HBV is a noncytopathic hepadnavirus and major human pathogen that causes immune-mediated acute and chronic hepatitis. The immune response to HBV antigens is age dependent: viral clearance occurs in most adults, while neonates and children usually develop chronic infection and liver disease. Here, we characterize an animal model for HBV infection that recapitulates the key differences in viral clearance between early life and adulthood and find that IL-21 may be part of an effective primary hepatic immune response to HBV. In our model, adult mice showed higher HBV-dependent IL-21 production in liver, compared with that of young mice. Conversely, absence of the IL-21 receptor in adult mice resulted in antigen persistence akin to that of young mice. In humans, levels of IL-21 transcripts were greatly increased in blood samples from acutely infected adults who clear the virus. These observations suggest a different model for the dichotomous, agedependent outcome of HBV infection in humans, in which decreased IL-21 production in younger patients may hinder generation of crucial CD8 + T and B cell responses. These findings carry implications for therapeutic augmentation of immune responses to HBV and potentially other persistent liver viruses.

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Hepatitis B virus (HBV) is a hepadnavirus that is a major cause of acute and chronic hepatitis in humans. Hepatitis B viral infection itself is noncytopathic, and it is the immune response to the viral antigens that is thought to be responsible for hepatic pathology. Previously, we developed a transgenic mouse model of primary HBV infection and demonstrated that the acute liver injury is mediated by nonclassical natural killer (NK)T cells, which are CD1d-restricted, but nonreactive to ␣-GalCer. We now demonstrate a role for NKG2D and its ligands in this nonclassical NKT cell-mediated immune response to hepatitis B virus and in the subsequent acute hepatitis that ensues. Surface expression of NKG2D and one of its ligands (retinoic acid early inducible-1 or RAE-1) are modulated in an HBV-dependent manner. Furthermore, blockade of an NKG2D-ligand interaction completely prevents the HBV-and CD1d-dependent, nonclassical NKT cell-mediated acute hepatitis and liver injury. This study has major implications for understanding activation of NKT cells and identifies a potential therapeutic target in treating hepatitis B viral infection.I nfection with hepatitis B virus (HBV) is a major cause of liver disease worldwide. More than 350 million people are persistently infected with HBV (1, 2). Hepatitis B viral infection itself is noncytopathic, and it is the immune response to the viral antigens that is thought to be responsible for the necroinflammatory process involved in chronic infection, cirrhosis, and hepatocellular carcinoma (3, 4). Thus, understanding the pathogenesis of acute and chronic HBV infection mandates understanding the immune responses underlying these processes. Unfortunately, the study of HBV immunopathogenesis has been problematic because natural hepadnaviral infections occur only in outbred species whose immune systems are difficult to experimentally manipulate.We have established a transgenic mouse model of primary HBV infection that allows the study of mechanisms underlying the immunopathogenesis of hepatitis B virus-induced disease.To generate this mouse model of primary HBV infection, we used either mice that express the small, middle, and large envelope proteins of HBV as transgenes in the liver by using an albumin promoter (hereafter designated HBV-Env ϩ ) (5) or mice that express a terminally redundant HBV DNA construct as a transgene and display intrahepatic HBV replication (hereafter designated HBV-Replication ϩ ) (6). We ablated the resident adaptive immune system of these HBV-transgenic mice, in which the B and T cells are tolerant to HBV, by crossing the HBVtransgenic mice to mice with mutations in the recombinase activating genes (RAG-1) (7). We then reconstituted the immune system in these mice by the adoptive transfer of unimmunized splenocytes isolated from syngeneic, wild-type mice. In this way, a liver with HBV-expressing hepatocytes is exposed to a healthy, untolerized, naïve immune system, just as in acute HBV infection of humans. This system results in a biphasic illness, with a rapi...

Background & AimsAltered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA).MethodsTo identify potential susceptibility loci, Caucasian children, normal (controls) and with BA (cases) at two US centers were compared at >550000 SNP loci. Systems biology analysis was carried out on the data. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 2-5-day post-fertilization zebrafish embryos after morpholino-antisense oligonucleotide knockdown of the candidate gene ADP ribosylation factor-6 (ARF6, Mo-arf6).ResultsAmong 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3’ flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10-7, OR 2.66; 0.286 vs. 0.13, P = 5.57x10-7, OR 2.66). Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58x10-2, OR = 2.66). Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x10-7), ERK/MAPK and CREB canonical pathways (p<1 x10-34), and functional networks for cellular development and proliferation (p<1 x10-45), further supporting the role of EGFR-ARF6 signaling in BA. In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos. Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA.ConclusionsThe BA-associated SNPs identify a chromosome 14q21.3 susceptibility locus encompassing the ARF6 gene. arf6 knockdown in zebrafish implicates early biliary dysgenesis as a basis for BA, and also suggests a role for EGFR signaling in BA pathogenesis.

Acyl CoA Oxidase 2 (ACOX2) encodes branched-chain acyl-CoA oxidase, a peroxisomal enzyme believed to be involved in the metabolism of branched-chain fatty acids and bile acid intermediates. Deficiency of this enzyme has not been described previously. We report an 8-y-old male with intermittently elevated transaminase levels, liver fibrosis, mild ataxia, and cognitive impairment. Exome sequencing revealed a previously unidentified homozygous premature termination mutation (p.Y69*) in ACOX2. Immunohistochemistry confirmed the absence of ACOX2 expression in the patient’s liver, and biochemical analysis showed marked elevation of intermediate bile acids upstream of ACOX2. These findings define a potentially treatable inborn error of bile acid biosynthesis caused by ACOX2 deficiency.