Mesenchymal Stem Cells Alleviate Sj(?)gren's Syndrome Through Regulating IL-12 Production

Background:Primary Sj(?)gren's Syndrome(pSS)is an autoimmune disease mainly characterized by the secretory dysfunctions of exocrine glands,manifested by typical clinical symptoms such as dry mouth and dry eyes.It could also lead to systemic damages and functional impairment of exo-gland organs.So far,it is still unclear which factors trigger or accelerate the immunopathogenesis of pSS and there were no effective therapeutic effects in treating this disease.Mesenchymal Stem Cells(MSC),known for their low immunogenicity,multi-linage differentiation capability and strong ability in immunomodulation,have been used to treat a wide range of autoimmune diseases.Although several reports have demonstrated the efficacy of MSC in treating pSS patients and mice,the underlying mechanisms remain largely unknown.Researches have shown that Interleukin 12(IL-12),produced by activated antigen presenting cells(APC),contributes to the pathogenesis of pSS.MSC are capable of modulating APC functions.Objectives:We aim to explore whether the therapeutic effects of MSC on pSS are mediated via modulating IL-12 production,which would provide new evidence for elucidating of mechanisms of MSC in treating pSS and promoting of MSC therapies in future clinical applications.Methods:1)IL-12 levels in serum from pSS patients and healthy controls(HC)were quantified by ELISA,correlations of IL-12 with patients' clinical manifestations were analyzed by Spearman's test.CD14+monocytes were isolated from SS and HC peripheral blood,and were induced toward macrophages(M?)or dendritic cells(DC)with M-CSF or GM-CSF plus IL-4,respectively.LPS was used to induce maturation and promote cytokine production of Mcp or DC.IL-12 mRNA levels in monocytes,M? and DC from SS and HC were measured by real-time quantitative PCR(qPCR).Percentages of Thl,Th2,Thl7,Treg,Tfh,B cells and plasma cells in PBMCs from SS and HC were detected by flow cytometry,correlations of IL-12 with patients'lymphocyte subset proportions were analyzed by Spearman's test.5 pSS patients received umbilical cord derived MSC(UCMSC)transplantation,and their whole blood was collected before and 1 week after MSC transplantation.Serum were collected for the quantification of IL-12 levels by ELISA.2)Female NOD mice were used as pSS animal models.Mice were randomly divided into three groups as follows:(?)control group,which were injected i.p with PBS,(?)IL-12 treatment group,which were injected i.p with recombinant murine IL-12,and(?)IL-12 antibody treatment group,which were injected i.p with IL-12 monoclonal antibodies.One week later,saliva flows were detected.Then mice were sacrificed and submandibular glands,lacrimal glands,lungs,kidneys and livers were collected for histological evaluation with hematoxylin-eosin(HE)staining.3)Female NOD mice were randomly divided into groups as follows:(?)UCMSC treatment group,(?)Fibroblast-Like Synoviocyte(FLS)treatment group,and(?)control group.Mice were treated with 106 UCMSC,106 FLS or PBS,respectively.Mice were sacrificed 4 weeks later.The parameters detected in the above section also determined in these mice.CD11c+and CD11b+ cells of each mouse were sorted by magnetic beads.IL-12 mRNA expression in these cells were detected by qPCR.4)The purified splenic CD11c+and CD11b+cells were co-cultured with UCMSC for 24 hours.IL-12 levels were quantified by qPCR.Human THP-1 cells served as monocytes.THP-1 cells were stimulated with PMA to generate Mcp.Human CD 14+cells were induced to generate DC.Monocytes,M? and DC were all co-cultured with UCMSC for 24 hours before RNAs were extracted.IL-12 mRNA levels were detected by qPCR.UCMSC co-cultured with the above cells were detected for the expression of functional molecules,such as CD40,CD40L,CD137,4-1BBL,IFN-?,IFN-?,IFN-y and C5.Results:Serum IL-12 levels were significantly elevated in pSS.Monocytes,M? andDC from pSS patients generated more IL-12 than that from HC.A positive correlation was found between IL-12 levels and the patients' disease scores(ESSDAI).Thl,Th17 and Tfh cell percentages elevated in pSS patients.The Treg percentage was slightly decreased.Besides,plasma cell percentage was also higher in pSS.These results indicated disturbance of immune balances in lymphocyte subtypes.In vivo treatment of NOD mice with IL-12 significantly decreased saliva volume and increased salivary gland swollen severity,accompanying by significantly more lymphocyte infiltrations in the glands.Lymphocytic infiltrations in the parabronchus region,renal interstitium sites and around small bile ducts in the IL-12 treatment group were also observed,while none or little infiltrations were detected in the control group or IL-12 antibody group.On the other hand,percentages and absolute numbers of Th1,Tfh and Th17 cells were significantly lower in the IL-12 antibody treatment group,with less infiltrations in the affected organs.IL-12 levels decreased in pSS patients 1 week after MSCT.Salivary flow was slightly higher in the MSC group.Lymphocytic infiltrations in the submandibular glands were comparable between groups.Th17 and Tfh cell percentages were decreased while Treg and Trl cell percentages were increased in the MSC group,suggesting MSC could alleviate the SS-like symptoms and lymphocyte imbalances in the NOD mice.Four weeks after the MSC treatment,IL-12 mRNA expression levels in CD11c+and CD11b+cells and IL-12 levels in the serum were significantly decreased.When co-cultured with MSC,human monocytes,M? and DC expressed significantly reduced levels of IL-12.MSC produced significantly higher levels of CD 137,CD40 and CD40L when co-cultured with monocytes,M? and DC,respectively.Conclusions:Disturbance of lymphocyte balances and lymphocyte infiltrations in the salivary glands could be observed in pSS patients.IL-12 correlated with clinical manifestations and pheripheral lymphocyte subsets.MSC alleviate SS symptoms probably by reducing IL-12.All these findings suggest that MSC might achieve its therapeutic effects on pSS by inhibiting IL-12 production and correcting lymphocyte balances.