Study Of SOCS In The Pathogenesis Of Primary Biliary Cirrhosis

Primary biliary cirrhosis(PBC) is a progressive autoimmune liver disease characterized by destruction of the small intrahepatic bile ducts,portal inflammation,and the presence of antimitochondrial antibodies(AMA),eventually leading to cirrhosis and hepatic failure.The pathogenesis and aetiology of PBC remains obscure.Genetic, environmental risk factors,pollution,host susceptibility may be possible PBC triggers that initiate the immunopathological cascade.Some observations suggest that the generation of immune responsiveness to self-antigen can result in pathogenic autoimmune damage of the intrahepatic biliary epithelial cells mediated by both humoral and cellular immune responses.The pathogenetic mechanism is believed to be caused by the breakdown of immunologic tolerance,resulting in cholestasis and the development of PBC.But the mechanism of initiation of immune responsiveness and the self-tolerance breakdown is unclear.During the last ten years,the research of cellular immunity of PBC has been mainly focused on different T subsets,such as Th1,Th17,and cytotoxicity T lymphocyte,etc. Dendritic cells(DCs),the most potent antigen-presenting cell(APC),linking innate and adaptive immune responses,and thus play a crucial role in the initiation and modulation of an appropriate response of our immune system to danger signals,induction of immune tolerance,and stimulate the proliferation of T cells,and in a much stronger way than other APC such as B cells and macrophages.But there is a paucity of information regarding the function of DC in PBC.This has mainly been due to the limitation of amount of DCs of PBC patients and technical limitation of isolating the trace population of DCs from peripheral blood.Janus kinase(JAK)-signal transducer and activator of transcription(STAT) pathways is a cross-talk among many kinds of cytokines,and involved in the procession of different kinds of chronic inflammatory diseases including the autoimmune diseases.Suppressor of cytokine signaling(SOCS) proteins are inhibitors of cytokine signaling pathways.Most SOCS proteins are induced by cytokines and therefore act in a classical negative-feedback of the cytokine-induced JAK-STAT signaling cascade and the toll like receptor(TLR) pathway.Studies have shown that SOCS proteins are key physiological regulators of both innate and adaptive immunity to avoid overshooting activation.These molecules positively and negatively regulate macrophage and dendritic cell activation and are essential for T-cell development and differentiation.Evidence is also emerging of the involvement of SOCS proteins in diseases of the immune system.SOCS are the necessary negative regulation proteins of Th1 and Th2-cell differentiation and maintenance of immune tolerance by DC.Since SOCS proteins are involved in a wide range of many immunological processes, the use of SOCS proteins to suppress cytokine signaling could be a useful therapy for the treatment of inflammatory diseases and autoimmune diseases of hyper-cytokine signaling involved.SOCS protein mimetic combined with drug-delivery systems have been proved useful for the treatment of inflammatory diseases by animal experiment.Up to now,there is not any research about the role of JAK-STAT-SOCS signaling in the pathogenesis of PBC.Is there any relationship between altered levels of cytokines and the negative regulation of SOCS in PBC? Is the breakdown of immune tolerance of PBC associated with the changes in regulatory function of dendritic cells?On the basis of status described above,in order to explore these hypothesis,four sections were included in our experiments:1) to determine the serum levels of pro-inflammatory and anti-inflammatory cytokines in PBC patients;2) to detect the Toll like receptors gene expression levels,SOCS protein and gene expression levels in PBMCs; 3) to measure the SOCS protein expression levels in PBMCs-derived DC from PBC patients,meanwhile,to observe the changes of function of DC;4) to explore the role of SOCS in DC by using Western blot,flow cytometry(FCM) and SiRNA methods through the effects of mitochondrial M2 protein in vitro detritic cell culture.Part 1 Detection of the serum levels of pro-inflammatory and anti-inflammatory cytokines in PBC patientsThe onset and pathological consequences of autoimmune diseases are often associated with disturbances in the functional balance between immunoregulatory and pro-inflammatory cytokines.Cytokines can exert distinct signals on a wide variety of T cells and influence the inflammatory status.It is thought that the manipulation of cytokine pathways may represent a valid mean to re-establish a balance between immunity and tolerance.78 patients with PBC,30 patients with chronic hepatitis B(CHB) and 60 healthy controls(HC) were enrolled in this study.Serum levels of pro-inflammatory cytokines [IL-6,IL-17,(Interferon-gamma,IFN-γ)]and anti-inflammatory cytokines(IL-10,TGF-β) were determined enzyme-linked immunosorbent assay(ELISA).Hs-CRP,as an important biomarker of chronic inflammation,was assayed by routine clinical chemical methods.The results showed that the serum levels of IL-6,IL-17A,IFN-γand IL-10 were significantly elevated in PBC patients compared to healthy controls,there were no significant changes of TGF-βlevel between the two groups.Compared to CHB group,IL-6 and TGF-βlevels were found to be decreased in PBC patients,IL-17A is increased,no significant changes of IL-10 and IFN-γ.The serum hs-CRP level was elevated significantly in both groups compared to healthy controls(P<0.05).In contrast,hs-CRP in CHB patients was raised significantly higher than that in PBC patients.These results suggest that there was chronic inflammatory in the progression of PBC.Part 2 Measurement of expression of TLR and SOCS in PBMCs from PBC patientsTLRs signaling not only can induce high-level inflammatory cytokines release and autoimmunity,but might also regulate tolerance.The SOCS1 and SOCS3 mediated modulation in signaling from cytokine receptors therefore has profound effects on the regulation of immunity and inflammation to avoid overshooting of TLR stimulation.In this part,to explore the expression of TLR and SOCS in PBMCs from PBC patients,using real-time fluorescent quantitative TR-PCR,the levels of TLR4,TLR9, SOCS1 and SOCS3 mRNA in PBMCs were respectively detected from 32 PBC patients, 30 patients with CHB as disease controls and 30 healthy controls.The levels of SOCS1 and SOCS3 proteins in PBMCs were determined by Western blot.It was demonstrated that the levels of TLR4,TLR9,SOCS1 and SOCS3 mRNA were significantly higher in the PBC group than that in the HC(1.48,2.51,3.43 and 1.48 folds, P<0.05) respectively.The expression of TLR9 and SOCS1 mRNA levels in PBC group were 6.85 and 2.99 folds that of CHB group.The proteins levels of SOCS1 and SOCS3 in PBMCs from PBC group were increased significantly than that in HC group.A real-time quantitative RT-PCR method for detecting the expression of TLR and SOCS gene in PBMCs has been successfully established;TLR have the important role of the pathogenesis of PBC,SOCS as an important negative regulator of TLR signaling maybe can't inhibit the excessive activation of TLR and cytokines signaling completely.Part 3 Study on expression of SOCS in PBMCs-derived Dendritic cells from PBC patients and its significanceOur previous studies have shown that the inflammatory status consists in the progression of PBC.The levels of SOCS mRNA and protein in PBMCs increased significantly,which cant inhibit the excessive activation of TLR and cytokines signaling completely.Since SOCS is highly expressed and deeply involved in the development, maturation and activation of DC? Is there any relationship between SOCS and DC function? Therefore,in this part,by means of FCM,ELISA and Western blot methods,the levels of SOCS protein and the function of DC were respectively observed from 10 PBC patients and 8 healthy individuals.1.Phenotypic analysis of DCs by FCMPhenotypic analysis of cultured PBMCs-derived DC was performed by FCM,the results showed that the expression of CD83,CD86 and HLA-DR in PBC patients were (79.4±4.8)%,(86.5±6.3)%and(90±3.5)%,higher significantly than the control group[(68.3±4.1)%,(74.2±6.3)%和(83.6±7.6)%],respectively.2.Levels of cytokine excreted of DC by ELISAThe levels of IL-12 and IFN-γin culture supernatant fluid of DCs in PBC patients were(53.5±11.1) pg/ml and(32.0±9.0) pg/ml,significantly higher than those in HC group[(32.1±10.7) pg/ml and(15.4±8.1) pg/ml)],respectively.There were not any significant difference of IL-10 level between the two group(7.0±4.6) pg/ml and(5.8±4.2) pg/ml.3.Expression of SOCS1 and SOCS3 proteins of DC by Western blotThe proteins levels of SOCS1 and SOCS3 in PBMCs-derived DCs from PBC group were decreased significantly than those in HC group.The results in this part suggest that the PBMCs-derived DCs in PBC patients had greater ability of potent maturation than healthy individual.Maybe increased expression of T cell stimulatory molecules,such as the HLA-DR and CD86 molecules can help increase the antigen presentation function of DC.DCs in PBC patients secreted larger amounts of IL-12 and IFN-γ,which induced optimal Th1 cell responses,and resulted in the excessive immunological reaction and the breakdown of self-tolerance.Part 4 Study on effects of mitochondrial M2 in PBMCs-derived Dendritic cells by SOCS1 signaling pathwaySerologically,PBC is characterized by the presence of serum anti-mitochondrial antibody(AMA).especially anti-M2 antibody,positive rate of which is over 95%.RNA interference(RNAi) is a phenomenon in which the introduction of endogenous or exogenous double-stranded RNA(dsRNA) into cells causes degradation of the complementary mRNA.RNAi is an effective tool used to study gene function due to its high performance,sequence specificity and high stability.In the third part,the level of SOCS protein in the PMBC-derived DCs in PBC patients was decreased than the control group,which was not coincident with the up-regulation expression in PBMCs.So,which pathway was SOCS involved in the pathogenesis of PBC? Whether the mitochondrial M2 protein could involve in the JAK-STAT-SOCS pathway and activate DC,which result in the Th1 polarization and cytokine secretion? By FCM,ELISA and Western blot,various effects of M2 on PBMCs-derived DCs from healthy blood donors were analyzed before or after silencing SOCS1 signaling pathway.1.Transfection and inhibition efficiency of SOCS1 SiRNATransfection efficiency of FAM-siRNA was measured by FCM and fluorescence microscope,which is about 95%.Inhibition efficiency was calculated by the detection of SOCS1 expression using real-time RT-PCR.The results showed that the inhibition efficiency of SOCS1-156 at mRNA level can reach 85%.2.Expression of CD83 and CD86 of DC by FCMThe expression of CD83 and CD86 of DC under stimulation of mitochondrial M2 at 70μg/ml after 24 h,at 35μg/ml after 24 and 48 h were higher than the control.After silencing SOCS1 by siRNA,the levels of CD83 and CD86 in DC in the presence of various concentrations of M2 at different times were all increased significantly than those only in the presence of M2.3.Levels of IL-10 and IL-12 excreted of DCs by ELISAThe levels of IL-10 and IL-12 in culture supernatant fluid of DCs under stimulation of mitochondrial M2 at 70 ug/ml after 24 h,at 35 ug/ml after 24,48 and 72 h were higher than the control.After silencing SOCS1 by siRNA,the levels of IL-12 excreted of DC in the presence of various concentrations of M2 at different times were all increased significantly than those only in the presence of M2,especially in the presence of 35μg/ml M2 after 48 h reached the peak.However,there were not any significant difference of IL-10 levels of DCs between siRNA+M2 group and M2 group at any stimulating concentrations and times.4.Expression of SOCS1 and SOCS3 proteins of DC by Western blotAfter silencing SOCS1 by siRNA on DC in the presence of various concentrations of M2 at different times,the levels of S0CS1 were decreased obviously than those only in the presence of M2.The levels of p-STATl were increased obviously after silencing SOCS1 by siRNA.In the presence of different concentrations of M2 protein at different times, there were not significant changes of levels of SOCS3 and p-STAT3 proteins before and after siRNA.In this part,the results indicated that the PBMCs-derived DC after stimulation of M2 protein at high concentration in vitro had increased capacity to activate Th1 subset proliferation and antigen presentation.After silencing SOCS1 by siRNA,DCs were secreted higher level of pro-inflammatory IL-12 and expressed more co-stimulatory molecules,CD86,which was identical with the changes of functions of DC in PBC patients.Therefore,we supposed that SOCS1-siRNA-treated dentritic cells in vitro had enhanced ability of antigen-presenting and Th1 cell activation,induced DC maturation and activation,hyper-activated STAT1,which contributed to the broken of self-tolerance.We studied the role of SOCS in the pathogenesis of PBC and found that the level of SOCS in the peripheral blood was increased passively by activated cytokine and TLR, which may not inhibit the overshooting of immune responsive.The decreased levels of SOCS in PBMCs-derived DC of PBC may have relationship with the capability of maturation,antigen presentation and polarization of Thl cell,which may result the in the breakdown of self-tolerance and the excessive immunological reaction and onset of PBC.