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IMLET 5522 1–7 ARTICLE IN PRESS


Immunology Letters xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

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

1 Dynamics of interleukin-21 production during the clinical course of


2 primary and secondary dengue virus infections
3 Q1 H. Vivanco-Cid a,∗,1 , M.J. Maldonado-Rentería a,1 , L.A. Sánchez-Vargas a ,
4 I.Y. Izaguirre-Hernández a , K.G. Hernández-Flores a , R. Remes-Ruiz b,c
5 Q2 a
Instituto de Investigaciones Medico-Biológicas, Universidad Veracruzana, Veracruz, Mexico
b
6 Hospital Regional de Alta Especialidad de Veracruz, Servicios de Salud de Veracruz, Mexico
c
7 Universidad del Valle de México, campus Villa Rica, Facultad de Medicina “Dr. Porfirio Sosa Zárate”, Mexico
8

9
22 a r t i c l e i n f o a b s t r a c t
10
11 Article history: Previous studies have revealed the clinical relevance of pro-inflammatory cytokine production during
12 Received 29 December 2013 dengue virus (DENV) infections. In this study, we evaluated the production of interleukin-21 (IL-21), a
13 Received in revised form 22 April 2014 key soluble mediator mainly produced by CD4+ T cells.
14 Accepted 7 May 2014
The aim of this study was to investigate the role of IL-21 production during the clinical course of
15 Available online xxx
primary and secondary DENV infections and the potential association of IL-21 serum levels with the
16
disease pathogenesis. Blood samples from DENV-infected patients were collected on different days after
17 Keywords:
the onset of symptoms. Patients were classified according to their phase of disease (acute vs. convalescent
18 Dengue
19 Interleukin 21
phases), the type of infection (primary vs. secondary), and the clinical severity of their disease (dengue
20 Antibody response fever (DF) vs. dengue hemorrhagic fever (DHF)). IL-21 levels were measured using a quantitative capture
21 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 Published by Elsevier B.V.

23 1. Introduction of both protection and the pathogenesis of the severe forms of this 32

viral disease. A cascade of pro-inflammatory cytokines is released 33

24Q4 Dengue fever is the most common transmitted arboviral disease during acute dengue infections, including cytokines from innate 34

25 worldwide. The infection is caused by one of the four serotypes immunity cell sources, such as TNF-␣, IL-1, IL-6, and IFN-␣, and 35

26 of dengue virus, the etiologic agents, all of which are members of chemokines, such as MIP1-␣, IP-10 and IL-8. Pro-inflammatory 36

27 the Flavivirus group into the family Flaviviridae [1]. The pathophy- cytokines are implicated in host protection roles such as activat- 37

28 siology of DF/DHF/DSS in humans is complex and multi-factorial, ing innate and adaptive immune cells, chemotaxis and inhibition of 38

29 involving the interplay of host and viral factors that influence dis- viral replication. Some detrimental roles for the high inflammatory 39

30 ease severity and the clinical outcome [2]. With respect to host cytokine levels that are often observed in severe disease include the 40

31 factors, the immune response to DENV plays a dual role in terms induction of cell death (apoptosis and necrosis), endothelial activa- 41

tion, vascular leak and shock [3–7]. Cytokines produced by adaptive 42

immune cells, such as CD4+ and CD8+ T cells, also play a major role 43

with respect to viral spread and further support the development 44


∗ Corresponding author at: Laboratorio Multidisciplinario en Ciencias Biomédicas, of effector functions, including humoral, cell-specific and memory 45
Instituto de Investigaciones Medico-Biológicas, Universidad Veracruzana, Avenida immune responses. During the CD4+ T cell response to DENV, Th1 46
Q3 Iturbide S/N, Col. Centro, C.P. 91700 Veracruz, Mexico. Tel.: +52 2299318011x120;
and Th2 cells participate in the viral disease with the production 47
fax: +52 2299318011.
E-mail addresses: hvivanco@uv.mx, vivacid@hotmail.com (H. Vivanco-Cid). of IFN-␥, TNF-␤, and IL-2 or IL-4, IL-5, IL-6 and IL-10, respectively 48
1
These authors contributed equally to this work. [8]. A shift from the Th1 to Th2 response has been suggested in the 49

http://dx.doi.org/10.1016/j.imlet.2014.05.006
0165-2478/© 2014 Published by Elsevier B.V.

Please cite this article in press as: Vivanco-Cid H, et al. Dynamics of interleukin-21 production during the clinical course of primary and
secondary dengue virus infections. Immunol Lett (2014), http://dx.doi.org/10.1016/j.imlet.2014.05.006
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IMLET 5522 1–7 ARTICLE IN PRESS
2 H. Vivanco-Cid et al. / Immunology Letters xxx (2014) xxx–xxx

50 clinical pathogenesis of DHF on the basis of in vitro studies and the of the Instituto de Investigaciones Medico-Biológicas, Universidad 110

51 ex vivo analysis of the cytokine patterns in the serum samples of Veracruzana (Veracruz, México). The study protocol was approved 111

52 patients with severe disease [5,8–10]. by the institutional ethics review board. 112

53 In this study, we evaluated the interleukin-21 (IL-21) produc-


54 tion in sera from DENV infected patients, a key pro-inflammatory 2.3. Dengue diagnosis 113

55 and soluble mediator mainly produced by CD4+ T cells. To date,


56 IL-21 has not previously been studied during the clinical course Serum samples were tested and diagnosed by reverse 114

57 of DENV infections. IL-21 is a cytokine member of the common ␥- transcription-polymerase chain reaction (RT-PCR) and three refer- 115

58 chain family, which includes IL-2, IL-4, IL-7, IL-9 and IL-15 [11]. ence anti-dengue enzyme-linked immunosorbent assays (ELISA): 116

59 IL-21 is produced by various subsets of activated CD4+ T cells, Platelia Dengue NS1 Ag Test (BIORAD Laboratories, France), Dengue 117

60 which include Th17 cells, T-follicular helper (Tfh) and CD4+ natu- IgM (Panbio, Australia) and, to discriminate among primary and 118

61 ral killer (NK) T cells, and exerts multiple and pleiotropic effects on secondary infections, a commercial kit (capture IgG ELISA, Pan- 119

62 various innate and adaptive immune cells, such as NK cells, mono- bio) that is used for the quantitative detection of elevated titers of 120

63 cytes, macrophages, B cells, and CD4+ and CD8+ T lymphocytes, IgG antibodies to dengue virus in patients with secondary infection 121

64 as well as on non-immune cells such as keratinocytes [12], fibro- according to previously established criteria [16]. 122

65 blasts [13,14] and vascular endothelial cells [15]. The aim of this
66 work was to investigate whether the serum levels of IL-21 are ele- 2.4. Classification of samples 123

67 vated in dengue-infected patients, analyzing the production during


68 primary and secondary infections in comparison with healthy indi- Based on the time of clinical illness, we classified dengue 124

69 viduals and the potential association of IL-21 serum levels with the patients into acute (1–7 days after disease onset) and convales- 125

70 disease pathogenesis. cent (samples collected on days 8–10 after disease onset) phases. 126

Primary dengue infection was defined for patients with positive 127

results for RT-PCR, NS1 and/or IgM tests and negative IgG ELISA 128
71 2. Materials and methods
results. Secondary dengue infection was defined for IgG ELISA pos- 129

itive results (equivalent to a hemagglutination inhibition titer of 130


72 2.1. Subjects
>2560) regardless of RT-PCR, NS1 and IgM results. 131

73 A clinical cohort of patients with suspected cases of dengue


2.5. Measurement of serum IL-21 levels 132
74 were recruited in the city of Veracruz México during outbreaks that
75 occurred from June 2010 through November 2012. A total of 360
The IL-21 concentrations in the sera from dengue-positive 133
76 febrile patients were laboratory confirmed for dengue infection and
patients and healthy controls were measured by sandwich ELISA 134
77 enrolled in our study. Blood samples from patients were collected
kit from PeproTech Inc. (Rocky Hill, NJ) according to the manufac- 135
78 in three urban health centers (“Anastasio Iturralde”, “21 de Abril”,
turer’s instructions. The optical density at 405 nm with a reference 136
79 and “El Coyol”), Hospital Regional de Alta Especialidad de Veracruz
filter at 650 nm was measured by using a microplate reader (Aware- 137
80 and Hospital Naval de Veracruz. Dengue patients had not received
ness Technology Inc., Palm City, FL, USA). 138
81 any drug or treatments before the sample collection. Blood samples
82 were tested and diagnosed by reverse transcription-polymerase
2.6. Measurement of serum DENV-specific IgM and IgG levels 139
83 chain reaction (RT-PCR) and ELISA techniques to confirm the cases.
84 To rule out other viral or bacterial infections, further serological
The serum samples from dengue-infected patients were tested 140
85 tests for leptospirosis, influenza A, hepatitis A and Salmonella typhi
for DENV IgM and IgG antibodies by ELISA at a 1:100 dilution. 141
86 were carried out at the clinical admission time. Patients were classi-
Polysorb 96-well microplates (Nalge Nunc International, Rochester, 142
87 fied as having dengue fever or dengue hemorrhagic fever according
NY) coated with recombinant antigens provided in the Dengue IgM 143
88 to clinical and laboratory criteria of the World Health Organiza-
kit and the commercial kit capture IgG ELISA (Panbio, Australia) 144
89 tion (WHO 1997). Clinical case definition of dengue fever included
were used to measure the DENV specific antibody levels. The serum 145
an acute febrile illness with two or more of the following mani-
samples were incubated for 1 h at 37 ◦ C. After incubation, serum
90
146
91 festations: headache, retro-orbital pain, arthralgia, myalgia, rash,
samples were removed, and horseradish peroxidase-conjugated 147
leukopenia and supportive molecular or serology diagnosis. Clin-
monoclonal anti-human IgM or IgG were added for 1 h at 37 ◦ C, fol-
92
148
ical case definition of dengue hemorrhagic fever included all of
lowed by the addition of 3,3 ,5 -tetramethyl benzidine chromogen
93
149
94 the above criteria for DF plus evidence of hemorrhagic tenden-
solution. One hundred microliters of Stop Solution was added to 150
95 cies (epistaxis, gingival bleeding, petechiae, a positive tourniquet
each well plate. The antibodies levels of each serum samples were 151
96 test). Vascular leakages were documented by chest X-ray and esti-
represented as an index value, according to the manufacturer’s 152
97 mating hypovolemia from multiple hematocrit determinations.
instructions. The OD results were corrected by subtracting the 153
98 Other clinical and laboratory findings included thrombocytopenia
OD values of blank samples which are included in every test kit. 154
99 (<100,000 counts/mm3 ), hypotension and hypoproteinemia. No
The ratio with a standard positive sample, the cut-off calibrator, is 155
100 fatal cases were included in the present study.
expressed as an index value (IV). Index value is calculated dividing 156
101 Seventy-eight serum samples from healthy individuals without
the optical density of the sample by the cutoff value. 157
102 clinical signs or manifestations suggestive of dengue (no febrile
103 symptoms) or other apparent illnesses in the previous 3 months
2.7. Statistical analysis 158
104 were included as controls. All serum samples were frozen at −70 ◦ C
105 until analysis.
The data were represented as the means ± SD or as medians. 159

The Chi square test was used to compare categorical variables. 160

106 2.2. Ethics statement The non-parametric Kruskal–Wallis test was used to comparison 161

more than two groups. Comparisons among each patient group 162

107 Written consent to participate in the study was obtained from were performed using Dunn’s multiple comparison tests. Statis- 163

108 each participant after a full explanation of the study was provided tical significance between two individual groups was determined 164

109 and in accordance with the guidelines of the Ethics Committee with the Mann–Whitney U test using GraphPad Prism software V 165

Please cite this article in press as: Vivanco-Cid H, et al. Dynamics of interleukin-21 production during the clinical course of primary and
secondary dengue virus infections. Immunol Lett (2014), http://dx.doi.org/10.1016/j.imlet.2014.05.006
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IMLET 5522 1–7 ARTICLE IN PRESS
H. Vivanco-Cid et al. / Immunology Letters xxx (2014) xxx–xxx 3

Table 1
Classification of dengue patients according to phase and severity disease.

Phase Dengue fever (n = 235) Dengue hemorrhagic fever (n = 125)

Primaryn (%) Secondaryn (%) Primaryn (%) Secondaryn (%)

Acute 71 (75.5%) 100 (70.9%) 20 (71.4%) 49 (50.5%)


Convalescent 23 (24.5%) 41 (29.1%) 8 (28.6%) 48 (49.5%)

Total (n) 94 141 28 97

166 5.0. P values <0.05 were considered statistically significant. Correla- 3.3. The highest serum levels of IL-21 are produced during the 196

167 tion between IL-21 and Dengue IgM and IgG levels among different convalescent phase of primary infections 197

168 groups was evaluated using Spearman’s correlation test.


Next, the serum levels of IL-21 were analyzed after classi- 198

fying all dengue patients according to their clinical evolution 199

(acute and convalescent phases) and type of infection (primary 200


169 3. Results
or secondary). Interestingly, we observed that IL-21 was signifi- 201

cantly elevated during the convalescent phase of primary infections 202


170 3.1. Patient characteristics
(mean 230.5 ± 225.6 pg/ml, median 167.4 pg/ml) compared with 203

acute primary infections (mean 108.8 ± 152.2 pg/ml, median 204


171 The mean age of dengue patients was 29.9 years-old (range:
74.56 pg/ml), acute secondary infections (mean 160 ± 226.9 pg/ml, 205
172 5–74 years-old). The gender ratio was 1:1.6 (male: female). From
median 72.58 pg/ml), and convalescent secondary infections (mean 206
173 the 360 dengue-positive patients, 235 (65.28%) and 125 (34.72%)
136.1 ± 178.3 pg/ml, median 86.4 pg/ml), Fig. 1B. 207
174 were classified as having DF and DHF, respectively, accordingly
175 to the World Health Organization clinical criteria (WHO 1997)
176 described before. DHF Patients were clinically managed in the
3.4. The stratification of dengue-infected patients according to 208
177 hospital for a median of 4 days. All DHF patients had laboratory
the IL-21 concentrations, clinical phase and type of infection 209
178 evidence of a rising hematocrit (evidence of plasma leakage) and
179 thrombocytopenia (<100,000 counts/mm3 ). 122 patients were con-
Given the previous findings about the heterogeneity in the con- 210
180 firmed to have primary dengue infection, and 238 were confirmed
centrations of IL-21 detected among patients, we were interested 211
181 to have secondary dengue infections, following the laboratory crite-
in determining how the clinical stage of infection can influence the 212
182 ria described in the materials and methods section. Based on the
production pattern for this cytokine. Thus, we stratified all of the 213
183 time of clinical illness, a total of 240 patients were classified in the
patients into three categories based on their IL-21 serum concen- 214
184 acute phase (days 1–7 after disease onset) and 120 patients were
trations. Patients were classified in tertiles as low, medium, and 215
185 classified in the convalescent phase (days 8–10 after disease onset)
high IL-21 producers. The cutoff values for each category were set 216
186 (Table 1).
considering the mean value for the healthy controls group plus one 217

or two standard deviations. Concentrations below the first cutoff 218

(≤73.9 pg/ml) were classified as low IL-21 producers. Concentra- 219


187 3.2. The serum levels of IL-21 are increased in dengue-infected tions between the first and second cutoff (74–133.3 pg/ml) were 220
188 patients compared with healthy controls classified as medium IL-21 producers, and concentrations above the 221

second cutoff (≥133.4 pg/ml) were classified as high IL-21 produc- 222
189 To investigate whether IL-21 is produced during DENV infection, ers. We related these categories with the type of infection (primary 223
190 the serum levels of IL-21 were measured by enzyme immunoassay. or secondary) and the course of the disease (acute or convalescent 224
191 In a first analysis, we observed very low serum levels of IL-21 in the phases). A high percent (61.3%) of patients with primary infections 225
192 healthy control group (mean 14.5 ± 59.4 pg/ml, median 1.6 pg/ml) in the convalescent phase produced the highest levels of IL-21. High 226
193 and a higher and heterogeneous production of IL-21 in dengue- concentrations of IL-21 were also detected in 40.3% of patients with 227
194 infected patients (mean 147.2 ± 200.4 pg/ml, median 83.37 pg/ml), secondary infections in the acute phase. In contrast, 1.3% of the 228
195 P < 0.0001, Fig. 1A. healthy controls were classified as high IL-21 producers, whereas 229

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).

Please cite this article in press as: Vivanco-Cid H, et al. Dynamics of interleukin-21 production during the clinical course of primary and
secondary dengue virus infections. Immunol Lett (2014), http://dx.doi.org/10.1016/j.imlet.2014.05.006
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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 APn (%) CPn (%) ASn (%) CSn (%) HCn (%) P value

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.

230 97.4% of the control group was classified as low IL-21 producers
231 (Table 2).

232 3.5. The serum levels of IL-21 are associated with the severity of
233 dengue infection during the acute phase of secondary infections

234 To evaluate whether there was a relationship among the het-


235 erogeneity in the serum concentrations of IL-21 and the severity
236 of disease, we grouped the patients into two groups based on their
237 clinical classification as DF or DHF. There was a trend toward higher
238 IL-21 serum levels in DHF patients with respect to DF patients.
239 However, in this first analysis, there was no statistically signifi-
240 cant difference between the groups (P > 0.05, Fig. 2A). Next, we
241 decided to analyze the sample in more detail by comparing among
242 the same clinical stages. Because almost 80% of the DHF cases
243 recruited in our study correspond to secondary infections, we com-
244 pared the serum levels of IL-21 considering just the cases of DF
245 and DHF classified within the secondary infection group (Fig. 2B
246 and C). As shown in Fig. 2B, IL-21 was significantly elevated in the
247 serum of DHF patients during the acute phase of secondary infec-
248 tions (mean 165 ± 259.1 pg/ml, median 131.8 pg/ml) compared
249 with DF patients in the acute phase of secondary infections (mean
250 149.6 ± 141.6 pg/ml, median 62.84 pg/ml; P = 0.02).

251 3.6. The stratification of dengue-infected patients according to


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

253 We performed the same analysis as previously described in


254 Table 2 to determine the distribution of DF or DHF patients stratified
255 by their IL-21 serum concentrations (Table 3). Interestingly, 49.0%
256 of DHF patients with secondary infections in the acute phase were
257 classified as high IL-21 producer, 16.3% were classified as medium
258 producers and 34.7% were classified as low IL-21 producers. In con-
259 trast, 36% of DF patients with secondary infections in the acute
260 phase were classified as high IL-21 producers, 6% were classified as
261 medium producers and 58% were classified as low IL-21 producers.
262 When we compared the convalescence phases of DF and DHF, we
263 observed a similar distribution of the patients independent of the
264 severity of the disease. A remarkable result for the convalescent
265 phases was the high percentage of DF patients (60.9%) and DHF
266 patients (62.5%) in the convalescent phase of primary infections
267 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
268 3.7. The serum IL-21 levels in dengue patients correlate with patients with dengue fever (DF) (n = 235), dengue hemorrhagic fever (DHF) (n = 125)
269 DENV-specific IgM and IgG levels and healthy controls (HC) (n = 78). (B) Comparison of IL-21 levels in patients with
secondary acute DF infections (n = 100) and secondary acute DHF infections (n = 49).
(C) Comparison of IL-21 levels in patients with secondary convalescent DF infections
270 Finally, we identified possible correlations between the serum
(n = 41) and secondary convalescent DHF infections (n = 48). Statistical confidence
271 IL-21 levels and the humoral response raised against DENV. The was analyzed in figure A by Kruskall–Wallis test. Mann–Whitney U test was used
272 strength of association between the serum IL-21 levels and the for statistical analysis in figures B and C. The IL-21 levels are represent in pg/ml.
273 anti-Dengue IgM or IgG levels were examined using the Spear- *P < 0.05, ****P < 0.0001.
274 man’s correlation test. Fig. 3 shows the scatter plots for the levels
275 of anti-dengue IgM and IgG against the serum IL-21 levels in
276 primary and secondary dengue infections, respectively. Dengue
277 patients in the acute phase of primary infections, days 1–7 showed

Please cite this article in press as: Vivanco-Cid H, et al. Dynamics of interleukin-21 production during the clinical course of primary and
secondary dengue virus infections. Immunol Lett (2014), http://dx.doi.org/10.1016/j.imlet.2014.05.006
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Table 3
Q6 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.

278 undetectable or low levels of DEN-specific IgM antibodies (IgM 4. Discussion 293

279 ratio, 2.052 ± 2.641) with no significant correlation values with the
280 serum IL-21 levels (Fig. 3A). The levels of dengue-specific IgM anti- Cytokine levels differ dramatically from steady state conditions 294

281 bodies increase in the convalescent phase of primary infections, in healthy controls compared with patients exhibiting active dis- 295

282 on days 8–10 to a mean ratio of 4.598 ± 2.599. The serum lev- ease. In dengue infections, there is a highly heterogeneous host 296

283 els of IL-21 were significantly and positively correlated with the immune response to the viral infection depending of the severity of 297

284 increase of anti-dengue IgM levels (r = 0.4260, P < 0.0189) in the the disease. Here, we present for the first time evidence of IL-21 pro- 298

285 convalescent phase of primary infections (Fig. 3B). For secondary duction during dengue infections. It is apparent from our findings 299

286 infections we found a significant and positive correlation between that the levels of IL-21 increase significantly during DENV infection 300

287 the serum levels of IL-21 and the increase of anti-dengue IgG lev- compared with the levels in healthy individuals. In our study, the 301

288 els (IgG ratio, 5.818 ± 1.881) during the acute phase (r = 0.2928, highest IL-21 concentrations were measured in patients during the 302

289 P < 0.0097). However, no significant correlation value was found convalescence phase of primary infections. This particular produc- 303

290 between dengue-IgG levels and the serum levels of IL-21 during the tion pattern of IL-21 was demonstrated for both clinical forms: DF 304

291 convalescent phase of secondary infections (r = 0.2110, P < 0.1026) and DHF. A high percent of DF (60.9%) and DHF patients (62.5%) 305

292 (Fig. 3D). in the convalescent phase of primary infections were classified as 306

Please cite this article in press as: Vivanco-Cid H, et al. Dynamics of interleukin-21 production during the clinical course of primary and
secondary dengue virus infections. Immunol Lett (2014), http://dx.doi.org/10.1016/j.imlet.2014.05.006
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307 the highest IL-21 producers. Interestingly, we found in the same MIP-1␣, Eotaxin, and IP-10, by human NK cells [33,34], monocytes 373

308 clinical stage that the serum IL-21 levels correlated well with the [35], macrophages [36], dendritic cells [37] and T cells [38–40]. 374

309 DENV-specific IgM production. These observations are consistent Although the IL-21 receptor is expressed on vascular endothelial 375

310 with the role IL-21 plays in plasma cell differentiation [17]. cells, a direct effect of this cytokine on vascular activation and vas- 376

311 High IL-21 levels during the convalescent phase correlate well cular leakage has not been reported. 377

312 with the time when the dengue-infected patients began to recover.
313 The recovery phase is characterized by improving general well 5. Conclusions 378
314 being and hemodynamic stability, which suggest a protective role
315 of this cytokine in this clinical stage of primary infections. Fur- Here, we report for the first time the role of IL-21 during the 379
316 thermore, during the acute phase of secondary dengue infections, clinical course of DENV infections. Our present study is the first 380
317 a positive correlation between the serum IL-21 levels and DENV- to report that there is a relationship between the elevated serum 381
318 specific IgG levels was found. This association also is consistent with levels of IL-21 and the production of DENV-specific IgM and IgG 382
319 the critical role IL-21 plays as a potent inducer of IgG production antibodies. 383
320 [18–20] and promoting the activation of memory B cells by T cells We speculate that IL-21 may play a protective role in the context 384
321 [17]. of the convalescent phase of primary infections and the acute phase 385
322 Because of the cellular-restricted origins of IL-21, this cytokine of secondary infections. Given the different biological effects and 386
323 could be considered a good potential biomarker of the protection the pleiotropic activities of IL-21, our results open a very attractive 387
324 mediated by CD4+ T or NKT cells against dengue virus in pri- field of study for this cytokine in a very complex infectious disease 388
325 mary infections. Interestingly, a recent study has identified that an such as dengue fever. 389
326 important proportion of DENV-specific CD4+ T cells isolated from
327 PBMCs from convalescent dengue virus patients displayed a pheno-
Funding 390
328 type characteristic of circulating follicular helper T cells (TFH cells),
329 suggesting that they are interacting with B cells in vivo [21]. A frac-
This study was supported by Fondo Sectorial en Ciencia Básica Q5 391
330 tion of DENV-specific CXCR5+ cells was capable of producing IL-21
SEP-CONACyT, project ID 157274 and Fondo Mixto CONACyT- 392
331 or IFN-␥ following stimulation with DENV peptides. The evidence
Gobierno del Estado de Veracruz, project ID 109270 and 128001. 393
332 provided by Rivino et al., and our own results, support the idea
Maldonado-Rentería MJ, Sánchez-Vargas LA, Izaguirre-Hernández 394
333 that a fraction of DENV-specific CD4+ T cells could be involved in
IY and Hernández-Flores KG gratefully acknowledge the scholar- 395
334 supporting B cell antibody production through an IL-21 dependent
ship from CONACyT to pursue their postgraduate studies. 396
335 mechanism.
336 The biological effects and clinical relevance of the production
337 of IL-21 in primary dengue infections could be related to the Ethical approval 397

338 importance of IL-21 in the acute phase to enhance the cytotoxic


339 activity of NK cells and establish viral control early. In the convales- The study protocol was approved in accordance with the guide- 398

340 cence phase, IL-21 could be very important to support the humoral lines of the Ethics Committee of the Instituto de Investigaciones 399

341 response through the proliferation and differentiation of B cells Medico-Biológicas, Universidad Veracruzana. 400

342 into plasma cells, enhancing immunoglobulin production and iso-


343 type class switch and promoting the activation of cellular responses Authors’ contributions 401

344 mediated by CD8+ T cells and the development of immunological


345 memory. Clinical and basic research has established the protective Conceived and designed the study: VCH and RRR. Performed 402

346 role of IL-21 in other viral diseases, including HIV [22–25], HBV the experiments, enrolled patients, recorded clinical and data 403

347 [26], herpes simplex virus [27], and respiratory syncytial virus [28]. analysis: MRMJ, SVLA, IHIY, HFKG, VCH, and RRR. Contributed 404

348 Recently, a protective role for IL-21 has been demonstrated during reagents/materials/analysis tools: VCH and RRR. Wrote the paper: 405

349 the course of the chronic Hepatitis C infections in humans [29]. Hep- VCH, MRMJ, and SVLA. All authors read and approved the final 406

350 atitis C virus is a member of the Flaviviridae family and is closely manuscript. 407

351 related to the dengue fever virus. Although IL-21 has been mainly
352 associated with protective roles and sustaining T cell responses Competing interests 408
353 during chronic viral infections, little is known about the role of this
354 cytokine in the course of acute viral infections such as dengue. The We declare that the authors have not any financial or personal 409
355 role of IL-21 during secondary infection by DENV is more intrigu- relationship with other people or organizations that could create 410
356 ing than in primary infections. In our study, IL-21 levels indeed a potential conflict of interest or the appearance of a conflict of 411
357 were significantly higher in DHF patients than in patients with DF interest with regard to the work. 412
358 in the acute phase of secondary infections. However, the higher lev-
359 els of IL-21 in secondary infections of DHF patients compared to DF
Acknowledgments 413
360 patients probably are a reflection of the more intense immune acti-
361 vation in DHF rather than a cause of the severity of disease. It is well
We are grateful for the support from Health Centers “El Coyol”, 414
362 known that secondary infections with a distinct DENV serotype led
“Anastasio Iturralde”, and “21 de Abril” and the Hospital Regional 415
363 to the activation of serotype-cross-reactive T cells, resulting in ele-
de Alta Especialidad de Veracruz, Servicios de Salud de Veracruz 416
364 vated levels of adaptive immune response-derived cytokines such
for providing access to dengue-infected patients. We also thank the 417
365 as IFN-␥, IL-2, IL-4, IL-10 and TNF-␣, which have been reported to be
Centro Estatal de la Transfusión Sanguínea del Estado de Veracruz 418
366 more pronounced in DHF compared to DF in some studies [30–32].
for providing access to healthy controls. 419
367 A potential detrimental role for IL-21 during the acute phase of
368 DHF secondary infections could be through the activation of dif-
369 ferent immune cell populations, such as monocytes, macrophages, References 420

370 dendritic cells and T cells. IL-21 has been reported to induce the
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secondary dengue virus infections. Immunol Lett (2014), http://dx.doi.org/10.1016/j.imlet.2014.05.006
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Please cite this article in press as: Vivanco-Cid H, et al. Dynamics of interleukin-21 production during the clinical course of primary and
secondary dengue virus infections. Immunol Lett (2014), http://dx.doi.org/10.1016/j.imlet.2014.05.006

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