The Role And Mechanism Of SMS1-SM In The B Cell Function And The Progression Of SLE

Objetive: Systemic lupus erythematosus(SLE)is a systemic autoimmune syndrome characterized by autoantibodies production and multiple organs damage,the central pathogenesis of which is due to the breakthrough of immune tolerance and over-activation of B-lymphocytes.The specific mechanisms leading to B cell dysfunction remain unclear.Lipid rafts(LRs)are cholesterol and sphingomyelin(SM)-enriched microdomains on cellular membranes,involved in various signal transduction and lymphocytes activation.SM can be catalyzed by sphingomyelin synthase 1(SMS1)and various biological functions of lipid have been gradually discovered in recent years.To explore the role of SMS1-SM in B cell activation is expected to provide possibilities for the new intervention targets of SLE treatment.Therefore,the present study was designed to elucidate the role and mechanism of SMS1-SM in B cell dysfunction and SLE development.Methods: 1.B lymphocytes from peripheral circulation of SLE patients and age and gender-matched healthy controls were isolated by means of immunomagnetic cell sorting.RT-PCR to analyze the m RNA expression of SMS1 of isolated B cells.BCA was performed to test a panel of serum cytokines titers.The Spearman's correlation was analyzed between the above results and other laboratory indicators as well as disease activity.The comparisons between the above results were analyzed using two-tailed unpaired Student's t-test and Spearman's correlation analysis.2.B cells of splenic mononuclear cells(SMCs)from SMS1-/-and WT groups were purified using MACS isolation and then stimulated in vitro.The activation,proliferation and differentiation of B cells were detected by flow cytometry.Western blot was used to observe the phosphorylation of key protein molecules in upstream signal transduction pathway.3.Using age and gender-matched SMS1-/-and WT mice as recipients while Bm12 mice as donors,we constructed Bm12-induced c GVHD models to simulate the pathological state of SLE.Serum and urine specimens were collected on the day of injection(as day 0)and every 2weeks thereafter until experiment termination.The serum ANA titers were detected by ELISA.Urinary creatinine levels were tested by quantitative colorimetric determination and urine protein levels were detected by the BCA method.The activation states of splenic B-lymphocytes in mice were evaluated by flow cytometry.In addition,the spleen-derived B cells of WT mice were sorted and then transferred i.v.into SMS1-/-and WT mice at the same day before the induction of c GVHD.The changes of the above indexes were observed after 4 weeks of induction.4.SMS1-/-and WT mice were intraperitoneal injected with pristane for lupus induction.The serum titers of autoantibodies,pathological score and Ig G deposition of kidney as well as B cell activation were observed after 6 months of induction.Results: 1.The sphingomyelin synthase 1(SMS1)m RNA of B-lymphocytes from SLE patients was significantly increased and positively correlated with serum titers of IL-1??IFN-??TNF-??IL-10?IL-12 as well as IL-23.There also existed positive correlation between SMS1 m RNA of B-lymphocytes and serum Ig G and globulin titers,though we didn't found the correlation of SMS1 expression and SLEDAI index.2.SMS1 knockout can also significantly attenuate the expression of activation markers CD69,CD80 and CD86 of splenic B-lymphocytes in vitro stimulation,but did not change the CTB-labeled GM1 overall levels.The BCR-mediated early signaling was partially blocked due to the reduced phosphorylation of Fyn and Syk in SMS1-/-mice.Additionally,there existed significant inhibition on the ability of proliferation and differentiation of SMS1-/-B cells.3.SMS1-/-mice were significantly protected from the ANA production and 24 h urine protein excretion after 4 weeks of c GVHD induction.The suppression of c GVHD response in KO mice was associated with the depression of B cell activation,and the expression of CD69,CD80 and CD86 on B cells were significantly lower than WT group.While adoptive transfer of normal-function B cells before c GVHD induction can partially exacerbated the ANA production,24 h urine protein excretion and B-lymphocytes hyper-activation of SMS1-/-mice.4.Similarly,we also utilized the pristane-induced SLE model to double elaborate that SMS1 deficiency can significantly decrease auto-Abs titers,as well as the pathological score and Ig G deposition of kidney.Pathological improvement was also associated with inhibition of B cell over-activation.Conclutions: In conclusion,our present research identified the critical role of SMS1 in B-lymphocytes activation and development of SLE and may serve as a promising mechanism therapeutic target for the treatment of SLE.