Association of HBsAg levels with differential gene expression in NK, CD8 T, and memory B cells in treated patients with chronic HBV.

Background & Aims: HBsAg secretion may impact immune responses to chronic HBV infection. Thus, therapeutic approaches to suppress HBsAg production are being investigated. Our study aims to examine the immunomodulatory effects of high and low levels of circulating HBsAg and thereby improve our understanding of anti-HBV immunity. Methods: An optimized 10x Genomics single-cell RNA sequencing workflow was applied to blood samples and liver fine-needle aspirates from 18 patients undergoing tenofovir/entecavir (NUC) treatment for chronic HBV infection. They were categorized based on their HBsAg levels: high (920-12,447 IU/ml) or low (1-100 IU/ml). Cluster frequencies, differential gene expression, and phenotypes were analyzed. Results: In the blood of HBV-infected patients on NUC, the proportion of KLRC2+ "adaptive" natural killer (NK) cells was significantly lower in the HBsAg-high group and, remarkably, both KLRC2+ NK and KLRG1+ CD8 T cells display enrichment of lymphocyte activation-associated gene sets in the HBsAg-low group. High levels of HBsAg were associated with mild immune activation in the liver. However, no suppression of liver-resident CXCR6+ NCAM1+ NK or CXCR6+ CD69+ CD8 T cells was detected, while memory B cells showed signs of activation in both the blood and liver. Conclusions: Among NUC-treated patients, we observed a minimal impact of HBsAg on leukocyte populations in the blood and liver. However, for the first time, we found that HBsAg has distinct effects, restricted to NK-, CD8 T-, and memory B-cell subsets, in the blood and liver. Our findings are highly relevant for current clinical studies evaluating treatment strategies aimed at suppressing HBsAg production and reinvigorating immunity to HBV. Impact and implications: This study provides unique insight into the impact of HBsAg on gene expression levels of immune cell subsets in the blood and liver, particularly in the context of NUC-treated chronic HBV infection. It holds significant relevance for current and future clinical studies evaluating treatment strategies aimed at suppressing HBsAg production and reinvigorating immunity to HBV. Our findings raise questions about the effectiveness of such treatment strategies and challenge the previously hypothesized immunomodulatory effects of HBsAg on immune responses against HBV.

Single-cell RNA sequencing of liver fine-needle aspirates captures immune diversity in the blood and liver in chronic hepatitis B patients.

BACKGROUND AND AIMS: HBV infection is restricted to the liver, where it drives exhaustion of virus-specific T and B cells and pathogenesis through dysregulation of intrahepatic immunity. Our understanding of liver-specific events related to viral control and liver damage has relied almost solely on animal models, and we lack useable peripheral biomarkers to quantify intrahepatic immune activation beyond cytokine measurement. Our objective was to overcome the practical obstacles of liver sampling using fine-needle aspiration and develop an optimized workflow to comprehensively compare the blood and liver compartments within patients with chronic hepatitis B using single-cell RNA sequencing. APPROACH AND RESULTS: We developed a workflow that enabled multi-site international studies and centralized single-cell RNA sequencing. Blood and liver fine-needle aspirations were collected, and cellular and molecular captures were compared between the Seq-Well S 3 picowell-based and the 10× Chromium reverse-emulsion droplet-based single-cell RNA sequencing technologies. Both technologies captured the cellular diversity of the liver, but Seq-Well S 3 effectively captured neutrophils, which were absent in the 10× dataset. CD8 T cells and neutrophils displayed distinct transcriptional profiles between blood and liver. In addition, liver fine-needle aspirations captured a heterogeneous liver macrophage population. Comparison between untreated patients with chronic hepatitis B and patients treated with nucleoside analogs showed that myeloid cells were highly sensitive to environmental changes while lymphocytes displayed minimal differences. CONCLUSIONS: The ability to electively sample and intensively profile the immune landscape of the liver, and generate high-resolution data, will enable multi-site clinical studies to identify biomarkers for intrahepatic immune activity in HBV and beyond.

Differential gene expression, irrespective of circulating Hepatitis B Surface Antigen levels, between Inactive Carrier and Nucleos(t)ide Analogue-Treated Hepatitis B Virus patients.

Long-term viremia control in chronic HBV patients occurs either spontaneously in inactive carrier (IC) patients or therapy-induced by nucleos(t)ide analogues (NUC). To better understand the characteristics of viremia control, we evaluated gene expression in purified leukocyte subsets from IC versus NUC-treated patients, and evaluated the putative modulatory effects of hepatitis B surface antigen (HBsAg). We observed that gene expression in NUC-treated patients differed markedly from IC patients, especially in dendritic cells, monocytes, and CD8+ T cells, while serum HBsAg levels had little effect. Nevertheless, based on our findings it cannot be excluded that HBsAg may act locally in the infected liver or preferentially affects HBV-specific cells.

Treatment induced clearance of hepatitis E viruses by interferon-lambda in liver-humanized mice.

BACKGROUND: Hepatitis E viruses (HEV) are an underestimated global cause of enterically transmitted viral hepatitis, which may persist in immunocompromised hosts, posing a risk for progressive liver fibrosis with limited treatment options. We previously established liver-humanized mice as a model for chronic HEV infections, which can be cleared by a 2-week pegylated (peg)-Interferon(IFN)α treatment course. However, severe side effects may hamper the use of IFNα in immunocompromised transplant recipient patients. IFNλ may be a valuable alternative, as its receptor is less ubiquitously expressed. AIMS: In this study, we assess the in vitro and in vivo potency of pegIFNλ to induce innate immune signalling in liver cells and to clear a persistent HEV infection in liver-humanized mice. METHODS & RESULTS: We found that human liver cells expressed the IFNλ receptor (IFNLR1) and are responsive to pegIFNλ. Treatment with pegIFNλ of liver-humanized mice persistently infected with HEV genotype 3 showed that pegIFNλ was well tolerated. Dose escalation studies showed that although HEV was not cleared at pegIFNλ doses up to 0.12 mg/kg for a maximum of 8 weeks, a dose of 0.3 mg/kg pegIFNλ treatment resulted in complete clearance of HEV antigen and HEV RNA from the liver in 8 out of 9 liver-humanized mice. CONCLUSIONS: PegIFNλ is well tolerated in mice and leads to clearance of a persistent HEV infection in liver-humanized mice.

Sexual Dimorphism in Hepatocyte Xenograft Models.

Humanized liver mouse models are crucial tools in liver research, specifically in the fields of liver cell biology, viral hepatitis and drug metabolism. The livers of these humanized mouse models are repopulated by 3-dimensional islands of fully functional primary human hepatocytes (PHH), which are notoriously difficult to maintain in vitro. As low efficiency and high cost hamper widespread use, optimization is of great importance. In the present study, we analyzed experimental factors associated with Hepatitis E virus (HEV) infection and PHH engraftment in 2 xenograft systems on a Nod-SCID-IL2Ry-/- background: the alb-urokinase plasminogen activator mouse model (uPA-NOG, n=399); and the alb-HSV thymidine kinase model (TK-NOG, n = 198). In a first analysis, HEV fecal shedding in liver humanized uPA-NOG and TK-NOG mice with comparable human albumin levels was found to be similar irrespective of the mouse genetic background. In a second analysis, sex, mouse age at transplantation and hepatocyte donor were the most determinant factors for xenograft success in both models. The sexual imbalance for xenograft success was related to higher baseline ALT levels and lower thresholds for ganciclovir induced liver morbidity and mortality in males. These data call for sexual standardization of human hepatocyte xenograft models, but also provide a platform for further studies on mechanisms behind sexual dimorphism in liver diseases.

Hepatitis B core-related antigen monitoring during peginterferon alfa treatment for HBeAg-negative chronic hepatitis B.

Serum Hepatitis B core-related antigen (HBcrAg) level moderately correlates with cccDNA. We examined whether HBcrAg can add value in monitoring the effect of peginterferon (PEG-IFN) therapy for HBeAg-negative chronic hepatitis B (CHB) infection. Thus, serum HBcrAg level was measured in 133 HBeAg-negative, mainly Caucasian CHB patients, treated with 48 weeks of PEG-IFN alfa-2a. We assessed its association with response (ALT normalization & HBV DNA < 2000 IU/mL) at week 72. HBcrAg level strongly correlated with HBV DNA level (r = 0.8, P < 0.001) and weakly with qHBsAg and ALT (both r = 0.2, P = 0.01). At week 48, mean HBcrAg decline was -3.3 log U/mL. Baseline levels were comparable for patients with and without response at week 72 (5.0 vs 4.9 log U/mL, P = 0.59). HBcrAg decline at week 72 differed between patients with and without response (-2.4 vs -1.0 log U/mL, P = 0.001), but no cut-off could be determined. The pattern of decline in responders resembled that of HBV DNA, but HBcrAg decline was weaker (HBcrAg -2.5 log U/mL; HBV DNA: -4.0 log IU/mL, P < 0.001). For early identification of nonresponse, diagnostic accuracy of HBV DNA and qHBsAg decline at week 12 (AUC 0.742, CI-95% [0.0.629-0.855], P < 0.001) did not improve by adding HBcrAg decline (AUC 0.747, CI-95% [0.629-0.855] P < 0.001), nor by replacing HBV DNA decline by HBcrAg decline (AUC 0.754, CI-95% [0.641-0.867], P < 0.001). In conclusion, in Caucasian patients with HBeAg-negative CHB, decline of HBcrAg during PEG-IFN treatment was stronger in patients with treatment response. However, HBcrAg was not superior to HBV DNA and qHBsAg in predicting response during PEG-IFN treatment.

Diagnostic and analytical performance of the hepatitis B core related antigen immunoassay in hepatitis B patients.

BACKGROUND: Novel serological markers for Hepatitis B virus (HBV) infection are needed for prognosis and guidance of therapy. OBJECTIVE: We evaluated the diagnostic performance of the Fujirebio Lumipulse G HBcrAg immunoassay on the Fujirebio LUMIPULSE G1200 analyzer. STUDY DESIGN: Analytical performance was examined using three HBeAg positive HBV samples. Diagnostic specificity was assessed using subpanels of 54 confirmed acute HAV, HCV, HEV, B19, CMV and EBV infections. Diagnostic sensitivity was investigated in well-defined HBV positive patient groups, both treated and untreated, including immunocompromised patients. RESULTS: The Lumipulse G HBcrAg immunoassay provided a linear measurement at a dilution between 1:100 and1:10,000. Six out of 54 samples showed non-specific reactivity in sera from acute CMV, EBV and HEV infections, of which 2 of them >3 log U/ml. The highest levels of HBcrAg were measured in HBeAg positive patients, in both treated and untreated as well as in immunocompromised patients. Untreated patients had relatively low serum HBcrAg levels in the inactive carrier phase, which increased upon progression into the HBeAg-negative hepatitis phase. Also, we showed that the applicability of HBcrAg to distinguish between patients with resolved HBV infection and false-positive reactivity to solitary anti-HBc is limited. CONCLUSIONS: Our study demonstrated significant differences in HBcrAg levels depending on HBeAg status, the clinical phase, as well as the treatment status. Specificity of the assay is good; only 2 out of 54 samples showed reactivity above 3 log U/ml. Before implementing the assay in clinical practice, additional research in larger patient cohorts should be carried out.

Relationship between hepatitis B core-related antigen levels and sustained HBeAg seroconversion in patients treated with nucleo(s)tide analogues.

Hepatitis B e antigen (HBeAg) seroconversion experienced during nucleo(s)tide analogue (NUC) therapy is often not sustained. We aimed to study whether hepatitis B core-related antigen (HBcrAg) levels predict sustained HBeAg seroconversion in patients treated with NUCs. We studied HBeAg-positive patients treated with NUCs for at least 6 months. We quantified HBcrAg at baseline and at the time of HBeAg seroconversion and studied the relationship with HBeAg seroconversion and subsequent relapse. HBcrAg was quantified at baseline in 196 patients; levels varied significantly by HBV genotype and correlated with HBsAg, HBV DNA and HBeAg. Baseline HBcrAg levels were lower in patients who achieved HBeAg seroconversion than in those who did not; the unadjusted hazard ratio (HR) was 0.802 (95% CI: 0.656-0.980, P = 0.031); and this association was not sustained in multivariate analysis. HBcrAg remained detectable in all patients at the time of HBeAg seroconversion. Higher HBcrAg at the time of seroconversion was an independent predictor of relapse (adjusted HR: 1.855 (95% CI: 1.099-3.133, P = 0.021), and none of the patients with HBcrAg < 4.90 log U/mL experienced relapse. Baseline HBcrAg is not an independent predictor of HBeAg seroconversion during NUC therapy. HBcrAg remains detectable in patients after HBeAg seroconversion. Patients with lower levels at the time of seroconversion have a higher probability of sustained HBeAg seroconversion.

Gene expression profiling of human tissue-resident immune cells: Comparing blood and liver.

In this study, we describe a method to reliably characterize intrahepatic leukocyte populations using flow cytometry and next-generation RNA sequencing on fresh human liver biopsies. Over the last decades, immune responses of viral hepatitis patients, and of other liver diseases, have been incompletely characterized. Most studies include peripheral blood samples only, foregoing the possibility to investigate the site of inflammation directly. Here, we show that with an optimized protocol that combines cell sorting and RNA sequencing, we can perform a side by side comparison of both intrahepatic and peripheral immune cells. Using core liver biopsies from chronic hepatitis B virus patients, we show that the expression levels of IFN-stimulated genes and leukocyte-specific genes are markedly different in the liver compartment as compared to the peripheral blood. These observations emphasize the need to sample the liver directly. The variation of gene expression profiles in these chronic hepatitis B patients was considerable, despite the uniform treatment with nucleotide analogs and absence of liver inflammation in these patients. Finally, we show that this method can provide a detailed characterization of previously undetected liver-specific effects of novel candidate therapeutic compounds.

Mucosal-associated invariant T-cell frequency and function in blood and liver of HCV mono- and HCV/HIV co-infected patients with advanced fibrosis.

BACKGROUND & AIMS: Mucosal-associated invariant T (MAIT) cells are important innate T cells with antimicrobial and immunoregulatory activity, recently found to be depleted in blood of patients with HIV and HCV mono-infections. In this study, we assessed the impact of HIV, HCV and HCV/HIV co-infection on circulating and intrahepatic MAIT-cells and correlations with liver fibrosis. METHODS: In this cross-sectional study, nine healthy subjects, nine HIV, 20 HCV and 22 HCV/HIV co-infected patients were included. Blood and liver fine needle aspirate biopsies were studied using flowcytometry for CD3+ CD161+ Vα7.2+ MAIT-cell frequency, phenotype and function in HCV mono-infected and HCV/HIV co-infected patients without or with mild fibrosis (Metavir-score F0-F1) or severe fibrosis to cirrhosis (Metavir-score F3-F4). RESULTS: Circulating MAIT-cells were decreased in blood of HCV, HIV and HCV/HIV patients with F0-F1. In HCV/HIV co-infected individuals with severe fibrosis to cirrhosis, the frequency of circulating MAIT-cells was even further depleted, whereas their function was comparable to HCV/HIV co-infected patients with low or absent fibrosis. In contrast, in HCV mono-infected patients, MAIT-cell frequencies were not related to fibrosis severity; however, MAIT-cell function was impaired in mono-infected patients with more fibrosis. More advanced liver fibrosis in HCV or HCV/HIV-infected patients was not reflected by increased accumulation of MAIT-cells in the affected liver. CONCLUSIONS: Severe liver fibrosis is associated with dysfunctional MAIT-cells in blood of HCV mono-infected patients, and lower MAIT frequencies in blood of HCV/HIV co-infected patients, without evidence for accumulation in the liver.

Mucosal-Associated Invariant T Cells Are More Activated in Chronic Hepatitis B, but Not Depleted in Blood: Reversal by Antiviral Therapy.

Background: Mucosal-associated invariant T (MAIT) cells might play a role in control of viral replication during chronic hepatitis B (cHBV) infection, but little is known of their number, phenotype, or function in cHBV patients. Methods: We performed flow cytometry on CD3+Vɑ7.2+CD161+ MAIT cells in blood of 55 cHBV patients. Nine patients were sampled before and on entecavir treatment. Six patients on therapy underwent a liver biopsy for flow cytometric analysis. Measurements included MAIT cell frequency, phenotype, and cytokine-producing capacity. Results: The MAIT cells were not deleted in blood or liver of cHBV patients compared with healthy controls, but they had higher percentages of CD38+ MAIT cells in blood, which declined on entecavir treatment. Peripheral MAIT cells of patients in the HBeAg-negative phase were least activated. Cytokine-producing MAIT cells were as frequent, but granzyme B-producing MAIT cells were more frequent upon stimulation with Escherichia coli compared with healthy controls. Conclusions: We demonstrate that, in sharp contrast to hepatitis C virus and human immunodeficiency virus patients, MAIT cells isolated from HBV patients are not deleted but are more activated, which can be normalized by nucleoside analog therapy. These observations may aid in deciphering the role of MAIT cells in immune responses to HBV.

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.

NK cell phenotypic and functional shifts coincide with specific clinical phases in the natural history of chronic HBV infection.

BACKGROUND: Chronic HBV infection can be divided into 4 distinct clinical phases: immune tolerant, immune active, inactive carrier, and HBeAg-negative hepatitis. Using a systems biology approach, we recently identified innate immune response components, specifically NK cells as a distinctive factor of specific HBV clinical phases. To expand on this study and identify the underlying immunological mechanisms, we performed a comprehensive profiling of NK cells in chronic HBV infection. METHODS: Peripheral blood from untreated chronic HBV patients was used to analyze phenotypic markers, as well as cytokine production and cytoxicity of NK cells. RESULTS: The overall composition, phenotype, and cytolytic activity of the NK cells remained constant across all clinical phases, with the exception of a few specific markers (KIRs, NKp46). CD56bright NK cells of chronic HBV patients differed in their ability to produce IFN-γ between the clinical phases pre- and post-HBeAg seroconversion. CONCLUSION: This depicts a shift in NK cell characteristics between the immune active, under heavy viral or immune pressure, and inactive carrier phases, that coincides with HBeAg seroconversion. Although these changes in NK cells do not appear to be completely responsible for differences in liver damage characteristic of specific clinical phases, they could provide a step toward understanding immune dysregulation in chronic HBV infection.

Frequencies of Circulating MAIT Cells Are Diminished in Chronic HCV, HIV and HCV/HIV Co-Infection and Do Not Recover during Therapy.

OBJECTIVE: Mucosal-associated invariant T (MAIT) cells comprise a subpopulation of T cells that can be activated by bacterial products and cytokines to produce IFN-γ. Since little is known on MAIT cells during HCV infection, we compared their phenotype and function in comparison to HIV and HCV/HIV co-infected patients, and determined the effect of IFN-α-based and direct-acting antiviral therapy on MAIT cells of HCV patients. METHODS: Blood samples from patients with chronic HCV (CHCV), virologically suppressed HIV, acute HCV/HIV co-infection (AHCV/HIV) and healthy individuals were examined by flowcytometry for phenotype and function of MAIT and NK cells. RESULTS AND CONCLUSIONS: Compared to healthy individuals, the frequency of CD161+Vα7.2+ MAIT cells was significantly decreased in patients with CHCV, HIV and AHCV/HIV co-infection. CD38 expression on MAIT cells was increased in AHCV/HIV patients. MAIT cells were responsive to IFN-α in vitro as evidenced by enhanced frequencies of IFN-γ producing cells. IFN-α-based therapy for CHCV decreased the frequency of IFN-γ+ MAIT cells, which was still observed 24 weeks after successful therapy. Importantly, even after successful IFN-α-based as well as IFN-α-free therapy for CHCV, decreased frequencies of MAIT cells persisted. We show that the frequencies of MAIT cells are reduced in blood of patients with CHCV, HIV and in AHCV/HIV co-infection compared to healthy individuals. Successful therapy for CHCV did not normalize MAIT cell frequencies at 24 weeks follow up. The impact of HIV and HCV infection on the numbers and function of MAIT cells warrant further studies on the impact of viral infections and the antimicrobial function of MAIT cells.

Similar frequencies, phenotype and activation status of intrahepatic NK cells in chronic HBV patients after long-term treatment with tenofovir disoproxil fumarate (TDF).

BACKGROUND: Currently, much effort is directed at further improving treatment for chronic hepatitis B patients by assessing the effect of immunomodulatory agents during therapy with nucleotide analogues (NUC). Although there are some reports on the effect of NUC therapy on peripheral natural killer (NK) cells, no studies investigated the long-term effects of NUC treatment on intrahepatic NK cells of chronic HBV patients. We aimed to prospectively investigate cell frequencies, phenotype, and activation status of intrahepatic NK cells of CHB patients on prolonged treatment with TDF. METHODS: Fine needle aspiration biopsies were collected from 11 chronic HBV patients at baseline, and at 12, 24, and 48 weeks of treatment with a daily 245 mg dose of TDF. Four patients underwent an additional aspiration biopsy after appoximately 6 years of treatment. RESULTS: Longitudinal evaluation of these patients during tenofovir therapy showed that all patients achieved a viral load reduction with undetectable DNA load after 48 weeks of therapy. Repeated sampling of the liver during therapy showed that the frequency of distinct lymphocyte populations in the liver remained unchanged despite viral load reduction. During the course of therapy, no modulation of the expression levels and frequencies of CD69, HLA-DR, NKG2A and NKG2D on liver NK cells were detected. However, evaluation of intrahepatic NK cell activation after continuous TDF therapy for 6 years demonstrated a mild increase in 3 out of 4 patients. CONCLUSIONS: Our findings provide a unique insight in the intrahepatic NK cell compartment in chronic HBV patients during prolonged treatment. We observed that long-term NUC-induced viral suppression, accompanied by gradual decrease of HBsAg levels, had no or only a limited effect on the frequencies, phenotype, and activation status of intrahepatic NK cells.

Longitudinal analysis of peripheral and intrahepatic NK cells in chronic HCV patients during antiviral therapy.

INTRODUCTION: A strong immune response is integral to the clearance of HCV infection. NK cells are specialized cells that are able to inhibit replication of HCV in infected hepatocytes. Previous studies have correlated therapy outcome in HCV to the expression of various markers on NK cells. However, the effect of viral load reduction on NK cell function during therapy is still largely unknown, particularly in the liver. Therefore we investigated NK cell phenotype and effector function in both the peripheral and intrahepatic compartments during the course of antiviral therapy in chronic HCV patients. METHODS: Chronic HCV patients were treated for 24 or 48weeks with triple therapy consisting of telaprevir, pegIFN-α and ribavirin. Blood and fine needle aspiration (FNA) biopsies of the liver were collected at start and 6h, 1week and 12weeks during therapy. Flowcytometry was performed for expression of different markers (NKG2A, NKG2D, NKp46, and CD69). RESULTS: Our results demonstrate a highly activated phenotype of NK cells in liver compared to blood in chronic HCV patients. Six hours after start of triple therapy, no activation of intrahepatic NK cells was observed in the liver as compared to baseline. At 1week after start of triple therapy, the frequency of NK cells with the activating receptor NKp46 was increased in blood, whereas at week 12, the frequencies of the inhibitory receptor NKG2A was increased. No alterations were observed during therapy in liver NK cell phenotype. CONCLUSION: IFN-based therapy for chronic HCV affects NK cell phenotype in peripheral blood more than in the liver.

Monocytes from chronic HBV patients react in vitro to HBsAg and TLR by producing cytokines irrespective of stage of disease.

Individuals who are chronically infected with the hepatitis B virus (HBV) are highly heterogeneous with respect to serum levels of HBV DNA, HBV particles and viral proteins. Since circulating leukocytes, such as monocytes, are constantly exposed to these viral components, it is likely that the functionality of these cells is affected. However, at present, little information is available on the consequences of the interaction between monocytes and viral components. Therefore, we examined the in vitro effects of HBV surface antigen (HBsAg) on monocytes and evaluated whether these effects were reflected in vivo. We observed that in vitro HBsAg exposure of monocytes induced robust production of IL-6 and TNF. However, between chronic HBV patients with distinct levels of serum HBsAg, HBV early antigen (HBeAg), and HBV DNA, TLR-induced monocyte cytokine production did not differ. Importantly, HBsAg-induced cytokine production by monocytes was similar between patients and healthy controls showing that earlier in vivo exposure to HBsAg does not affect the in vitro response. Additionally, we show that IL-10 is able to inhibit cytokine production by HBsAg-induced monocytes. In conclusion, we demonstrate that monocytes can recognize and respond to HBsAg, resulting in vigorous pro-inflammatory cytokine production in vitro. However, phenotype and function of the monocyte compartment in chronic HBV patients are not influenced by differences in levels of serum viral components, suggesting that regulatory mechanisms are active to avoid excessive in vivo monocyte activation.

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

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.

Restoration of TLR3-activated myeloid dendritic cell activity leads to improved natural killer cell function in chronic hepatitis B virus infection.

There is increasing evidence that the function of NK cells in patients with chronic hepatitis B (CHB) infection is impaired. The underlying mechanism for the impaired NK cell function is still unknown. Since myeloid dendritic cells (mDC) are potent inducers of NK cells, we investigated the functional interaction of mDC and NK cells in CHB and the influence of antiviral therapy. Blood BDCA1(+) mDC and NK cells were isolated from 16 healthy controls or 39 CHB patients at baseline and during 6 months of antiviral therapy. After activation of mDC with poly(I · C) and gamma interferon (IFN-γ), mDC were cocultured with NK cells. Phenotype and function were analyzed in detail by flow cytometry and enzyme-linked immunosorbent assay. Our findings demonstrate that on poly(I · C)/IFN-γ-stimulated mDC from CHB patients, the expression of costimulatory molecules was enhanced, while cytokine production was reduced. In cocultures of poly(I · C)/IFN-γ-stimulated mDC and NK cells obtained from CHB patients, reduced mDC-induced NK cell activation (i.e., CD69 expression) and IFN-γ production compared to those in healthy individuals was observed. Antiviral therapy normalized mDC activity, since decreased expression of CD80 and CD86 on DC and of HLA-E on NK cells was observed, while poly(I · C)/IFN-γ-induced cytokine production by mDC was enhanced. In parallel, successful antiviral therapy resulted in improved mDC-induced NK cell activation and IFN-γ production. These data demonstrate that CHB patients display a diminished functional interaction between poly(I · C)/IFN-γ activated mDC and NK cells due to impaired mDC function, which can be partially restored by antiviral therapy. Enhancing this reciprocal interaction could reinforce the innate and thus the adaptive T cell response, and this may be an important step in achieving effective antiviral immunity.

Viral load reduction improves activation and function of natural killer cells in patients with chronic hepatitis B.

BACKGROUND & AIMS: Natural killer (NK) cells play a major role in anti-viral immunity as first line defense and regulation of virus-specific T cell responses. This study aimed to investigate phenotype and function of NK cells in patients with chronic hepatitis B virus (HBV) infection and to study the effect of anti-viral therapy. METHODS: Peripheral blood NK cells from 40 chronic HBV patients were compared to NK cells of 25 healthy controls. The effect of entecavir-induced viral load reduction on NK cell phenotype and function was investigated in 15 chronic HBV patients. RESULTS: NK cell numbers and subset distribution did not differ between HBV patients and normal subjects. In chronic HBV patients, the cytotoxic capacity was retained, but NK cell activation and subsequent IFNγ and TNFα production, especially of the CD56(dim) subset, were strongly hampered. This functional dichotomy was paralleled by an altered activation state, elevated expression of NKG2A, and downregulated expression of CD16 and NKp30, which correlated with serum HBV-DNA load. Anti-viral therapy partially restored NK cell phenotype, as shown by NKG2A downregulation. Moreover, viral replication inhibition improved IFNγ production as a result of an increased ability of CD56(dim) NK cells to become activated de novo. This improved NK cell activation and function which correlated with therapy-induced reduction in serum ALT levels, but not HBV-DNA load. CONCLUSIONS: The specific defect in CD56(dim) NK cell activation and the reduced capacity to produce anti-viral and Th1-skewing cytokines may play a role in HBV persistence. Restoration of this NK cell cytokine-producing capacity, as achieved by viral load reduction, could therefore contribute to definite clearance of the virus.