Stefania Mantovani

Impaired intrahepatic natural killer cell cytotoxic function in chronic hepatitis C virus infection

Hepatitis C virus (HCV) persistence in the host results from inefficiencies of innate and adaptive immune responses. Most studies addressing the role of innate immunity concentrated on peripheral blood (PB) natural killer (NK) cells, whereas only limited information is available on intrahepatic (IH) NK cells. We therefore examined phenotypic and functional features of IH and PB NK cells in paired liver biopsy and venous blood samples from 70 patients with chronic HCV infection and 26 control persons subjected to cholecystectomy for gallstones as controls. Ex vivo isolated IH NK cells from HCV‐infected patients displayed unique phenotypic features, including increased expression of NKp46‐activating receptor in the face of reduced tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) and cluster of differentiation (CD) 107a expression, which resulted in impaired degranulation compared with controls. To gain insights into the effect of HCV on NK cells, we exposed peripheral blood mononuclear cells (PBMCs) from patients and healthy donors to cell‐culture–derived HCV (HCVcc) and measured NK cell degranulation, TRAIL, and phosphorylated extracellular signal‐regulated kinase 1/2 (pERK1/2) expression. Exposure of PBMCs to HCVcc significantly boosted NK degranulation, pERK1/2, and TRAIL expression in healthy donors, but not in patients with chronic HCV infection, a defect that was completely reversed by interferon‐alpha. Purified NK cells showed a minimal, though significant, increase in degranulation and TRAIL expression, both in patients and controls, after exposure to HCVcc. Conclusions: These findings indicate dysfunctional IH NK cell cytotoxicity associated with TRAIL down‐regulation in chronic HCV infection, which may contribute to virus persistence. PB NK cell impairment upon exposure to HCVcc suggests the existence of an accessory cell‐dependent NK cell lytic defect in chronic HCV infection predominantly involving the TRAIL pathway. (HEPATOLOGY 2012;56:841–849)

Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection

BACKGROUND & AIMS A substantial proportion of patients with chronic hepatitis C virus infection treated with pegylated interferon α/ribavirin fail to achieve sustained virological response (SVR). Since growing evidence suggests that innate immunity may influence treatment responses, we examined natural killer (NK) cell phenotypic and functional changes during standard antiviral therapy. METHODS Expression of several NK-cell regulatory molecules was evaluated by flow cytometry in 37 consecutive patients with chronic HCV infection at baseline and at different time points during and after discontinuation of treatment. Cytokine production was evaluated by intracellular staining. Cytolytic potential was assessed as degranulation and as antibody-dependent cytotoxicity. RESULTS Baseline frequencies of CD56(dim) NK cells and perforin content were significantly higher, whereas CD16 expression was lower in SVR vs. non-responder subjects. Analysis by linear regression for repeated measures during the first 12 weeks showed significantly increased frequencies of activated (CD69(+)) NK cells in rapid virological responders (RVR) and identified a typical NK cell profile associated with SVR, featuring higher NK perforin content, lower CD16 expression, and higher proportion of CD56(dim)/CD16(-) cells. Moreover, SVR patients displayed higher natural and antibody-dependent NK cell cytotoxicity. IL28B rs12979860 CC homozygosis was significantly associated with SVR, independently of NK-cell phenotype and function. CONCLUSIONS Different NK-cell phenotypic and functional features, in patients with chronic hepatitis C treated with standard therapy, were observed between non-responder vs. SVR patients, suggesting a potential role of NK cells in the response to treatment.

Natural killer cell functional dichotomy: a feature of chronic viral hepatitis?

Natural killer (NK) cells are involved in innate immune responses to viral infections either via direct cytotoxicity which destroys virus-infected cells or production of immunoregulatory cytokines which modulate adaptive immunity and directly inhibit virus replication. These functions are mediated by different NK subpopulations, with cytotoxicity being generally performed by CD56dim NK cells, whereas CD56bright NK cells are mainly involved in cytokine secretion. NK functional defects are usually combined so that impaired degranulation is often associated with deficient cytokine production. Innate immunity is thought to be relevant in the control of hepatitis virus infections such as hepatitis B virus (HBV) and hepatitis C virus (HCV), and recent findings reproducibly indicate that NK cells in chronic viral hepatitis are characterized by a functional dichotomy, featuring a conserved or enhanced cytotoxicity and a reduced production of interferon (IFN)-γ and tumor necrosis factor-α. In chronic HCV infection this appears to be caused by altered IFN-α signaling resulting from increased signal transducer and activator of transcription 1 (STAT1) phosphorylation, which polarizes NK cells toward cytotoxicity, and a concomitantly reduced IFN-α induced STAT4 phosphorylation yielding reduced IFN-γ mRNA levels. These previously unappreciated findings are compatible on the one hand with the inability to clear HCV and HBV from the liver and on the other they may contribute to understand why these patients are often resistant to IFN-α-based therapies.

Lack of Siglec-7 expression identifies a dysfunctional natural killer cell subset associated with liver inflammation and fibrosis in chronic HCV infection

Objective Sialic-acid-binding immunoglobulin-like lectin-7 (Siglec-7) is a natural killer (NK) cell inhibitory receptor associated with NK phenotypic and functional abnormalities in HIV-1 infection. We investigated the significance of NK-expressed and serum soluble Siglec-7 in relation to NK functional ability and parameters of liver necroinflammation and fibrosis in chronic HCV infection. Design NK-expressed and serum Siglec-7 were evaluated in 130 and 166 HCV-infected individuals by flow cytometry and ELISA, respectively. NK cell degranulation and cytokine secretion were determined by flow cytometry. 65 patients with chronic HBV infection, 84 with chronic biliary disorders and 168 healthy donors served as controls. Results Expression of Siglec-7 was significantly decreased on NK cells from HCV-infected and HBV-infected patients and, conversely, serum Siglec-7 was significantly increased in these patients compared with controls. The frequency of Siglec-7pos NK cells was significantly higher at baseline in sustained virological responders to pegylated interferon-α/ribavirin treatment than in non-responders. Activating receptor expression was significantly higher in Siglec-7pos NK cells and was associated with increased degranulation and cytokine secretion compared with Siglec-7neg cells. In chronic HCV infection, there was an inverse correlation between Siglec-7 expression and serum aminotransferases, γ-glutamyl transpeptidase, liver stiffness, aspartate aminotransferase to platelet ratio index and fibrosis-4 scores, and a positive correlation between serum Siglec-7 and the same clinical parameters, including histological staging. Conclusions These findings identify Siglec-7neg NK cells as a dysfunctional subpopulation associated with severe liver disease in chronic HCV infection.

NKp30 isoforms in patients with chronic hepatitis C virus infection

Natural killer (NK) cells play an important role in virus infection, their action being regulated by several activating and inhibitory receptors. The NKp30 activating receptor and its isoforms have recently emerged as important determinants of efficient NK cell responses. We determined the relative proportions of NKp30 isoforms in patients with chronic hepatitis C virus (HCV) infection and healthy donors (HD). NK cell function (degranulation and cytokine production) and correlations with clinical parameters were assessed following unsupervised hierarchical clustering of patients according to isoform expression. NKp30 receptor expression on NK cells and all isoforms were reduced in HCV‐infected patients. Patients were clustered into two groups: the HCV‐1 group had similar isoform expression to the HD group, whereas the HCV‐2 group had lower expression. The latter showed a better functional activity, and a higher proportion of the activating a isoform and of the NKp30 isoform a/c ratio compared with the HCV‐1 cluster. There was a positive correlation between the activating a isoform and liver stiffness and an inverse relationship between the immunosuppressive c isoform and the fibrosis 4 score, suggesting a potentially important role of NKp30 isoforms in influencing liver damage and ensuing fibrosis.

Hepatitis C virus-induced NK cell activation causes metzincin-mediated CD16 cleavage and impaired antibody-dependent cytotoxicity

BACKGROUND & AIMS The Fc receptor family for immunoglobulin (Ig)G type III (FcγRIII, CD16) is an activating receptor on natural killer (NK) cells and an essential mediator of antibody-dependent cellular cytotoxicity (ADCC). There is only limited information on its role during chronic hepatitis C virus (HCV) infection. We studied CD16 expression in relation to NK cell functional activity in HCV-infected patients and sought mechanistic insights into virus-induced modulation. METHODS NK cell CD16 expression and activation status were evaluated ex vivo by flow cytometry in HCV-infected patients and healthy controls (HC) as well as in vitro after co-culture with HCV-infected HuH7.5 cells. Rituximab-mediated ADCC was assessed in HC and HCV-infected patients using Daudi cells as a target. The role of metzincins in CD16 down-modulation was assessed using specific inhibitory molecules and by evaluating intracellular mRNA levels. RESULTS HCV-infected patients exhibited increased frequencies of ex vivo activated NK cells and a concomitantly decreased NK CD16 expression, which resulted in impaired ADCC activity. Moreover, exposure of NK cells to culture-derived HCV recapitulated the ex vivo findings of decreased CD16 expression and increased NK cell activation. Importantly, blockade of metzincin-mediated shedding activity, including selective a disintegrin and metalloproteinase 17 (ADAM-17) inhibition, restored NK CD16 expression. Successful treatment with direct-acting antivirals partially improved NK ADCC function despite delayed CD16 reconstitution. CONCLUSION Chronic HCV infection induces NK cell activation resulting in ADAM-17-dependent CD16 shedding and consequent impaired ADCC function. Altered ADCC may contribute to failure to eradicate HCV-infected hepatocytes. LAY SUMMARY We show here that hepatitis C virus (HCV) activates natural killer (NK) lymphocytes which, as a consequence, loose their Fc receptor for IgG (CD16), an essential molecule for antibody binding. We show that this occurs through the action of enzymes named metzincins, resulting in altered NK-mediated antibody-dependent killing (ADCC) of target cells. This mechanism may contribute to HCV persistence and may represent a general phenomenon whereby some viruses can escape hosts immune responses.

Hepatitis C virus inhibits CD4 T cell function via binding to Toll-like receptor 7

Toll-like receptor 7 (TLR7) is a ssRNA receptor that activates dendritic cells and macrophages upon ssRNA binding; however, little is known of its role in CD4+ T cells. We show here that hepatitis C virus (HCV) induces a dose dependent inhibition of cytokine production and expression of activation markers in CD4 T cells, which were restored by a TLR7-specific antagonist. These findings indicate that HCV induces CD4 T cell impairment via TLR7 which may contribute to failure of virus eradication, casting doubts on the use of TLR7 agonists to boost innate immunity in chronic RNA virus infections.

Monocytes inhibit hepatitis C virus-induced TRAIL expression on CD56bright NK cells

BACKGROUND & AIMS Natural killer (NK) cells play an important role in the pathogenesis of hepatitis C virus (HCV) infection. We have previously shown that culture-derived HCV (HCVcc) enhance tumor necrosis-factor-related apoptosis-inducing ligand (TRAIL) expression on healthy NK cells, but not on those from patients infected with HCV, which was likely dependent on accessory cells. Here we sought to elucidate the mechanisms involved in altered TRAIL upregulation in this setting. METHODS Peripheral blood mononuclear cells (PBMC) from controls and patients infected with HCV were exposed to HCVcc. Cell depletions were performed to identify cells responsible for NK cell activation. Flow cytometry and ELISA were used to identify the cytokines involved in the NK activation process. RESULTS In patients infected with HCV, soluble factors secreted by control PBMC restored the ability of NK cells to express TRAIL. Of note, CD14+ cell depletion had identical effects upon virus exposure and promoted increased degranulation. Moreover, increased concentrations of interleukin (IL)-18 binding protein a (IL-18BPa) and IL-36 receptor antagonist (IL-36RA) were observed after PBMC exposure to HCVcc in patients with HCV. HCVcc-induced NK cell TRAIL expression was inhibited by IL-18BPa and IL-36RA in control subjects. There were statistically significant correlations between IL-18BPa and indices of liver inflammation and fibrosis, supporting a role for this protein in the pathogenesis of chronic HCV infection. CONCLUSIONS During chronic HCV infection, monocytes play a key role in negative regulation of NK cell activation, predominantly via secretion of inhibitors of IL-18 and IL-36. LAY SUMMARY Coordination and collaboration between immune cells are essential to fight pathogens. Herein we show that during HCV infection monocytes secrete IL-18 and IL-36 inhibitory proteins, reducing NK cell activation, and consequently inhibiting their ability to express TRAIL and kill target cells.

Deficient natural killer cell NKp30‐mediated function and altered NCR3 splice variants in hepatocellular carcinoma

The activating natural cytotoxicity receptor NKp30 is critical for natural killer (NK) cell function and tumor immune surveillance. The natural cytotoxicity receptor‐3 (NCR3) gene is transcribed into several splice variants whose physiological relevance is still incompletely understood. In this study, we investigated the role of NKp30 and its major ligand B7 homolog 6 (B7‐H6) in patients with hepatocellular carcinoma (HCC). Peripheral blood NK cell phenotype was skewed toward a defective/exhausted immune profile with decreased frequencies of cells expressing NKp30 and natural killer group 2, member D and an increased proportion of cells expressing T‐cell immunoglobulin and mucin‐domain containing‐3. Moreover, NKp30‐positive NK cells had a reduced expression of NCR3 immunostimulatory splice variants and an increased expression of the inhibitory variant in patients with advanced tumor, resulting in deficient NKp30‐mediated functionality. Tumor‐infiltrating lymphocytes showed a prevalent inhibitory NKp30 isoform profile, consistent with decreased NKp30‐mediated function. Of note, there were significant differences in the cytokine milieu between the neoplastic and the surrounding non‐neoplastic tissue, which may have further influenced NKp30 function. Exposure of NK cells to B7‐H6‐expressing HCC cells significantly down‐modulated NKp30, that was prevented by small interfering RNA–mediated knockdown, suggesting a role for this ligand in inhibiting NKp30‐mediated responses. Interestingly, B7‐H6 expression was reduced in HCC tissue and simultaneously augmented as a soluble form in HCC patients, particularly those with advanced staging or larger nodule size. Conclusion: These findings provide evidence in support of a role of NKp30 and its major ligand in HCC development and evolution.

Altered natural killer cell cytokine profile in type 2 autoimmune hepatitis

Type 2 autoimmune hepatitis (AIH-2) is a rare disease presenting in early childhood. The immunopathogenetic mechanisms are poorly characterized, although a defect of regulatory T cells (Treg) has been shown. There is virtually no information on innate immune responses and natural killer (NK) cells in particular. We have performed an extended immunophenotypic and functional analysis of NK cells in children with AIH-2. We show that NK cell frequency is reduced in this setting and that the balance between NK activating and inhibitory receptors is skewed toward activation. More importantly, NK cells display an altered cytokine pattern characterized by increased IFNγ and reduced IL2 production which could contribute to impaired Treg function. Exposure of mononuclear cells to IL2 resulted in normalization of NK IFNγ production. Thus, our findings support treatment of AIH-2 with low-dose IL2, which would result in normalization of NK cell function and expansion of the Treg cell subset.