Number and activation status of immune cells
DENV has been shown to infect a wide range of cells including dendritic cells (DCs),
monocytes, lymphocytes, hepatocytes, endothelial cells (ECs) and mast cells in vitro6]. Although the role of these cells in DENV infection remains less clear in vivo, activation of memory T cells resulting in cascades of inflammatory cytokines and
other chemical mediators that trigger death of target cells through apoptosis is a
critical element contributing to severe dengue 11]. DCs and macrophages are the primary targets of DENV infection 12], 13]. Both the absolute number and frequency of circulating myeloid DCs (mDCs) and plasmacytoid
DCs (pDCs) were decreased early in acute viral illness in children but not in adults
who subsequently developed DHF and decreased level of pDCs was associated with higher
viremia levels 14], 15]. Activated DCs may contribute to vascular leak through the production of TNF-?, IFN-?
and matrix metalloproteases-2, 3 and 9 16], 17]. Studies show that CD4 T cell, CD8 T cell, NK cell and ?? T cell counts were significantly
decreased in DHF compared to DF early in the course of illness 18]. The CD8 T cells and NK cells from dengue patients displayed activation markers such
as CD69, HLA-DR, CD38 and cytotoxic granule TIA-1 and cell adhesion molecules CD44
and CD11a during the acute phase 19], 20]. The decreased numbers of lymphocytes could be due to increased apoptosis of peripheral
blood mononuclear cells observed during DENV infection evidenced by the presence of
increased plasma levels of soluble CD95, a mediator of apoptosis, and down-regulation
of the antiapoptotic protein Bcl-2 in these cells 21], 22]. Cross-reactive memory T cells that are highly activated with increased levels of
cytokine producing capacity was observed in patients with acute dengue disease. Activation
of cross-reactive low affinity T-cells results in copious amounts of cytokine and
chemokine production such as IFN-?, TNF-?, IL-1, IL-6, IL-8, IL-10, CCL2 (MCP-1) and
CCL5 (RANTES) 23], 24].
Activation of mast cells and increased levels of urinary histamine, which is a major
product of mast cells were observed in dengue patients and the levels correlated with
disease severity 25]. Several data indicated that virus stimulated mast cells selectively produced and
secreted a variety of mediators including chemokines, cytokines, lipid mediators,
and granule associated products. Elevated levels of secreted CCL3 (MIP-1?), CCL4 (MIP-1?)
and CCL5 were observed following infection of human mast cell lines 26], 27]. Substantial levels of tryptase and chymase were found in mast cells and these proteases
are considered to be selective markers of mast cell activation. Plasma levels of both
tryptase and chymase were increased significantly in DHF/DSS compared with DF. 28] Thrombocytopenia is one of the clinical hallmarks for dengue patients. There are
many mechanisms leading to the depletion of platelets in affected subjects, these
include direct infection of megakaryocytes by DENV as well as platelet destruction
due to nonstructural protein 1 (NS1) binding and platelet-associated antibodies 29]–31]. Thrombocytopenia is best used as a marker of severe disease particularly when it
is 100,000 cells/c.mm and serve as an indicator of prognosis during the course of
the disease 18], 32], 33]. Since thrombocytopenia is seen in both DF and DHF patients, a platelet count of
60,000 cells/c.mm serves as a better cut-off in identifying more severe cases 7].
Increased levels of cytokines and chemokines
Patients with DHF/DSS present a ‘cytokine storm’, with high levels of circulating
cytokines and chemokines. Therefore, serum cytokine and chemokine levels can serve
as a laboratory tool for predicting severe disease. T cells, NK cells, monocytes,
macrophages, hepatocytes and ECs have been shown to contribute to the increased production
of cytokines and chemokines. Increased levels of IFN-?, TNF-?, IL-1?, IL-4, IL-6,
IL-7, IL-8, IL-10, IL-13, IL-15, IL-17, IL-18, macrophage migration inhibitory factor
(MIF) and chemokines CCL2, CCL4, CCL5, and CXCL10 (IP-10) have been reported in patients
with DHF when compared to DF. 34]–38] Studies show that elevated levels of IL-6, IL-10, IFN-?, MIF, and CCL-4 could be
used as potential predictors of severe dengue 39]–46].
Complement, antibodies and other soluble factors
Complement activation and an increase in complement protein products correlate with
severe dengue disease 47]. Large amounts of C3, C3a and C5a have been detected in DENV-infected patients and
determining their levels in serum is important since these anaphylatoxins direct the
lysis of infected cells and mast cell degranulation leading to histamine release 48], 49]. An increase in the number of B lymphocytes was demonstrated in DHF. 18] Total and dengue-specific IgE antibody levels were higher in patients with DHF and
DSS compared with those with DF. 50] NS1 is an immunogen and high concentrations of anti-NS1 antibodies have been found
in severe disease. Antibodies to NS1 can cross-react with human ECs and platelets
and cause vascular permeability with production of nitric oxide (NO) and apoptosis
51], 52]. Determining the levels of dengue-specific IgE, anti-platelet and anti-EC antibodies
might be used as biomarkers of severe dengue disease. Soluble factors are more stable
and have the potential to serve as biomarkers. Increased levels of soluble receptors
such as sTNFRII, sCD4, sCD8, sIL-2R were reported in DHF patients when compared to
those with DF and that their levels correlated with disease severity 18], 53]. Release of sTNFR may be an early and specific marker of the endothelial changes
that cause DSS 42]. The IL-1 receptor-like-1 protein (IL1RL1), also known as ST2, is a member of the
IL1R/Toll-like receptor (TLR) superfamily. Increased serum sST2 was found in patients
having secondary infection and DHF patients compared to DF patients and may be a predictive
marker of dengue severity 44], 54].