Elisavet Serti

Successful Interferon-Free Therapy of Chronic Hepatitis C Virus Infection Normalizes Natural Killer Cell Function

BACKGROUND & AIMS Chronic hepatitis C virus infection activates an intrahepatic immune response, leading to increased expression of interferon (IFN)-stimulated genes and activation of natural killer (NK) cells-the most prevalent innate immune cell in the liver. We investigated whether the elimination of hepatitis C virus with direct-acting antiviral normalizes expression of IFN-stimulated genes and NK cell function. METHODS We used multicolor flow cytometry to analyze NK cells from the liver and blood of 13 HCV-infected patients who did not respond to treatment with pegylated interferon and ribavirin. Samples were collected before and during IFN-free treatment with daclatasvir and asunaprevir and compared with samples from the blood of 13 healthy individuals (controls). Serum levels of chemokine C-X-C motif ligand (CXCL) 10 or CXCL11 were measured by enzyme-linked immunosorbent assay. RESULTS Before treatment, all patients had increased levels of CXCL10 or CXCL11 and a different NK cell phenotype from controls, characterized by increased expression of HLA-DR, NKp46, NKG2A, CD85j, signal transducer and activator of transcription 1 (STAT1), phosphorylated STAT1, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). NK cells from patients also had increased degranulation and decreased production of IFNγ and tumor necrosis factor α compared with NK cells from controls. Nine patients had an end-of-treatment response (undetectable virus) and 4 had virologic breakthrough between weeks 4 and 12 of therapy. A rapid decrease in viremia and level of inflammatory cytokines in all patients was associated with decreased activation of intrahepatic and blood NK cells; it was followed by restoration of a normal NK cell phenotype and function by week 8 in patients with undetectable viremia. This normalized NK cell phenotype was maintained until week 24 (end of treatment). CONCLUSIONS Direct-acting antiviral-mediated clearance of HCV is associated with loss of intrahepatic immune activation by IFNα, which is indicated by decreased levels of CXCL10 and CXCL11 and normalization of NK cell phenotype and function.

IL-29 Is the Dominant Type III Interferon Produced by Hepatocytes During Acute Hepatitis C Virus Infection

Early, vigorous intrahepatic induction of interferon (IFN)‐stimulated gene (ISG) induction is a feature of hepatitis C virus (HCV) infection, even though HCV inhibits the induction of type I IFNs in vitro. To identify the cytokines and cells that drive ISG induction and mediate antiviral activity during acute HCV infection, type I and III IFN responses were studied in (1) serial liver biopsies and plasma samples obtained from 6 chimpanzees throughout acute HCV infection and (2) primary human hepatocyte (PHH) cultures upon HCV infection. Type I IFNs were minimally induced at the messenger RNA (mRNA) level in the liver and were undetectable at the protein level in plasma during acute HCV infection of chimpanzees. In contrast, type III IFNs, in particular, interleukin (IL)‐29 mRNA and protein, were strongly induced and these levels correlated with ISG expression and viremia. However, there was no association between intrahepatic or peripheral type III IFN levels and the outcome of acute HCV infection. Infection of PHH with HCV recapitulated strong type III and weak type I IFN responses. Supernatants from HCV‐infected PHH cultures mediated antiviral activity upon transfer to HCV‐replicon–containing cells. This effect was significantly reduced by neutralization of type III IFNs and less by neutralization of type I IFNs. Furthermore, IL‐29 production by HCV‐infected PHH occurred independently from type I IFN signaling and was not enhanced by the presence of plasmacytoid dendritic cells. Conclusion: Hepatocyte‐derived type III IFNs contribute to ISG induction and antiviral activity, but are not the principal determinant of the outcome of HCV infection. (HEPATOLOGY 2012;56:2060–2070)

Early changes in interferon signaling define natural killer cell response and refractoriness to interferon‐based therapy of hepatitis C patients

Natural killer (NK) cells exhibit a polarized phenotype with increased cytotoxicity and decreased interferon gamma (IFN‐γ) production in chronic hepatitis C virus (HCV) infection. Here, we asked whether this is caused by type I interferon (IFN)‐induced expression and phosphorylation levels of signal transducer and activator of transcription (STAT) molecules in NK cells and whether it affects the response and refractoriness of NK cells to IFN‐α‐based therapy of HCV. STAT1 levels in NK cells were significantly higher in patients with chronic HCV infection than in uninfected controls. STAT1 levels and induction of phosphorylated STAT1 (pSTAT1) increased further during IFN‐α‐based therapy with preferential STAT1 over STAT4 phosphorylation. Induction of pSTAT1 correlated with increased NK cytotoxicity (tumor necrosis factor–apoptosis‐inducing ligand [TRAIL] expression and degranulation) and decreased IFN‐γ production. NK cells from patients with a greater than 2 log10 first‐phase HCV RNA decline to IFN‐α‐based therapy (>99% IFN effectiveness) displayed strong pSTAT1 induction in vivo and were refractory to further stimulation in vitro. In contrast, NK cells from patients with a less than 2 log10 first‐phase HCV RNA decline exhibited lower pSTAT1 induction in vivo (P = 0.024), but retained greater IFN‐α responsiveness in vitro (P = 0.024). NK cells of all patients became refractory to in vivo and in vitro stimulation by IFN‐α during the second‐phase virological response. Conclusion: These data show that IFN‐α‐induced modulation of STAT1/4 phosphorylation underlies the polarization of NK cells toward increased cytotoxicity and decreased IFN‐γ production in HCV infection, and that NK cell responsiveness and refractoriness correlate to the antiviral effectiveness of IFN‐α‐based therapy. (Hepatology 2012)

Monocytes Activate Natural Killer Cells via Inflammasome-Induced Interleukin 18 in Response to Hepatitis C Virus Replication

BACKGROUND & AIMS Production of interferon (IFN)-γ by natural killer (NK) cells is attenuated during chronic infection with hepatitis C virus (HCV). We investigated whether this is due to intrinsic or extrinsic mechanisms of NK cells. METHODS Peripheral blood mononuclear cells (PBMCs) were collected from patients with chronic HCV infection or uninfected blood donors (controls); NK cells and monocytes were isolated or eliminated. We cultured hepatoma cells that express luciferase-tagged subgenomic HCV replicons (Huh7/HCV replicon cells) or their HCV-negative counterparts (Huh7) with NK cells in the presence or absence of other populations of PBMCs. Antiviral activity, cytotoxicity, and cytokine production were assessed. RESULTS NK cells produced greater amounts of IFN-γ when PBMC were cocultured with Huh7/HCV replicon cells than with Huh7 cells; NK cells and PBMCs from controls suppressed HCV replication to a greater extent than those from patients with chronic HCV infection. This antiviral effect was predominantly mediated by tumor necrosis factor (TNF)-α and IFN-γ. The antiviral activity of NK cells and their production of IFN-γ were reduced when they were used in coculture alone (rather than with PBMC), or after depletion of CD14(+) monocytes, after knockdown of the inflammasome in monocytes, or after neutralization of interleukin-18, which is regulated by the inflammasome. These findings indicate a role for monocytes in NK cell activation. Compared with control monocytes, monocytes from patients with chronic HCV infection had reduced TNF-α-mediated (direct) and reduced NK cell-mediated (indirect) antiviral effects. Control monocytes increased the antiviral effects of NK cells from patients with chronic HCV infection and their production of IFN-γ. CONCLUSIONS Monocytes sense cells that contain replicating HCV and respond by producing interleukin-18 via the inflammasome and by activating NK cells. Patients with chronic HCV infection have reduced monocyte function, attenuating NK cell IFN-γ-mediated responses.

Rapid decrease in hepatitis C viremia by direct acting antivirals improves the natural killer cell response to IFNα

Objective Chronic HCV infection is characterised by innate immune activation with increased interferon-stimulated genes (ISG) expression and by an altered phenotype of interferon-responsive natural killer (NK) cells. Here, we asked whether a rapid reduction in viremia by daclatasvir (DCV) and asunaprevir (ASV) improves the response to pegylated interferon (PegIFN) in patients who had previously failed a standard course of PegIFN/ribavirin (RBV) therapy. Design Twenty-two HCV-infected non-responders to previous PegIFN/RBV therapy were studied for IFN-responsiveness of NK cells during quadruple (QUAD) therapy with DCV, ASV, PegIFN and RBV. A direct comparison of early NK cell responses in PegIFN/RBV therapy and QUAD therapy was performed for seven patients using paired cryopreserved peripheral blood mononuclear cells (PBMC) from both treatment courses. As a validation cohort, nine DCV/ASV-treated patients were studied for their NK cell response to in vitro stimulation with IFNα. Results The 24 h virological response to QUAD therapy correlated with an increase in signal transducer and activator of transcription 1 (STAT1), phosphorylated STAT1 (pSTAT1) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) expression in NK cells, and the STAT1/pSTAT1/TRAIL induction was greater during QUAD therapy than during previous PegIFN/RBV therapy. Successful QUAD therapy as well as successful IFN-free DCV/ASV regimen resulted in an improved functional NK cell response (degranulation and TRAIL expression) to in vitro stimulation with IFNα. Conclusions IFN-responsiveness can be improved by inhibiting HCV replication and reducing the HCV-induced activation of the innate immune response. This may provide a rationale for clinical trials of a brief period of direct acting antiviral therapy followed by PegIFN/RBV therapy to reduce the overall treatment costs in low-income and middle-income countries. Trial registration numbers NCT01888900 and NCT00718172.

Ribavirin improves the IFN‐γ response of natural killer cells to IFN‐based therapy of hepatitis C virus infection

Ribavirin (RBV) is an important component of interferon (IFN)‐based and direct antiviral treatment regimens for hepatitis C virus (HCV) infection. Immunomodulation, in particular improvement of the host IFN response, has been proposed as RBVs mechanism of action. Natural killer (NK) cells are sensitive biomarkers for IFN‐α/β receptor signaling, as NK cell cytotoxicity and IFN‐γ production are regulated by signal transducer and activator of transcription (STAT)1‐ and STAT4‐phosphorylation, respectively. Specifically, pSTAT1‐dependent NK cell cytotoxicity increases and pSTAT4‐dependent IFN‐γ production decreases in response to endogenous, virus‐induced IFN‐α and during IFN‐α‐based therapy. To assess whether RBV has a direct effect on NK cells and/or improves the IFN‐γ response of NK cells in the presence of IFN‐α, we prospectively studied 22 HCV patients with and 32 patients without 4 weeks of RBV pretreatment, who all received subsequent pegylated (Peg)IFN/ribavirin combination therapy. During RBV pretreatment, both the frequency of CD56dim NK cells with cytotoxic effector functions and the frequency of CD56bright NK cells with the capacity to produce IFN‐γ decreased (P = 0.049 and P = 0.001, respectively). In vitro or in vivo exposure of NK cells to RBV improved the pSTAT4 (P < 0.01) but not pSTAT1 response of NK cells to subsequent stimulation with IFN‐α. This was associated with an increase in IFN‐γ production but not cytotoxicity of NK cells during subsequent IFN‐α‐based therapy. The frequency of IFN‐γ‐producing NK cells was greater in fast second‐phase virological responders than in slow responders. Conclusion: RBV enhances the pSTAT4 and IFN‐γ response of NK cells to IFN‐α‐stimulation. (Hepatology 2014;60:1160–1169)

Hepatitis B virus evades innate immunity of hepatocytes but activates cytokine production by macrophages

Hepatitis B virus (HBV) infects hepatocytes specifically and causes immune‐mediated liver damage. How HBV interacts with the innate immunity at the early phase of infection, either with hepatocytes or other cells in the liver, remains controversial. To address this question, we utilized various human cell‐culture models and humanized Alb‐uPA/SCID mice. All these models were unable to mount an interferon (IFN) response despite robust HBV replication. To elucidate the mechanisms involved in the lack of IFN response, we examined whether HBV actively inhibits innate immune functions of hepatocytes. By treating HBV‐infected cells with known inducers of the IFN signaling pathway, we observed no alteration of either sensing or downstream IFN response by HBV. We showed that the DNA innate sensing pathways are poorly active in hepatocytes, consistent with muted innate immune recognition of HBV. Upon exposure to high‐level HBV, human macrophages could be activated with increased inflammatory cytokine expressions. Conclusion: HBV behaves like a “stealth” virus and is not sensed by, nor actively interferes with, the intrinsic innate immunity of infected hepatocytes. Macrophages are capable of sensing HBV, but require exposure to high HBV titers, potentially explaining the long “window period” during acute infection and HBVs propensity to chronic infection. (Hepatology 2017;66:1779–1793)

Hepatitis B virus evades innate immunity of hepatocytes but activates macrophages during infection

Hepatitis B virus (HBV) infects hepatocytes specifically and causes immune‐mediated liver damage. How HBV interacts with the innate immunity at the early phase of infection, either with hepatocytes or other cells in the liver, remains controversial. To address this question, we utilized various human cell‐culture models and humanized Alb‐uPA/SCID mice. All these models were unable to mount an interferon (IFN) response despite robust HBV replication. To elucidate the mechanisms involved in the lack of IFN response, we examined whether HBV actively inhibits innate immune functions of hepatocytes. By treating HBV‐infected cells with known inducers of the IFN signaling pathway, we observed no alteration of either sensing or downstream IFN response by HBV. We showed that the DNA innate sensing pathways are poorly active in hepatocytes, consistent with muted innate immune recognition of HBV. Upon exposure to high‐level HBV, human macrophages could be activated with increased inflammatory cytokine expressions. Conclusion: HBV behaves like a “stealth” virus and is not sensed by, nor actively interferes with, the intrinsic innate immunity of infected hepatocytes. Macrophages are capable of sensing HBV, but require exposure to high HBV titers, potentially explaining the long “window period” during acute infection and HBVs propensity to chronic infection. (Hepatology 2017;66:1779–1793)

Aberrant tRNA processing causes an autoinflammatory syndrome responsive to TNF inhibitors

Objectives To characterise the clinical features, immune manifestations and molecular mechanisms in a recently described autoinflammatory disease caused by mutations in TRNT1, a tRNA processing enzyme, and to explore the use of cytokine inhibitors in suppressing the inflammatory phenotype. Methods We studied nine patients with biallelic mutations in TRNT1 and the syndrome of congenital sideroblastic anaemia with immunodeficiency, fevers and developmental delay (SIFD). Genetic studies included whole exome sequencing (WES) and candidate gene screening. Patients’ primary cells were used for deep RNA and tRNA sequencing, cytokine profiling, immunophenotyping, immunoblotting and electron microscopy (EM). Results We identified eight mutations in these nine patients, three of which have not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly interleukin (IL)-6, interferon gamma (IFN-γ) and IFN-induced cytokines were elevated in the serum, whereas tumour necrosis factor (TNF) and IL-1β were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients’ fibroblasts showed significant deficiency of mature cytosolic tRNAs. EM of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for blood transfusions and improved growth. Conclusions Mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease consequences.

Baseline intrahepatic and peripheral innate immunity are associated with hepatitis C virus clearance during direct‐acting antiviral therapy

Hepatitis C virus (HCV) infection induces interferon (IFN)‐stimulated genes (ISGs) and downstream innate immune responses. This study investigated whether baseline and on‐treatment differences in these responses predict response versus virological breakthrough during therapy with direct‐acting antivirals (DAAs). Thirteen HCV genotype 1b–infected patients who had previously failed a course of pegylated IFN/ribavirin were retreated with asunaprevir/daclatasvir for 24 weeks. After pretreatment biopsy, patients were randomized to undergo a second biopsy at week 2 or 4 on therapy. Microarray and NanoString analyses were performed on paired liver biopsies and analyzed using linear mixed models. As biomarkers for peripheral IFN responses, peripheral blood natural killer cells were assessed for phosphorylated signal transducer and activator of transcription 1 (pSTAT1) and tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) expression and degranulation. Nine of 13 (69%) patients achieved sustained virological response at 12 weeks off therapy (SVR12), and 4 experienced virological breakthroughs between weeks 4 and 12. Patients who achieved SVR12 displayed higher ISG expression levels in baseline liver biopsies and a higher frequency of pSTAT1 and TRAIL‐expressing, degranulating natural killer cells in baseline blood samples than those who experienced virological breakthrough. Comparing gene expression levels from baseline and on‐therapy biopsies, 408 genes (±1.2‐fold, P < 0.01) were differentially expressed. Genes down‐regulated on treatment were predominantly ISGs. Down‐regulation of ISGs was rapid and correlated with HCV RNA suppression. Conclusion: An enhanced IFN signature is observed at baseline in liver and blood of patients who achieve SVR12 compared to those who experience a virological breakthrough; the findings suggest that innate immunity may contribute to clearance of HCV during DAA therapy by preventing the emergence of resistance‐associated substitutions that lead to viral breakthrough during DAA therapy.