Virology Reports (2017) 9–19 Contents lists available at ScienceDirect Virology Reports journal homepage: www.elsevier.com/locate/virep Induced nitric oxide synthase (iNOS) and indoleamine 2,3-dioxygenase (IDO) detection in circulating monocyte subsets from Brazilian patients with Dengue-4 virus Luciana Gomes Fialho a, Amanda Torrentes-Carvalho a, Rivaldo Venâncio Cunha c, Nieli Faria a, Mariana Gandini b, Márcio Cipitelli a, Luzia Maria de-Oliveira-Pinto a, Elzinandes Leal Azeredo a,1, Claire Fernandes Kubelka a,⁎,1 a b c Laboratório de Imunologia Viral, Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro, Brazil Laboratório de Microbiologia Celular, Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro, Brazil Departamento de Clinica Medica, FM, Universidade Federal Mato Grosso Sul, Campo Grande, MS, Brazil a r t i c l e i n f o Article history: Received 18 October 2016 Received in revised form February 2017 Accepted February 2017 Available online 07 February 2017 a b s t r a c t Among viral diseases transmitted by mosquitoes, dengue is characterized by its rapid dispersion around the world Dengue severity is associated to a cytokine “storm” leading to vascular hemorrhagic manifestations, plasma leakage and shock, but also producing viral clearance Macrophage/monocyte activation occurs during infection Monocyte lineage cells are among those that allow virus replication We investigated circulating human monocyte subsets - classical CD14+ CD16− and non-classical CD14+ CD16+ - during DENV-4 infection in patients Intracellular inducible nitric oxide synthase (iNOS) and indoleamine 2,3–dioxygenase (IDO) were detected in both monocyte subsets Circulating CD14+ CD16+ monocyte frequency is mildly increased during DENV-4 infection INOS is more intensely detected in CD14+ CD16− than in CD16+ monocytes and IDO in CD14+ CD16+ DENV-4 patients show increase in NO, TNF-α, IFN-y, IP-10/CXL10, IL-10 and MCP-1/CCL2 plasma levels when compared to healthy individuals The classical monocyte subset, CD14+ CD16− was shown to be inversely correlated with IL-10 and IP-10/CXCL10 levels, while the non-classical CD14+ CD16+ is positively correlated with IL-10 cytokine TNF-α, IL-10 cytokines and IP-10/CXL10 chemokine are positively correlated with the CD14+ iNOS+ monocyte population Both CD14+ cells - CD16− iNOS+ and CD16+ iNOS+ subsets - presented positive correlation with IL-10, IP-10/CXL10 and MCP-1/ CCL2, besides TNF-α associated with CD16− iNOS+ cells CD14+ CD16− IDO+ and CD16+ IDO+ populations correlated positively with IL-10 Furthermore, CD16− IDO+ monocyte subset also presented a positive correlation with TNF-α and IP-10/CXCL10 According to these data, we considered that iNOS and IDO are activated in monocyte CD16− and CD16+ subsets, likely exerting both antiviral effects and modulating exacerbated immunological responses during dengue fever © 2017 The Authors Published by Elsevier B.V This is an open access article under the CC BYNC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction In the last decade, the emergence of new recognized arboviruses and the re-emergence of others have been reported Among arboviruses transmitted by mosquitoes, Dengue virus (DENV) spreads more quickly around the world During the last 50 years, there was a 30% increase in incidence, with geographical expansion to new countries (WHO, 2009) In Brazil, N1.5 million ⁎ Corresponding author at: Laboratório de Imunologia Viral, Instituto Oswaldo Cruz/FIOCRUZ, Av Brasil, 4365, Rio de Janeiro, RJ CEP 21045900, Brazil E-mail address: claire@ioc.fiocruz.br (C.F Kubelka) Authors contributed equally to the work http://dx.doi.org/10.1016/j.virep.2017.02.001 2214-6695/© 2017 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/4.0/) 10 L.G Fialho et al / Virology Reports (2017) 9–19 cases were reported until the 51th epidemiological week of 2016, with 844 confirmed cases of severe dengue, and 8237 cases of dengue fever with warning signs and 629 deaths by dengue (Brazilian-Federal-Government, 2017) The DENV is transmitted mainly by Aedes aegypti mosquitoes (Gubler, 2002), belonging to the genus Flavivirus, family Flaviviridae with four specific serotypes (DENV-1 to -4) (Mukhopadhyay et al., 2005) of greater medical importance in the world among human arboviruses, causing an acute febrile disease, of wide clinical spectrum that ranges from an unapparent infection to severe and fatal forms (WHO, 2009) The DENV-4 serotype, detected for the first time in Brazil in 1982, re-emerged in 2010 when it was disseminated to several states (Figueiredo et al., 2008; Temporao et al., 2011) Several features such as the hosts, viral and epidemiological factors have been linked to the pathogenesis of the disease The significant increase in soluble inflammatory mediator's levels, known as “cytokine storm” is present in the most severe forms of the disease (Srikiatkhachorn and Green, 2010) The exacerbated production of various inflammatory mediators may promote platelet and endothelial activation, resulting in thrombocytopenia, vascular permeability, hypotension and shock, as observed in severe cases However, some cytokines, such as interferons, play a protective role during DENV infection In this study, we investigated patient's samples from a DENV-4 epidemic occurred during the year of 2013, in Mato Grosso Sul (MS), an endemic area for dengue viruses The introduction of a new serotype (DENV-4) in this state has been observed with extreme concern due to the high vector density and population susceptibility to this dengue serotype thus offering potential to cause epidemics (Bertolacci-Rocha et al., 2014) The first cases of DENV-4 detected in Mato Grosso Sul occurred in Campo Grande, the state capital and the most populous city in MS After DENV-4 introduction, a large dengue epidemic occurred in 2013 with 67.150 notifications reported (Brazilian-Federal-Government, 2014) In vitro studies indicate that monocytic lineage cells, such as monocytes and immature dendritic cells are main target cells allowing viral replication (Kou et al., 2008; Wu et al., 2000) In the peripheral blood, the human monocyte population may be divided into two subsets, according to expression of CD14 and CD16 markers (Passlick et al., 1989; Ziegler-Heitbrock, 1996) In healthy physiological conditions, most of the total CD14+ monocyte population is composed by the so-called CD16− cells, corresponding to 90%, while the remaining 10% correspond to CD16+ population These monocyte subsets may produce different cytokines, exhibiting distinct phenotypes, functions and inflammatory profiles (Skrzeczynska-Moncznik et al., 2008; ZieglerHeitbrock, 1996) In several infectious and inflammatory conditions when CD16+ monocytes are elevated they may play proinflammatory roles (Ziegler-Heitbrock, 2007) Enzymes indoleamine 2,3-dioxygenase (IDO) and induced nitric oxide synthase (iNOS) are among molecules expressed upon monocyte activation IDO participates in tryptophan catabolism occurring in liver (WHO, 2009) and may be induced by IFN-γ in monocytes (Ozaki et al., 1987) It may exert antimicrobial and immunosuppressive activities (Mellor and Munn, 2004) In dengue patients' sera a decrease in the tryptophan/kynurenine rate was observed, indicating tryptophan catabolism by IDO activity (Becerra et al., 2009); no significant differences in IDO levels between severe and uncomplicated dengue patients were observed (Villar et al., 2013) The role of IDO in the severity of the disease and its role in dengue is not entirely clear NOS enzymes contribute to transmit signals in different body cell systems, via NO synthesis Induced isoform (iNOS), may be stimulated by cytokines and other inflammatory processes (MacMicking et al., 1997) After DENV infection, activated monocytes display iNOS detected in patient's cells or after in vitro infection There are also evidences that nitric oxide (NO), the NOS product, may exert antiviral activity in DENV infection Considering that iNOS and IDO enzymes may be involved in dengue infection and that DENV activate specific monocyte subsets inducing production of some pro- and anti-inflammatory cytokines/chemokines, we aimed to verify whether CD16− and CD16+ monocyte subsets would express iNOS and IDO also investigating their associations with cytokines/chemokines produced during a DENV-4 infection Materials and methods 2.1 Sample collection and laboratorial diagnosis The study was carried out in Campo Grande, MS, Brazil, during a DENV-4 epidemic occurred in 2013 Patients were assisted at Professora Hesterina Corsini Dia-Hospital, at Mato Grosso Sul Federal University, during the months of February–April, in collaboration with Dr Rivaldo Venâncio da Cunha Samples were collected during the acute phase of the disease and data from history and detailed clinical examination were registered immediately after admission Cases included during the study were submitted to investigation Blood from patients was collected for complete cell counts, aminotransferase and albumin determinations, among others All these laboratory determinations were performed at the Hospital facilities and obtained from patient's records For diagnosis of the suspected dengue cases the following assays were performed: the IgM capture test (Panbio dengue virus IgM capture ELISA, Alere); NS1 antigen detection, by PLATELIA™ Dengue NS1 Ag kit (Biorad Laboratories), and the molecular diagnosis by RT-PCR In some patients, PCR assay was not performed and in others, negative results were obtained due to the advanced course of the disease Such patients were confirmed by the IgM or NS1 ELISA, or by both assays After diagnosis confirmation, patients were classified according to their clinical symptoms and were then grouped according to parameters established by the World Health Organization (WHO, 2009) The adopted classification was: dengue without warning signs (DF without WS) characterized by vomiting, pain, leucopenia, positive bowel test, dengue with warning signs (DF with WS) characterized by abdominal pain, persistent vomiting, mucosal bleeding, enlarged liver or severe dengue (SD) characterized by severe plasma leakage and bleeding and severe organ involvement Ten individuals were enrolled in the study as healthy donors considering they showed no episodes of fever for N months or a history of other diseases L.G Fialho et al / Virology Reports (2017) 9–19 11 2.2 Blood mononuclear cell preparation and cryopreservation About 20 ml of peripheral blood from each patient in acute phase was collected into citrate or heparin tubes Peripheral blood mononuclear cells (PBMCs) were purified by centrifugation on density gradient (Ficoll-Paque, Sigma), resuspended in freezing solution (FBS containing 10% dimethyl sulfoxide) and cryopreserved in liquid nitrogen for further studies Plasma and sera samples were aliquoted and stored at −70 °C for subsequent measurement of soluble mediators 2.3 Soluble factors quantification by immunoassays Plasma and sera samples were obtained from DENV patients and control subjects and stored at −70 °C Thirty-five patients and ten control samples were tested for cytokine/chemokine content Stored plasmas were thawed and IFN-γ, TNF-α and IL-10 cytokines were quantified by High-Sensitivity (0.13–2000 pg/ml) multiplex microbead biometric immunofluorescent assay kits (eBioscience), according to the manufacturer's instructions and analyzed by a multiplex array reader (Bio-Plex Workstation & Bio-Plex Manager Software from Bio-Rad Laboratories) Levels of MCP-1/CCL2 and IP-10/CXL10 chemokines levels were detected by ELISA (Peprotech 900-M31) in sera samples diluted in 1:10 Standard curves were prepared by serial dilutions (15–1000 pg/ml) of the aliquot corresponding to cytokine pattern supplied by the manufacturer Determination of the optical density of samples and standards was performed using SpectraMax spectrophotometer with a wavelength of 620 nm Five-Parameter Logistic Regression was used to estimate circulating levels from the different factors 2.4 NO−2 detection by Griess reaction Griess reagent (Sigma #G4410) was mixed with equal volume of plasma samples (100 μl/well) in a 96-well microplate After 15 incubation, the determination of nitrite levels was performed in a plate reader (540 nm) from a standard curve with values between and 80 μM As a positive control, it was used a NO donor was used (SNAP-10 μg/ml) and a NO inhibitor (L-NMMA-10 μg/ml) was used as a negative control 2.5 Extra and intracellular labeling for flow cytometry analysis of PBMCs Cryopreserved PBMCs' aliquots from patients and healthy donors, were thawed in water bath at 37 °C, centrifuged (350 g, min) and washed once with ml PBS pH 7.4 with 2% FBS and 0.01% NaN3 Cells were counted and dead cells excluded by trypan blue dye Each aliquot contained 0.3 × 106–4.2 × 106 viable cells, corresponding to a 33–89% of cell viability Multiple labeling of PBMCs was performed for flow cytometry analysis Cells were firstly blocked with PBS pH 7.4 containing 1% BSA, 0.1% NaN3 and 5% inactivated human plasma and extracellularly labeled with an anti-CD14 (PercP) and anti-CD16 (APCeFluor780) conjugated antibodies Then, they were permeabilized and labeled for intracellular markers with anti-iNOS (AlexaFluor488) and anti-IDO (PE) conjugated antibodies After a few washing sessions, the cells were fixed and stored at °C until acquisition At least 10.000 events were acquired on a FACSAria (BD) cytometer and analyses were performed with the FlowJo version 7.6.1 software (Tree Star Inc., San Diego, CA) Isotype antibody control for CD16 marker was performed in CD14 labeled cells with isotypes for INOS and IDO antibodies For INOS and IDO MFI nonspecific determination, isotype antibodies were used in CD14 and CD16 labeled cells and isotype MFI values were subtracted from samples labeled with anti-INOS and anti-IDO antibodies 2.6 Statistical analysis Data obtained from assays that presented normal distribution were analyzed by unpaired t-test Nonparametric Mann–Whitney U test was used to evaluate differences between tested groups of controls and total DF Correlations between cytokine/ chemokine production and the monocyte subsets were estimated by Spearman's correlation P-values b 0.05 were considered statistically significant Statistical analyses were performed using GraphPad Prism software, version 6.0 2.7 Ethics statement Procedures performed in this study are part of a project approved by the Ethics Committees of FIOCRUZ Research, Ministry of Health (CAAE: 13318113.7.0000.5248) All participating patients agreed with the Project by signing a consent form, free and well informed Results 3.1 Clinical, laboratorial and demographic characteristics Thirty-five patients with positive diagnosis of dengue infection at the acute phase (2–9 days after disease onset) during a DENV-4 outbreak in Mato Grosso Sul, Brazil, in 2013 were included in this study Among them, 25 (71%) patients were classified as dengue fever (DF) without warning signs (WS) and 10 (29%) as dengue fever with WS, according to the classification adopted by WHO (2009) Demographical, clinical and laboratorial data of the study population are shown in Table 12 L.G Fialho et al / Virology Reports (2017) 9–19 Most of subjects were male, although there was no difference between groups neither regarding genre nor age of the study group with a median age of 36 years Considering manifestations associated with risks of unfavorable outcomes, DENV patients with WS presented plasma leakage (one) or hemorrhagic manifestations (four) such as melena, hematuria and/or metrorrhagia, representing 14% of total patients Data reflect mild characteristics recorded for the infection by DENV serotype in MS at this period (Faria et al., 2016) Thrombocytopenia was one of the most common hematologic alterations observed during the acute phase of DENV infection As expected, significantly lower platelet counts were found in the group with WS Changes in laboratorial parameters such as leukopenia, higher hematocrit and circulating NS1 detection besides elevated AST and ALT levels are commonly observed in patients with severe DF compared with mild cases (Ferreira et al., 2015) In our study group, there were No statistical differences between groups DF without WS and DF with WS were observed in this study Although circulating AST and ALT levels were not significantly different between these two groups (Table 1), DF patients with WS showed considerable increase in these enzyme levels, when compared to the patients without WS Monocyte counts also showed no significant difference in the DF group with WS comparing to DF without WS (Table 1) More than half of patients were confirmed for DENV infection through a positive IgM assay, by ELISA Among them, 11 patients were only IgM positive, while the remaining were confirmed detecting RNA, by a PCR technique, or viral NS1, by ELISA, or even by the association of two or more assays Patients that were not tested by the PCR assay or showed a negative result, were already at advanced stage of the disease One patient from DF group without WS presented positive PCR for DENV-1, one patient from DF group with WS presented positive PCR for DENV-2 and the n = patients were positive for DENV-4 The Campo Grande epidemic in 2013 where these patients originated was predominantly marked by Dengue virus serotype 4, based on laboratorial diagnosis of all patients analyzed during the collection period (Faria et al., 2016) 3.2 Monocyte subset CD14+ CD16+ is mildly expanded during DENV-4 infection PBMC thawed samples obtained from dengue patients, as well as from healthy donors, were used for the study of monocyte subsets, applying immunofluorescent techniques and flow cytometry analysis (Fig 1A e 1B) Monocyte subsets defined as classic monocytes (CD14+ CD16−) and non-classic or inflammatory monocytes (CD14+ CD16+) were evaluated for their frequencies among PBMCs from DENV-4 infected patients and compared to control individuals (Fig 2) Frequency of the classic monocyte subset, CD16−, within CD14+ cells ranged from 96 to 98% (95% CI of mean) for the control group and from 91 to 95% for patients infected with DENV It was observed a mild statistically significant decrease in this monocyte subset in DF patients (Fig 2A) On the other hand, the frequency of non-classic monocytes subset CD16+ cells within CD14+, was of approximately 2.2–3.5%.% (95% CI of mean) for controls and 5.1–8.4% for patients A mild significant increase in this population was observed in DF group as compared to controls (Fig 2B) Table Clinical, laboratorial and demographic parameters of dengue patients Classification WHO (2009) Characteristicsa Demographic data Age in years Genre (M:F) Days of diseaseb Hemorrhagic manifestations Bleedingc Plasma leakaged Laboratorial parameters Lowest platelets (counts × 103/mm3) Hematocrit (%) AST/TGO peak (U/L) ALT/TGP peak (U/L) Total leukocytes (counts × 103/mm3) Monocytes (%) Anti-dengue IgM (%) NSI dosage (ng/mL) DENV-1:DENV-2:DENV-4:Negative PCR DF without WS n = 25 DF with WS n = 10 35 (25–60) 14:11 4.0 (4.0–6.0) 41 (23–55) 05:05 6.5 (3.0–8.0) 0/25 0/25 04/10 01/10 137 (103–182) 44 (41–46) 55 (36–149) 66 (48–110) 4100 (2900–5200) 4.8 (3.3–6.4) 52% 3.4 ± 1.9 1:0:7:13 60 (17–221)* 44 (38–50) 128 (53–149) 126 (61–198) 3800 (1800–5000) 3.0 (1.3–7.0)* 60% 2.2 ± 1.3 0:1:2:6 Statistical differences between the DF without WS and DF with WS groups were analyzed by Mann Whitney U test Statistical differences were observed only in the platelets and monocytes counts * (P b 0.05) a DF, dengue fever WS, warning signs Median (95% CI, confidence interval) b Days of disease, day of symptoms onset until the patient interview c Bleeding, includes rash, epistaxis, gingival bleeding, melena, hematuria and/or metrorrhagia d Plasma leakage includes signs as ascites, pleural effusion, and/or from pericardium L.G Fialho et al / Virology Reports (2017) 9–19 Fig Contour and dot plots from flow cytometry analysis representing monocyte gates and iNOS/IDO histograms within CD14+ CD16− or CD16+ subsets in dengue patients PBMCs from dengue patients or from healthy donors were isolated and labeled as described in Materials and methods Scatter dot plots represent data from PBMCs in one healthy donor and one DENV patient, analyzed by flow cytometry for CD14 and CD16 expression Monocyte gated cells were analyzed for CD14-PerCP and CD16- -Alexa Fluor780 labeling and CD16 isotype control is shown in CD14 labeled cells The representative contour plots show the percentages of double CD14+ CD16− and CD14+ CD16+ cell subsets in a healthy donor (A) or in a DENV patient (B) 13 14 L.G Fialho et al / Virology Reports (2017) 9–19 Fig Frequencies of circulating CD14+ CD16−/CD16+ cells in dengue patients PBMCs from DENV patients were thawed, labeled with anti-CD14PerCP, anti-CD16Alexa Fluor780 and the CD16 isotype control and analyzed by flow cytometry Control - group of healthy individuals; DF - dengue fever; WS - warning signs (A) CD16− cell frequencies within CD14+ cells; (B) CD16+ cell frequencies within CD14+ cells Statistical differences between groups were analyzed by Unpaired t-test ** P b 0.01 were considered significant 3.3 Intensity of iNOs and IDO enzymes detected during DENV-4 monocyte infection are increased and associated with specific CD16+ or CD16− subsets Firstly, the presence of iNOS and IDO enzymes within the total CD14+ monocyte population was evaluated during DENV-4 infection in PBMCs from infected patients and control subjects Within total CD14+ monocyte population, iNOS and IDO enzymes were quantified regarding their mean fluorescence intensity (MFI) DENV-4 infected patients increased in both iNOS and IDO detection when compared to healthy controls (Fig 3A and B, respectively) However, increase in IDO enzyme in infected patients was not so intense as detected for iNOS Furthermore, the presence of iNOS and IDO was evaluated in different monocyte subsets As demonstrated in Fig 3C, the iNOS detection presented higher MFI levels in the CD14+ CD16− subset than in CD16+ cells both in control subjects as well as in DENV-4 infected patients iNOS intensity was significantly increased in monocytes from infected patients in both CD16+ and CD16− subsets when compared to healthy controls Opposing to what was observed for iNOS, IDO MFI detection was higher in the non-classic monocyte subset, CD14+ CD16+, both in cells from control subjects and DENV-4 infected patients (Fig 3D) A significant increase in IDO intensity was observed in monocyte subsets, CD14+ CD16− and CD14+ CD16+, in patients when compared to healthy controls 3.4 DENV-4 patients show an increase in NO plasma levels NO produced by iNOS induction in monocytes has several physiological effects in the organism such as regulator of blood pressure, immunological agent and also as an important antiviral factor including DENV infections (Chareonsirisuthigul et al., 2007; Levy et al., 2010; Dusse et al., 2003) Therefore, NO presence was evaluated in plasma of dengue patients and compared to those from healthy controls Previously frozen plasma samples from patients and control individuals were used for detection of nitrite, a product originated from NO reaction with oxygen, since the stability of NO molecule is low in biological samples When compared to control subjects (Fig 4), patients infected with DENV-4 showed a significant increase in nitrite production, with levels similar to the positive control - a NO donor (SNAP) used in the assay 3.5 Different monocyte subsets are correlated with plasmic cytokine/chemokine levels in DENV-4 patients It is well known that cytokines and inflammatory mediators are involved in the immunological response to DENV infection and some are associated with more severe clinical manifestations (Bozza et al., 2008; Srikiatkhachorn and Green, 2010) On the other hand, some factors may play a protective role during infection and are involved in viral clearance (Fagundes et al., 2011) Cytokines and inflammatory mediators can also be involved in cell activation related to immune system regulation, acting on monocytes and leading to induction of enzymes, such as iNOS and IDO (Eleftheriadis et al., 2016; Neves-Souza et al., 2005; Pang et al., 2016) In order to evaluate inflammatory and anti-inflammatory factors produced by monocyte subsets during the course of DENV-4 infection in Brazilian patients and related to iNOS and IDO activation, cytokines and chemokines were detected in patients' plasma by immunoassays Circulating levels of IFN-γ and TNF-α cytokines significantly increased in DENV-4 patients when compared to controls (Table 2) Anti-inflammatory cytokine IL-10 was also detected as increased significantly in the studied patients Both chemokines MCP-1/CCL2 and IP-10/CXCL10 also presented higher circulating levels in DENV-4 patients when compared to healthy individuals The association of cytokine production with classical (CD14+ CD16−) or non-classical (CD14+ CD16+) monocytes subsets, besides the positive subpopulations for iNOS and IDO were evaluated L.G Fialho et al / Virology Reports (2017) 9–19 15 Fig Mean fluorescence intensity (MFI) of iNOS or IDO within CD14+ cells in dengue patients according to monocyte subsets PBMCs from DENV infected patients and healthy controls were labeled with anti-CD14, anti-CD16, anti-iNOS and anti-IDO antibodies and analyzed by flow cytometry MFIs for iNOS (A) and IDO labeling (B) within total CD14+ monocytes were analyzed and infected patients (DENV) were compared with healthy subjects MFIs of (C) iNOS and (D) IDO positive cells were also evaluated according to monocyte subsets MFI from isotype labeled samples were subtracted from the MFI obtained in specific INOS and IDO antibodies (see Materials and methods) The statistical differences observed between groups were evaluated by Mann Whitney U test * P b 0.05, ** P b 0.01 and *** P b 0.001 were considered significant Fig Nitrite (NO− ) level in plasma from dengue patients The nitric oxide (NO) concentration was indirectly quantified in plasma samples from dengue patients and healthy subjects The samples were thawed and used for nitrite (NO− ) determination, a product of NO reaction with the oxygen, by Griess reaction, as described in Materials and methods As a positive control, NO donor (SNAP) was used and a NO inhibitor (L-NMMA) was used as a negative control, both at concentrations of 10 μg/ml The statistical differences between NO− values from healthy subjects and dengue patients were analyzed by Mann Whitney U test ** P b 0.01 were considered significant 16 L.G Fialho et al / Virology Reports (2017) 9–19 Table Determination of circulating cytokines/chemokines in dengue patients Cytokines/chemokines and inflammatory mediators Control IFN-γ TNF-α MCP-1/CCL2 IL-10 IP-10/CXL10 Dengue fever 0.73 (0.17–0.73) 3.39 (2.63–6.20) 137 (26–340) 0.46 (0.34–0.81) 64 (24–166) 313 (135–411)⁎⁎⁎ 9.00 (7.18–11.75)⁎⁎⁎ 659 (320–800)⁎⁎⁎ 2.38 (0.97–3.45)⁎⁎⁎ 770 (414–811)⁎⁎ a a Median (95% CI, confidence interval); cytokines/chemokines were measured in Brazilian patients with dengue and compared to healthy individuals Statistical differences between controls × DENV groups were analyzed by Mann Whitney U test ⁎⁎ P b 0.01 ⁎⁎⁎ P b 0.001 The classical monocyte subset, CD14+ CD16− was negatively correlated with the IL-10 cytokine and IP-10/CXL10 chemokine The non-classical CD14+ CD16+ monocytes correlated positively with IL-10 (Table 3) With respect to monocyte subsets expressing iNOS, both CD14+ CD16− iNOS+ and CD14+ CD16+ iNOS+ subsets presented a positive correlation with IL-10, IP-10/CXL10 and MCP-1/CCL2 mediators, with TNF-α also associated with CD16− iNOS+ cells On the other hand, among the CD14+ monocyte subsets expressing IDO, CD16− IDO+ correlated positively with TNF-α, IL-10 and IP-10/ CXL10; the CD16+ IDO+ subset correlated positively only with IL-10 (Table 3) Discussion Dengue has several well-established clinical and laboratory features such as increased hematocrit and liver enzyme levels, thrombocytopenia, among others (Ferreira et al., 2015) Here we investigated patients infected mostly with DENV-4 during an epidemic in Campo Grande (MS, Brazil) and evaluated, besides their clinical parameters, specific monocyte circulating subsets expressing iNOS and IDO, and cytokines/chemokines produced in response to infection For the first time, the increased intensity in INOS and IDO was determined in different monocyte subsets originated from patients However, it was not possible to determine statistical differences between patients with different outcomes (DF showing no warning signs compared to those with warning signs; data not shown) In opposition to what had been described in infections of other DENV serotypes, DENV-4 presented a mild profile with respect to demographic and clinical characteristics of patients (Faria et al., 2016) Monocytes play an important role in dengue infection, but specific monocyte subset functions remain unclear During several inflammatory and infectious conditions, CD16+ monocytes are increased and proposed to play pro-inflammatory roles (ZieglerHeitbrock, 2007) Earlier studies demonstrate that both monocyte subsets – CD16− and CD16+ are infected with DENV (Azeredo et al., 2010; Wong et al., 2012) Despite the fact that the virus load was not detected within monocytes by immunofluorescence in flow cytometry analysis (data not shown), viral NS1 and RNA was present in sera samples from patients We showed that during DENV-4 infection, monocytes were activated, as demonstrated by iNOS and IDO higher expression and acquired a non-classical profile by increased CD14+ CD16+ co-expression During infection monocytes are activated producing various inflammatory mediators (Torrentes-Carvalho et al., 2009) Increase in iNOS intensity in different monocyte subsets originated from DENV-4 infected patients was carefully investigated A previous study has demonstrated the presence of this enzyme in cells characterized only by monocyte gate regarding size and granularity and Table Correlation among circulating monocyte subsets and cytokine/chemokine levels in dengue patients CD14+ CD16− cells (%) CD14+ CD16+ cells (%) CD14+ iNOS+ cells (MFI) CD14+ IDO+ cells (MFI) CD14+ CD16− iNOS+ cells (MFI) CD14+ CD16+ iNOS+ cells (MFI) CD14+ CD16− IDO+ cells (MFI) CD14+ CD16+ IDO+ cells (MFI) TNF-α IL-10 IP-10/CXL10 MCP-1/CCL2 IFN-γ r = −0.1607 P b 0.4139 r = 0.0958 P b 0.6277 r = 0.4213 P b 0.0163 r = 0.3100 P b 0.0843 r = 0.4154 P b 0.0279 r = 0.3010 P b 0.1195 r = 0.4258 P b 0.0239 r = 0.1078 P b 0.5850 r = −0.4277 P b 0.0232 r = 0.3786 P b 0.0470 r = 0.4545 P b 0.0090 r = 0.2207 P b 0.2248 r = 0.4674 P b 0.0121 r = 0.4269 P b 0.0235 r = 0.4346 P b 0.0208 r = 0.5556 P b 0.0021 r = −0.5678 P b 0.0112 r = 0.3607 P b 0.1293 r = 0.6105 P b 0.0055 r = 0.2940 P b 0.2218 r = 0.5860 P b 0.0084 r = 0.6877 P b 0.0011 r = 0.4561 P b 0.0497 r = 0.3719 P b 0.1169 r = −0.3624 P b 0.1273 r = 0.4186 P b 0.0745 r = 0.3789 P b 0.1096 r = 0.1878 P b 0.4413 r = 0.5123 P b 0.0249 r = 0.6298 P b 0.0039 r = 0.3211 P b 0.1802 r = 0.0701 P b 0.7753 r = 0.1777 P b 0.5997 r = −0.1640 P b 0.6278 r = 0.1818 P b 0.5926 r = −0.2909 P b 0.3855 r = 0.0000 P b 1.0090 r = −0.1958 P b 0.5431 r = −0.1469 P b 0.6509 r = −0.4965 P b 0.1006 Monocyte CD14+ CD16− and CD14+ CD16+ subsets analyzed for correlations with cytokines/chemokines measured in plasma of DENV patients by Spearman correlation test R and P values are represented in the table above P b 0.05 were considered significant and are highlighted in bold L.G Fialho et al / Virology Reports (2017) 9–19 17 originated from patients infected with DENV-1 (Neves-Souza et al., 2005) INOS is induced by various infectious stimuli and responsible for NO molecule production (Dusting and Macdonald, 1995; Frucht et al., 2001) NO has a variety of functions on dilation of blood vessels, platelet aggregation, fight against infections and tumors, inflammation among others (Bogdan, 1998; Moncada and Higgs, 1993; Moncada et al., 1991) On the other hand, NO plays an important antiviral role in dengue during infections by different DENV serotypes, reducing viral proteins and RNA in several infection models (Chareonsirisuthigul et al., 2007; Fagundes et al., 2011; Levy et al., 2010; Neves-Souza et al., 2005; Takhampunya et al., 2006) Moreover, the association of increased NO levels with mild clinical manifestations of the disease suggests a protective role of NO in dengue fever (Levy et al., 2010) We may hypothesize that iNOS plays role in controlling virus load in the disease For the first time, IDO induction was detected within monocytes in dengue patients and we wonder about its involvement in the disease This enzyme is known to play an immunosuppressive role, creating an environment that inhibits antigen-specific T cell responses Tryptophan depletion by IDO and kynurenine proapoptotic activity leads to inhibition of T cell proliferation, apoptosis and anergy (Barth and Raghuraman, 2014; Fallarino and Grohmann, 2011; Moffett and Namboodiri, 2003) The role of this enzyme in dengue disease remains unclear Few studies demonstrate the action of this enzyme in the disease and its reported antiviral activity is still not well defined (Becerra et al., 2009) In other diseases, such as leprosy, IDO plays an immunosuppressive role in inhibiting lymphocyte proliferation and expression of CTLA-4 receptor-like (de Souza Sales et al., 2011; Moura et al., 2012) Moreover, IDO activation by Epstein-Bar virus (EBV) in human monocyte-derived macrophages (MDM) suppressed T cell proliferation and impaired CD8+ T cell cytotoxic activity (Liu et al., 2014) Patients with primary Sjögren's syndrome have their high IDO activity associated with increase in T regulatory cell frequencies (Maria et al., 2016) We identified iNOS more intensely distributed in the classical circulating monocyte subset (CD14+ CD16−) from dengue patients while IDO was found more intense in non-classical monocytes (CD14+ CD16+) (Ziegler-Heitbrock, 2014) Data obtained may be explained by the fact that NO, induced after iNOS expression, can inhibit IDO activity (Thomas et al., 2007) Both iNOS and IDO were found in different monocyte subpopulations under other clinical conditions (Furuzawa-Carballeda et al., 2010; Lopez-Moratalla et al., 1997; Szaflarska et al., 2004), but this is the first time that their association with specific CD16 expressing subsets during dengue fever was demonstrated CD14+ CD16+ cells, known as non-classical monocytes, are among peripheral blood cells expressing IDO that may be expressed in CD14+ CD16−, CD14+ CD16+ and also CD14− CD16+ Further characterization of iNOS and IDO expressing monocytes in dengue are deserved Whether the IDO effect on T regulatory cells may play an important role in controlling virus replication and being somehow not efficient in inhibiting iNOS, it remains to be determined On the other hand, the increased IDO presence in the CD16+ subset appears to be a response of the immune system trying to control the inflammatory response, using a feedback mechanism (Fallarino et al., 2006) The acute infection by DENV-4 could stimulate the production of immunological mediators such as NO, IFN-γ, TNF-α, MCP-1/ CCL2, IL-10 and IP-10/CXL10 The NO and IFN-γ molecules have important antiviral functions in the response to infection by different DENV serotypes NO has antiviral properties in DENV infections and its product, nitrite, was detected in plasma of DENV-4 patients Besides the association between increased NO levels with mild dengue fever (Levy et al., 2010), during in vitro DENV infection within monocytes, a NO inhibitor increased virus detection while a NO donor inhibited DENV (Neves-Souza et al., 2005), indicating the involvement of iNOS and NO in antiviral effects NO production by DENV-infected monocytic lineages THP-1 decreased viral RNA in a number of detected copies (Chareonsirisuthigul et al., 2007) NO increase in patients infected with serotype from DENV could be an explanation for the mild characteristic of this infection, together with increased IFN-y production CD14+ iNOS+ cells associated with cytokines/chemokines, such as TNF-α, IP-10/CXL10 and MCP-1/CCL2, likely indicate cell activation and IL-10 correlation reflects possibly the inflammatory control mechanisms Considering CD14+ CD16+ IDO+ subset associated with the increased IL-10 plasma levels observed in DENV-4 patients, we can speculate that the IDO detection rather in this monocyte subset, may generate an immune regulating environment, linked to induction of T-cell differentiation into T regulatory cell phenotype (Fallarino et al., 2006; Maria et al., 2016), essential in regulating the immune system and producing regulatory cytokines such as IL-10 As known, IL-10 is involved in maintaining the balance between pathogen elimination and immunopathology during viral infections (Suvas et al., 2004) Besides that, T-regulatory cell frequencies are increased in mild cases of dengue (Luhn et al., 2007), and could explain the mild feature of DENV-4 infection, since IDO increase in these patients Furthermore, we have observed in preliminary results an increased Treg cell frequency in mild DENV-4 infected patients compared with severe infected DENV-2 ones (unpublished data) CD16− IDO+ population is also positively correlated with IL-10 cytokine production, indicating a regulator mechanism of this enzyme in both monocyte subsets Furthermore, IDO induction in classical population, CD14+ CD16−, may be associated with the production of TNF-α, since this cytokine is IDO inducer and has been positively correlated with this subpopulation (Babcock and Carlin, 2000) In summary, we report that DENV-4 patients presented an increase in the iNOS and IDO detection in different monocyte subsets, besides the expansion of the CD14+ CD16+ cells These patients also showed increase in NO and some cytokines/chemokines production during the acute phase of infection Conclusion Our results showed for the first time in dengue fever, the association of IDO enzyme with monocyte subset CD14+ CD16+, and the iNOS detection in the CD14+ CD16− population We also determined their association with the production of inflammatory 18 L.G Fialho et al / Virology Reports (2017) 9–19 mediators, indicating that the monocyte subsets expressing iNOS and IDO may play important roles during dengue disease that may have implications for immunopathogenesis We may conclude that iNOS activation may be associated with TNF- α, IP-10/ CXL10 and MCP-1/CCL2 IL-10 production may result from a feedback mechanism after cell activation The fact that IDO+ cells are also associated with IL-10 may indicate that these cells may have been induced by DENV into a modulation mechanism during virus infection and clearance Acknowledgements We gratefully thank Dr Ana Rita Motta Castro at UFMS (Campo Grande MS) and her team, for their help and assistance with patient recruitment and sample collection We are indebted to the Flavivirus Laboratory team at FIOCRUZ-RJ, especially to Dr Ana Maria Bispo Fillips LGF and MC were Ph.D student fellows at POSGBP, IOC, FIOCRUZ This work was supported by PAEF/IOC, RPT08A Citometria de Fluxo – RJ and PAPES all from FIOCRUZ; and CNPq/DECIT and FAPERJ References Azeredo, E.L., Neves-Souza, P.C., Alvarenga, A.R., Reis, S.R., Torrentes-Carvalho, A., Zagne, S.M., Nogueira, R.M., Oliveira-Pinto, L.M., Kubelka, C.F., 2010 Differential regulation of toll-like receptor-2, toll-like receptor-4, CD16 and human leucocyte antigen-DR on peripheral blood monocytes during mild and severe dengue fever Immunology 130, 202–216 Babcock, T.A., Carlin, J.M., 2000 Transcriptional activation of indoleamine dioxygenase by interleukin and tumor necrosis factor alpha in interferon-treated epithelial cells Cytokine 12, 588–594 Barth, H., Raghuraman, S., 2014 Persistent infectious diseases say - IDO Role of indoleamine-2,3-dioxygenase in disease pathogenesis and implications for therapy Crit Rev Microbiol 40, 360–368 Becerra, A., Warke, R.V., Martin, K., Xhaja, K., de Bosch, N., Rothman, A.L., Bosch, I., 2009 Gene expression profiling of dengue infected human primary cells identifies secreted mediators in vivo J Med Virol 81, 1403–1411 Bertolacci-Rocha, L.G., Cunha, R.V., Lichs, G.G., Dal Fabbro, M.M., Motta-Castro, A.R., 2014 Introduction of the dengue virus type in the State of Mato Grosso Sul, Brazil Cad Saude Publica 30, 1789–1792 Bogdan, C., 1998 The multiplex function of nitric oxide in (auto)immunity J Exp Med 187, 1361–1365 Bozza, F.A., Cruz, O.G., Zagne, S.M., Azeredo, E.L., Nogueira, R.M., Assis, E.F., Bozza, P.T., Kubelka, C.F., 2008 Multiplex cytokine profile from dengue patients: MIP-1beta and IFN-gamma as predictive factors for severity BMC Infect Dis 8, 86 Brazilian-Federal-Government, 2014 Dengue: monitoramento das primeiras Semanas Epidemiológicas (SE) de 2014, Brasil http://portalsaude.saude.gov.br/images/ pdf/2014/maio/29/BE-2014-45–3–primeiras-semanas.pdf (Acessed 19 December 2017) Brazilian-Federal-Government, 2017 Monitoramento dos casos de dengue, febre de chikungunya e febre pelo vírus Zika até a Semana Epidemiológica 51, 2016 http:// portalsaude.saude.gov.br/images/pdf/2017/janeiro/12/2017_001%20-%20Dengue%20SE51_publicacao.pdf (Acessed 19 December 2017) Chareonsirisuthigul, T., Kalayanarooj, S., Ubol, S., 2007 Dengue virus (DENV) antibody-dependent enhancement of infection upregulates the production of antiinflammatory cytokines, but suppresses anti-DENV free radical and pro-inflammatory cytokine production, in THP-1 cells J Gen Virol 88, 365–375 de Souza Sales, J., Lara, F.A., Amadeu, T.P., de Oliveira Fulco, T., da Costa Nery, J.A., Sampaio, E.P., Pinheiro, R.O., Sarno, E.N., 2011 The role of indoleamine 2,3dioxygenase in lepromatous leprosy immunosuppression Clin Exp Immunol 165, 251–263 Dusse, L.M.S., Vieira, L.M., Carvalho, M.G., 2003 Revisão sobre óxido nítrico J Bras Patol Med Lab 39, 343–350 Dusting, G.J., Macdonald, P.S., 1995 Endogenous nitric oxide in cardiovascular disease and transplantation Ann Med 27, 395–406 Eleftheriadis, T., Pissas, G., Antoniadi, G., Tsogka, K., Makri, P., Liakopoulos, V., Stefanidis, I., 2016 Increased Indoleamine 2,3-dioxygenase in monocytes of patients on hemodialysis Iran J Kidney Dis 10, 91–93 Fagundes, C.T., Costa, V.V., Cisalpino, D., Amaral, F.A., Souza, P.R., Souza, R.S., Ryffel, B., Vieira, L.Q., Silva, T.A., Atrasheuskaya, A., Ignatyev, G., Sousa, L.P., Souza, D.G., Teixeira, M.M., 2011 IFN-gamma production depends on IL-12 and IL-18 combined action and mediates host resistance to dengue virus infection in a nitric oxide-dependent manner PLoS Negl Trop Dis 5, e1449 Fallarino, F., Grohmann, U., 2011 Using an ancient tool for igniting and propagating immune tolerance: IDO as an inducer and amplifier of regulatory T cell functions Curr Med Chem 18, 2215–2221 Fallarino, F., Grohmann, U., You, S., McGrath, B.C., Cavener, D.R., Vacca, C., Orabona, C., Bianchi, R., Belladonna, M.L., Volpi, C., Santamaria, P., Fioretti, M.C., Puccetti, P., 2006 The combined effects of tryptophan starvation and tryptophan catabolites down-regulate T cell receptor zeta-chain and induce a regulatory phenotype in naive T cells J Immunol 176, 6752–6761 Faria, N.R., Solorzano, V.E., Nogueira, R.M., Chouin-Carneiro, T., Nunes, P.C., Simoes, J.B., de Bruycker Nogueira, F., Lima Mda, R., de Oliveira Pinto, L.M., Kubelka, C.F., da Cunha, R.V., de Azeredo, E.L., dos Santos, F.B., 2016 Dengue epidemics in two distinct periods reveal distinct epidemiological, laboratorial and clinical aspects in a same scenario: analysis of the 2010 and 2013 epidemics in Mato Grosso Sul, Brazil Trans R Soc Trop Med Hyg 110, 228–236 Ferreira, R.A.X., de Oliveira, S.A., Gandini, M., Ferreira, L.D.C., Correa, G., Abiraude, F.M., Reid, M.M., Cruz, O.G., Kubelka, C.F., 2015 Circulating cytokines and chemokines associated with plasma leakage and hepatic dysfunction in Brazilian children with dengue fever Acta Trop 149, 138–147 Figueiredo, R.M., Naveca, F.G., Bastos, M.S., Melo, M.N., Viana, S.S., Mourao, M.P., Costa, C.A., Farias, I.P., 2008 Dengue virus type 4, Manaus, Brazil Emerg Infect Dis 14, 667–669 Frucht, D.M., Fukao, T., Bogdan, C., Schindler, H., O'Shea, J.J., Koyasu, S., 2001 IFN-gamma production by antigen-presenting cells: mechanisms emerge Trends Immunol 22, 556–560 Furuzawa-Carballeda, J., Lima, G., Uribe-Uribe, N., Avila-Casado, C., Mancilla, E., Morales-Buenrostro, L.E., Perez-Garrido, J., Perez, M., Cardenas, G., Llorente, L., Alberu, J., 2010 High levels of IDO-expressing CD16+ peripheral cells, and Tregs in graft biopsies from kidney transplant recipients under belatacept treatment Transplant Proc 42, 3489–3496 Gubler, D.J., 2002 Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century Trends Microbiol 10, 100–103 Kou, Z., Quinn, M., Chen, H., Rodrigo, W.W., Rose, R.C., Schlesinger, J.J., Jin, X., 2008 Monocytes, but not T or B cells, are the principal target cells for dengue virus (DV) infection among human peripheral blood mononuclear cells J Med Virol 80, 134–146 Levy, A., Valero, N., Espina, L.M., Anez, G., Arias, J., Mosquera, J., 2010 Increment of interleukin 6, tumour necrosis factor alpha, nitric oxide, C-reactive protein and apoptosis in dengue Trans R Soc Trop Med Hyg 104, 16–23 Liu, W.L., Lin, Y.H., Xiao, H., Xing, S., Chen, H., Chi, P.D., Zhang, G., 2014 Epstein-Barr virus infection induces indoleamine 2,3-dioxygenase expression in human monocyte-derived macrophages through p38/mitogen-activated protein kinase and NF-kappaB pathways: impairment in T cell functions J Virol 88, 6660–6671 Lopez-Moratalla, N., Gonzalez, A., Aymerich, M.S., Lopez-Zabalza, M.J., Pio, R., de Castro, P., Santiago, E., 1997 Monocyte inducible nitric oxide synthase in multiple sclerosis: regulatory role of nitric oxide Nitric Oxide 1, 95–104 Luhn, K., Simmons, C.P., Moran, E., Dung, N.T., Chau, T.N., Quyen, N.T., Thao le, T.T., Van Ngoc, T., Dung, N.M., Wills, B., Farrar, J., McMichael, A.J., Dong, T., Rowland-Jones, S., 2007 Increased frequencies of CD4+ CD25(high) regulatory T cells in acute dengue infection J Exp Med 204, 979–985 MacMicking, J., Xie, Q.W., Nathan, C., 1997 Nitric oxide and macrophage function Annu Rev Immunol 15, 323–350 L.G Fialho et al / Virology Reports (2017) 9–19 19 Maria, N.I., van Helden-Meeuwsen, C.G., Brkic, Z., Paulissen, S.M., Steenwijk, E.C., Dalm, V.A., van Daele, P.L., Martin van Hagen, P., Kroese, F.G., van Roon, J.A., Harkin, A., Dik, W.A., Drexhage, H.A., Lubberts, E., Versnel, M.A., 2016 Association of increased Treg cell levels with elevated indoleamine 2,3-dioxygenase activity and an imbalanced kynurenine pathway in interferon-positive primary Sjogren's syndrome Arthritis Rheumatol 68, 1688–1699 Mellor, A.L., Munn, D.H., 2004 IDO expression by dendritic cells: tolerance and tryptophan catabolism Nat Rev Immunol 4, 762–774 Moffett, J.R., Namboodiri, M.A.A., 2003 Tryptophan and the immune response Immunol Cell Biol 81, 247–265 Moncada, S., Higgs, A., 1993 The L-arginine-nitric oxide pathway N Engl J Med 329, 2002–2012 Moncada, S., Palmer, R.M., Higgs, E.A., 1991 Nitric oxide: physiology, pathophysiology, and pharmacology Pharmacol Rev 43, 109–142 Moura, D.F., de Mattos, K.A., Amadeu, T.P., Andrade, P.R., Sales, J.S., Schmitz, V., Nery, J.A., Pinheiro, R.O., Sarno, E.N., 2012 CD163 favors mycobacterium leprae survival and persistence by promoting anti-inflammatory pathways in lepromatous macrophages Eur J Immunol 42, 2925–2936 Mukhopadhyay, S., Kuhn, R.J., Rossmann, M.G., 2005 A structural perspective of the flavivirus life cycle Nat Rev Microbiol 3, 13–22 Neves-Souza, P.C., Azeredo, E.L., Zagne, S.M., Valls-de-Souza, R., Reis, S.R., Cerqueira, D.I., Nogueira, R.M., Kubelka, C.F., 2005 Inducible nitric oxide synthase (iNOS) expression in monocytes during acute dengue fever in patients and during in vitro infection BMC Infect Dis 5, 64 Ozaki, Y., Edelstein, M.P., Duch, D.S., 1987 The actions of interferon and antiinflammatory agents of induction of indoleamine 2,3-dioxygenase in human peripheral blood monocytes Biochem Biophys Res Commun 144, 1147–1153 Pang, X., Song, H., Zhang, Q., Tu, Z., Niu, J., 2016 Hepatitis C virus regulates the production of monocytic myeloid-derived suppressor cells from peripheral blood mononuclear cells through PI3K pathway and autocrine signaling Clin Immunol 164, 57–64 Passlick, B., Flieger, D., Ziegler-Heitbrock, H.W., 1989 Identification and characterization of a novel monocyte subpopulation in human peripheral blood Blood 74, 2527–2534 Skrzeczynska-Moncznik, J., Bzowska, M., Loseke, S., Grage-Griebenow, E., Zembala, M., Pryjma, J., 2008 Peripheral blood CD14high CD16+ monocytes are main producers of IL-10 Scand J Immunol 67, 152–159 Srikiatkhachorn, A., Green, S., 2010 Markers of dengue disease severity Curr Top Microbiol Immunol 338, 67–82 Suvas, S., Azkur, A.K., Kim, B.S., Kumaraguru, U., Rouse, B.T., 2004 CD4 + CD25+ regulatory T cells control the severity of viral immunoinflammatory lesions J Immunol 172, 4123–4132 Szaflarska, A., Baj-Krzyworzeka, M., Siedlar, M., Weglarczyk, K., Ruggiero, I., Hajto, B., Zembala, M., 2004 Antitumor response of CD14+/CD16+ monocyte subpopulation Exp Hematol 32, 748–755 Takhampunya, R., Padmanabhan, R., Ubol, S., 2006 Antiviral action of nitric oxide on dengue virus type replication J Gen Virol 87, 3003–3011 Temporao, J.G., Penna, G.O., Carmo, E.H., Coelho, G.E., Socorro Silva Azevedo, R., Teixeira Nunes, M.R., da Costa Vasconcelos, P.F., 2011 Dengue virus serotype 4, Roraima State, Brazil Emerg Infect Dis 17, 938–940 Thomas, S.R., Terentis, A.C., Cai, H., Takikawa, O., Levina, A., Lay, P.A., Freewan, M., Stocker, R., 2007 Post-translational regulation of human indoleamine 2,3dioxygenase activity by nitric oxide J Biol Chem 282, 23778–23787 Torrentes-Carvalho, A., Azeredo, E.L., Reis, S.R., Miranda, A.S., Gandini, M., Barbosa, L.S., Kubelka, C.F., 2009 Dengue-2 infection and the induction of apoptosis in human primary monocytes Mem Inst Oswaldo Cruz 104, 1091–1099 Villar, L.A., Gelvez, R.M., Rodriguez, J.A., Salgado, D., Parra, B., Osorio, L., Bosch, I., 2013 Biomarkers for the prognosis of severe dengue Biomedica 33 (Suppl 1), 108–116 WHO, 2009 Dengue: guidelines for diagnosis, treatment, prevention and control: new edition WHO Guidelines Approved by the Guidelines Review Committee; Geneva Wong, K.L., Chen, W., Balakrishnan, T., Toh, Y.X., Fink, K., Wong, S.-C., 2012 Susceptibility and response of human blood monocyte subsets to primary dengue virus infection PLoS One 7, e36435 Wu, S.J., Grouard-Vogel, G., Sun, W., Mascola, J.R., Brachtel, E., Putvatana, R., Louder, M.K., Filgueira, L., Marovich, M.A., Wong, H.K., Blauvelt, A., Murphy, G.S., Robb, M.L., Innes, B.L., Birx, D.L., Hayes, C.G., Frankel, S.S., 2000 Human skin Langerhans cells are targets of dengue virus infection Nat Med 6, 816–820 Ziegler-Heitbrock, H.W., 1996 Heterogeneity of human blood monocytes: the CD14+ CD16+ subpopulation Immunol Today 17, 424–428 Ziegler-Heitbrock, L., 2007 The CD14+ CD16+ blood monocytes: their role in infection and inflammation J Leukoc Biol 81, 584–592 Ziegler-Heitbrock, L., 2014 Reprint of: monocyte subsets in man and other species Cell Immunol 291, 11–15