The Role And The Regulatory Mechanism Of Monocytes/Macrophages In Liver Failure

Liver failure (LF) is a highly complex clinical syndrome characterized by massive hepatocyte death, sudden cessation of normal hepatic function (e.g., synthesis, detoxification, excretion and biotransformation functions) and multiple organ failure (e.g., coagulation disorder, jaundice, hepatic encephalopathy and ascites). The internal clinical management of LF is still a tough problem. The poor prognosis and the high mortality of LF have greatly threatened the lives of the patients and affected the social stability. The pathogenesis of LF is very complex, which has become a hot topic of hepatology. Monocyte/macrophage, the first cellular protective line in innate immune system, plays a key role in the development of LF. The proportion and the functional status of macrophages, as well as the profile of inflammatory cytokines may largely determine the main process of hepatic immune response and the clinical outcome of LF. If the intensive studies of dynamic phenotype and functional status of hepatic macrophages are made, it could prevent from the aggregation of deleterious inflammatory response in the early phase of LF. However, the dynamic functional profile of hepatic macrophages and the factors regulating macrophage early activation are virtually unexplored. The aims of the present study are to determine the dynamic phenotype and functional status of monocytes/macrophages in LF, to investigate the roles of intracellular high-mobility group box protein1(HMGB1) in modulating the pro-inflammatory properties of macrophages, to elucidate the possible signaling pathways involved in the regulation of HMGB1on macrophages and to provide novel ideas for developing rational strategies to predict, prevent and treat LF. Methods:1. Experiments in vivo:LF in mice was established by intravenously injection with Con A. The mice were divided into the exposure group exposed to Con A (20mg/kg) and control group exposed to normal saline for1h,3h,6h,12h and24h, respectively. Serum and hepatic tissues were collected, and the changes in liver injury were assessed by histological examination of Haematoxylin&Eosin stained hepatic tissues and by serum ALT and AST level detection. Peripheral blood mononuclear cells (PBMCs) and hepatic macrophages were isolated from normal or Con A-treated mice. The proportions of peripheral Ly6C+CD11b+monocytes and hepatic F4/80+macrophages were determined by flow cytometry. RT-PCR was performed to detect the levels of TNF-α、IL-6and HMGB1mRNA in hepatic macrophages. The apoptotic rates of hepatic macrophages were determined by Annexin V and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling. The expressions of HMGB1in hepatic tissue and the translocation of HMGB1in hepatic macrophages were examined by Western blot and immunofluorescence. The levels of serum HMGB1were measured with ELISA.2. Experiments in vitro:Hepatic macrophages were isolated from healthy mice and purified by extensive washing with medium. The siRNA against HMGB1was utilized to interfere with the expression of HMGB1in hepatic macrophages. Cells were exposed to Con A at the does of5μg/ml for24h. The expressions of MHCⅡ in hepatic macrophages were examined by flow cytometry. The levels of TNF-a and IL-6in cell cultures were measured with ELISA. The phosphorylation levels of ERK and p38MAPK proteins were examined by Western blot.3. Clinical research:One hundred and forty-seven subjects consisted of63cases with mild/medium chronic hepatitis B (MCHB),39cases with acute-on-chronic liver failure (ACLF) in early stage,27asymptomatic HBV carriers (ASC) and18healthy controls. There is no significant difference in age and gender between the case groups and the control group (P>0.05). Serum virologic indicators and HMGB1were measured with ELISA. The alanine aminotransferase, aspartate aminotransferase, total bilirubin and prothrombin time activity percentage were also measured. The potential associations of serum HMGB1with the hepatic function indicators were analyzed.Results:1. The results of experiments in vivo(1) The proportion of pro-inflammatory Ly6C+CDllb+monocytes in total CDllb+monocytes increased markedly during the development of LF.(2) The expressions of TLR4and MHCⅡ as well as the levels of TNF-a and IL-6mRNA in hepatic macrophages increased significantly at the early stage of LF, which rapidly decreased later.(3) The proportion of F4/80+hepatic macrophages in hepatic mononuclear cells decreased significantly with the progression of LF and the apoptotic rates of freshly isolated F4/80+hepatic macrophages significantly increased with the exposure time of Con A.(4) The levels of HMGB1in serum and hepatic tissues increased obviously with the development of LF.(5) The enhanced expression of HMGB1and the translocation of nuclear HMGB1from the nucleus into the cytoplasm were observed after the onset of LF, and HMGB1protein was exported outside the cells as the disease develops.2. The results of experiments in vivo(1) Pretreatment with HMGB1-siRNA to hepatic macrophages could not reverse the enhanced expression of MHC II on hepatic macrophages induced by Con A, there was not a statistical difference as compared with the control-siRNA group (P>0.05).(2) The blockade of intracellular HMGB1caused by siRNA inhibited the production of TNF-a and IL-6in hepatic macrophages exposed to Con A, there was a statistical difference as compared with the control-siRNA group (P<0.05).(3) The blockade of HMGB1led to a down-regulation of ERK and p38MAPK phosphorylation in hepatic macrophages exposed to Con A, there was a statistical difference as compared with the control-siRNA group (P<0.05).3. The results of clinical research(1) The levels of serum HMGB1in MCHB and ACLF groups were significantly higher than those in ASC and N.C. Group (P<0.05), and there was a significant difference between MCHB and ACLF groups (P<0.05).(2) The elevated levels of serum HMGB1is positively correlated with the increased levels of serum ALT/AST and total bilirubin in patients with CHB (P<0.05), and the levels of serum HMGB1in negatively correlated with the prothrombin activity (P<0.05)Conclusions:(1) In brief, there is, at least, a biphasic immunologic status of hepatic macrophages during the development of Con A-induced liver failure. The initial pro-inflammatory activity of hepatic macrophages was suppressed at the later stage of LF, which is in parallel with cell apoptosis.(2) The intracellular HMGB1is involved in the early activation of hepatic macrophages at least partly through the ERK and p38MAPK signaling pathway, contributing to hepatic pathological injury in LF.(3) HMGB1may be a crucial indicator of LF progression, which has an important role in early diagnosis and treatment of LF...