The role of inducible nitric oxide synthase in a murine acute hepatitis B virus (HBV) infection model induced by hydrodynamics-based in vivo transfection of HBV-DNA

Abstract

Background/Aims: Inducible nitric oxide synthase (iNOS) is found to have antiviral activity. Its role is evaluated using a murine acute hepatitis B virus (HBV) infection model.

Methods: pHBV3.6 plasmid containing HBV genome was injected into mice by hydrodynamics-based in vivo transfection. HBV antigenemia and serum HBV-DNA were detected by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction, respectively. HBV replication in liver was analyzed by Northern and Southern blot. Intrahepatic leukocytes were isolated and analyzed with flow cytometry.

Results: After hydrodynamics injection of pHBV3.6, HBV genome was synthesized in the liver and HBV-DNA, as well as hepatitis B surface antigen and hepatitis B e antigen were secreted into the blood. Anti-HBV antibody responses developed afterward. A murine acute HBV infection model was established with hydrodynamics injection of non-transponase based HBV-DNA. Using this protocol in iNOS deficient or wild type B6 mice, the level of HBV transcript, replicative intermediate, and antigenemia were higher in iNOS^−/− than in B6 mice. The intrahepatic leukocytes in iNOS^−/− mice were also affected after transfection.

Conclusions: Our data suggests that the iNOS expression not only affects the HBV clearance, but also modulates the infiltrating leukocytes response to HBV antigens.

Introduction

Hepatitis B virus (HBV) infection in human is associated with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma [1], [2], [3]. One third of the world population has been infected with HBV and about 350 million people are chronic carriers of HBV [4]. HBV is non-cytopathic to hepatocytes and the hepatitis it causes is thought to be mediated by immune mechanism. Inducible nitric oxide synthase (iNOS) can be induced by tumor necrosis factor (TNF)-α or interferon (IFN)-γ in many immune cells [5], [6], [7]. NO synthesized by iNOS can function as an antimicrobial agent able to kill or reduce replication of viruses, bacteria or protozoa [8], and plays an important role in immune regulation [5]. In chronic HBV infection, the iNOS expression was found to correlate to the disease progress [9], [10], [11]. Using HBV transgenic mice, Guidotti et al. also found NO-mediated inhibition of HBV replication [12]. But its role in acute HBV infection has not been addressed yet.

Hydrodynamics-based in vivo transfection can induce expression of naked DNA primarily in the liver [13], [14], [15]. Recently, a murine acute HBV infection model was generated by hydrodynamics-based injection of transponase-based HBV DNA [16]. By hydrodynamics injection of pHBV3.6, a non-transponase based plasmid containing whole HBV genome into iNOS knock out mice, the role of iNOS in acute HBV replication and intrahepatic leukocyte response was reported in this study.