IFN-γ secretion of PBMC from non-drug-allergic control persons: Considerations for the validity of a positive lymphocyte transformation test.

BACKGROUND: The lymphocyte transformation test (LTT) is used for the in vitro detection of a drug sensitization in assumed drug allergic patients. It is based on the detection of antigen (drug)-specific activation of T cells indicated by e.g. proliferation or cytokine secretion. However, occasional stimulatory effects of the drug unrelated to specific drug-allergic mechanisms can only be detected if a larger number of non-drug allergic control persons are tested with this specific drug. In this respect, the overall specificity of the LTT with ELISA read-out is summarized in several review articles, but the impact of a specific drug on the specificity has not yet been analyzed in a larger set of control persons. OBJECTIVE: Do amoxicillin, cefuroxime and clindamycin induce an interferon (IFN)-y or interleukin (IL)-5 secretion of PBMC from control persons using the LTT with ELISA read-out? METHODS: We performed LTTs with amoxicillin, cefuroxime and clindamycin and determined drug-specific IFN-γ and IL-5 secretion measured by ELISA read-out. We included PBMC from 60 non-drug allergic control persons, who were unexposed to the tested drug at the time of blood donation. RESULTS: PBMC from 12 out of 23 control persons tested with amoxicillin gave a positive stimulation index (SI > 3.0) for IFN-γ resulting in a specificity of 47.8%. The corresponding specificity was 75% for cefuroxime (5/20 if SI > 3.0) and 58.8% for clindamycin (7/17, if SI > 2.0), respectively. In a next step, we calculated the Δ IFN-γ concentration by subtracting the background IFN-γ concentration in the unstimulated sample from the stimulated sample. After stimulation with amoxicillin, a mean concentration of 21.0 pg/mL IFN-γ was secreted. The less outlier prone median concentration was 7.4 pg/mL and much higher than for cefuroxime (1.7 pg/mL) and clindamycin (1.0 pg/mL). Remarkably, IL-5 concentrations were below the detection limit (< 1 pg/mL) for all drugs in all control persons who responded to TT. CONCLUSION: Consideration of these observations may be helpful since a positive LTT result in a control patient may challenge the validity of a positive LTT result in the same experiment for a patient with assumed drug allergy.

Lymphocyte transformation test for drug allergy detection: When does it work?

BACKGROUND: The lymphocyte transformation test (LTT) is an in vitro test system for the detection of a sensitization in the context of allergies to drugs. Its reported sensitivity varies largely and seems to be affected by different parameters. In review articles, the average LTT performance was often calculated by combining overall mean sensitivities of various published studies, but without considering different patient characteristics or varying patient numbers per publication. OBJECTIVE: To investigate the impact of different patient-specific and methodological parameters on the sensitivity of the LTT based on data on the level of the individual patient extracted from single studies. METHODS: We performed an advanced literature search in PubMed and screened the identified publications according to previously defined inclusion criteria. In total, individual patient data from 721 patients were extracted from 30 studies. Random-effects meta-regression analyses were performed. RESULTS: The analysis indicate that the enzyme-linked immunosorbent assay-based read-out is more sensitive compared with the classical radioactivity method (enzyme-linked immunosorbent assay: 80% vs radioactivity: 66%; P = .08). Interestingly, drug reaction with eosinophilia and systemic symptoms/drug-induced hypersensitivity syndrome is associated with a higher probability of a positive LTT test result compared with other investigated clinical phenotypes ("drug reaction with eosinophilia and systemic symptoms/drug-induced hypersensitivity syndrome" vs "bullous reaction"; odds ratio, 2.52; P value = .003). Our analysis also revealed an impact of the time to testing period after the occurrence of the allergic event ("< 2 weeks" vs "2 weeks-2 months"; odds ratio, 2.12; P value = .03). CONCLUSION: The read-out method and relevant clinical parameters affect the sensitivity of the LTT. These findings are based on a meta-analysis providing a higher level of evidence than a single study or previous reviews not considering individual patient data.

Future perspectives on in-vitro diagnosis of drug allergy by the lymphocyte transformation test.

This article aims to envisage future perspectives of the lymphocyte transformation test (LTT). We describe the select innovative techniques, which can be integrated at different stages of the LTT to potentially improve the sensitivity, specificity, or practicability of the LTT. We first focus upon the cell sorting techniques comprising immunomagnetic cell separation and flow cytometry, which can be implemented prior and after the LTT culturing step to concentrate and quantify specific immune cell types. Further, we elaborate upon three important omics techniques such as transcriptomics, proteomics, and metabolomics, which can be integrated downstream of the LTT to analyze molecular changes in specific immune cells following drug induced activation and proliferation. We also develop visions, how state of the art techniques used in other scientific fields, can be transferred and applied in the context of in-vitro detection of drug allergy.

Lymphocyte transformation test: History and current approaches.

Drug-induced hypersensitivity reactions encompass a variety of different clinical phenotypes ranging from harmless rashes to fatal reactions. They can be classified into allergic (i.e. drug allergy) and non-allergic reactions (i.e. non-allergic hypersensitivity). Drug allergies in turn can either be antibody (e.g. IgE) or T cell-mediated. One of the diagnostic tools for the in vitro detection of drug allergy is the lymphocyte transformation test (LTT) which is based on the activation and expansion of the drug-specific memory T cells following co-incubation of the patient's peripheral mononuclear cells (PMBC) with the suspected drug in vitro. The read-out parameter in the classical LTT is T cell proliferation which can be measured as counts per minute following the addition of radiolabeled thymidine to the cell culture. However, in the course of time different modifications of the classical LTT with regard to the read-out parameters and methods have been proposed. Likewise, variations of the LTT platform itself have been described in the literature. This review article describes the development of the classical LTT and its use in the context of drug allergy detection and summarizes the modifications which have been published over time.

Compartment-specific distribution of human intestinal innate lymphoid cells is altered in HIV patients under effective therapy.

Innate lymphocyte cells (ILCs), a novel family of innate immune cells are considered to function as key orchestrators of immune defences at mucosal surfaces and to be crucial for maintaining an intact intestinal barrier. Accordingly, first data suggest depletion of ILCs to be involved in human immunodeficiency virus (HIV)-associated damage of the intestinal mucosa and subsequent microbial translocation. However, although ILCs are preferentially localized at mucosal surfaces, only little is known regarding distribution and function of ILCs in the human gastrointestinal tract. Here, we show that in HIV(-) individuals composition and functional capacity of intestinal ILCs is compartment-specific with group 1 ILCs representing the major fraction in the upper gastrointestinal (GI) tract, whereas ILC3 are the predominant population in ileum and colon, respectively. In addition, we present first data indicating that local cytokine concentrations, especially that of IL-7, might modulate composition of gut ILCs. Distribution of intestinal ILCs was significantly altered in HIV patients, who displayed decreased frequency of total ILCs in ileum and colon owing to reduced numbers of both CD127(+)ILC1 and ILC3. Of note, frequency of colonic ILC3 was inversely correlated with serum levels of I-FABP and sCD14, surrogate markers for loss of gut barrier integrity and microbial translocation, respectively. Both expression of the IL-7 receptor CD127 on ILCs as well as mucosal IL-7 mRNA levels were decreased in HIV(+) patients, especially in those parts of the GI tract with reduced ILC frequencies, suggesting that impaired IL-7 responses of ILCs might contribute to incomplete reconstitution of ILCs under effective anti-retroviral therapy. This is the first report comparing distribution and function of ILCs along the intestinal mucosa of the entire human gastrointestinal tract in HIV(+) and HIV(-) individuals.

Alterations of the NK cell pool in HIV/HCV co-infection.

BACKGROUND: A relevant proportion of human immunodeficiency virus (HIV) infected patients is co-infected with the hepatitis C virus (HCV). HCV co-infection in HIV-positive patients is associated with faster progression of liver disease in comparison to HCV mono-infection. Natural killer (NK) cells critically modulate the natural course of HCV infection. Both HIV and HCV mono-infection are associated with alterations of the NK cell pool. However, little data is available concerning phenotype and function of NK cells in HIV/HCV co-infection. METHODS: A total of 34 HIV/HCV co-infected, 35 HIV and 39 HCV mono-infected patients and 43 healthy control persons were enrolled into this study. All HIV-positive patients were under effective antiretroviral therapy. NK cell phenotype, IFN-γ production and degranulation were studied by flow cytometry. RESULTS: NK cell frequency in HIV/HCV co-infection was significantly lower than in healthy individuals but did not differ from HIV and HCV mono-infection. HIV/HCV co-infection was associated with significantly decreased expression of the maturation/differentiation markers CD27/62L/127 on NK cells but increased expression of CD57 compared to healthy controls. Of note, expression also differed significantly from HCV mono-infection but was similar to HIV mono-infection, suggesting a pronounced impact of HIV on these alterations. Similar findings were made with regard to the NK cell receptors NKG2A/C and NKp30. More importantly, NK cells in co-infection displayed a highly impaired functional activity with significantly lower IFN-γ production and degranulation than in healthy donors as well as HIV and HCV mono-infection, suggesting a synergistic effect of both viruses. CONCLUSIONS: Our data indicate that HIV/HCV co-infection is associated with significant alterations of the NK cell pool, which might be involved in the rapid progression of liver disease in co-infected patients and which mainly reflect alterations observed in HIV mono-infection.

IL-28B Genetic Variants Determine the Extent of Monocyte-Induced Activation of NK Cells in Hepatitis C.

BACKGROUND: Immuno-genetic studies suggest a functional link between NK cells and λ-IFNs. We recently showed that NK cells are negative for the IFN-λ receptor IFN-λR1 and do not respond to IFN-λ, suggesting a rather indirect association between IL-28B genotype and NK cell activity. METHODS: A total of 75 HCV(+) patients and 67 healthy controls were enrolled into this study. IL-28B (rs12979860) and IFNL-4 (rs368234815) genotypes were determined by rtPCR. Total PBMC, monocytes, and NK cells were stimulated with IL-29, the TLR-7/8 agonist R848, or a combination of both. NK cell IFN-γ response was analysed by FACS. IL-12 and IL-18 secretion of monocytes was studied by ELISA. In blocking experiments anti-IL-12/anti-IL-18 were used. RESULTS: Following stimulation of total PBMCs with R848 we found NK cell IFN- γ responses to vary with the IL-28B genotype, with carriers of a T/T genotype displaying the lowest frequency of IFN-γ(+)NK cells. When isolated NK cells were studied no such associations were observed, indicating an indirect association between IL-28B genotype and NK cell activity. Accordingly, we found R848-stimulated monocytes of patients with a T/T genotype to be significantly less effective in triggering NK cell IFN- γ production than monocytes from carriers of a non-T/T genotype. In line with these findings we observed monocytes from T/T patients to secrete significantly lower concentrations of IL-12 than monocytes from non-T/T individuals. CONCLUSIONS: Our data indicate that monocytes from carriers of an IL-28B T/T genotype display a reduced ability to stimulate NK cell activity and, thus, provide a link between IL-28B genotype and NK functions.

HIV mono-infection is associated with an impaired anti-hepatitis C virus activity of natural killer cells.

OBJECTIVE: Hepatitis C virus (HCV) infection in HIV(+) patients is associated with faster liver disease progression compared with HCV mono-infection. HIV-associated immune defects are considered to play an important role in this context. Here, we analyzed the effects of HIV infection on natural killer (NK)-cell-mediated anti-HCV activity. DESIGN: NK cell phenotype and interferon gamma (IFN-γ) production, NK cell-mediated inhibition of HCV replication and CD4 T-cell/NK cell interactions were studied in treatment naive HIV (n = 22), and HIV patients under combined antiretroviral therapy (n = 29), compared with healthy controls (n = 20). METHODS: NK cell-mediated inhibition of HCV replication was analyzed using the HuH7A2HCVreplicon model. IFN-γ production of NK cells as well as interleukin-2 secretion of CD4 T lymphocytes were studied by flow cytometry. RESULTS: Peripheral blood mononuclear cells from HIV(+) patients displayed a significantly impaired anti-HCV activity, irrespective of combined antiretroviral therapy. This could in part be explained by HIV-associated decline in NK cell numbers. In addition, NK cell IFN-γ production was significantly impaired in HIV infection. Accordingly, we found low frequency of IFN-γ(+) NK cells in HIV(+) patients to be associated with ineffective inhibition of HCV replication. Finally, we show that CD4 T-cell-mediated stimulation of NK cell IFN-γ production was dysregulated in HIV infection with an impaired interleukin-2 response of NK cells. CONCLUSION: HIV infection has a strong suppressive effect on anti-HCV activity of NK cells. This may contribute to low spontaneous clearance rate and accelerated progression of HCV-associated liver disease observed in HIV(+) patients.

CD3(+)CD56(+) Natural Killer-Like T Cells Display Anti-HCV Activity but Are Functionally Impaired in HIV(+) Patients With Acute Hepatitis C.

OBJECTIVE: To analyze the role of CD3(+)CD56(+) natural killer (NK)-like T cells in HIV(+) patients with acute hepatitis C. DESIGN: Frequency, phenotype, and anti-hepatitis C virus (HCV) activity of CD3(+)CD56(+) NK-like T cells were studied in 36 HIV(+) patients with acute hepatitis C. As controls, 12 patients with chronic HCV/HIV coinfection, 8 HIV monoinfected patients, and 12 healthy donors were enrolled in this study. METHODS: CD3(+)CD56(+) NK-like T-cell-mediated inhibition of HCV replication was analyzed using the HuH7A2HCVreplicon model. The CD3(+)CD56(+) NK-like T-cell phenotype and interferon (IFN)-γ secretion were studied by flow cytometry. RESULTS: Interleukin 12/interleukin 15 stimulated CD3(+)CD56(+) NK-like T cells from healthy donors effectively block HCV replication in vitro in an IFN-γ dependent manner. Accordingly, we found that blocking of IFN-γ with a specific antibody significantly reduced the antiviral activity of CD3(+)CD56(+) NK-like T cells. However, when CD3(+)CD56(+) NK-like T cells from HIV(+) patients were studied, we found HIV infection to be associated with a significantly impaired IFN-γ production, irrespective of HCV coinfection. Accordingly, CD3(+)CD56(+) NK-like T cells from HIV(+) patients were significantly less effective in blocking HCV replication in vitro than cells from healthy individuals. CONCLUSIONS: Taken together, our data indicate that HIV infection is associated with an impaired anti-HCV activity of CD3(+)CD56(+) NK-like T cells, which might represent a novel mechanism of dysregulated immune response in HIV/HCV-coinfected patients.

Hypoxia impairs anti-viral activity of natural killer (NK) cells but has little effect on anti-fibrotic NK cell functions in hepatitis C virus infection.

BACKGROUND & AIMS: Natural killer (NK) cells have been shown to exert anti-viral as well as anti-fibrotic functions in hepatitis C virus (HCV) infection. Previous studies, however, analyzed NK cell functions exclusively under atmospheric oxygen conditions despite the fact that the liver microenvironment is hypoxic. Here, we analyzed the effects of low oxygen tension on anti-viral and anti-fibrotic NK cell activity. METHODS: Peripheral (n=34) and intrahepatic (n=15) NK cells from HCV(+) patients as well as circulating NK cells from healthy donors (n=20) were studied with respect to anti-viral and anti-fibrotic activity via co-culture experiments with HuH7 replicon cells and hepatic stellate cells, respectively. RESULTS: Anti-viral activity of resting NK cells from healthy controls was not affected by hypoxia. However, hypoxia significantly reduced the response of healthy NK cells to cytokine stimulation. In contrast to healthy controls, we observed resting and cytokine activated peripheral NK cells from HCV patients to display a significantly decreased anti-viral activity when cultured at 5% or 1% oxygen, suggesting HCV NK cells to be very sensitive to hypoxia. These findings could be confirmed when intrahepatic NK cells were tested. Finally, we show that anti-fibrotic NK cell activity was not affected by low oxygen tension. CONCLUSIONS: Our results show that anti-viral function of NK cells from HCV(+) patients is critically affected by a hypoxic microenvironment and, therefore, indicate that in order to obtain an accurate understanding of intrahepatic NK cell anti-HCV activity, the laboratory modelling should take into account the liver specific levels of oxygen.

The ratio of calprotectin to total protein as a diagnostic and prognostic marker for spontaneous bacterial peritonitis in patients with liver cirrhosis and ascites.

BACKGROUND: Diagnosis of spontaneous bacterial peritonitis (SBP) is based on a differential ascites leukocyte count which does not provide prognostic information. We performed a pilot study to assess calprotectin in ascites as an alternative diagnostic and prognostic marker. METHODS: We collected ascites from patients with liver cirrhosis from March 2012 to July 2013. Routine clinical and laboratory data of the patients were recorded. Ascites calprotectin levels were determined by ELISA. RESULTS: Overall, we collected 120 ascites samples from 100 patients with liver cirrhosis and from eight patients with malignant peritoneal effusion as disease control. Samples without infection had significantly lower calprotectin levels (median 34 ng/mL, range 5-795) than SBP samples (median 928 ng/mL, range 21-110,480; p<0.001) and malignant effusions (median 401, range 47-2596 ng/mL; p<0.001). In non-infected ascites, calprotectin levels were higher in Child-Pugh stage B versus C (median 57 ng/mL vs. 17 ng/mL; p<0.001) and in alcoholic versus viral cirrhosis (median 37 ng/mL vs. 10 ng/mL; p=0.015). The ratio of ascites calprotectin to total protein was a better marker for SBP than calprotectin alone (AUROC=0.93; p<0.001; sensitivity 93%, specificity 79%; positive predictive value 60%; negative predictive value 97%). In addition, high levels of the ratio to total protein were associated with poor 30-day survival (p=0.042). CONCLUSIONS: The ratio of ascites calprotectin to total protein may be a promising new diagnostic and prognostic marker in patients with liver cirrhosis and SBP and should be evaluated further.

Regulatory CD4+ T cells modulate the interaction between NK cells and hepatic stellate cells by acting on either cell type.

BACKGROUND & AIMS: NK cells regulate liver fibrosis by killing activated hepatic stellate cells (HSCs) and are controlled themselves by immune cells and/or soluble factors. Here, we analysed if CD4(+) regulatory T cells (Tregs) modify the interaction between NK cells and HSCs. METHODS: The modification of NK cell activity against HSCs was studied in CD56(high)CD16(-) NK cells, using a flow cytometric CD107a degranulation assay and co-cultures with Tregs from healthy donors and patients with hepatitis C, respectively. We studied the underlying mechanisms in detail, applying Treg supernatants, Treg pretreated HSCs, and recombinant IL-8, TGF-ß1, and IL-10 as well as blocking experiments with neutralizing antibodies and analysed Treg-associated changes in the expression of NK cell receptor ligands on HSCs. RESULTS: Tregs suppressed NK cell activation during HSC co-culture in a cell-contact-dependent manner involving the cytotoxic T-lymphocyte antigen 4 (CTLA-4). NK cell degranulation was further reduced, when HSCs had been pretreated with Tregs (p=0.043), Treg supernatants (p=0.001) or recombinant IL-8 (R=0.630, p=0.001) and TGF-ß1 (R=0.608, p=0.002), respectively. This additional inhibitory effect corresponded to the IL-8/TGF-ß1-mediated downregulation of MIC-A/B and HLA class-I on HSCs. Tregs from hepatitis C likewise inhibited NK cell activity, which was reversed significantly in specific blocking experiments. CONCLUSIONS: Our data indicate that Tregs interfere with NK cell regulation of fibrogenesis via both direct cell-contact-dependent inhibition of NK cells and release of soluble factors, downregulating activating NK cell receptor ligands on HSCs. Our data may be particularly relevant for the intrahepatic accumulation of Tregs in chronic hepatitis C because downregulated NK cell activity against HSCs may blunt their control of fibrogenesis.

CD27(+)CD56Bright natural killer cells may be involved in spontaneous clearance of acute hepatitis C in HIV-positive patients.

OBJECTIVE: The objective of this study was to analyse the potential role of CD27 in natural killer (NK) cell-mediated control of hepatitis C virus (HCV) infection in HIV-positive patients. DESIGN: Frequency of CD27-expressing CD56 NK cells was analysed in HIV mono-infected individuals and HIV-positive patients with acute or chronic hepatitis C. Anti-HCV activity of CD27(+) and CD27(-) NK cells was compared. METHODS: NK cell mediated inhibition of HCV replication was analysed using the HUH7 HCV Replicon model. NK cell phenotype and interferon (IFN) secretion was studied by flowcytometry. RESULTS: High frequency of CD27(+)CD56 NK cells is associated with spontaneous clearance of acute hepatitis C in HIV-positive patients. Accordingly, we found CD27(+)CD56 NK cells to display strong anti-HCV activity. CONCLUSION: Our results underline the important role of NK cells in modulating outcome of HCV infection.

An effective interferon-gamma-mediated inhibition of hepatitis C virus replication by natural killer cells is associated with spontaneous clearance of acute hepatitis C in human immunodeficiency virus-positive patients.

UNLABELLED: Hepatitis C virus (HCV) coinfection is an increasing health problem in human immunodeficiency virus-positive (HIV(+) ) individuals. However, a considerable proportion of HIV(+) patients manage to overcome acute hepatitis C (AHC) spontaneously. In the present study, we analyzed the role of natural killer (NK) cells in modulating the course of AHC in HIV(+) patients. Twenty-seven HIV(+) patients with AHC (self-limited course: n = 10; chronic course: n = 17), 12 HIV(+) patients with chronic hepatitis C (CHC), 8 HIV monoinfected individuals, and 12 healthy controls were studied. NK cells were phenotypically analyzed by flow cytometry. Interferon-gamma (IFN-γ) secretion, degranulation (CD107a), and anti-HCV (= inhibition of HCV replication) activity of NK subpopulations were analyzed using the HuH7A2 HCVreplicon cell system. NK cell frequency did not differ significantly between HIV(+) patients with chronic and self-limited course of AHC. However, we found NK cells from patients with self-limiting infection to be significantly more effective in inhibiting HCV replication in vitro than NK cells from patients developing CHC. Of note, antiviral NK cell activity showed no significant correlation with NK cell degranulation, but was positively correlated with IFN-γ secretion, and blocking experiments confirmed an important role for IFN-γ in NK cell-mediated inhibition of HCV replication. Accordingly, NK cells from patients that spontaneously cleared the virus displayed a stronger IFN-γ secretion than those developing chronic infection. Finally, we observed high expression of NKG2D and NKp46, respectively, to be associated with self-limiting course of aHCV. Accordingly, we found that blocking of these NK cell receptors significantly impaired antiviral NK cell activity. CONCLUSION: Our data suggest a strong IFN-γ-mediated antiviral NK cell response to be associated with a self-limited course of AHC in HIV(+) patients.

Influence of the CXCL1 rs4074 A allele on alcohol induced cirrhosis and HCC in patients of European descent.

BACKGROUND AND AIMS: CXCL1 (CXC chemokine-ligand-1) is a ligand for CXC chemokine receptor 2 expressed on hepatic stellate cells (HSC). Thus, CXCL1 might contribute to HSC activation and fibrogenesis. In the present study, we investigated the influence of the CXCL1 rs4074 polymorphism on the occurrence of alcohol induced liver cirrhosis and hepatocellular carcinoma (HCC). METHODS: The study involved 458 patients with alcoholic cirrhosis (170 with HCC), 115 alcoholics without liver disease and 342 healthy controls. All subjects were genotyped for the CXCL1 rs4074 polymorphism and CXCL1 serum levels of 132 patients were measured. In vitro CXCL1 secretion in TLR-transfected cell lines were studied by ELISA. RESULTS: Distribution of the CXCL1 genotypes (GG/GA/AA) was 159/219/80 in patients with alcoholic cirrhosis, 52/44/19 in alcoholic controls and 158/140/44 in healthy controls. Patients with alcohol-induced cirrhosis were significantly more often carriers of the CXCL1 rs4074 A allele (65.3%) than alcoholics without liver disease (54.8%, OR=1.55; 95%CI=1.025-2.350; p=0.04) and healthy controls (53.8%, OR=1.62; 95%CI=1.212-2.151; p=0.001). Accordingly, the frequency of the CXCL1 rs4074 A allele was significantly higher in the cirrhotic patients than in the subjects without cirrhosis (41.4% vs. 33.9%, OR=1.38, 95% CI:1.14-1.66, p=0.001). Furthermore cirrhotic carriers of the CXCL1 rs4074 A allele had significantly higher CXCL1 serum levels than carriers of the GG genotype. In contrast to sera from healthy controls, sera from patients with alcoholic cirrhosis induced CXCL1 secretion in TLR2- (p=0.016) and TLR4- (p=0.008) transfected HEK293 cells. This finding indicates that sera from patients with alcoholic cirrhosis contain soluble ligands that can induce CXCL1 production via stimulation of TLRs. CONCLUSION: The enhanced CXCL1 serum levels in carriers of the rs4074 A allele together with their increased frequency in patients with alcohol induced cirrhosis suggest the CXCL1 rs4074 A allele as a genetic risk factor for alcoholic cirrhosis.

Impaired CD4⁺ T cell stimulation of NK cell anti-fibrotic activity may contribute to accelerated liver fibrosis progression in HIV/HCV patients.

BACKGROUND & AIMS: HIV/HCV co-infection is characterized by a faster progression to liver fibrosis compared to HCV mono-infection. Epidemiologic studies found an association between low CD4(+) T cell counts and advanced stages of liver fibrosis. However, the mechanisms underlying this association remain unclear. CD4(+) T cells critically modulate NK cell activity. Of note, NK cells have been shown to display anti-fibrotic activity via killing of activated hepatic stellate cells (HSC). Thus, we speculated that CD4(+) T cells might modulate fibrosis progression by interacting with NK cells. METHODS: NK cells from HCV(+) (n=35), HIV(+)/HCV(+) (n=28), HIV(+) (n=8) patients, and healthy controls (n=30) were used in this study. NK cells were cultured in the presence or absence of supernatants from CD3/CD28-stimulated CD4(+) cells. Then, NK cells were co-incubated with activated HSC and studied for degranulation, IFN-γ secretion, and induction of HSC apoptosis. RESULTS: Following incubation with CD4(+) T cell supernatants, NK cells displayed a significantly increased activity against primary HSC as compared to unstimulated NK cells. This effect was, at least in part, mediated via an IL-2 dependent upregulation of NKG2D expression. HCV/HIV co-infection was associated with an impaired IL-2 secretion of CD4(+) T cells resulting in an ineffective stimulation of anti-fibrotic NK cell function. CONCLUSIONS: Here, we show that CD4(+) T cells are able to stimulate anti-fibrotic NK cell activity via IL-2 mediated upregulation of NKG2D. HIV-induced loss of CD4(+) T cells together with an impaired activity of CD4(+) T cells may contribute to accelerate progression of liver fibrosis observed in co-infection.

The CXCR3(+)CD56Bright phenotype characterizes a distinct NK cell subset with anti-fibrotic potential that shows dys-regulated activity in hepatitis C.

BACKGROUND: In mouse models, natural killer (NK) cells have been shown to exert anti-fibrotic activity via killing of activated hepatic stellate cells (HSC). Chemokines and chemokine receptors critically modulate hepatic recruitment of NK cells. In hepatitis C, the chemokine receptor CXCR3 and its ligands have been shown to be associated with stage of fibrosis suggesting a role of these chemokines in HCV associated liver damage by yet incompletely understood mechanisms. Here, we analyzed phenotype and function of CXCR3 expressing NK cells in chronic hepatitis C. METHODS: Circulating NK cells from HCV-infected patients (n = 57) and healthy controls (n = 27) were analyzed with respect to CXCR3 and co-expression of different maturation markers. Degranulation and interferon-γ secretion of CXCR3(+) and CXCR3(-) NK cell subsets were studied after co-incubation with primary human hepatic stellate cells (HSC). In addition, intra-hepatic frequency of CXCR3(+) NK cells was correlated with stage of liver fibrosis (n = 15). RESULTS: We show that distinct NK cell subsets can be distinguished based on CXCR3 surface expression. In healthy controls CXCR3(+)CD56Bright NK cells displayed strongest activity against HSC. Chronic hepatitis C was associated with a significantly increased frequency of CXCR3(+)CD56Bright NK cells which showed impaired degranulation and impaired IFN-γ secretion in response to HSC. Of note, we observed intra-hepatic accumulation of this NK cell subset in advanced stages of liver fibrosis. CONCLUSION: We show that distinct NK cell subsets can be distinguished based on CXCR3 surface expression. Intra-hepatic accumulation of the functionally impaired CXCR3(+)CD56Bright NK cell subset might be involved in HCV-induced liver fibrosis.

Natural killer p46High expression defines a natural killer cell subset that is potentially involved in control of hepatitis C virus replication and modulation of liver fibrosis.

UNLABELLED: Natural killer (NK) cells play a role in the early control and natural course of hepatitis C virus (HCV) infection. NK cell function is regulated by a multitude of receptors, including activating NKp46 receptor. However, reports on NKp46 in hepatitis C are controversial. Therefore, we investigated the hepatic recruitment and function of NKp46(+) NK cells, considering differential surface expression of NKp46 resulting in NKp46(High) and NKp46(Dim) subsets. Intra- and extrahepatic NK-cell subsets from HCV-infected patients were characterized by flow cytometry. Cytotoxic activity and interferon-gamma (IFN-γ) secretion were studied using K-562, P815, and primary hepatic stellate cells as targets. Anti-HCV activity of NK-cell subsets was studied using the replicon system. Density of NKp46 surface expression clearly segregated NKp46(Dim) and NKp46(High) subsets, which differed significantly with respect to the coexpression of maturation markers and NK-cell receptors. More important, NKp46(High) NK cells showed a higher cytolytic activity and stronger IFN-γ secretion than NKp46(Dim) NK cells. Accordingly, NKp46(High) NK cells efficiently blocked HCV replication in vitro. Blocking experiments confirmed an important role for the NKp46 receptor. Furthermore, we found an intrahepatic accumulation of NKp46(High) NK cells. Of note, high cytolytic activity of NKp46(High) NK cells was also confirmed in the intrahepatic NK-cell population, and the frequency of intrahepatic NKp46(High) NK cells was inversely correlated with HCV-RNA levels and fibrosis stage. CONCLUSIONS: NKp46(High) expression defines a specific NK-cell subset that may be involved in both the suppression of HCV replication and HCV-associated liver damage underpinning the role of NK cells in the immunopathogenesis of HCV.