The Macrophage Regulation Mechanism And Non-invasive Diagnosis Of Hepatitis B-related Hepatic Cirrhosis

Background Hepatic cirrhosis caused by various chronic liver diseases is a pathological process of diffuse and excessive deposition of extracellular matrix(ECM)in the liver.Chronic hepatitis B(CHB)is an important cause of hepatic cirrhosis.If hepatitis B virus(HBV)continues to replicate,the persistent inflammatory response induced by HBV and its related components will accelerate the continuous progression of hepatic cirrhosis,which will further lead to portal hypertension and severe clinical complications,such as esophageal varices(EV),hypersplenism,and ascites.Currently,there is still a lack of safe and effective treatments to reverse hepatic cirrhosis in clinical practice.Therefore,it is of great clinical significance for the prevention and treatment of hepatic cirrhosis to study the the mechanism of hepatic cirrhosis in depth and diagnose hepatic cirrhosis and its related complications timely and accurately.Hepatic cirrhosis is characterized by the deposition of extracellular matrix proteins and increased intrahepatic angiogenesis induced by the activation of hepatic stellate cells(HSC)and hepatic sinusoidal endothelial cells(HSEC).The activation of HSC plays a key role in this process.HSC transdifferentiated into myofibroblast-like cells with the stimulation of various etiological factors.Activated HSC are characterized with proliferation,contraction,chemotaxis,and the secretion of ECM.In hepatic microenvironment,extracellular signals from the surrounding cells can act in a paracrine manner to promote the activation of HSC.Macrophages are one of the major regulators of hepatic cirrhosis and remodeling of deposited ECM.Accumulating evidence indicates that activated macrophages can produce pro-fibrotic mediators,such as growth factors,cytokines and chemokines etc,contributing to the activation and recruitment of HSC,and further accelerate the deposition of ECM.However,the biological role and the mechanism of macrophages have not been fully elucidated in hepatitis B related cirrhosis.After HBV entry into hepatocytes and begin to replicate,virus related proteins can be detected in the liver and blood.HBV-related proteins are mainly composed of hepatitis B surface antigen(HBsAg),hepatitis B core-related antigen(HBcrAg)and HBx proteins[3].HBcrAg consists of three proteins coded by the precore/core region including hepatitis B core antigen(HBcAg),hepatitis B e antigen(HBeAg)and a 22-kDa precore protein.At present,the biological functions of hepatitis B virus-related proteins have not yet been fully revealed.Our recent work has proved that,compared with HBcAg and HBsAg,HBeAg is the most important element in HBV-associated antigens inducing macrophage activation.However,it is still unclear whether HBeAg can activate HSC to promote the occurrence and progression of hepatic cirrhosis directly or in a macrophage dependent manner.The research in mechanism can help us understand the course of CHB deeply,and find new therapeutic targets.In addition,for CHB patients,timely and accurate diagnosis of hepatic cirrhosis and related complications is also accompanied with important clinical significance.Liver biopsy and upper gastrointestinal endoscopy are gold standards for evaluating hepatic cirrhosis and EV,respectively.However,the disadvantages of these inspections lie in their invasiveness,high cost,and concomitant risk of adverse events.Therefore,it is difficult to perform the above-mentioned invasive examinations repeatedly during the course of continuous disease development for CHB patients.An accurate and effective non-invasive detection method is the key to solving this problem.Two-dimensional shear wave elastography(2D-SWE)is a non-invasive,two-dimensional ultrasound technique.2D-SWE uses focused ultrasonic beams to create shear waves in the liver tissue,the propagation of which is then captured in real time by a high frame-rate ultrasound imaging sequence.The liver stiffness(LS)measured by 2D-SWE is closely related to the stage of hepatic cirrhosis.Since the degree of hepatic cirrhosis is positively correlated with portal pressure to a certain extent,this technique has also been applied to detect EV non-invasively.Multi-center,prospective studies have shown that 2D-SWE displays ideal accuracy in diagnosing hepatic cirrhosis and EV.However,it is worth noting that for cirrhotic patients with different etiologies,the LS values detected by 2D-SWE are not the same.Therefore,the cut-off of LS measured with 2D-SWE for the diagnosis of HRV still needs to be determined in patients with hepatitis B-related cirrhosis.In addition,the LS measured by 2D-SWE will also be affected by some factors,such as histologic inflammatory activity and intrahepatic cholestasis.Overall,it still needs further research to assess hepatic cirrhosis and EV in patients with hepatitis B related hepatic cirrhosis.The whole study includes three parts.In the first part,on the basis of previous research,we found HBeAg activated macrophages by directly binding to TLR-2,thereby promoting the release of inflammatory cytokines through NF-κB signaling pathway.Meanwhile,C-terminal peptide(122-143 aa.)of core domain in HBeAg was critical for macrophages activation.Arginine-rich domain of HBcAg hided this function,although HBcAg and HBeAg shared the same core domain.Moreover,HBeAg promoted the proliferation,motility,and contraction of HSC in a macrophage dependent manner,but not alone.In terms of mechanism,PI3K-AKT-mTOR and p38 MAPK signaling pathway were responsible for motility phenotype of HSC,while SMAD dependent TGF-β signaling pathway for proliferation and contraction of them.In the second part,we evaluated the diagnostic efficacy of 2D-SWE in evaluating hepatic cirrhosis.On this basis,we further analyzed the influence of hepatic steatosis(HS)in LS values as well as its diagnostic efficacy.We found that CHB patients with HS>10%were more likely to be overestimated as hepatic cirrhosis.In the third part,we firstly analyzed the optimal cut-off value and its diagnostic efficacy of LS measured by 2D-SWE for evaluating HRV in patients with compensated hepatitis B related cirrhosis.Furthermore,we established that the combination of LS and PLT can improve the diagnostic efficacy of 2D-SWE,and is not affected by the antiviral therapy history.In summary,this study clarifies a new mechanism of hepatic cirrhosis and its complications.It also provides a new clinical idea and optimization method for 2D-SWE to estimate hepatitis B related cirrhosis and its complications accurately,which has an extensive clinical significance.Part 1 HBeAg mediates the secretion of cytokines from macrophages by TLR-2 contributing to hepatic cirrhosisAIM We have proved that HBeAg can obviously induce the production of inflammatory cytokines from macrophages compared with HBsAg and HBcAg.We aimed to further analyze the functional domain and receptors of HBeAg,and clarify its effects and mechanisms on hepatic cirrhosis.Methods 1.To analyze the recognition receptor of HBeAg in macrophages,we firstly detected the kinetics of typical cytokines at the dose of 2μg/mL so as to determine the best detection time point.The expression of TNF-α and IL-6 in macrophages was detected by qRT-PCR assay.To understand the regulatory role of HBeAg on macrophage comprehensively,RNAseq analysis was applied.The expression of key receptor family with significant modulation was further verified in mouse and human macrophages by qRT-PCR assay.2.We blocked the possible recognition receptors by receptor inhibitors,RNA interference,or neutralizing antibodies.Then the changes of cytokines or signal pathways were detected to screen out the target receptors by qRT-PCR,ELISA,or Western blot analysis.Finally,the direct interaction between the target receptor and HBeAg was confirmed by Co-IP assay in vivo and in vitro.3.The different ability for HBcAg and HBeAg to activate macrophages was compared by qRT-PCR and ELISA analysis.Based on the differences in domains between HBcAg and HBeAg,recombinant domain proteins were constructed and used to stimulate macrophages in order to analyze the functional domains.Moreover,we performed bioinformatics analysis for the surface accessible peptides of HBeAg.Recombinant HBeAg proteins in which the accessible peptides were deleted were used to confirm their abilities in activating macrophages.4.We added conditioned medium from HBeAg activated macrophages(CM-E)to HSC for 24h as compared with the effect of HBeAg alone.The levels of α-SMA and ECM components(collagen and fibronectin)were detected.The proliferaton,motility,and contractile phenotypes of HSC were detected by CCK-8、Transwell and collagen gel lattice assays,separately.5.To investigate mechanisms that are regulated by CM-E for the activation of HSC,we used a Phospho Explorer antibody micro array to detect and analyze phosphorylation events under specific conditions.The key signaling intermediates were verified using western blots.KEGG and pathway mapping analysis was further used to analyze these pivotal pathways.Finally,specific inhibitors of the corresponding pathways were used to verify the key pathways.6.To analyze the soluble factors responsible for the phenotype,CM-E was screened for the levels of various cytokines,chemokines and growth factors using the Magnetic Luminex Performance Assay.7.HBeAg was injected through the tail vein in C57BL/6 mice,and the mice were sacrificed at different time points.HE staining was performed on mouse liver tissues to evaluate the degree of hepatic inflammatory necrosis and inflammatory cell infiltration.Serum ALT and AST were detected to evaluate liver damage.qRT-PCR assay was used to detect the expression of inflammatory factors in the liver.8.The effect of HBeAg on fibrogenesis was assessed in acute CCL4 model in C57BL/6 mice.The above-mentioned methods were used to evaluate hepatic inflammatory necrosis,inflammatory cell infiltration and liver damage.Co-immunofluorescence of F4/80 and α-SMA was used to analyze macrophage infiltration and HSC activation.Co-immunofluorescence of ki67 and Desmin was used to analyze the proliferation and migration of HSC.9.To further elucidate the roles of macrophages in vivo,macrophages were depleted by injecting clodronate-containing liposomes before HBeAg or CCL4 treatment.The depletion efficiency of macrophages(F4/80+)was confirmed by IHC assay.qRT-PCR assay was used to detect the expression of inflammatory factors,ECM components and α-SMA in the liver.10.To verify the roles of TLR-2 in vivo,mice were pretreated with C29 before the administration of HBeAg.qRT-PCR assay was used to detect the expression of inflammatory factors in the liver.11.To verify our results in patients infected with HBV,we recruited 61 patients with acute hepatitis B(AHB)and 151 patients with CHB.Various clinical data and related examination results of patients were collected.In patients with acute hepatitis B,the correlation between serum HBeAg content and HBV DNA,ALT or AST was analyzed.In patients with chronic hepatitis B,we analyzed the correlation between serum HBeAg and the related indexes of hepatic cirrhosis and inflammatory activity,respectively.Resuts 1.Four hours were the best time period for HBeAg to stimulate macrophages.Except for growth factors,cytokines and chemokines,a significant change of the expression of TLRs was also observed,especially for TLR-2 and TLR-3.2.According to cell functional experiments,the activation of macrophages was weakened most significantly by blocking TLR-2.Moreover,HBeAg could directly interact with TLR-2 in vitro or in vivo.3.Compared with HBcAg,HBeAg also had a stronger ability to activate human macrophages.Accumulating data had shown that the radically different molecular structure between HBeAg and HBcAg was determined by N-terminal propeptied of HBeAg and arginine-rich domain(ArD)of HBcAg.Thus we synthesized recombinant protein ArD and core domain(N-terminal 10-residue propeptide deleted HBeAg),and then treated macrophage with them.According to cell functional experiments,the core domain is the functional domain of HBeAg to activate macrophages.Moreover,we performed bioinformatics analysis for the surface accessible peptides,showing that the accessible peptides for HBeAg were scattered in three sections of the core domain(aa.1-17,29-96,122-143).The recombinant HBeAg in which aa.122-143 was deleted was minimally stimulatory for macrophages as compared with the others.4.HBeAg promoted the activation of hepatic stellate cells in a macrophage dependent manner.5.A more predominant effect was detected in PI3K-AKT-mTOR and p38 MAPK signaling pathway for motility,and SMAD dependent TGF-β signaling pathway for proliferation and contraction of HSC.6.Magnetic Luminex Performance Assay indicated that multiple soluble factors,such as CCL-2、CCL-5、CXCL-10、TNF-α、PDGF-BB,may be key components of the activation of HSC.7.There is a few small inflammatory infiltrates,but no observable hepatic necrosis areas were detected at 4-12h,which was in line with the transient expression of cytokines.Concomitantly,both serum ALT and AST were increased mildly at different detection time points.8.In the acute liver injury model induced by CCL4,HBeAg can further aggravate liver damage,and promote macrophage infiltration.HBeAg can also promote the activation,proliferation,and migration of HSC.9.Nearly all macrophages were depleted at day 1 and day 4 by clodronate as evidenced by reduced protein expression of F4/80.Macrophage depletion led to a significant reduction in the expression of growth factors,cytokines,chemokines,α-SMA and collagen no matter in HBeAg treatment alone or in the development of cirrhosis induced by CCL4.10.After blocking TLR-2 in vivo,the expression of pro-inflammatory cytokines and chemokines decreased in the liver,and the expression of anti-inflammatory cytokines increased.11.As an index of viral replication,the level of HBeAg was positively correlated with HBV DNA load in AHB patients.Moreover,the levels of serum ALT,AST were increased with the HBeAg in these patients.In CHB patients,hepatic inflammation grades were higher in HBeAg+ patients than HBeAg-patients,nevertheless statistical significance could only be achieved between G=0 and G ≥1(p=0.030).Meanwhile,a significant correlation was observed between the CD68+ cells infiltration and the level of HBeAg(r=0.557,p=0.003).Moreover,hepatic fibrosis was more severe in HBeAg+ patients than HBeAg-patients(p=0.004).Consistently,HBeAg+patients showed higher incidence of the cirrhosis related complications such as esophageal varices(EV),ascites,and splenomegaly,even though only rates of EV achieved statistical significance.Furthermore,the positive stainning areas of α-SMA was correlated with the level of HBeAg positively(r=0.515,p=0.014).Conclusion HBeAg activated macrophages via TLR-2/NF-κB signal pathway,and further exacerbated hepatic cirrhosis by facilitating motility,proliferation and contraction of HSC.Part 2 The effect of hepatic steatosis on the diagnostic efficiency of 2D-SWE in diagnosing hepatitis B related cirrhosisAIM LS measured by 2D-SWE has been used to assess hepatic cirrhosis.We aimed to determine whether HS affected the diagnostic accuracy for 2D-SWE to assess hepatic cirrhosis in patients with CHB.Methods 1.Serum parameters,clinical specimens and LS values were obtained from 161 patients with CHB.2.The stages of hepatic cirrhosis and inflammatory activity were estimated based on the METAVIR Cooperative Study Group criteria,and the extent of HS was defined as the percentage of hepatocytes containing fat droplets using oil red staining.Results 1.We found that LS values were independently correlated with HS in the early stages of hepatic fibrosis(F0-F2 or F0-3).2.Furthermore,LS values in patients with significant steatosis(S≥10%)were higher than the counter part in fibrosis stages F0-2(6.82±1.57 vs.7.92 ±1.99,p=0.010)and F0-3(7.18±1.84 vs.8.25 ± 1.91,p=0.007).3.Therefore,false positive rates in the diagnosis of advanced fibrosis(16.00%vs.37.04%,p=0.037)and cirrhosis(6.67%vs.21.62%,p=0.030)were higher in patients with significant steatosis.Conclusion The use of 2D-SWE in the measurement of LS overestimates the stage of hepatic cirrhosis in CHB patients with HS>10%.This should be taken into consideration to combine LS results with other non-invasive parameters to improve its accuracy.Part 3 The combination of two-dimensional shear wave elastography and platelet counts to predict high-risk esophageal varices in patients with hepatitis B-realted cirrhosisAIM EV is an important complication of hepatitis B related cirrhosis.Early diagnosis of high risk varices(HRV)can reduce the risk of upper gastrointestinal bleeding and prolong the survival of patients.We aimed to prospectively analyze the cut-off of the combination of LS measured by 2D-SWE and PLT in predicting HRV and the influence of antiviral therapies in its efficacy.Methods 1.Serum parameters,LS,and endoscopy results were obtained from 160 compensated hepatitis B related cirrhotic patients.2.In the whole cohort,we analyzed the cut-off value and diagnostic efficacy of the combination of LS measured by 2D-SWE and PLT to predict HRV.3.The accuracy of the combined algorithm was further assessed in the subgroups with or without consecutive antiviral therapies in the past 6 months.Resuts 1.In the whole cohort,the optimal cut-off value of LS for HRV was 14.5 kPa.2.According to recommendations of the expanded Baveno VI guidelines,the cut-off of PLT for HRV is adopted as 110×109/L in order to further reduce the invasive detection of upper gastrointestinal endoscopy.Patients who meet the criteria(LS<14.5 kPa and PLT>110×109/L)were less likely to be diagnosed with HRV(NPV=99.07%).One hundred and eight out of the 160 patients(67.50%)fulfilled the criteria and,consequently,avoided endoscopy screening.3.By combining LS(≥14.5 kPa)and PLT(≤110×109/L)to assess HRV,patients can be diagnosed with 82.35%certainty,and 17(10.63%)patients can avoid endoscopy screening.4.Antiviral therapy had no significant effect on the accuracy and rates saved from further endoscopy screening,when comparing patients with or without antiviral therapies(all p values>0.05).Conclusion The combination of LS(14.5 kPa)measured by 2D-SWE and PLT(110×109/L)can predict HRV accurately in compensated hepatitis B related cirrhotic patients without significant interference of antiviral therapy histories.