Study On The Requirement Of TLR4 For Effects Of Mitochondrial M2 In Pathogenesis Of Primary Biliary Cirrhosis

Primary biliary cirrhosis(PBC) is a slowly progressive cholestatic autoimmune disease of the liver,characterized by destruction of intrahepatic bile ducts.So far,the etiology and pathogenesis of PBC have remained to be understood.Seroiogically, PBC is characterized by the presence of serum anti-mitochonchial antibody(AMA), especially anti-M2 antibody,positive rate of which is over 95%.M2 contains various autoantigens,of which the major autoantigen is dihydrolipoamide acetyltransferase, the E2 subunit of pyruvate dehydrogenase complex(PDC-E2).More than 95%of patients with PBC have a high titer of sera AMA directed against PDC-E2.Other autoantigens include E2 subunit of the 2-oxoglutarate dehydrogenase complex (OGDC-E2) and E2 subunit of the branched chain 2-oxo-acid dehydrogenase complex(BCOADC-E2),etc.According to the special association between PDC-E2 and PBC,PDC-E2 has been further studied.It has been reported that PDC-E2 could be located on the surface of intrahepatic biliary epithelial cells(BECs) in patients with PBC.However,what is the effect of PDC-E2 on BECs remains to be clarified.Toll-like receptors(TLRs) are pattern recognition receptor(PRR) for components of various pathogenic microorganism,which could bridge innate immunity and adaptive immunity.TLR4 is the first one of TLRs discovered and identified by Medzhitov,et al in 1997,which can recognize its natural ligand,namely component of cell wall from gram-negative bacteria---lipopolysaccharide(LPS).There are so many endogenous ingredients as heat shock protein(HSP)60,HSP22,hyaluronic acid,HMGB,neutrophil elastase,myeloid related protein(Mrp) 8 and 14,etc,which can activate TLR4 signaling pathway,therefore,can be involved in the pathogenesis of various autoimmune diseases.Recently,it has been demonstrated that:the expression level of TLR4 was significantly up-regulated on IBECs from PBC patients;In vitro,peripheral blood monocytes from PBC patients showed hypersensitivity to LPS;LPS could promote BECs to secret inflammatory factors or chemokines IL-6,MCP-1 and IL-8 via NF-κB and MAPK dependent pathway. We can draw two points from demonstrations above.One is that TLR4 can be expressed on BECs,the other is that innate immunity involving TLR4 can play a certain role in the pathogenesis of PBC.However,pathogen or LPS was not found in sera from a large number of PBC patients including the ones recruited by current study.What is the cause of higher expression of TLR4 in PBC patients? Are there other endogenous ingredients for TLR4,which can up-regulate the expression of TLR4 and activate TLR4 signaling pathway in PBC patients? For these questions, there has been no answer.On the basis of status described above,we propose such a hypothesis that mitochondrial M2 could promote adhesion molecules(e.g.ICAM-Ⅰ) expression, chemokines(e.g.MCP-1,IL-8,MIP-1α,etc.) secretion of human intrahepatic biliary epithelial cell(HIBEC),which can recruit various inflammatory cells and immune cells including monocytes/macrophages etc.M2 could also activate monocytes/macrophages via TLR4 signaling pathway and promote them to secret inflammatory factors and cytokines(e.g.TNF-α,IL-12,sTRAIL,etc.),which could cause the damage of HIBEC,involved in the pathogenesis of PBC.To confirm this hypothesis,we performed some studies as follows.Firstly,the expression level of TLR4 in peripheral blood leukocytes was determined from PBC patients,clinically to explore the association of TLR4 with PBC.Secondly,through in vitro culture cell, the effects of M2 on HIBEC via TLR4 signaling pathway were analyzed.Lastly,the effects of M2 on monocytes and the interaction between M2 and TLR4 were explored.Part 1 Study on expression of TLR4 in peripheral blood leukoeytes from PBC patients and its significance.In this part,to explore the association of the expression change of TLR4 in peripheral blood leukocytes from PBC patients,the expression level of TLR4 in peripheral blood from PBC patients was determined,with the patients with liver cirrhosis induced by hepatitis B and healthy population as controls.Using real-time fluorescent quantitative RT-PCR and flow cytometry(FCM),the levels of TLR4 mRNA and protein in peripheral blood leukocytes were respectively detected from 30 PBC patients,20 disease controls and 30 healthy individuals.The levels of serum cytokines were measured by ELISA.GGT and ALP were automatically analyzed by automatic biochemical analyzer.The results indicated that the positive rate of TLR4 protein on peripheral blood monocytes and the expression level of TLR4 mRNA in peripheral blood mononuclear cell(PBMC) are significantly higher in patients with PBC or liver cirrhosis induced by hepatitis B than in healthy controls(P＜0.05).The positive rate of TLR4 on monocytes closely correlated with the expression level of TLR4 mRNA in PBMCs in all the population.The serum levels of TNF-αand IL-12 were significantly higher in patients with PBC or liver cirrhosis induced by hepatitis B than in healthy controls,while the serum levels of ALP and GGT were significantly higher in PBC patients than in patients with liver cirrhosis induced by hepatitis B and healthy controls.In PBC patients,the expression levels of TLR4 mRNA and protein clearly correlated with the serum levels of TNF-α,IL-12 and sTRAIL,but did not with ALP and GGT.These results suggested that the abnormality of TLR4 signaling pathway might be closely associated with pathogenesis of PBC.Part 2 Study on effects of mitoehondrial M2 in human intrahepatie biliary epithelial cell via TLR4 signaling pathwayTo explore the effects of mitochondrial M2 in HIBEC as well as their association with TLR4,HIBECs were cultured in vitro in present study.Firstly,the influence of various concentrations of mitochondrial M2 on the expression of TLR4 on HIBEC was analyzed by FCM.Furthermore,using FCM,ELISA,Western blot and EMSA,various effects of M2 on HIBEC including HIBEC apoptosis;adhesion molecule(e.g.ICAM-Ⅰ) expression and chemokines(e.g.MCP-1,IL-8 and MIP-1α) secretion of HIBEC,IκB and pJNK contents and activity of NF-κB in HIBEC,were analyzed before or after silencing TLR4 signaling pathway,with Chang' hepatoeytes as control.The results were demonstrated as follows.1) TLR4 could be expressed on both HIBEC and Chang' hepatocyte,and the positive rate of TLR4 on HIBEC was significantly higher than that on Chang' hepatocyte((70.3±2.3)%virsus(9.6±0.4)%). The positive rates of TLR4 on HIBCEs and hepatocytes were significantly increased just at 24h after stimulation of 2μg/ml mitochondrial M2(P＜0.05),and showed a trend in M2 dose-dependent fashion.2) The early apoptotic rate of HIBECs was significantly enhanced in the presence of various concentrations of M2 after 24h in M2 dose-independent fashion.The early apoptotic rate of HIBECs at 24h after stimulation of 2μg/ml M2 did not show significant difference between the presence and absence of siRNA for TLR4 gene.However,after silencing TLR4 by siRNA,the early apoptotic rate was significantly increased in the presence of 10μg/ml and 50μg/ml M2 for 24h,especially in the presence of 50μg/ml M2.Different from HIBEC,the early apoptotic rate of Chang's hepatocytes was not significantly increased in the presence of various concentrations of M2 but slightly decreased. Interestingly,after silencing TLR4 by siRNA,the early apoptotic rate of Chang's hepatocytes was significantly increased in the presence of various concentrations of M2,especially in the presence of 10μg/ml and 50μg/ml M2.3) After 24h,the expression level of ICAM-Ⅰon HIBECs was significantly elevated in the presence of 2μg/ml M2.When the concentration of M2 reached 10μg/ml and 50μg/ml,the expression level of ICAM-Ⅰon HIBECs showed a decreased trend,but still remained a higher level than that in absence of M2.The expression level of ICAM-Ⅰon Chang's hepatocytes was significantly higher than that on HIBECs in absence of M2.After 24h,the expression level of ICAM-Ⅰon Chang's hepatocytes did not basically varied in presence or absence of 2μg/ml and 10μg/ml M2.Only if the concentration of M2 reached 50μg/ml,the expression level of ICAM-Ⅰon Chang's hepatocytes started to go up.Interestingly,after silencing TLR4 by siRNA, the expression level of ICAM-Ⅰon Chang's hepatocytes was significantly increased in presence of M2,compared with that in absence of M2.4) In presence or absence of M2,both HIBEC and Chang's hepatocytes secreted IL-8,but not MIP-1α,and Chang's hepatocytes also did not secret MCP-1.After 24h,the concentration of IL-8 in HIBEC culture supernatant was basically not influenced in presence of 2μg/ml M2.When the concentration of M2 reached 10μg/ml,IL-8 was significantly up-regulated in dose-dependent fashion.After silencing TLR4 by siRNA,IL-8 was significantly decreased again.After 24h,the concentration of IL-8 in Chang's hepatocytes culture supernatant did not varied clearly in presence of various concentrations of M2.Furthermore,the high concentration of M2(50μg/ml) showed an inhibitory effect on IL-8 secretion of Chang's hepatocytes.After 24h,the concentration of MCP-1 was significantly increased in presence of M2 in M2 dose-dependent fashion,and after silencing TLR4 by siRNA,MCP-1 was significantly decreased again.5) The results in Western blot assay indicated that after stimulation of 50μg/ml M2,pJNK was not detected in HIBECs and Chang's hepatocytes,and content of IκB-αwas significantly diminished in HIBEC,but significantly increased again after silencing TLR4 by siRNA.After stimulation of 50μg/ml M2,content of IκB-αdid not clearly varied in presence or absence of TLR4 gene siRNA.6) In this part,we also blocked NF-κB by PDTC,and repeated above protocols.Similar to silencing TLR4 by siRNA,the effects of M2 on HIBECs was inhibited at least partly by blockage of NF-KB by PDTC.The results in this part demonstrated that the expression of TLR4 on HIBECs could be up-regulated by M2,and adhesion molecule expression,chemokine secretion of HIBEC could be enhanced via activating NF-KB-dependent TLR4 signaling pathway.Therefore,various immune cells including monocyte/macrophage, etc could be recruited around HIBEC,to cause the damage of HIBEC,which is involved pathogenesis of PBC.However,the pro-apoptotic effect of M2 itself on HIBEC is inhibited through activating NF-KB-dependent TLR4 signaling pathway, and the detailed mechanism still remained to be further studied.Part 3 Study on effects of mitochondrial M2 in monocytes via TLR4 signaling pathwayIt has been reported in previous studies that there are a great number of inflammatory cells including lymphocytes,monocyte/macrophages,etc infiltrating the portal tracts in the liver from patients with PBC.There have been accumulating evidences indicating that some epitopes of PDC-E2(e.g.PDC-E2_159-167 and PDC-E2_163-176) could be recognized by auto-reactive T cells,and PDC-E2 could also be recognized by antibody IgA secreted by B cells.However,the role of monocyte/macrophage in pathogenesis and the association between monocyte/macrophage and mitochondrial M2 still remained to be clarified. Therefore,in this part,we used U937 cell line as monocytic model to explore the effects of M2 on monocytes and the role of TLR4 signaling pathway.Firstly, regulation of TLR4 on U937 by mitochondrial M2 was analyzed by FCM.And then, by ELISA,Western blot and EMSA assay,the effects of M2 on TNF-α,IL-12 and sTRAIL secretion of monocytes,the contents of IκB and pJNK and activity of NF-κB in monocytes were analyzed in presence or absence of TLR4 gene siRNA.The results indicated that 1) the positive rate of TLR4 on U937 was significantly increased after 24h in presence of 2μg/ml M2 in M2 dose-independent fashion.2) in western blot assay,IκB was significantly decreased and even disappeared after 1h in presence of 50μg/ml M2.After 2h,IκB was gradually increased,but still remained lower than control.After 4h,IκB was restored to the level of control.After blockage of TLR4 by HTA125,IκB was significantly increased in presence of 50μg/ml M2 at 1h and 2h.However,at 4h,IκB did not vary before or after blockage of TLR4.In presence of 50μg/ml M2,pJNK was not detected in U937 at various time point(1h, 2h and 4h),and activity of NF-κB was significantly increased after 1h,and gradually decreased after 2h,and significantly decreased after 4h.After blockage of TLR4 by HTA125,the activity of NF-κB was significantly decreased in presence of 50μg/ml M2 at 1h,2h and 4h respectively,compared with that in absence of blockage of TLR4.Especially at 4h,nearly no activity of NF-κB was detected.3) after 24h,the TNF-αand sTRAIL but not IL-12 in U937 culture supernatant was significantly increased in presence of 50μg/ml M2,and after 48h,three kinds of cytokines were all significantly increased in time-dependent fashion.After blockage of TLR4 by HTA125,the three kinds of cytokines was significantly decreased at various time points,compared with that in absence of blockage of TLR4.It has been concluded through the results in this part that the expression of TLR4 on monocytes could be up-regulated by mitochondrial M2,and cytokine(including TNF-α,IL-12 and sTRAIL) secretion of moncytes could be enhanced via activating NF-κB-dependent TLR4 signaling pathway,which can cause the damage of HIBEC directly or through regulating other immune cells,therefore,involved in pathogenesis of PBC.