ORIGINAL PAPER

Chronic hepatitis C virus infection triggers spontaneousdifferential expression of biosignatures associated with T cellexhaustion and apoptosis signaling in peripheral bloodmononucleocytes

Muttiah Barathan • Kaliappan Gopal • Rosmawati Mohamed • Rada Ellegard •

Alireza Saeidi • Jamuna Vadivelu • Abdul W. Ansari • Hussin A. Rothan •

M. Ravishankar Ram • Keivan Zandi • Li Y. Chang • Ramachandran Vignesh •

Karlhans F. Che • Adeeba Kamarulzaman • Vijayakumar Velu • Marie Larsson •

Tunku Kamarul • Esaki M. Shankar

� Springer Science+Business Media New York 2015

Abstract Persistent hepatitis C virus (HCV) infection

appears to trigger the onset of immune exhaustion to

potentially assist viral persistence in the host, eventually

leading to hepatocellular carcinoma. The role of HCV on

the spontaneous expression of markers suggestive of

immune exhaustion and spontaneous apoptosis in immune

cells of chronic HCV (CHC) disease largely remain elu-

sive. We investigated the peripheral blood mononuclear

cells of CHC patients to determine the spontaneous

recruitment of cellular reactive oxygen species (cROS),

immunoregulatory and exhaustion markers relative to

healthy controls. Using a commercial QuantiGenePlex�

2.0 assay, we determined the spontaneous expression pro-

file of 80 different pro- and anti-apoptotic genes in per-

sistent HCV disease. Onset of spontaneous apoptosis

significantly correlated with the up-regulation of cROS,

indoleamine 2,3-dioxygenase (IDO), cyclooxygenase-2/

prostaglandin H synthase (COX-2/PGHS), Foxp3, Dtx1,

Blimp1, Lag3 and Cd160. Besides, spontaneous differential

surface protein expression suggestive of T cell inhibition

viz., TRAIL, TIM-3, PD-1 and BTLA on CD4? and

CD8? T cells, and CTLA-4 on CD4? T cells was also

evident. Increased up-regulation of Tnf, Tp73, Casp14,

Tnfrsf11b, Bik and Birc8 was observed, whereas FasLG,

Fas, Ripk2, Casp3, Dapk1, Tnfrsf21, and Cflar were

moderately up-regulated in HCV-infected subjects. Our

Part of this work was presented at the International HIV Science and

Infectious Diseases Congress (HIV Science 2014) held at YRG

Centre for AIDS Research and Education (YRG CARE), Chennai,

India between January 30 and February 1, 2014.

Muttiah Barathan and Kaliappan Gopal contributed equally to the

work.

M. Barathan � A. Saeidi � J. Vadivelu � M. Ravishankar Ram �K. Zandi � L. Y. Chang � E. M. Shankar (&)

Tropical Infectious Disease Research and Education Center

(TIDREC), Department of Medical Microbiology, Faculty of

Medicine, University of Malaya, Lembah Pantai,

50603 Kuala Lumpur, Malaysia

e-mail: shankarem@um.edu.my; shivamsarvam@gmail.com

K. Gopal � T. Kamarul

National Orthopedics Center for Excellence in Research and

Learning (NOCERAL), Department of Orthopedics surgery,

Tissue engineering group (TEG), Faculty of Medicine,

University of Malaya, Lembah Pantai, 50603 Kuala Lumpur,

Malaysia

R. Mohamed � A. Kamarulzaman

Department of Medicine, University of Malaya, Lembah Pantai,

50603 Kuala Lumpur, Malaysia

R. Ellegard � M. Larsson

Division of Molecular Virology, Department of Clinical and

Experimental Medicine, Linkoping University, Linkoping,

Sweden

A. W. Ansari � A. Kamarulzaman � E. M. Shankar

Center for of Excellence for Research in AIDS (CERiA),

University of Malaya, Lembah Pantai, 50603 Kuala Lumpur,

Malaysia

H. A. Rothan

Department of Molecular Medicine, University of Malaya,

Lembah Pantai, 50603 Kuala Lumpur, Malaysia

R. Vignesh

YRG Center for AIDS Research and Education, Voluntary

Health Services Hospital Campus, Taramani, Chennai 600113,

India

123

Apoptosis

DOI 10.1007/s10495-014-1084-y

Author's personal copy

observation suggests the spontaneous onset of apoptosis

signaling and T cell exhaustion in chronic HCV disease.

Keywords Apoptosis � Caspase � Hepatitis C �Spontaneous immune exhaustion � TRAIL

Introduction

Persistent viral infection (PVI) due to human immunode-

ficiency virus (HIV), hepatitis B (HBV) and C viruses

(HCV), and human papillomavirus (HPV) represents a

leading global cause of morbidity and mortality from

infectious diseases [1]. Recent estimates show that *170

million persons are chronically infected with HCV world-

wide. HCV is an enveloped, single-stranded, positive-sense

RNA virus belonging to the Flaviviridae family [2, 3].

Sequencing analysis of HCV genome indicates the exis-

tence of *11 genotypes, of which genotype 1 is the most

prevalent in the United States, whereas genotypes 1 and 3

in Malaysia [4]. A vast majority of acutely infected indi-

viduals develop chronic hepatitis C (CHC), and eventually

progress to develop cirrhosis and hepatocellular carcinoma

(HCC) [5].

The biology of HCV in the host is poorly understood and

the most challenging attribute is the mechanism underlying

persistence [6]. During host-virus encounter, only *15 % of

infected individuals spontaneously clear the virus [7].

Intriguingly, in individuals where HCV is not cleared, the

virus appears to promote functional exhaustion of virus-

specific T cells, including anergy and deletion owing to

sustained up-regulation of death markers, lack of positive

costimulation [8, 9], and expansion of regulatory T cells

(Tregs) [10]. A recent study suggests a role for elevated

immune cell apoptosis with the onset of HCV disease [11].

The increase in immune cell death has also been reported in

clinical/chronic HIV, SIV and HCV diseases [12–14].

Apoptosis represents a genetic program key to development

and differentiation of normal cellular functions whereby

senescent cells undergo programmed death [15, 16]. Immune

exhaustion, especially due to PD-1 ligation appears to trigger

Fas(CD95)-mediated apoptosis of immune cells. Growing

evidence suggest that spontaneous apoptosis of peripheral

blood mononuclear cells (PBMCs) could be one mechanism of

potential immune impairment, especially by HIV, where the

involvement of diverse apoptosis pathways has been proposed

[17–20]. Death pathways can be activated in host cells via

receptor-mediated (extrinsic) and non-receptor-mediated

(intrinsic) pathways [21, 22]. Current studies indicate that PVIs

program apoptosis via the FasL–Fas interactions [21] TNF-a,

mitochondrial, and Bcl-2 family death pathways potentially

leading to viral persistence [23, 24]. A resurgence of interest in

HCV has led to investigations prompted at deciphering the role

of reactive oxygen species (ROS) released via the intrinsic

pathway [25], and spontaneous apoptosis of primed immune

cells in PVIs [20, 26, 27]. Despite its importance, far less is

known about the role of inhibitory molecules and spontaneous

apoptosis in chronic HCV-primed immune cells. Here, we

observed evidence of signatures of spontaneous immune

exhaustion, and intrinsic and extrinsic apoptosis pathways

highlighting their possible association with chronic HCV dis-

ease. The data described here represents a survey of mediators

associated with spontaneous immune exhaustion and apopto-

sis, and will aid to better understand HCV immunopathogenesis

to be able for developing improved intervention strategies

against viral persistence.

Materials and methods

Human subjects

Chronically HCV infected patients (n = 7) and healthy

controls (n = 7) were recruited for this investigation. The

study was carried out following approval of the protocols by

the Medical Ethics Committee (MEC) of University of

Malaya Medical Center, for ethical issues. Written, informed

consent was obtained from all the participants before study

enrolment. The study was carried out in compliance with

good clinical practice, including the International Confer-

ence on Harmonization Guidelines and the Declaration of

Helsinki. All chronic HCV-infected patients were positive

for HCV antibodies and HCV RNA, and negative for HIV

antibodies and hepatitis B surface antigen (HBsAg). At the

time of recruitment, none of the patients were receiving or

had received any pegylated interferon-a or immunosup-

pressive therapy within 6 months. Additional exclusion

criteria were recent illness and/or vaccination within

4 weeks prior to phlebotomy, diabetes mellitus, hyperten-

sion/cardiovascular disease, pregnancy and treatment for

any form of inflammatory manifestations. The demographics

and clinical characteristics of participants are summarized in

Table 1.

Liver transaminases and HCV plasma viral load

Plasma aspartate transaminase (AST) levels were estimated

using a commercial ELISA (IBL America, Minneapolis,

K. F. Che

Unit for Lung and Airway Research, Division of Physiology,

Institute for Environmental Medicine, Karolinska Institute,

Solna, Stockholm, Sweden

V. Velu

Department of Microbiology and Immunology, Emory Vaccine

Center, Atlanta, USA

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MN), according to manufacturer’s instructions. Plasma

alanine transaminase (ALT) levels were determined with a

Hitachi7050 Automatic Analyzer (Hitachi Corp, Tokyo,

Japan) using a commercial ALT assay kit (Wako Pure

Chemicals, Osaka, Japan). The cut-off values were set at

20 ng/ml. HCV plasma viral loads (PVLs) were measured

using a commercial COBAS AMPLICOR HCV test, ver-

sion 2.0 (Roche Molecular Systems, Branchburg, NJ,

USA). The analytical sensitivity (95 % threshold) of the

COBAS AMPLICOR HCV 2.0 was 60 and 100 HCV IU/

ml with EDTA plasma and serum, respectively. The total

lymphocyte counts (TLCs) for each HCV positive partici-

pant were determined by flow cytometry.

Peripheral blood mononuclear cells

Isolation of PBMCs from buffy coats was carried out as

previously described [28]. Briefly, 9 ml of whole blood

was obtained per patient or control in a BD Vacutainer�

heparin lithium tubes (BD Biosciences, Franklin Lakes, NJ,

USA), and PBMCs were prepared by density-gradient

centrifugation over Ficoll-PaqueTM (Amersham Pharmacia,

Piscataway, NJ, USA). The cells were resuspended in a

freezing media (10 % DMSO in 90 % fetal bovine serum

(FBS) (Gibco, Carlsbad, CA, USA) and cryopreserved in

liquid nitrogen until use in the experiments.

Primary cell culture

Culture of PBMCs was made in RPMI1640 medium supple-

mented with HEPES buffer (25 mM), L-glutamine (2 mM),

penicillin (100U/ml), streptomycin (100 lg/ml), sodium

pyruvate (1 mM), gentamicin (5 lg/ml) (all procured from

Life Technologies, Victoria, Australia), and 10 % of FBS

(Gibco). Cells were cultured at 37 �C in a 5 % CO2 incubator.

Indoleamine 2,3-dioxygenase (IDO) assay

Serum Indoleamine 2,3-dioxygenase (IDO) was measured

using a commercial Sandwich ELISA (Uscn Life Science

Inc, Wuhan, Hubei, PRC) according to the manufacturer’s

instructions with a detection range of 1.563–100 ng/ml.

This assay has a sensitivity of 0.61 ng/ml for detection of

IDO from serum, plasma, tissue homogenates and other

body fluids. Briefly, blood from CHC-infected patients and

healthy controls were centrifuged at 3,000 rpm for 10 min

and dispensed into microtiter wells immobilized with a

biotin-conjugated anti-IDO. For IDO evaluation, the

enzyme-substrate reaction mixture was terminated by

adding 20 % (w/v) trichloroacetic acid. IDO concentration

was estimated by comparing the optical density of samples

with the standard curve.

Cyclooxygenase-2 (COX-2)/prostaglandin H synthase

(PGHS-2) assay

Serum COX-2/PGHS-2 was measured using a commercial

ELISA (R&D Systems, Abingdon, United Kingdom).

Blood samples from CHC-infected individuals and controls

were centrifuged at 3,000 rpm for 10 min, and serum

COX-2 levels were evaluated as per the supplier’s

instructions. The reaction was terminated by adding 100 ll

of 8 % formaldehyde in phosphate buffered saline (PBS)

solution (v/v), and the mixture was read at 450 nm.

Multicolor flow cytometry

PBMCs were stained for spontaneous expression of surface

protein markers associated with immune exhaustion and

death ligand, TRAIL (ApoL2). Cells were stained with

peridinin chlorophyll protein-cyanin-5.5 (PerCP-Cy5.5)-

conjugated antibody to CD3 (clone SK7), phycoerythrin-

Table 1 Demographic and

clinical characteristics of

chronic hepatitis C-infected

individuals

Plasma HCV viral load in

healthy controls and CHC

patients

N/A non-applicable, ALT

alanine aminotransferase (SGPT

serum glutamic pyruvic

transaminase), AST aspartate

aminotransferase (SGOT serum

glutamic oxaloacetic

transaminase), PVL plasma viral

load, IU/l international units per

liter

Characteristics Healthy controls (n = 7) Chronic HCV-infected (n = 7)

Sex (M/F) 5/2 3/4

ALT (IU/l) 11.20 ± 3.43 52.6 ± 9.31

AST (IU/l) 13.00 ± 3.54 57.43 ± 28.8

PVL (105 IU/ml) N/A 3.2

Patient no. ALT (IU/l) AST (IU/l) Plasma viral load (IU/l) Total lymphocyte

count (9102/ll)

1 54 37 1,154,000 11.2

2 50 36 \15 18.1

3 54 43 898,000 13.2

4 69 63 191,400 15.2

5 39 51 \15 17.2

6 46 119 320 16.8

7 39 51 15 17.9

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cyanin7 (PE-Cy7)-conjugated antibody to CD4 (clone

RPA-T4), allophycocyanin-H7 (APC-H7)-conjugated

antibody to CD8 (clone SK-1) (all from BD Pharmin-

genTM), APC-conjugated antibody to TIM-3 (clone

344823, R & D Systems), PE-conjugated antibody to

BTLA (clone J168-540, BD PharmingenTM), PE-conju-

gated antibody to TRAIL (clone RIK-2), fluorescein iso-

thiocyanate (FITC)-conjugated antibody to PD-1 (clone

MIH-4), and Brilliant VioletTM 421 (BV421)-conjugated

antibody to CTLA-4 (clone BNI3) (all from BD Pharm-

ingenTM) were used for immunophenotyping of T cells.

CD3-PerCP-Cy5.5 cells were co-stained with TRAIL-PE,

TIM-3-APC, PD-1-FITC, BTLA-PE, and CTLA-4-BV421.

CD4-PE-Cy7 T cells were co-stained with TRAIL-PE,

TIM-3-APC, BTLA-PE, PD-1-FITC, and CTLA-4-BV421.

In addition, CD8-APC-H7 T cells were also co-stained

with TRAIL-PE, TIM-3-APC, BTLA-PE, PD-1-FITC, and

CTLA-4-BV421. Isotype controls were used to set the

background for selection of positive cells viz., mouse

IgG2a FITC (MAB349051), mouse IgM j FITC

(MAB555583), PE-labeled mouse IgG1 j PE-CYTM7

mouse IgG1 j (PMG557872), mouse IgM j Alexa 647

(PMG560806), IgG1 j APC-H7 (PMG560167), PerCP-

CYTM5.5 mouse IgG1, j (PMG552834), APC mouse IgG

2b j (PMG555745), mouse IgG2a PerCP (MAB349054),

mouse IgG2b j FITC (MAB555742) and normal goat IgG

fluorescein control (GZ-IC108F). All isotype controls were

procured from R & D Systems, USA). Data were acquired

on a 7-color FACS Canto IITM (BD Immunocytometry

Systems, San Jose, CA, USA) using the FCAP array (BD

Biosciences) and analyzed using FlowJo software (Tree-

Star, Ashland, OR, USA).

Measurement of cellular reactive oxygen species

(cROS)

cROS formation was measured using a commercial oxi-

dation-sensitive fluorescent probe, 20,70-dichlorofluorescein

diacetate (DCFH-DA) ROS detection assay kit (Mito-

sciences�, Cambridge, UK) as previously described [29]

after 2 days of culture of PBMCs according to the manu-

facturer’s instructions. The kit employs a fluorogenic dye

20,70-dichlorofluoresceindiacetate (DCFDA), which mea-

sures hydroxyl, peroxyl and any other intracellular ROS.

Following diffusion, DCFDA is deacetylated by cellular

esterases to a non-fluorescent compound, which is subse-

quently oxidized by intracellular ROS to form a highly

fluorescent 20,70-dichlorofluorescin (DCF), detectable by

fluorescence spectroscopy. Briefly, the cells were seeded in

100 ll culture medium overnight in a 96-well plate in the

dark. On the day of experiment, cells were washed with

PBS and incubated with 25 lM DCFH-DA for 45 min in

the dark. Later, the cells were washed with PBS to remove

residual DCFH-DA. Cellular fluorescence was measured at

485 nm (excitation) and 535 nm (emission) using a Vari-

oskan� Flash microplate reader (Thermo-Scientific, Wal-

tham, MA, USA).

mRNA isolation and one-step quantitative real-time

PCR

Total RNA was extracted from PBMCs of CHC-infected

patients and healthy controls using a commercial Spin

technology (RNeasy Mini Kit, Qiagen, Solna, Sweden)

according to the manufacturer’s protocol. DNase treatment

was carried out to remove any contaminating DNA using

an RNase-Free DNase Set (Qiagen). RNA concentration

was determined using a NanoDropTM spectrophotometer.

qRT-PCR was performed in a total volume of 20 ll

including 2 ll of RNA, forward and reverse primers

(0.2 mM each) and 25 ll of SYBR�Green PCR mix

(SYBR� Green: Applied Biosystems, Stockholm, Swe-

den). Reactions were run on a iQ5 Thermal cycler (BioRad,

Hercules, CA, USA) using the universal thermal cycling

parameters. Results were obtained using the sequence

detection software iQ5 thermal cycler and analyzed using

MS Excel software. For all samples, melting curves were

acquired for quality control purposes. For gene expression

quantification, we used the comparative Ct method. Ini-

tially, gene expression levels for each sample were nor-

malized against the average Ct values of two endogenous

controls (b-actin and tubulin), according to manufacturer’s

protocol.

The primers were designed using the NCBI Primer-

BLAST (Basic Local Alignment Search Tool) online

software at http://www.ncbi.nlm.nih.gov/tools/primer-

blast/index.cgi?LINKLOC=BlastHome. The primers used

against the corresponding genes were synthesized by First

Base Labs (First Base, Kuala Lumpur, Malaysia), and are

as follows: Blimp1, forward 50-CAGCTCGCCCACCTGC

AGAA-30 and reverse 50-GCCGCAGCGCAGTTCCCTTT-

30; Btla, forward 50-TGCCTGGTTTGTTTTCTTCCAG

GC-30 and reverse 50-TGGGTCATACCGCTGTTCTGC

AA-30; Cd160, forward 50-GCCTTGTGGCCCTTCAAGC

TTTGT-30 and reverse 50-TCCCCTGTGCCCTGTTG

CAT-30; Ctla4, forward 50-GGGCATAGGCAACGGAAC

CCA-30 and reverse 50-GGGGGCATTTTCACATAGACC

CCTG-30; Foxp3, forward 50-CAGCACATTCCCAGAGT

TCCTC-30 and reverse 50-GCGTGTGAACCAGTGGTA

GATC-30; Lag3, forward 50 -CTAGCCCAGGTGCCCA

ACGC-30 and reverse 50-GCCTGCGGAGGGTGAATCC

C-30; Pd1, forward 50-CTCAGGGTGACAGAGAGAAG-

30 and reverse 50-GACACCAACCACCAGGGTTT-30;Tim3, forward 50-AGGGGACATGGCCCAGCAGA-30 and

reverse 50-GCCAGCCCAGCACAGATCCC-30; Trail, for-

ward 50 -CTTTACCAACGAGCTG-30 and reverse 50-

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GTTATGTGAGCTGCTAC-3; b-actin, forward 50-AGAG

GGAAATCGTGCGTGAC-30 and reverse 50-CAATAGT

GATGACCTGGCCGT-30; and tubulin, forward 50-AACA

CGGGATCGACTTGGC-30 and reverse 50-CTCGGGGC

ACATATTTCCTAC-30.

QuantiGene Plex� 2.0 assay

Total RNA from PBMCs of participants was processed by

means of QuantiGene Plex� 2.0 assay (Affymetrix, Santa

Clara, CA, USA) according manufacturer’s instructions. The

QuantiGene Plex� 2.0 is a hybridization-based assay using

the xMAP� Luminex� magnetic beads and performed on

96-well plates designed to quantify 80 different RNA targets

of the extrinsic and intrinsic apoptosis pathways. The assay

directly quantifies the RNA targets using xMAP Luminex

beads for multiplexing of 3–80 RNA targets and branched

DNA (bDNA) signal amplification technology. Briefly, a

working bead mix was prepared containing lysis mixture,

blocking reagent, capture beads, and 2.0 probe set. The bead

mix was dispensed into the hybridization plate, and 20 ll of

total RNA was added to each well. For background control

wells, 20 ll of sterile nuclease-free water was added to the

bead mix. The hybridization plate was placed in a shaking

incubator (600 rpm) for 22 h at 54 �C. After hybridization,

the wash solution, pre-amplifier, amplifier, label probe, and

streptavidin–phycoerythrin (SAPE) solutions were pre-

pared. Later, the hybridized samples were transferred to a

magnetic separation plate. Samples were washed and incu-

bated for 1 h each (50 �C at 600 rpm) with the pre-made

amplifier solutions. Later, the SAPE solution was added and

the samples were incubated at 25 �C for 30 min at 600 rpm

in the dark. The unbound SAPE was washed, and 130 ll of

SAPE wash buffer was added to each RNA sample. The plate

was shaken at 25 �C for 3 min at 800 rpm to resuspend the

beads. The plate was read immediately using a Bio-Plex�

200 system (BioRad, Hercules, CA, USA). Fluorescent

readings from blank wells were subtracted from fluorescent

values for each mRNA of interest. The values were nor-

malized against the geometric mean expression of two

internal control genes, Ppib (peptidylpropyl isomerase B)

and Hprt1 (hypoxanthine–guanine phosphoribosyltransfer-

ase) for each sample that was run in triplicates.

Statistical analysis

Statistical significance was analysed using MS Excel and

GraphPad Prism 6.0 softwares (GraphPad, La Jolla, CA,

USA). The levels of significance for comparisons between

two or more independent samples were determined using a

two-tailed unpaired Student’s t test. Data are presented as

mean ± SD, where n refers to the number of independent

experiments. For QuantiGene Plex� 2.0 assays, expression

values for each gene were multiplied by a constant, and

compared between CHC-infected patients and healthy

controls. The values were normalized against the geo-

metric mean expression of two internal control genes Ppib

and Hprt1 for each sample, and expressed as fold change

[2-fold, highly up-regulated; 1.5- to 2-fold, moderately

up-regulated; 1- to 1.5-fold, mildly up-regulated. For

expression of immune exhaustion molecules by FACS

analysis, the data were transformed and normalized, and a

two-tailed non-parametric paired t test was used for

revealing significance. The level of significance was con-

sidered at P \ 0.05, and measures represented by

*P \ 0.05, **P \ 0.005, ***P \ 0.001 and

****P \ 0.0001.

Results

Demographic analysis

Our study consisted of 14 participants that included seven

CHC-infected individuals and seven healthy controls. Table 1

shows the mean age of participants, TLCs, HCV PVLs, and

serum ALT and AST liver enzyme levels of CHC-infected

individuals. Of the seven CHC subjects, three were males and

four were females with a mean PVL of 3.2 9 105 HCV IU/ml

of blood. In contrast, the healthy controls included five males

and two females. We observed that the TLCs decreased with

increasing frequency of spontaneous apoptosis.

Serum indoleamine 2,3-dioxygenase levels were

elevated in chronic HCV-infected individuals

IDO is an intracellular enzyme that deprives immune cells

of tryptophan, an amino acid essential for T cell prolifer-

ation [30], facilitating T cell apoptosis aided by inhibitory

molecules and induction of oxidative stress [31]. IDO

activity increases with disease severity in patients with

PVIs, especially HIV disease [32]. COX-2/PGHS-2 repre-

sents an inflammatory metabolite occurring in most tissues

[33] usually at extremely low or undetectable levels, and

could be associated with tumorigenesis [34, 35]. Here, we

assessed the serum levels of IDO and COX-2 and their

association with spontaneous apoptosis in PBMCs derived

from CHC-infected individuals. Our investigations showed

that serum IDO (Fig. 1a) and COX-2/PGHS-2 levels

(Fig. 1b) were significantly increased among CHC-infected

individuals relative to healthy controls (Table 2) although

there was no association between PVL and IDO levels

(data not shown). We speculated that the elevated serum

levels of IDO and COX-2/PGHS-2 could likely influence

spontaneous apoptosis of immune cells in chronic HCV-

infected individuals.

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Cellular reactive oxygen species levels were increased

in chronic HCV-infected subjects

Next, we determined the cROS levels in PBMCs of CHC-

infected subjects and healthy controls as the level of cROS

reflects ongoing oxidative stress and apoptosis via the

caspase cascades [36, 37], and most likely the onset of

intrinsic apoptosis pathways. COX-2/PGHS-2 indicates an

ongoing inflammation and ROS levels contribute to oxi-

dative stress in HCV infection [38]. Our findings showed a

significant increase in cROS levels in CHC-infected indi-

viduals compared to healthy controls (Fig. 1c; Table 2).

The observed increase in cROS indicates increased intra-

cellular oxidative stress that could likely be the mediators

of ongoing apoptosis in CHC disease.

Immune cells of chronic HCV-infected subjects showed

up-regulated expression of TRAIL and certain other

biosignatures of immune exhaustion

PVIs appears to facilitate immune exhaustion in CD8? T

cells [39] and impairment of immune cells [40]. For

instance, increased intrahepatic expression of Trail-

induced apoptosis in primed immune cells [41]. The

association between increased levels of cROS and TRAIL

prompted us to investigate the possible role of molecules

associated with immune exhaustion in CHC disease. We

initiated this by studying the expression levels of mRNA of

immune inhibitory molecules by qRT-PCR. Our investi-

gations showed that the gene expression of inhibitory

molecules in CHC-infected patients was marginally

increased as compared to healthy controls. We observed

relatively increased gene expressions of Tim3, Pd1, Cd160,

Ctla4, Trail, Btla, and Lag3 in CHC-infected individuals

(Fig. 2a, b) (Table 3). Conversely, the increased expression

of inhibitory molecules in CHC-infected individuals sug-

gest a role for immune exhaustion molecules, especially

Trail (14.7-fold) with accentuated apoptosis in CHC cases.

CD4? T cells of chronic HCV-infected individuals

displayed increased surface expression of TRAIL

and differential expression of immune exhaustion

molecules

Next, we investigated the surface expression levels of

selected immune inhibitory molecules on CD4? and CD8?

T cells in PBMCs from CHC-infected subjects by flow

cytometry (Fig. 3a–f; Table 4). Our findings indicated that

the surface expression of TRAIL, TIM-3, PD-1, and CTLA-4

in CD4? T cells of CHC-infected subjects were up-regulated

relative to healthy controls. On CD8? T cells, we observed an

up-regulation in TRAIL, TIM-3, and PD-1 in CHC-infected

individuals relative to healthy controls (CD8? CTLA-4?

Table 2 Expression profile of immunoregulatory (IDO & COX-2/

PGHS-2) and apoptosis (cROS) molecules in chronic hepatitis

C-infected individuals and healthy controls

Molecular

expression

Healthy

controls

(n = 7)

Chronic HCV-

infected (n = 7)

Significance

Serum IDO 2.5 9.80 P \ 0.005

Serum COX-2/PGHS-2 2.1 9.74 P \ 0.001

Cellular ROS 1.1 9.65 P \ 0.001

0

5

10

15

0

5

10

15

0

5

10

15

IDO

COX-2/PGHS-2

ROS

Rel

ativ

e flu

ores

cenc

e in

tens

ity

Healthy controlsCHC patients

A

B

C

Fig. 1 Chronic HCV infection results in increased levels of serum

indoleamine 2,3-dioxygenase (IDO), cyclooxygenase-2/prostaglandin

H synthase (COX-2) and cellular reactive oxygen species (cROS).

a Serum levels of IDO in chronic HCV disease (n = 7) and healthy

controls (n = 7). Serum IDO was measured using a commercial

ELISA (Uscn Life Science Inc, Hubei, PRC). b Serum levels of COX-

2 measured using a commercial ELISA (R&D, Abingdon, UK).

c cROS formation was measured using a commercial oxidation-

sensitive fluorescent probe, 20,70-dichlorofluorescein diacetate

(DCFH-DA) ROS detection assay kit (Mitosciences�, Cambridge,

UK) after 2 days of culture of PBMCs. The levels of significance for

comparisons between two or more independent groups were deter-

mined using a two-tailed unpaired Student’s t test. Data are presented

as mean ± SD, where n refers to the number of independent

experiments. Bars represent mean values ± SEM. *P \ 0.05,

**P \ 0.005, ***P \ 0.001 and ****P \ 0.0001

Apoptosis

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data not shown). We found that BTLA expression was sig-

nificantly decreased in CHC-infected subjects in CD4? and

CD8? T cells (Fig. 3f; Table 4). Of note, we found that

CD4? T cells of chronic HCV-infected individuals displayed

increased surface expression of TRAIL and differential

expression of other immune exhaustion molecules.

Increased expression of inhibitory molecules

was consistent with up-regulation of Blimp-1, Dtx1

and FoxP3 in immune cells of chronic HCV disease

Transcription factors, Dtx1, Foxp3 and Blimp1 reportedly

limit T cell survival and functionality in PVIs [42].

Decreased Blimp1 expression results in improved CD8? T

cell survival owing to reduced expression of Pd1, Lag3, and

Cd160 on virus-specific CD8? T cells in PVIs [13]. More-

over, Foxp3 has recently been identified as a pro-apoptotic

protein [23]. Hence, we broadly surveyed the spontaneous

expression of transcriptional factors in PBMCs of the study

population. Our results showed marginal up-regulation of

Foxp3, and Dtx1, and Blimp1 in CHC-infected individuals

relative to healthy controls (Fig. 4) (Table 3), suggesting a

likely role of these transcription factors with the increase of

co-inhibitory molecules in chronic HCV disease.

Extrinsic and mitochondrial pathways regulated

the spontaneous onset of expression of apoptosis

markers in immune cells of chronic HCV-infected

subjects

Given the increased expression of TRAIL both at the gene

and protein levels in PBMCs hinting the likely role of

apoptosis in persistent HCV disease, next we asked if the

genes encoding the extrinsic and intrinsic apoptosis path-

ways played any role in the CHC disease. Spontaneous

apoptosis is induced simultaneously by both extrinsic and

intrinsic pathways, and therefore we surveyed the expres-

sion of an array of apoptotic genes, using a commercial

QuantiGene Plex� 2.0 assay. Our results clearly showed

that CHC infection modulates the spontaneous expression

of a large array of apoptotic genes in immune cells. High

up-regulation of Tnf (24.1-fold), Bcl2a1, Tp73, Bcl2l10,

Casp14, Tnfrsf11b, Bik and Birc8 was observed in CHC-

infected cases (Fig. 5). The profile of expression of apop-

tosis-related genes has been shown in Table 5. Together,

we demonstrated that spontaneous onset of apoptosis in

PBMCs of CHC-infected individuals was likely co-regu-

lated by both extrinsic and intrinsic pathways.

Discussion

The cell death mechanisms operational in HCV-pre-

primed immune cell phenotypes remain ambiguous

despite that apoptosis of hepatocytes has been well-

described in clinical HCV disease [43]. Cell death char-

acteristically follows one of two patterns: apoptosis (ATP-

Table 3 Gene expression profile of molecules associated with

immune exhaustion in chronic hepatitis C-infected individuals and

healthy controls

Gene (PBMCs)

qRT-PCRaHealthy

controls

(n = 7)

Chronic HCV-

infected (n = 7)

Significance

Tim3 2 2.2 ?

Pd1 2 2.1 ?

Cd160 2 6.6 ????

Ctla4 2 3.9 ???

Trail 2 14.7 ????

Btla 2 22.9 ????

Lag3 2 6.1 ????

Foxp3 2 14.6 ????

Dtx1 2 2.6 ?

Blimp1 2 2.2 ?

a Fold change cut-off set at 2-fold in healthy controls and compared

with HCV-infected patients (2- to 3-fold, mildly up-regulated; 3- to

4-fold, moderately up-regulated; [4-fold, highly up-regulated)

0

10

20

Lag3 Ctla4 Btla0

5

10 Healthy controlsCHC patients

Fold

cha

nge

A

B

Cd160 Trail Tim3 Pd1

Fig. 2 Chronic HCV infection leads to increased spontaneous

expression of Trail and certain other biosignatures of immune

exhaustion. Expression of mRNA of a Lag3, Ctla4 and Btla, and

b Cd160, Trail, Tim3 and Pd1 in peripheral blood mononuclear cells

was performed by qRT-PCR on chronic hepatitis C infected (n = 7)

and controls (n = 7). Fold change cut-off set at twofold in healthy

controls and compared with HCV-infected patients (2- to 3-fold, mild

up-regulation; 3- to 4-fold, moderate up-regulation;[4-fold, high up-

regulation)

Apoptosis

123

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dependent) or necrosis (ATP-independent) [44, 45]. Sev-

eral hypotheses have been proposed to explain PVI-

induced cell death [46]. A growing body of literature

suggests that HBV- [47, 48], SIV- [13] and HIV- [49]

infected PBMCs undergo apoptosis. Nevertheless, the

contribution of spontaneous T cell exhaustion and apop-

tosis of PBMCs during PVIs remain unclear. Here, we

show evidence of spontaneous T cell exhaustion and

apoptosis in immune cells of CHC-infected subjects. Our

current findings are in accordance with previous findings

of markers of oxidative stress in HCV infection [43].

Interestingly, significant expression of inhibitory mole-

cules, transcription factors and immunoregulatory

enzymes were clearly evident among CHC-infected indi-

viduals indicating the likely association between immune

exhaustion and immune cell apoptosis.

IDO is a T cell proliferation-limiting enzyme [30], and

increased IDO activity has been linked to disease severity

A

UnstainedLymphocytes

CD3+ CD4/CD8+

Fig. 3 Persistent HCV disease results in differential surface protein

expression of TRAIL, TIM-3, PD-1, CTLA-4 and BTLA on CD4?

and CD8? T cells. a Depiction of the gating strategy used throughout:

lymphocytes were selected on the basis of forward- and side-scatter

characteristics, dead cells excluded, then CD3?, CD4 and CD8? T

cells selected. Surface expression of b TRAIL, c TIM-3, d PD-1,

e CTLA-4 and f BTLA on CD4? and CD8? T cells of CHC-infected

subjects and healthy controls (Top panel, healthy controls; Bottom

panel, chronic HCV infection). PBMCs were stained with the

following antibody combinations: TRAIL: anti-PerCP-Cy5.5-CD3,

PE-Cy7-CD4, APC-H7-CD8, and PE-TRAIL; TIM-3: anti-PerCP-

Cy5.5-CD3, PE-Cy7-CD4, APC-H7-CD8, and APC-TIM-3; PD-1:

anti-PerCP-Cy5.5-CD3, PE-Cy7-CD4, APC-H7-CD8, and FITC-PD-

1; CTLA-4: anti-PerCP-Cy5.5-CD3, PE-Cy7-CD4, APC-H7-CD8,

and BV421-CTLA-4; and BTLA: anti-PerCP-Cy5.5-CD3, PE-Cy7-

CD4, APC-H7-CD8, and PE-BTLA mAbs, and acquired on a 7-color

FACS Canto IITM and analyzed using FlowJoTM software. Data were

transformed and normalized, and paired t tests were used for revealing

statistical significance. Data are presented as mean ± SD, where n

refers to the number of independent experiments. Bars represent mean

values ± SEM. *P \ 0.05, **P \ 0.005, ***P \ 0.001 and

****P \ 0.0001

Apoptosis

123

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in PVIs, especially HIV infection [32]. The other attribute

in our research is the elevation of serum COX-2 in CHC-

infected subjects. COX-2 catalyzes the conversion of ara-

chidonic acid to prostaglandins and thromboxanes [33].

Under normal conditions COX-2 is low or undetectable,

but can be readily induced in response to cellular activation

by physiologic stimuli. Up-regulation of COX-2 leads to

chronic inflammation, limiting lymphocyte expansion [34,

35]. HCV is known to up-regulate ROS via inducible nitric

oxide synthase (iNOS) and COX-2 in chronic HCV

infection [43]. Oxidative stress involves generation of

superoxide anions, H2O2, singlet oxygen, and hydroxyl-

peroxy radicals triggering caspase activation [50]. Our

finding of increased cROS levels in CHC indicates acti-

vation of the intrinsic pathway [51]. Although, there were a

few anti-apoptotic genes that were elevated, this likely

could be to sustain cellular homeostasis [52, 53] and

requires additional investigation.

Immune exhaustion has also been linked to spontane-

ous apoptosis [40], which is regulated by a balance

between anti- and pro-apoptotic molecules via Fas-FasL

interactions and/or the mitochondrial pathway [54]. In

agreement with this, here we found that Cd160, Trail,

Lag3, Tim3, Pd1, and Ctla4 were up-regulated in HCV-

primed PBMCs suggesting ongoing immune exhaustion in

parallel with immune cell apoptosis in CHC infection.

The most intriguing finding is the significantly down-

regulated surface expression of BTLA in spite of up-

regulated gene expression, which is in line with our recent

investigations on HIV-infected T cells in vitro [32], and

others also support this finding [54, 55]. Dysregulation of

B cells could also be associated with decreased BTLA in

HIV viremic individuals [56], and therefore the functional

implications of decreased BTLA in CHC disease warrants

further evaluation.

Exhausted CD8? T cells during PVIs specifically

increases the expression of Cd160 [57, 58], which is in line

with our current finding. Another study showed that PD-1?

CD8? T cells undergo both spontaneous and Fas-mediated

apoptosis [21]. One study showed that Trail triggers

Healthy CHC30

35

40

45

50

55

Healthy CHC0

10

20

30

40

Healthy CHC0

10

20

30

40

50

B

TIM-3

Healthy CHC30

40

50

60

70

% C

D4+

TRAIL

+

% C

D8+

TRAIL

+

C

* ns ns ns

TRAIL

CD4

CD8

CD4

CD8

Chronic Hepatitis C Infection

Healthy Controls

% C

D4+

TIM

-3+

% C

D8+

TIM

-3+

Fig. 3 continued

Apoptosis

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apoptosis in fresh liver explants of patients with viral

hepatitis [59]. Further, lack of Lag3 in mice markedly

delays T cell apoptosis [34]. Likewise, TIM-3 generates an

inhibitory signal leading to reduced secretion of antiviral

cytokines [41]. Blimp1 is a transcriptional repressor of

memory differentiation in CD8? T cells, which also

appears to intrinsically regulate Pd1, Lag3, and Cd160

expression in virus-specific CD8? T cells during PVIs

[60]. Hence, the expression of these molecules may likely

be linked to one another to modulate the functions of each

other. HCV infection reportedly elevates the turn-over

frequencies of TIM-3? and PD-1? HCV-specific T cells,

which has been closely associated with liver disease pro-

gression [61]. Besides, HCV-HIV co-infected patients also

appear to show increased expression of PD-1 and Fas that

consequently has been correlated with stages of HCV liver

disease [14]. One recent finding showed that intrahepatic

CD8? T cells of CHC patients expressed increased levels

of TIM-3 and PD-1 [62]. Besides, enhanced neutrophil

apoptosis via activation of caspase 10 has also been linked

to HCV infection although its role with disease progression

needs elaborate discussion [63]. Together, the concerted

role played by immune exhaustion molecules and immune

cell apoptosis still remains a gray area of investigation.

The extrinsic pathway is triggered by ligation of death

receptors, such as Fas/CD95 or the TRAIL receptors,

TRAIL-R1 and TRAIL-R2 or TNFR-1 or 2 via their cognate

ligands FasL or TRAIL or TNF-a respectively, resulting in

receptor trimerization, assembly of death domains, and

recruitment of adaptor molecules, Fas-associated death

domain (FADD) via homophilic contact facilitated by the

death domain [64, 65]. FADD recruits caspase-8 to the

activated Fas to form the Fas death-inducing signaling

complex (DISC). Oligomerization of caspase-8 upon DISC

formation drives its activation via self-cleavage. Subse-

quently, caspase-8 activates downstream effector caspases 3,

6, and 7 (all up-regulated). Eventually, caspase-8 initiates

activation of BH3 interacting-domain death agonist (Bid).

D E % C

D4+

PD-1

+

H e a lth y C H C0

20

40

60 ns

% C

D8+

PD-1

+

H e a lth y C H C0

10

20

30

40

50 *

% C

D4+

CTLA

-4+

Healthy CHC0

2

4

6

8 ns

PD-1 CTLA-4

CD4

CD8

CD4

CD8

Chronic HCV Infection

Healthy Controls

Fig. 3 continued

Apoptosis

123

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Caspase 10 also appears to bind to Fas and CFLAR that were

all up-regulated in CHC infection in the current study. While

it is also well-known that CD27 can bind to TRAF2 and

TRAF3 in addition to TNF (all up-regulated) and mediate T

cell apoptosis in CHC-infected subjects, up-regulation of

anti-apoptotic genes, Bcl-2A1 and Bcl-2L10, and a mild up-

regulation of Bcl-2L2, and Mcl-1 in CHC patients indicate

that HCV preserves some cells (preferably unprimed cells)

from undergoing apoptosis. However, since other pro-

apoptotic genes were up-regulated in immune cells of CHC

patients inducing spontaneous apoptosis, the cROS-induced

cell death is likely an alternate death pathway resulting from

caspase activation [55]. Our data suggest that elevated cROS

levels activate the intrinsic pathway involving the release of

mitochondrial intermembrane space proteins, including Cyt

c, and AIF into the cytosol. Later, cytosolic Cyt c activates

the apoptosome complex, eventually cleaving procaspase-9

and effector caspases 3, 6, and 7 resulting in apoptosis [66,

67]. The onset of spontaneous apoptosis is further fortified by

the up-regulation of Bcl-2 family members in the PBMCs of

CHC-infected patients. The ‘Bcl-2-regulated pathway’ is a

vital trigger of intrinsic and extrinsic apoptosis, which

BTLA

F

% C

D4+

BTLA

+

Healthy CHC0

20

40

60

80 *

% C

D8+

BTLA

+

Healthy CHC30

40

50

60

70

80 ns

CD4

CD8

Chronic HCV Infection

Healthy ControlsFig. 3 continued

Apoptosis

123

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synergistically regulates the mediators of apoptosis. Here,

we also hypothesized that cROS owing to persistent HCV

infection initiates specific BH3-only proteins, which then

inactivates Bcl-2-like pro-survival molecules [68] as sup-

ported by our current findings. Our current findings are also

in agreement with the up-regulation of apoptosis pathways in

chronic HCV disease and HCV-HIV co-infection [69].

Our investigation has shown that an elevated expression

of molecules that induce spontaneous apoptosis and T cell

inhibition in immune cells of CHC-infected subjects,

although it still remains to be seen if the functional attri-

butes of the immune cells studied here were compromised.

Our observations showed evidence of increased levels of

oxidative stress and immune exhaustion in CHC-infected

patients, triggering the potential recruitment of apoptotic

genes. Furthermore, up-regulation of pro-apoptotic factors

functioning via the extrinsic and intrinsic pathways sug-

gests their utility for intracellular viral persistence, which

however, remains to be investigated. There are also certain

limitations in the current investigation, which remain

unresolved. Our original aim was to investigate the

potential association between immune exhaustion and

apoptosis. Nonetheless, we were only partly successful in

linking both the spontaneous entities. One of the major

Table 4 Expression profile of

surface protein molecules

associated with immune

exhaustion on CD4? and

CD8? T cells in chronic

hepatitis C-infected individuals

and healthy controls

Protein expression Healthy controls (n = 7) Chronic HCV-infected (n = 7) Significance

CD4? T cells

TIM-3 27.49 ± 2.9 31.43 ± 3.6 P = 0.06

PD-1 36.74 ± 2.8 42.39 ± 3.0 P = 0.1

CTLA-4 4.456 ± 0.4 5.241 ± 0.3 P = 0.1

BTLA 62.31 ± 3.6 43.46 ± 6.9 *P = 0.03

TRAIL 39.97 ± 1.4 46.36 ± 2.4 *P = 0.006

CD8? T cells

TIM-3 49.66 ± 2.6 56.06 ± 3.0 P = 0.1

PD-1 27.91 ± 3.4 34.80 ± 3.5 P = 0.1

BTLA 62.39 ± 4.7 54.99 ± 3.4 P = 0.2

TRAIL 21.19 ± 2.5 20.74 ± 2.9 P = 0.3

0

5

10

15

20

Foxp3 Dtx1 Blimp1

Fold

cha

nge

Healthy controlsCHC patients

Fig. 4 Chronic HCV infection results in increased expression of

transcription factors Foxp3, Dtx1 and Blimp1. Expression of mRNA

expression was performed by qRT-PCR on chronic hepatitis C

infected (n = 7) and healthy controls (n = 7). Fold change cut-off set

at 2-fold in healthy controls and compared with HCV-infected

patients (2- to 3-fold, mild up-regulation; 3- to 4-fold, moderate up-

regulation; [4-fold, high up-regulation)

0

1

2

3

4

5

6

Bcl2a1 Tp73 Bcl2l10 Casp14 Tnfrsf11b Bik Birc8

Non-HCV controlChronic Hepatitis C

Fold

cha

nge

Tnf

Fold

cha

nge

0

5

10

15

20

25

30

Fig. 5 Differential expression of apoptotic genes in peripheral blood

mononuclear cells of chronic HCV infection and healthy controls

determined by a commercial QuantiGene Plex� 2.0 assay. Highly up-

regulated genes measured from mRNA of PBMCs of chronic hepatitis

C infected (n = 7) and healthy controls (n = 7). Total RNA from

PBMCs of participants was processed by a QuantiGene Plex� 2.0

assay (Affymetrix, Santa Clara, CA, USA). The values were

normalized against the geometric mean expression of two internal

control genes, Ppib (peptidylpropyl isomerase B) and Hprt1 (hypo-

xanthine–guanine phosphoribosyltransferase) for each sample (Fold

change [2-fold, high up-regulation)

Apoptosis

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limitations is the sample size due to poor turnover of

individuals with HCV infection in the setting, and use of

cells for other investigations. Further, there was limited

clinical data that correlated with markers of apoptosis

studied. In addition, it is also not clear if the ongoing

inflammation in chronic HCV patients originated from

ongoing liver cell damage, and therefore use of controls,

for instance, inclusion of subjects with non-alcoholic ste-

atohepatitis (NASH) is warranted. It remains to be seen

how the immune cells program the expression of the bio-

signatures investigated herein, following stimulation with

standard HCV protein antigens to clearly underpin the

concrete association of virus-specific immune exhaustion

with apoptosis signaling in viral persistence.

Acknowledgments We thank all the participants, clinical, para-

clinical and laboratory staff of University of Malaya Medical Center

for assistance with patient recruitment, specimen collection and

cooperation. This work was financially supported by the High Impact

Research (UM.C.625/1/HIR/139), University of Malaya to Esaki M.

Shankar for a study titled ‘Mechanisms of T cell dysfunctions in

hepatitis C infection’. Tunku Kamarul is supported by the HIRG-

MOHE A000003-50001 Grant of University of Malaya. Marie Lars-

son is supported by Grant No. AI52731 from the Swedish Research

Council, the Swedish Physicians against AIDS Research Foundation,

the Swedish International Development Cooperation Agency; SIDA

SARC, VINNMER for Vinnova, Linkoping University Hospital

Research Fund, CALF and the Swedish Society of Medicine. We also

acknowledge financial support from the University of Malaya

Research Grant (UMRG) RG448-12HTM of the Health and Trans-

lational Medicine Research Cluster, University of Malaya to Esaki M.

Shankar.

Conflict of interest The authors declare that they have no conflict

of interest to disclose.

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