MicroRNA‐155 controls Toll‐like receptor 3‐ and hepatitis C virus‐induced immune responses in the liver

The hepatitis C virus (HCV) establishes persistent infections despite strong activation of the innate immune system through TLR3 and other sensors. Therefore, we analysed regulatory mechanisms of TLR3‐induced immune responses in nonparenchymal liver cells (NPCs). Effects of Interleukin‐10 (IL‐10), transforming growth factor beta (TGF‐β) and immunoregulatory miR‐155 on poly I:C‐activated murine (C57BL/6) Kupffer cells (KC) and sinusoidal endothelial cells (LSEC) were assessed in vitro. NPCs were assayed for inflammatory and antiviral cytokines and T‐cell (Balb/c)‐activating factors. Gene expression of miR‐155, IL‐10, TGF‐β and interferon sensitive genes (ISGs) in biopsies of patients with HCV was determined by qrt‐PCR. TLR3‐induced antiviral activity in murine NPCs was potently suppressed by IL‐10 and TGF‐β which correlated with decreased TLR3 expression and inhibition of NF‐κB and IRF‐3 activation. T‐cell activation, induced by TLR3‐activated NPCs, was also suppressed by IL‐10 and TGF‐β, which was associated with a down‐regulation of CD80 and CD86. Pretreatment with IL‐10 or TGF‐β suppressed TLR3‐induced miR‐155 expression, which itself positively regulated poly I:C‐mediated immune responses, thus counteracting IL‐10 or TGF‐β‐induced immunosuppression. In addition, hepatic expression of miR‐155 was elevated in chronically infected patients with HCV, was associated with an IL‐28B SNP (rs12979860) and was inversely correlated with HCV serum load and ISG expression levels. As miR‐155 is a key regulator of anti‐inflammatory mechanisms that control innate and adaptive hepatic immune responses during HCV infection, miR‐155 based therapies may represent a novel mechanism to control HCV in the future.     

Correlates and characteristics of hepatitis C virus‐specific T‐cell immunity in exposed uninfected high‐risk prison inmates

Some hepatitis C (HCV)‐uninfected, high‐risk individuals have HCV‐specific cellular immunity without viraemia or seroconversion. The characteristics of these responses and the risk behavioural associations were studied in 94 subjects in a prospective cohort of recently seronegative prisoners reporting injecting drug use (IDU). Detailed behavioural data were collected. HCV antibody and PCR testing were performed. ELISpot assays for HCV‐induced interferon (IFN)‐γ and interleukin (IL)‐2 production by T lymphocytes, as well as multiplex in vitro cytokine production assays, were performed. Seventy‐eight subjects remained antibody and PCR negative and 16 seroconverted. Of the seronegative group, 22 (28%) had IFN‐γ ELISpot responses in comparison with 13 of the 16 seroconverters (82%). This seronegative immune status was associated positively with injecting anabolic steroids and negatively with a recent break from IDU. The IFN‐γ ELISpot responses involved both CD4 and CD8 T lymphocytes and were comparable in magnitude, but narrower in specificity, in uninfected subjects than in seroconverters. A subset of seronegative subjects had HCV‐induced cytokine production patterns comparable with the seroconverters with increased production of IFN‐γ, IL‐2 and tumour necrosis factor (TNF)‐α and reduced IL‐10 in response to nonstructural peptides. In conclusion, comparable patterns of HCV‐specific cellular immunity are found in recently infected subjects and in a minority of high‐risk, uninfected subjects. Further characterization of these responses and their protective efficacy will inform HCV vaccine development.     

Hepatitis C‐specific effector and regulatory CD4 T‐cell responses are associated with the outcomes of primary infection

Clearance of primary hepatitis C virus (HCV) infection has been associated with strong and broadly targeted cellular immune responses. This study aimed to characterize HCV‐specific CD4+ effector and regulatory T‐cell numbers and cytokine production during primary infection. Antigen‐specific CD4+ T‐cell responses were investigated in a longitudinal cohort of subjects from pre‐infection to postoutcome, including subjects who cleared [n=12] or became chronically infected [n=17]. A cross‐sectional cohort with previously cleared, or chronic infection [n=15 for each], was also studied. Peripheral blood mononuclear cells were incubated with HCV antigens and surface stained for T‐effector (CD4+CD25^highCD134+CD39‐) and T‐regulatory (CD4+CD25^highCD134+CD39+) markers, and culture supernatants assayed for cytokine production. Contrary to expectations, the breadth and magnitude of the HCV‐specific CD4+ T‐cell responses were higher in subjects who became chronically infected. Subjects who cleared the virus had HCV‐specific CD4+ T‐cell responses dominated by effector T cells and produced higher levels of IFN‐γ, in contrast to HCV‐specific CD4+ T‐cell responses dominated by regulatory T cells and more IL‐10 production in those who became chronically infected. Better understanding of the role of antigen‐specific CD4+ T‐cell responses in primary HCV will further define pathogenesis and help guide development of a preventative vaccine.     

Hepatitis C virus (HCV) specific immune responses in anti-HCV positive patients without hepatitis C viraemia

BACKGROUND/AIMS: Most patients infected with hepatitis C virus (HCV) develop chronic infection and persistent viraemia. The immune mechanisms responsible for resolution of viraemia remain poorly understood. HCV specific humoral and cellular immune responses in patients with and without viraemia were investigated. METHODS: In vitro T helper (TH) lymphocyte responses to structural and non-structural HCV proteins were determined by means of proliferative response and cytokine production in 35 anti-HCV positive/HCV RNA negative patients and in 31 patients with chronic HCV infection and persistent viraemia. Humoral responses were determined by measuring HCV specific antibody quantity and specificity. RESULTS: A TH response to two or more HCV proteins was present in 18 of 35 patients with serological viral clearance compared with just one of 31 viraemic patients (p = 0.00001). HCV specific interferon-gamma production was increased only in the former group. In contrast, the antibody levels were significantly lower and directed at fewer HCV antigens in patients with undetectable HCV RNA. CONCLUSIONS: Patients without viraemia after HCV infection frequently have strong TH lymphocyte responses of the TH1 type to multiple HCV antigens many years after the onset of infection, whereas antibody responses are less marked. These results suggest that control of HCV replication may depend on effective TH lymphocyte activation.     

Impact of oral silymarin on virus‐ and non‐virus‐specific T‐cell responses in chronic hepatitis C infection

Silymarin displays anti‐inflammatory effects on T lymphocytes in vitro. The immunomodulatory properties of oral silymarin in vivo in humans with chronic hepatitis C have not previously been characterized. We hypothesized that silymarin would suppress T‐cell proliferation and pro‐inflammatory cytokine production of virus‐ and non‐virus‐specific T cells while increasing anti‐inflammatory IL‐10 production in vivo. Patients from one site of the SyNCH‐HCV double‐masked, placebo‐controlled study of oral silymarin in prior interferon nonresponders with chronic hepatitis C provided blood samples at baseline and treatment week 20. Mononuclear cells were stimulated with recombinant HCV proteins and controls in ³H‐thymidine proliferation assays, IFNγ ELISPOT and IL‐10 ELISPOT. The frequency of CD4⁺CD25^hi and CD4⁺foxp3⁺ regulatory T cells, serum cytokine levels, serum IP‐10 and lymphocyte interferon‐stimulated gene expression were also quantified at baseline and week 20. Thirty‐two patients were recruited (10; placebo, 11; 420 mg three times a day, 11; 700 mg three times a day). Serum ALT and HCV RNA titres did not change in any group. HCV‐specific CD4⁺ T‐cell proliferation and the frequency of IFNγ‐ and IL‐10‐producing T cells were not significantly changed in silymarin‐treated subjects. However, C. albicans‐induced T‐cell IFNγ and phytohaemagglutinin‐induced T‐cell proliferation were suppressed by silymarin therapy. A trend towards augmentation of interferon‐induced ISG15 expression was present in the high‐dose silymarin group. While no effect on HCV‐specific T cells was identified, these data confirm that high‐dose oral silymarin exerts modest nonspecific immunomodulatory effects in vivo. The impact of this anti‐inflammatory effect on long‐term liver health in chronic hepatitis C merits future clinical investigation.     

IL28B genetic variation and hepatitis C virus–specific CD4+ T‐cell responses in anti‐HCV‐positive blood donors

Summary. Epidemiological, viral and host factors are associated with the outcome of hepatitis C virus (HCV) infection, and strong host immune responses against HCV favour viral clearance. Recently, genome‐wide association studies have shown a strong correlation between single‐nucleotide polymorphisms (SNPs) near the interleukin‐28B (IL28B) gene and spontaneous or treatment‐induced HCV clearance. We have investigated whether protective IL28B genetic variants are associated with HCV‐specific T‐cell responses among Spanish blood donors. The rs12979860 IL28B haplotype was determined in 69 anti‐HCV‐positive blood donors (21 HCV RNA negative and 48 HCV RNA positive) and 30 seronegative donors. In all cases, HCV‐specific CD4⁺ T‐cell responses to HCV recombinant proteins (core, NS3 and NS3 helicase) were assessed by ex vivo interferon‐γ ELISpot assay. The rs12979860‐CC genotype was highly overrepresented in donors with spontaneous HCV clearance when compared to those with chronic infection (76.2%vs 29.2%, P < 0.001; odds ratio, 7.77; 95% confidence interval, 2.4–25.3, P < 0.001). HCV‐specific CD4⁺ T‐cell responses were detected in 16 (76.2%) spontaneous resolvers especially towards nonstructural proteins, but with no correlation with IL28B genotype. Chronic individuals had a significantly lower overall T‐cell response again irrespective of IL28B genotype. When spontaneous resolvers and chronic individuals were stratified according to their IL28B genotype, significantly stronger T‐cell responses were only observed among those with non‐CC haplotypes. Although the protective rs12979860 IL28B CC genotype is associated with spontaneous HCV clearance, stronger CD4⁺ T‐cell responses towards NS3 were only evident among those with non‐CC haplotypes.     

Expression of programmed cell death protein 1 and T‐cell immunoglobulin‐ and mucin‐domain‐containing molecule‐3 on peripheral blood CD4+CD8+ double positive T cells in patients with chronic hepatitis C virus infection and in subjects who spontaneously cleared the virus

Chronic hepatitis C virus (HCV) infection is characterized by increased proportion of CD4+CD8+ double positive (DP) T cells, but their role in this infection is unclear. In chronic hepatitis C, immune responses to HCV become functionally exhausted, which manifests itself by increased expression of programmed cell death protein 1 (PD‐1) and T‐cell immunoglobulin‐ and mucin‐domain‐containing molecule‐3 (Tim‐3) on T cells. The aim of our study was to determine PD‐1 and Tim‐3 phenotype of DP T cells in subjects with naturally resolved and chronic HCV infection. Peripheral blood mononuclear cells from 16 patients with chronic infection and 14 subjects who cleared HCV in the past were stained with anti‐CD3, anti‐CD4, anti‐CD8, anti‐PD‐1 and anti‐Tim‐3 antibodies and, in 12 HLA‐A*02‐positive subjects, MHC class I pentamer with HCV NS3₁₄₀₆ epitope. In chronic and past HCV infection, proportions of total DP T cells and PD‐1+ DP T cells were similar but significantly higher than in healthy controls. DP T cells were more likely to be PD‐1+ than either CD4+ or CD8+ single positive (SP) T cells. HCV‐specific cells were present in higher proportions among DP T cells than among CD8+ SP T cells in both patient groups. Furthermore, while the majority of HCV‐specific DP T cells were PD‐1+, the proportion of HCV‐specific CD8+ T cells which were PD‐1+ was 4.9 and 1.9 times lower (chronic and past infection, respectively). PD‐1 and Tim‐3 were predominantly expressed on CD4^highCD8^low and CD4^lowCD8^high cells, respectively, and co‐expression of both markers was uncommon.     

Anti‐envelope antibody responses in highly exposed seronegative individuals may be associated with protection from HCV infection

In rare cases, individuals with a history of long‐term injecting drug use remain seronegative and aviraemic, despite prolonged and likely repeated exposure to Hepatitis C virus (HCV) through high‐risk behaviour. We describe anti‐HCV Envelope (E) antibody responses in a prospective cohort of carefully defined highly exposed but uninfected subjects (HESN) and comparison subjects who were also high risk and uninfected, but rapidly became HCV infected (Incident). Longitudinally collected samples from HESN cases (n = 22) were compared to Incident controls (n = 22). IgG, IgM and IgA from sera were tested by ELISA to genotype 1a and 3a E glycoproteins, and recombinant genotype 1a E2 antigen. IgG subclass isotyping was performed for those positive for IgG. Virus‐neutralizing activity was assessed on HCV pseudoparticles, and HCV E–specific B cells analysed using flow cytometry. A significant minority of HESN cases (n = 10; 45%) had anti‐E, predominantly in the IgG2 subclass, which was not found in the pre‐infection time point of the Incident cases (n = 1; 5%). A subset of the HESN subjects also had neutralizing activity and HCV‐specific B cells detected significantly more than Incident cases pre‐infection. In conclusion, the HESN phenotype is associated with IgG2 anti‐E antibodies, neutralization activity and HCV E–specific memory B cells. These findings suggest that HESN subjects may be resistant to HCV infection through humoral immune‐mediated mechanisms.     

Association of inflammatory and anti‐inflammatory cytokines with insulin resistance in chronic hepatitis C

Background: The pathogenetic basis for the association between hepatitis C virus (HCV) infection and type‐2 diabetes remains uncertain. It has been reported that insulin resistance (IR) plays an essential role. We investigated the association of inflammatory [tumour necrosis factor (TNF)‐α, interleukin (IL)‐6] and anti‐inflammatory cytokines (adiponectin and IL‐10) with IR in chronic HCV infection. Methods: Eighty‐one consecutive non‐diabetic chronic hepatitis C patients (37 men and 44 women, mean age of 51.9±12.2 years) and 40 age, sex and body mass index (BMI)‐matched healthy individuals were collected. IR was evaluated by the homoeostasis model assessment (HOMA). Serum levels of cytokines were measured by enzyme‐linked immunosorbent assay. Results: Patients with chronic hepatitis C have a higher HOMA‐IR, TNF‐α, IL‐6, adiponectin and IL‐10, as compared with controls. By multiple linear regression analysis, moderate/severe steatosis grade, total cholesterol level and adiponectin was significantly associated with HOMA‐IR, whereas, TNF‐α, IL‐6 and IL‐10 was not. Male gender, BMI and HOMA‐IR was inversely correlated with the serum adiponectin level. Serum adiponectin was positively correlated with TNF‐α level, which was significantly associated with higher degree of hepatic necroinflammation. Conclusion: Our data suggest that chronic HCV infection is associated with increased IR, which is correlated inversely with the serum adiponectin level. The complex role of adiponectin in the pathogenesis of IR and hepatic necroinflammation in chronic HCV infection merit further investigation.     

Acquirement and disappearance of HBsAg and anti‐HCV in an aged population: a follow‐up study in an endemic township

Background: HBsAg and anti‐hepatitis C virus (anti‐HCV) are stable markers and widely used. The seroconversion and seroclearance of HBsAg and anti‐HCV are important for disease control and prognosis of diseases. Aims: To investigate acquirement and disappearance of HBsAg and anti‐HCV in an endemic area. Methods: Seven years after a community screening, 1002 of 2909 residents of Tzukuan Township were recruited. HBsAg, anti‐HCV and alanine transaminase (ALT) were checked in all who participated and hepatitis B virus (HBV) DNA, anti‐HBs, anti‐HBc, HCV RNA, anti‐HDV and upper abdominal ultrasonography were studied in different groups. Results: There were 461 male and 541 female residents with a mean age of 66.7±8.6 years. No new HBsAg carrier was noted and the HBsAg clearance rate was 1.58% per year. One of the 17 cases with HBsAg clearance had positive HBV DNA, three had ALT elevation, two had cirrhosis and seven had anti‐HBs seroconversion. Quantitative of HBsAg and HBV DNA were concordant and 78.1% subjects had low levels of titration. Anti‐HBc alone contributed to 32.1% and was prominent in old age and the anti‐HCV‐positive group. The anti‐HCV seroconversion rate was only 0.74% per year and household transmission was the only risk factor. Only 37.5% of cases with anti‐HCV seroconversion had HCV viraemia and the anti‐HCV seroreversion rate was 0.63% per year. The anti‐HDV seroconversion rate was 0.72% per year and no subject showed anti‐HDV clearance. Conclusions: Much higher rates of HBsAg seroclearance, anti‐HCV seroreversion and anti‐HBc alone were noted in this endemic area and no subject showed anti‐HDV clearance.     

Stronger hepatitis C virus‐specific CD8+ T‐cell responses in HIV coinfection

Summary. Hepatitis C virus (HCV) is a widespread chronic infection that shares routes of transmission with human immunodeficiency virus (HIV). Thus, coinfection with these viruses is a relatively common and growing problem. In general, liver disease develops over years with HIV coinfection, when compared to decades in HCV monoinfection. The role of the immune system in the accelerated pathogenesis of liver disease in HIV/HCV coinfection is not clear. In this study, we compared the frequency, magnitude, breadth and specificity of peripheral blood CD4⁺ and CD8⁺ T‐cell responses between HCV‐monoinfected and HCV/HIV‐coinfected individuals and between HIV/HCV‐coinfected subgroups distinguished by anti‐HCV antibody and HCV RNA status. While HIV coinfection tended to reduce the frequency and breadth of anti‐HCV CD8⁺ T‐cell responses in general, responses that were present were substantially stronger than in monoinfection. In all groups, HCV‐specific CD4⁺ T‐cell responses were rare and weak, independent of either nadir or concurrent CD4⁺ T‐cell counts of HIV‐infected individuals. Subgroup analysis demonstrated restricted breadth of CD8⁺ HCV‐specific T‐cell responses and lower B‐cell counts in HIV/HCV‐coinfected individuals without anti‐HCV antibodies. The greatest difference between HIV/HCV‐coinfected and HCV‐monoinfected groups was substantially stronger HCV‐specific CD8⁺ T‐cell responses in the HIV‐coinfected group, which may relate to accelerated liver disease in this setting.     

Production of Interleukin‐10 by Combining a Granulocyte and Monocyte Adsorption Carrier With Ulinastatin

Interleukin (IL)‐10 is an anti‐inflammatory cytokine mainly produced by monocytes and is essential for the induction of anti‐inflammatory intestinal macrophages with macrophage colony‐stimulating factor (M‐CSF). Thus, IL‐10‐ and M‐CSF‐rich conditions in colonic tissues seem to contribute to the improvement of pathological conditions in patients with inflammatory bowel diseases (IBD). We have already reported that ulinastatin, a serine protease inhibitor, increases M‐CSF production during granulocyte/monocyte (GM) adsorption to cellulose acetate (CA) beads (carriers for Adacolumn therapy). However, the effects of ulinastatin on IL‐10 production have not been clarified. The aim of the present study was to clarify the effects of ulinastatin on IL‐10 production during GM adsorption by in vitro experiments. Peripheral blood was divided into four groups: (Control) no ulinastatin added, no contact with CA beads; (1) no ulinastatin added, contact with CA beads; (2) ulinastatin added, no contact with CA beads; and (3) ulinastatin added, contact with CA beads. After incubation, IL‐10 in the plasma was measured. Compared with the level in the Control group, plasma IL‐10 was significantly higher only in group 3, in which ulinastatin was added in the presence of CA beads, but did not increase in the absence of CA beads. These results suggest that ulinastatin synergistically increases IL‐10 production with monocyte adsorption stimuli. By increasing not only M‐CSF but also IL‐10, a combination of ulinastatin and Adacolumn therapy may improve clinical efficacy for the treatment of IBD in terms of the induction of anti‐inflammatory intestinal macrophages.     

Effect of four rounds of annual school‐wide mass praziquantel treatment for schistosoma mansoni control on schistosome‐specific immune responses

This study evaluated potential changes in antischistosome immune responses in children from schools that received 4 rounds of annual mass drug administration (MDA) of praziquantel (PZQ). In a repeated cross‐sectional study design, 210 schistosome egg‐positive children were recruited at baseline from schools in western Kenya (baseline group). Another 251 children of the same age range were recruited from the same schools and diagnosed with schistosome infection by microscopy (post‐MDA group). In‐vitro schistosome‐specific cytokines and plasma antibody levels were measured by ELISA and compared between the 2 groups of children. Schistosome soluble egg antigen (SEA) and soluble worm antigen preparation (SWAP) stimulated higher IL‐5 production by egg‐negative children in the post‐MDA group compared to the baseline group. Similarly, anti‐SEA IgE levels were higher in egg‐negative children in the post‐MDA group compared to the baseline group. Anti‐SEA and anti‐SWAP IgG4 levels were lower in egg‐negative children in the post‐MDA group compared to baseline. This resulted in higher anti‐SEA IgE/IgG4 ratios for children in the post‐MDA group compared to baseline. These post‐MDA immunological changes are compatible with the current paradigm that treatment shifts immune responses to higher antischistosome IgE:IgG4 ratios in parallel with a potential increase in resistance to reinfection.     

Oral anti‐CD3 immunotherapy for HCV‐nonresponders is safe,promotes regulatory T cells and decreases viral load and liver enzyme levels: results of a phase‐2a placebo‐controlled trial

Orally administered anti‐CD3 antibodies are biologically active in the gut through induction of regulatory T cells, exert an immune‐modulatory effect, and alleviate insulin resistance and liver damage in patients with NASH. Aims: To determine the safety of oral anti‐CD3 monoclonal antibody (MAb) immunotherapy in chronic HCV patients with associated immune dysfunction. Methods: Four groups (n = 9) of chronic HCV patients who were nonresponders to interferon plus ribavirin therapy received oral placebo (group A) or anti‐CD3 MAb at one of three dosage levels for 30 days. Patients were followed for safety parameters and serum levels of liver enzymes, virus, cytokines and regulatory T cells. Results: Oral anti‐CD3 immunotherapy was safe and well tolerated; no treatment‐related adverse events were noted. The following improvements were noted relative to pretreatment levels: HCV viral load and AST and ALT levels decreased in the low‐ and high‐dose groups following 30 days of therapy. In two of the treated groups, an increase in regulatory T cells (CD4⁺ CD25⁺) was noted. The positive effects were somewhat more apparent in subjects with initially elevated liver enzyme levels. Conclusions: Oral anti‐CD3 MAb immunotherapy for nonresponder HCV patients was safe and well tolerated. Trends and statistically significant improvements were observed as reductions in viral load and liver enzyme levels, along with an increase in regulatory T‐cell levels. These data support a role for the immune system in the pathogenesis of HCV infection and suggest that this immunotherapy is worthy of evaluation in combination with HCV antiviral drugs.     

HIV co‐infection accelerates decay of humoral responses in spontaneous resolvers of HCV infection

Acute hepatitis C virus (HCV) infection is primarily followed by chronic infection, while spontaneous recovery of HCV infection (SR‐HCV) occurs in a minority of those infected. Identification of SR‐HCV clinically depends on two combined indicators, persistently undetectable peripheral HCV RNA and positivity for anti‐HCV. However, the characteristics of dynamic variation in anti‐HCV antibodies in SR‐HCV, especially in those patients co‐infected with HIV, are still undefined. In this study, a cohort of patients infected with HCV through commercial blood collection practices was studied. We found that the annual decreasing rate of anti‐HCV presented a gradually accelerated process in HCV resolvers. However, the variation in the decline of anti‐HCV presented a slowly accelerated process within the early decrease stage and a gradually decelerated process within the latter decrease stage. In addition, we deduced that it expended approximately 16 years from natural HCV recovery to undetectable peripheral anti‐HCV in HCV resolvers co‐infected with HIV, while this time was estimated to be 20 years in SR‐HCV without HIV co‐infection. Our data indicated that the decay of anti‐HCV was accelerated by HIV‐related impairment of immune function. The prevalence of HCV infection may be severely underestimated in this large‐scale retrospective epidemiologic investigation in an HIV‐infected population.     

IL‐28B (IFN‐λ3) and IFN‐α synergistically inhibit HCV replication

Genetic variation in the IL‐28B (interleukin‐28B; interferon lambda 3) region has been associated with sustained virological response (SVR) rates in patients with chronic hepatitis C treated with peginterferon‐α and ribavirin. However, the mechanisms by which polymorphisms in the IL‐28B gene region affect host antiviral responses are not well understood. Using the HCV 1b and 2a replicon system, we compared the effects of IFN‐λs and IFN‐α on HCV RNA replication. The anti‐HCV effect of IFN‐λ3 and IFN‐α in combination was also assessed. Changes in gene expression induced by IFN‐λ3 and IFN‐α were compared using cDNA microarray analysis. IFN‐λs at concentrations of 1 ng/mL or more exhibited concentration‐ and time‐dependent HCV inhibition. In combination, IFN‐λ3 and IFN‐α had a synergistic anti‐HCV effect; however , no synergistic enhancement was observed for interferon‐stimulated response element (ISRE) activity or upregulation of interferon ‐ stimulated genes (ISGs). With respect to the time course of ISG upregulation, the peak of IFN‐λ3‐induced gene expression occurred later and lasted longer than that induced by IFN‐α. In addition, although the genes upregulated by IFN‐α and IFN‐λ3 were similar to microarray analysis, interferon‐stimulated gene expression appeared early and was prolonged by combined administration of these two IFNs. In conclusion, IFN‐α and IFN‐λ3 in combination showed synergistic anti‐HCV activity in vitro. Differences in time‐dependent upregulation of these genes might contribute to the synergistic antiviral activity.     

Long‐term stimulation of Toll‐like receptor 3 in primary human hepatocytes leads to sensitization for antiviral responses induced by poly I:C treatment

Chronic hepatitis C infection is associated with increased expression of interferon‐sensitive genes (ISGs) in the liver, which is, paradoxically, correlated with the nonresponse to interferon (IFN)‐based therapies. In the present study PHHs were isolated from HCV‐infected or uninfected patients and stimulated with the TLR1‐9 ligands for 6–24 h. Expression of cytokines and ISGs was determined by ELISA and qRT‐PCR. A comparative analysis was performed for TLR3 signalling, which was also correlated with single nucleotide polymorphisms (SNPs) related to HCV pathogenesis. TLR‐activated PHHs produced pro‐inflammatory and anti‐inflammatory cytokines, whereas IFNs were exclusively induced by TLR3 stimulation. Here, IL‐29 and IL‐28A were significantly highly expressed than IFN‐α and IFN‐β. TLR3‐induced IFN response was enhanced in hepatocytes isolated from patients with HCV infection. This hyper‐responsiveness could be mimicked in naïve PHHs consistently stimulated with low dose of poly I:C, but not Guardiquimod. The higher responsiveness in PHH isolated from HCV‐infected patients could be partially explained by higher frequencies of unfavourable SNP alleles of different SNPs associated with HCV progression and treatment outcome. These data suggest that durable activation of TLR3 but not TLR7, by low doses of viral replicative intermediates, increases the sensitivity to viral invasion. These findings shed new light on the relevance of TLR3 in the pathogenesis of HCV and may provide a possible explanation for the increased ISG expression during chronic HCV infection, the so‐called IFN paradox.