cells in the spleen and PB of BIS mice were lower than the control groups,the percentages of Treg cells in the spleen decreased significantly(p=0.027,p=0.002).After treatment with anti-IL-6 receptor antibodies,the skin thickness was thinner,hydroxyproline levels decreased,and the expression of CD31 andα-SMA increased.Treg cell percentages were increased.Conclusion1.The percentage and absolute number of Tang cells in PB of SSc patients and difference with different clinical subtypes.The increased proportion of CD8~+Tang cells in PB may be an important characteristic of SSc associated PAHs and may serve as a potential biomarker for SSc-PAH.2.The level of VEGF,IL-6 in serum,and the level of molecules expressed on Tang were all abnormal in PB of SSc patients and HC.Tang cells inhibited the proliferation of Teff cells and had enhanced effects with Treg cells.IL-6 and IL-6R antibodies can affect the percentage and number of Tang cells in SSc PB,suggesting that IL-6 may participate in the angiogenesis of SSc by affecting Tang cells.3.Anti-IL-6 receptor antibodies may affect BIS by down-regulating Tang cells and up-regulating Treg in vivo.