Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Cytokine Expression Profile of Dengue Patients at Different Phases of Illness

  • Anusyah Rathakrishnan,

    Affiliation Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia

  • Seok Mui Wang,

    Affiliation Institute of Medical Molecular Biotechnology, Faculty of Medicine, Universiti Teknologi MARA, Selangor, Malaysia

  • Yongli Hu,

    Affiliation Perdana University Graduate School of Medicine, Serdang, Selangor, Malaysia

  • Asif M. Khan,

    Affiliations Perdana University Graduate School of Medicine, Serdang, Selangor, Malaysia, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America

  • Sasheela Ponnampalavanar,

    Affiliation Department of Infectious Diseases, University Malaya Medical Centre, Kuala Lumpur, Malaysia

  • Lucy Chai See Lum,

    Affiliation Department of Paediatrics, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia

  • Rishya Manikam,

    Affiliation Department of Trauma and Emergency Medicine, University Malaya Medical Centre, Kuala Lumpur, Malaysia

  • Shamala Devi Sekaran

    shamalamy@yahoo.com

    Affiliation Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia

Cytokine Expression Profile of Dengue Patients at Different Phases of Illness

  • Anusyah Rathakrishnan, 
  • Seok Mui Wang, 
  • Yongli Hu, 
  • Asif M. Khan, 
  • Sasheela Ponnampalavanar, 
  • Lucy Chai See Lum, 
  • Rishya Manikam, 
  • Shamala Devi Sekaran
PLOS
x

Abstract

Background

Dengue is an important medical problem, with symptoms ranging from mild dengue fever to severe forms of the disease, where vascular leakage leads to hypovolemic shock. Cytokines have been implicated to play a role in the progression of severe dengue disease; however, their profile in dengue patients and the synergy that leads to continued plasma leakage is not clearly understood. Herein, we investigated the cytokine kinetics and profiles of dengue patients at different phases of illness to further understand the role of cytokines in dengue disease.

Methods and Findings

Circulating levels of 29 different types of cytokines were assessed by bead-based ELISA method in dengue patients at the 3 different phases of illness. The association between significant changes in the levels of cytokines and clinical parameters were analyzed. At the febrile phase, IP-10 was significant in dengue patients with and without warning signs. However, MIP-1β was found to be significant in only patients with warning signs at this phase. IP-10 was also significant in both with and without warning signs patients during defervescence. At this phase, MIP-1β and G-CSF were significant in patients without warning signs, whereas MCP-1 was noted to be elevated significantly in patients with warning signs. Significant correlations between the levels of VEGF, RANTES, IL-7, IL-12, PDGF and IL-5 with platelets; VEGF with lymphocytes and neutrophils; G-CSF and IP-10 with atypical lymphocytes and various other cytokines with the liver enzymes were observed in this study.

Conclusions

The cytokine profile patterns discovered between the different phases of illness indicate an essential role in dengue pathogenesis and with further studies may serve as predictive markers for progression to dengue with warning signs.

Introduction

In certain infectious diseases, shock may occur due to excessive plasma leakage and this leakage is often postulated to be caused by endothelial sieves created by inappropriate cytokine responses in the host. Dengue, traditionally classified as Dengue Fever (DF), Dengue Haemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) is one such disease where a key feature of DHF is vascular leakage which then leads to hypovolemic shock (DSS), inevitably increasing the chances of fatality. Recently, WHO has suggested a new classification for this disease, which includes dengue with or without warning signs and severe dengue [1].

Dengue, although with a low mortality rate, is one of the highest morbidity rated arthropod diseases. It is endemic in more than 120 countries around the world, with 55% of the world’s population at risk of being infected [1]. Despite being around for centuries, there have not been any effective vaccines, therapeutics or anti-viral drugs for this disease. The lack of such “cure” can be attributed to firstly, to an incomplete understanding of dengue immunopathogenesis, secondly, a lack of a suitable animal model and finally the inherent dangers of live vaccines [2].

Some of the postulated hypotheses on dengue immunopathogenesis include (i) the antibody enhancement theory [3], [4], (ii) cross-reactive memory T cells activation [5] and (iii) the original antigenic sin [6], where all in a way cause either an over production or a skewed profile of cytokine release, hence the term cytokine storm/cytokine tsunami. This cytokine storm has a direct effect on the vascular endothelial cells by increasing capillary permeability and causing leakage [7]. Cytokines also exhibit synergism, where for example, tumor necrosis factor-alpha (TNF-α), interferon-γ (IFN-γ) and interleukin-1 (IL-1) together can increase the capillary permeability compared to when the cytokine is acting alone [8]. There is also the inapparent ability of the endothelium to repair itself which could be a result of some aspect of endothelial dysfunction, though this has not been shown.

The study of permeability and leakage in dengue is often applied with the use of human umbilical vein endothelial cell (HUVEC) line where in one such study, Anderson et al. observed that these cells are activated when exposed to culture fluids from dengue virus (DENV)-infected peripheral blood mononuclear cells (PBMCs) [9]. In another study, the vascular permeability of HUVECs was found to be increased when exposed to either recombinant human monocyte chemo-attractive protein-1 (rhMCP-1) or to the culture supernatant of DENV2-infected human monocytes [10]. Moreover, certain cell lines, such as DENV-infected primary human monocytes and epithelial cell lines have shown increased production of cytokines [11]. DENV infections of HepG2 and primary dendritic cells (DCs) have also shown the ability to induce the production of cytokines such as IL-8, RANTES, macrophage inhibitory protein-1-alpha (MIP-1α) and MIP-1β [12].

The cytokine storm hypothesis has also been studied by analyzing sera of DHF/DSS patients in Vietnam, India and Cuba, which indeed showed the presence of elevated levels of IFN-γ, TNF-α and IL-10 [13], [14], [15]. A recent study on dengue infected Venezuelan patients had documented significant increased levels of MCP-2, IP-10 and TRAIL in patients’ serum during the febrile period [16].

Despite extensive research on the role of cytokines in the progression of severe dengue [17], [18], [19], [20], the cytokine profiles, especially at the defervescence stage, and the synergy between them that leads to continued plasma leakage is not clearly understood. Thus, in this study, not only did we attempt cytokine profiling of dengue patients, but also set to establish patient and cytokine kinetics throughout the phases of illness. We opted to gain preliminary insights on differences in the levels of cytokines in primary and secondary dengue infections. With that, we endeavoured to analyse the relationship between the cytokines and the clinical parameters of dengue patients.

Materials and Methods

Ethics Statement and Study Population

Five millilitres of blood were obtained from 44 DENV infected patients at the University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia from January 2005 to June 2009 with written informed consent. Blood was drawn at three time point of illness- febrile, defervescence and convalescence for each patient. The febrile phase usually lasts for 2–7 days and often has indistinguishable clinical symptoms, whereas defervescence is the critical stage where patient may develop severe signs and the convalescence stage is when the patient starts to recover. The 2009 WHO dengue classification scheme and case definition [1] was used to diagnose patients. Data on demographic characteristics (i.e. age, gender and race), clinical features (i.e. day of fever, body temperature, bleeding manifestation, leakage, abdominal pain and hypotension) and routine haematological and biochemical laboratory test findings (i.e. full blood count, liver function tests) were also collected. Healthy donors’ blood samples that are of age, gender and race matched with patients, were obtained from Blood Bank, UMMC as controls. All data analysed were anonymized and ethical clearance for this work was approved by the Scientific and Ethical Committee of UMMC (Ethics Committee/IRB Reference No: 321.4).

Sera Isolation and Dengue Confirmatory Tests

Blood serum was collected by centrifugation of blood containing tubes at 1500 rpm for 10 minutes and the serum was stored at −80°C until further use. All patients were further confirmed to have dengue by detection of (i) DENV via virus isolation; (ii) DENV RNA via real-time SYBR-Green based RT-PCR assay [21]; (iii) DENV antigen via NS1 assay (Pan-E dengue early ELISA kit; Panbio, Queensland, Australia); (iv) DENV-specific antibodies via in-house capture IgM Enzyme-Linked Immunosorbent Assay (ELISA) [22] and haemagglutination inhibition (HI) test [23]. The HI assay was also used to define primary and secondary DENV infection based on the total antibody in paired sera.

Identification and Quantification of Cytokines

Twenty-nine different cytokines, namely IL-1β, IL-1ra, IL-2, IL-4, IL-5, Il-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IP-10, ICAM-1, IFN-γ, MCP-1, MIP-1α, MIP-1β, Eotaxin, basic-fibroblast growth factor (FGF-Basic), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), RANTES, TNF-α were evaluated. The cytokine levels in the serum of all patients and controls, at all 3 different phases of illness (febrile, defervescence, and convalescence) were analyzed using the Bio-Plex human cytokine 27-plex panel, 8-plex panel and 2-plex panel kits (Bio-Plex Human Cytokine Assay; Bio-Rad Inc., Hercules, CA, USA). Briefly, patients’ serum samples were mixed with beads coated with antibodies (Abs) to various cytokines, having unique fluorescent intensity. Subsequently, the mixtures were incubated with biotinylated anti-cytokine Abs. Finally, PE-conjugated streptavidin was added, and the fluorescent signals were detected using the multiplex array reader Bio-Plex 200 System (Bio-Rad Laboratories). Raw data was initially measured as the relative fluorescence intensity and then converted to cytokines concentration based on the standard curve generated from the reference concentrations supplied in the kit (Bio-Rad Laboratories).

Statistical Analysis

The Kruskal-Wallis one way analysis of variance (ANOVA), followed by Dunn’s multiple comparison test were used to evaluate differences between raw cytokine levels in the different groups of dengue patients compared to control groups. The Mann-Whitney U Test was applied to assess differences in the cytokine levels of primary and secondary infections. To establish the correlation between cytokine levels and clinical parameters/findings, the correlation matrix was applied. Results are given as correlation coefficient, r (ranges from −1 to +1). Two-tailed P value of less than 0.05 was considered to be significant for all test performed. All three statistical analyses performed were done using GraphPad Prism 5 for Windows, Version 5.01 (San Diego, California, USA).

Results

Characteristics of Study Population

Forty-four adult patients with laboratory confirmed dengue virus infection were investigated for their cytokine profiles. These patients were classified by the WHO 2009 guideline into 11 with “Dengue without Warning Signs (DwoWS)”, 29 with “Dengue with Warning Signs (DwWS) and 4 with “Severe Dengue (SD)”. The 24 males and 20 females study cohort consisted of 24 Malays, 4 Chinese, 13 Indians and 3 of other ethnicities. The demographics and clinical parameters (age, duration of illness, temperature, platelet count, and hematocrit) as well as clinical symptoms (bleeding manifestation, plasma leakage, abdominal pain and hypotension) are described in Table 1 and Table 2.

The liver function tests (Table 3) included measurement of total bilirubin (TB), total albumin (TA), aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and gamma glutamyl transferase (GGT). Whereas, the white blood cell (WBC) profile (Table 4) of this study population includes WBC, neutrophils, lymphocytes, monocytes and atypical lymphocytes.

From the lab diagnostic tests performed (Table 5), DENV was successfully isolated from 17 patients and DENV RNA was detected in 31 patients. All the 4 DENV serotypes were detected in our study cohort, with the highest percentage to be DENV-1 (43.3%) and the rest ranging from 13.3%–23.3%. DENV NS1 antigen was detected 27 patients. Serologically, 41 patients were found to be IgM positive and amongst them, 37 had IgM seroconversion during the defervescence or convalescence stage. From the HI test, 16 patients had primary infections whereas the rest were experiencing either presumptive or confirmed secondary infections.

Levels of Cytokines at Different Phases of Illness

The levels of 29 different types of cytokines in dengue patients were determined at the febrile, defervescence and convalescent phases. In this analysis, the 4 severe dengue patients were excluded due to the small sample size. We then categorized the cytokines into 3 groups: (i) inflammatory cytokines; (ii) chemokines; (iii) adhesion molecules and growth factors. The cytokines which demonstrated significant differences between the different groups of patients and controls are as summarized in Table 6.

thumbnail
Table 6. Significant cytokines at different phases of illness when compared to the controls.

https://doi.org/10.1371/journal.pone.0052215.t006

i. Inflammatory cytokines (Figure 1A).

Fifteen different inflammatory cytokines were analyzed where the majority of them were pro-inflammatory while only 4 were anti-inflammatory. Of these, various trends were observed, notably, among the pro-inflammatory cytokines, with only the mean cytokine levels of IL-18 were elevated for both groups of patients at all three phases of illness. The levels of pro-inflammatory cytokines, IL-5 and IL-12 as well as anti-inflammatory cytokine IL-4 were lower throughout the illness in both groups when compared to the healthy controls.

thumbnail
Figure 1. Cytokine trends over the three different phases of illness.

The mean levels of (A) inflammatory cytokines, (B) chemokines and (C) adhesion molecules and growth factors in dengue patients without warning signs (DwoWS) (red symbol), dengue patients with warning signs (DwWS) (blue symbol) and healthy controls (black line) at different phase of illness (Feb = Febrile; Def = Defervescence; Conv = Convalescence). Error bars indicate standard error mean (SEM).

https://doi.org/10.1371/journal.pone.0052215.g001

The IFN-γ levels were generally lower in DwoWS patients and were higher in DwWS patients with decreasing trend as patients recovered. The cytokines IL-1β and TNF-α displayed a similar trend where lower levels were detected during febrile phase, which then peaked during defervescence. The levels of IL-6 were generally higher in DwWS patients, but during defervescence, DwoWS patients had high levels similar to the warning signs group. Both groups displayed higher levels of IL-9 than controls, but the patients with warning signs had peak levels during defervescence. Anti-inflammatory cytokines, IL-10 and IL-13 showed mixed patterns, whereas IL-1ra in both groups displayed similar a trend throughout the disease, however with the warning signs group having higher levels than the DwoWS patients. Two cytokines (IL-2 and IL-17) had insufficient patient response in both groups. Despite the various trends observed, none of the inflammatory cytokines were significantly different between the groups studied at any time point.

ii. Chemokines (Figure 1B).

More than half of the chemokines analysed, namely IP-10, MCP-1, MIP-1β, and RANTES, had elevated mean cytokine levels in both patient with and without warning signs across the three time points. Notably, IP-10 levels in both groups were significantly different from healthy donors at febrile (DwoWS: P<0.01; DwWS: P<0.001) and defervescence (DwoWS: P<0.001; DwWS: P<0.01) phases (Figure 2A). In the case of MIP-1β, a significant difference was noted in the DwoWS during defervescence (P<0.01) and in the DwWS patients in the febrile phase (P<0.05) of disease compared to the controls (Figure 2B). Further, the MCP-1 was significantly higher (P<0.01) in the warning signs patients during the febrile stage when compared to the controls (Figure 2C). Eotaxin was generally lower in DwoWS patients and higher in DwWS patients and IL-8 was higher in patients without warning signs compared to the DwWS.

thumbnail
Figure 2. Distribution of raw cytokine response values in dengue without warning signs (DwoWS) and dengue with warning signs (DwWS) compared with healthy controls.

Levels of (A) IP-10, (B) MIP-1β, (C) MCP-1 and (D) G-CSF in DwoWS (red symbol), DwWS (blue symbol) patients at different phase of illness compared with control subjects (black symbol). The SEM are indicated by the error bars in the scatter plot. Statistical significance based on the Kruskal-Wallis, Dunn’s multiple comparison test where *-P<0.05; **-P<0.01, and ***-P<0.001.

https://doi.org/10.1371/journal.pone.0052215.g002

iii. Adhesion molecules and growth factors (Figure 1C).

The only adhesion molecule studied, ICAM-1, had mean levels that were slightly elevated in both groups of patients. In contrast, the mean levels of growth factors, FGF-Basic and G-CSF, were decreased in both groups relative to normal groups at all the three phases of illness, with a significant decrease for G-CSF in the patients’ without warning signs (P<0.05) (Figure 2D) during defervescence. Other growth factors, IL-7, PDGF and VEGF were generally lower in patients with warning signs, relative to both patients without warning signs and the normal group. Growth factor GM-CSF lacked data for in group without warning signs.

Levels of Cytokines in Primary Versus Secondary Infections

The differences in cytokines levels were also assessed between the 16 primary infected patients and the 28 patients who were experiencing secondary infections. Of the 29 cytokines analyzed, most displayed relatively similar levels in both infection statuses. However, at the febrile phase, we found that 3 cytokines differed significantly where eotaxin, IP-10 and ICAM-1 were significantly higher in patient with secondary infections (Figure 3). At the defervescence phase, on the other hand, pro-inflammatory cytokine IFN-γ, chemokine RANTES and growth factors, PDGF as well as G-CSF, were found to be significantly higher in primarily infected patients than those suffering from secondary infections (Figure 3).

thumbnail
Figure 3. Comparison of mean cytokine levels in primary and secondary dengue infections.

Primary and secondary infection status was determined by haemagglutination inhibition assay. Mean with SEM of seven cytokines (three at febrile phase of illness and 4 at defervescence). Statistical significance based on the Mann-Whitney U test where *-P<0.05 and **-P<0.01.

https://doi.org/10.1371/journal.pone.0052215.g003

Relationship between Cytokines and Clinical Parameters

The possible associations between the levels of cytokines with clinical presentation of our study cohort are described in Table 7. Generally, in patients without warning signs, we found that the decreased levels of platelet were associated with the decreasing levels of VEGF and RANTES during defervescence. At this phase, the augmented liver enzymes were associated with several cytokines where AST was associated with increased levels of IL-1ra and IL-10; ALT with IP-10 and Gamma-GT with IL-4, IL-12 and IL-9. In the convalescence phase, the increased levels of AST were conversely related to the decreasing levels of MCP-1. Whereas, the increased levels of VEGF at this time point, was associated with the decreased levels of neutrophils and higher state of lymphocytes.

thumbnail
Table 7. Association of cytokines with clinical parameters in the study cohort.

https://doi.org/10.1371/journal.pone.0052215.t007

Significant associations between the decreased levels of IL-7, IL-12 and PDGF with the decreased levels of platelet were observed in patients with warning signs during the febrile phase, whereas in the defervescence, another 3 cytokines (IL-5, RANTES and VEGF) were also associated with platelet levels. In defervescing DwWS patients, we also observed an association between the increased levels of atypical lymphocytes and the decreased levels of G-CSF. While during convalescence, an association was noted between the atypical lymphocytes and the decreasing levels of IP-10 in these patients. The convalescing patients with warning signs also showed association between the increased levels of IL-7 and monocytes. At all 3 phases of illness, the increased levels of liver enzyme AST was associated with various cytokines where in febrile, ALT was correlated to increased levels of ICAM-1 and decreased levels of FGF-Basic, IL-13, IL-4, IL-12 and VEGF. During defervescence, AST was associated with decreased levels of PDGF, whereas at convalescence, it was linked with the increased levels of IL-10, and IFN-γ.

Discussion

Generally, most hypotheses explaining dengue immunopathogenesis conclude that the overproduction and/or a skewed cytokine response during the critical phase of disease causes plasma leakage and hence, a more severe manifestation of dengue. In this study, an analysis of various cytokines and their correlation with dengue disease was performed.

Despite finding no significant difference in the levels of inflammatory cytokines among patients with and without warning signs as well as with the healthy controls, we showed trends of various cytokines at the three different phases of illness. Outstandingly, IL-15 which was higher in patients with warning signs, has been known to be involved in T cell activation and proliferation, and has been shown to be required for memory CD8+ T cells division. In the absence of IL-2 (as noted in our study, where many patients had undetectable levels of IL-2), the levels of IL-15 is increased [24]. This could possibly enhance proliferation of dengue memory T cells.

Interleukins-4, -5, -12 and -13 were clearly lower in dengue patients than controls throughout the illness. IL-13, an effector cytokine, synergizes with IL-2 to regulate IFN-γ production [25], and low levels of this cytokine could be attributed to the low level of IFN-γ in our cohort especially in patients without warning signs. Regulatory cytokine IL-4, previously found to be increased in DHF/DSS patients [26] has been indicated to play a role in vascular permeability and with the exclusion of severe dengue patients in our study, this may reflect the lower levels of IL-4 observed. This cytokine has also been known for immunoglobulin class and subclass switch [27], and shift from Th1 to Th2 responses in severe dengue, and this could possibly explain the lower levels in patients without warning signs as they remain in a mild state of infection.

Interferon-γ has been shown to be increased in severe dengue cases [28], [29], and this is echoed in our study cohort with lower number of severe dengue cases, where IFN-γ was only slightly higher in patients with warning signs. Another possibility of these lower levels could be attributed to the low levels of IL-12, where an in vivo study showed that IL-12 (p40 chain) - deficient mice had decreased IFN-γ production [30]. IL-12 and IL-18 together, augment IFN-γ production by activating Th1 cells [31], and in our study, despite IL-18 being higher in dengue patients, still had interferon levels that were negligible, implying that IFN-γ production by IL-12 is a co-induction with IL-18 and IL-18 induces IFN-γ only when its receptor is upregulated by IL-12 [30].

Interleukin-10 showed a decreasing trend in patients without warning signs, however remained high in DwWS patients throughout the disease in concordance with several studies that have suggested IL-10 in dengue pathogenesis [15], [32]. An important modulator in vascular leakage, platelet-activating factor (PAF) and T cell apoptosis, the over-expression of IL-10 in transgenic mice have demonstrated inhibition of TNF-α production, where in our study TNF-α remained generally at a lower level.

A study in Brazil, demonstrated a correlation between MIP-1β and NK cells, suggesting its role in dengue protective mechanism [33]. This was again shown in another study where MIP-1β was higher in mild dengue than severe dengue [29], which is in line with our findings where this chemokine was significantly higher in patients without warning signs.

IP-10, an important mediator in inflammatory response, was shown to inhibit dengue infection through competitive binding of heparan sulphate on host cell membrane [34], [35]. Initially during the febrile and defervescence stage, both groups of patients demonstrated significant high levels of IP-10, however, the levels declined steadily for patients without warning signs throughout the phases. However, it remained high in patients with warning signs, offering a possibility that it may be affecting vascular permeability as IP-10 is a potent inhibitor of angiogenesis in vivo [36].

An MCP-1 deficient mice model was unable to switch into subclass Th2 responses [37] and this chemokine has been associated with permeability changes in endothelial cells, where alterations occur to the tight junctions of vascular endothelial cells and leading to plasma leakage in dengue patients [38], [39], [40]. In this study, significantly elevated levels of MCP-1 were found at the febrile phases of patients with warning signs compared to healthy individuals suggesting this chemokine as a possible biomarker in dengue patients who are going to develop more severe clinical outcome.

The infection status (primary versus secondary infections) of an individual has also been disputed to be involved in the pathogenesis of dengue, where most of the postulated theories revolve around secondary infections. In our study, the main limitation was the small sample size, hence we could not categorize infection status by the respective dengue classification and hence only decipher the cytokine levels of primary and secondary infected dengue patients as a whole at different time point of illness. Eotaxin, IP-10 and ICAM-1 were significantly higher in secondary infected dengue patients during the febrile phase of illness. Increased levels ICAM-1 have been indicated in endothelium damage and activation [41], [42], and 75% of the secondary cases in our cohort were of patients with warning signs and/or with severe dengue. Likewise, eotaxin levels which were higher in patients with warning signs have been demonstrated to increase permeability of human coronary artery endothelial cells by downregulating tight junction proteins [43]. IP-10 levels were significantly higher in the overall dengue patients, indicating a more vigorous inflammatory response in secondary infections.

During the defervescence phase, 4 other cytokines displayed significantly lower levels in secondary dengue cases, which were IFN-γ, RANTES, PDGF and G-CSF. Interferon-γ is a critical cytokine in the innate and adaptive immunity against viral infections. Lower levels of this cytokine during a secondary infection indicate defective ability to inhibit viral replication or to be immunomodulatory. RANTES recruits lymphocytes and NK cells to sites of inflammation, and in an influenza mice model deficient in RANTES/CCL5, delayed viral clearance and excessive inflammation occurred [44]. PDGF which promotes cellular proliferation and inhibits apoptosis and is an integral component for maintaining the vascular networks [45] is lower in secondary infections offering a possible explanation as to why secondary infected patients suffered from vascular leakage. G-CSF, a WBC stimulating factor, is lower in secondary infections in line with the occurrence of leukopenia and neutropenia in such cases [46], [47].

In dengue patients without warning signs, a decrease in platelets was noted during defervescence, and this was correlated strongly with RANTES and VEGF. Both RANTES, a chemokine stored in α-granules of platelets, secreted upon platelet activation [48], and VEGF, a growth factor released by platelets, would be expected to decrease upon thrombocytopenia. The defervescing DwoWS patients also had increased levels of liver enzymes AST, ALT and Gamma-GT. Raised AST levels during acute liver damage, has been associated with secreted IL-1ra which is an acute phase protein [49] produced by liver cells and also with IL-10, an anti-inflammatory cytokine which have previously correlated to necroinflammatory activity in liver damaged hepatitis C patients [50]. ALT, an enzyme present in hepatocytes was linked to IP-10 which is known to be induced in the liver. This cytokine plays a specific role in the intralobular accumulation of mononuclear cells and/or the death of hepatocytes in chronic hepatitis [51]. The gamma-GT levels, on the other hand were associated with IL-4, IL-12 and IL-9. Schistosomal patients with hepatic damage were found to have high levels of IL-4 [52] indicating an active Th2 immune response which could also be the situation in dengue patients. Elevated levels of IL-12 have been shown to be associated with liver damage in various studies conducted [53], [54]. At the convalescence phase, these without warning signs patients who had high levels of monocytes were associated with the increased levels of VEGF which possibly could be due to the involvement of VEGF in monocytes activation [55]. Surprisingly though, VEGF was inversely correlated to neutrophils. These patients also had high AST levels which was negatively associated with the decreasing levels of MCP-1 which have been implicated in the liver injury process [56].

Dengue patients with warning signs also exhibited thrombocytopenia, however, this clinical feature began earlier during the febrile phase and lasted till defervescence. A total of 6 different cytokines were thought to have possible association with platelet destruction, with 3 (IL-7, IL-12 and PDGF) occurring at both febrile and defervescence. IL-12 has been known to stimulate platelet-activating factor (PAF) [57] and the decrease of the cytokine probably disallowed normal platelet aggregation and degranulation. As mentioned earlier, the platelets are also known to release several growth factors, which probably explain the decreased levels of both VEGF and PDGF in the event of thrombocytopenia. Elevated levels of IL-7, conversely, have been previously shown to be involved in thrombocytosis [58]. During defervescence, the low levels of IL-5 probably deregulated functional PAF on eosinophils [59]. The presence of atypical lymphocytes in patients with warning signs from defervescence onwards was associated with G-CSF and IP-10.

The AST enzyme in dengue patients with warning signs was raised throughout the illness, and this enzyme was associated (i) at the febrile phase- ICAM-1, FGF-Basic, IL-13, IL-4, IL-12 and VEGF; (ii) defervescence: PDGF and (iii) convalescence- IFN-γ and IP-10. ICAM-1 has been suggested to play a role in inflammatory liver diseases [60] by recruiting leukocytes which can injure tissue by releasing various proteases and oxidants. The anti-inflammatory cytokines, IL-13 and IL-4 has been known to have hepatoprotective effects [61], [62]. IL-12 overexpression, as pointed out earlier, had been involved in liver damage, and many of its effects has been implied to be mediated by IFN-γ [53]. IFN-γ has also been suggested to be a negative regulator in liver cell proliferation and also to aggravate hepatitis viral-induced liver damage [63]. In an adult liver, endothelial cells provide nutritional and trophic support [64] and these cells are activated by VEGF and PDGF, whereby an association of decreased levels of VEGF was noticed with elevated levels of AST.

The main limitation in our study was the sample size number, and even though all patients were accounted and tested for cytokine levels at every phase of illness, some patients had undetected response towards certain cytokines. Furthermore, we did not include the severe dengue cases in our analyses as there were only four of them, hence losing out on valuable information as we could not establish cytokine trends/profile for severe dengue patients. Despite all these, our findings managed to re-establish the roles and dynamism of multiple cytokines at different phases of illness according to the new WHO dengue classification. The cytokine profiles from this study not only may have provided probable prognostics markers for but also shed new insights in dengue pathogenesis and this warrants further study. With the recent advancement of cytokine adjuvants and anti-cytokine therapies, our findings may serve towards better management in the field of dengue which is currently lacking a vaccine.

Author Contributions

Selection of patients for study: LCSL SP. Demarcation of patient category: LCSL SP. Demarcation to new WHO classification: RM. Conceived and designed the experiments: SDS SMW. Performed the experiments: AR SMW. Analyzed the data: AMK YH AR SDS. Contributed reagents/materials/analysis tools: SDS. Wrote the paper: AR AMK SDS SMW.

References

  1. 1. TDR/WHO (2009) Dengue: guidelines for diagnosis, treatment, prevention and control; World Health O, Special Programme for R, Training in Tropical D, World Health Organization. Department of Control of Neglected Tropical D, World Health Organization E et al., editors. Geneva: World Health Organization.
  2. 2. Webster DP, Farrar J, Rowland-Jones S (2009) Progress towards a dengue vaccine. Lancet Infect Dis 9: 678–687.
  3. 3. Halstead SB, Nimmannitya S, Cohen SN (1970) Observations related to pathogenesis of dengue hemorrhagic fever. IV. Relation of disease severity to antibody response and virus recovered. Yale J Biol Med 42: 311–328.
  4. 4. Halstead SB, Porterfield JS, O’Rourke EJ (1980) Enhancement of dengue virus infection in monocytes by flavivirus antisera. Am J Trop Med Hyg 29: 638–642.
  5. 5. Kurane I, Ennis FE (1992) Immunity and immunopathology in dengue virus infections. Semin Immunol 4: 121–127.
  6. 6. Mongkolsapaya J, Dejnirattisai W, Xu XN, Vasanawathana S, Tangthawornchaikul N, et al. (2003) Original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever. Nat Med 9: 921–927.
  7. 7. Mathew A, Rothman AL (2008) Understanding the contribution of cellular immunity to dengue disease pathogenesis. Immunol Rev 225: 300–313.
  8. 8. Burke-Gaffney A, Keenan AK (1993) Modulation of human endothelial cell permeability by combinations of the cytokines interleukin-1 alpha/beta, tumor necrosis factor-alpha and interferon-gamma. Immunopharmacology 25: 1–9.
  9. 9. Anderson R, Wang S, Osiowy C, Issekutz AC (1997) Activation of endothelial cells via antibody-enhanced dengue virus infection of peripheral blood monocytes. J Virol 71: 4226–4232.
  10. 10. Lee YR, Liu MT, Lei HY, Liu CC, Wu JM, et al. (2006) MCP-1, a highly expressed chemokine in dengue haemorrhagic fever/dengue shock syndrome patients, may cause permeability change, possibly through reduced tight junctions of vascular endothelium cells. J Gen Virol 87: 3623–3630.
  11. 11. Bosch I, Xhaja K, Estevez L, Raines G, Melichar H, et al. (2002) Increased production of interleukin-8 in primary human monocytes and in human epithelial and endothelial cell lines after dengue virus challenge. J Virol 76: 5588–5597.
  12. 12. Talavera D, Castillo AM, Dominguez MC, Gutierrez AE, Meza I (2004) IL8 release, tight junction and cytoskeleton dynamic reorganization conducive to permeability increase are induced by dengue virus infection of microvascular endothelial monolayers. J Gen Virol 85: 1801–1813.
  13. 13. Nguyen TH, Nguyen TL, Lei HY, Lin YS, Le BL, et al. (2005) Association between sex, nutritional status, severity of dengue hemorrhagic fever, and immune status in infants with dengue hemorrhagic fever. Am J Trop Med Hyg 72: 370–374.
  14. 14. Chakravarti A, Kumaria R (2006) Circulating levels of tumour necrosis factor-alpha & interferon-gamma in patients with dengue & dengue haemorrhagic fever during an outbreak. Indian J Med Res 123: 25–30.
  15. 15. Perez AB, Garcia G, Sierra B, Alvarez M, Vazquez S, et al. (2004) IL-10 levels in Dengue patients: some findings from the exceptional epidemiological conditions in Cuba. J Med Virol 73: 230–234.
  16. 16. Becerra A, Warke RV, Martin K, Xhaja K, de Bosch N, et al. (2009) Gene expression profiling of dengue infected human primary cells identifies secreted mediators in vivo. J Med Virol 81: 1403–1411.
  17. 17. Chaturvedi UC, Agarwal R, Elbishbishi EA, Mustafa AS (2000) Cytokine cascade in dengue hemorrhagic fever: implications for pathogenesis. FEMS Immunol Med Microbiol 28: 183–188.
  18. 18. Mustafa AS, Elbishbishi EA, Agarwal R, Chaturvedi UC (2001) Elevated levels of interleukin-13 and IL-18 in patients with dengue hemorrhagic fever. FEMS Immunol Med Microbiol 30: 229–233.
  19. 19. Chaturvedi UC (2009) Shift to Th2 cytokine response in dengue haemorrhagic fever. Indian J Med Res 129: 1–3.
  20. 20. Priyadarshini D, Gadia RR, Tripathy A, Gurukumar KR, Bhagat A, et al. (2010) Clinical findings and pro-inflammatory cytokines in dengue patients in Western India: a facility-based study. PLoS One 5: e8709.
  21. 21. Yong YK, Thayan R, Chong HT, Tan CT, Sekaran SD (2007) Rapid detection and serotyping of dengue virus by multiplex RT-PCR and real-time SYBR green RT-PCR. Singapore Med J 48: 662–668.
  22. 22. Lam SK, Devi S, Pang T (1987) Detection of specific IgM in dengue infections. Southeast Asian J Trop Med Pub Health 18: 532–538.
  23. 23. Clarke DH, Casals J (1958) Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses. Am J Trop Med Hyg 7: 561–573.
  24. 24. Ku CC, Murakami M, Sakamoto A, Kappler J, Marrack P (2000) Control of Homeostasis of CD8+ Memory T Cells by Opposing Cytokines. Science 288: 675–678.
  25. 25. Minty A, Chalon P, Derocq JM, Dumont X, Guillemot JC, et al. (1993) Interleukin-13 is a new human lymphokine regulating inflammatory and immune responses. Nature 362: 248–250.
  26. 26. Chen J, Ng MM, Chu JJ (2008) Molecular profiling of T-helper immune genes during dengue virus infection. Virol J 5: 165.
  27. 27. Koraka P, Suharti C, Setiati TE, Mairuhu ATA, Van Gorp E, et al. (2001) Kinetics of Dengue Virus-Specific Serum Immunoglobulin Classes and Subclasses Correlate with Clinical Outcome of Infection. Journal of Clinical Microbiology 39: 4332–4338.
  28. 28. Kurane I, Innis BL, Nimmannitya S, Nisalak A, Meager A, et al. (1991) Activation of T lymphocytes in dengue virus infections. High levels of soluble interleukin 2 receptor, soluble CD4, soluble CD8, interleukin 2, and interferon-gamma in sera of children with dengue. The Journal of Clinical Investigation 88: 1473–1480.
  29. 29. Bozza F, Cruz O, Zagne S, Azeredo E, Nogueira R, et al. (2008) Multiplex cytokine profile from dengue patients: MIP-1beta and IFN-gamma as predictive factors for severity. BMC Infectious Diseases 8: 86.
  30. 30. Dinarello CA (1999) Interleukin-18. Methods 19: 121–132.
  31. 31. Kohno K, Kataoka J, Ohtsuki T, Suemoto Y, Okamoto I, et al. (1997) IFN-gamma-inducing factor (IGIF) is a costimulatory factor on the activation of Th1 but not Th2 cells and exerts its effect independently of IL-12. J Immunol 158: 1541–1550.
  32. 32. Green S, Vaughn DW, Kalayanarooj S, Nimmannitya S, Suntayakorn S, et al. (1999) Elevated plasma interleukin-10 levels in acute dengue correlate with disease severity. J Med Virol 59: 329–334.
  33. 33. Azeredo EL, De Oliveira-Pinto LM, Zagne SM, Cerqueira DI, Nogueira RM, et al. (2006) NK cells, displaying early activation, cytotoxicity and adhesion molecules, are associated with mild dengue disease. Clin Exp Immunol 143: 345–356.
  34. 34. Chen LC, Lei HY, Liu CC, Shiesh SC, Chen SH, et al. (2006) Correlation of serum levels of macrophage migration inhibitory factor with disease severity and clinical outcome in dengue patients. Am J Trop Med Hyg 74: 142–147.
  35. 35. Hsieh MF, Lai SL, Chen JP, Sung JM, Lin YL, et al. (2006) Both CXCR3 and CXCL10/IFN-inducible protein 10 are required for resistance to primary infection by dengue virus. J Immunol 177: 1855–1863.
  36. 36. Angiolillo AL, Sgadari C, Taub DD, Liao F, Farber JM, et al. (1995) Human interferon-inducible protein 10 is a potent inhibitor of angiogenesis in vivo. J Exp Med 182: 155–162.
  37. 37. Gu L, Tseng S, Horner RM, Tam C, Loda M, et al. (2000) Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. Nature 404: 407–411.
  38. 38. Dewberry RM, Crossman DC, Francis SE (2003) Interleukin-1 receptor antagonist (IL-1RN) genotype modulates the replicative capacity of human endothelial cells. Circ Res 92: 1285–1287.
  39. 39. Dahinden CA, Geiser T, Brunner T, von Tscharner V, Caput D, et al. (1994) Monocyte chemotactic protein 3 is a most effective basophil- and eosinophil-activating chemokine. J Exp Med 179: 751–756.
  40. 40. Sierra B, Perez AB, Vogt K, Garcia G, Schmolke K, et al. (2010) MCP-1 and MIP-1alpha expression in a model resembling early immune response to dengue. Cytokine 52: 175–183.
  41. 41. Cardier JE, Rivas B, Romano E, Rothman AL, Perez-Perez C, et al. (2006) Evidence of vascular damage in dengue disease: demonstration of high levels of soluble cell adhesion molecules and circulating endothelial cells. Endothelium 13: 335–340.
  42. 42. Khongphatthanayothin A, Phumaphuti P, Thongchaiprasit K, Poovorawan Y (2006) Serum levels of sICAM-1 and sE-selectin in patients with dengue virus infection. Jpn J Infect Dis 59: 186–188.
  43. 43. Jamaluddin MS, Wang X, Wang H, Rafael C, Yao Q, et al. (2009) Eotaxin Increases Monolayer Permeability of Human Coronary Artery Endothelial Cells. Arteriosclerosis, Thrombosis, and Vascular Biology 29: 2146–2152.
  44. 44. Tyner JW, Uchida O, Kajiwara N, Kim EY, Patel AC, et al. (2005) CCL5-CCR5 interaction provides antiapoptotic signals for macrophage survival during viral infection. Nat Med 11: 1180–1187.
  45. 45. Cao R, Brakenhielm E, Pawliuk R, Wariaro D, Post MJ, et al. (2003) Angiogenic synergism, vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2. Nat Med 9: 604–613.
  46. 46. Watt G, Jongsakul K, Chouriyagune C, Paris R (2003) Differentiating dengue virus infection from scrub typhus in Thai adults with fever. Am J Trop Med Hyg 68: 536–538.
  47. 47. Gonzalez D, Castro OE, Kouri G, Perez J, Martinez E, et al. (2005) Classical dengue hemorrhagic fever resulting from two dengue infections spaced 20 years or more apart: Havana, Dengue 3 epidemic, 2001–2002. Int J Infect Dis 9: 280–285.
  48. 48. Gear ARL, Camerini D (2003) Platelet Chemokines and Chemokine Receptors: Linking Hemostasis, Inflammation, and Host Defense. Microcirculation 10: 335–350.
  49. 49. Gabay C, Smith MF, Eidlen D, Arend WP (1997) Interleukin 1 receptor antagonist (IL-1Ra) is an acute-phase protein. The Journal of Clinical Investigation 99: 2930–2940.
  50. 50. Bruno CM, Valenti M, Bertino G, Ardiri A, Amoroso A, et al. (2011) Relationship between circulating interleukin-10 and histological features in patients with chronic C hepatitis. Ann Saudi Med 31: 360–364.
  51. 51. Narumi S, Tominaga Y, Tamaru M, Shimai S, Okumura H, et al. (1997) Expression of IFN-inducible protein-10 in chronic hepatitis. J Immunol 158: 5536–5544.
  52. 52. Elsammak MY, Al-Sharkaweey RM, Ragab MS, Amin GA, Kandil MH (2008) IL-4 and reactive oxygen species are elevated in Egyptian patients affected with schistosomal liver disease. Parasite Immunology 30: 603–609.
  53. 53. Leifeld L, Cheng S, Ramakers J, Dumoulin F-L, Trautwein C, et al. (2002) Imbalanced intrahepatic expression of interleukin 12, interferon gamma, and interleukin 10 in fulminant hepatitis B. Hepatology. 36: 1001–1008.
  54. 54. Tung K-H, Huang Y-S, Yang K-C, Perng C-L, Lin H-C, et al. (2010) Serum Interleukin-12 Levels in Alcoholic Liver Disease. Journal of the Chinese Medical Association 73: 67–71.
  55. 55. Heil M, Clauss M, Suzuki K, Buschmann IR, Willuweit A, et al. (2000) Vascular endothelial growth factor (VEGF) stimulates monocyte migration through endothelial monolayers via increased integrin expression. Eur J Cell Biol 79: 850–857.
  56. 56. Czaja MJ, Geerts A, Xu J, Schmiedeberg P, Ju Y (1994) Monocyte chemoattractant protein 1 (MCP-1) expression occurs in toxic rat liver injury and human liver disease. Journal of Leukocyte Biology 55: 120–126.
  57. 57. Bussolati B, Rollino C, Mariano F, Quarello F, Camussi G (2000) IL-10 stimulates production of platelet-activating factor by monocytes of patients with active systemic lupus erythematosus (SLE). Clin Exp Immunol 122: 471–476.
  58. 58. Wasilewska A, Zoch-Zwierz WM, Tomaszewska B, Zelazowska B (2005) Relationship of serum interleukin-7 concentration and the coagulation state in children with nephrotic syndrome. Pediatrics International 47: 424–429.
  59. 59. Kishimoto S, Shimadzu W, Izumi T, Shimizu T, Fukuda T, et al. (1996) Regulation by IL-5 of expression of functional platelet-activating factor receptors on human eosinophils. The Journal of Immunology 157: 4126–4132.
  60. 60. Kono H, Uesugi T, Froh M, Rusyn I, Bradford BU, et al. (2001) ICAM-1 is involved in the mechanism of alcohol-induced liver injury: studies with knockout mice. American Journal of Physiology - Gastrointestinal and Liver Physiology 280: G1289–G1295.
  61. 61. Yoshidome H, Kato A, Miyazaki M, Edwards MJ, Lentsch AB (1999) IL-13 Activates STAT6 and Inhibits Liver Injury Induced by Ischemia/Reperfusion. The American Journal of Pathology 155: 1059–1064.
  62. 62. Seki T, Kumagai T, Kwansa-Bentum B, Furushima-Shimogawara R, Anyan WK, et al. (2012) Interleukin-4 (IL-4) and IL-13 Suppress Excessive Neutrophil Infiltration and Hepatocyte Damage during Acute Murine Schistosomiasis Japonica. Infection and Immunity 80: 159–168.
  63. 63. Horras CJ, Lamb CL, Mitchell KA (2011) Regulation of hepatocyte fate by interferon-γ. Cytokine & Growth Factor Reviews 22: 35–43.
  64. 64. Coultas L, Chawengsaksophak K, Rossant J (2005) Endothelial cells and VEGF in vascular development. Nature 438: 937–945.