Interferon Alpha Induces Sustained Changes in NK Cell Responsiveness to Hepatitis B Viral Load Suppression In Vivo

This work was supported by funding from The NIHR Academic Clinical Fellowship scheme and a Wellcome Trust Clinical Research Training fellowship (107389/Z/15/Z) awarded to USG; a Wellcome Trust Senior Investigator award (101848/Z/ 13/Z) to MKM and a Barts and The London Charity award (No. 723/1795) to PTFK

Comparative analysis of the lambda-interferons IL-28A and IL-29 regarding their transcriptome and their antiviral properties against hepatitis C virus.

Specific differences in signaling and antiviral properties between the different Lambda-interferons, a novel group of interferons composed of IL-28A, IL-28B and IL-29, are currently unknown. This is the first study comparatively investigating the transcriptome and the antiviral properties of the Lambda-interferons IL-28A and IL-29. Expression studies were performed by microarray analysis, quantitative PCR (qPCR), reporter gene assays and immunoluminometric assays. Signaling was analyzed by Western blot. HCV replication was measured in Huh-7 cells expressing subgenomic HCV replicon. All hepatic cell lines investigated as well as primary hepatocytes expressed both IFN-λ receptor subunits IL-10R2 and IFN-λR1. Both, IL-28A and IL-29 activated STAT1 signaling. As revealed by microarray analysis, similar genes were induced by both cytokines in Huh-7 cells (IL-28A: 117 genes; IL-29: 111 genes), many of them playing a role in antiviral immunity. However, only IL-28A was able to significantly down-regulate gene expression (n = 272 down-regulated genes). Both cytokines significantly decreased HCV replication in Huh-7 cells. In comparison to liver biopsies of patients with non-viral liver disease, liver biopsies of patients with HCV showed significantly increased mRNA expression of IL-28A and IL-29. Moreover, IL-28A serum protein levels were elevated in HCV patients. In a murine model of viral hepatitis, IL-28 expression was significantly increased. IL-28A and IL-29 are up-regulated in HCV patients and are similarly effective in inducing antiviral genes and inhibiting HCV replication. In contrast to IL-29, IL-28A is a potent gene repressor. Both IFN-λs may have therapeutic potential in the treatment of chronic HCV

Deciphering the Interleukin 28B Variants That Better Predict Response to Pegylated Interferon-α and Ribavirin Therapy in HCV/HIV-1 Coinfected Patients

Previous works have documented the contribution of different IL28B-associated SNPs to spontaneous HCV clearance. This study investigated the effect of different interleukin (IL) 28B genetic variants on interferon (IFN)-based therapy response. We genotyped eight IL28B single-nucleotide polymorphisms (SNPs) in a cohort of 197 hepatitis C virus (HCV)/human immunodeficiency virus type 1 (HIV-1) coinfected patients from our clinic unit who received combined pegylated (peg)-IFN-α and ribavirin (RBV) therapy. This analysis included the two strongest tag predictors for HCV clearance, rs8099917 and rs12979860, and four causal variants (rs4803219, rs28416813, rs8103142, and rs4803217) located in the IL28B promoter, coding, and 3′-untranslated regions. Haplotypes carrying the major alleles at IL28B SNPs were highly associated with sustained virological responses (SVRs) after treatment with peg-IFN-α and RBV [odds ratio (OR) = 2.5, 95% confidence interval (CI) = 1.6–4.0, 4.0×10−5]. Three causal SNP genotypes (rs28416813, rs8103142, and rs4803217) displayed the highest association with SVRs (OR = 3.7, 95% CI = 2.0–6.7, p = 1.3×10−5). All four causal variants were in high linkage disequilibrium, both among themselves (r2≥0.94) and with the rs12979860 variant (r2≥0.92). In contrast, rs8099917 was in low linkage disequilibrium with the four causal variants (r2≤0.45) and with the rs12979860 variant (r2 = 0.45). These results demonstrate that rs12979860, compared to rs8099917, may be a better predictor of response to the peg-IFN/RBV treatment among HCV/HIV-1 coinfected patients. Moreover, causal IL28B variants are strongly associated with treatment SVRs

TGF-β and Iron Differently Alter HBV Replication in Human Hepatocytes through TGF-β/BMP Signaling and Cellular MicroRNA Expression

The nature of host-virus interactions in hepatitis B virus infection is incompletely understood. Since soluble factors, e.g., cytokines and metals, may exacerbate liver injury in chronic hepatitis, we considered that defining the effects of receptor-mediated signaling upon viral replication will be significant. Consequently, we studied effects of iron or TGF-β-induced TGF-β/BMP signaling in the HepG2 2.2.15 cell model of hepatitis B virus replication. We found iron and TGF-β increased hepcidin mRNA expression or TGF-β receptor kinase activity, respectively, which indicated that 2.2.15 cells responded appropriately to these substances. However, iron increased but TGF-β decreased hepatitis B virus mRNA and DNA expression. TGF-β induced expression at the mRNA level of multiple TGF-β/BMP pathway genes. This change was not observed in iron-treated cells. On the other hand, presence of SMAD proteins in iron or TGF-β-treated cells, including of SMAD4, did confirm convergence of TGF-β/BMP signaling pathways under these conditions. Since transcription factors in TGF-β/BMP signaling pathways could not have directly targeted hepatitis B virus itself, we studied whether iron or TGF-β exerted their effects through alternative mechanisms, such as by involvement of antiviral cellular microRNAs. We discovered cellular microRNA expression profiles were significantly different in iron or TGF-β-treated cells compared with untreated control cells. In many cases, exposure to iron or TGF-β changed microRNA expression in opposite directions. Introduction in cells of sequences representing such differentially expressed microRNAs, e.g., hsa-miR-125a-5p and -151-5p, even reproduced effects on virus replication of iron- or TGF-β. We surmised that TGF-β/BMP pathway members, i.e., SMADs, likely governed iron or TGF-β-induced microRNA expression. Iron may have mediated Drosha/DGCR8/heme-mediated processing of microRNAs. In turn, cellular microRNAs regulated replication of hepatitis B virus in iron or TGF-β-treated cells. This knowledge should advance studies of mechanisms in viral-host interactions, hepatic injury, and therapeutic developments for hepatitis B

An IL28B Genotype-Based Clinical Prediction Model for Treatment of Chronic Hepatitis C

BACKGROUND:Genetic variation in IL28B and other factors are associated with sustained virological response (SVR) after pegylated-interferon/ribavirin treatment for chronic hepatitis C (CHC). Using data from the HALT-C Trial, we developed a model to predict a patient's probability of SVR based on IL28B genotype and clinical variables. METHODS:HALT-C enrolled patients with advanced CHC who had failed previous interferon-based treatment. Subjects were re-treated with pegylated-interferon/ribavirin during trial lead-in. We used step-wise logistic regression to calculate adjusted odds ratios (aOR) and create the predictive model. Leave-one-out cross-validation was used to predict a priori probabilities of SVR and determine area under the receiver operator characteristics curve (AUC). RESULTS:Among 646 HCV genotype 1-infected European American patients, 14.2% achieved SVR. IL28B rs12979860-CC genotype was the strongest predictor of SVR (aOR, 7.56; p<.0001); the model also included HCV RNA (log10 IU/ml), AST:ALT ratio, Ishak fibrosis score and prior ribavirin treatment. For this model AUC was 78.5%, compared to 73.0% for a model restricted to the four clinical predictors and 60.0% for a model restricted to IL28B genotype (p<0.001). Subjects with a predicted probability of SVR <10% had an observed SVR rate of 3.8%; subjects with a predicted probability >10% (43.3% of subjects) had an SVR rate of 27.9% and accounted for 84.8% of subjects actually achieving SVR. To verify that consideration of both IL28B genotype and clinical variables is required for treatment decisions, we calculated AUC values from published data for the IDEAL Study. CONCLUSION:A clinical prediction model based on IL28B genotype and clinical variables can yield useful individualized predictions of the probability of treatment success that could increase SVR rates and decrease the frequency of futile treatment among patients with CHC

Activation of Toll-Like Receptor 3 Impairs the Dengue Virus Serotype 2 Replication through Induction of IFN-β in Cultured Hepatoma Cells

Toll-like receptors (TLRs) play an important role in innate immunity against invading pathogens. Although TLR signaling has been indicated to protect cells from infection of several viruses, the role of TLRs in Dengue virus (DENV) replication is still unclear. In the present study, we examined the replication of DENV serotype 2 (DENV2) by challenging hepatoma cells HepG2 with different TLR ligands. Activation of TLR3 showed an antiviral effect, while pretreatment of other TLR ligands (including TLR1/2, TLR2/6, TLR4, TLR5 or TLR7/8) did not show a significant effect. TLR3 ligand poly(I∶C) treatment prior to viral infection or simultaneously, but not post-treatment, significantly down-regulated virus replication. Pretreatment with poly(I∶C) reduced viral mRNA expression and viral staining positive cells, accompanying an induction of the type I interferon (IFN-β) and type III IFN (IL-28A/B). Intriguingly, neutralization of IFN-β alone successfully restored the poly(I∶C)-inhibited replication of DENV2. The poly(I∶C)-mediated effects, including IFN induction and DENV2 suppression, were significantly reversed by IKK inhibitor, further suggesting that IFN-β is the dominant factor involved in the poly(I∶C) mediated antiviral effect. Our study presented the first evidence to show that activation of TLR3 is effective in blocking DENV2 replication via IFN-β, providing an experimental clue that poly(I∶C) may be a promising immunomodulatory agent against DENV infection and might be applicable for clinical prevention

IL28B and IL10R -1087 polymorphisms are protective for chronic genotype 1 HCV infection and predictors of response to interferon-based therapy in an East-Central European cohort.

BACKGROUND: Previous studies have shown that single nucleotide polymorphisms (SNP) in IL28B and IL10R are associated with sustained virological response (SVR) in chronic hepatitis C patients treated with pegilated interferon plus ribavirin (P/R). The present study extends our earlier investigations on a large East-Central European cohort. The allele frequencies of IL28B and IL10R in genotype 1 HCV infection were compared with that of healthy controls for the purpose of examining the relationship between the polymorphisms and the SVR to P/R treatment. METHODS: A total of 748 chronic HCV1 infected patients (365 male, 383 female; 18-82 years) and 105 voluntary blood donors as controls were enrolled. Four hundred and twenty HCV patients were treated with P/R for 24-72 weeks, out of them 195 (46.4%) achieved SVR. The IL28 rs12979860 SNP was determined using Custom Taqman SNP Genotyping Assays. The IL10R -1087 (also known as IL10R -1082 (rs1800896) promoter region SNP was determined by RT-PCR and restriction fragment length polymorphism analysis. RESULTS: The IL28B CC genotype occurred with lower frequency in HCV patients than in controls (26.1% vs 51.4%, p<0.001). P/R treated patients with the IL28B CC genotype achieved higher SVR rate, as compared to patients with CT (58.6% vs 40.8%, p=0.002). The prevalence of IL10R -1087 GG genotype was lower in patients than in controls (31.8 % vs 52.2%, p<0.001). Among patients achieving SVR, the IL10R -1087 GG genotype occurred with higher frequency than the AA (32.0% vs 17.4%, p=0.013). The IL28B T allele plus IL10R A allele combination was found with higher prevalence in patients than in controls (52% vs 20.7%, p<0.001). The IL28B CC plus IL10R A allele combination occurred with higher frequency among patients with SVR than in non-responders (21.3% vs 12.8%, p=0.026). Both the IL28B CC plus IL10R GG and the IL28B CC plus IL10R A allele combinations occurred with lower frequency in patients than in controls. CONCLUSIONS: In our HCV1 patients, both the IL28B CC and IL10R GG genotypes are associated with clearance of HCV. Moreover, distinct IL28B and IL10R allele combinations appear to be protective against chronic HCV1 infection and predictors of response to P/R therapy

Age-associated impaired plasmacytoid dendritic cell functions lead to decreased CD4 and CD8 T cell immunity

Increased susceptibility to infections, particularly respiratory viral infections, is a hallmark of advancing age. The underlying mechanisms are not well understood, and there is a scarcity of information regarding the contribution of the innate immune system, which is the first line of defense against infections. In the present study, we have investigated the effect of advancing age on plasmacytoid dendritic cell (PDC) function because they are critical in generating a robust antiviral response via the secretion of interferons (IFN). Our results indicate that PDCs from the aged are impaired in their capacity to secrete IFN-I in response to influenza virus and CPG stimulation. Additionally, we observed a severe reduction in the production of IFN-III, which plays an important role in defense against viral infections at respiratory mucosal surfaces. This reduction in IFN-I and IFN-III were a result of age-associated impaired phosphorylation of transcription factor, IRF-7. Furthermore, aged PDCs were observed to be impaired in their capacity to induce perforin and granzyme in CD8 T cells. Comparison of the antigen-presenting capacity of aged PDC with young PDC revealed that PDCs from aged subjects display reduced capacity to induce proliferation and IFN-gamma secretion in CD4 and CD8 T cells as compared with PDCs from young subjects. In summary, our study demonstrates that advancing age has a profound effect on PDC function at multiple levels and may therefore, be responsible for the increased susceptibility to infections in the elderly

The role of the proteasome in the generation of MHC class I ligands and immune responses

The ubiquitin–proteasome system (UPS) degrades intracellular proteins into peptide fragments that can be presented by major histocompatibility complex (MHC) class I molecules. While the UPS is functional in all mammalian cells, its subunit composition differs depending on cell type and stimuli received. Thus, cells of the hematopoietic lineage and cells exposed to (pro)inflammatory cytokines express three proteasome immunosubunits, which form the catalytic centers of immunoproteasomes, and the proteasome activator PA28. Cortical thymic epithelial cells express a thymus-specific proteasome subunit that induces the assembly of thymoproteasomes. We here review new developments regarding the role of these different proteasome components in MHC class I antigen processing, T cell repertoire selection and CD8 T cell responses. We further discuss recently discovered functions of proteasomes in peptide splicing, lymphocyte survival and the regulation of cytokine production and inflammatory responses