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

Treatment with tenofovir disoproxil fumarate or entecavir in chronic hepatitis B virus-infected patients with renal impairment: results from a 7-year, multicentre retrospective cohort study.

BackgroundLimited data exist regarding tenofovir disoproxil fumarate (TDF) safety and effectiveness in chronic hepatitis B virus-infected (CHB) patients with renal impairment (RI).AimsTo compare real-world data on renal safety and effectiveness of TDF vs entecavir (ETV) in CHB patients with moderate-to-severe RI.MethodsRetrospective, non-interventional, cohort study analysing medical records for TDF/ETV-treated CHB patients (54 European centres). Included patients experienced moderate-to-severe RI (creatinine clearance 20-60 mL/min [Cockcroft-Gault]) either before TDF/ETV initiation ('before' subgroup [baseline = treatment initiation]) or after TDF/ETV initiation ('after' subgroup [baseline = first RI occurrence]). The primary objective was TDF safety, particularly renal-related adverse events of special interest (AESI). TDF and ETV safety and effectiveness were compared and multivariate analyses were performed using inverse probability treatment weighting.Results'Before' subgroup included 107 TDF- and 91 ETV-treated patients; 'after' subgroup included 212 TDF- and 77 ETV-treated patients. Mean baseline creatinine clearance was higher for TDF- vs ETV-treated patients (both subgroups). Median follow-up was 3.1 years (both treatments). AESI were more frequent with TDF vs ETV ('before': 18.7% vs 8.8%; 'after': 9.9% vs 3.9%); however, differences were not significant by multivariate analysis. Only TDF-treated patients experienced renal tubular dysfunction (6.5% 'before'; 1.9% 'after') as well as renal adverse events leading to treatment discontinuation (8.4% 'before'; 7.1% 'after'). Effectiveness was similar between treatments.ConclusionsOverall safety was similar for TDF vs ETV (both subgroups). Given that renal tubular dysfunction occurred with TDF and not with ETV, renal safety concerns may be greater with TDF in CHB patients with RI

HBV inhibits cytokine production and pDC-induced NK cell activation.

<p><b>ABCD</b>: Purified pDC were cultured with CpG in the presence or absence HepG2.215-derived HBV. Supernatants were analysed for TNFα (A), IP-10 (B), IL-6 (C) and IL-8 (D). Data demonstrate mean±SEM of 8 independent experiments. <b>EFG</b>: Purified pDC were stimulated with CpG in the presence or absence of patient serum-derived HBV. Healthy control serum was treated in a similar way and added in the same volume to pDC. After 24h, supernatants were harvested and tested for the presence of IFNα (E), TNFα (F) and IL-6 (G) by ELISA. Shown is the mean±SD of triplicate cultures from one out of 2 experiments with different donors. <b>HI</b>: NK cells were cultured with or without pDC and with or without HepG2.215-derived HBV in medium containing IL-3 and CpG. Data show mean±SEM CD25 expression on CD56+ cells (E) and IFNγ production (F) of 7 independent experiments. *p<0.05, Wilcoxon signed rank test.</p

HBV and HBsAg inhibit CpG-induced S6 phosphorylation.

<p><b>A</b>: pDC were cultured with CpG in the presence or absence of HepG2.215-derived HBV. Data show the expression of phosphorylated S6 and the mean fluorescence intensity (MFI) and are representative for 8 independent experiments. <b>B</b>: pDC were cultured with or without CpG in the presence or absence of HBcAg, HBeAg, or HBsAg. Data show expression of phosphorylated S6 and the MFI and are representative for 3 independent experiments. <b>C</b>: Data show mean±SEM expression of phosphorylated S6 in BDCA4⁺CD123⁺ pDC in PBMC exposed to CpG or Lox in the presence or absence of HepG2.215-derived HBV from 6 independent experiments. *p<0.05, Wilcoxon signed rank test.</p

HBV does not activate pDC.

<p><b>A</b>: PBMC were stimulated with CpG and analysed for IFNα production by pDC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015324#s4" target="_blank"><i>Materials and Methods</i></a>. Shown is a representative FACS plot of PBMC stained for CD123 and BDCA-4 to identify pDC and the detection of IFNα positive cells within this pDC-gate. <b>B</b>: IFNα and TNFα producing pDC within PBMC cultured in the presence or absence of HepG2.215-derived HBV, CpG, HSV-1, Lox or Influenza are presented as mean±SEM of 10 independent experiments.</p

HBV dose-dependently inhibits CpG-induced transcription of IFNα in pDC.

<p><b>A</b>: IFNα production by pDC cultured in medium, CpG, HSV-1, Lox or Influenza with or without HepG2.215-derived HBV was determined by flow cytometry or ELISA. Mean±SEM of 3 independent experiments. <b>B</b>: CpG-activated pDC were cultured with different doses of HepG2.215-derived HBV. IFNα production determined by ELISA is presented as mean±SEM of 16 independent experiments with different donors. <b>C</b>: pDC were cultured with CpG in the presence or absence of HepG2.215-derived HBV for 3h. Data present the mean±SEM percentage of cells positive for phosphorylated IRF7 from 5 independent experiments. <b>DE</b>: pDC were cultured with CpG in the presence or absence of HepG2.215-derived HBV for 2 or 4h. Data represent IFNα2 (<b>DE</b>) and IFNα8 (<b>E</b>) mRNA levels normalized to GAPDH and are representative for 6 experiments (<b>D</b>) or show the mean±SEM IFNα mRNA levels relative to cultures without HBV (n = 6) (<b>E</b>). *p<0.05, **p<0.01, p<0.001, Wilcoxon signed rank test.</p

HBsAg and HBeAg inhibit cytokine production by pDC.

<p>pDC were cultured with CpG in the presence or absence of increasing doses or 5 µg/ml of HBcAg, HBeAg or HBsAg. Supernatants were harvested and analysed for IFNα (A), TNFα (B), IP-10 (C), IL-6 (D), and IL-8 (E). Data presented are mean±SEM of at least 8 independent experiments. *P<0.05, Wilcoxon signed rank test compared to control.</p

Effect of HBV on pDC maturation.

<p>To investigate possible pDC activation by HepG2.215-derived HBV in comparison with other pDC stimuli, PBMC were cultured in the presence or absence of HBV (100 geq/cell), CpG, HSV-1, Lox or Influenza (see ‘ctr’ lane). To investigate the immune regulatory effect of HBV on pDC maturation induced by other known pDC activating ligands, PBMC were cultured with or without CpG, HSV-1, Lox or Influenza in the presence (‘HBV’ lane) or absence (‘ctr’ lane) of HBV (100 geq/cell). After 24h, cells were harvested and the expression of CD40, CD80, CD86 and HLA-DR on pDC was determined by flow cytometry. Data are presented as mean±SEM fluorescent intensity of 3 independent experiments with different donors. Similar data were observed for purified pDC (not shown).</p><p>*p<0.05, paired t-test; nd = not done.</p

Hepatitis B virus surface antigen impairs myeloid dendritic cell function: a possible immune escape mechanism of hepatitis B virus

Chronic hepatitis B virus (HBV) infection is the result of an inadequate immune response towards the virus. Myeloid dendritic cells (mDC) of patients with chronic HBV are impaired in their maturation and function, resulting in more tolerogenic rather than immunogenic responses, which may contribute to viral persistence. The mechanism responsible for altered mDC function remains unclear. The HBV-infected patients display large amounts of HBV particles and viral proteins in their circulation, especially the surface antigen HBsAg, which allows multiple interactions between the virus, its viral proteins and DC. To assess whether HBV directly influences mDC function, the effects of HBV and HBsAg on human mDC maturation and function were investigated in vitro. As already described for internalization of HBV by DC, the present study shows that peripheral blood-derived mDC of healthy controls also actively take up HBsAg in a time-dependent manner. Cytokine-induced maturation in the presence of HBV or HBsAg resulted in a significantly more tolerogenic mDC phenotype as demonstrated by a diminished up-regulation of costimulatory molecules and a decreased T-cell stimulatory capacity, as assessed by T-cell proliferation and interferon-γ production. In addition, the presence of HBV significantly reduced interleukin-12 production by mDC. These results show that both HBV particles and purified HBsAg have an immune modulatory capacity and may directly contribute to the dysfunction of mDC in patients with chronic HBV. The direct immune regulatory effect of HBV and circulating HBsAg particles on the function of DC can be considered as part of the mechanism by which HBV escapes immunity