Cellular Immunology 262 (2010) Contents lists available at ScienceDirect. Cellular Immunology

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1 Cellular Immunology 262 (2010) Contents lists available at ScienceDirect Cellular Immunology journal homepage: Secondary heterologous dengue infection risk: Disequilibrium between immune regulation and inflammation? Beatriz Sierra a, *, Ana B. Perez a, Katrin Vogt b, Gissel Garcia a, Kathrin Schmolke b, Eglys Aguirre a, Mayling Alvarez a, Florian Kern b, Gustavo Kourí a, Hans-Dieter Volk b, Maria G. Guzman a a Virology Department, PAHO/WHO Collaborating Center for the Study of Dengue and its Vector, Institute of Tropical Medicine, Pedro Kouri Autopista Novia del Mediodia, Km 6[1/2], La Lisa, Habana, Cuba b Institute for Medical Immunology, Charité-Universitätsmedizin Berlin, CCM, Charitéplatz 1,D Berlin, Germany article info abstract Article history: Received 9 September 2009 Accepted 4 February 2010 Available online 10 February 2010 Keywords: Cytokines Immunopathogenesis Dengue Increased serum levels of cytokines released by cells of the immune response have been detected in patients suffering from dengue disease. Likewise, secondary infections by a different dengue virus serotype result in a highest risk of development of the severe dengue disease. Both findings suggest that the memory immune response is one of the key players in the pathogenesis of this disease. Here we take advantage of the particular Cuban epidemiological situation in dengue to analyze a broad spectrum of cell-mediated immune response mediators at mrna and protein level. Evidences for a regulatory immune pattern in homologous (TGF-b, IL-10) vs. pro-inflammatory pattern (IFN-c, TNF-a) in heterologous dengue virus re-challenge were found, suggesting a possible association with the higher incidence of severe dengue cases in the latter case. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Dengue is an emerging arboviral disease caused by any of the four dengue viruses (DENV-1 4), grouped in the dengue complex of the Flaviviridae family. The morbidity and mortality associated with the severe DENV infection is a major increasing public health problem throughout the tropical and subtropical regions of the world [1,2]. DENV infection can be asymptomatic or can shows two main clinical syndromes, dengue fever (DF), an acute febrile disease accompanied by headache, arthralgia and rash, from which patients usually recover without complications, and Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS) characterized by plasma leakage resulting in hypovolemia and circulatory system collapse in some patients [3]. T cells and a cytokine storm are associated with secondary infections and severe disease in DHF/ DSS patients suggesting the cellular immune response is a contributor to the pathogenesis of DHF/DSS [4 6]. Pro-inflammatory cytokines released by DENV serotype cross-reactive memory T cells could explain the severe course in some patients after DENV reinfection, since it has been suggested that plasma leakage could be related to malfunction of vascular endothelial cells induced by * Corresponding author. Address: Cellular Immunology Lab. Department of Virology, Institute for Tropical Medicine Pedro Kourí, Avenida Novia del Mediodia, Km 6[1/2], Marianao 13, Apto 601, La Lisa, Ciudad Habana, Cuba. Fax: address: siebet@ipk.sld.cu (B. Sierra). cytokines or chemical mediators rather than by destruction of the small vessels [7 10]. Contrary to most dengue endemic countries where several dengue serotypes co-circulate, the Cuban epidemiological situation is considered unique. Epidemics caused by only one serotype have been fully controlled, allowing the study of the role of the secondary infection as a major risk factor for DHF/DSS. In the Cuban epidemics of 1981, 1997 and , the secondary infection was considered the main host risk factor for the severe disease. In each epidemic, a the secondary infection was demonstrated in more than 97% of the DHF/DSS clinically classified patients [11,12]. Taking advantage of the Cuban epidemiological situation, we addressed the role of the regulatory/pro-inflammatory mediators in the dengue infection immunopathogenesis. 2. Materials and methods 2.1. Patients and controls The study enrolled 20 Cuban individuals from Havana City (12 female and 8 male, ages 20 60, mean of 35.8) with levels of antidengue IgG of 1/40 or higher as tested by ELISA. [13]. Ten individuals were classified like primary cases of dengue 1 (anti-dengue antibodies able to neutralize only dengue 1 virus) and 10 like primary cases of dengue 2 (anti-dengue antibodies able to neutralize only dengue 2 virus) as shown using plaque reduction neutralization: sera were tested by neutralization test at dilution s between /$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi: /j.cellimm

2 B. Sierra et al. / Cellular Immunology 262 (2010) /10 and 1/1000 for their ability to neutralize dengue virus by semimicro methods in BHK21 cells. As average all of them showed titles of neutralizing antibodies of 1/100 [14 16]. These individuals developed DF infection without hemorrhagic manifestations [17] during the 1977, the 1981 and the 2001 Cuban epidemics. The etiology of infection in the above-mentioned epidemics was established by virus isolation in Aedes albopictus, C6/36 cells [18], RT-PCR [19] or by serological evidence of recent infection by demonstrating the presence of IgM-capture dengue antibodies by ELI- SA. [20]. Ten individuals from Havana City (3 female and 7 male, ages 20 60, mean of 33.4), that had not been previously exposed to dengue infection, without antibodies against to DENV as tested by ELISA, were included as negative controls. This study was conducted according to the Declaration of Helsinki as a statement of ethical principles for medical research involving human subjects, and has been approved by the Institutional Ethical Review Committee of the Institute of Tropical Medicine Pedro Kourí and by the Ethical Committee the Cuban National Academy of Sciences. Written informed consent was obtained from each individual upon enrollment in the study Dengue virus preparation DENV antigens were prepared as previously described [21]. C636 cell lines from A. albopictus were grown to confluence, infected at a multiplicity of infection of 0.1 particle forming unit (pfu) per cell with the dengue strains DENV Peru 1990, DENV-2 A15 Cuba 1981 and DENV Cuba 2000, and cultured in Minimum Essential Medium (MEM) supplemented with 2% fetal calf serum. When a higher than 50% cytopathic effect was noted, culture supernatant was clarified by centrifugation at 10,000 rpm for 30 min at 4 C. Non-infected C636 cells supernatant were used as negative control antigen (mock control). BHK21, clone 15 cell line was used for virus titration as previously described [16]. The presence in the viral preparations of contaminating lipopolysaccharide was evaluated by the Limulus Amebocyte Lysate test (Bio- Whittaker Inc., Walkersville, MD). All viral preparations used in the present study were lipopolysaccharide-free Dengue virus stimulation of PBMC for RT-PCR and CBA assays Peripheral venous blood (20 ml) was collected from the individuals included in the study. PBMC were isolated from 15 ml of citrated venous blood by standard density gradient centrifugation using Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden), and sera was obtained from 5 ml of peripheral venous blood without anticoagulant. [22] Cells were adjusted to in 1000 ll of RPMI 1640 supplemented with 5% of autologous sera, 2 mm glutamine, 100 lg of streptomycin/ml, and 100 U of penicillin, and cultured for 24 h at 37 C in the presence of DENV-1, -2 or -3 at a multiplicity of 0.1 pfu or mock (supernatant of uninfected C636 cells), respectively. Phytohaemagglutinin (PHA) (Sigma, Poole, United Kingdom), 5 lg/ml, served as a positive assay control. After 24 h, cells were separated from supernatants and both frozen at 80 C Gene expression analysis DNase-treated total RNA was isolated from stimulated PBMC by means of RNeasy Mini kit (Qiagen, Hilden, Germany) and evaluated by using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The cdna was synthesized from mrna with poly(dt) primers and Superscript II reverse transcriptase (Life Technologies, Rockville, MD, USA) and quantified by real-time PCR analysis using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA, USA), according to the manufacturer s protocols. Samples were analyzed in triplicate for the expression of IL-2, IL-4, IL-5, IL-10 tumor necrosis factor (TNF-a), interferon gamma (IFN-c) and tumor grow factor beta (TGF-b), respectively, as well as for the housekeeping gene hypoxanthine phosphoribosyltransferase-1 (HPRT-1). Specific expression was calculated in relation to that of HPRT-1, by using the delta/delta Ct method as recommended by ABI Quantification of cytokine proteins by Cytometry Beads Assay (CBA) The concentrations of IFN-c, TNF-a, IL-10, IL-2, IL-4, IL-5 in the supernatants from PBMC cells stimulated with dengue antigen or control antigen for 24 h were measured using a multiplex bead array, the human Th1/Th2 cytokine CBA kit I (BD PharMingen, San Diego, CA, USA). Briefly, microbead populations with distinct fluorescence intensities were coated with capture antibody specific for each cytokine. Fifty microliter of capture beads, 50 ll of phycoerythrin-conjugated detection antibodies, and 50 ll recombinant standards (calibrators) or test samples were incubated for 3 h at room temperature. The samples are then washed with wash buffer and recovered by centrifugation (200 g for 5 min). After careful aspiration of the supernatant, the beads were re-suspended in 300 ll of wash buffer, vortexed briefly, and analyzed by FACScalibur (Becton Dickinson, San Jose, CA, USA) and BD CBA analysis software. The fluorescence intensity measured is proportional to the concentration of the cytokine in the sample, which is quantified from a calibration curve, generated according to the recombinant standards (calibrators). Each assay had a sensitivity range of pg/ml ELISPOT assay Ninety-six-well ELISPOT plates (Millipore, Eschborn, Germany) coated with primary IFN-c antibody (Perbio Science, Bonn, Germany) were blocked with RPMI 1640 containing 10% heat-inactivated fetal calf serum, 2 mm glutamine, 100 lg of streptomycin/ ml, and 100 U of penicillin) for 2 h. A total of PBMC were disposed in 100 ll of supplemented RPMI per well, and DENV-1, -2 or -3, or negative control antigen (non-infected C636 cells supernatant) were subsequently added. After an overnight incubation at 37 C and 5% CO 2 in air, plates were washed with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (Sigma). Next, 100 ll of PBS containing 1 lg of biotinylated anti-ifn-c monoclonal antibody 7-B6-1-biotin (PerbioScience, Bonn, Germany)/ml was added for 4 h at room temperature, then streptavidin-horse radish peroxidase was added for 90 min, and the plates were finally developed using 3-amino-9-ethylcarbazol (Sigma Aldrich, Munich, Germany). The number of spot-forming cells (SFCs) in each well was counted with the aid of a dissecting microscope. Wells containing PBMC stimulated with phytohemagglutinin (Sigma, Poole, United Kingdom) served as a positive assay control. The average number of spots per well was used to express each experimental value as SFCs per 300,000 PBMC. Responses were considered positive when a minimum of five SFCs per 300,000 PBMC were present per well. This cut-off was determined based on the results of samples from dengue non-immune individuals tested against dengue virus antigens, which developed 0 5 SFC/ 300,000 PBMC Statistical analysis Means of quantitative variables (TNF-a, IFN-c, IL-10, TGF-b) were analyzed between groups of patients according to the preceding dengue immunity and the serotype of dengue virus used for stimulation, using the Mann Whitney U-test. Values obtained were interpreted in the following way: p < 0.05 = statistically significant differences (), p < 0.01 = very statistically significant differences

3 136 B. Sierra et al. / Cellular Immunology 262 (2010) (), and p > 0.05 non-statistically significant differences (ns). The Mann Whitney U-test was also used for comparison of T cell frequencies between subgroups of individuals. Spearman s rank correlation was used to calculate bivariate correlations. The SPSS software package SPSS, version 10 (SPSS, Inc., Chicago, Il), was used for all analyses. 3. Results 3.1. DENV-specific long-term memory immune response The existence of a long-term memory cellular immune response to DENV 20 years after a single exposure was previously demonstrated [23]. Those results were corroborated in the present work, in which PBMC collected from individuals with a history of a primary infection by DENV-1 or -2 during the outbreaks of 1977 and 1981, respectively, were ex vivo stimulated for 24 h (detecting memory cell response only) with DENV-1, -2 and -3, in presence of autologous sera, resembling the situation of an in vivo secondary DENV infection (Figs. 1 3). A stronger support to the existence of a long-term memory cellular immune response to dengue virus after a natural primary infection came from the results of the IFN-c ELISPOT assay, performed to investigate the frequency of IFN-c producing cells to dengue viral antigens in PBMC from individuals with a history of a primary infection by DENV-1 or -2 during the outbreaks of 1977 and Since these IFN-c spots signify the cytokine production by single cells, i.e., the measurements reflect actual frequencies of IFN-c-producing cells responding to dengue virus antigen. Individuals immune to DENV-1 and DENV-2 had significantly higher ELISPOT frequencies compared with dengue non-immune individuals (Fig. 4). The magnitude of responses to dengue virus antigens ranged from 15 to 80 SFCs/ PBMC Distinct pattern of long-term memory T cell response to secondary challenge by serotype-specific (homologous) versus serotype crossreactive (Heterologous) DENV Individuals exposed to a particular DENV serotype long before are at particular high risk to develop DHF/DSS after exposure to a heterologous but not to a homologous DENV serotype [24,25]. Our ex vivo model of DENV-1 3 stimulation of PBMC from individuals exposed to DENV-1 in 1977 or DENV-2 in 1981 simulates this situation. Remarkably, exposure to serotype cross-reactive DENV triggered a significantly higher IFN-c and TNF-a gene expression than serotype-specific stimulation (Fig. 1). At protein level, IFN-c levels in DENV-1 immune individuals were 2.2- and 4.1-fold higher following exposure to DENV-2 and -3, respectively, compared to homologous DENV-1 stimulation (p < 0.01) (Fig. 2). Similarly, DENV-2 immune individuals secreted 3.2- and 3.1-fold more IFNc in response to heterologous DENV-1/3 re-exposure, respectively, compared with homologous DENV-2 stimulation (p < 0.01) (Fig. 2). On the contrary, the analysis of the expression of the regulatory cytokines IL-10 and TGF-b in response to heterologous stimulation showed no significant differences compare to mock (Fig. 3). Fig. 1. Enhanced TNF-a and IFN-c response to heterologous vs. homologous DENV re-challenge at gene expression level. PBMC of dengue-seropositive healthy individuals exposed more than 20 years ago to DENV-1 (n = 10) or DENV-2 (n = 10) were re-exposed ex vivo to DENV-1 virus (D1 challenge), to DENV-2 virus (D2 challenge), and to DENV- 3 virus (D3 challenge) in presence of autologous serum to simulate homologous and heterologous re-infection. Dengue non-immune individuals were used as negative controls. After 24 h culture, total RNA was extracted for real-time RT-PCR analysis and TNF-a and IFN-c /housekeeping ratio was determined according to the delta/delta Ct method. C, Controls (dengue non-immune individuals); D1, dengue 1 immune individuals; D2, dengue 2 immune individuals. Data are shown as mean levels and box-plots. Each box plot shows statistic data about the gene expression determination. The central horizontal line in the box marks the median of the samples, the box edges (hinges) the first and third quartile. The interquartile range within the box includes the central 50% of the values. The whiskers show the maximum and minimum values. Statistical significant differences (Mann Whitney rank sum test) in cytokine production among groups are indicated. A p < 0.01 was considered statistically significant. A p > 0.01 was considered not statistically significant (NS).

4 B. Sierra et al. / Cellular Immunology 262 (2010) Fig. 2. Enhanced inflammatory memory immune response to heterologous vs. homologous re-challenge at protein levels. PBMC of dengue-seropositive healthy individuals exposed >20 years ago to DENV-1 (n = 10) or DENV-2 (n = 10) were re-exposed ex vivo to DENV-1, -2, -3 serotype in presence of autologous serum to simulate homologous and heterologous re-infection. Dengue na individuals were used as negative control. After 24 h culture, cytokines released into the supernatant (IFN-c, IL-10 and IL-2) were quantified using a human Th1/Th2 cytokine bead array (CBA) assay. C, Control (dengue non-immune) individuals; D1, dengue 1 immune individuals; D2, dengue 2 immune individuals. Data are shown as mean levels and box-plots. Each box plot shows statistic data about the gene expression determination. The central horizontal line in the box marks the median of the samples, the box edges (hinges) the first and third quartile. The interquartile range within the box includes the central 50% of the values. The whiskers show the maximum and minimum values. Statistical significant differences (Mann Whitney rank sum test) in cytokine production among groups are indicated. A p < 0.01 was considered statistically significant. A p > 0.01 was considered not statistically significant (NS). In contrast, response to homologous DENV re-challenge showed significantly higher expression of IFN-c and TNF-a compared to the mock, but also a higher up-regulation of TGF-b and IL-10 gene expression in for both DENV-1 and DENV-2 immune individuals (Fig. 3). At protein level, IL-10 was about twofold higher expressed, following homologous vs. heterologous re-challenge, and confirming the PCR data (Fig. 2). Other markers, like IL-2, showed not significant differences among heterologous and homologous re-challenge at mrna and at protein level (Fig. 2). IL-4/IL-5 up-regulation was not detectable at either mrna or protein level following homologous as well as heterologous response (data not shown). 4. Discussion The significant increased levels of IFN-c and IL-2 in seropositive individuals vs. seronegative age-matched controls indicates a strong and specific cellular memory response to the three DENV serotypes even 30 years after a natural primary infection in the absence of any in vivo exposition to DENV in between. The population in Cuba suffered two large epidemics due to DENV-1 (1977) and DENV-2 (1981) and the data recorded from 1982 to the present demonstrates that no dengue 1 or dengue 2 viruses had circulated in Havana during this 30 years [26]. The dengue 2 epidemic occurred in Santiago de Cuba in 1997 showed that a primary heterotypic infection is a risk for the developing of the severest clinical presentation, even 20 years after the primary infection. Due to the intensive dengue infection surveillance carried out from 1981 to 1997 it became clear that during the Santiago de Cuba outbreak DHF/DSS cases occurred in persons infected initially with DENV-1 in [3,4]. Our results confirm the existence of memory cell response that exhibits serotype-specific but also cross-reactive response to dengue virus 30 years after the primary dengue infection. This imply a high risk for the development of the severest dengue clinical picture for the individuals with antecedents of an old dengue infection by dengue 1 and dengue 2 of Havana city after a heterologous DENV outbreak as already occurred during the DENV Havana outbreak. The study of dengue serologic responses of all but 3 of the 78 patients with DHF/DSS in the DENV-3 Havana outbreak in sera obtained months after clinical dengue illnesses showed a past infection with DENV-1 and DENV-2 prior to infection with DENV-3 [27]. The pathogenesis of the most severe complications of DENV infection, the DHF/DSS, is not very well understood. The detection of increased serum levels of cytokines and chemokines in dengue severe patients and the observation that secondary infections by

5 138 B. Sierra et al. / Cellular Immunology 262 (2010) Fig. 3. Enhanced TGF-b and IL-10 response to homologous vs. heterologous DENV re-challenge. PBMC of dengue-seropositive healthy individuals exposed more than 20 years ago to DENV-1 (n = 10) or DENV-2 (n = 10) were re-exposed ex vivo to DENV-1 virus (D1 challenge), to DENV-2 virus (D2 challenge), and to DENV-3 virus (D3 challenge) in presence of autologous serum to simulate homologous and heterologous re-infection. Dengue non-immune individuals were used as negative controls. After 24 h culture, total RNA was extracted for real-time RT-PCR analysis and TGF-b and IL-10 /housekeeping ratio was determined according to the delta/delta Ct method. C, Controls (dengue non-immune individuals); D1, dengue 1 immune individuals; D2, dengue 2 immune individuals. Data are shown as mean levels and box-plots. Each box plot shows statistic data about the gene expression determination. The central horizontal line in the box marks the median of the samples, the box edges (hinges) the first and third quartile. The interquartile range within the box includes the central 50% of the values. The whiskers show the maximum and minimum values. Statistical significant differences (Mann Whitney rank sum test) in cytokine production among groups are indicated. A p < 0.01 was considered statistically significant. A p > 0.01 was considered not statistically significant (NS). a different (heterologous) DENV serotype express the highest risk of DHF/DSS development suggest that the immune response is one of the key players in the pathogenesis of this disease [28]. By using an ex vivo model simulating different sequences of DENV-(re)challenges and taking in advantage the exceptional Cuban epidemiological situation, different patterns of cellular immune response were observed in the markers studied that could be associated to protection or pathogenesis of DENV infection. Our study shows that heterologous ex vivo re-challenge with DENV serotypes 1 3 in cells collected from DENV-1 or DENV-2-immune individuals infected decades before induced a dominant inflammatory (IFN-c, TNF-a) response with a weak expression of regulatory cytokines (TGF-b, IL-10). On the contrary, the homologous re-challenge triggered an opposite pattern dominated by regulatory cytokines, besides significant expression of IFN-c/TNF-a compare to mock. IFN-c/TNF-a are key players in cell-mediated inflammation, both in infectious and non-infectious diseases, being controlled by anti-inflammatory cytokines, like TGF-b and IL-10 [8,29 31]. The equilibrium between these two branches of the immune response is important for a balanced and effective control of the dengue infection. The incontrollable inflammatory response (IFN-c/ TNF-a) in the absence of an adequate regulatory control (TGF-b/ IL-10) could explain the enhanced risk of DHF/DSS after a heterologous re-infection in individuals exposed to DENV long before. It have been reported that CD4+ T cells produce TNF-a, IFN-c and TNF-b after DENV stimulation, which could contribute to the pathogenesis of the secondary DENV infection. [32,33] An increase in IFN-c levels results in an increase in the expression of Fc receptor, leading to a higher number of DENV-infected monocytes through the ADE mechanism and consequently to a higher T cell activation, and, in turn, to the release of TNF-a, IFN-c and interleukin-2 (IL-2) [30,31,34 36]. Besides lymphocytes, monocytes/macrophages, mast cells, endothelial cells, liver and dendritic cells are targets of DENV and could produce cytokines and chemokines after the infection as it has been reported in dengue patients [5,37]. This tsunami of cytokines over a short period induces the disruption of vascular endothelial cells and deregulation of the haemocoagulation system, conducting to plasma leakage, shock, and hemorrhagic manifestations [8]. Since DHF has been associated with a heterotypic secondary infection [38 42], the significantly higher pro-inflammatory DENV cellular immune response found in our study, in the serotype cross-reactive re-challenge, can be related to the cascade of events generated during a heterologous infection in vivo contributing to the pathogenesis of DHF/DSS. The higher levels of TGF-b1 and IL-10 found in the homologous DENV re-challenge constitute an interesting finding, taking into account that the role of both mediators as regulatory and antiinflammatory cytokines, has not been clearly associated to the serotype-specific response. It is accepted that a primary infection induces a long lasting protective homologous immunity [43 46]. Several studies correlate the over expression of pro-inflammatory cytokines and over-activation of the immune system with dengue severity [34,47 49], thus expecting that a protective immune status be associated to an anti-inflammatory environment [50 52].

6 B. Sierra et al. / Cellular Immunology 262 (2010) Fig. 4. Frecuence of IFN-c dengue-specific T cells. PBMC of dengue-seropositive healthy individuals exposed more than 20 years ago to DENV-1 (n = 10) or DENV-2 (n = 10) were re-exposed ex vivo to DENV-1 virus (D1 challenge), to DENV-2 virus (D2 challenge), and to DENV-3 virus (D3 challenge) in presence of autologous serum to simulate homologous and heterologous re-infection. Controls: dengue non-immune individuals, D1 immune: dengue 1 immune individuals, D2 immune: dengue 2 immune individuals. NC: Controls (supernatant of uninfected C636 cells), D1: dengue 1 virus antigen, D2: dengue 2 virus antigen, D3: dengue 3 virus antigen. Data are shower as Scatter dot plot. Mean and single values of frequencies of IFN-c-producing cells in dengue immune individuals with versus dengue non-immune individuals are showed. Statistical Mann Whitney rank sum test was employed. A p < 0.01 was considered statistically significant. A p > 0.01 was considered not statistically significant (NS). The higher levels of IL-10 found after the homologous re-challenge could be in apparent contradiction with the pathogenic role attributed to this cytokine, taking into consideration that high IL- 10 levels have been reported in DHF patients [53 55]. However, this apparent contradiction is explainable as the high concentrations of inflammatory mediators like TNF-a and IFN-c, released during the clinical course of the severe disease, could induce the regulatory induction of high levels of IL-10 and TGF-b1. The regulatory activities of both cytokines are probably crucial in controlling the inflammatory response during dengue disease, preventing its associated immunopathogenesis and their actions could be related to the recovery of most patients with a secondary infection [56 58]. Interestingly, Lühn et al. explored the role of regulatory T cells in acute dengue infection showed that T reg cells suppressed the production of vasoactive cytokines after denguespecific stimulation. Furthermore, T reg cell frequencies and also T reg cell/effector T cell ratios were increased in patients with acute infection, being significantly increased in patients with mild disease in contrast to severe cases. It is a strong indication that a relative rise of T reg cell/effector T cell ratios is beneficial for dengue disease outcomes [59]. Our data suggest that the presence of an effective antiviral inflammatory response under an adequate immune regulation could be associated to protection in dengue secondary infection. Further studies are needed to characterize the contribution of the different immune cell subsets to the inflammatory-regulatory response. Acknowledgments The authors acknowledge the contribution of Dr. Christian Meisel by discussing the methods, and Christa Liebenthal and Kristin Neuhaus for their technical assistance. The authors thank Dr. Armando Martinez-Cambray and Dr. Linda Lloyd for the helpful reviewing of this paper. References [1] E.A. Henchal, J.R. Putnak, The dengue viruses, Clin. Microbiol. Rev. 3 (1990) [2] E.B. Hayes, D.J. Gubler, Dengue and dengue hemorrhagic fever, Pediatr. Infect. Dis. J. 11 (1992) [3] T.P. Monath, Dengue: the risk to developed and developing countries, Proc. Natl. Acad. Sci. USA 91 (1994) [4] I. Kurane, Dengue hemorrhagic fever with special emphasis on immunopathogenesis, Comp. Immunol. Microbiol. Infect. Dis. 30 (2007) [5] U.C. Chaturvedi, R. Agarwal, E.A. Elbishbishi, A.S. Mustafa, Cytokine cascade in dengue hemorrhagic fever: implications for pathogenesis, FEMS Immunol. Med. Microbiol. 28 (2000) [6] A. Mathew, A.L. Rothman, Understanding the contribution of cellular immunity to dengue disease pathogenesis, Immunol. Rev. 225 (2008) [7] C. Suharti, E.C. van Gorp, T.E. Setiati, W.M. Dolmans, R.J. Djokomoeljanto, C.E. Hack, C.H. Ten, J.W. van der Meer, The role of cytokines in activation of coagulation and fibrinolysis in dengue shock syndrome, Thromb. Haemost. 87 (2002) [8] A.L. Rothman, Immunology and immunopathogenesis of dengue disease, Adv. Virus Res. 60 (2003)

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