Marianna Hösel

Not interferon, but interleukin‐6 controls early gene expression in hepatitis B virus infection

With about 350 million virus carriers, hepatitis B virus (HBV) infection remains a major health problem. HBV is a noncytopathic virus causing persistent infection, but it is still unknown whether host recognition of HBV may activate an innate immune response. We describe that upon infection of primary human liver cells, HBV is recognized by nonparenchymal cells of the liver, mainly by liver macrophages (Kupffer cells), although they are not infected. Within 3 hours, this recognition leads to the activation of nuclear factor kappa B (NF‐κB) and subsequently to the release of interleukin‐6 (IL‐6) and other proinflammatory cytokines (IL‐8, TNF‐α, IL‐1β), but does not induce an interferon response. The activation of proinflammatory cytokines, however, is transient, and even inhibits responsiveness toward a subsequent challenge. IL‐6 released by Kupffer cells after activation of NF‐κB controls HBV gene expression and replication in hepatocytes at the level of transcription shortly after infection. Upon binding to its receptor complex, IL‐6 activates the mitogen‐activated protein kinases exogenous signal‐regulated kinase 1/2, and c‐jun N‐terminal kinase, which inhibit expression of hepatocyte nuclear factor (HNF) 1α and HNF 4α, two transcription factors essential for HBV gene expression and replication. Conclusion: Our results demonstrate recognition of HBV patterns by nonparenchymal liver cells, which results in IL‐6‐mediated control of HBV infection at the transcriptional level. Thus, IL‐6 ensures early control of the virus, limiting activation of the adaptive immune response and preventing death of the HBV‐infected hepatocyte. This pattern recognition may be essential for a virus, which infects a new host with only a few virions. Our data also indicate that therapeutic neutralization of IL‐6 for treatment of certain diseases may represent a risk if the patient is HBV‐infected. (HEPATOLOGY 2009:50:1773–1782.)

A Role for the Human Nucleotide-binding Domain, Leucine-rich Repeat-containing Family Member NLRC5 in Antiviral Responses

Proteins of the nucleotide-binding domain, leucine-rich repeat (NLR)-containing family recently gained attention as important components of the innate immune system. Although over 20 of these proteins are present in humans, only a few members including the cytosolic pattern recognition receptors NOD1, NOD2, and NLRP3 have been analyzed extensively. These NLRs were shown to be pivotal for mounting innate immune response toward microbial invasion. Here we report on the characterization of human NLRC5 and provide evidence that this NLR has a function in innate immune responses. We found that NLRC5 is a cytosolic protein expressed predominantly in hematopoetic cells. NLRC5 mRNA and protein expression was inducible by the double-stranded RNA analog poly(I·C) and Sendai virus. Overexpression of NLRC5 failed to trigger inflammatory responses such as the NF-κB or interferon pathways in HEK293T cells. However, knockdown of endogenous NLRC5 reduced Sendai virus- and poly(I·C)-mediated type I interferon pathway-dependent responses in THP-1 cells and human primary dermal fibroblasts. Taken together, this defines a function for NLRC5 in anti-viral innate immune responses.

Interferon-γ and Tumor Necrosis Factor-α Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis

BACKGROUND & AIMS Viral clearance involves immune cell cytolysis of infected cells. However, studies of hepatitis B virus (HBV) infection in chimpanzees have indicated that cytokines released by T cells also can promote viral clearance via noncytolytic processes. We investigated the noncytolytic mechanisms by which T cells eliminate HBV from infected hepatocytes. METHODS We performed a cytokine enzyme-linked immunosorbent assay of serum samples from patients with acute and chronic hepatitis B. Liver biopsy specimens were analyzed by in situ hybridization. HepG2-H1.3 cells, HBV-infected HepaRG cells, and primary human hepatocytes were incubated with interferon-γ (IFNγ) or tumor necrosis factor-α (TNF-α), or co-cultured with T cells. We measured markers of HBV replication, including the covalently closed circular DNA (cccDNA). RESULTS Levels of IFNγ and TNF-α were increased in serum samples from patients with acute vs chronic hepatitis B and controls. In human hepatocytes with stably replicating HBV, as well as in HBV-infected primary human hepatocytes or HepaRG cells, IFNγ and TNF-α each induced deamination of cccDNA and interfered with its stability; their effects were additive. HBV-specific T cells, through secretion of IFNγ and TNF-α, inhibited HBV replication and reduced cccDNA in infected cells without the direct contact required for cytolysis. Blocking IFNγ and TNF-α after T-cell stimulation prevented the loss of cccDNA. Deprivation of cccDNA required activation of nuclear APOBEC3 deaminases by the cytokines. In liver biopsy specimens from patients with acute hepatitis B, but not chronic hepatitis B or controls, hepatocytes expressed APOBEC3A and APOBEC3B. CONCLUSIONS IFNγ and TNF-α, produced by T cells, reduce levels of HBV cccDNA in hepatocytes by inducing deamination and subsequent cccDNA decay.

Dendritic cells take up viral antigens but do not support the early steps of hepatitis B virus infection

Dendritic cells (DC) of hepatitis B virus (HBV) carriers have been reported to exhibit functional impairment. Possible explanations for this phenomenon are infection of HBV by DC or alteration of DC function by HBV. We therefore analyzed whether DC support the different steps of HBV infection and replication: uptake, deposition of the HBV genome in the nucleus, antigen expression, and progeny virus release. When HBV genomes were artificially introduced into monocyte‐derived DC by adenoviral vectors, low‐level expression of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) but no HBV replication was detected. When monocyte‐derived DC were subjected to wild‐type HBV or a recombinant HBV expressing Renilla luciferase under a non–liver‐specific promoter, intracellular HBV DNA was detected in a low percentage of cells. However, neither nuclear cccDNA was formed nor luciferase activity was detected, indicating that either uncoating or nucleocytoplasmic transport were blocked. To verify our observation in the in vivo situation, myeloid and plasmacytoid DC were isolated from blood of high viremic HBV carriers, and analyzed by quantitative polymerase chain reaction (PCR) and electron microscopy. Although circulating DC had in vivo been exposed to more than 104 HBV virions per cell, HBV genomic DNA was hardly detected, and no nuclear cccDNA was detected at all. By using electron microscopy, subviral particles were found in endocytic vesicles, but virions were undetectable as were viral capsids in the cytoplasm. In conclusion, circulating DC may take up HBV antigens, but neither support nucleocytoplasmic transport nor replication of HBV. (HEPATOLOGY 2006;43:539–547.)

A concerted action of HNF4alpha and HNF1alpha links hepatitis B virus replication to hepatocyte differentiation.

Hepatitis B virus (HBV) is an important human pathogen, which targets the liver extremely efficient, gaining access to hepatocytes by a so far unknown receptor and replicating in a hepatocyte‐specific fashion. Cell differentiation seems to determine HBV replication. We here show that the level of hepatocyte differentiation, as indicated by hepatocyte polarization and metabolic activity, is closely correlated to the transcription of the HBV RNA pregenome. Pregenome transcription determined the level of HBV replication in various cell lines of hepatocellular origin and in primary human hepatocytes. A variety of hepatocyte‐enriched nuclear factors have been described to regulate transcription of the pregenome, but it remained unknown which factors link HBV replication to hepatocyte differentiation. We determined that high expression levels of HNF4α but not its potential cofactors or other hepatocyte‐enriched transcription factors were essential for efficient HBV replication, and link it to hepatocyte differentiation. HNF1α contributed to the control of HBV replication because it regulated the expression of HNF4α. Thus, a concerted action of HNF4α and HNF1α, which also determines morphological and functional differentiation of hepatocytes, links HBV replication to hepatocyte differentiation.

Toll‐like receptor 2–mediated innate immune response in human nonparenchymal liver cells toward adeno‐associated viral vectors

Adeno‐associated viral vectors (rAAV) are frequently used in gene therapy trials. Although rAAV vectors are of low immunogenicity, humoral as well as T cell responses may be induced. While the former limits vector reapplication, the expansion of cytotoxic T cells correlates with liver inflammation and loss of transduced hepatocytes. Because adaptive immune responses are a consequence of recognition by the innate immune system, we aimed to characterize cell autonomous immune responses elicited by rAAV in primary human hepatocytes and nonparenchymal liver cells. Surprisingly, Kupffer cells, but also liver sinusoidal endothelial cells, mounted responses to rAAV, whereas neither rAAV2 nor rAAV8 were recognized by hepatocytes. Viral capsids were sensed at the cell surface as pathogen‐associated molecular patterns by Toll‐like receptor 2. In contrast to the Toll‐like receptor 9–mediated recognition observed in plasmacytoid dendritic cells, immune recognition of rAAV in primary human liver cells did not induce a type I interferon response, but up‐regulated inflammatory cytokines through activation of nuclear factor κB. Conclusion: Using primary human liver cells, we identified a novel mechanism of rAAV recognition in the liver, demonstrating that alternative means of sensing rAAV particles have evolved. Minimizing this recognition will be key to improving rAAV‐mediated gene transfer and reducing side effects in clinical trials due to immune responses against rAAV. (Hepatology 2012;55:287–297)

Intrinsic phospholipase A2 activity of adeno-associated virus is involved in endosomal escape of incoming particles.

The unique region of the VP1 capsid protein of adeno-associated viruses (AAV) in common with autonomously replicating parvoviruses comprises a secreted phospholipase A2 (sPLA2) homology domain. While the sPLA2 domain of Minute Virus of Mice has recently been shown to mediate endosomal escape by lipolytic pore formation, experimental evidence for a similar function in AAV infection is still lacking. Here, we explored the function of the sPLA2 domain of AAV by making use of the serotype 2 mutant (76)HD/AN. The sPLA2 defect in (76)HD/AN, which severely impairs AAVs infectivity, could be complemented in trans by co-infection with wild-type AAV2. Furthermore, co-infection with endosomolytically active, but not with inactive adenoviral variants partially rescued (76)HD/AN, providing the first evidence for a function of this domain in endosomal escape of incoming AAV particles.

Apoptosis of hepatitis B virus-infected hepatocytes prevents release of infectious virus.

ABSTRACT Apoptosis of infected cells is critically involved in antiviral defense. Apoptosis, however, may also support the release and spread of viruses. Although the elimination of infected hepatocytes is required to combat hepatitis B virus (HBV) infection, it is still unknown which consequences hepatocyte apoptosis has for the virus and whether or not it is advantageous to the virus. To study this, we designed a cell culture model consisting of both HBV-producing cell lines and primary human hepatocytes serving as an infection model. We showed that the release of mature, enveloped virions was 80% to 90% reduced 24 h after the induction of apoptosis in HBV-replicating hepatoma cells or HBV-infected hepatocytes. Importantly, HBV particles released from apoptotic hepatocytes were immature and nonenveloped and proved not to be infectious. We found an inverse correlation between the strength of an apoptotic stimulus and the infectivity of the virus particles released: the more potent the apoptotic stimulus, the higher the ratio of nonenveloped capsids to virions and the lower their infectivity. Furthermore, we demonstrated that HBV replication and, particularly, the expression of the HBx protein transcribed from the viral genome during replication do not sensitize cells to apoptosis. Our data clearly reject the hypothesis that the apoptosis of infected hepatocytes facilitates the propagation of HBV. Rather, these data indicate that HBV needs to prevent the apoptosis of its host hepatocyte to ensure the release of infectious progeny and, thus, virus spread in the liver.

PtdIns(4,5)P‐restricted plasma membrane localization of FAN is involved in TNF‐induced actin reorganization

The WD‐repeat protein factor associated with nSMase activity (FAN) is a member of the family of TNF receptor adaptor proteins that are coupled to specific signaling cascades. However, the precise functional involvement of FAN in specific cellular TNF responses remain unclear. Here, we report the involvement of FAN in TNF‐induced actin reorganization and filopodia formation mediated by activation of Cdc42. The pleckstrin‐homology (PH) domain of FAN specifically binds to phosphatidylinositol‐4,5‐bisphosphate (PtdIns(4,5)P), which targets FAN to the plasma membrane. Site‐specific mutagenesis revealed that the ability of FAN to mediate filopodia formation was blunted either by the destruction of the PtdIns(4,5)P binding motif, or by the disruption of intramolecular interactions between the PH domain and the adjacent beige and Chediak‐Higashi (BEACH) domain. Furthermore, FAN was shown to interact with the actin cytoskeleton in TNF‐stimulated cells via direct filamentous actin (F‐actin) binding. The results of this study suggest that PH‐mediated plasma membrane targeting of FAN is critically involved in TNF‐induced Cdc42 activation and cytoskeleton reorganization.

Identification of Specific Cellular Genes Up-Regulated Late in Adenovirus Type 12 Infection

ABSTRACT The infection of human cells by adenoviruses leads to a gradual reduction in the activity of host cell functions while viral gene expression progresses in a regulated way. We used the DNA microarray technique to determine the transcriptional activity profiles of cellular genes upon infection with adenovirus type 12 (Ad12). The microarray data were validated by quantitative real-time PCR for genes which showed significant alterations after Ad12 infection. At 12 h postinfection, there is a striking up-regulation between 10- and 30-fold in the expression of the G1P2, IFIT1, and IFIT2 cellular immune response genes compared to mock-infected cells. At later stages of infection, when the majority of regulated cellular genes has been turned down, a limited number of cellular genes exhibit increased activities by factors of 3 or less. These genes belong to the signal transduction or transcriptional regulator classes or are active in protein degradation, like ANPEP, an aminopeptidase. The SCD and CYP2S1 genes function in lipid metabolism. The eucaryotic translation initiation factor 4 is up-regulated, and one of the major histocompatibility complex genes is diminished in activity. For two of the genes, one up-regulated (CTSF gene) and one down-regulated (CYR61 gene), alterations in gene activity were confirmed at the protein level by Western blotting experiments. Increased genetic activity of cellular genes late in adenovirus infection has not been reported previously and demonstrates that Ad12 has a sustained control of host cell gene expression well into the late phase of infection.