Response And Signal Pathway Of Interferon In HBeAg-positive Patients Treated With Nudeotide Analogues

[BACKGROUND&OBJECTIVE]There is a large number of chronic hepatitis B (CHB) patients in our country, their health were threatened by serious end-stage liver disease. Effective anti-viral therapy can reduce the incidence of cirrhosis, liver cancer and other complications and significantly improve the prognosis. Current anti-HBV therapy is mainly divided into two categories, one is the nucleoside analogues (nucleotide analogues, NAs), there are four kinds of NAs in China:lamivudine (LAM), adefovir dipivoxil (ADV), telbivudine (LDT), entecavir (ETV). The other is interferon (IFN), including IFN and Peg-IFN. NAs and IFN.have different characteristics. NAs exert a strong inhibitory effect on viral replicationand are characterized by oral administration, convenient to carry and use, less side effects.However, it is difficult to obtain a desired therapeutic endpoint (HBeAg/HBsAg seroconversion or HBsAg clearance) and is required long time medication which easily lead to the risk of drug resistance,and relapse frequently occurs when treatment is discontinued. Advantages of IFN treatment are finite teatment course (usually12months or extended to18months), high HBeAg/HBsAg seroconversion/HBsAg clearance rate, sustained immunological control. The disadvantage of IFN is side effects which contribute to treatment interruption.Meanwhile, evolution of the virus itself may develop various strategies to inhibit the activation of IFN signaling pathway, resulting in decreased efficacy of IFN. Since the two types of drugs have their advantages and disadvantages, many scholars have tried to use their strengths to get better clinical outcomes, such as combined/sequential treatment.Morever, optimizing or individualized treatment has become a hot issue in HBV research.Acorrding to the molecular mechanism, IFN is the only agent displaying both antiviral and direct immunomodulatory effects in hepatitis B antiviral treatment. Vitro studies have shown that IFN exerts its antiviral effect by the following mechanism:IFN bind to its receptors in cell surface and activate a variety of signaling molecules such as STAT1(signal transducer and activator of transcription1),then activating JAK/STAT signaling pathway and induce downstream antiviral effect gene expression. Among these Genes, myxovirus resistance protein (MxA),2’,5’-oligoadenylate synthetase (2’,5’-OAS), and protein kinase R (PKR) are utmost important. IFN-induced MxA protein is distributed in the cytoplasm and exert broad-spectrum antiviral activity. MxA has a strong inhibitory effect on orthomyxovirus,rod virus, small RNA viruses and hepatitis B virus. Activated2’,5’-OAS nucleolytic enzyme can selectively induce the degradation of the viral mRNA. For the JAK/STAT pathway, there are some negative regulatory genes to inhibit its signaling activation. When JAK/STAT signaling is activated, the negative regulatory genes will be activated by negative feedback to avoid excessive activation of signaling pathways. Among these genes,SOCS3(suppressor of cytokine signaling3), PIAS (protein inhibitor of activated STAT1) etl are most important. SOCS3can bind to phosphorylated cytokine receptor, then inhibit JAKs activation and signal transduction. Compared to SOCS3, PIAS is more likely to be the buffers which regulate the transcription of target genes by controlling the amount of activated STATs.For IFN treatment,lower response rate may hint that IFN resistance could occur in patients with CHB. However,molecular mechanisms of IFN exerts its antiviral and immunomodulatory effects in the treatment of CHB are incompletely understood,and the detailed mechanism of viral resistance to IFN have not been fully elucidated. OSST clinical study (optimizing seroconversion sequential treatment)was designed to answer whether HBeAg-positive patient who had maintained HBV DNA suppression with ETV could achieve primary endpoint of HBeAg/HBsAg seroconversion or HBsAg clearance when safely switching to Peg-IFN. Based on the result of clinical research, we aim to explore the relationship between expression of signaling molecules and differential clinical endpoints. Our study provide new insight into optimizing treatment and further deepens the understanding of IFN response and signaling pathways in CHB patients.The details are as follows:1HBeAg-positive chronic hepatitis B patients (patient who had maintained HBV DNA suppression with ETV) switched to Peg-IFN180ug for48weeks(group A) or continued on ETV for a further48weeks(group B). Clinical index(HBV DNA, HBeAg, HBsAg, ALT)were observed during treatment. Dynamic changes of clinical index in two groups were analyzed. Response rates between the two groups were compared at48weeks/96weeks.2By using Real Time PCR, western blot and IHC,IFN signaling related-gene expression were detected in liver tissue and PBMC from patients. Dynamic changes.of target gene expression were compared in A/B group and responders/non-responders from A group.Correlation of gene expression and differential clinical outcomes were analyzed and established[METHODS]1Fifty-four E antigen-positive CHB patients with a maintained virologic response to ETV were recruited in Tongji Hospital (a part of OSST study). Inclusion criteria:patient treated with ETV9-36months, HBV DNA undetectable(≤103copies/mL) and low levels of HBeAg (<100PEIU/ml. Patients were randomly assigned to group A and group B. Group A switched to Peg-IFN(40KD) therapy (combination therapy of ETV+IFN at initial8weeks) for48weeks. Group B continued to ETV for48weeks. Response rate of two groups were compared at48weeks and96weeks.2We collected blood samples from54patients recruited in A, B group at different time points (0,4,12,24,36,48,72,96weeks). PBMC isolation were performed. RNA and protein were extracted from PBMC. Real Time PCR were performed to detect STAT1, MxA,2’5’-OAS and SOCS3mRNA level expression in PBMC. Western-Blot were used to measure fold induction of phosphorylated STAT1(key factor of activated IFN signaling pathway) from0-4weeks in group A.3Pre and post-treatment (0and48weeks) liver biopsies were collected from a part of patients in group A. Real Time PCR were peformed to detect STAT1, MxA,2’,5’-OAS and SOCS3mRNA level expression in liver. Immunohistochemistry chemical (IHC) were used to measure phosphorylated-STAT1and SOCS3expression in liver tissue.[RESULTS]1Baseline characteristics of group A and B were no difference, while a significant difference of HBsAg baseline level was observed in the responders and non-responders from group A. A more pronounced HBsAg decline was observed in group A when compared to B group,and the same trend was true for responder when compared to non-responder from A group. At48weeks and96weeks, significantly higher rates of HBeAg/HBsAg seroconversion or HBsAg clearance was observed in A group when compared to B group(22.2%vs3%;37%vs3%).2Compared to baseline level,STAT1, MxA,2’,5’-OAS, and SOCS3mRNA levels were significantly increased in PBMCs from patients in A group,whereas the gene expression in group B were not changed. Further analysis in group A showed that baseline SOCS3mRNA level in responders were lower than in non-responders. Fold induction of STAT1(0-4weeks) and MxA (0-4weeks,0-12weeks) mRNA level in responders were markably higher than in non-responders,and fold induction of phosphorylated STAT1protein levels(0-4weeks) was significantly different between responder and non-responders in group A. HBsAg decline (0-48weeks) was positively correlated with STAT1mRNA level fold induction from0to4weeks and MxA mRNA level fold induction from0to12weeks,respectively 3At48weeks, representive immunohistochemical staining showed that phosphorylated STAT1expression was significantly higher in responder than in non-reponder. Compared to non-responder, lower baseline phosphorylated STAT1level was observed in reponder. Immunohistochemical staining of liver from patient with virus breakthrough showed that phosphorylated STAT1expression over-activated. Real Time PCR detection showed that fold induction of STAT1and MxA mRNA levels from0-48weeks were significantly higher in reponders than in non-responders,wheras fold induction of SOCS3mRNA levels was lower in responder than in non-responders. Immunohistochemical staining of further confirmed this result.[CONCLUSION]1Patients with sustained virological response to ETV can achive significantly higher rates of HBeAg/HBsAg seroconversion or HBsAg clearance when switched to the finite course of peg-IFN compared with remaining on ETV. According to our results, this sequential treatment was better than NAs treatment alone. Our results provide theoretical basis and new insight into optimizing treatment of CHB.2SOCS3baseline level, higher fold induction of STAT1and MxA mRNA levels at early stage were correlated to the response of IFN treatment. Fold induction of STAT1and MxA mRNA level were positively correlated with HBsAg decline. Our results suggested that these genes may predict the activity and efficacy of IFN, to some extent. Our findings provide predictive factors and therapeutic targets to IFN treatment.3Higher baseline level of phosphorylated STAT1in liver may contribute to poor response of IFN therapy. Fold induction of liver STAT1, MxA, SOCS3from0to48weeks play predictive roles in IFN treatment. Our results deepens the understanding of IFN signaling-related molecular mechanisms as well as viral resistance to IFN.