The Mechanism Of IL-21in The Development Of Sjogren’s Syndrome

Sjogren’s syndrome (SjS) is considered an autoimmune disease in which the immune system targets the exocrine glands and leads to the loss of secretory function. SjS is classified as either primary SjS or secondary SjS. Primary SjS is a chronic autoimmune attack involving both lacrimal and salivary glands in the absence of other autoimmune diseases, whereas secondary SjS is an autoimmune attack against the lacrimal and/or salivary glands in the presence of other autoimmune diseases, most often rheumatoid arthritis, systemic lupus erythmatosus (SLE), or scleroderma. SjS is one of the three most common autoimmune diseases, along with SLE and systemic sclerosis. SjS affects0.5-3%of the population, and predominates in females with a ratio of9:1versus males, suggesting hormonal involvement. The pathogenesis of SjS is complicated with many respects remaining elusive.IL-21is a pleiotropic type Ⅰ cytokine and plays critical roles in lymphocyte development, proliferation, and differentiation, especially in antibody production and the maturation of B cells into primary antibody-producing plasma cells. Accumulated research of IL-21has associated IL-21with the development of many autoimmune diseases, such as inflammatory bowel disease, rheumatoid arthritis, diabetes and systemic lupus erythematosus. So IL-21is considered as a key factor and therapeutic agent in the treatment of autoimmune diseases. The role of IL-21in the development of Sjogren’s syndrome remained elusive. Many studies have shown that the level of serum IL-21in patients with SjS is higher than that in healthy control, and minor salivary glands biopsy specimens revealed positive staining for both IL-21and IL-21receptor within lymphocytic foci and periductal area whereas only minimal expression was seen in samples of control. However, the role of IL-21in the pathogenesis of SjS remains elusive and its mechanism is not clear so far.In order to figure out the role of IL-21in SjS, SjS model animal nonobese diabetic (NOD) mouse was used in our study. We blocked IL-21pathway in NOD mice to evaluate the role of IL-21in the development of SjS and figure out the mechanism.Part Ⅰ. The role of IL-21blockage played in development of SjS of NOD miceObject:To evaluate the role of IL-21played in development of SjS, fusion protein IL-21R.Fc was used to block IL-21signal in NOD mice and the changes of SjS symptoms were monitored.Methods:The extracellular domain of IL-21R and Fc domain of IgG2a of mice were amplified from mouse cDNA following the molecular cloning methods. The235,318,320and322aa of Fc domain were mutated to minimize Fc binding and complement fixation. DNA segments of murine IL-21R extracellular domain and mutated IgG2a Fc domain were connected and cloned into plasmid pcDNA3.1+to construct plasmid pcDNA3.1-IL-21R.Fc. Plasmid pcDNA3.1-IL-21R.Fc was purified and transfected into CHO cells with Lipofectamine2000. IL-21R.Fc fusion protein was purified through Protein A affinity columns with CHO cell lysates and conditioned medium. The protein was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NOD mice were administered intravenuously in tail with purified IL-21R.Fc. Saliva flow rate and lymphocytic infiltration in submandibular glands of NOD mice were evaluated every4weeks.Result:Plasmid pcDNA3.1-IL-21R.Fc was constructed successfully, and fusion protein IL-21R.Fc was purified. Blockage of IL-21signal by IL-21R.Fc in NOD mice alleviated symptoms of SjS. The development of secretory dysfunction and lymphocytic infiltration were retarded in submandibular glands.Conclusions:IL-21played a critical role in the development of SjS in NOD mice. IL-21pathway could be used as a promising therapeutic target in the treatment of SjS.Part Ⅱ. The role of local suppression of IL-21in submandibular glands of NOD mice played in development of SjS and mechanism researchObject:The aim of this study was to verify the validity of IL-21local suppression in submandibular glands of preventing the development of SjS in non-obese diabetic (NOD) mice and try to figure out the mechanism.Methods:IL-21levels in submandibular glands were suppressed by ductal cannulation of IL-21shRNA lentivirus. Then, saliva flow rates and histopathologic changes of submandibular glands were measured to assess the severity of disease development. Real-time PCR, flow cytometry, and immunohistochemistry were used to detect the changes of T helper cells and related cytokines.Result:The reduction in SFRs in NOD mice was significantly alleviated from9to17weeks of age along with the suppression of IL-21in submandibular glands. Lymphocytic infiltration was also milder than control NOD mice. Moreover, the lower level of IL-21led to the down-regulation of follicular helper T (Tfh) cells.Conclusions:Local suppression of IL-21alleviated secretory disfunction and disrupted lymphocyte infiltration in SMG of NOD mice and thus retarded the development of SjS-like symptoms of NOD mice. Suppression of IL-21might interfere with the development of GC and maturation of B cells through Tfh cells.In summary, our study proved the important role of IL-21in the development of SjS, and found out that local suppression of IL-21is effective in the treatment of SjS. IL-21could contribute to the development of SjS through Tfh cells to improve the formation of GC and the maturation of B cells in submandibular glands. Further research is needed on IL-21that is a promising target in the treatment of SjS.