Study On Function And Mechanism Of Follicular Helper T 17 Cells In Immunoglobulin A Vasculitis

Objective:Immunoglobulin A vasculitis（IgAV）or Henoch-Sch（?）nlein purpura（HSP）is a systemic inflammatory autoimmune disease characterized by the deposition of immunoglobulin A1（IgA1）-dominant immune complexes in small vessels.IgAV is the most common childhood vasculitis and generally self-limiting,whereas concomitant nephritis is the most serious complication that may developed into chronic kidney disease or end-stage renal failure.However,the treatment approaches for IgAVN including corticosteroids and immunosuppressant therapies are not satisfactory and often accompanied by serious side effects,which have a remarkable influence on the growth and development of children.Therefore,it is urgent to search for more efficient and safe therapeutics for IgAVN patients.The pathogenesis of IgAV remains largely elusive.Most researchers believe that genetic susceptibility,infection and dysregulation of the immune response are closely related to the occurrence and development of this disease.Immune abnormalities in IgAV mainly are resulted from excessive activation of B cells,leading to the production of profound IgA-related autoantibodies.T follicular helper（Tfh）cells,considered to be the most important type of T cells that assist B cells to produce immunoglobulin and promote inflammatory response,interact with B cells within the germinal centers（GCs）of secondary lymphoid tissues to initiate B cell activation,antibody responses,and the generation of memory B cells and plasmablasts.Abnormal differentiation and proliferation of Tfh cells can lead to the occurrence of several autoimmune diseases.Recently,expansion of Tfh cells have also been demonstrated in the circulation of IgAV patients and associated with renal injury.Tfh cells can be divided into 3 different functional subtypes which are correlated with the production of different immunoglobulin.Tfh17 cells are thought to help GC B cells switch to high affinity IgA production.Therefore,targeted therapy against Tfh17 cells may specifically inhibit the secretion of IgA by B cells,thus allowing the development of tailored medicines for treating IgAV.The mechanisms that underlie the differentiation of Tfh17 cells remain obscuring.Interleukin-6（IL-6）plays a pathological effect on chronic inflammation and autoimmunity.IL-6 signaling can facilitate Tfh cell development and ongoing IL-6signaling consolidates the Tfh lineage and is required for its maintenance and function.What’s more,IL-6 signaling is also a determinant of Th17 cell fate.Therefore,we infer that IL-6 may be the cytokine with the most impact on the differentiation of Tfh17.Tocilizumab,a humanised anti-IL-6R monoclonal antibody,targets IL-6R to block IL-6signaling.Worldwide clinical trials and off-label use of tocilizumab have proved its outstanding effects with a favorable safety and tolerability profile on immune diseases which characterized by IL-6 inflammation such as rheumatoid arthritis,juvenile idiopathic arthritis,and Castleman disease,leading to the approval of tocilizumab for the treatment of these diseases.Serum IL-6 concentration was also significantly increased in patients with IgAV.To date,tocilizumab has not been indicated for IgAV,and reports about the effects of tocilizumab on IgAV were not available.The present study was designed to define the crucial role of IL-6 on the differentiation Tfh17 cells,thereby providing promising therapeutic targets for IgAV.Methods:Part Ⅰ:Forty IgAV subjects were included.Health controls（HCs）（n=40）were age and sex matched.Peripheral blood mononuclear cells（PBMCs）were collected and isolated for flow cytometry to detect the proportion of CD4⁺T cell subtypes such as Th1,Th2,Th17 and Tfh cells and further the proportion of Tfh cell subtypes（Tfh1,Tfh2 and Tfh17 cells）.Serum were obtained from people to measure the level of IL-6,IL-17A,and IL-21 with Elisa kits.The baseline demographics and clinical characteristics of patients with IgAV were collected to analyze the correlation between Tfh17 cells and disease severity.Part Ⅱ:1.PBMCs from patients were isolated and cultured in the presence of either tocilizumab or IL-6 for 72 h.The proportions of Tfh cells,Tfh17 cells and plasma cells in PBMCs were measured using flow cytometry.Elisa was performed to detect the concentration of IL-21 in the culture supernatant.2.PBMCs from HCs were isolated and further total CD4⁺T cells were purified and cultured with IL-6,TGF-β,or a combination of both for 96 h.The proportions of Tfh cells,Tfh17 cells in the vitro culture of CD4⁺T cells,as well as the expression level of phosphorylated signal transducer and activator of transcription（STAT）1 and STAT3 in Tfh cells were measured using flow cytometry.3.PBMCs from HCs were isolated and further total CD4⁺T cells and dendritic cells were purified and cultured with IL-6,anti-TGF-βantibody for 96 h.Part Ⅲ:Effect of tocilizumab on the onset of IgAV rats.Male Sprague Dawley（SD）rats（5-6 weeks）were used in this study.SD rats were randomly assigned to three groups including IgAV,Tocilizumab（IgAV+Tocilizumab）and HC group（n=6 in each group）.The body weight was measured every other day,and the changes of skin rash,urine,stool,food intake and activity were obtained.Urine was collected to measure urinary protein and creatinine,and the ratio of urinary protein to creatinine was calculated.Serum IL-6levels were measured by Elisa.IgA deposition in kidney was measured by immunofluorescence staining.The skin and gastrointestinal tissues were stained with hematoxylin and eosin（HE）to observe vascular inflammatory changes.The kidney tissues were stained with HE and periodic acid Schiff（PAS）to analysis the degree of mesangial proliferation.The general appearance of spleen was observed,body weight and spleen weight were measured,and spleen index was calculated.The gross morphology of the spleen tissues was collected and the spleen weight was measured to calculate spleen index.The expression of PNA in spleen was detected using immunofluorescence and flow cytometry.Flow cytometry was used to detect the proportions of CD4⁺T cell subtypes（Th1,Th2,Th17,Tfh）,Tfh cell subtypes（Tfh1,Tfh2,Tfh17）and plasma cells,as well as the phosphorylation levels of STAT1 and STAT3 in Tfh cells in the peripheral blood and spleen of rats.The proportions of GC-CD4⁺T cells,GC-Tfh cells and GC-B cells in spleen were also measured using flow cytometry.Results:Part Ⅰ:1.Percentages of CD4⁺CXCR5⁺PD-1⁺Tfh-like cells and CD69⁺activated Tfh cells were significantly higher in patients with IgAV than in HCs.What’s more,we noticed a dramatically increasing in the frequency of CD4⁺CXCR5⁺CCR6⁺Tfh17-like cells whereas only a slight increasing in the frequency of CD4⁺CXCR3^-CCR6⁺Th17-like cells in IgAV patient compared to HCs.Serum levels of IL-17A and IL-21 were also upregulated in IgAV patients.2.The frequency of B cells showed a significant and positive correlation with the frequency of circulating Tfh-like cells and a correlative trend with Tfh17 cells in patients with IgAV.However,no significant correlations between Tfh17 or Tfh cells and circulating IgA were observed.Positive correlations of the frequency of Tfh17 or Tfh cells with inflammation indicator C-reactive protein（CRP）were also maintained.We compared the proportion of Tfh17 cells in IgAV patients with multiple organ involvement and that with simply skin involvement.Results showed that patients with multiorgan involvement had higher frequency of Tfh17 cells compared to patients with simply skin involvement.Besides,the proportion of Tfh17 cells was much higher in patients with gastrointestinal bleeding.3.We assessed the serum level of IL-6in IgAV patients.Significantly increasing of serum IL-6 level was observed in IgAV patients compared with HCs.Positive correlation of levels of IL-6 with CRP and erythrocyte sedimentation rate（ESR）was demonstrated.Importantly,serum levels of IL-6 were positively associated with the frequency of Tfh17-like,Tfh-like cells,as well as CD19⁺B cells.Part Ⅱ:1.We investigated whether the blockage of IL-6 pathway could suppress Tfh17 cells on IgAV patients.PBMCs from IgAV patients were cultured under the stimulation of anti-CD3 in the presence of IL-6 or tocilizumab for 3 days.There was a significant increase in CXCR5 and Bcl-6 expression by IL-6 stimulation and dramatically reduction of that by IL-6R neutralization using tocilizumab.What’s important,the percentage of Tfh17 cell subset,but not Tfh1 cell subset,was affected by IL-6 and tocilizumab.The frequency of CD27⁺CD38^hiplasmablasts and the level of IL-21 in culture supernatant were also affected by IL-6.2.IL-6 stimulation caused a significant increase in the expression of p STAT1 and p STAT3 in CD4⁺T cells which was reversed by tocilizumab.Results showed that IL-6 stimulation can significantly enhance the expressions of CXCR5 and Bcl-6 while IL-6R blockage using tocilizumab reduced the expressions of them in CD4⁺T cells.However,CD4⁺T cells under IL-6 condition failed to enhance the proportion of Tfh17 cells.The synergy of IL-6 and TGF-βsignificantly up-regulated the proportion of Tfh17 cells which was reversed by tocilizumab.The level of TGF-βwas increased in the presence of IL-6.RORg T⁺Bcl-6⁺Tfh17-like cells were induced under the stimulation of IL-6 while suppressed by anti-TGF-βantibodies.Part Ⅲ:1.In order to further study the in vivo role of IL-6 signaling in IgAV,rat models were generated.IgAV rats recapitulated clinical features of the human IgAV as evidenced by hemorrhagic rash,bloody stool,increased protein-to-creatinine ratio and renal IgA deposition.Decreased food intake,body weight and activity were also observed in IgAV rats.Notably,serum IL-6 level was significantly increased in IgAV rats.Hemorrhagic rash severity was reduced after the administration of tocilizumab.Histopathological analysis of IgAV rat skin lesions revealed extravagated erythrocytes,thickened vessel walls,and inflammatory cell infiltration which was alleviated by tocilizumab.Tocilizumab alleviated the symptoms of gastrointestinal bleeding.Pathological features of gastric and intestinal tissue from tocilizumab-treated group displayed decreased hemangiectasis,hyperemia and hemorrhage compared to IgAV group.Tocilizumab reduced renal IgA deposition which was detected in IgAV rats.Protein-to-creatinine ratio was reduced upon tocilizumab treatment.In addition,renal pathology displayed significantly decreased in the proliferation of mesangial cells and mesangial matrix in the glomerulus by tocilizumab intervention.2.IL-6 exerts effects on CD4⁺T cell differentiation through the STAT1 and STAT3 signaling pathways.We observed that an up regulation in the expression of phosphorylated STAT3（p STAT3）and p STAT1 in CD4⁺CXCR5^hiTfh cells in IgAV rats and substantially down regulation of that after tocilizumab administration.However,tocilizumab exerts its effect through binding IL-6R without reducing serum IL-6 level.The percentages of ICOS⁺CXCR5^hiTfh cells from spleen and PBMCs of IgAV rats was higher compared to HCs.IL-17-producing Tfh17 cells,but not IL-4-producing Tfh2 cells or IFN-γ-producing Tfh1cells,significantly increased in IgAV rats.Elisa analysis confirmed the up-regulation of serum IL-17A in IgAV rats.In addition,the transcription of Bcl6,Icos and Il17 were also up-regulated in the spleen of IgAV rats.In contrast,treatment with tocilizumab reduced proportions of ICOS⁺CXCR5^hiTfh cells and IL-17-producing Tfh17 subsets,accompanied by severely compromised Bcl6 and Icos expressions and IL-17A production in IgAV.We found that the frequency of PNA⁺CD4⁺T（GC-CD4⁺T）cells and PNA⁺CXCR5^hiTfh-like（GC-Tfh）cells were also elevated in IgAV rats.As expected,treatment with tocilizumab reduced the proportions of GC-CD4⁺T and GC-Tfh cells.3.Compared to control rat,the spleen of IgAV rat was significantly enlarged and spleen index was elevated.A greater number of GCs was formed in the spleen of IgAV rats as indicated by immunofluorescence staining of spleen with PNA,while only a few PNA-positive GCs were observed in the spleen of control rats and tocilizumab-treated rats.The percentage of PNA-positive cells in IgAV rats was also elevated as demonstrated by flow cytometry.More importantly,the proportion of PNA⁺B220⁺GC-B cells was higher in IgAV rats,and decreased by tocilizumab treatment.IgAV rats exhibited a higher percentage of CD27^hiCD38^hiB220⁺plasmablast compared to control rats,while tocilizumab treatment reduced the increasing.Elevated serum levels of IgA in IgAV rats was observed and can be decreased by tocilizumab treatment.Conclusions:1.Tfh17 cells was the main CD4⁺T cells that involved in the pathogenesis of IgAV disease.2.IL-6 is essential for the accumulation and function of pathogenic Tfh17 cells in IgAV.IL-6 can support DCs to produce TGF-βand promote Tfh17differentiation in coordination with TGF-β.3.Tocilizumab was effective in preventing the development of IgAV through reducing Tfh17 cells,Ab-secreting cells and serum IgA in IgAV.