Zwier M. A. Groothuismink

Role for IL-10 in inducing functional impairment of monocytes upon TLR4 ligation in patients with chronic HCV infections

The consequences of chronic infection with the HCV on immunity to distinct pathogens are not fully appreciated, despite the potent modulatory effects of HCV on the immune system. We observed that upon TLR4 ligation, monocytes from chronic HCV patients demonstrated three to five times lower TNF and IL‐12p40 production as compared with healthy individuals. However, augmented production of TNF, IL‐12p40, and IL‐12p70 by monocytes was observed upon stimulation with R848. Importantly, we observed that the levels of IL‐10 in chronic HCV patients are higher in serum and that more IL‐10 is produced by monocytes as compared with healthy individuals. The inhibitory effect of IL‐10 on the production of proinflammatory cytokines by monocytes was only observed upon LPS stimulation but not upon R848 stimulation, showing that only the TLR4 pathway in monocytes is sensitive to the suppressive effects of IL‐10. Interestingly, monocytes stimulated with the TLR4 agonist, but not TLR8 agonist, produced higher levels of IL‐10 when exposed to patient serum as compared with serum from healthy individuals. Our results indicate that by differentially affecting TLR4 and TLR8 pathways, IL‐10 may mediate highly selective modulation of the function of monocytes observed in chronic HCV patients. This suggests that there is no overall increased susceptibility to pathogens but a specific suppression of the functionality of TLR4 signaling pathway in monocytes, which is, at least partly, mediated via IL‐10.

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.

Potent immune activation in chronic hepatitis C patients upon administration of an oral inducer of endogenous interferons that acts via Toll-like receptor 7

BACKGROUND ANA773, an oral prodrug of a small-molecule Toll-like receptor (TLR)7 agonist, induces a dose-related decrease in serum HCV RNA levels in chronic hepatitis C patients. METHODS The prodrug ANA773 was administered to healthy individuals and chronic hepatitis C patients. At different time points during the course of treatment, modulation of the phenotype and function of peripheral leukocytes were evaluated to determine the role of distinct immune cells on the clinical outcome of therapy. RESULTS Early after administration of the TLR7 agonist, a mild transient reduction of the number of lymphocytes was observed in both healthy individuals and chronic hepatitis C patients. Moreover, repeated administration of ANA773 resulted in transiently reduced numbers of myeloid and plasmacytoid dendritic cells (DC) in blood. Interestingly, reduced plasmacytoid DC numbers as well as increased serum interferon (IFN)-α and IFN-γ inducible protein (IP)-10 levels were observed only in virological responders (≥1 log(10) IU/ml reduction of HCV RNA levels upon ANA773 treatment), but were absent in virological non-responders. In vitro stimulation of peripheral blood mononuclear cells from virological responders showed a high frequency of IFN-α-producing plasmacytoid DC upon stimulation in vitro with ANA773, whereas no IFN-α was induced in non-responders. CONCLUSIONS These findings indicate that the viral load decline in chronic hepatitis C patients treated with the TLR7 agonist ANA773 is likely due to intrinsic differences in the induction of endogenous IFNs and IFN-stimulated gene products (IFN-α and IP-10) upon TLR7 ligation.

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 heterogenous 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.

IFN-λ is able to augment TLR-mediated activation and subsequent function of primary human B cells

During the past decade, increased emphasis has been placed on finding alternatives to IFN‐α‐based therapies. One such alternative, IFN‐λ, has shown therapeutic promise in a variety of diseases, but research of this family of cytokines has been primarily focused on their antiviral activities. The goal of the present study was to investigate the role of IFN‐λ in the regulation and modulation of B cell function. We show that, similar to IFN‐α, IFN‐λ1 is able to augment TLR‐mediated B cell activation, partially attributed to an upregulation of TLR7 expression, and that both naϊve and memory B cells express the limiting type III IFN receptor component, IFN‐λR1. Furthermore, this IFN‐λ‐enhanced B cell activation resulted in increased cytokine and Ig production during TLR7 challenge, most prominently after the addition of helper T cell signals. Ultimately, these elevated cytokine and Ig levels could be partially attributed to the increase in proliferation of TLR7‐challenged B cells by both type I and type III IFNs. These findings demonstrate the ability of IFN‐λ to boost humoral immunity, an important attribute to consider for further studies on immunity to pathogens, vaccine development, and ongoing advancement of therapeutic strategies aimed at replacing IFN‐α‐based treatments with IFN‐λ.

Gene Expression Profiling To Predict and Assess the Consequences of Therapy-Induced Virus Eradication in Chronic Hepatitis C Virus Infection

ABSTRACT Systems biology has proven to be a powerful tool to identify reliable predictors of treatment response in chronic hepatitis C virus (HCV) infection. In the present study, we studied patients with chronic HCV infection who responded to interferon (IFN)-based therapy, as evidenced by an absence of HCV RNA at the end of treatment, and focused on two issues that have not received much attention. First, we evaluated whether specific genes or gene expression patterns in blood were able to distinguish responder patients with a viral relapse from responder patients who remained virus negative after cessation of treatment. We found that patients with chronic HCV infection who were sustained responders and relapsers after IFN-based therapy showed comparable baseline clinical parameters and immune compositions in blood. However, at baseline, the gene expression profiles of a set of 18 genes predicted treatment outcome with an accuracy of 94%. Second, we examined whether patients with successful therapy-induced clearance of HCV still exhibited gene expression patterns characteristic of HCV or whether normalization of their transcriptome was observed. We observed that the relatively high expression levels of IFN-stimulated genes (ISGs) in patients with chronic HCV infection prior to therapy were reduced after successful IFN-based antiviral therapy (at 24 weeks of follow-up). These ISGs included the CXCL10, OAS1, IFI6, DDX60, TRIM5, and STAT1 genes. In addition, 1,428 differentially expressed non-ISGs were identified in paired pre- and posttreatment samples from sustained responders, which included genes involved in transforming growth factor beta (TGF-β) signaling, apoptosis, autophagy, and nucleic acid and protein metabolism. Interestingly, 1,424 genes with altered expression levels in responder patients after viral eradication were identified, in comparison to normal expression levels in healthy individuals. Additionally, aberrant expression levels of a subset of these genes, including the interleukin-32 (IL-32), IL-16, CCND3, and RASSF1 genes, were also observed at baseline. Our findings indicate that successful antiviral therapy for patients with chronic HCV infection does not lead to normalization of their blood transcriptional signature. The altered transcriptional activity may reflect HCV-induced liver damage in previously infected individuals. IMPORTANCE Tools to predict the efficacy of antiviral therapy for patients with HCV infection are important to select the optimal therapeutic strategy. Using a systems biology approach, we identify a set of 18 genes expressed in blood that predicts the recurrence of HCV RNA after cessation of therapy consisting of peginterferon and ribavirin. This set of genes may be applicable as a useful biomarker in clinical decision-making, since the number of genes included in the predictor is small and the correct prediction rate is high (94%). In addition, we observed that the blood transcriptional profile in patients with chronic HCV infection who were successfully treated is not normalized to the status observed in healthy individuals. Even 6 months after therapy-induced elimination of HCV RNA, gene expression profiles in blood are still altered in these patients with chronic HCV infection, strongly suggesting long-term modulation of immune parameters in previously infected patients.

Negative Regulation of Hepatitis C Virus Specific Immunity Is Highly Heterogeneous and Modulated by Pegylated Interferon-Alpha/Ribavirin Therapy

Specific inhibitory mechanisms suppress the T-cell response against the hepatitis C virus (HCV) in chronically infected patients. However, the relative importance of suppression by IL-10, TGF-β and regulatory T-cells and the impact of pegylated interferon-alpha and ribavirin (PegIFN-α/ribavirin) therapy on these inhibitory mechanisms are still unclear. We revealed that coregulation of the HCV-specific T-cell responses in blood of 43 chronic HCV patients showed a highly heterogeneous pattern before, during and after PegIFN-α/ribavirin. Prior to treatment, IL-10 mediated suppression of HCV-specific IFN-γ production in therapy-naive chronic HCV patients was associated with higher HCV-RNA loads, which suggests that protective antiviral immunity is controlled by IL-10. In addition, as a consequence of PegIFN-α/ribavirin therapy, negative regulation of especially HCV-specific IFN-γ production by TGF-β and IL-10 changed dramatically. Our findings emphasize the importance of negative regulation for the dysfunctional HCV-specific immunity, which should be considered in the design of future immunomodulatory therapies.

Analysis of the transcriptome and immune function of monocytes during IFNα-based therapy in chronic HCV revealed induction of TLR7 responsiveness

Although in vitro studies have been performed to dissect the mechanism of action of IFNα, detailed in vivo studies on the long-term effects of IFNα on monocytes have not been performed. Here we examined peripheral blood from 14 chronic HCV patients at baseline and 12 weeks after start of IFNα-based therapy. Monocytes were phenotyped by flow-cytometry and their function evaluated upon TLR stimulation and assessed by multiplex cytokine assays. During therapy of HCV patients, monocytes displayed a hyperactive state as evidenced by increased TLR-induced pro-inflammatory cytokine levels, as well as enhanced CD69 and CD83 mRNA and protein expression. Moreover, monocytes from 8 patients at baseline and 12 weeks after start of IFNα-based therapy were transcriptomically profiled by high throughput RNA-sequencing. Detailed RNA-seq analysis of monocytes showed significant ISG mRNA induction during therapy. Importantly, IFNα-based therapy activated TLR7 signaling pathways, as demonstrated by up-regulated expression of TLR7, MyD88, and IRF7 mRNA, whereas other TLR family members as well as CD1c, CLEC4C, and CLEC9A were not induced. The induction of TLR7 responsiveness of monocytes by IFNα in vivo in HCV patients is relevant for the development of TLR7 agonists that are currently under development as a promising immunotherapeutic compounds to treat chronic viral hepatitis.

Natural killer cell activity and function in chronic HCV-infected patients during peg interferon and ribavirin: Early effects of active substance use

In Western countries, chronic hepatitis C virus (HCV)-infection mostly affects former and active substance users. The effect of active substance use on interferon (IFN)-responsiveness and therapy efficacy is not well understood. In this study, we compared natural killer (NK) cell activity and function in healthy controls and chronic HCV-infected patients with and without active substance use, as well as the early effects of antiviral therapy with peg-IFN and ribavirin. No differences were observed between chronic HCV patients and healthy individuals in the number and frequencies of CD56(dim) and CD56(bright) NK cells. Also, IL-12/18-induced IFN-gamma production by NK cells was comparable between all groups, whereas the cytotoxic ability of NK cells (granzyme and CD107a levels) was more potent in HCV-infected patients as compared to healthy controls, and highest in non-substance users. Moreover, at baseline, the activation of NK cells was significantly lower in HCV-infected patients who used substances, when compared to healthy individuals. Therapy-induced viral load reduction assessed early at day 7 showed a similar decline in substance users and non-substance use HCV patients, with 25% substance users and 17% non-substance users testing HCV-RNA negative at day 7. Furthermore, early during IFN-based therapy, NK cells from HCV patients remained responsive to IFN, and only a minor decline in the degree of STAT-1 phosphorylation was observed irrespective of substance use. These findings were further supported by comparable in vitro p-STAT-1 induction in all three experimental groups. Despite subtle differences at baseline between healthy individuals and chronic HCV patients, we observed that active substance use in chronic HCV-infected patients did not affect the immune responsiveness to IFN early after start of treatment, and thus, we found no evidence - from an immunological point of view - that antiviral therapy of our cohort of HCV-infected patients with active substance use is less efficient.

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‐&ggr; 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. HIGHLIGHTSThe frequency of circulating NK cells is stable during the course of chronic HBV.Between the clinical phases of chronic HBV IFN&ggr; production by NK cells differs.The transition to the IA phase is characterized by a reduction of CD57+ NK cells.