Elucidating the mechanisms involved in liver damage and hepatitis B virus inhibition by hepatitis delta virus infection

Resumen

Hepatitis delta virus (HDV) infection represents the most severe form of viral hepatitis; however, the specific reasons for the severity of the disease remain unknown. The absence of mouse models in which HDV-induced liver damage occurs has prevented the study of this important clinical aspect of the disease. We developed an adeno-associated virus (AAV)- mediated HDV/HBV co-infection model in which, for the first time, liver damage associated with HDV was detected in mice. Using blocking agents and KO mice, we found that hepatocyte death was, at least partially, associated with TNF-α production. Here, we report that TNF-α was mainly produced by liver macrophages and that the death of HDV-hepatocytes was mediated predominantly by caspase 8 induced-apoptosis. The receptor-interacting protein kinase 1 (RIPK1) has been shown to play a role in TNF-α-induced apoptosis but also in cell survival. In order to evaluate the role of RIPK1 in the fate of HDV-infected hepatocytes, we developed a hepatocyte-specific CRISPR/Cas9 system to eliminate/reduce its expression in these cells. Upon AAV-HBV/HDV co-injection, RIPK1-edited mice presented a significantly higher liver damage than mice receiving control CRISPR/Cas9, reflected by higher transaminase serum levels and an increased percentage of apoptotic hepatocytes. This indicated that RIPK1 plays a significant role in hepatocytes survival in an HDV infected liver. The elimination of both caspase 8 and RIPK1 from hepatocytes resulted in the almost complete absence of hepatocyte death after AAV-HBV/HDV injection, which further corroborated caspase 8-induced apoptosis as the main cell death mechanism in RIPK1-edited mice. Surprisingly, the absence of TNF-α or the depletion of macrophages had no effect on the exacerbation of liver disease associated with the downregulation of RIPK1 in hepatocytes. Interestingly, depletion of macrophages had a significant detrimental effect, indicating that these cells play a protective role. Our investigations underline the protective role of RIPK1 in HDV-induced liver pathology and strongly suggest that the RIPK1-caspase-8 axis influences the pathogenesis of HDV infection. On the other hand, the replication of hepatitis B virus (HBV) does not significantly activate the innate immune response and is strongly affected by co-infection with HDV, which induces a type I interferon (IFN) response. The second aim of this study was to analyse the potential role of the IFN response on the HBV inhibition in HDV co- and super-infection mouse models. Although AAV-HBV/HDV co-injection results in inhibition of HBV replication, the essential requirement of AAV-HBV genomes for HBV replication in the AAV-HBV/HDV mouse model represents a strong limitation since AAV genomes are lost independently of the type I IFN response because of HDV-induced hepatocyte death. Thus, in order to investigate the role of the IFN-β response in HBV repression upon HDV infection, we developed a HBVtg x IFN-α/βR KO mouse model in which HBV replication does not depend on AAV-HBV genomes. Interestingly, HBcAg expression in the liver and HBV viremia increased in HBVtg x IFN-α/βR KO mice under steady state conditions. Importantly, while AAV-HDV injection induces the decrease of liver HBcAg expression, HBV-DNA levels and HBV viremia in HBVtg mice, this effect disappeared in HBVtg x IFN-α/βR KO mice. Therefore, the inhibitory effect on HBV in the AAV-HDV super-infection model is completely dependent on the type I IFN response Our results underline the essential role of the type I IFN response in HDV-mediated inhibition of HBV replication. A better characterization of the molecular mechanisms involved in the liver pathology will help to identify novel therapeutic strategies for HDV-infected patients.