Potential conflict of interest: Nothing to report.
The authors thank Dr. Takaji Wakita (National Institute of Infectious Diseases) for kindly providing the JFH-1 plasmid. The authors thank the Colorado Center for AIDS Research Laboratory Core for access to FACS sorting.
Major racial and gender differences have been documented in the natural history and treatment responses of chronic hepatitis C virus (HCV) infection; however, distinct mechanisms have remained enigmatic. We hypothesized that racial- and gender-related differences in natural killer (NK) cell populations may explain altered natural history and treatment responses. Our study cohort consisted of 29 African-American (AA; 55% male) and 29 Caucasian-American (CA; 48% male) healthy uninfected control subjects. Multiparameter flow cytometric analysis was used to characterize levels, phenotype with respect to 14 NK receptors, and lymphokine-activated killing (LAK) function. Gene expression was assessed by real-time reverse-transcriptase polymerase chain reaction after 6-hour in vitro stimulation with Toll-like receptor (TLR) ligands. The ability to control HCV infection was assessed in the Huh-7.5/JFH-1 coculture system. NK expression of natural cytotoxicity receptor NKp46 was strongly associated with CA race and female gender and correlated positively with LAK activity (P = 0.0054). NKp46high NKs were more efficient at controlling HCV than their NKp46low counterparts (P < 0.001). Similarly, ligation of NKp46 on isolated NK cells resulted in a significant reduction in the HCV copy number detected in Huh-7.5/JFH-1 coculture (multiplicity of infection: 0.01) at an effector:target ratio of 5:1 (P < 0.005). After TLR stimulation, genes involved in cytotoxicity, but not cytokine genes, were significantly up-regulated in NKp46high NKs. Cytokine stimulation (interleukin [IL]-12 and IL-15) demonstrated that NKp46high NK cells have significantly higher interferon-gamma production than NKp46low cells. TLR stimulation significantly induced degranulation as well as tumor necrosis factor alpha (TNF-α)-related apoptosis-inducing ligand, Fas, and TNF-α protein expression in NKp46high NKs. NKp46 ligand was induced on HCV-infected hepatocytes. Conclusions: NKp46 expression may contribute to differential HCV responses. NKp46 expression correlates with anti-HCV activity in vitro and thus may prove to be a useful therapeutic target. (HEPATOLOGY 2012)
Natural killer (NK) cells constitute the first line of host defense against viral pathogens1, 2; they eliminate virus-infected cells both directly through cytolytic mechanisms and indirectly by secreting cytokines, such as interferon-gamma (IFN-γ).3, 4 NK cell activity is stringently controlled by inhibitory NK receptors (NKRs), which, in steady-state conditions, override signals provided by the engagement of activating receptors. NKRs include the predominantly inhibitory killer immunoglobulin (Ig)-like receptors, C-type lectin-like receptors of the CD94/NKG2 family comprising inhibitory (NKG2A) and activatory (NKG2C/D) isoforms, as well as the natural cytotoxicity receptors (NCRs), such as NKp30 (NCR3/CD337), NKp44 (NCR2/CD336), and NKp46 (NCR1/CD335), that deliver activatory signals.5-7
The NKp46 receptor, expressed on both resting and activated NK cells, is considered the major human NCR involved in NK cell cytotoxicity.8, 9 Levels of expression of this NCR have been shown to differ significantly among donors and to correlate directly with natural cytotoxicity in these individuals.8 In addition to playing a significant role in antitumor immunity, NKp46 is clearly important for antiviral immunity. Originally reported to interact with influenza hemaggglutinin,9 NKp46 is involved in the NK response against influenza-infected monocyte-derived dendritic cells (mDCs).10 As further corroboration, a murine model demonstrates a critical function for NKp46 in the in vivo eradication of influenza virus.11 Although the cellular ligand has yet to be identified, recent work demonstrated the dominant contribution of this receptor to the activation of NK cells in response to human cytomegalovirus (HCMV)-infected mDCs.12 Down-regulation of NKp46 has been implicated in human papillomavirus (HPV-16) infection and cervical cancer progression.13 Down-regulation of NCRs, including NKp46, has been implicated in the attenuation of NK cell activity in human immunodeficiency virus (HIV) infection.14 To date, there are limited data on NKp46 expression in HCV-infected individuals. A decrease in NKp46 has been demonstrated in acute and chronic HCV in some studies15, 16; however, others have reported increased expression.17 A recent study suggests that up-regulation of NKp46 in response to interferon-alpha (IFN-α) is predictive of SVR in chronic HCV infection.18
Toll-like receptors (TLRs) constitute a family of conserved pattern-recognition sensors that play a prominent role during early antiviral response through the induction of type I IFNs and inflammatory cytokines.19 Several viruses have been shown to activate the TLR-signaling cascade.20 NK cells express pattern-recognition receptors, including several members of the TLR family, although not all are functional. Human NK cells express functional TLR2,21 TLR3,22 TLR7/8,23 and TLR9.24 Thus, in addition to classical NKRs, TLRs are likely to be important for effective NK antiviral responses.
Considerable evidence indicates that the risk of viral persistence, natural history, and response to antiviral therapy in chronic HCV infection varies among racial groups.25, 26 In a large study of the natural history of HCV, patients with spontaneous viral clearance were more likely to be nonblack and female.25 Symptomatic females have an 8-fold greater chance of clearing HCV infection in the acute infection setting.27 African Americans (AAs) with chronic hepatitis C genotype 1 infection have lower rates of virologic response to pegylated IFN (Peg-IFN) and ribavirin (RBV) than Caucasian Americans (CAs), and these differences are not explained by disease characteristics, baseline viral levels, or amount of medication taken.28 Taken together, the above-mentioned studies provide evidence that both gender and race influence the natural history of, and treatment efficacy in some human viral infections. In this study, we show that race- and gender-related differences in the expression of the NCR, NKp46, correlates with increased antiviral cytolytic activity and gene transcription. Our data suggest that expression patterns of NKp46 on NK cells may explain some of the differences observed for HCV natural history and treatment responses. NKp46 expression correlates with anti-HCV activity in vitro and thus may prove to be a useful therapeutic target.
This study utilized 58 uninfected control subjects equally distributed by AA (n = 29) or CA (n = 29) race. Median age for AAs was 33.5 years (range, 21-62), and 48.28% were male. For CA subjects, median age was 34 years (range, 21-60), and 55.17% were male. This research was conducted in accord with the Helsinki principles: All patients gave informed, written consent before their participation and was approved by the University of Colorado Denver Institutional Review Board.
Sample Collection and Storage.
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by cellular preparation tubes (anticoagulant sodium citrate; Becton-Dickinson, Franklin Lakes, NJ). PBMCs were viably frozen in 80% fetal bovine serum (BioWhittaker, Walkersville, MD), 10% dimethyl sulfoxide, and 10% RPMI 1640 media (Life Technologies, Grand Island, NY) and stored in liquid nitrogen for subsequent analyses.
Antibodies for Detection and Fluorescence-Activated Cell Sorting Analysis of Antigen Expression.
Four-color multiparameter flow cytometry was performed using a BD FACSCanto II or BD FACScan instrument (BD Biosciences, San Jose, CA) compensated with single fluorochromes and analyzed using Diva or CellQuest software (BD Biosciences). Lymphocyte populations were identified by their characteristic forward scatter/side scatter properties. Fluorochrome-labeled (peridinin chlorophyll protein/allophycocyanin [PerCP/APC]) monoclonal antibodies (mAbs) specific for CD3 and CD56 (BD Biosciences) were used to identify NK (CD3−CD56+) and NT (CD3+CD56+) cells within the overall lymphocyte population. Anti-NKR antibodies (fluorescein isothiocyanate/phycoerythrin [FITC/PE]) CD161, CD94, CD95, CD16, CD158a, CD158b, CD158e, and NKG2D were obtained from BD Biosciences. Anti-NKG2C-PE and TNF-α-related apoptosis-inducing ligand (TRAIL)-PE mAbs were purchased from R&D Systems (Minneapolis, MN). Anti-NKG2A-PE, NKp30-PE, NKp44-PE, and NKp46-PE were obtained from Immunotech (Beckman Coulter, Fullerton, CA). Anti–Fas ligand (FasL)/PE was purchased from eBioscience (San Diego, CA). Thawed PBMCs (1-2 × 106) were stained for cell-surface antigen expression at 4°C in the dark for 30 minutes, then washed twice in 2 mL of phosphate-buffered saline containing 1% bovine serum albumin and 0.01% sodium azide (FACS wash) and subsequently fixed in 200 uL of 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO). Isotype-matched control antibodies were used to determine background levels of staining.
Thawed mononuclear cell suspensions were enriched for NKs using the NK Isolation Kit II from Miltenyi Biotec (Gladbach, Germany), according to the manufacturer's instructions. Median purity of NKs was >90% in all cases. After isolation, NKs were cultured in the presence or absence of IL-2 (25 ng/mL; R&D Systems) for 48 hours at 37°C and 5% CO2. After culture, carboxy fluorescein succinimidyl ester–labeled target cells (K562s) were added to the NKs at effector:target (E:T) concentrations of 0:1 (negative control) and 10:1 (test) and were incubated at 37°C for 4 hours. After incubation, cytotoxicity was measured using the flow-cytometry–based Total Cytotoxicity and Apoptosis Detection Kit from Immunochemistry (Bloomington, MN). Immediately before acquisition, 7-aminoactinomycin D was added to E:T populations and incubated for 15 minutes on ice. Cells treated with 0.1% Triton-X served as positive controls.
Hepatocyte Cytotoxicity Assay.
NKs were enriched using magnetic beads and surface stained for CD3, CD56, and NKp46 as described above. NKs (CD3−CD56+) were fluorescence-activated cell sorting (FACS) sorted on the expression of NKp46 using a FACSAria instrument (BD Biosciences). Huh-7.5 cells (Apath LLC, St. Louis, MO) were seeded at a concentration of 1.25 × 105 cells/well in 24-well plates. After 24 hours, NKp46high and NKp46low/neg fractions of NKs were added at a ratio of 5 NK to 1 Huh-7.5 cell (5:1) or 1:1. Cells were infected simultaneously with Japanese fulminant hepatitis type 1 (JFH-1; National Institute of Infectious Diseases, Tokyo, Japan) at a multiplicity of infection (MOI) of 0.01. Five days postinfection, cells were harvested for RNA extraction (RNeasy mini Kit; Qiagen, Valencia, CA). RNA was transcribed to complementary DNA (cDNA) using the QuantiTect Reverse Transcription Kit (Qiagen), and HCV transcripts were detected using a Step One Plus Real-Time polymerase chain reaction (PCR) instrument (Applied Biosystems, Foster City, CA). A standard curve was created using JFH-1 plasmid stock (range, 1 × 107 to 1 × 101). PCR Taqman Master Mix, primers, and probes were purchased from Applied Biosystems. Primer and probe sequences were as follows: HCV-forward GCA CAC TCC GCC ATC AAT CAC T; HCV-reverse CAC TCG CAA GCG CCC TAT CA; HCV-probe 6FAM AGG CCT TTC GCA ACC CAA CGC TAC T TAMRA.
NK cells were enriched from PBMCs (n = 4) and stained for the expression of CD56/CD3 and NKp46, as described above. FACS sorting (Aria; BD Biosciences) was used to isolate NK cells based on the expression of NKp46. Sorted populations were cultured for 6 hours at a concentration of 1 million/mL in the presence or absence of a TLR-stimulation cocktail (100 ug/mL of polyinosonic-polycytidylic acid [TLR3], 5 uM of loxoribine [TLR7], and 5 um of deoxycytidylate-phosphate-deoxyguanylate [TLR9]). After culture, NK cells were washed and RNA was extracted from cell pellets using the RNeasy RNA Isolation Kit (Qiagen). cDNA was transcribed using 500 ng of RNA in a 20-uL reaction using the Quantitect Reverse Transcription Kit (Qiagen). For the detection of TLR responses at a protein level, PBMCs (n = 5) were incubated with the TLR cocktail, as described above, for 24 hours, and cell-surface expressions of TRAIL, Fas, and Fas-L were assessed by flow cytometric analysis. Intracellular flow cytometic staining was used to measure TNF-α and IFN-γ responses after 6-hour stimulation. For some experiments, cytokine stimulation (IL-12/IL-15) was used.
Gene expression was assessed using the Step One Plus Real-Time PCR system (Applied Biosystems) using the Quantifast SYBR Green protocol (Qiagen). QuantiTect primer assays for use with SYBR Green detection were purchased from Qiagen/Superarray.
Immunofluorescent Staining of Hepatocytes.
Huh-7.5 cells (Apath LLC) were seeded onto coverslips (1.25 × 105 cells/well in 24-well plates) and incubated overnight. Cells were then infected with JFH-1 (National Institute of Infectious Diseases) at an MOI of 0.01. Five days postinfection, coverslips were removed, washed, fixed, and stained for the expression of NKp46 ligands using an Fc fusion protein of the NKp46 receptor (R&D Systems) and detected using an antibody coupled to FITC. Images were acquired with a ×20 objective lens at constant exposure. An isotype control was used to exclude background staining.
Results are expressed as the median (range). Mann Whitney's non-parametric U test was used to compare differences between patient groups. Significance was defined as a P value of <0.05. The JMP 6.0 (SAS Institute, Inc., Cary NC) statistical software package was used.
NKp46 Expression Is Associated With Female Gender and Caucasian Race.
We hypothesized that racial- and gender-related differences in NK cell populations may explain the altered natural history of chronic viral infection and disparate treatment responses to antiviral therapy. To address this question, we used a cohort of 58 age- and gender-matched uninfected control subjects. Multiparameter flow cytometric analysis was used to characterize NK cell levels and phenotype with respect to 14 different activating and inhibitory NKRs. Total NK cell levels did not differ according to race or gender (Table 1). The phenotype of NK cells was remarkably similar in our four test groups (Table 2). However, NK expression of the activatory NCR, NKp46, was strongly associated with CA race and female gender. Female CAs had significantly higher expression than all other groups tested (Fig. 1).
NK cells eliminate virus-infected cells directly by cytolytic mechanisms; therefore, we examined the ability of bead-purified NK cells to kill the NK-sensitive cell line, K562, using a standard flow-based assay.29 LAK activity mirrored the pattern observed for NKp46 expression, with female CAs demonstrating the highest activity and male AAs being the weakest. NK cells isolated from females had greater activity than males (35.93% versus 17%; P = 0.0105). In addition, CA subjects had significantly higher LAK activity than AA subjects (39.08% versus 16.92%; P = 0.0004). NKp46 expression correlated positively with LAK activity (P = 0.0054), suggesting that increased NKp46 expression contributes to more effective cytolytic activity of NK cells (Fig. 2). We confirmed this by testing the ability of FACS-purified NKp46high/low subsets of NK cells to lyse NK-cell–sensitive targets (K562) in the presence (LAK) or absence (natural cytotoxicity) of IL-2. NK cells expressing high levels of NKp46 have increased LAK activity, compared to NKp46low NK cells. No difference was observed in natural cytotoxicity (data not shown).
NKp46 Expression Is Associated With Increased Antiviral NK Cell Activity.
Because NKp46 is important for cytotoxic function and because expression on NK cells was significantly correlated with LAK activity, we next tested the functional significance of NKp46 expression in a more-relevant viral model. We used the Huh-7.5/JFH-1 in vitro HCV infection system to compare the ability of FACS-sorted NKp46low/neg and NKp46high subsets of NK cells to attenuate the infection of hepatocytes by HCV. NKp46high NKs were more efficient at controlling HCV copy number in this system than their NKp46low counterparts, even at an E:T of 1:1 (Fig. 3A). Similarly, direct ligation of NKp46 using an agonist antibody on isolated NK cells resulted in a significant reduction in the HCV copy number detected in Huh-7.5/JFH-1 coculture at an E:T of 5:1 (Fig. 3B).
NKp46 Level Is Associated With Increased NK Cell Expression of Death Receptor Genes and Proteins.
TLRs expressed by NK cells play a prominent role during the early antiviral response by the induction of type I IFNs and inflammatory cytokines19; therefore, we tested the ability of FACS-purified NKp46high/low subsets of NK cells to respond to stimulation by TLR pathways. Gene expression in NK cell subsets was assessed by real-time reverse-transcription PCR after 6-hour in vitro stimulation with TLR ligands. Several genes involved in cytotoxicity are up-regulated in NK cells expressing high levels of NKp46, in contrast to the expression of the IFN-γ gene, which was comparable (Fig. 4A). No difference was observed in the expression of type I and type III IFN or suppressor of cytokine signaling 3 in the NK cell subsets. Because the production of IFN-γ represents a major function of NK cells, in addition to cytotoxicity, we wanted to assess whether NKp46 expression was related to the NK cell response to cytokines. IFN-γ expression in NK cell subsets, based on the expression of NKp46, was assessed by flow cytometry after 6-hour in vitro stimulation with varying concentrations of IL-12 and IL-15. After stimulation, a higher percentage of NKp46high NKs expressed IFN-γ than NKs expressing low levels of this receptor (Fig. 4B). As has been reported on previously,30 we saw no enhancement of IFN-γ on the ligation of NKp46 with an agonist antibody. These data suggest that the ability to produce IFN-γ is higher in NK cells expressing high levels of NKp46, but is not enhanced by the activation of TLR pathways or NKp46 itself. Because TLR stimulation resulted in the up-regulation of genes involved in NK cytolytic activity, we wanted to see whether this up-regulation was also evident at a protein level. After 24-hour stimulation, Fas protein was significantly up-regulated on total NK cells with the TLR cocktail or cytokine stimulation. Up-regulation of TRAIL was also observed, but to a much greater extent with TLR stimulation, compared to cytokine activation (Fig. 4C). Of note, in these cultures, the expression of both Fas and TRAIL was more prominent for the NKp46high NK cell subset, compared to NK cells expressing low levels of NKp46 (Fig. 4D). Short-term (i.e., 6-hour) stimulation with the TLR cocktail resulted in increased TNF-α production, predominantly in NK cells expressing high levels of NKp46 (Fig. 4E and 4F). Fas-L expression was unchanged by TLR stimulation (data not shown). A functional correlate of this gene and protein up-regulation is suggested by the significantly higher induction of degranulation (CD107a expression) by TLR on NKp46high NK cells in the same cultures (Fig. 5).
HCV Infection Up-regulates Expression of NKp46-Ligand On Hepatocytes.
Next, taking advantage of the availability of soluble NCR-Fc fusion proteins, we determined whether replication within the Huh7.5 cell line induced an up-regulation of the NKp46 ligand. HCV-infected hepatocytes demonstrated a cell-surface pattern of staining for NKp46 ligand (Fig. 6). Although uninfected control hepatocytes also stained positive for the ligand for NKp46, the intensity of the staining was much lower. In contrast to the staining pattern observed for infected hepatocytes, NKp46 ligand staining was more diffuse and, for the most part, localized to the cytoplasm. These data suggest that not only is NKp46 ligand up-regulated, but is also transported to the cell surface upon HCV infection.
NK cells comprise a central component of the innate immune response, representing the first line of defense against a variety of microbrial pathogens, including viruses, bacterial, fungi, as well as tumors.31 The surface density of NCRs on NK cells is associated with the magnitude of cytolytic activity against NK-susceptible target cells. Our comprehensive analysis of NCRs (Table 2) identified race- and gender-related differences. Although the ligands recognized by NCRs remain incompletely defined, it is known that soluble NKp46-Ig fusion protein binds to both the hemagglutinin of influenza virus and the hemagglutinin-neuraminidase of parainfluenza virus.9 For the first time, we demonstrate that HCV infection in the Huh7.5 replicon cell line induces expression of the NKp46 ligand on the surface of hepatocytes. Whether this is the result of a specific viral component or a stress response to the infection is the focus of ongoing work. Regardless, the findings that NKp46 is associated with increased expression of killing molecules after TLR stimulation, increased IFN-γ production, LAK, degranulation, and in vitro control of HCV replication corroborate the strong epidemiological data that both race and gender are associated with spontaneous recovery from HCV infection.25 The fact that the outcome of other viral infections do not consistently track with race and gender as in HCV infection may be related to differences in viral tropism or expression of the NKp46 ligand(s) in target tissues. Our collective findings suggest that, for HCV infection, NKp46 may represent a useful therapeutic target.