Immunology Letters 161 (2014) 89–95

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Immunology Letters
journal homepage: www.elsevier.com/locate/immlet

Dynamics of interleukin-21 production during the clinical course of

primary and secondary dengue virus infections
H. Vivanco-Cid a,c,∗,1 , M.J. Maldonado-Rentería a,1 , L.A. Sánchez-Vargas a ,
I.Y. Izaguirre-Hernández a , K.G. Hernández-Flores a , R. Remes-Ruiz b
a
Instituto de Investigaciones Medico-Biológicas, Universidad Veracruzana, Veracruz, México
b
Hospital Regional de Alta Especialidad de Veracruz, Servicios de Salud de Veracruz, México
c
Universidad del Valle de México, campus Villa Rica, Facultad de Medicina “Dr. Porfirio Sosa Zárate”, México

a r t i c l e i n f o a b s t r a c t

Article history: Previous studies have revealed the clinical relevance of pro-inflammatory cytokine production during
Received 29 December 2013 dengue virus (DENV) infections. In this study, we evaluated the production of interleukin-21 (IL-21), a
Received in revised form 22 April 2014 key soluble mediator mainly produced by CD4+ T cells.
Accepted 7 May 2014
The aim of this study was to investigate the role of IL-21 production during the clinical course of
Available online 22 May 2014
primary and secondary DENV infections and the potential association of IL-21 serum levels with the
disease pathogenesis. Blood samples from DENV-infected patients were collected on different days after
Keywords:
the onset of symptoms. Patients were classified according to their phase of disease (acute vs. convalescent
Dengue
Interleukin 21
phases), the type of infection (primary vs. secondary), and the clinical severity of their disease (dengue
Antibody response fever (DF) vs. dengue hemorrhagic fever (DHF)). IL-21 levels were measured using a quantitative capture
Cellular immunity ELISA assay. The levels of IL-21 were significantly elevated in the disease group compared with the
control group. IL-21 was detected in primary and secondary DENV infections, with a significantly higher
concentration in the convalescent phase of primary infections. IL-21 levels were significantly higher
in patients with secondary acute DHF infections when compared with those with secondary acute DF
infection. There was a relationship between the elevated serum levels of IL-21 and the production of
DENV-specific IgM and IgG antibodies. Taking together, our results show for the first time the involvement
of IL-21 during the clinical course of DENV infections.
We speculate that IL-21 may play a protective role in the context of the convalescent phase of primary
infections and the acute phase of secondary infections.
© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction
Dengue fever is the most common transmitted arboviral disease
worldwide. The infection is caused by one of the four serotypes
of dengue virus, the etiologic agents, all of which are members of
the Flavivirus group into the family Flaviviridae [1]. The pathophy-
siology of DF/DHF/DSS in humans is complex and multi-factorial,
involving the interplay of host and viral factors that influence dis-
ease severity and the clinical outcome [2]. With respect to host
factors, the immune response to DENV plays a dual role in terms
∗ Corresponding author at: Laboratorio Multidisciplinario en Ciencias Biomédicas,
Instituto de Investigaciones Medico-Biológicas, Universidad Veracruzana, Avenida
Iturbide S/N, Col. Centro, C.P. 91700 Veracruz, México. Tel.: +52 2299318011x120;
fax: +52 2299318011.
E-mail addresses: hvivanco@uv.mx, vivacid@hotmail.com (H. Vivanco-Cid).
1
These authors contributed equally to this work.
of both protection and the pathogenesis of the severe forms of this
viral disease. A cascade of pro-inflammatory cytokines is released
during acute dengue infections, including cytokines from innate
immunity cell sources, such as TNF-␣, IL-1, IL-6, and IFN-␣, and
chemokines, such as MIP1-␣, IP-10 and IL-8. Pro-inflammatory
cytokines are implicated in host protection roles such as activat-
ing innate and adaptive immune cells, chemotaxis and inhibition of
viral replication. Some detrimental roles for the high inflammatory
cytokine levels that are often observed in severe disease include the
induction of cell death (apoptosis and necrosis), endothelial activa-
tion, vascular leak and shock [3–7]. Cytokines produced by adaptive
immune cells, such as CD4+ and CD8+ T cells, also play a major role
with respect to viral spread and further support the development
of effector functions, including humoral, cell-specific and memory
immune responses. During the CD4+ T cell response to DENV, Th1
and Th2 cells participate in the viral disease with the production
of IFN-␥, TNF-␤, and IL-2 or IL-4, IL-5, IL-6 and IL-10, respectively
[8]. A shift from the Th1 to Th2 response has been suggested in the

http://dx.doi.org/10.1016/j.imlet.2014.05.006
0165-2478/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
90 H. Vivanco-Cid et al. / Immunology Letters 161 (2014) 89–95

clinical pathogenesis of DHF on the basis of in vitro studies and the
ex vivo analysis of the cytokine patterns in the serum samples of
patients with severe disease [5,8–10].
In this study, we evaluated the interleukin-21 (IL-21) produc-
tion in sera from DENV infected patients, a key pro-inflammatory
and soluble mediator mainly produced by CD4+ T cells. To date,
IL-21 has not previously been studied during the clinical course
of DENV infections. IL-21 is a cytokine member of the common ␥-
chain family, which includes IL-2, IL-4, IL-7, IL-9 and IL-15 [11].
IL-21 is produced by various subsets of activated CD4+ T cells,
which include Th17 cells, T-follicular helper (Tfh) and CD4+ natu-
ral killer (NK) T cells, and exerts multiple and pleiotropic effects on
various innate and adaptive immune cells, such as NK cells, mono-
cytes, macrophages, B cells, and CD4+ and CD8+ T lymphocytes,
as well as on non-immune cells such as keratinocytes [12], fibro-
blasts [13,14] and vascular endothelial cells [15]. The aim of this
work was to investigate whether the serum levels of IL-21 are ele-
vated in dengue-infected patients, analyzing the production during
primary and secondary infections in comparison with healthy indi-
viduals and the potential association of IL-21 serum levels with the
disease pathogenesis.
2. Materials and methods
2.1. Subjects
of the Instituto de Investigaciones Medico-Biológicas, Universidad
Veracruzana (Veracruz, México). The study protocol was approved
by the institutional ethics review board.
2.3. Dengue diagnosis
Serum samples were tested and diagnosed by reverse
transcription-polymerase chain reaction (RT-PCR) and three refer-
ence anti-dengue enzyme-linked immunosorbent assays (ELISA):
Platelia Dengue NS1 Ag Test (BIORAD Laboratories, France), Dengue
IgM (Panbio, Australia) and, to discriminate among primary and
secondary infections, a commercial kit (capture IgG ELISA, Pan-
bio) that is used for the quantitative detection of elevated titers of
IgG antibodies to dengue virus in patients with secondary infection
according to previously established criteria [16].
2.4. Classification of samples
Based on the time of clinical illness, we classified dengue
patients into acute (1–7 days after disease onset) and convales-
cent (samples collected on days 8–10 after disease onset) phases.
Primary dengue infection was defined for patients with positive
results for RT-PCR, NS1 and/or IgM tests and negative IgG ELISA
results. Secondary dengue infection was defined for IgG ELISA pos-
itive results (equivalent to a hemagglutination inhibition titer of
>2560) regardless of RT-PCR, NS1 and IgM results.

A clinical cohort of patients with suspected cases of dengue

2.5. Measurement of serum IL-21 levels
were recruited in the city of Veracruz México during outbreaks that
occurred from June 2010 through November 2012. A total of 360
The IL-21 concentrations in the sera from dengue-positive
febrile patients were laboratory confirmed for dengue infection and
patients and healthy controls were measured by sandwich ELISA
enrolled in our study. Blood samples from patients were collected
kit from PeproTech Inc. (Rocky Hill, NJ) according to the manufac-
in three urban health centers (“Anastasio Iturralde”, “21 de Abril”,
turer’s instructions. The optical density at 405 nm with a reference
and “El Coyol”), Hospital Regional de Alta Especialidad de Veracruz
filter at 650 nm was measured by using a microplate reader (Aware-
and Hospital Naval de Veracruz. Dengue patients had not received
ness Technology Inc., Palm City, FL, USA).
any drug or treatments before the sample collection. Blood samples
were tested and diagnosed by reverse transcription-polymerase
2.6. Measurement of serum DENV-specific IgM and IgG levels
chain reaction (RT-PCR) and ELISA techniques to confirm the cases.
To rule out other viral or bacterial infections, further serological
The serum samples from dengue-infected patients were tested
tests for leptospirosis, influenza A, hepatitis A and Salmonella typhi
for DENV IgM and IgG antibodies by ELISA at a 1:100 dilution.
were carried out at the clinical admission time. Patients were classi-
Polysorb 96-well microplates (Nalge Nunc International, Rochester,
fied as having dengue fever or dengue hemorrhagic fever according
NY) coated with recombinant antigens provided in the Dengue IgM
to clinical and laboratory criteria of the World Health Organiza-
kit and the commercial kit capture IgG ELISA (Panbio, Australia)
tion (WHO 1997). Clinical case definition of dengue fever included
were used to measure the DENV specific antibody levels. The serum
an acute febrile illness with two or more of the following mani-
samples were incubated for 1 h at 37 ◦ C. After incubation, serum
festations: headache, retro-orbital pain, arthralgia, myalgia, rash,
samples were removed, and horseradish peroxidase-conjugated
leukopenia and supportive molecular or serology diagnosis. Clin-
monoclonal anti-human IgM or IgG were added for 1 h at 37 ◦ C, fol-
ical case definition of dengue hemorrhagic fever included all of
lowed by the addition of 3,3 ,5 -tetramethyl benzidine chromogen
the above criteria for DF plus evidence of hemorrhagic tenden-
solution. One hundred microliters of Stop Solution was added to
cies (epistaxis, gingival bleeding, petechiae, a positive tourniquet
each well plate. The antibodies levels of each serum samples were
test). Vascular leakages were documented by chest X-ray and esti-
represented as an index value, according to the manufacturer’s
mating hypovolemia from multiple hematocrit determinations.
instructions. The OD results were corrected by subtracting the
Other clinical and laboratory findings included thrombocytopenia
OD values of blank samples which are included in every test kit.
(<100,000 counts/mm3 ), hypotension and hypoproteinemia. No
The ratio with a standard positive sample, the cut-off calibrator, is
fatal cases were included in the present study.
expressed as an index value (IV). Index value is calculated dividing
Seventy-eight serum samples from healthy individuals without
the optical density of the sample by the cutoff value.
clinical signs or manifestations suggestive of dengue (no febrile
symptoms) or other apparent illnesses in the previous 3 months
2.7. Statistical analysis
were included as controls. All serum samples were frozen at −70 ◦ C
until analysis.
The data were represented as the means ± SD or as medians.
The Chi square test was used to compare categorical variables.
2.2. Ethics statement The non-parametric Kruskal–Wallis test was used to comparison
more than two groups. Comparisons among each patient group
Written consent to participate in the study was obtained from were performed using Dunn’s multiple comparison tests. Statis-
each participant after a full explanation of the study was provided tical significance between two individual groups was determined
and in accordance with the guidelines of the Ethics Committee with the Mann–Whitney U test using GraphPad Prism software V
 H. Vivanco-Cid et al. / Immunology Letters 161 (2014) 89–95
Table 1
Classification of dengue patients according to phase and severity disease.
Phase Dengue fever
(n = 235)
Primary
n (%)
Acute 71 (75.5%)
Convalescent 23 (24.5%)
Total (n) 94
5.0. P values <0.05 were considered statistically significant. Correla-
tion between IL-21 and Dengue IgM and IgG levels among different
groups was evaluated using Spearman’s correlation test.
3. Results
3.1. Patient characteristics
The mean age of dengue patients was 29.9 years-old (range:
5–74 years-old). The gender ratio was 1:1.6 (male: female). From
the 360 dengue-positive patients, 235 (65.28%) and 125 (34.72%)
were classified as having DF and DHF, respectively, accordingly
to the World Health Organization clinical criteria (WHO 1997)
described before. DHF Patients were clinically managed in the
hospital for a median of 4 days. All DHF patients had laboratory
evidence of a rising hematocrit (evidence of plasma leakage) and
thrombocytopenia (<100,000 counts/mm3 ). 122 patients were con-
firmed to have primary dengue infection, and 238 were confirmed
to have secondary dengue infections, following the laboratory crite-
ria described in the materials and methods section. Based on the
time of clinical illness, a total of 240 patients were classified in the
acute phase (days 1–7 after disease onset) and 120 patients were
classified in the convalescent phase (days 8–10 after disease onset)
(Table 1).
3.2. The serum levels of IL-21 are increased in dengue-infected
patients compared with healthy controls
To investigate whether IL-21 is produced during DENV infection,
the serum levels of IL-21 were measured by enzyme immunoassay.
In a first analysis, we observed very low serum levels of IL-21 in the
healthy control group (mean 14.5 ± 59.4 pg/ml, median 1.6 pg/ml)
and a higher and heterogeneous production of IL-21 in dengue-
infected patients (mean 147.2 ± 200.4 pg/ml, median 83.37 pg/ml),
P < 0.0001, Fig. 1A.
Fig. 1. The serum levels of IL-21 are elevated in dengue-infected patients. (A) IL-21 concentrations in the sera of healthy controls (HC) (n = 78) and dengue-infected patients
(DEN) (n = 360). (B) Comparison of the serum levels of IL-21 in acute (n = 91) and convalescent phases (n = 31) of primary dengue infections vs. acute (n = 149) and convalescent
phases (n = 89) of secondary dengue infections. Statistical confidence was analyzed in figure A by Mann–Whitney U test. The Kruskall–Wallis test was used for statistical
analysis in figure B. The IL-21 levels are represent in pg/ml. **P < 0.01, ***P < 0.001, ****P < 0.0001. Abbreviations: AP (acute primary), CP (convalescent primary), AS (acute
secondary), CS (convalescent secondary).
91
Dengue hemorrhagic fever
(n = 125)
Secondary Primary Secondary
n (%) n (%) n (%)
100 (70.9%) 20 (71.4%) 49 (50.5%)
41 (29.1%) 8 (28.6%) 48 (49.5%)
141 28 97
3.3. The highest serum levels of IL-21 are produced during the
convalescent phase of primary infections
Next, the serum levels of IL-21 were analyzed after classi-
fying all dengue patients according to their clinical evolution
(acute and convalescent phases) and type of infection (primary
or secondary). Interestingly, we observed that IL-21 was signifi-
cantly elevated during the convalescent phase of primary infections
(mean 230.5 ± 225.6 pg/ml, median 167.4 pg/ml) compared with
acute primary infections (mean 108.8 ± 152.2 pg/ml, median
74.56 pg/ml), acute secondary infections (mean 160 ± 226.9 pg/ml,
median 72.58 pg/ml), and convalescent secondary infections (mean
136.1 ± 178.3 pg/ml, median 86.4 pg/ml), Fig. 1B.
3.4. The stratification of dengue-infected patients according to
the IL-21 concentrations, clinical phase and type of infection
Given the previous findings about the heterogeneity in the con-
centrations of IL-21 detected among patients, we were interested
in determining how the clinical stage of infection can influence the
production pattern for this cytokine. Thus, we stratified all of the
patients into three categories based on their IL-21 serum concen-
trations. Patients were classified in tertiles as low, medium, and
high IL-21 producers. The cutoff values for each category were set
considering the mean value for the healthy controls group plus one
or two standard deviations. Concentrations below the first cutoff
(≤73.9 pg/ml) were classified as low IL-21 producers. Concentra-
tions between the first and second cutoff (74–133.3 pg/ml) were
classified as medium IL-21 producers, and concentrations above the
second cutoff (≥133.4 pg/ml) were classified as high IL-21 produc-
ers. We related these categories with the type of infection (primary
or secondary) and the course of the disease (acute or convalescent
phases). A high percent (61.3%) of patients with primary infections
in the convalescent phase produced the highest levels of IL-21. High
concentrations of IL-21 were also detected in 40.3% of patients with
secondary infections in the acute phase. In contrast, 1.3% of the
92 H. Vivanco-Cid et al. / Immunology Letters 161 (2014) 89–95

Table 2
Stratified analysis of IL-21 levels in dengue-infected patients, according to the clinical stage and the type of infection.

IL-21 production AP CP AS CS HC P value

n (%) n (%) n (%) n (%) n (%)

Low 45 (49.5%) 7 (22.6%) 75 (50.3%) 40 (44.9%) 76 (97.4%) <0.0001

Medium 20 (22.0%) 5 (16.1%) 14 (9.4%) 19 (21.4%) 1 (1.3%) 0.0001
High 26 (28.5%) 19 (61.3%) 60 (40.3%) 30 (33.7%) 1 (1.3%) <0.0001

Total (n) 91 31 149 89 78

Low 0–73.9 pg/ml, medium 74–133.3 pg/ml, high >133.3 pg/ml.

AP = acute primary, CP = convalescent primary, AS = acute secondary, CS = convalescent secondary, HC = healthy controls.

healthy controls were classified as high IL-21 producers, whereas

97.4% of the control group was classified as low IL-21 producers
(Table 2).

3.5. The serum levels of IL-21 are associated with the severity of
dengue infection during the acute phase of secondary infections

To evaluate whether there was a relationship among the het-

erogeneity in the serum concentrations of IL-21 and the severity
of disease, we grouped the patients into two groups based on their
clinical classification as DF or DHF. There was a trend toward higher
IL-21 serum levels in DHF patients with respect to DF patients.
However, in this first analysis, there was no statistically signifi-
cant difference between the groups (P > 0.05, Fig. 2A). Next, we
decided to analyze the sample in more detail by comparing among
the same clinical stages. Because almost 80% of the DHF cases
recruited in our study correspond to secondary infections, we com-
pared the serum levels of IL-21 considering just the cases of DF
and DHF classified within the secondary infection group (Fig. 2B
and C). As shown in Fig. 2B, IL-21 was significantly elevated in the
serum of DHF patients during the acute phase of secondary infec-
tions (mean 165 ± 259.1 pg/ml, median 131.8 pg/ml) compared
with DF patients in the acute phase of secondary infections (mean
149.6 ± 141.6 pg/ml, median 62.84 pg/ml; P = 0.02).

3.6. The stratification of dengue-infected patients according to

the IL-21 concentration, clinical phase and severity of disease

We performed the same analysis as previously described in

Table 2 to determine the distribution of DF or DHF patients stratified
by their IL-21 serum concentrations (Table 3). Interestingly, 49.0%
of DHF patients with secondary infections in the acute phase were
classified as high IL-21 producer, 16.3% were classified as medium
producers and 34.7% were classified as low IL-21 producers. In con-
trast, 36% of DF patients with secondary infections in the acute
phase were classified as high IL-21 producers, 6% were classified
as medium producers and 58% were classified as low IL-21 pro-
ducers. When we compared the convalescence phases of DF and
DHF, we observed a similar distribution of the patients indepen-
dent of the severity of the disease. A remarkable result was the
high percentage of DF patients (60.9%) and DHF patients (62.5%) in
the convalescent phase of primary infections who were classified
as high IL-21 producers. Fig. 2. The serum levels of IL-21 and clinical disease severity. (A) IL-21 levels in
patients with dengue fever (DF) (n = 235), dengue hemorrhagic fever (DHF) (n = 125)
and healthy controls (HC) (n = 78). (B) Comparison of IL-21 levels in patients with
3.7. The serum IL-21 levels in dengue patients correlate with
secondary acute DF infections (n = 100) and secondary acute DHF infections (n = 49).
DENV-specific IgM and IgG levels (C) Comparison of IL-21 levels in patients with secondary convalescent DF infections
(n = 41) and secondary convalescent DHF infections (n = 48). Statistical confidence
Finally, we identified possible correlations between the serum was analyzed in figure A by Kruskall–Wallis test. Mann–Whitney U test was used
IL-21 levels and the humoral response raised against DENV. The for statistical analysis in figures B and C. The IL-21 levels are represent in pg/ml.
*P < 0.05, ****P < 0.0001.
strength of association between the serum IL-21 levels and the
anti-Dengue IgM or IgG levels were examined using the Spear- patients in the acute phase of primary infections, days 1–7 showed
man’s correlation test. Fig. 3 shows the scatter plots for the levels undetectable or low levels of DEN-specific IgM antibodies (IgM
of anti-dengue IgM and IgG against the serum IL-21 levels in ratio, 2.052 ± 2.641) with no significant correlation values with
primary and secondary dengue infections, respectively. Dengue the serum IL-21 levels (Fig. 3A). The levels of dengue-specific IgM
 H. Vivanco-Cid et al. / Immunology Letters 161 (2014) 89–95 93

Table 3
Stratified analysis of IL-21 levels in dengue fever and dengue hemorrhagic fever patients, according to the clinical stage and the type of infection.

IL-21 production AP CP AS CS HC P value

n (%) n (%) n (%) n (%) n (%)

Dengue fever patients

Low 36 (50.7%) 5 (21.7%) 58 (58.0%) 19 (46.3%) 76 (97.4%) <0.0001
Medium 13 (18.3%) 4 (17.4%) 6 (6.0%) 8 (19.5%) 1 (1.3%) 0.002
High 22 (31.0%) 14 (60.9%) 36 (36.0%) 14 (34.2%) 1 (1.3%) <0.0001

Total (n) 71 23 100 41 78

Dengue hemorrhagic fever patients

Low 9 (45.0%) 2 (25%) 17 (34.7%) 21 (43.8%) 76 (97.4%) <0.0001
Medium 7 (35.0%) 1 (12.5%) 8 (16.3%) 11 (22.9%) 1 (1.3%) 0.003
High 4 (20%) 5 (62.5%) 24 (49.0%) 16 (33.3%) 1 (1.3%) <0.0001

Total (n) 20 8 49 48 78

Low 0–73.9 pg/ml, medium 74–133.3 pg/ml, high >133.3 pg/ml.

AP = acute primary, CP = convalescent primary, AS = acute secondary, CS = convalescent secondary, HC = healthy controls.

Fig. 3. Correlation between the Dengue-specific IgM antibodies levels and the IL-21 serum levels in the acute (A) or convalescent phases (B) of primary dengue infections.
The serum levels of IL-21 were significantly and positively correlated with the increase of anti-dengue IgM levels (r = 0.4260, P < 0.0189), just in the convalescent phase of
primary infections. Correlation between the Dengue-specific IgG antibodies levels and the IL-21 serum levels in the acute (C) or convalescent phases (D) of secondary dengue
infections. A significant and positive correlation between the serum levels of IL-21 and the increase of anti-dengue IgG levels was found during the acute phase (r = 0.2928,
P < 0.0097). Each dot represents one subject.

antibodies increase in the convalescent phase of primary infec-
tions, on days 8–10 to a mean ratio of 4.598 ± 2.599. The serum
levels of IL-21 were significantly and positively correlated with the
increase of anti-dengue IgM levels (r = 0.4260, P < 0.0189) in the
convalescent phase of primary infections (Fig. 3B). For secondary
infections we found a significant and positive correlation between
the serum levels of IL-21 and the increase of anti-dengue IgG lev-
els (IgG ratio, 5.818 ± 1.881) during the acute phase (r = 0.2928,
P < 0.0097) (Fig. 3C). However, no significant correlation value was
found between dengue-IgG levels and the serum levels of IL-21
during the convalescent phase of secondary infections (r = 0.2110,
P < 0.1026) (Fig. 3D).
4. Discussion
Cytokine levels differ dramatically from steady state conditions
in healthy controls compared with patients exhibiting active
disease. In dengue infections, there is a highly heterogeneous host
immune response to the viral infection depending of the severity of
the disease. Here, we present for the first time evidence of IL-21 pro-
duction during dengue infections. It is apparent from our findings
that the levels of IL-21 increase significantly during DENV infection
compared with the levels in healthy individuals. In our study, the
highest IL-21 concentrations were measured in patients during
the convalescence phase of primary infections. This particular pro-
duction pattern of IL-21 was demonstrated for both clinical forms:
DF and DHF. A high percent of DF (60.9%) and DHF patients (62.5%)
in the convalescent phase of primary infections were classified as
the highest IL-21 producers. Interestingly, we found in the same
clinical stage that the serum IL-21 levels correlated well with the
DENV-specific IgM production. These observations are consistent
with the role IL-21 plays in plasma cell differentiation [17].
High IL-21 levels during the convalescent phase correlate well
with the time when the dengue-infected patients began to recover.
94 H. Vivanco-Cid et al. / Immunology Letters 161 (2014) 89–95
The recovery phase is characterized by improving general well
being and hemodynamic stability, which suggest a protective role
of this cytokine in this clinical stage of primary infections. Fur-
thermore, during the acute phase of secondary dengue infections,
a positive correlation between the serum IL-21 levels and DENV-
specific IgG levels was found. This association also is consistent with
the critical role IL-21 plays as a potent inducer of IgG production
[18–20] and promoting the activation of memory B cells by T cells
[17].
Because of the cellular-restricted origins of IL-21, this cytokine
could be considered a good potential biomarker of the protection
mediated by CD4+ T or NKT cells against dengue virus in pri-
mary infections. Interestingly, a recent study has identified that an
important proportion of DENV-specific CD4+ T cells isolated from
PBMCs from convalescent dengue virus patients displayed a pheno-
type characteristic of circulating follicular helper T cells (TFH cells),
suggesting that they are interacting with B cells in vivo [21]. A frac-
tion of DENV-specific CXCR5+ cells was capable of producing IL-21
or IFN-␥ following stimulation with DENV peptides. The evidence
provided by Rivino et al., and our own results, support the idea
that a fraction of DENV-specific CD4+ T cells could be involved in
supporting B cell antibody production through an IL-21 dependent
mechanism.
The biological effects and clinical relevance of the production
of IL-21 in primary dengue infections could be related to the
importance of IL-21 in the acute phase to enhance the cytotoxic
activity of NK cells and establish viral control early. In the con-
valescence phase, IL-21 could be very important to support the
humoral response through the proliferation and differentiation of
B cells into plasma cells, enhancing immunoglobulin production
and isotype class switch and promoting the activation of cellu-
lar responses mediated by CD8+ T cells and the development of
immunological memory. Clinical and basic research has estab-
lished the protective role of IL-21 in other viral diseases, including
HIV [22–25], HBV [26], herpes simplex virus [27], and respira-
tory syncytial virus [28]. Recently, a protective role for IL-21 has
been demonstrated during the course of the chronic Hepatitis C
infections in humans [29]. Hepatitis C virus is a member of the Fla-
viviridae family and is closely related to the dengue fever virus.
Although IL-21 has been mainly associated with protective roles
and sustaining T cell responses during chronic viral infections, little
is known about the role of this cytokine in the course of acute viral
infections such as dengue. The role of IL-21 during secondary infec-
tion by DENV is more intriguing than in primary infections. In our
study, IL-21 levels indeed were significantly higher in DHF patients
than in patients with DF in the acute phase of secondary infec-
tions. However, the higher levels of IL-21 in secondary infections
of DHF patients compared to DF patients probably are a reflec-
tion of the more intense immune activation in DHF rather than
a cause of the severity of disease. It is well known that secondary
infections with a distinct DENV serotype led to the activation of
serotype-cross-reactive T cells, resulting in elevated levels of adap-
tive immune response-derived cytokines such as IFN-␥, IL-2, IL-4,
IL-10 and TNF-␣, which have been reported to be more pronounced
in DHF compared to DF in some studies [30–32]. A potential detri-
mental role for IL-21 during the acute phase of DHF secondary
infections could be through the activation of different immune cell
populations, such as monocytes, macrophages, dendritic cells and
T cells. IL-21 has been reported to induce the production of many
pro-inflammatory cytokines, such as GM-CSF, IL-1, IL-2, IL-7, IL-15,
IFN-␥, and chemokines, such as IL-8, RANTES, MIP-1␣, Eotaxin, and
IP-10, by human NK cells [33,34], monocytes [35], macrophages
[36], dendritic cells [37] and T cells [38–40]. Although the IL-21
receptor is expressed on vascular endothelial cells, a direct effect
of this cytokine on vascular activation and vascular leakage has not
been reported.
5. Conclusions
Here, we report for the first time the role of IL-21 during the
clinical course of DENV infections. Our present study is the first
to report that there is a relationship between the elevated serum
levels of IL-21 and the production of DENV-specific IgM and IgG
antibodies.
We speculate that IL-21 may play a protective role in the context
of the convalescent phase of primary infections and the acute phase
of secondary infections. Given the different biological effects and
the pleiotropic activities of IL-21, our results open a very attractive
field of study for this cytokine in a very complex infectious disease
such as dengue fever.
Funding
This study was supported by Fondo Sectorial en Ciencia Básica
SEP-CONACyT, project ID 157274 and Fondo Mixto CONACyT-
Gobierno del Estado de Veracruz, project ID 109270 and 128001.
Maldonado-Rentería MJ, Sánchez-Vargas LA, Izaguirre-Hernández
IY and Hernández-Flores KG gratefully acknowledge the scholar-
ship from CONACyT to pursue their postgraduate studies.
Ethical approval
The study protocol was approved in accordance with the guide-
lines of the Ethics Committee of the Instituto de Investigaciones
Medico-Biológicas, Universidad Veracruzana.
Authors’ contributions
Conceived and designed the study: VCH and RRR. Performed
the experiments, enrolled patients, recorded clinical and data
analysis: MRMJ, SVLA, IHIY, HFKG, VCH, and RRR. Contributed
reagents/materials/analysis tools: VCH and RRR. Wrote the paper:
VCH, MRMJ, and SVLA. All authors read and approved the final
manuscript.
Competing interests
We declare that the authors have not any financial or personal
relationship with other people or organizations that could create
a potential conflict of interest or the appearance of a conflict of
interest with regard to the work.
Acknowledgments
We are grateful for the support from Health Centers “El Coyol”,
“Anastasio Iturralde”, and “21 de Abril” and the Hospital Regional
de Alta Especialidad de Veracruz, Servicios de Salud de Veracruz
for providing access to dengue-infected patients. We also thank the
Centro Estatal de la Transfusión Sanguínea del Estado de Veracruz
for providing access to healthy controls.
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