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Abstract

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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 NKp46High and NKp46Dim 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 NKp46Dim and NKp46High subsets, which differed significantly with respect to the coexpression of maturation markers and NK-cell receptors. More important, NKp46High NK cells showed a higher cytolytic activity and stronger IFN-γ secretion than NKp46Dim NK cells. Accordingly, NKp46High 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 NKp46High NK cells. Of note, high cytolytic activity of NKp46High NK cells was also confirmed in the intrahepatic NK-cell population, and the frequency of intrahepatic NKp46High NK cells was inversely correlated with HCV-RNA levels and fibrosis stage. Conclusions: NKp46High 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. (HEPATOLOGY 2012)

Infection with the hepatitis C virus (HCV) often results in chronic liver disease. Elimination of HCV infection requires the coordinated function of the innate and the adaptive immune system.

Natural killer (NK) cells constitute a major component of the intrahepatic lymphocyte pool. In contrast to the peripheral blood, which contains approximately 5%-10% NK cells, intrahepatic lymphocytes comprise approximately 30% NK cells, and the percentage of intrahepatic NK cells may increase >50% in liver diseases.1

Immunogenetic analyses indicate that NK cells may influence the outcome of acute HCV infection as well as immunopathogenesis in chronic hepatitis C (CHC).2 Accordingly, in vitro studies suggest that NK cells are able to recognize and kill HCV-infected hepatocytes in vivo.3 However, published data on phenotype and function of NK cells in HCV infection are controversial.4-13

Based on the expression of CD56, human NK cells have been divided into two functional subsets.14 In humans, approximately 90% of circulating NK cells belong to the CD56Dim subset, which is characterized by moderate to high expression levels of Fc-γ RIIIA (CD16) and perforin, as well as high cytotoxic capability. CD56Bright NK cells, in contrast, display rather poor natural cytolytic activity and are considered to function as immunoregulatory cells through the secretion of cytokines, such as interleukin (IL)-15.14

The function of NK cells is tightly orchestrated by a balance between signals derived from inhibitory and activating receptors.15

NKp46, a member of the natural cytotoxicity receptor family, is a main activating NK-cell receptor.16 Although its ligand(s) remain elusive, recent studies demonstrated NKp46 to be critically involved in lymphoma and melanoma eradication. Moreover, NKp46 plays an important role in the killing of virus-infected cells by interacting with viral proteins.17-21 Studies on NKp46 in HCV infection are controversial, because both reduced and increased surface expression have been reported.5, 7-9

In this context, it is of interest that earlier studies showed that the surface expression of NKp46 may vary in different human peripheral blood NK cells, and that the NKp46 phenotype of NK clones correlates with cytolytic activity.22

In the present study, we compared the ex vivo phenotypic and functional characteristics of circulating and intrahepatic NKp46Dim and NKp46High NK cells, and demonstrate that NKp46 surface density defines, phenotypically and functionally, different human NK-cell subsets. Moreover, we show the intrahepatic accumulation of NKp46High cells and present first data suggesting a role of NKp46High NK cells in the control of viral replication and hepatic fibrogenesis in hepatitis C.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Patients.

A total of 57 Caucasian patients, all from the Bonn area in Germany, were studied, including 36 patients with CHC genotype 1 infection, 12 patients with nonalcoholic steatohepatitis (NASH), and 9 patients with autoimmune (AI)-mediated hepatitis. Liver biopsies were available from 41 patients (23 HCV patients, 12 NASH patients, and 9 AI patients). Staging and grading of liver biopsies were performed as part of the routine diagnostic work-up.

As an additional control, we studied 27 healthy donors (Table 1).

Table 1. Patient Characteristics
CharacteristicsAINASHHCVHealthy Controls
  • Abbreviations: AST, aspartate aminotransferase; γ-GT, gamma-glutamyl transferase; NA, not analyzed.

  • *

    Number of cases (number/total in %).

  • Mean (range).

Number9123627
Female sex,* (%)6 (66)6 (50)15(41.6)14 (51.8)
Age, years51.3 (23-76)51.0 (29-69)49.6 (18-81)29.4 (21-45)
Clinical data    
ALT, U/L176.8 (24-653)78.9 (10-286)135.8 (23-1,857)NA
AST, U/L122.7 (20-485)89.9 (15-318)83.2 (16-860)NA
γ-GT348.7 (37-1,136)365 (40-1,478)110.6 (25-716)NA
HCV status    
HCV load(× 105 IU/mL)169.9 (<0.02-1,740)
HCV genotypes (%)    
 1*36 (100)
 2*
 3*
 4*

Informed consent was obtained from all patients. The study had been approved by the local ethics committee of the University of Bonn (Bonn, Germany).

Flow Cytometry.

For fluorescence-activated cell sorting (FACS) analysis, the following fluorochrome-labeled antibodies were used: anti-CD3, anti-CD27, anti-CD56, anti-CD57, anti-CD62L, anti-CD69, anti-CD107a, anti-CD127, and anti-CD161 (all purchased from BD Biosciences, Heidelberg, Germany); anti-NKG2A, anti-NKG2C, anti-NKG2D, anti-NKp30, anti-NKp44, anti-NKp46, and anti-INF-γ (interferon-gamma) were purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany). Samples were analyzed on a FACSCanto flow cytometer using the CellQuest Pro (BD Biosciences) and FlowJo 7.2.2 software packages (TreeStar Inc., Ashland, OR).

Liver Specimens.

Liver biopsy specimens were obtained from routine liver biopsies. Fresh liver samples were washed twice in fresh medium and shaken gently to avoid blood contamination. After mechanical disruption, the fragments were homogenized on a cell strainer (BD Labware; BD Biosciences). The resulting cell suspension was washed and resuspended in RPMI 1640 medium. Intrahepatic cells were then directly analyzed by flow cytometry.

NK-Cell Separation.

NK cells were immunomagnetically separated from total peripheral blood mononuclear cells (PBMCs) by depletion of non-NK cells using MACS cell separation kits, following the manufacturer's recommendations (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of NK cells was >95%.

Isolation of NKp46Dim and NKp46High NK-Cell Subsets.

Separated NK cells were stained with fluorescein isothiocyanate–conjugated anti-NKp46, phycoerythrin-conjugated anti-CD56, and allophycocyanin-conjugated anti-CD3. Then, NKp46HighCD56+CD3 and NKp46DimCD56+CD3 were sorted using a BD FACSARIA III cell sorter (BD Biosciences). Purified cell subsets were cultured in the presence of 100 U/mL of interleukin (IL)-2 for 14 hours.

IFN-γ Secretion.

NK cells were cultured in the presence or absence of recombinant human (rh)IL-12 (0.1-10 ng/mL; eBioscience, San Diego, CA) and rhIL-15 (1-100 ng/mL; eBioscience) for 16 hours. Then, brefeldin A (BFA; 10 μg/mL; Sigma-Aldrich, St. Louis, MO) was added for another 4 hours, followed by intracellular staining with anti-IFN-γ and FACS analysis. Alternatively, NK cells were preincubated with IL-12 (0.1 ng/mL) and IL-15 (1 ng/mL) for 12 hours, washed two times with phosphate-buffered saline, resuspended in RPMI medium (10% fetal calf serum [FCS] and 1% penicillin/streptomycin), and cocultured with Huh7A2HCV replicon cells for 6 hours in the presence of BFA.

CD107a Degranulation Assay and NKp46-Mediated Redirected Killing Assay.

Purified NK cells were coincubated with either major histocompatibility complex–deficient K562 cells or Huh7A2HCV replicon cells at different effector/target (E:T) ratios in the presence of anti-CD107a to assess degranulation, as described before.22

For NKp46-mediated redirected killing assay, sorted NK-cell subsets were cultured with IL-2 (100 U/mL). The next day, NK cells were coincubated with carboxyfluorescein succinimidyl ester (CFSE)-labeled FcγR+P815 cells loaded with unconjugated anti-NKp46 (10 μg/mL; R&D Systems) for 30 minutes (E:T ratios: 1:1, 1:5, and 1:10). After 4 hours, 7-aminoactinomycin D (7-AAD; 2.5 μg/mL) was added for an additional 30 minutes in the dark, washed, and analyzed by FACS. Specific lysis was calculated as (% 7AAD+CSFE+ [dead targets] − % spontaneous 7-AAD+CSFE+ [dead targets])/(100 − % spontaneous 7-AAD+CSFE+ [dead targets]).

Huh7A2HCV Replicon Cells.

Huh7A2HCV replicon cells23 were kindly provided by V. Lohmann and R. Bartenschlager (Department of Molecular Virology, University of Heidelberg, Heidelberg, Germany). Cells were grown in high glucose (4.5 g/L) Dulbecco's modified Eagle's medium supplemented with glutamine (PAA Laboratories, Cölbe, Germany), 10% FCS, nonessential amino acids (Biochrom AG, Berlin, Germany), and 1% penicillin/streptomycin (PAA Laboratories). Blasticidin S hydrochloride (3 μg/mL) and G418 (1 mg/mL) (PAA Laboratories) were added to cells containing subgenomic replicons. Huh7A2HCV cells were passaged twice a week and were seeded at a dilution of 1:4.

Luciferase Assay.

Huh7-HCV replicon cells (1 × 105) were seeded in 24-well plates. After 3 hours, media were removed and replicon cells were cocultured with sorted NK-cell subsets at different E:T ratios for 24 hours in the presence of IL-2 (25 U/mL). In addition, Huh7-HCV replicon cells (1 × 105) were cultured for 24 hours with the supernatants of the respective direct coculture experiments. The assay was performed using the Steady-Glo Luciferase Assay System (Promega, Mannheim, Germany) and measured with Tecan infinite M200 (Tecan Group Ltd., Männedorf, Switzerland).

Primary Human Hepatic Stellate Cells.

Isolated primary activated human hepatic stellate cells (HSCs; ScienCell, San Diego, CA)24-26 were cultured for two to four passages in defined Stellate Cell Medium (SteCM; ScienCell) supplemented with 2% fetal bovine serum, 5-mL stellate cell growth supplement, 10 U/mL of penicillin, and 10 μg/mL of streptomycin (all ingredients obtained from ScienCell) at 37°C with 5% CO2 and cryopreserved until further use.

Two days before HSCs were used in an experiment, cells were thawed and cultured in SteCM medium. Then, cells were harvested, washed, checked for viability using trypan blue, and then used in the respective experiments. Activated status of HSC was verified by immunofluorescence staining of alpha-smooth muscle actin.

Blocking Experiments.

In blocking experiments, NK cells were preincubated with either anti-NKp46 (10 μg/mL; R&D Systems), anti-IFN-γ (10 μg/mL; BioLegend, San Diego, CA), or an isotype control.

Transcriptomic Profiling of NKp46High and CD56Bright Populations.

Total RNA (100 ng) from sorted NK-cell subpopulations obtained from 3 healthy donors were subjected to a single round of in vitro transcription and biotin labeling (Total Prep RNA amplification kit; Ambion, Austin, TX). Complementary RNA was hybridized on Human HT12 v4 Expression BeadChips (Illumina, San Diego, CA), according to the manufacturer's instructions. Expression data were exported from the Illumina BeadStudio software and analyzed using R/Bioconductor and limma. Data were quality-weighed, background-corrected, quantile-normalized, log-transformed, and explored for differentially expressed genes, with a false discovery rate <0.05 using a paired comparison. The expression sets for all biological replicates were assessed for similarities and dissimilarities using a principal components analysis.

Statistical Analysis.

Statistical analyses were performed using GraphPad Prism software (version 5.0a; GraphPad Software, Inc., San Diego, CA) and the SPSS 17.0 (SPSS, Inc., Chicago, IL) statistical package. Mann-Whitney's U tests were used to compare NK-cell phenotype, cytolytic response, cytokine levels, and luciferase activity of HUH7-HCV replicon cells between samples. Paired Student t tests were used to assess differences in NK-cell phenotypes between paired liver and blood samples. Linear regression analysis was used to examine correlations. A two-sided P value <0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

NKp46High Surface Expression Defines a Specific Human NK-Cell Subsets That Is Enriched in Hepatitis C.

First, we gated on CD3CD56+ NK cells for the assessment of NKp46 expression.

Density of NKp46 surface expression clearly segregated NKp46Negative, NKp46Dim, and NKp46High subsets, which in blood from healthy donors accounted for 21.1% ± 2.7%, 65.8% ± 2.4%, and 13.1% ± 6.1% of NK cells, respectively (Fig. 1A). The vast majority of NKp46Dim NK cells was found in the CD56Dim population, whereas the NKp46High subset was constituted of both CD56Dim and CD56Bright NK cells (Fig. 1B,C).

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Figure 1. Dissection of human NK-cell subsets by NKp46 surface expression. (A) Freshly isolated PBMCs were stained with anti-CD3, anti-CD56, and anti-Nkp46. CD3(−)CD56(+) NK cells were then gated for the quantification of NKp46 surface density. (B and C) Distribution of NKp46High NK cells between the CD56Bright and CD56Dim subset. Columns represent mean and standard error of the mean. Data shown are representative for 10 healthy donors. (D) Frequency of circulating NKp46Dim (left graph) and NKp46High (right graph) in patients with CHC (n = 17), in comparison to healthy controls (n = 8), patients with NASH (n = 10), and AI-mediated liver disease (n = 8). *P < 0.05.

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Because surface expression of NK receptors often corresponds to NK maturation and function,12, 13, 27 we next analyzed the expression patterns of several receptors in NKp46High and NKp46Dim NK-cell subsets (Supporting Fig. 1A). Compared with the NKp46Dim NK-cell subsets, the NKp46High subset displayed higher expression of CD127, CD62L, and CD27, but lower expression of CD57. In addition, there was a greater fraction of the NKp46High subpopulation expressing the NK-cell receptors, NKp44, NKG2A, and NKG2C.

Of note, HCV infection was associated with a significantly lower proportion of NKp46Dim, but higher frequency of NKp46High NK cells, as compared to healthy controls, the NASH controls, and patients with AI-mediated liver disease (P < 0.05 versus each; Fig. 1D). Coexpression of maturation/differentiation markers (e.g., CD127, CD94, CD62L, and CD27) on circulating NKp46High and NKp46Dim subsets did not differ significantly between HCV patients and healthy controls (data for review only). Analysis of intrahepatic NK cells confirmed that NKp46High and NKp46Dim NK cells differ phenotypically (Supporting Fig. 1B). Of note, microarray analysis revealed that more than 800 genes were differentially regulated between NKp46High and CD56Bright NK cells (Supporting Table 2), and we did not find any significant correlation between frequency of CD56Bright and NKp46High NK cells (Supporting Fig. 3), indicating that the NKp46High NK-cell subset is different from CD56Bright NK cells.

Circulating NKp46High NK Cells Have a Strong Cytotoxic Potential and Show High IFN-γ Secretion.

Next, we compared the functional activity of NKp46High and NKp46Dim NK-cell subsets. The NKp46High cell subset displayed a significantly higher cytolytic activity than the NKp46Dim subpopulation against K562 cells (Fig. 2A). Importantly, also within the cytolytic CD56Dim subset, high expression of NKp46 marked cells with strong cytotoxic activity (Supporting Fig. 2A).

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Figure 2. Peripheral blood NKp46High NK cells are highly cytotoxic. (A) Lysis of K562 cells by sorted NKp46Dim (open triangles) and NKp46High NK cells at different E:T cell ratios. Data are given for 3 healthy donors as mean and SEM, as indicated. (B) Cytolytic activity of the NKp46High and NKp46Dim NK-cell subsets derived from healthy controls (n = 3) against FcR+ P815 cells loaded without monoclonal antibody (left graph), an immunoglobulin G–specific control antibody (middle graph), or with anti-NKp46 (right graph). (C) Activity of NKp46Dim and NKp46High NK cells from HCV(+) patients (n = 4), in comparison to healthy controls (n = 3), against NKp46-loaded P815 cells. *P < 0.05. (D) IFN-γ secretion of purified NK-cell subsets from healthy donors after stimulation with either IL-12 (10 ng/mL) or IL-15 (100 ng/mL) or a combination of both cytokines. Bar graphs represent mean and SEM for at least four independent experiments. (E) IFN-γ secretion after stimulation with increasing concentrations of IL-12 and IL-15 in healthy controls (n = 4) and patients with CHC (n = 4). (F) Comparison of IFN-γ secretion between healthy controls and HCV(+) patients (n = 4). *P < 0.05. SEM, standard error of the mean.

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In addition, we also analyzed the NKp46-specific cytotoxic activity of NKp46High and NKp46Dim NK cells in a redirected killing assay, as described before28 (Fig. 2B). As could be expected, NKp46High NK cells displayed a significantly higher NKp46-specific cytolytic activity against anti-NKp46-loaded P815 targets than the NKp46Dim subset (Fig. 3B: 5:1: P < 0.05; 10:1: P < 0.05). However, median lytic activity of NKp46High NK cells exceeded that of the NKp46Dim subset, even when unloaded or isotype-loaded P815 cells were used (P < 0.05).

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Figure 3. NKp46High NK-cell subsets effectively block HCV replication and preincubation with anti-IFN-γ significantly blocks anti-HCV activity. (A) Huh7-HCV replicon cells were cocultured with sorted NKp46High and NKp46Dim NK-cell subsets, respectively, at different E:T ratios (B) or were cultured with the supernatants of the direct coculture experiments. Then, luciferase activity was measured. The graph shows inhibition of luciferase activity (LU) in relation to Huh7-HCV replicon cells cultured alone (n = 4). (C) IFN-γ secretion of NK-cell subsets after stimulation with HUH7replicon cells in healthy controls (n = 8) and HCV RNA(+) patients (n =). (D) Effect of IFN-γ blockade on anti-HCV activity in NK46High and NKp46Dim NK cells (n = 4), respectively.

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Of note, functional analysis of circulating NK cells from HCV(+) individuals confirmed these findings. Interestingly, cytolytic potential of both NK-cell subsets was higher in HCV-infected patients, as compared to healthy individuals (Fig. 2C: P < 0.05, NKp46High versus NKp46Dim; P < 0.05, HCV[+] versus HCV[−]).

Next, we compared IFN-γ production from monokine-activated NKp46Dim and NKp46High NK-cell subsets. The fraction of IFN-γ(+) cells was significantly greater in the NKp46High than in the Nkp46Dim subset (P < 0.05; Fig. 2D). Titration experiments confirmed that this difference in IFN-γ production remained significant, even after stimulation with high doses of IL-12 and IL-15 in both healthy (Fig. 2E, left graph) and HCV-infected (Fig. 2E, right graph) individuals (P < 0.05). Interestingly, NKp46Dim NK cells from HCV(+) patients displayed significantly lower IFN-γ secretion than NKp46Dim NK cells obtained from healthy controls (P < 0.05), whereas no differences were observed in the NKp46High subset (Fig. 2F).

NKp46 Is Involved in anti-HCV Activity of NK Cells.

Next, we studied the anti-HCV activity of both NK-cell subsets using Huh7-HCV replicon cells. Of note, NKp46High NK cells were significantly more effective in blocking HCV replication in vitro, as compared to the NKp46Dim NK-cell subset (Fig. 3A: P < 0.05). Similar findings were observed after incubation of Huh7-HCV replicon cells with supernatants of the respective direct coculture experiments, suggesting that soluble factors may be involved (Fig. 3B: P < 0.05). Accordingly, NKp46High NK cells showed significantly higher IFN-γ secretion after coincubation with HUH7replicon cells than the NKp46Dim subset (Fig. 3C). Moreover, the addition of an IFN-γ-specific antibody effectively blocked NK-cell-mediated inhibition of HCV replication (Fig. 3D).

In summary, these data showed that NKp46High NK cells effectively block HCV replication in vitro, suggesting a role for NKp46 in the control of HCV infection. In line with this hypothesis, we found that preincubation of NK cells with anti-NKp46 significantly reduced anti-HCV activity of both NK-cell subsets (Fig. 4A). Moreover, blocking of NKp46 not only impaired the inhibition of HCV replication (Fig. 4B), but also resulted in reduced NK-cell degranulation and IFN-γ secretion, irrespective of HCV infection (Fig. 4C,D).

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Figure 4. NKp46 is involved in anti-HCV activity of NK cells from HCV(+) patients and healthy donors. (A) Preincubation with anti-NKp46 significantly blocks anti-HCV activity in both NK46High and NKp46Dim NK cells (n = 6). Effect of anti-NKp46 on anti-HCV activity (B) or NK-cell degranulation (C) and IFN-γ secretion after coincubation with Huh7A2HCV replicon cells (D) in HCV(+) patients (n = 8) and healthy controls (n = 7). *P < 0.05; **P < 0.01.

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Intrahepatic Accumulation of NKp46High NK Cells.

The liver represents the main site of viral replication in hepatitis C. Thus, we next compared the percentages of NKp46High and NKp46Dim NK cells between peripheral blood and the intrahepatic compartment: The liver was enriched for NKp46High NK cells, whereas the frequency of NKp46Dim cells was significantly lower in the liver than in peripheral blood in hepatitis C (Fig. 5B). This differential compartmentalization of both NK-cell subsets was also found in patients with NASH (Fig. 5C) and AI-mediated liver disease (Fig. 5D).

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Figure 5. Intrahepatic accumulation of NKp46High NK cells. (A) Example for staining of intrahepatic NK-cell subsets. Frequency of extrahepatic (EH) and intrahepatic (IH) NKp46Dim and NKp46High NK cells was analyzed in patients with CHC (n = 17) (B), patients with NASH (n = 11) (C), or patients with AI-mediated liver diseases (n = 8) (D).

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Intrahepatic Accumulation of NKp46High NK Cells Is Correlated With Viral Load.

To corroborate our data, we finally analyzed the cytotoxic activity of intrahepatic NK cells. Of note, the finding of strong cytolytic potential of circulating NKp46High NK cells (Fig. 6A, right graph) could also be confirmed in the intrahepatic NK-cell pool (Fig. 6A, left graph: P < 0.05).

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Figure 6. Function of NKp46High NK cells in patients with HCV infection. (A) Cytolytic activity of intrahepatic NKp46High and NKp46Dim NK cells (n = 4), in comparison to circulating NK-cell subsets (n = 4), was assessed by flow cytometric analysis of CD107 expression after coincubation with K562 target cells. (B) Huh7-HCV replicon cells were cocultured with intrahepatic NK cells from HCV(+) patients (n = 6) in the absence or presence of anti-NKp46. Then, luciferase activity was measured. The graph shows inhibition of luciferase activity (LU) in relation to Huh7-HCV replicon cells cultured alone. Correlation between intrahepatic frequency of NKp46High NK cells (left graphs) or NKp46 surface expression density (RFI) on intrahepatic NK cells (right graphs) and HCV serum loads (IU/mL) (C), ALT serum levels (IU/mL) (D), and HAI score (E) in CHC.

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Moreover, preincubation with anti-NKp46 significantly reduced the ability of NK cells isolated from HCV-infected livers to block HCV replication in vitro, which further supports a role for NKp46 in the regulation of HCV (Fig. 6B).

Therefore, we sought for potential associations between the frequency of intrahepatic NK cells and demographic and clinical data.

The frequency of intrahepatic NKp46High NK cells (Fig. 6C, left panel: r = −0.795; P = 0.0006) as well as the density of NKp46 surface expression on intrahepatic NK cells (Fig. 6C, right panel: r = −0.52; P = 0.02) showed a negative correlation with HCV load, whereas no such correlations were found for circulating NKp46High cells.

No correlation was found for alanine aminotransferase (ALT) levels (Fig. 6D) or liver inflammation (hepatitis activity index [HAI] score; Fig. 6E).

However, we found the frequency of intrahepatic NKp46High NK cells to be inversely associated with stage of liver fibrosis, with significantly higher frequencies in patients with no or only mild liver fibrosis (F = 0/1), as compared to patients displaying more advanced fibrosis stages (P = 0.047; Fig. 7A). Therefore, we finally analyzed the role of NKp46 in interactions of NK cells with primary human HSCs. We found NKp46High NK cells to show significantly higher cytotoxic activity against HSCs, as compared to the NKp46Dim subset in both HCV(+) patients and healthy controls (Fig. 7B). Moreover, blocking experiments confirmed a role for NKp46 in NK-cell activity against HSCs (Fig. 7C,D).

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Figure 7. Function of NKp46High NK cells in patients with HCV infection. (A) Correlation between intrahepatic frequency of NKp46High NK cells and fibrosis stage. (B) Cytolytic activity of NK-cell subsets from HCV(+) patients (n = 5) and healthy controls (n = 6) against primary HSCs. Effect of NKp46 blockade on NK-cell degranulation (C) and IFN-γ secretion (D) after coincubation with HSCs in HCV(+) patients (n = 5) and healthy controls (n = 7).

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Discussion

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

There is accumulating evidence that NK cells are critically involved in the control of infection by many viruses,2, 29-32 and immunogenetic analyses indicate that NK cells may influence the outcome of HCV infection.2 Moreover, in vitro studies suggest that NK cells should also be able to recognize and kill HCV-infected hepatocytes in vivo.3 The exact role of NK cells in HCV infection, however, remains elusive, because current studies have yielded inconsistent results regarding phenotype and function of NK cells.4-12

For example, surface expression and function of NKp46, a major activating receptor, is discussed controversially.5, 7-9, 33 These discrepancies might, in part, be explained by different characteristics of study cohorts and/or methodological issues.28 Moreover, it is important to note that previous reports not only demonstrated varying expression of NKp46 in different NK-cell clones, but also suggested that density of NKp46 expression may correlate with cytolytic activity in vitro.22, 34

Therefore, we put forward the hypothesis that the NKp46 phenotype (i.e., NKp46Dim versus NKp46High) defines different NK-cell subsets and studied the function of these NK-cell subpopulations as well as the role of NKp46 in hepatitis C.

First, we compared the ex vivo phenotypic and functional characteristics of circulating NKp46Dim and NKp46High NK cells.

NK-cell subsets characterized by dim or high expression of NKp46, partially overlapped with “classical” subpopulations defined on the basis of CD56 expression. Most of the NKp46Dim NK cells were CD56Dim, whereas the majority of NKp46High NK cells were CD56Bright. Of note, microarray analysis demonstrated that more than 800 genes were differentially regulated between NKp46High and CD56Bright NK cells (Supporting Table 2), and there was no significant correlation between frequency of CD56Bright and NKp46High NK cells (Supporting Fig. 3), indicating that the population of NKp46High NK cells is different from CD56Bright NK cells.

Moreover, a relevant proportion of NKp46High cells was also found in the CD56Dim subset. Different expression levels of maturation markers, activating and inhibitory NK receptors, and, more important, the difference in IFN-γ secretion and cytolytic activity corroborated that NKp46Dim and NKp46High NK cells are separate subsets.

It has long been debated whether CD56Bright and CD56Dim NK cells correspond to distinct, terminally differentiated cell types or rather represent a continuum of NK-cell differentiation. However, increasing evidence indicates that NK-cell development proceeds from a CD56Bright to CD56Dim phenotype, which is accompanied by changes in the expression of specific surface molecules.35-39 NKp46High NK cells expressed higher levels of “immature” NK cell markers, such as CD62L, CD27, and CD94, than the NKp46Dim subset, suggesting a dynamic regulation of NKp46 expression during NK-cell maturation.

Functional studies demonstrated NKp46High NK cells to be “bifunctional,” in that they are capable of producing IFN-γ and yet have a high cytolytic activity, compared to the NKp46Dim subset. Of note, high cytolytic activity of the NKp46High subset could also be confirmed in the intrahepatic NK-cell pool. In addition, we observed that a significant accumulation of NKp46High NK cells in the liver, which is the main site of viral replication, in HCV infection was an intrahepatic accumulation. Thus, NKp46High NK cells are not only in the right location, but also display the functional capacity needed to control a hepatotropic virus such as HCV.

Accordingly, we showed that NKp46High NK cells efficiently control HCV replication in vitro. Moreover, we found that intrahepatic frequency of NKp46High NK cells inversely correlated with HCV loads. Whether NKp46 recognizes a HCV-derived protein17-21 or interacts with a yet unknown NKp46 ligand, as has been suggested for malignant transformed cells,40, 41 remains to be clarified. However, blocking of NKp46 significantly reduced the capability of both circulating and intrahepatic NK cells to block HCV replication, suggesting a role of NKp46 in the regulation of anti-HCV immune responses.

Interestingly, there was no significant association between intrahepatic frequency and liver inflammation, suggesting an important role for noncytolytic effector functions. Accordingly, we found that anti-IFN-γ significantly blocked the antiviral activity of both NKp46Dim and NKp46High NK cells, indicating a role of IFN-γ secretion in NK-cell-mediated inhibition of viral replication. This would be in line with data recently published by Jo et al. who showed that in vitro antiviral activity of HCV-specific CD8+ T cells is critically dependent on noncytolytic effector functions.42

In addition, our findings suggest that NKp46 also may mediate antifibrotic effects, because we found the frequency of intrahepatic NKp46High NK cells to be inversely associated with stage of fibrosis. Moreover, we could show that NK-cell degranulation and IFN-γ secretion after coincubation with human HSCs involved NKp46, because the blocking of this receptor significantly reduced NK-cell activity. These observations are in line with recent findings showing that the killing of human HSCs is NKp46 dependent.43 Because activated HSCs critically contribute to the establishment of hepatic fibrosis, the killing of HSCs by immune cells may represent an important antifibrotic mechanism. Accordingly, dysregulated NK-cell function has been shown to be associated with an accelerated progression of liver fibrosis in mice.44, 45

However, there was a tendency toward higher numbers of NKp46High NK cells in livers with more severe liver inflammation, suggesting that these NK cells may also mediate detrimental effects.

In this context, it is of interest that intrahepatic accumulation of NKp46High NK cells was not specific for hepatitis C, indicating a potential role for NKp46-expressing NK cells in the immunopathogenesis of NASH and AI-mediated liver diseases. At the moment, little is known regarding the expression and function of NKp46 in these inflammatory liver diseases. However, there is clear evidence suggesting a role for NK cells. For instance, Kahraman et al. recently reported NASH to be associated with an increase in hepatic NK cells, and proposed that a dysregulated expression of NK-cell receptor ligands ?MICA/B? may affect the progression of liver injury.46

Taken together, we present the first data indicating that NKp46High NK cells represent a specific “bifunctional” NK-cell subset that may be involved both in the control of viral replication and inhibition of liver fibrosis in hepatitis C. Moreover, our data clearly support an important role of the NKp46 receptor in HCV infection.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Claudia Zwank and Monika Michalk for their perfect technical assistance and Robert Thimme (University of Freiburg, Freiburg, Germany) for his helpful discussions and comments.

References

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  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_25804_sm_SuppFig1A.tif3067KSupporting Information Figure 1A
HEP_25804_sm_SuppFig1B.tif2956KSupporting Information Figure 1B
HEP_25804_sm_SuppFig2.tif2488KSupporting Information Figure 2
HEP_25804_sm_SuppFig3.tif2229KSupporting Information Figure 3
HEP_25804_sm_SuppTab1.doc1100KSupporting Information Table 1

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