Study On The Regulatory Effects Of CD38 In Inflammatory Reaction Of Rheumatoid Arthritis And Its' Mechanisms

Objective:Rheumatoid arthritis is a chronic autoimmune disease with high rate of teratogenesis and high rate of disability.The main features of rheumatoid arthritis were synovial inflammation of joint,cartilage degeneration and destrcution of bone.Despite significant break throughs have been made in treatment for rheumatoid arthritis,there are no curative therapy avaliable for rheumatoid arthritis at the moment.Traditional treatments,including TNF-?antagonists or sterocorticoids,have 50%of irresponsivness and can potentially induce immuno-supression,which increases the risk of developing cancer.Hence,securing a novel therapeutic target for rheumatoid arthritis is an important research question for autoimmune diseases.Dentritic cells are the most powerful antigen-presenting cells in our immune system,they play important roles not only in the initiation of immune response,but also in inducing immune tolerance,modulating regulatory-T cell activity.CD38 gene expression has been reported increased in synovium of rheumatoid arthritis patients and the chemotaxis ability of neutrophils and dendritic cells is decreased in CD38^-/-mice.But,there were no reports on the effects of CD38 on regulating of RA and its mechanisms.In a previous research study we observed the compromised development of spleenic B-cells and the decreased expression of RelB gene expression,and the collagen induced inflammatory response of joints were alleviated when treated with RelB gene silenced DCs.Wild-type,CD38^-/-mice and their collagen alternative-induced arthritis?CAIA?mice models were set up,and our study aim at:1)the effcts of CD38deficiency on the expression of RelB and on the development and function of bone marrow-derived dendritic cells,2)the effcts of CD38 deficiency on the pathologic change of joints in CAIA mice,3)the effcts of CD38 deficiency on the production of proinflammatory cytokines in dendritic cells,and the underlying molecular mechanisms.The results of this study will illuminate the effects of CD38 denificency on alleviating inflammatory response of rheumatoid arthritis and the regulatory mechanisms of NF-?B signaling pathway.Our study will also be useful in providing theoretical basis for the treatment strategies in rheumatoid arthritis with CD38antagonist or its antibodies.Methods:1.Extract DNA from tails of mice,and detect the expression levels of CD38and Neo genes by polymerase chain reaction?PCR?.2.Bone marrow-derived DCs were isolated from 8 weeks old male WT?C57BL/6?mice and were cultured in medium supplemented with IL-4 and GM-CSF.7days later,DCs were stained with cell surface antibodies?CD11C PE PerCP Cy5?and the purity of cultured DCs were identified by Fluorescence activated cell sorter?FACS?technique.3.Bone marrow-derived DCs were isolated and cultured for 7 days and stained with surface antibodies?MHC-II FITC,CD40 PE,CD11C PE PerCP Cy5 and CD80APC?.The maturation of mouse bone marrow-derived DCs was detected by FACS.4.Take the draining lymph nodes of Balb/c mice to make a single cell suspension.T cells were isolated and purified by Ficoll.5.Mixed lymphocyte cultures of mouse bone marrow-derived DCs and T-cells derived from the draining lymph nodes of the heterologous mouse were performed on the 7^th days.72 hours later,the differences in T cell proliferation were compared by CCK8 assay,which means the antigen presentation function of DCs.6.CAIA mouse models were set up with 8 weeks old WT and CD38^-/-male mice with chicken collagen II?CII?.7.The knee joints of WT and CD38^-/-mice were used to observe the pathological changes of their joints by hematoxylin-eosin staining?HE staining?.8.Real-time PCR was used to detect the mRNA expression of RelB gene in bone marrow-derived DCs of WT and CD38^-/-mice,Th1 pro-inflammatory cytokines TNF-?,IL-1?and Th2 anti-inflammatory cytokines IL-4 and IL-10.The mRNA levels of TNF-?,IL-1?,IL-4 and IL-10 in bone marrow derived DCs of WT and CD38^-/-CAIA mice were detected too.9.The protein expression of p65,P-p65,p-105,P-p105 and Sirt1 in bone marrow derived DCs of WT and CD38^-/-CAIA mice was detected by Western blotting.Results:1.The CD38 gene was positive in the tail DNA of the WT mice,and the Neo gene was negative.In the CD38^-/-mice,the CD38 gene were negative and the Neo gene was positive.2.7 days after pure culture,CD11C⁺cells in bone marrow-derived DCs is about97%with verified by FACS,and this meets the requirements of experimental purity.3.Compared with WT mice,the mRNA expression level of RelB gene in bone marrow-derived DCs of CD38^-/-mice was decreased significantly?P<0.01?.4.Compared with WT mice,the maturation of bone marrow-derived DCs in CD38^-/-mice showed a significantly decrease in MHC II expression?from 50.8%to34.0%??P<0.01?.5.Compared with WT mice,the antigen-presenting ability of bone marrow-derived DCs in splenic T cells of heterologous mice was significantly decreased?P<0.01?.6.Compared with WT mice,10 days after the set up of CAIA mice modeling,the mRNA expression levels of Th1 proinflammatory cytokine IL-1?is increased significantly?P<0.01?,but the expression level of TNF-?mRNA shows no change.The mRNA expression levels of anti-inflammatory cytokines IL-4 and IL-10 were decreased significantly?P<0.01?.7.HE staining results of mice knee joints showed that,when induced by collagen,the articular cartilage of WT mice became thinner and the gap of cavum articular were enlarged.While the joint damage of CD38^-/-mice was inhibited,it shows almost no difference with normal mice.8.Compared with WT CAIA mice,the mRNA expression of Th1 type pro-inflammatory cytokine IL-1?in CD38^-/-CAIA mice was significantly decreased?P<0.01?,and the mRNA expression level of TNF-?mRNA shows no change.The mRNA expression levels of anti-inflammatory cytokines IL-4 and IL-10 were decreased significantly?P<0.01?.9.Compared with WT CAIA mice,the expression of Sirt1 in bone marrow-derived DCs of CD38^-/-CAIA mice was significantly increased?P<0.01?,the expression of P-p105 was significantly decreased?P<0.01?,while p50,p65 and P-p65show no significantly change.Conclusion:1.The CD38^-/-mice were identified,the purity of cultured bone marrow-derived DCs match the experimental requirements,which ensured the reliability of the experimental results.2.CD38 deficiency can inhibit the expression of RelB gene in bone marrow-derived DCs of mouse.3.CD38 deficiency can inhibit the maturation and antigen presentation of bone marrow derived DCs in mice.4.CD38 deficiency can inhibit collagen-induced arthritis in mice;5.CD38 deficiency can promote the Th1-Th2 transition of inflammatory cytokines in bone marrow-derived DCs of CAIA mice;6.CD38 deficiency upregulates Sirt1 expression and decreases phosphorylation of NF-?B1?p105?;In summary,CD38 deficiency can inhibit collagen-induced arthritis in mice by inhibiting the phosphorylation of NF-?B1?p105?,and Sirt 1 may be its'target.