The Mechanism Of Hepatocyte Derived Exosomal MiR-27b-3p Regulating Macrophage RIG-I/TBK1 Signaling Pathway In Response To IFN

Background & Aims: Interferon(IFN)plays a crucial antiviral role in the treatment of patients with chronic hepatitis B(CHB).Clinical studies such as OSST and New Swtich study have found that for CHB patients treated with nucleoside,switching to interferon treatment can significantly improve the rate of clinical cure and reduce hepatitis B surface antigen(HBs Ag)quantification.It has been reported that the therapeutic efficacy of interferon is related with host immunity,and exosomes derived from hepatocyte can regulate the function of immune cells,including DC cells,NK cells,etc.The mechanism of activated IFN signaling pathways in macrophages mediated by hepatic exosomes in response to IFN is unclear.This study aims to define the association of the levels expression of miRNA in hepatatic exosomes and the antiviral effect of interferon,to explore the role of exosomal miRNAs derived from hepatocytes on interferon synthesis pathway in macrophages,and further clarify the role of interferon in the anti-HBV immune system.Methods: 41 nucleoside-experienced patients whose HBV DNA was negative and HBs Ag quantification was lower than 3000 IU/m L were enrolled to receive entecavir combined with Peg-IFNα treatment for 48 weeks.Responders were defined as those who achieved a decline in HBs Ag levels of greater than 0.5 log10 IU/ml at week 12 compared with baseline,or else were defined as non-responders.The exosomal miRNA sequencing and bioinformatics analysis found the key miRNAs related to the clinical antiviral efficacy of Peg-IFNα in the treatment of CHB patients and the possible signaling pathways of these differentially expressed miRNAs.The differentially expressed miRNAs were further verified by quantitative real-time polymerase chain reaction(PCR).The morphology,size and surface markers of exosomes were identified by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and western blot(WB).Serum exosomes from CHB patients treated with Peg-IFNα and exosomes from the supernatant of Hep AD38 stimulated by interferon were cultured with macrophages,and the secretion of interferon was detected by enzyme-linked immunosorbent assay(ELISA),and the expression of phosphorylation of TANK-binding kinase 1(TBK1)and interferon regulatory factor 3/7(IRF3/7)were detected PCR,WB and immunofluorescence.Hep AD38 cells pretreated with miR-27b-3p mimics or inhibitors were co-cultured with macrophages,and the expression of IFNα,TBK1 and IRF3/7 in macrophages were also detected.The target genes of miR-27b-3p were predicted from four databases,namely Target Scan,miRWalk,Star Base and Diana Tools,and the target genes were determined by PCR,WB and luciferase experiments.Results: By sequencing the miRNAs in serum exosomes from responders and nonresponders,six differentially expressed miRNAs were identified,including miR-493-5p,miR-27b-3p,miR-339-5p,miR-769-5p,miR-15b-3p and miR-23b-3p.Specifically,the six exosomal miRNAs in responders were distinctly reduced at week 12 than baseline,and these miRNAs were lower in the responders compared to the non-responders at week 12.Analysis of the six differentially expressed miRNAs by kyoto encyclopedia of genes and genomes(KEGG)and gene ontology(GO)showed they might be related with host immune status and viral clearance.In vivo,it showed that the levels of miR-27b-3p in serum exosomes of patients with CHB were associated with positively HBs Ag quantification,and the levels of miR-27b-3p in peripheral blood mononuclear cells(PBMC)of patients in responders were lower while IFNα m RNA levels were higher than those at baseline.The serum exosomes from responders could be taken up by macrophages and increased the levels of phosphorylation of TBK1 and IRF3/7 in macrophages and activated the immune response.In vitro,it was found that the levels of exosomal miR-27b-3p were significantly reduced in the supernatant of Hep AD38 cells stimulated by interferon,and the exosomes could also increase the phosphorylation of TBK1 and IRF3/7 in macrophages,thereby promoting endogenous IFNα synthesis.Furthermore,when Hep AD38 cells pretreated with miR-27b-3p mimics or inhibitors co-cultured with macrophages,it was found that miR-27b-3p overexpressed in Hep AD38 cells could be transferred from Hep AD38 cells to macrophages via exosomes,inhibiting endogenous IFNα synthesis in macrophages.The addition of exosome release inhibitors GW4869 and Spiroepoxide blocked the delivery of miR-27b-3p and its inhibitory effect on macrophages.Knockdown of miR-27b-3p expression in Hep AD38 cells could activate the endogenous IFNα synthesis pathway in macrophages,thereby inhibiting HBV replication in hepatocytes.Four databases including Target Scan,miRWalk,Star Base and Diana Tools predicted that retinoic acid-inducible gene I(RIG-I)and TBK1 were the target genes of miR-27b-3p.Mi R-27b-3p inhibited endogenous interferon production by directly inhibiting the expression of RIG-I and TBK1.The dualluciferase reporter gene experiment further verified that miR-27b-3p counld bind to the 94-100 sites of the 3’ untranslated coding regions(3’UTR)of RIG-I m RNA and bind to the 172-178 sites of the 3’UTR of TBK1 m RNA.Conclusions: The results showed that the levels of serum exosomal miR-27b-3p in CHB responders treated with Peg-IFNα were significantly reduced,and hepatic exosomes with decreased levels of miR-27b-3p could activate RIG-I/TBK1/IRF signaling pathway in macrophages and further activate innate immunity to exert antiviral effect.Serum exosomal miR-27b-3p might serve as a potential biomarker or host therapeutic target in CHB patients.