Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol.

110(2): 267-271, April 2015 267

Spontaneous hepatitis C viral clearance and hepatitis C chronic infection

are associated with distinct cytokine profiles in Mexican patients
Nora A Fierro1,2, Karina González-Aldaco3,4, Rafael Torres-Valadez3,4, Maria E Trujillo-Trujillo3,4,
Sonia Roman3,4, Jorge L Trujillo-Ochoa1,2, Arturo Panduro3,5/+
1
Unidad de Inmunovirología 3Servicio de Biología Molecular en Medicina, Hospital Civil de Guadalajara Fray Antonio Alcalde,
Guadalajara, Jalisco, México 2Departamento de Fisiologia 4Departamento de Biología Molecular 5Departamento de Clínicas Médicas,
Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, Jalisco, México

The mechanisms related to the spontaneous clearance of hepatitis C virus (HCV) have been primarily studied in
regions where the infection is endemic. Results of prior studies have been extrapolated to populations with low en-
demicity, such as Mexico. Herein, we determined the cytokine profiles in serum samples from Mexican patients who
spontaneously cleared HCV and patients chronically infected with HCV genotype 1a. Chronic HCV-infected patients
displayed increased interleukin (IL)-8 and regulated upon activation, normal T-cell expressed and secreted (CCL-5)
secretion, whereas patients who spontaneously cleared HCV showed augmented levels of IL-1 alpha, tumour necro-
sis factor-alpha, transforming growth factor-beta, monocyte chemoattractant protein-2 (CCL-8), IL-13 and IL-15.
Our study suggests that cytokine profiles may predict disease outcome during HCV infection.

Key words: hepatitis C virus - cytokines - viral clearance - genotype 1a

Hepatitis C virus (HCV) is an enveloped RNA virus common and the results have been extrapolated to regions
belonging to the Flaviviridae family. Each year, HCV in- of low endemicity. The exact mechanisms responsible for
fects approximately three-four million individuals world- viral clearance and recovery in humans are unknown. In
wide. It is estimated that 170 million people are chroni- this context, efforts to determine whether biomarkers, in-
cally infected with HCV and at risk of developing chronic cluding serum cytokines, can accurately predict the out-
liver disease and 350,000 deaths occur each year due to come of HCV infection are valuable for establishing bet-
HCV-related causes (Hanafiah et al. 2013). HCV is present ter HCV control strategies in Latin American regions.
worldwide, but its distribution pattern is not uniform. In In the present study, 33 serum samples from patients
Latin America, HCV genotype 1 is the most prevalent (> 18 years of age) admitted to the Molecular Biology
and the overall prevalence of HCV antibody is estimated Service of the Fray Antonio Alcalde Civil Hospital of
to be 1.5% (Alvarado-Mora & Pinho 2013). In Mexico, Guadalajara (AHCFAA) from 2011-2013 were retrospec-
an identified region with low HCV prevalence, the most tively analysed. Hepatitis was defined as described in
prevalent viral genotypes are 1a and 1b followed by 2a and a previously published study (Escobedo-Meléndez et al.
3b (Panduro et al. 2011). In recent years, chronic diseases 2012). Informed consent was obtained from all patients
associated with liver malfunction have gained increasing involved in the study. The local ethical committee of the
importance in the world (Fierro et al. 2014); thus, the study AHCFAA approved the study protocol.
of causal agents of liver damage is of great significance. Samples from patients with liver disease and under
HCV is able to establish lifelong persistent infection treatment with a hepatotoxic drug, patients with chronic
in most individuals by successfully evading the immune hepatitis associated with an aetiological agent distinct to
system. However, approximately 30% of patients sponta- viral hepatitis, patients with autoimmune hepatitis and
neously clear the virus (Rehermann & Bertoletti 2015). patients identified as overweight or obese were excluded
The immunopathology associated with HCV infection has from the study. Clinical history and demographic data were
been predominantly studied in regions where infection is collected for all participants through a structured question-
naire, as previously reported (Escobedo-Meléndez et al.
2012). Serum alanine aminotransferase (ALT) and aspar-
tate aminotransferase (AST) levels were measured using an
enzymatic method (Human, Germany) with an automatic
analyser. The abnormal cut-off values for ALT and AST
doi: 10.1590/0074-02760140377
enzymes were 40 UI/mL and 50 UI/mL, respectively.
Financial support: CONACYT (127229, 188240) Clinical evaluation of HCV-infected patients -
RT-V, MET-T and JLT-O were supported by PhD scholarships from Chronic HCV infection was defined as a positive anti-
the CONACYT, NAF, SR and AP are sponsorship recipients of the
Red Temática de Colaboración Académica en Fisiopatología de las
HCV test (ELISA Third-Generation, AxSYM) result
Enfermedades Hepáticas (SEP-Promep). and the presence of serum HCV RNA for more than six
+ Corresponding author: [email protected] months (COBAS® AmpliPrep and COBAS® TaqMan 48).
Received 13 October 2014 Samples from patients who spontaneously cleared HCV
Accepted 26 January 2015 tested positive for anti-HCV antibody in the absence of

online | memorias.ioc.fiocruz.br
268 Cytokines and HCV • Nora A Fierro et al.

HCV RNA six months after the initial test, in accor- The protocol was conducted in accordance with the Hel-
dance with the definition of spontaneous viral clearing sinki Declaration of 1975, as revised in 1983.
(Hanafiah et al. 2013, EASL 2014). AST and ALT levels
Statistical analysis - The data are reported as indi-
were determined by dry chemistry on a Vitros 250 anal-
vidual densitometry analyses for each patient for each
yser (Ortho Clinical Diagnostics, Johnson & Johnson,
cytokine. The mean ± standard deviation (SD) for study
USA). Viral genotyping was performed at the Bayesian
groups is indicated. The data are presented as the mean
Markov model through a conventional line probe assay
± SD. Statistical comparisons were performed using
(VERSANT HCV Genotype 2.0 Assay LiPA, Germany)
GraphPad Prism v.5.01. A nonparametric Mann-Whitney
after HCV RNA extraction (QIAamp RNA mini kit) and
RNA amplification (VERSANT HCV LiPA 2.0 Ampli-
fication Kit) following the manufacturer’s instructions.
Patients with positive viral RNA were stratified into
four groups according to their fibrosis stage, as determined
by transitional elastography using a FibroScan® instrument
(Echosens, France). As validated by the manufacturer, the
stage of fibrosis in patients with chronic HCV infection
was defined as follows: F1, initial fibrosis, F2, moderate
fibrosis, F3, advanced fibrosis, and F4, cirrhosis stage.
Analysis of serum cytokines - As reported previ-
ously, a standardised (Fierro et al. 2011) dot blot-based
assay was used according to the manufacturer’s instruc-
tions (Ray-Biotech, USA) to detect the relative expres-
sion levels of 23 cytokines in serum samples collected
from patients. The following cytokines were analysed:
pro-inflammatory cytokines [interleukin (IL)-8, tumour
necrosis factor-alpha (TNF-α), IL-2, IL-1 and IL-6], an-
ti-inflammatory cytokines [tumour growth factor-beta
(TGF-β) and IL-10], fibrogenic (IL-13), immunoregula-
tory cytokines [IL-5, IL-15, interferon-gamma (IFN-γ),
IL-7 and TNF-β], chemokines [monocyte chemoattrac-
tant protein (MCP)-1 (CCL-2), MCP-2 (CCL-8), MCP-3
(CCL-7), monokine-induced by IFN-γ (MIG) (CXCL-9)
and regulated upon activation, normal T-cell expressed
and secreted (RANTES) (CCL-5)] and growth factors
[growth-regulated oncogene-alpha (GRO-α), GRO,
IL-3, granulocyte-macrophage colony-stimulating fac-
tor (GMCSF) and granulocyte colony-stimulating fac-
tor (GCSF)] (Fierro et al. 2011). Briefly, the membranes
were blocked with a blocking buffer and 1 mL of a 1:500
dilution of the patient serum was added. Next, the mem-
branes were incubated at room temperature (RT) for 2
h. After the membranes were washed, 1 mL of primary
biotin-conjugated antibodies was added to detect the The cytokine profiles during hepatitis C virus (HCV) infection dis-
23 cytokines previously described and the membranes tinguish viral clearance from persistent infection. Representative dot
were incubated at RT for 2 h. The membranes were incu- blots of the study groups are shown (A: chronically HCV-infected pa-
tients with detectable viral RNA titres; B: patients who spontaneously
bated with 2 mL of horseradish peroxidase-conjugated cleared HCV; C: an array map). The dot blot assay was used according
streptavidin at RT for 1 h, which was developed using to the manufacturers’ instructions to detect the cytokine levels in serum
an enhanced chemiluminescence-type solution subse- samples collected from patients (Ray-Biotech). For each membrane, the
quently exposed to film and processed by autoradiogra- individual background levels were subtracted. Individual densitometry
phy. The densitometry analysis was performed with an analyses and averages for each group are shown [D: interleukin-1 alpha
Alpha-Innotech FluorChem Imaging System. For each (IL-1α); E: tumour necrosis factor-alpha (TNF-α); F: tumour growth
membrane, the individual background value obtained factor-beta (TGF-β); G: IL-10; H: IL-8; I: monocyte chemoattractant
protein (MCP)-2; J: regulated upon activation, normal T-cell expressed
from the densitometry values for the blank controls in- and secreted (RANTES); K: IL-13; L: IL-15]. Chronically HCV-infect-
cluded in each assay (A in Figure) were subtracted from ed patients were defined as patients with positive anti-HCV test results
the densitometry values corresponding to each cytokine. and detectable HCV genotype 1a viral RNA titres (HCV+) and patients
The densitometry units corresponding to the relative ex- who spontaneously cleared HCV presented positive anti-HCV test re-
pression levels of each cytokine are shown. sults and undetectable HCV RNA titres (HCV-). Differences with p <
0.05 were considered statistically significant (*: p < 0.05; **: p < 0.001).
Ethics - Blood from patients and controls was ob- DU: densitometry units (refer to the relative expression levels of de-
tained by venipuncture with approval from the local eth- tected cytokines); IFN-γ: interferon-gamma; MIG: monokine-induced
ical committee of the HCFAA (IRB: HCG/CI-883/09). by IFN-γ; TNF-β: tumour necrosis factor-beta.
 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 110(2), April 2015 269

TABLE
Clinical and demographic characteristics of the patients

HCV+ HCV-
Characteristics (n = 16) (n = 17) p

Gender [female (%)] 68.75 76.4 -

Mean age (years ± SD) 53.13 ± 10.3 49.12 ± 12.83 0.332
Mean ALT (UI/L ± SD) 64.43 ± 41.14 29.8 ± 15.07 0.005
Mean AST (UI/L ± SD) 61.86 ± 42.45 29.8 ± 16.74 0.012
Positive anti-HCV 16 17 -
Viral genotype 1a Undetectable -
Mean viral titre (UI/L ± SD) 1.064 x 10^7 ± 1.56 x 10^7 Undetectable -

Lipid profile (mg/dL)

COL 170.2 ± 52.67 177.3 ± 59.42 0.724
TG 157.3 ± 76.96 148.6 ± 63.4 0.728
HDL 38.27 ± 9.74 40.29 ± 7.6 0.514
LDL 100.5 ± 50.78 119.9 ± 34 0.210
VLDL 31.4 ± 15.28 30.29 ± 13 0.826

Body mass index (kg/m 2) 22.94 ± 1.38 22.9 ± 1.26 0.925

Grade of fibrosis (n)
F1 1 - -
F1-F2 1 - -
F2 1 - -
F2-F3 2 - -
F4 1 - -

ALT: alanine aminotransferase; AST: aspartate aminotransferase; COL: cholesterol; HCV-: patients who exhibited positive anti-
hepatitis C virus test results and undetectable HCV RNA titres for more than six months. Averages of quantitative variables are
expressed as mean ± standard deviation (SD); HCV+: chronically HCV-infected patients who exhibited positive anti-HCV test
results and detectable HCV genotype 1a viral RNA titres for more than six months; HDL: high density lipoprotein; LDL: low
density lipoprotein; TG: triglyceride; VLDL: very low density lipoprotein.

U test was used to calculate the statistical significance of In an attempt to rule out the influence of overweight
the assay results. A p value ≤ 0.05 was considered sta- and obesity on the clearance or evolution of chronic HCV
tistically significant. Significant p values were corrected infection, this study only included samples from patients
using the Bonferroni method to ensure that there were with a normal body mass index. No differences in the
differences between the compared groups. lipid profiles of the analysed groups were found (Table).
Representative dot blots showing the relative expression
RESULTS
of cytokines in the study groups are shown in A, B in Fig-
Based on HCV antibody and RNA viral profiles, ure. A low detection rate of certain cytokines, including
patients were stratified into two groups: patients who IL-2, IL-6, IL-5, IFN-γ, IL-7, TNF-β, MCP-1 (CCL-2),
spontaneously cleared HCV and patients with chronic MCP-3 (CCL-7), MIG (CCL-9), GRO-α, GRO, IL-3,
HCV infection who continued to have detectable RNA GMCSF and GCSF, was observed in our study groups
titres. There were no significant gender or age differ- (A-C in Figure). Interestingly, compared with chronic
ences found between the analysed groups. However, HCV-infected patients with detectable RNA, the patients
the patients with chronic HCV infection and detectable who spontaneously cleared HCV displayed increased lev-
viral RNA presented significantly increased ALT and els of IL-1α, TNF-α, TGF-β, MCP-2 (CCL-8), IL-13 and
AST levels compared with the patients who had sponta- IL-15 (D-F, I, K, L in Figure, respectively). In contrast,
neously cleared HCV. Genotype 1a was found in all of the chronic HCV-infected patients with detectable RNA
the chronically infected patients with detectable RNA. showed increased levels of IL-8 and RANTES (CCL-5)
Fibrosis staging determined by elastography was con- compared with patients who spontaneously cleared HCV
ducted in a small number of participants (6) from the (H, J in Figure, respectively). No significant differences
chronic HCV group. Patients with distinct stages of liver were found in IL-10 levels. However, there was a trend
disease were included in the study and cirrhosis (F4) was towards increased IL-10 levels in the chronic HCV-in-
found exclusively in one of these patients (Table). fected patients with detectable RNA (G in Figure).
270 Cytokines and HCV • Nora A Fierro et al.

Viral and host factors are related to the progression B virus infection in native Mexican groups (Fierro et al.
of HCV infection. In 2009, it was demonstrated that 2011). Herein, our findings suggest that cytokine expres-
HCV viral clearance is associated with genetic varia- sion can influence the extent of HCV development and
tion in the IL-28B gene (Ge et al. 2009, Thomas et al. provide important insights into cytokine-mediated mech-
2009, Rauch et al. 2010). Numerous studies have been anisms underlying the long-term persistence of HCV. In
conducted on this family of cytokines and have led to this context, efforts to determine whether biomarkers, in-
several inconsistencies and controversies, including the cluding serum cytokines, can accurately predict the out-
possible correlation between serum protein levels and come of HCV infection are valuable for establishing bet-
disease outcomes in chronic viral hepatitis patients (Tor- ter HCV control strategies in Latin American regions.
res et al. 2014). Thus, it is accepted that variations in the ACKNOWLEDGEMENTS
profile of cytokines involved in the immune response
may contribute to the ability to clear HCV. These studies To Flor P Castro and Jesus Meza, for the technical assistance.
have mainly been conducted in regions where the virus REFERENCES
is endemic (Shi et al. 2012), thus information on popu-
lations in regions of low endemicity is scarce. Herein, Alvarado-Mora MV, Pinho JR 2013. Epidemiological update of hepa-
titis B, C and delta in Latin America. Antivir Ther 18: 429-433.
we characterised serum cytokine expression profiles in
Mexican HCV-infected patients and found subtle differ- Castro-Garcia FP, Corral-Jara KF, Escobedo-Meléndez G, Sandoval-
ences in the cytokine profiles between distinct clinical Hernandez MA, Rosenstein Y, Roman S, Panduro A, Fierro NA
courses of HCV infection. In our study, persistent infec- 2014. Conjugated bilirubin adjusts cytokine profiles in hepatitis
tion with detectable genotype 1a viral content resulted A virus infection by modulating function of signal transducer
and activator of transcription factors. Immunology 143: 578-587.
in a proinflammatory profile characterised by increased
levels of IL-8 and RANTES (CCL-5) and a trend towards EASL - European Association for Study of Liver 2014. EASL Clinical
increased IL-10 levels. These findings correlate with in Practice Guidelines: management of hepatitis C virus infection.
vitro studies that have shown that natural killer cells J Hepatol 60: 392-420.
from HCV-infected patients are impaired in their capac- Escobedo-Meléndez G, Fierro NA, Roman S, Maldonado-González
ity to activate dendritic cells due to the overproduction M, Zepeda-Carrillo E, Panduro A 2012. Prevalence of hepatitis
of IL-10 (Jinushi et al. 2004). Likewise, the increased A, B and C serological markers in children from western Mexico.
levels of IL-8 found in the chronic HCV group in the Ann Hepatol 2: 194-201.
present study may be related to the association between Fierro NA, Escobedo-Meléndez G, de Paz L, Realpe M, Roman S,
IL-8 production and IFN-α inhibition described in both Panduro A 2012. Cytokine expression profiles associated with
in vivo and in vitro studies (Khabar et al. 1997, Jia et al. distinct clinical courses in hepatitis A virus-infected children.
2007). Moreover, the analysis of cytokines in each pa- Pediatr Infect Dis J 31: 870-871.
tient revealed a trend towards higher serum concentra-
Fierro NA, Gonzalez-Aldaco K, Torres-Valadez R, Martinez-Lopez
tions of IL-10 and IL-8 in patients with more severe liver E, Roman S, Panduro A 2014. Immunologic, metabolic and ge-
damage (F2-F3 and F4) (data not shown). Altogether, netic factors in hepatitis C virus infection. World J Gastroenterol
our findings suggest that IL-10 and IL-8 overproduc- 20: 3443-3456.
tion may contribute to a lower probability of viral clear-
ance, whereas homeostasis between pro-inflammatory Fierro NA, Roman S, Realpe M, Hernandez-Nazara Z, Zepeda-Carrillo
EA, Panduro A 2011. Multiple cytokine expression profiles reveal
[IL-1 α, TNF-α, MCP-2 (CCL-8)], anti-inflammatory immune-based differences in occult hepatitis B genotype H-infected
(TGF-β), fibrogenic (IL-13) and immunoregulatory (IL- Mexican Nahua patients. Mem Inst Oswaldo Cruz 106: 1007-1013.
15) profiles allows for viral clearance, as observed in
patients with undetectable viral RNA levels. However, Ge D, Fellay J, Thompson AJ, Simon JS, Shianna KV, Urban TJ, Hein-
large-scale studies with adequate statistical power to zen EL, Qiu P, Bertelsen AH, Muir AJ, Sulkowski M, McHutchison
JG, Goldstein DB 2009. Genetic variation in IL-28B predicts hepa-
support this hypothesis are needed. Furthermore, given titis C treatment-induced viral clearance. Nature 461: 399-401.
that our data do not rule out the possibility that cytokine
levels may be a consequence and not the cause of HCV Hanafiah KM, Groeger J, Flaxman AD, Wiersma ST 2013. Global epi-
clearance, future studies aimed at analysing the possibil- demiology of hepatitis C virus infection: new estimates of age-spe-
ity of a reverse causation are necessary. cific antibody to HCV seroprevalence. Hepatology 4: 1333-1342.
The progression of HCV infection is associated with Jia Y, Wei L, Jiang D, Wang J, Cong X, Fei R 2007. Antiviral action of
the characteristics of lipid profiles in chronic HCV pa- interferon-alpha against hepatitis C virus replicon and its modu-
tients. In fact, it is accepted that lipid components as- lation by interferon-gamma and interleukin-8. J Gastroenterol
sociated with HCV infection are finely modulated in the Hepatol 22: 1278-1285.
Mexican population (Fierro et al. 2014). In the present Jinushi M, Takehara T, Tatsumi T, Kanto T, Miyagi T, Suzuki T, Ka-
study, no association between lipid profiles and viral nazawa Y, Hiramatsu N, Hayashi N 2004. Negative regulation of
clearance was found. Future studies that include over- NK cell activities by inhibitory receptor CD94/NKG2A leads to
weight and obese individuals will allow the determina- altered NK cell-induced modulation of dendritic cell functions in
tion of the exact role of lipids in viral clearance. chronic hepatitis C virus infection. J Immunol 173: 6072-6081.
We previously reported the influence of cytokines on Khabar KS, Al-Zoghaibi F, Al-Ahdal MN, Murayama T, Dhalla M,
the development of distinct clinical courses in children Mukaida N, Taha M, Al-Sedairy ST, Siddiqui Y, Kessie G, Mat-
infected with hepatitis A (Fierro et al. 2012, Castro-Gar- sushima K 1997. The alpha chemokine, interleukin 8, inhibits the
cia et al. 2014) and on the development of occult hepatitis antiviral action of interferon alpha. J Exp Med 1867: 1077-1085.
 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 110(2), April 2015 271

Panduro A, Escobedo-Meléndez G, Fierro NA, Ruiz-Madrigal B, Shi X, Pan Y, Wang M, Wang D, Li W, Jiang T, Zhang P, Chi X, Jiang
Zepeda-Carrillo EA, Román S 2011. Epidemiology of viral hepa- Y, Gao Y, Zhong J, Sun B, Xu D, Jiang J, Niu J 2012. IL28B ge-
titis in Mexico. Salud Publica Mexico 53: 37-45. netic variation is associated with spontaneous clearance of hepa-
titis C virus, treatment response, serum IL-28B levels in Chinese
Rauch A, Kutalik Z, Descombes P, Cai T, Di Iulio J, Mueller T, Bochud population. PLoS ONE 7: e37054.
M, Battegay M, Bernasconi E, Borovicka J, Colombo S, Cerny A,
Dufour JF, Furrer H, Günthard HF, Heim M, Hirschel B, Malinverni Thomas DL, Thio CL, Martin MP, Qi Y, Ge D, O’Huigin C, Kidd J,
R, Moradpour D, Müllhaupt B, Witteck A, Beckmann JS, Berg T, Khakoo SI, Alexander G, Goedert JJ, Kirk GD, Donfield SM,
Bergmann S, Negro F, Telenti A, Bochud PY, Swiss Hepatitis C Rosen HR, Tobler LH, Busch MP, McHutchison JG, Goldstein
Cohort Study, Swiss HIV Cohort Study 2010. Genetic variation in DB, Carrington M 2009. Genetic variation in IL28B and sponta-
IL28B is associated with chronic hepatitis C and treatment failure: a neous clearance of hepatitis C virus. Nature 461: 798-801.
genome-wide association study. Gastroenterology 138: 1338-1345.
Torres C, Brahm J, Venegas M 2014. Lambda interferon serum levels
Rehermann B, Bertoletti A 2015. Immunological aspects of antiviral in patients with chronic hepatitis C virus infection according to
therapy of chronic hepatitis B virus and hepatitis C virus infec- their response to therapy with pegylated interferon and ribavirin.
tions. Hepatology 61: 712-721. J Interferon Cytokine Res 34: 106-110.