Background
Dengue virus (DENV) infection remains the most common arthropod-borne viral infection worldwide [
1,
2], with a recent global estimate suggesting that ~390 million clinical DENV cases occur annually [
3]. Nearly ~75% of the global DENV disease burden is concentrated across South East Asia and Western Pacific [
4], and the incidence had been steadily increasing over the past decade at the rate of ~threefold from 1.2 million in 2008 to 3.2 million in 2015 [
3]. In 2015 alone, 111,000 DENV infection cases were reported from Malaysia [
3]. Though the mortality rates due to dengue is relatively low, the widespread distribution of DENV has led to significant morbidity across the country warranting a strong demand for hospital beds, finances and personnel thereby diverting recourses from other medical areas [
5]. However, not all DENV infections develop complications requiring hospitalization. According to the WHO classification 2009, dengue has been classified as with or without warning signs and severe dengue [
6] based on clinical symptoms and laboratory tests. Nonetheless, the initial clinical symptoms of dengue cannot distinguish between mild dengue from severe disease, and warning signs may only develop eventually as the disease progresses [
7,
8]. Furthermore, these warning signs usually occur only a day prior to clinical deterioration [
9,
10] rendering effective interventions challenging [
11]. Hence, there is a pressing need to identify biomarkers that will triage dengue patients into those that are most or least likely to develop severe forms of dengue disease, allowing best supportive care prioritised to individuals in need, especially during epidemics [
12].
Cytokine storm has been shown to affect vascular endothelial permeability [
13], and has also been associated with dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) [
14‐
16], as per the classical dengue disease classification. However, the role of cytokine storm and its association with the WHO 2009 classification i.e. dengue without warning signs, dengue with warning signs and severe dengue have seldom been studied. One recent study showed that products associated with microbial translocation, i.e. lipopolysaccharide (LPS) and the monocyte activation marker soluble CD14 (sCD14) were elevated among patients with severe dengue disease. This suggests that apart from viral factors, cytokine storm may also be at least in part due to the elevation of LPS in plasma and perhaps other danger-associated molecule patterns (DAMPs). Here, we investigated plasma markers including cytokines, chemokines as well as other inflammatory mediators that predict the severity of dengue disease with respect to the WHO 2009 classification. Our results indicate that markers associated with inflammasome activation (IL-18), microbial translocation, monocyte activation (sCD14) and LPS binding protein (LBP) were among the best predictors in clinical DENV infection.
Discussion
Dengue has become a public health concern. With the new dengue classification by WHO in 2009, studies in the past that predict DHF and DSS according to the WHO 1997 classification may not be applicable to this new classification. Early prediction of DWS+ and SD is of pivotal importance towards identifying dengue patients who will need the best supportive care, thereby helping to optimize the management of severe cases [
12]. An ideal biomarker needs to identify individuals who are at risk of developing DWS+ or SD when they first present at the hospital, typically during the febrile stage. The present study is the first to investigate the association of biomarkers using the new WHO classification. We investigated 20 plasma biomarkers and identified that IL-18 at both febrile and defervescence phases as well as LBP and sCD14 at febrile phase are of the best predictive value. In addition, decreased levels of IP-10 and IL-10 as well as increased levels of CXCL6 and VEGF were associated with development of SD. We also show that the platelet counts were correlated with VEGF and inversely correlated with sCD14.
Most hypotheses explaining dengue immunopathogenesis suggest 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 [
8]. Here we observed that the severity of dengue disease was associated with aberrant activation of monocyte or macrophages as evidenced by elevation of plasma IL-18, sCD14 and LBP particularly at the febrile phase. Furthermore, the free circulating IL-18 remained significantly elevated among patients with the more severe form of dengue disease.
IL-18 is a potent proinflammatory cytokine predominantly produced by activated monocytes/macrophages via the NLRP3 inflammasome pathway [
32,
33]. Interleukin-18 is present in monocytes or macrophages as a biologically inactive precursor form, proIL-18 [
32,
33]. Upon NLRP3 activation and assembly, the proIL-18 will be further processed by intracellular cysteine protease caspase-1 and released as a fully functional matured IL-18. The elevation of IL-18 among DWS+ and SD patients in this cohort suggest that aberrant activation of the inflammasome may play a role in dengue immunopathogenesis, consistent with previous reports [
7,
17,
34,
35]. IL-18 secreted by monocytes/macrophages will then induce activation and expression of both CC and CXC chemokines from a wide range of cells [
36] enhancing natural killer (NK) cell cytotoxicity, neutrophil activity, and IFN-γ production by T cells and NK cells [
36]. This is in line with our observation that CXCL6, a neutrophilic chemokine was elevated among DWS+ and SD patients at the defervescence phase.
However, the biological activity of IL-18 is regulated by a naturally occurring IL-18 binding protein (IL-18BP). To gain better insight into the regulation of biological activity of IL-18 during dengue disease, we measured the plasma levels of IL-18BPa and free circulating IL-18 levels were estimated. Our data demonstrated that free circulating IL-18 was associated with severity of the dengue disease. Furthermore, the correlation between IL-18 and IL-18BP was stronger in DWS− patients as compared to DWS+/SD patients. In contrast, Michels et al. [
37] showed no significant elevation of free circulating IL-18 among patients with severe dengue as compared to non-severe dengue. The exact reason behind this discrepancy is not known and may reflect the use of different assay methods or differences in patients’ characteristics and therefore require further investigation. Despite this, other studies have also found that dengue virus is capable to induce inflammasome activation via CLEC5A [
38]. Furthermore, NLRP-3 inflammasome in platelet was shown to be activated after expose to dengue virus in vitro and the level of inflammasome activation correlates with vascular permeability [
39]. These findings suggest that inflammasome activation may play a significant role in the immunopathogenesis of dengue disease.
Microbial translocation (MT) is a phenomenon characterized by translocation of microbial products e.g. LPS from the gut into blood stream under inflammatory conditions. Although MT has been studied extensively in HIV disease [
40], it’s also shown to play an important role in other diseases such as graft versus host disease after hematopoietic stem cell transplantation [
41], inflammatory bowel syndrome [
42], chronic liver disease [
43] and even end stage of kidney disease [
44]. Recently, MT has been observed among dengue patients [
25] and levels of LPS was associated with severity of dengue disease [
26]. Extending from these study, we sought to determine the values of MT markers i.e. LBP and sCD14 in predicting severity of dengue disease. The LBP is a soluble acute-phase protein that binds to bacterial LPS and triggers immune responses by transferring the LPS to sCD14 and TLR4 expressed on cell surface of monocyte/macrophages [
45], while sCD14 is secreted by monocyte and macrophages upon LPS stimulation [
46]. While our data showed that both LBP and sCD14 were predicted with DWS+ and SD, consistent with previous reports [
25,
47], by using a multivariate regression model, we showed IL-18, LBP and sCD14 were independently associated with increased risk of developing DWS+ and SD. Furthermore, we also showed sCD14 was inversely correlated with platelet counts. This data suggests that apart from inflammasome activation, MT also plays an important role in driving the exacerbated inflammation in dengue disease. Since LPS is also known for causing systemic immune activation and noncanonical inflammasome activation, thus elevation of plasma LPS among DWS+ and SD patients is thereby fuelling inflammatory responses.
Although DWS+ is associated with some forms of plasma leakage and spontaneous bleeding, this does not invariably lead to SD which is characterised by severe plasma leakage leading to the reduced pulse pressure, disseminated intravascular coagulation and multiple organ failure. We therefore sought to identify possible cytokines and immune factors that may contribute to the development of severe dengue. Our data showed that SD was associated with decreased levels of IL-10 and IP-10 and increased levels of CXCL6 at the defervescence phase. One study found that patients who developed severe dengue had significantly lower T cell counts when compared to non-severe dengue, whilst their serum IL-10 and IP-10 levels positively correlated with T cell apoptosis [
48]. Therefore, it is plausible that the decreased of IL-10 and IP-10 among patients with SD in our cohort was due to excessive loss of T cells. CXCL6 is a chemokine that is known to exert potent neutrophil activation and chemotaxis [
49], hence elevation of CXCL6 and VEGF among SD patients may suggest a role for neutrophils in the pathogenesis of SD.
To date, the causes of increased vascular permeability among dengue patients is not well understood. Several lines of evidence suggest that endothelial dysfunction rather than necrosis of the endothelium is likely to be the cause of vascular leak [
50,
51]. VEGF is a potent growth factor that has a role in promoting endothelial permeability and proliferation and it may contribute to inflammation and coagulation [
52]. Consistent with previous reports [
8,
53,
54], we found that the VEGF was consistently elevated at both febrile and defervescence phases. Since VEGF is a platelet derived growth factor [
55], hence elevation of VEGF among patients with severe dengue may also suggest aberrant platelet activation.
One limitation in our study was the sample size number especially in the severe dengue group as there were only six of them in the febrile phase and 4 in the defervescence phase, hence losing out some important details as we had to combine the SD patients with DWS+ patients in some of the analysis. Future study with bigger numbers of SD patients is warranted. Notwithstanding this limitation, our study has identified some plasma markers to allow for early and accurate prediction of DWS+ and SD, thereby serving as possible biomarkers especially during outbreaks.
Authors’ contributions
YKY, HYT, EMS, and SDS conceived and designed the experiments; YKY and HYT performed the experiments; YKY, HYT, EMS, SDS analyzed the data; SDS, SKN, SHJ and RM contributed reagents, materials, and analysis tools; and YKY, HYT, EMS and SDS wrote the manuscript. All authors read and approved the final manuscript.