Race- and gender-related variation in natural killer p46 expression associated with differential anti-hepatitis C virus immunity

Article (PDF Available)inHepatology 56(4):1214-22 · October 2012with59 Reads
DOI: 10.1002/hep.25771 · Source: PubMed
Abstract
Unlabelled: 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). NKp46(high) NKs were more efficient at controlling HCV than their NKp46(low) 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 NKp46(high) NKs. Cytokine stimulation (interleukin [IL]-12 and IL-15) demonstrated that NKp46(high) NK cells have significantly higher interferon-gamma production than NKp46(low) 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 NKp46(high) 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.
Race- and Gender-Related Variation in Natural Killer
p46 Expression Associated With Differential
Anti-Hepatitis C Virus Immunity
Lucy Golden-Mason,
1,2
Amy E.L. Stone,
1,2
Kiran M. Bambha,
1
Linling Cheng,
1
and Hugo R. Rosen
1,2,3
Major racial and gender differences have been documented in the natural history and
treatment responses of chronic hepatitis C virus (HCV) infection; however, distinct mecha-
nisms have remained enigmatic. We hypothesized that racial- and gender-related differen-
ces 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. Multi-
parameter 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 infec-
tion was assessed in the Huh-7.5/JFH-1 coculture system. NK expression of natural cyto-
toxicity receptor NKp46 was strongly associated with CA race and female gender and
correlated positively with LAK activity (P 5 0.0054). NKp46
high
NKs were more efficient
at controlling HCV than their NKp46
low
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 NKp46
high
NKs. Cytokine stimulation
(interleukin [IL]-12 and IL-15) demonstrated that NKp46
high
NK cells have significantly
higher interferon-gamma production than NKp46
low
cells. TLR stimulation significantly
induced degranulation as well as tumor necrosis factor alpha (TNF-a)-related apoptosis-
inducing ligand, Fas, and TNF-a protein expression in NKp46
high
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;56:1214-1222)
See Editorial on Page 1197
N
atural killer (NK) cells constitute the first line
of host defense against viral pathogens
1,2
;
they eliminate virus-infected cells both
directly through cytolytic mechanisms and indirectly
by secreting cytokines, such as interferon-gamma
(IFN-c).
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
Abbreviations: AA, African American; CA, Caucasian American; cDNA, complementary DNA; E:T, effector-to-target ratio; FACS, fluorescence-activated cell
sorting; Fas-L, Fas-ligand; FITC, fluorescein isothiocyanate; HCMV, human cytomegalovirus; HIV, human immunodeficiency virus; IFN-a, interferon-alpha; IFN-
c, interferon-gamma; Ig, immunoglobulin; JFH-1, Japanese fulminant hepatitis type 1; IL, interleukin; LAK, lymphokine activated killing; mAbs, monoclonal
antibodies; mDCs, monocyte-derived dendritic cells; MOI, multiplicity of infection; NCR, natural cytotoxicity receptor; NKR, natural killer receptor; PBMCs,
peripheral blood mononuclear cells; PCR, polymerase chain reaction; PE, phycoerythrin; Peg-IFN, pegylated IFN; RBV, ribavirin; TLR, Toll-like receptor; TNF-a,
tumor necrosis factor alpha; TRAIL, TNF-a-related apoptosis-inducing ligand.
From the
1
Division of Gastroenterology and Hepatology, Hepatitis C Center, Department of Medicine, University of Colorado Denver, Aurora, CO;
2
Integrated
Program in Immunolog y, University of Colorado Denver and National Jewish Hospital, Denver CO; and
3
Denver VA Medical Center, Denver, CO..
Received January 5, 2012; accepted March 14, 2012.
This work was supported by VA Merit Review Grant, and NIH grants U19 AI 1066328 and K24AI083742. 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.
1214
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 sig-
nals.
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 sig-
nificantly 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 hemagg-
glutinin,
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 recep-
tor 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 studies
15,16
; however, others have
reported increased expression.
17
A recent study suggests
that up-regulation of NKp46 in response to interferon-
alpha (IFN-a) 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 inflammator y cyto-
kines.
19
Several viruses have been shown to activate the
TLR-signaling cascade.
20
NK cells express pattern-rec-
ognition 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 anti-
viral 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 to-
gether, the above-mentioned studies provide evidence
that both gender and race influence the natural history
of, and treatment efficacy in some human viral infec-
tions. 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 nat-
ural history and treatment responses. NKp46 expres-
sion correlates with anti-HCV activity in vitro and
thus may prove to be a useful therapeutic target.
Materials and Methods
Study Population. This study utilized 58 unin-
fected control subjects equally distributed by AA (n ¼
29) or CA (n ¼ 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 participa-
tion 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 so-
dium citrate; Becton-Dickinson, Franklin Lakes, NJ).
Address reprint requests to: Hugo R. Rosen, M.D., Division of Gastroenterology and Hepatology, University of Colorado Denver, B-158, Academic Office
Building 1, 12631 East 17th Avenue, Room 7614, P.O. Box 6511, Aurora, CO 80045. E-mail: Hugo.Rosen@UCDENVER.edu; fax: 303-724-1891.
Copyright
V
C
2012 by the American Association for the Study of Liver Diseases.
View this article online at wileyonlinelibrary.com.
DOI 10.1002/hep.25771
Potential conflict of interest: Nothing to report.
HEPATOLOGY, Vol. 56, No. 4, 2012 GOLDEN-MASON ET AL. 1215
PBMCs were viably frozen in 80% fetal bovine serum
(BioWhittaker, Walkersville, MD), 10% dimethyl sulf-
oxide, 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 for-
ward scatter/side scatter properties. Fluorochrome-la-
beled (peridinin chlorophyll protein/allophycocyanin
[PerCP/APC]) monoclonal antibodies (mAbs) specific
for CD3 and CD56 (BD Biosciences) were used to
identify NK (CD3
CD56
þ
) and NT (CD3
þ
CD56
þ
)
cells within the overall lymphocyte population. Anti-
NKR antibodies (fluorescein isothiocyanate/phycoery-
thrin [FITC/PE]) CD161, CD94, CD95, CD16,
CD158a, CD158b, CD158e, and NKG2D were
obtained from BD Biosciences. Anti-NKG2C-PE and
TNF-a-related apoptosis-inducing ligand (TRAIL)-PE
mAbs were purchased from R&D Systems (Minneap-
olis, 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 10
6
) were
stained for cell-surface antigen expression at 4
Cin
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% para-
formaldehyde (Sigma-Aldrich, St. Louis, MO). Iso-
type-matched control antibodies were used to deter-
mine background levels of staining.
Cytotoxicity Assays. Thawed mononuclear cell sus-
pensions were enriched for NKs using the NK Isola-
tion Kit II from Miltenyi Biotec (Gladbach, Ger-
many), according to the manufacturer’s instructions.
Median purity of NKs was >90% in all cases. After
isolation, NKs were cultured in the presence or ab-
sence of IL-2 (25 ng/mL; R&D Systems) for 48 hours
at 37
C and 5% CO
2
. After culture, carboxy fluores-
cein succinimidyl ester–labeled target cells (K562s)
were added to the NKs at effector:target (E:T) concen-
trations 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). Immedi-
ately 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
þ
) 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 concen-
tration of 1.25 10
5
cells/well in 24-well plates. After
24 hours, NKp46
high
and NKp46
low/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 10
7
to 1 10
1
). 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
Table 1. Lymphocyte Distribution
Population F AA M AA F CA M CA
CD56
þ
cells* 17.5 (5.9-26.2)† 20.35 (7.5-55.66) 14.8 (6.3-33.95) 16.32 (3.9-30.12)
NK cells* 12.2 (4.6-24.2) 15.01 (6.3-38.64) 8.8 (2.6-27.81) 12.25 (3.2-20.6)
CD56
þ
NT cells* 2.9 (0.73-14.4) 3.36 (1.55-17.02) 3.7 (1.2-22.88) 2.32 (0.4-17.8)
CD56
bright
NKs‡ 6.61 (2.88-59.77) 5.7 (0.4-15.15) 7.56 (0.97-24.06) 6 (0.93-19.26)
Abbreviations: F, female; M, male.
*Percentage of total lymphocytes.
†Median (range).
‡Percentage of NK cells.
1216 GOLDEN-MASON ET AL. HEPATOLOGY, October 2012
CCC TAT CA; HCV-probe 6FAM AGG CCT TTC
GCA ACC CAA CGC TAC T TAMRA.
TLR Response. NK cells were enriched from
PBMCs (n ¼ 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 popu-
lations 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-poly-
cytidylic 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 ¼ 5) were incubated with the TLR cocktail, as
described above, for 24 hours, and cell-surface expres-
sions of TRAIL, Fas, and Fas-L were assessed by flow
cytometric analysis. Intracellular flow cytometic staining
was used to measure TNF-a and IFN-c responses after 6-
hour stimulation. For some experiments, cytokine stimu-
lation (IL-12/IL-15) was used.
Real-Time PCR. 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 10
5
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
Table 2. NK Cell Phenotype
Receptor* F AA M AA F CA M CA
NKp30 39.1 (12.4-79.67)† 40.16 (2.4-92.92) 44.4 (15.62-82.79) 45.3 (16.03-79.1)
NKp44 1.5 (0.5-10.1) 2.8 (1.0-29.79) 2.35 (1.2-4.43) 2.51 (0.65-7.3)
NKp46 57.6 (13.8-94.04) 42.18 (10.5-89.48) 81.64 (49.2-93.34) 67.49 (33.3-92.88)
NKG2A 51.3 (38.72-84.0) 58.5 (20.0-79.05) 49.73 (32.4-69.69) 51.27 (26.0-66.93)
NKG2C 9.8 (0.8-57.36) 12.2 (0.5-51.1) 6.22 (2.06-32.44) 5.61 (1.62-31.02)
NKG2D 88.2 (60.7-98.82) 91.02 (48.5-98.4) 93.5 (40.3-99.23) 89.24 (55.4-98.11)
CD158a 36.4 (8.4-61.93) 36.84 (9.23-55.69) 25.8 (5.12-60.23) 26.59 (9.0-64.44)
CD158b 25.8 (19.84-62.5) 33 (8.93-50.66) 32.02 (15.37-49.83) 26.69 (21.9-49.99)
CD158e 9.35 (3.9-21.65) 13.2 (0.0-48.7) 12.22 (0.0-47.2) 11.35 (0.0-27.4)
TRAIL 1.4 (0.6-22.2) 5.53 (0.75-58.39) 1.63 (0.68-8.7) 4.09 (0.52-26.6)
Fas 32.4 (4.1-82.08) 73.68 (11.2-86.75) 56.6 (15.5-82.95) 52.45 (11.0-80.5)
Fas-L 1.3 (0.5-7.9) 1.9 (0.48-29.92) 1.4 (0.59-8.42) 3.11 (0.6-14.4)
CD16 79 (37-92.2) 67.71 (20.1-88.27) 83.35 (40.53-92.6) 81.17 (29.8-95.15)
CD161 56.7 (18.53-95.44) 59.84 (22.8-91.39) 69.9 (12.28-97.34) 68.22 (22-94.77)
Abbreviations: F, female; M, male
*Percentage of NK cells.
†Median (range).
Fig. 1. Higher NKp46 expression in females
and Caucasians. Multiparameter flow cytomet-
ric analysis was used to assess the expression
of a range of NK receptors on NK cells from
58 healthy control subjects. Of the 14 recep-
tors assayed, only the NCR, NKp46, differed
significantly among donors. The highest
expression of NKp46 was detected in the
female (F) CA group, and the lowest was
detected in the male (M) AA group (A). Rela-
tively increased levels of expression of NKp46
were associated with female gender (B) and
Caucasian race (C).
HEPATOLOGY, Vol. 56, No. 4, 2012 GOLDEN-MASON ET AL. 1217
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.
Statistical Analysis. Results are expressed as the
median (range). Mann Whitneys 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.
Results
NKp46 Expression Is Associated With Female
Gender and Caucasian Race. We hypothesized that
racial- and gender-related differences in NK cell popula-
tions may explain the altered natural history of chronic
viral infection and disparate treatment responses to
Fig. 2. LAK activity correlates
with NKp46 expression. IL-2-
induced (25 ng/mL) LAK activity
was measured against K562 target
cells, as described in Materials and
Methods. The highest LAK activity
was detected in the female (F) CA
group and the lowest was detected
in the male (M) AA group (A). Rel-
atively increased LAK activity was
associated with female gender (B)
and Caucasian race (C). LAK activ-
ity correlated directly with expres-
sion levels of NKp46 (D).
Fig. 3. NKp46 expression is associated with increased antiviral NK cell activity. (A) The Huh-7.5/JFH-1 in vitro infection system was used to
assess the efficacy of FACS-sorted NKp46
low/neg
and NKp46
high
subsets of NK cells to attenuate the infection of hepatocytes by HCV.
Experiments were carried out in triplicate. NK cells expressing high levels of NKp46 (white bars) were more effective in controlling the infection
of Huh-7.5 cells than their NKp30
low/neg
counterparts (black bars) at E:T ratios of 1:1 or 5:1. (B) The same system was used to compare the
ability of bead-purified NK cells stimulated with an agonist antibody targeting NKp46 to attenuate infection. Infection of Huh-7.5 cells in the
presence of control immunoglobulin G at an MOI ¼ 0.01 results in robust infection after 5 days (black bars). NK cell activation through NKp46
in the absence of exogenous cytokine significantly reduces HCV copy number detected in hepatocytes.
1218 GOLDEN-MASON ET AL. HEPATOLOGY, October 2012
antiviral therapy. To address this question, we used a
cohort of 58 age- and gender-matched uninfected con-
trol 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, N K 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).
LAK Activity Correlates With NKp46 Expres-
sion. NK cells eliminate virus-infected cells directly by
cytolytic mechanisms; therefore, we examined the abil-
ity 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 ¼ 0.0105). In
addition, CA subjects had significantly higher LAK ac-
tivity than AA subjects (39.08% versus 16.92%; P ¼
0.0004). NKp46 expression correlated positively with
LAK activity (P ¼ 0.0054), suggesting that increased
NKp46 expression contributes to more effective cytoly-
tic activity of NK cells (Fig. 2). We confirmed this by
testing the ability of FACS-purified NKp46
high/low
sub-
sets of NK cells to lyse NK-cell–sensitive targets
(K562) in the presence (LAK) or absence (natural cy-
totoxicity) of IL-2. NK cells expressing high levels of
NKp46 have increased LAK activity, compared to
NKp46
low
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 impor-
tant 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 NKp46
low/neg
and
NKp46
high
subsets of NK cells to attenuate the
Fig. 4. NKp46 expression is associ-
ated with increased expression of killing
genes and proteins. (A) Six-hour stimula-
tion of FACS-sorted NKp46
high/low
NK cell
subsets with TLR ligands results in relative
up-regulation of genes encoding death re-
ceptor/ligands TRAIL, TNF-a, and Fas/Fas-
L, but not IFN-c, in the NK cell subset
expressing high levels (NKp46
high
). (B) In-
tracellular IFN-c production was assessed
by flow cytometric analysis of total NK
cells or NK subsets gated on the expres-
sion of high or low levels of NKp46 after
short-term cytokine stimulation in the
presence of varying concentrations of IL-
12 and IL-15. More-robust production of
IFN-c is evident in the NKp46 high-NK
cell subset (*P < 0.05; **P < 0.001;
***P < 0.0001). (C) Stimulation of
PBMCs (n ¼ 5) for 24 hours with TLR
ligands or IL-12/IL-15 (1 and 10 ng/mL,
respectively) results in up-regulation of
TRAIL to a greater extent on NK cells
treated with TLR agonists. (D) NK cells
subgated on the expression of NKp46
demonstrated that up-regulation of Fas
and TRAIL occurs to a statistically greater
extent in the NKp46
high
subset. (E) Six-
hour stimulation with TLR agonists results
in up-regulation of TNF-a in total NK cells,
but to a much greater extent in the
NKp46
high
NK cell subset. (F) Representa-
tive histograms for TNF-a expression in
NKp46
high/low
subsets are shown.
HEPATOLOGY, Vol. 56, No. 4, 2012 GOLDEN-MASON ET AL. 1219
infection of hepatocytes by HCV. NKp46
high
NKs
were more efficient at controlling HCV copy number
in this system than their NKp46
low
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 Pro-
teins. TLRs expressed by NK cells play a prominent
role during the early antiviral response by the induc-
tion of type I IFNs and inflammatory cytokines
19
;
therefore, we tested the ability of FACS-purified
NKp46
high/low
subsets of NK cells to respond to stimu-
lation 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-c gene, which
was comparable (Fig. 4A). No difference was observed
in the expression of type I and type III IFN or sup-
pressor of cytokine signaling 3 in the NK cell subsets.
Because the production of IFN-c 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- c
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 per-
centage of NKp46
high
NKs expressed IFN-c than NKs
expressing low levels of this receptor (Fig. 4B). As has
been reported on previously,
30
we saw no enhancement
of IFN-c on the ligation of NKp46 with an agonist
antibody. These data suggest that the ability to pro-
duce IFN-c 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 stimula-
tion 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
NKp46
high
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-a production, predomi-
nantly 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 corre-
late of this gene and protein up-regulation is suggested
by the significantly higher induction of degranulation
(CD107a expression) by TLR on NKp46
high
NK cells
in the same cultures (Fig. 5).
HCV Infection Up-regulates Expression of
NKp46-Ligand On Hepatocytes. Next, taking advant-
age of the availability of soluble NCR-Fc fusion pro-
teins, we determined whether replication within the
Huh7.5 cell line induced an up-regulation of the
NKp46 ligand. HCV-infected hepatocytes demon-
strated a cell-surface pattern of staining for NKp46
ligand (Fig. 6). Although uninfected control hepatocytes
also stained positive for the ligand for NKp46, the in-
tensity 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.
Discussion
NK cells comprise a central component of the
innate immune response, representing the first line of
Fig. 5. TLR stimulation induces NK cell degranulation. (A) Flow
cytometric analysis demonstrates that 6-hour stimulation of PBMCs (n
¼ 5) with TLR ligands results in increased degranulation (CD107a
expression) of NK cells, compared to control cultures. Mean fluores-
cent intensity (MFI), which correlates directly with the number of mole-
cules on the cell surface, is significantly higher for NK cells expressing
high levels of NKp46. (B) Representative histograms for CD107a
expression for NKp46
high/low
subsets are shown.
1220 GOLDEN-MASON ET AL. HEPATOLOGY, October 2012
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-sus-
ceptible target cells. Our comprehensive analysis of
NCRs (Table 2) identified race- and gender-related dif-
ferences. Although the ligands recognized by NCRs
remain incompletely defined, it is known that soluble
NKp46-Ig fusion protein binds to both the hemaggluti-
nin of influenza virus and the hemagglutinin-neuramini-
dase of parainfluenza virus.
9
For the first time, we dem-
onstrate that HCV infection in the Huh7.5 replicon cell
line induces expression of the NKp46 ligand on the sur-
face of hepatocytes. Whether this is the result of a spe-
cific 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-
c production, LAK, degranulation, and in vitro control
of HCV replication corroborate the strong epidemiolog-
ical 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 consis-
tently track with race and gender as in HCV infection
may be related to differences in viral tropism or expres-
sion of the NKp46 ligand(s) in target tissues. Our col-
lective findings suggest that, for HCV infection, NKp46
may represent a useful therapeutic target.
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1222 GOLDEN-MASON ET AL. HEPATOLOGY, October 2012
    • "Morikawa et al. (Morikawa et al., 1986) found that young men had more cytotoxicity toward K562 target cells than women, but there were no gender differences when other target cells were used. Men had lower NK cell expression of NKp46 and less IL-2- stimulated NK cell cytotoxicity than women (Golden-Mason et al., 2012). Another measure of NK cell function, antibody-dependent cellular cytotoxicity, was higher in men in one study (McCredie and MacDonald, 1980), but not in another (Penschow and Mackay, 1980). "
    [Show abstract] [Hide abstract] ABSTRACT: Immune gender differences have been reported, but are little studied in elderly humans. We compared monocyte and lymphocyte subsets, along with soluble immune mediators in healthy men and women over the age of 70. We also measured natural killer (NK) lymphocyte cytotoxic granule exocytosis, chemokine synthesis, and cytokine synthesis in response to a variety of stimuli. Elderly women had significantly more circulating B cells than men, whereas men had more CD4 central memory T cells and higher monocyte levels. Plasma adiponectin levels were higher in women, plasma retinol-binding protein 4 levels were higher in men, but there were no significant gender differences in C-reactive protein, IL-15, or sphingosine-1-phosphate. Women had a higher ratio of immature CD56(bright) NK cells to mature CD56(dim) NK cells, indicating a gender difference in NK cell maturation in the elderly. Comparing sexes, female mature NK cells had more vigorous cytotoxic granule responses to K562 leukemia cells and IFN-γ responses to NKp46 crosslinking. Moreover, female NK cells were more likely to produce MIP-1β in response to a variety of stimuli. These data show that gender influences NK cell activity in elderly humans.
    Full-text · Article · Apr 2016
    • "This functional dichotomy is especially clear during HCV infection, where the NK cell subtype NKp46 appears to be involved in HCV replication suppression, antifibrotic activity and HCV-associated liver damage [11]. Supporting the importance of NKp46 and NK cells in the immunopathogenesis of HCV, a pair of elegant studies performed by the laboratories of Jacob Nattermann and Hugo Rosen found that NKp46 High expression defined a specific NK cell subset that might be involved in both HCV replication suppression and HCV-associated liver damage111213. However, for HBV this remains a subject of debate. "
    [Show abstract] [Hide abstract] ABSTRACT: Natural killer (NK) cells play an important role in hepatitis B virus (HBV) infection control, and are regulated by a complex network of activating and inhibitory receptors. However, NK cell activity in HBV patients remains poorly understood. The objective of this study was to investigate the phenotypic and functional characteristics of circulating NK cells in patients during different chronic hepatitis B (CHB) infection stages. We investigated NK cell phenotypes, receptor expression and function in 86 CHB patients and 20 healthy controls. NK cells were purified and NK cell subsets were characterized by flow cytometry. Cytotoxic activity (CD107a) and interferon-gamma (IFN-γ) secretion were examined, and Natural Killer p46 (NKP46) blockade and spontaneous NK cell cytolytic activity against K562, HepG2 and HepG2.215 cell lines was studied. Activating NKp46 receptor expression was higher in inactive HBsAg carriers when compared with other groups (p = 0.008). NKp46 expression negatively correlated with HBV DNA (R = -0.253, p = 0.049) and ALT (R = -0.256, p = 0.045) levels. CD107a was higher in immune-activated groups when compared with immune-tolerant groups (p = 0.039). CD107a expression was related to viral load (p = 0.02) and HBeAg status (p = 0.024). In vitro NKp46 blockade reduced NK cell cytolytic activity against HepG2 and HepG2.215 cell lines (p = 0.02; p = 0.039). Furthermore, NK cells from high viral load CHB patients displayed significantly lower specific cytolytic activity against anti-NKp46-loaded K562 targets (p = 0.0321). No significant differences were observed in IFN-γ secretion (p > 0.05). In conclusion, NKp46 expression regulates NK cell cytolytic function. NKp46 may moderate NK cell activity during HBV replication suppression and HBV-associated liver damage and may be critical for NK cell activity during CHB infection.
    Full-text · Article · Aug 2015
    • "Overall, both genders of mice showed similar rates of persistent infection, but liver pathology progresses slightly faster, if any, in males (Supplementary information, Table S9). Whether sex hormones45, IL-28B polymorphism46, and natural killer p46 expression47, as suggested in the gender differences in humans, play a role in this mouse model remains to be investigated. "
    [Show abstract] [Hide abstract] ABSTRACT: The majority of hepatitis C virus (HCV) infection develops chronic infection, which causes steatosis, cirrhosis and hepatocellular carcinoma. However, understanding HCV chronicity and pathogenesis is hampered by its narrow host range, mostly restricted to human and chimpanzee. Recent endeavour to infect a variety of humanized mice has not been able to achieve persistent HCV infection unless the essential innate immune responsive genes are knocked out. Nevertheless, such immune-compromised humanized mice still lacked HCV infection-induced hepatopathogenesis. Here we report that transgenic mice in ICR background harboring both human CD81 and occludin genes (C/O(Tg)) are permissive to HCV infection at a chronicity rate comparable to humans. In this mouse model, HCV accomplishes its replication cycle, leading to sustained viremia and infectivity for more than 12 months post infection with expected fibrotic and cirrhotic progression. Host factors favorable for HCV replication, and inadequate innate immune-response may contribute to the persistence. Lastly, NS3/4 protease inhibitor telaprevir can effectively inhibit de novo RNA synthesis and acute HCV infection of C/O(Tg) mice. Thus, chronic HCV infection with complete replication cycle and hepatopathologic manifestations is recapitulated, for the first time, in immune-competent mice. This model will open a new venue to study the mechanisms of chronic hepatitis C and develop better treatments.Cell Research advance online publication 26 August 2014; doi:10.1038/cr.2014.116.
    Full-text · Article · Aug 2014
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