Expression Level Of TAM Receptors Signaling Pathway In Primary Biliary Cirrhosis And Its Regulatory Mechanism In Macrophage Polarization

Primary biliary cirrhosis(PBC) is an immune-mediated chronic liver disease characterized by intrahepatic bile-duct destruction, cholestasis, and fibrosis. It can lead to cirrhosis, liver cancer and eventually liver failure. Clinically, 95% of PBC patients have the presence of highly specific, mainly AMA-M2. This disease primarily affects women over 40 years of age(female preponderance(9–10: 1). The epidemiological studies reported an incidence of 0.9 to 5.8 per 100,000 people, with 92% of patients women. The prevalence of PBC ranges from 1.91 to 40.20 per 100 000 people. The increased prevalence is probably attributable to a combination of increased disease recognition, better data capture by electronic medical records, and increased survival after the introduction of UDCA. The population-based epidemiological study across China revealed an incidence of 49.2 per 100 000 people. Among women with more than 40 years, the incidence is about 155.8 per 100 000 people. Thus, PBC is not orphan disease in China or worldwide. The PBC researches could help us to better understand the pathogenesis of PBC, and develop more effective therapy methods and prevent strategyThe prevalence of primary biliary cirrhosis is higher in families with an affected member, and the odds ratio in a first-degree relative of a patient is 11. Several genetic factors including MHC, SNP and epigenetic modification have been reported to be associated with PBC development. Recent GWAS screened several risk genes such as STAT4, TNFSF15. In addition to genetic factors, several large-scale epidemiological studies have shown that E. coli, certain mycobacterial infections, exogenous chemicals such as nail polish, hair dye, and smoking history may contribute to the development of PBC. Therefore, similar to other autoimmune disease, the combination of susceptible genetic background and environmental trigger factors contribute to the initiation and promotion of PBC.Risk alleles in primary biliary cirrhosis tend to occur in genes associated with immune function, and cross many different immune pathways such as myeloid cell differentiation, antigen presentation, T-cell differentiation, B-cell antibody production, and NK cell pro-inflammatory cytokines secreting. Biliary epithelial cells(BEC) play a key role in the pathogenesis of PBC. Biliary epithelial cells are targeted in primary biliary cirrhosis, and express T-cell ligands that are thought to be essential for the induction of biliary epithelial autolysis. Hi BEC apotope is also reported to be associated with spefic destruction of human small intrahepatic. In addition, biliary epithelial cells might also act as antigen-presenting cells, amplifying the immune response.From the firstly described case of primary biliary cirrhosis, PBC researchers have made lots of achievements in PBC field. Several research achievements have been applied into clinical practice, for example, AMA for diagnosis, UDCA for treatment. However, there is no more effective diagnostic and therapeutic method except for AMA and UDCA. Based on the pathophysiological features, PBC pathogenesis is a dynamic process. In early stage, PBC is characterized the inflammatory destruction of intrahepatic biliary epithelial cells; In late stage, it developed into fibrosis and cirrhosis, similar to other type of liver cirrhosis. Therefore, we believed that the mechanism and pathogenesis seems to be different, which should be depended on its stage. We should explore its pathogenesis and specific potential therapeutic targets from developmental viewpoint, thus we could find an effective and targeted therapeutic tool.Tyro3/Axl/Mer(TAM) family receptors are tyrosine-kinase receptors that include Tyro3, Axl and Mer and Gas6, Protein S. It has been proposed that the TAM signaling pathway plays an important role in the engulfment of apoptotic cells. Defects in the clearance of apoptotic intrahepatic biliary epithelial cells, some intracellular antigen such as PDC-E2 will be released into immune system and cause autoimmunity inflammatory response. This mechanism also explained why human intrahepatic biliary epithelial cells could be specific damaged. From this point, it is unclear whether TAM signaling might play a role in PBC development. In addition, TAM signaling pathway also plays a role in the regulating of innate immunity. Previous studies reported TAM signaling activation could prohibit the secreting of pro-inflammatory cytokines and inflammatory immune response. Mer Knockdown mice exhibit more susceptible to endotoxin shock and the significant increase in TNF-alpha production appeared in macrophages from Mer Knockdown mice after LPS stimulation. This macrophages lacking Mer show high reactivity,and could produce more amount of pro-inflammatory cytokines. As a typical autoimmune disease, PBC is characterized by high inflammatory immune response. From this point, it seems also not clear whether TAM signaling might play a role in PBC development. Due to the key function of TAM signaling pathway in the engulfment of apoptotic cells and regulation of innate immunity, we speculate that TAM signaling pathway could participate in the pathogenesis of PBC.Under healthy condition, the apoptotic cells and cellular debris could be eliminated readily from tissues. During this process, dying cells are rapidly cleared at early stages of apoptosis underlying the mechanisms which involve several types of membrane receptors and serum bridging molecules, including TAM signaling pathway. In this case, the effective clearance of apoptotic cells could not activate macrophage and inflammatory immune response, which is essential to maintenance of immune tolerance. However, when apoptotic cells could not be cleared effectively, the cells would proceed to “second necrosis” and the secondary necrotic material including auto-antigens would be released to circulation. In this case, macrophage could be activated and produce several different types of pro-inflammatory cytokines, then inflammatory immune response would develop. As an essential component of innate immunity, macrophages are capable of differentiating into protean varieties with a range of function. In respond to various environmental cues(e.g., microbial products, damaged cells, activated lymphocytes) or under different pathophysiologic conditions, macrophages can acquire distinct functional phenotypes via undergoing different phenotypic polarization. Phenotypically polarized macrophages are now generally classified into two major subtypes termed proinflammatory M1 and anti-inflammatory M2. Macrophages exposed to microbial products and interferon(IFN)-g become classically activated macrophages(M1), which produce copious amounts of pro-inflammatory cytokines and chemokine and function predominantly in inflammation, tissue damage, killing of intracellular microbes, and increased tumoricidal activity. Alternative activation of macrophages is canonically defined as that induced by certain stimuli, such as interleukin(IL)-4, IL-10, IL-13. Alternatively activated macro- phages(M2) are characterized by the production of antiinflammatory cytokines and expression of scavenger receptor and are principally associated with the clearance of cell debris, tissue repairing and remodeling. It is also not clear about the role of macrophage polarization.Based on the possible effect of TAM receptor signaling, macrophage polarization on PBC development, we conducted the present study to elucidate this association. We mainly focused on four questions as following: First, what is the expression level of TAM signaling in autoimmune diseases including PBC. Second, what is the concentration of soluble TAM signaling molecules? Third, what is the association of TAM signaling pathway with macrophage polarization in PBC patients. Forth, how is the regulation mechanism of Mer in macrophage polarization.PART 1. m RNA expression levels of TAM signaling in autoimmune disease and its clinical significanceIn this part, patients primary biliary cirrhosis, rheumatoid arthritis, systematic lupus erythematous, primary Sj?gren’s syndrome and normal controls were recruited. We used RT-PCR to measure the expression levels of TAM signaling in PBMCs from those patients, and further analyzed the association of their expression level with clinical characteristics. Results showed that TAM signaling molecules were differentially expressed in several autoimmune diseases, whatever receptors or ligands. In RA patients, the expression levels of Gas6, Tyro-3, Axl were significantly lower than controls. In SLE patients, the expression levels of Protein S and Axl were lower and correlated with disease severity, inflammatory response. The decreased levels of Tyro-3 and Axl were found in both p SS and PBC patients. These results suggested that the low expression level of TAM signaling could be associated with the development of autoimmune disease.PART 2. The levels of soluble TAM signaling in PBC patients and its clinical significanceIn this part, we measured the concentration of soluble TAM signaling in sera from PBC patients. We found Pro S concentration was significantly decreased and s Mer, s Tyro-3 concentration were significantly increased in PBC. The correlation analysis showed that there is a positive correlation between s Mer concentration and AKP, GGT, TBA. Similarly, we also observed a positive correlation between s Tyro-3 and AKP, GGT, TBA. These data suggested higher s Mer, s Tyro-3 in PBC patients with poorer liver function. To distinguish PBC patients from normal controls, ROC/AUC analysis showed a sensitivity of 0.70(specificity of 0.78; AUC=0.77, 95%CI 0.69-0.85) at a cutoff value of 6.3 ug/ml s Tyro-3. ROC/AUC analysis showed a sensitivity of 0.63(specificity of 0.72; AUC=0.66, 95%CI 0.56-0.76) at a cutoff value of 17.7 ug/ml s Mer. ROC/AUC analysis showed a sensitivity of 0. 87(specificity of 0. 47; AUC=0. 68, 95%CI 0.59-0.78) at a cutoff value of 103.1 ug/ml s Tyro-3. ROC/AUC analysis revealed a better diagnostic value of s Tyro-3 in PBC patients.PART 3. The levels of membrance TAM receptors in peripheral monocytes/macrophage and their correlation with macrophage polarization in PBC patients.In this part, we used flow cytometry to detect the ration of M1 and M2 macrophage, as well as membrance TAM receptors in peripheral monocytes/macrophage. The results showed the amount of M1 macrophage(CD68+/CCR2+) was significantly higher in early stage, while that of M2 macrophage(CD163+/CX3CR1+) was significantly higher in late stage. As we known, M1/M2 macrophage could secret specific cytokines. Further, we measured the concentration of TNF-α、IL-12、TGF-β、IL-10 in PBC patients with early stage and late stage. The level of IL-12 was significantly higher in early stage, while TGF-β was relatively higher in late stage. Take together, results revealed the different macrophge polarization in patients with ealry and late stage, M1 polarization was increased in early stage and M2 polarization was higher in late stage. In addition, we also used flow cytometry to detect the level of membrane TAM receptors, the results showed m Mer was signficantly increased in late stage other than m Tyro-3, m Axl. The correlation analysis revealed that M2/M1 ratio was positively correlated with m Mer level(r=0.57, p<0.01). And correlation analysis for clinical parameter also showed that M2/M1 ratio was postiviely with DBIL, AKP(r=081, p<0.01; r=0.71, p<0.01).PART 4. The potential mechnism for regulaiton of Mer in macrophage polarizationIn this part, we mainly explore the pontential mechnism for regulaiton of Mer in macrophage polarization. Using a co-culture of apoptotic intrahepatic epithelial cells and M0 macriphages, M2 macrophage ratio(CD163+/CX3CR1+) would be decreased after si RNA-Mer stimulaiton. After incubating with si RNA-Mer, M2 macrophage specfic genes were also decreased such as CCL18, RANTES, as well as specific cytokines TGF-β, IL-10. Further, we analyzed the effect of si RNA-Mer on signaling pathway concerning macrophage polarization. After transfected different concentration of si RNA-Mer, the level of p-AKT was decreased other than total AKT in a dose-dependent manner. Using the co-culture of apoptotic intrahepatic epithelial cells and M0 macrophages, the concentrations of IL-10, IL-13 in supernatant were decreased after AKT activation(SC79) as well as m RNA. Taken together, the potential mechnism for the regulaiton of Mer in macrophage polarization is to decrease the production of IL-10, IL-13 through p-AKT signaling pathway, further contribute to a decrease in amount of M2 macrophage.