The Mechanism Of BDNF/TrkB Affecting TFH Cell Differentiation And Promoting The Pathogenesis Of Primary Sjogren’s Syndrome

Primary Sjogren’s Syndrome(pSS)is a chronic,slowly progressive autoimmune disease characterized by dry mouth and eyes caused by lymphocytes infiltrating lacrimal glands,salivary glands and other exocrine glands.PSS can occur at any age,and is most common in middle-aged women,with a female to male ratio of 20:1.The prevalence of pSS in China is 0.5%～1%.The pathogenesis and therapeutic targets of pSS remain unclear.Therefore,it has become a research topic to explore the pathogenesis of pSS and find targeted drugs.Brain-derived neurotrophic factor(BDNF)is a member of the neurotrophic factor family.Its high-affinity receptor is tyrosine protein kinase receptor B(TrkB).BDNF/TrkB plays a role in promoting neuronal growth,differentiation and survival.It is also widely expressed in different cell types,including microglia,vascular endothelial cells,T/B lymphocytes,etc.,and the expression of TrkB increases after lymphocyte activation.BDNF/TrkB can promote T and B cell proliferation and differentiation,but the specific mechanism remains unclear.BDNF/TrkB has not been reported in pSS.Follicular helper T(TFH)cells are a subgroup of CD4+ T cells involved in humoral immunity,mediating the differentiation of B cells in germinal centers(GCs)into plasma cells and memory B cells,thus participating in the immune response.B cell lymphoma protein(Bcl-6)is a characteristic transcription factor of TFH cells,while Interleukin-21(IL-21)is a crucial cytokine in the differentiation process of TFH cells.IL-21 secreted by TFH cells can cooperate with plasma cells to produce more autoantibodies.Increased IL-21 levels in the serum and labial glandular tissue of pSS patients are key factors in the development of pSS.Therefore,this study explored the differences in the expression levels of BDNF/TrkB in peripheral blood and labial gland of pSS patients,and explored the clinical significance of BDNF/TrkB in pSS.At the same time,whether BDNF/TrkB affects the proliferation and differentiation of TFH cells and whether IL-21 secretion plays a pathogenic role in pSS patients was determined,so as to reveal the pathogenesis of pSS.The specific research content is divided into the following three parts:Part I: Study on the expression of BDNF/TrkB in peripheral blood and labial gland tissues of pSS patientsObjective: To determine the expression level of BDNF/TrkB in peripheral blood and labial gland tissues of pSS patients and healthy controls.Methods:The fresh peripheral blood,labial gland tissue and clinical data of newly treated pSS patients were collected.Meanwhile,peripheral blood of age and sex matched healthy volunteers and labial glandular tissue of patients requiring debridement after maxillofacial trauma were collected as controls.The serum BDNF levels in the two groups were determined by ELISA,and the correlation between serum BDNF levels and clinical data of pSS patients was analyzed.The proportion of CD3+CD4+TrkB+ and CD3+CD8+TrkB+T cells in peripheral blood was detected by flow cytometry,and the correlation between the proportion of CD3+CD4+TrkB+ and CD3+CD8+TrkB+T cells in peripheral blood and clinical indicators of pSS patients was analyzed.The expression of CD4+T cells,CD8+T cells,BDNF and TrkB in labial glandular tissue were detected by immunohistochemistry.Results:1.The level of serum BDNF in patients with primary Sjogren’s syndrome was significantly lower than that in healthy controls(HCs).The higher the disease activity,the lower the serum BNDF level.The levels of serum BDNF in thrombocytopenia group,hyperglobulinemia group,purpura group and interstitium pneumonia group were all significantly decreased.Serum BDNF level was positively correlated with platelet count,CD4+T cell count,CD8+T cell count,and negatively correlated with disease activity score(ESSDAI)and Ig G level.2.The expression ratio of CD3+CD4+TrkB+ and CD3+CD8+TrkB+ T cells in peripheral blood of patients with pSS was higher than that of healthy controls.The expression ratio of CD3+CD4+TrkB+T cells was positively correlated with Ig G level and ESSDAI level,but negatively correlated with C4 level and serum BDNF level.The expression of CD3+CD8+TrkB+T cells was positively correlated with ESSDAI,but negatively correlated with C3 and C4 levels and serum BDNF levels.3.In the labial glandular tissue of pSS patients,there was focal interglandular lymphocyte infiltration,and the surrounding acinar tissue structure was normal,some of them were accompanied by gland atrophy or duct dilation.BDNF was mainly highly expressed on the duct epithelial cells of pSS patients,and TrkB was highly expressed on CD4+ and CD8+T cells.There was no lymphocyte infiltration and duct dilation in the labial gland tissues of healthy controls,and BDNF expression was low in the duct epithelial cells of healthy controls.BDNF/TrkB is involved in the destruction of the labial gland in pSS.Conclusion:The level of BDNF in peripheral blood of pSS patients is significantly lower than that of healthy controls,while the expression ratio of TrkB in peripheral blood lymphocytes is significantly higher than that of healthy controls.In the labial glandular tissue,BDNF was highly expressed in the ductal epithelial cells of pSS patients,while TrkB was highly expressed in CD4+ and CD8+T cells of pSS patients.The high expression of BDNF/TrkB is closely related to the pathogenesis and disease activity of pSS.Part II: Study on the expression of BDNF/TrkB in TFH cells of peripheral blood and labial gland of pSS patientsObjective: To investigate the expression and clinical significance of BDNF/TrkB in TFH cells of peripheral blood and labial gland of pSS patients.Methods: Fresh peripheral blood and labial glandular tissue of newly treated pSS patients were collected,while peripheral blood of age and sex matched healthy volunteers and labial glandular tissue of patients requiring debridement after maxillofacial trauma were collected as control.CD4+CXCR5+PD-1+T cells were defined as TFH cells for the study,CD4+CXCR5-PD-1+T cells and CD4+CXCR5-PD-1-T cells were defined as non-TFH for the study.The expression ratio of TFH cells,TFH+TrkB+ cells and non-TFH+TrkB+ cells in peripheral blood was detected by flow cytometry.The correlation between the expression ratio of TFH cells and TFH+TrkB+ cells in peripheral blood and clinical data was analyzed.Serum IL-21 levels in pSS patients and healthy controls were determined by ELISA.The expression of TFH+ TrkB+ cells in labial glandular tissue of pSS patients was detected by immunohistochemistry.Results:1.The expression ratio of TFH cells in peripheral blood of pSS patients was significantly higher than that of healthy control group.The expression ratio of TFH+TrkB+ cells was significantly higher than that of healthy control group.The proportion of TFH+TrkB+cells was positively correlated with serum Ig G level,rheumatoid factor(RF)and ESSDAI.Compared with healthy controls,the expression ratio of non-TFH+TrkB+ cells in peripheral blood of pSS patients was not statistically significant.2.Compared with the healthy control group,the serum IL-21 level of pSS patients was significantly increased,and was positively correlated with ESSDAI and serum Ig G level.3.A large number of lymphocytes could be seen in the labial glandular tissue of pSS patients around the catheter,and expressions of TFH+TrkB+ cells could be seen on the infiltrated lymphocytes.However,there was no lymphocyte infiltration and no expression of TFH+TrkB+ cells in the labial glandular tissue of healthy controls.Conclusions: The proportion of TFH cells in patients with primary Sjogren’s syndrome is significantly increased,and the expression proportion of TFH+TrkB+ cells in peripheral blood is also significantly increased in patients with pSS.The proportion of TFH+TrkB+ cells is positively correlated with Ig G,RF and ESSDAI.The serum IL-21 level was increased in pSS patients,and was positively correlated with serum Ig G level and ESSDAI level.A large number of CD4+CXCR5+PD-1+TrkB+ cells were expressed on lymphocytes around the duct tissue of the labial gland,and these lymphocytes attacked the duct and acinar tissue,resulting in the destruction of the labial gland structure.Part III: BDNF/TrkB affects TFH cell differentiation in pSS patients through JAK1-STAT3 pathwayObjective: To explore the effect of BDNF/TrkB on the proliferation and differentiation of TFH cells in peripheral blood of pSS patients and its mechanism.Methods: The peripheral blood of newly treated pSS patients and age-and sex-matched healthy volunteers were collected as control.CD4+T cells were isolated from pSS patients and healthy controls by magnetic beads for in vitro culture.After exogenous addition of different concentrations of ANA-12(TrkB antagonist)(5?M,10?M,20?M),the activity of CD4+T cells was detected by CCK8.CD4+T cells were divided into 3groups,including CD4 untreated group,CD4+ polarized group(5 ng/m L IL-12,10ng/m L IL-6;1 ng/m L TGF-β),and CD4+ polarization +10?M ANA-12 group.Flow cytometry was used to detect the proportion of TFH cells cultured in vitro,and q PCR was used to detect the levels of TFH cell-specific transcription factors Bcl-6 and Blimp-1.The phosphorylation levels of JAK1 and STAT3 were detected by Western Blot.ELISA was used to detect IL-21 levels in cell supernatants of different groups.Results:1.ANA-12(TrkB antagonist)at 10?M concentration had no effect on the activity of CD4+T cells,and the concentration of 10?M was selected as the research object.2.After 24 h in vitro culture,flow cytometry showed that compared with the untreated CD4 group,the expression proportion of TFH cells in the CD4+ polarized group increased,while the proportion of TFH cells in the CD4+ polarized +10?M ANA-12 group decreased.3.After 48 h in vitro culture,q PCR detection showed that compared with the untreated CD4 group,the expression level of BCL-6 m RNA in the CD4+ polarized group was increased,while the expression level of BCL-6 m RNA in the CD4+ polarized +10?M ANA-12 group was decreased,while the expression level of Blimp-1 m RNA was not statistically significant.4.After 72 h in vitro culture,Western Blot showed that compared with the untreated CD4 group,the expression levels of p-JAK1 and p-STAT3 in the CD4+ polarized group increased,while the expression levels of p-JAK1 and p-STAT3 decreased in the CD4+polarized+10?M ANA-12 group,inhibiting activation of the pathway.It affected the proliferation and differentiation of TFH cells.5.After 72 h in vitro culture,ELISA showed that the level of IL-21 in supernatant of CD4+ polarized group increased compared with that of the untreated CD4 group,while the level of IL-21 in supernatant of CD4+ polarized+10?M ANA-12 group decreased.Conclusion: BDNF/TrkB affects the proliferation and differentiation of TFH cells in pSS patients through JAK1-STAT3 pathway.