Mechanisms Of Interferon Beta Promoting Sj(?)gren Syndrome Mice By Regulating Tfh Cell Differentiation

Objective:In this study,we aimed to investigate the effects of Interferon beta（IFN-β）on disease progression and T follicular helper cells（Tfh cells）differentiation and function in mice experimental Sj（?）gren’s Syndrome（ESS）.To further investigate the potential molecular mechanisms of IFN-βregulation of Tfh cells differentiation.Methods:（1）We construct the mice model of ESS,and detect serum IFN-βlevels in the disease process of ESS mice by enzyme linked immunosorbent assay（ELISA）.Sterile PBS or IFN-βwere injected into the tail vein of ESS mice,and the salivary gland（SG）protein and anti-SSA autoantibody levels were detected by ELISA after 35 days of model treatment.The salivary glands of mice were prepared in frozen sections and immunofluorescence（IF）was used to detect the deposition of immunoglobulin G（Ig G）in salivary gland tissue.Paraffin sections of salivary glands of mice were prepared and stained with hematoxylin-eosin staining（HE）to observe the histopathological changes of salivary glands.The spleen（Spleen,SP）and local draining lymph nodes（d LN）of mice were examined by flow cytometry（FCM）for Tfh cells（CD4⁺PD-1⁺CXCR5⁺BCL6⁺）,germinal center B cells（GC B cells;CD19⁺Fas⁺GL-7⁺）and plasma cells（CD138⁺B220^-）ratios and numbers,as well as the Tfh cell effector molecules CD40 Ligand（CD40L）,Inducible costimulatory（ICOS）and interleukin-21（IL-21）expression.（2）We constructed an ESS mice model by using type Ⅰ interferon receptor knockout(Ifnar^-/-)mice.The levels of anti-SG and anti-SSA antibodies in mice serum were determined by ELISA,and the inflammatory cell infiltration and destruction of glandular ducts in HE-stained sections of mice salivary gland tissues were observed.To detect the ratio and number of Tfh cells,GC B cells and plasma cells in the spleen and local draining lymph nodes of mice,as well as the expression of Tfh cell effector molecules CD40L,ICOS and IL-21 by FCM.（3）Sorted Tfh precursors cells（Pre-Tfh cells）and Tfh cells from the spleens of wild type（WT）and ESS mouse were used to detect the m RNA expression levels of type Ⅰ interferon receptors（IFNAR1 and IFNAR2）in the cells by quantitative reverse transcription PCR（RT-PCR）.Sorted na（?）ve CD4⁺T cells from the spleen of WT mice to establish a culture system for IFN-β-induced Tfh cells.After 3 days of culture,FCM was performed to detect the proportion of Tfh cells（CD4⁺PD-1⁺CXCR5⁺）,and RT-PCR was performed to detect the expression levels of functional molecules such as B cell lymphoma 6（BCL6）,chemokine C-X-C receptor 5（CXCR5）,programmed cell death protein-1（PD-1）,IL-21,CD40L and ICOS in na（?）ve CD4⁺T cells.Sorted ESS mice spleen-derived Tfh cells were stimulated with IFN-β,the m RNA levels of functional molecules such as BCL6,CXCR5,PD-1,IL-21,CD40L and ICOS were measured by RT-PCR in Tfh cells.（4）Sorted na（?）ve CD4⁺T cells from the spleen of WT mice were used to establish a culture system for IFN-β-induced Tfh cells,and the expression of indoleamine-2,3-dioxygenase 1（IDO1）in na（?）ve CD4⁺T cells was detected by western blot（WB）and RT-PCR.Collection of supernatants from the culture system and ELISA to detect the level of kynurenine（Kyn）in the supernatant.Nuclear translocation of the aryl hydrocarbon receptor（AhR）in na（?）ve CD4⁺T cells was detected using confocal laser scanning microscopy（CLSM）and FCM.AhR inhibitor was added to the culture system of IFN-β-induced Tfh cells,and the proportion of Tfh cells was detected by FCM after 3 days of culture.（5）We constructed an ESS mice model and injected sterile PBS or IFN-βinto the tail vein of the mice.Tfh cells were sorted from the spleen of ESS mice.The m RNA expression levels of AhR-related target genes,such as AhR,aryl-hydrocarbon receptor repressor（AHRR）,aryl-hydrocarbon receptor nuclear transporter（ARNT）,cytochrome P450 1A1（CYP1A1）and cytochrome P450 1B1（CYP1B1）were detected by RT-PCR in Tfh cells.Meanwhile,we also detected the phenomenon of AhR nuclear translocation in Tfh cells in the spleen and local draining lymph nodes of mice by FCM,and the level of Kyn in the serum of ESS mice by ELISA.Results:（1）The results showed that serum IFN-βlevels increased as the disease progressed in ESS mice（p<0.05）.IFN-βsignificantly increased the severity of disease in ESS mice,as evidenced by increased impairment of salivary gland flow rate（p<0.01）,increased spleen and local draining lymph node volume and cells number（SP:p<0.05,p<0.05;d LN:p<0.01,p<0.05）,increased serum anti-SG antibody（p<0.01,p<0.001）and anti-SSA antibody（p<0.05,p<0.001）levels.Compared with ESS mice,interferon beta exacerbated Ig G deposition,inflammatory cell infiltration,and glandular duct destruction in salivary gland tissues of ESS mice,and the proportion and number of Tfh cells（SP:p<0.01,p<0.05;d LN:p<0.001,p<0.001）,GC B cells（SP:p<0.01,p<0.01;d LN:p<0.01,p<0.05）and plasma cells（SP:p<0.01,p<0.01;d LN:p<0.05,p<0.05）were significantly increased in the spleen and local draining lymph nodes of mice,while the expression of effector molecules CD40L（SP:p<0.01,p<0.01;d LN:p<0.001,p<0.05）,ICOS（SP:p<0.05,p<0.001;d LN:p<0.05,p<0.05）and IL-21（SP:p<0.05,p<0.05;d LN:p<0.001,p<0.05）were significantly upregulated in Tfh cells.（2）Compared to the ESS-WT mice group,the ESS-Ifnar1^-/-mice group showed partial restoration of salivary gland flow rate（p<0.05）,reduction in spleen and local draining lymph node volume and cells number（SP:p<0.05;d LN:p<0.05）,reduction in inflammatory cell infiltration in salivary gland tissue and destruction of glandular ducts,and reduction in serum anti-SG（p<0.001）and anti-SSA antibody（p<0.05）levels.At the same time,ESS-Ifnar1^-/-mice showed a decrease in the proportion and number of Tfh cells（SP:p<0.05;d LN:p<0.05）,GC B cells（SP:p<0.05;d LN:p<0.05）and plasma cells（SP:p<0.01;d LN:p<0.05）in spleen and local draining lymph nodes,as well as a downregulation of the expression of Tfh cell effector molecules CD40L（SP:p<0.05;d LN:p<0.05）,ICOS（SP:p<0.001;d LN:p<0.01）and IL-21（SP:p<0.05;d LN:p<0.05）.（3）The results showed that the expression levels of IFNAR1/IFNAR2 were higher in spleen-derived Pre-Tfh and Tfh cells from ESS mice compared with WT mice.In the Tfh cell induction system,IFN-βsignificantly promoted an increase in the proportion of Tfh cells（CD4⁺PD-1⁺CXCR5⁺;p<0.01）and upregulated the expression levels of Tfh cell-associated functional molecules such as BCL6,IL-21,CXCR5,PD-1,CD40L and ICOS in na（?）ve CD4⁺T cells.In addition,IFN-βalso significantly promoted the expression levels of functional molecules such as BCL6,IL-21,CXCR5,PD-1,CD40L and ICOS in Tfh cells.（4）In the Tfh induction system,IFN-βpromoted upregulation of IDO1 expression in na（?）ve CD4⁺T cells（p<0.001）as well as elevated levels of Kyn in culture supernatants（p<0.01）,and AhR nuclear translocation occurred in na（?）ve CD4⁺T cells.The use of AhR inhibitors in the IFN-β-induced Tfh cell system significantly down-regulated the ability of IFN-βto induce Tfh cell differentiation（p<0.001）.（5）The results showed that IFN-βsignificantly upregulated the expression of AhR,AHRR,ARNT,CYP1A1 and CYP1B1 AhR-related target genes in spleen-derived Tfh cells from ESS mice compared with ESS mice,while IFN-βpromoted the occurrence of AhR nuclear translocation in Tfh cells from the spleen and local draining lymph nodes of mice（p<0.05）,and additionally IFN-βupregulated serum Kyn levels in ESS mice（p<0.01）.Conclusion:（1）IFN-βaggravates the severity of ESS mice.（2）IFN-βpromotes the differentiation and function of Tfh cells in ESS mice.（3）IFN-βregulates Tfh cell differentiation through IDO1-Kyn-AhR pathway.