Hepatitis B Virus Lacks Immune Activating Capacity, but Actively Inhibits Plasmacytoid Dendritic Cell Function

Chronic hepatitis B virus (HBV) infection is caused by inadequate anti-viral immunity. Activation of plasmacytoid dendritic cells (pDC) leading to IFNα production is important for effective anti-viral immunity. Hepatitis B virus (HBV) infection lacks IFNα induction in animal models and patients and chronic HBV patients display impaired IFNα production by pDC. Therefore, HBV and HBV-derived proteins were examined for their effect on human pDC in vitro. In addition, the in vitro findings were compared to the function of pDC derived from chronic HBV patients ex vivo. In contrast to other viruses, HBV did not activate pDC. Moreover, HBV and HBsAg abrogated CpG-A/TLR9-induced, but not Loxoribine/TLR7-induced, mTOR-mediated S6 phosphorylation, subsequent IRF7 phosphorylation and IFNα gene transcription. HBV/HBsAg also diminished upregulation of co-stimulatory molecules, production of TNFα, IP-10 and IL-6 and pDC-induced NK cell function, whereas TLR7-induced pDC function was hardly affected. In line, HBsAg preferentially bound to TLR9-triggered pDC demonstrating that once pDC are able to bind HBV/HBsAg, the virus exerts its immune regulatory effect. HBV not only directly interfered with pDC function, but also indirectly by interfering with monocyte-pDC interaction. Also HBeAg diminished pDC function to a certain extent, but via another unknown mechanism. Interestingly, patients with HBeAg-positive chronic hepatitis B displayed impaired CpG-induced IFNα production by pDC without significant alterations in Loxoribine-induced pDC function compared to HBeAg-negative patients and healthy controls. The lack of activation and the active inhibition of pDC by HBV may both contribute to HBV persistence. The finding that the interaction between pDC and HBV may change upon activation may aid in the identification of a scavenging receptor supporting immunosuppressive effects of HBV and also in the design of novel treatment strategies for chronic HBV

Favorable effect of adefovir on the number and functionality of myeloid dendritic cells of patients with chronic HBV

In patients with chronic hepatitis B virus (HBV), 2 predominant precursor dendritic cell (DC) subtypes, the myeloid dendritic cell (mDC) and the plasmacytoid dendritic cell (pDC), were recently found to be functionally impaired. HBV DNA was found to be present in the DC subtypes, but no viral replication could be detected. The question remains whether simply the presence of the virus and viral proteins causes this dysfunction of DCs. To address this issue, the effect of viral load reduction resulting from treatment with the nucleotide analogue adefovir dipivoxil on the number and functionality of circulating DCs was studied during 6 months of treatment. Treatment resulted in a mean 5 log(10) decrease in the viral load and normalization of alanine aminotransferase within 3 months. The number of mDCs, but not of pDCs, increased significantly over 6 months of treatment to a level comparable to that of uninfected healthy controls. The allostimulatory capacity of isolated and in vitro matured mDCs increased significantly after 3 months of treatment. Accordingly, mDCs exhibited an increased capacity to produce tumor necrosis factor alpha and interleukin-12 after 3-6 months of treatment. There was no change in interferon alpha production by pDCs during treatment. In conclusion, adefovir treatment results in an improvement in the number and functionality of mDCs, but not of pDCs. Our findings provide clues for the reasons why current antiviral therapy does not lead to consistently sustained viral eradicatio

Intrahepatic natural killer cell activation, but not function, is associated with HBsAg levels in patients with HBeAg-negative chronic hepatitis B

Abstract: Background & Aims: Natural killer (NK) cells play an important role in the immune response to viruses. As the hepatitis B virus (HBV) replicates in hepatocytes, examination of the liver of chronic hepatitis B (CHB) patients is crucial to better understand the role of NK cells in HBV. HBeAg-negative CHB differs in many aspects from HBeAg-positive CHB, and until now little is known about the intrahepatic NK cell response in HBeAg-negative patients. Intrahepatic immune control might be different in HBeAg-negative as compared with HBeAg-positive patients. Methods: Liver NK cells were investigated in 21 HBeAg-positive and 35 HBeAg-negative CHB patients. Biopsy specimens were processed for routine histopathology and staging according to Ishak scores. Intrahepatic and blood NK cell frequencies, activation status and function of NK cells were analysed by flow cytometry. Results: In HBeAg-negative CHB patients, compared to blood, liver NK cells displayed a more activated phenotype and stimulation further increased the activation status, but production of IFN-γ was markedly less. There was no difference with HBeAg-positive CHB. Only in HBeAg-negative CHB, but not in HBeAg-positive CHB, NK cell activation was inversely correlated with HBsAg levels. Conclusions: The present study indicates that liver NK cells of CHB have a higher activation status compared to blood. However, they are not capable to increase cytokine production above levels reached by activated blood NK cells. In HBeAg-negative CHB, the levels of HBsAg may contribute to the incapacity of activated liver NK cells to increase cytokine production

The Effect of Chronic Hepatitis B Virus Infection on BDCA3+ Dendritic Cell Frequency and Function

<div><p>Chronic hepatitis B virus (HBV) infection results from inadequate HBV-specific immunity. BDCA3⁺ dendritic cells (DCs) are professional antigen presenting cells considered to be important for antiviral responses because of specific characteristics, including high interferon-λ production. BDCA3⁺ DCs may thus also have a role in the immune response against HBV, and immunotherapeutic strategies aiming to activate DCs, including BDCA3⁺ DCs, in patient livers may represent an interesting treatment option for chronic HBV. However, neither the effect of chronic hepatitis B (CHB) infection on the frequency and function of BDCA3⁺ DCs in liver and blood, nor the effect of the viral surface protein (HBsAg) that is abundantly present in blood of infected individuals are known. Here, we provide an overview of BDCA3⁺ DC frequency and functional capacity in CHB patients. We find that intrahepatic BDCA3⁺ DC numbers are increased in CHB patients. BDCA3⁺ DCs from patient blood are not more mature at steady state, but display an impaired capacity to mature and to produce interferon-λ upon polyI:C stimulation. Furthermore, in vitro experiments exposing blood and intrahepatic BDCA3⁺ DCs to the viral envelope protein HBsAg demonstrate that HBsAg does not directly induce phenotypical maturation of BDCA3⁺ DCs, but may reduce IFN-λ production via an indirect unknown mechanism. These results suggest that BDCA3⁺ DCs are available in the blood and on site in HBV infected livers, but measures may need to be taken to revive their function for DC-targeted therapy.</p></div

HBsAg diminishes IFN-λ1 production by BDCA3⁺ DCs.

<p>(A) PBMC from healthy subjects were incubated with or without fluorescently-labeled HBsAg for 2 hours at indicated temparatures and HBsAg binding/uptake by BDCA3⁺ DCs was measured by flow cytometry. Representative plots of 3 independent experiments and donors are shown. (B) PBMC from healthy subjects were stimulated for 6 hours with or without rHBsAg, pHBsAg or polyI:C and maturation marker-expression on BDCA3⁺ DCs was analyzed by flow cytometry (n = 3; mean±SEM; * p<0.05 by paired Student’s <i>t</i>-test). (C-D) PBMC from healthy subjects were stimulated for 7 hours with polyI:C in the presence or absence of rHBsAg and the production of IFN-λ1 by BDCA3⁺ DCs was measured by ICS. Representative flow cytometry plots (C) and the summarized percentage of IFN-λ1-producing BDCA3⁺ DCs (D; n = 7; mean±SEM) are shown. To determine the percentage of IFN-λ-producing BDCA3⁺ DCs in blood, a minimum threshold of 70 BDCA3⁺ DCs was used. **<i>p <</i> 0.01 by paired Student’s <i>t</i>-test. (E) Liver cells from peri-tumor liver tissue were stimulated for 5 hours with or without polyI:C in the presence or absence of rHBsAg and the production of IFN-λ1 by BDCA3⁺ DCs was measured by ICS. The percentage of IFN-λ1-producing BDCA3⁺ DCs is shown (n = 3; mean±SEM). **<i>p <</i> 0.01 by paired Student’s <i>t</i>-test.</p