Mouse Models for the Study of Viral Hepatitis : (intra)cellular innate immunity

Liver-residing leukocytes are essential in determining the outcome of infection with hepatitis viruses. Patient studies of liver innate immune cells during chronic viral hepatitis have been performed but are hampered by, amongst others, a lack of baseline data and unknown time of infection. Therefore, animal models are essential for the study of the innate immune response in viral hepatitis. In the studies presented in this thesis, we aimed to better understand the cellular innate immune responses in the liver during lymphocytic choriomeningitis virus (LCMV) clone 13 induced chronic hepatitis, and to establish the human-liver chimeric mice as model for chronic Hepatitis E virus (HEV) infections in order to study the intracellular innate immune mechanisms upon HEV infection. In chapter 2 and 3, we aimed to unravel how the cellular innate immune system deals with chronic viral infection in the liver using the LCMV infection mouse model. In chapter 2, we described that early after LCMV infection a functional dichotomy is observed for inflammatory monocytes and F4/80high-Kupffer cells with respect to endocytosis, but their activation and cytokine gene expression profiles exhibit a strong resemblance. In addition, inflammatory monocytes exhibit a huge capacity for recruitment to the liver and plasticity dependent on the nature of the inflammatory signal, either viral or sterile. This suggests that inflammatory monocytes play a crucial role in shaping the inflammatory environment in the liver early after infection. In chapter 3, we showed that LCMV induces chronic viral hepatitis with limited intrahepatic cytokine and interferon responses during the chronic infection. Furthermore, we identified KC and IM as distinct cell populations before, during, and after chronic infection, with important differences in activation status, antigen presentation, and gene ex

Interferon-alpha treatment rapidly clears Hepatitis e virus infection in humanized mice

Antiviral treatment options for chronic Hepatitis E Virus (HEV) infections are limited and immunological determinants of viral persistence remain largely unexplored. We studied the antiviral potency of pegylated interferon-α (pegIFNα) against HEV infections in humanized mice and modelled intrahepatic interferon stimulated gene (ISG) responses. Human gene expression levels in humanized mouse livers were analyzed by qPCR and Nanostring. Human CXCL10 was measured in mouse serum. HEV genotype 3 (gt3) infections were cleared from liver and feces within 8 pegIFNα doses in all mice and relapsed after a single pegIFNα injection in only half of treated animals. Rapid viral clearance by pegIFNα was confirmed in HEV gt1, but not in Hepatitis B Virus infected animals. No ISG induction was observed in untreated HEV gt3 and gt1 infected humanized livers compared to control chimeric mice, irrespective of the human hepatocyte donor, viral isolate or HEV infection duration. Human specific ISG transcript levels in mouse liver increased significantly after pegIFNα treatment and induced high circulating human CXCL10 in mouse serum. In conclusion, HEV gt1 and gt3 infections do not elicit innate intrahepatic immune responses and remain highly sensitive to pegIFNα in immunocompromised humanized mice

Liver monocytes and kupffer cells remain transcriptionally distinct during chronic viral infection

Due to the scarcity of immunocompetent animal models for chronic viral hepatitis, little is known about the role of the innate intrahepatic immune system during viral replication in the liver. These insights are however fundamental for the understanding of the inappropriate adaptive immune responses during the chronic phase of the infection. We apply the Lymphocytic Choriomenigitis Virus (LCMV) clone 13 mouse model to examine chronic virus-host interactions of Kupffer cells (KC) and infiltrating monocytes (IM) in an infected liver. LCMV infection induced overt cli

Hepatitis e virus shows more genomic alterations in cell culture than in vivo

Hepatitis E Virus (HEV) mutations following ribavirin treatment have been associated with treatment non-response and viral persistence, but spontaneous occurring genomic variations have been less well characterized. We here set out to study the HEV genome composition in 2 patient sample types and 2 infection models. Near full HEV genome Sanger sequences of serum- and feces-derived HEV from two chronic HEV genotype 3 (gt3) patients were obtained. In addition, viruses were sequenced after in vitro or in vivo expansion on A549 cells or a humanized mouse model, respectively. We show that HEV acquired 19 nucleotide mutations, of which 7 nonsynonymous amino acids changes located in Open Reading Frame 1 (ORF1), ORF2, and ORF3 coding regions, after prolonged in vitro culture. In vivo passage resulted in selection of 8 nucleotide mutations with 2 altered amino acids in the X domain and Poly-proline region of ORF1. Intra-patient comparison of feces- and serum-derived HEV gt3 of two patients showed 7 and 2 nucleotide mutations with 2 and 0 amino acid changes, respectively. Overall, the number of genomic alterations was up to 1.25× per 1000 nucleotides or amino acids in in vivo samples, and up to 2.84× after in vitro expansion of the same clinical HEV strain. In vitro replication of a clinical HEV strain is therefore associated with more mutations, compared to the minor HEV genomic alterations seen after passage of the same strain in an immune deficient humanized mouse; as well as in feces and blood of 2 immunosuppressed chronically infected HEV patients. These data suggest that HEV infected humanized mice more closely reflect the HEV biology seen in solid organ transplant recipients

Hepatitis E virus (HEV) genotype 3 infection of human liver chimeric mice as a model for chronic HEV infection

Genotype 3 (gt3) hepatitis E virus (HEV) infections are emerging in Western countries. Immunosuppressed patients are at risk of chronic HEV infection and progressive liver damage, but no adequate model system currently mimics this disease course. Here we explore the possibilities of in vivo HEV studies in a human liver chimeric mouse model (uPA+/+Nod-SCID-IL2Rγ-/-) next to the A549 cell culture system, using HEV RNA-positive EDTA-plasma, feces, or liver biopsy specimens from 8 immunocompromised patients with chronic gt3 HEV. HEV from feces- or liver-derived inocula showed clear virus propagation within 2 weeks after inoculation onto A549 cells, compared to slow or no HEV propagation of HEV RNA-positive, EDTA-plasma samples. These in vitro HEV infectivity differences were mirrored in human-liver chimeric mice after intravenous (i.v.) inoculation of selected samples. HEV RNA levels of up to 8 log IU HEV RNA/gram were consistently present in 100% of chimeric mouse livers from week 2 to week 14 after inoculation with human feces- or liver-derived HEV. Feces and bile of infected mice contained moderate to large amounts of HEV RNA, while HEV viremia was low and inconsistently detected. Mouse-passaged HEV could subsequently be propagated for up to 100 days in vitro. In contrast, cell culture-derived or seronegative EDTA-plasma-derived HEV was not infectious in inoculated animals. In conclusion, the infectivity of feces-derived human HEV is higher than that of EDTA-plasma-derived HEV

Inflammatory monocytes recruited to the liver within 24 hours after virus-induced inflammation resemble kupffer cells but are functionally distinct

Due to a scarcity of immunocompetent animal models for viral hepatitis, little is known about the early innate immune responses in the liver. In various hepatotoxic models, both pro- and anti-inflammatory activities of recruited monocytes have been described. In this study, we compared the effect of liver inflammation induced by the Toll-like receptor 4 ligand lipopolysaccharide (LPS) with that of a persistent virus, lymphocytic choriomeningitis virus (LCMV) clone 13, on early innate intrahepatic immune responses in mice. LCMV infection induces a remarkable influx of inflammatory monocytes in the liver within 24 h, accompanied by increased transcript levels of several proinflammatory cytokines and chemokines in whole liver. Importantly, while a single LPS injection results in similar recruitment of inflammatory monocytes to the liver, the functional properties of the infiltrating cells are dramatically different in response to LPS versus LCMV infection. In fact, intrahepatic inflammatory monocytes are skewed toward a secretory phenotype with impaired phagocytosis in LCMV-induced liver inflammation but exhibit increased endocytic capacity after LPS challenge. In contrast, F4/80high-Kupffer cells retain their steady-state endocytic functions upon LCMV infection. Strikingly, the gene expression levels of inflammatory monocytes dramatically change upon LCMV exposure and resemble those of Kupffer cells. Since inflammatory monocytes outnumber Kupffer cells 24 h after LCMV infection, it is highly likely that inflammatory monocytes contribute to the intrahepatic inflammatory response during the early phase of infection. Our findings are instrumental in understanding the early immunological events during virus-induced liver disease and point toward inflammatory monocytes as potential target cells for future treatment options in viral hepatitis