Immune responses and dengue virus pathogenesis
DENV infection is a systemic and dynamic disease with a wide clinical spectrum. Gross pathological findings in cases of DHF or DSS include hemorrhages in the skin, subcutaneous tissues, gastrointestinal tract, and heart (129). Hemorrhage, dilatation and congestion of vessels, and edema of arterial walls are commonly found, and hemorrhagic manifestations in other organs combined with fluid accumulations in body cavities may be substantial (130, 131).
However, the underlying mechanisms of vascular leakage and hemorrhage are not well characterized. Elevated plasma levels of pro-inflammatory and vasoactive cytokines before and at the time of plasma leakage in patients with DHF suggest that excessive cytokine production (a ‘cytokine storm’) induce vascular permeability. Available data propose that the outcome of a DENV infection depends on a balance between favorable and unfavorable immune responses; the former providing control of viral replication, whereas the latter enhancing inflammatory and vascular permeability. The lack of reliable immunological markers for either protective or pathological immune responses to DENV and the lack of a suitable animal model for dengue disease hamper the understanding of dengue pathogenesis. Insights into the immune response against DENV infection rely primarily on clinical and epidemiological studies.
Tropism
Identification of the primary target cells of DENV replication has proven to be extremely difficult. Existing data are based on virus detection by immunohistochemical (IHC) analysis with antibodies against viral structural proteins, or by in situ hybridization to the positive-strand vRNA. However, it is difficult to prove direct infection of specific target cells by these methods as a positive signal could be due to virus endocytosed or phagocytosed by uninfected cells. Detection of negative-strand vRNA and/or DENV NS proteins would provide much stronger evidence of active DENV replication.
After inoculation by an infected mosquito, the initial round of viral replication is believed to occur in the subdermal Langerhans DCs (132-135). These infected cells become activated and migrate to draining lymph nodes (136). The activated DCs elicit a robust IFNα/β and TNFα response together with a strong pro-inflammatory response to limit contiguous spread (133). Viral replication continues in still undefined cells in the lymph node. There is a general consensus that candidate cell types belong to the macrophage-monocyte lineage. Autopsies and human biopsies confirm that cells from the mononuclear phagocyte linage probably are the primary targets of DENV infection following initial dissemination from the local skin site. Infiltrating mononuclear cells in affected tissues have been shown to contain DENV antigen (137, 138) and DENVs can occasionally be isolated from peripheral blood leukocyte fractions (139). Similar observations have been made in rhesus macaques where DENV was recovered from leukocyte-rich tissues such as regional lymph nodes, systemic lymphatic tissues, and disseminated skin sites.
Infection is amplified within the lymph nodes and viremia can be detected when the infectious virus enters the circulation via the efferent lymphatic system and thoracic duct. Circulating monocytes in the blood are believed to be infected due to the viremia facilitating spread to secondary visceral organs where macrophages within the spleen, liver, and bone marrow are infected (140-145).
There has been limited and inconsistent dissemination to solid organs (146); DENV antigen has been detected in lymphocytes (140, 147) hepatocytes (147-150), endothelium (140, 147, 151, 152), and cerebral neurons and astrocytes (147, 152). There are in addition other studies with contradicting results where the same tissues have been examined without any detected DENV antigen (140, 150, 151).
A further controversy surrounds the role of endothelial cells as the target for DENV infection. Severe dengue disease is characterized by systemic endothelial dysfunction accompanied by vascular leakage, even though destructive vascular lesions are generally absent in fatal cases (83). Primary human endothelial cells and human endothelial cell lines are permissive for DENV infection (47, 153), but endothelial infection, however, does not seem to be required for severe pathologic changes in individual tissues (154). Their contribution in vivo remains to be established.
The presence of DENV antigens in various organs and cell types suggest that the host receptor(s) is broadly distributed. Host receptors for DENV are believed to include mannose binding protein, heparan sulfate, chondroitin sulfate, and DC-SIGN (26, 155, 156). Following DENV infection natural antibodies (IgM), complement, and possibly NK cells control the initial levels of viremia and to certain extent tissue dissemination. Upon recognition by cytotoxic T lymphocytes, infected cells are targeted by the cellular immune system (discussed below).
The humoral immune response
The humoral immune response is hypothesized to be vital for controlling DENV infection and dissemination, and infection with one serotype provides long-lasting protection to that specific serotype (homotypic immunity). Subsequent infection by another serotype results in short-lived protection (heterotypic immunity), and may eventually be harmful and increase the risk of severe dengue disease. The transient nature of heterotypic immunity is believed to be due to cross-reactive viral E protein–specific antibodies which are protective above a certain concentration threshold (157).
The principal targets of the antibody response to DENV infection in humans are the prM, the E structural proteins, and the NS1 protein. Weak antibody responses to other NS proteins, for example, NS3 and NS5, have also been detected (158, 159). Neutralizing antibodies are directed against the viral E protein and inhibit viral attachment, internalization, and replication within cells. There are multiple epitopes residing within each of the three E domains (160, 161), but not all are equally accessible for antibody binding due to the dimeric conformation of the E protein on the virion surface, and its tight packing in the mature form (162-164),
Domain III of the E protein, which contains the putative host receptor-binding site, is the most variable in amino acid sequence between serotypes. As a result, antibodies specific for this domain show the greatest degree of serotype specificity (165). However, mutations in domain III of the E protein are common for escaping neutralizing antibody (166, 167). Loss of an effective neutralizing antibody response due to sequence variation has also been detected for the C and NS2B proteins (168, 169).
Antibodies against DENV may also bind to complement proteins and promote their activation. Anti-prM and/or E protein antibody–mediated complement fixation to virions can inhibit viral infection (170). As for other host immune responses to dengue, complement involvement may also be pathological. Complement activation is a feature of severe dengue and is temporally related to plasma leakage. This suggests that complement activation constitutes a major factor in the pathogenesis of dengue hemorrhagic shock (171, 172). Increased complement activation at endothelial cell surfaces could contribute to the vascular leakage, and the viral protein NS1 is proposed to be a modulator of the complement pathway. By promoting efficient degradation of C4 to C4b, NS1 may protect DENV from complement-dependent neutralization in solution (47).
The cellular immune response
In addition to the humoral immune response, cellular immune responses are also crucial in dengue pathogenesis. The DENV can infect both CD4+ T-cells and CD8+ T-cells (173), and similar to DENV-specific antibodies, the cellular immune responses can be either protective or harmfully reactive. DENV-specific T-cells respond with a diverse set of effector functions, including proliferation, target cell lysis, and the production of a range of cytokines. CD4+ T-cells produce IFNγ, TNFα, TNFβ, interleukin (IL)-2, and CC-chemokine ligand 4 (CCL4; also known as MIP1β) which may contribute to pathogenesis (174). The production of T helper type-2 cytokines, such as IL-4, is less common (175-178). In uncomplicated DENV infections, relatively more CD8+ T-cells are present resulting in lower levels of IFNγ and TNFα (179). CD8+ T-cell clones specific for DENV partially protect mice from lethal DENV challenge (180). The role of T-regulatory cells is unclear in dengue, but there is a study suggesting they are functional and expand in acute DENV infection (181).
Following primary infection, both serotype-specific and serotype cross-reactive memory T-cells are generated. Upon secondary exposure, both the protective and cross-reactive memory T-lymphocytes are activated and the non-protective memory T-cells will augment infection (182). Activated memory T-cells recognize both conserved and altered peptide ligand epitopes. The antigen sequence differences depend on the specific DENV epitope but will nevertheless affect the quality of the effector T-lymphocyte response. This in turn modifies the immunological repertoire and is suggested to be involved in the development of plasma leakage (183). A full agonist peptide will induce a full range of T-cell responses including production of multiple cytokines (e.g. IFNγ, TNF, and CCL4) and lysis of the infected cell. A partial agonist peptide, that is, one which varies at one residue, will cause cross-reactivity in memory T-cells and induce a skewed functional response, involving production of some cytokines but little of other cytokines and inefficient cell lysis. Thus, because of sequence diversity between DENV serotypes, the memory T-cells (and B cells) that are re-activated during a secondary DENV infection may not have optimal avidity for the epitopes of the new infecting virus. The ‘memory’ of the primary DENV infection alters the immune response to the secondary infection influencing the clinical outcome. There is a correlation between the level of T-cell responses and disease severity (184, 185). The phenomenon of low affinity for the current infecting serotype but a high affinity for a past infection with a different serotype is referred to as Original Antigenic Sin, and is the net effect of an altered balance between a protective and pathological outcome (185, 186). The pattern of antibody/T-cell responses in secondary DENV infections is also influenced by the sequence and interval between DENV infections (67, 116, 187-190). As for the ADE scenario, memory T-cell responses exhibiting serotype cross-reactive proliferative activity decades after the primary infection could potentially alter the balance from a protective immune response toward an improper and non-protective immune response. Interestingly, most of the identified CD4+ and CD8+ T-cell epitopes reside in the NS3 protein, which represents only ∼20% of the DENV amino acid coding sequence (179).
Cytokines in dengue pathogenesis
Viral recognition by the host cell occurs immediately upon virus entry to raise an appropriate antiviral response. Two main families of pathogen recognition receptors mediate DENV sensing; the extracellular/endosomal toll-like receptors (TLRs) (191, 192) and the cytoplasmic receptor family of DExD/H box RNA helicases (e.g. retinoic acid inducible gene 1 [RIG-1] and melanoma differentiation-associated gene-5 [MDA5]) (193) Binding to a TLR leads to activation of two families of transcriptional factors: the interferon regulatory factors (IRFs) and the nucleic factor-kappa B (NF-kB). These signaling cascades activate production of IFNα/β and proinflammatory cytokines that stimulate maturation of DCs and elicits an antiviral response (133, 194).
DENV is believed to primarily infect cells of the DC/macrophage/monocyte lineage via receptor-mediated endocytosis and/or enhanced uptake via antibody–virus complexes attached to Fc-gamma receptors (FcγR) (195). The exact mechanisms behind DHF/DSS are not understood, but the consensus is that infected cells and activated endothelial cells produce TNFα (196, 197), and NO (198, 199) increasing vascular wall permeability (200). The coincidence of severe disease manifestations with abatement of fever and virus control suggests that the symptoms may be a consequence of the immune response against the virus rather than virus-induced cytopathology. Consistent with this hypothesis is the increased levels of many different cytokines that have been observed in DENV infection (201). Elevated serum levels of cytokines and chemokines include IL-2 (202-204), IL-6 (205, 206), IL-8 (207), IL-10 (109, 202), IL-13, IL-18 (105), IFNγ (102, 103), TNFα (102, 205, 208, 209), and monocyte chemotactic protein-1 (MCP-1) (210). Furthermore, these cytokines are associated with secondary infections and severe dengue implying immunopathogenesis. However, it is not fully understood how these cytokines cause malfunction of vascular endothelial cells leading to plasma leakage. A Th1-type response is linked to recovery from acute infection, whereas a Th2-type response is associated with exacerbation of infection and a poor clinical outcome. Patients with non-severe dengue predominantly have a Th1-type response. Cross-regulation of Th1 and Th2 is primarily mediated by IL-10 and IFNγ, respectively (211). In addition, activated macrophages recruit CD4+ T-cells that produce human cytotoxic factor (hCF), which in turn induces a cytokine cascade that leads to a Th1-type or Th2-type response. Levels of hCF can be elevated in severe dengue cases and hCF autoantibodies protect against severe disease (201). As the severity of the illness increases, the response shifts to a Th2-type response, characterized by secretion of IL-4, IL-5, IL-6, IL-10, and IL-13. Infections primarily eliciting a humoral immune response induce a higher expression of Th2-related cytokines (201, 211).
Pathological features of hemorrhagic dengue are increased capillary permeability in the absence of morphological damage to the capillary endothelium, altered number and functions of leukocytes, increased haematocrit and thrombocytopenia. Thrombocytopenia is accompanied by plasma leakage and deregulated coagulation, and the latter is likely to be mediated by cytokines, for example, TNFα (212)]. Increased levels of IL-6 and IL-8 are associated with deregulated coagulation and fibrinolysis in dengue (213, 214). Thus, it is believed that indirect effects of virus infection render the vascular wall permeable. Secreted TNFα from activated, infected cells promotes increased endothelial permeability and increases the expression of adhesion molecules on endothelial cells (215-218), whereas increased IL-10 levels correlate to reduced levels of platelets and reduced platelet function (109, 219). This could contribute to the development of bleeding complications. Extensive plasma leakage into various serous cavities of the body, including the pleural, pericardial, and peritoneal cavities, may result in profound shock.
The immunopathogenesis of dengue has to a large extent been correlative in nature describing temporal associations between cytokine concentrations and the clinical events of plasma leakage. There is a need to identify causal immunopathogenic mechanisms compared to the abundance of descriptive studies of dengue pathogenesis. It is worth noting that other infectious diseases and inflammatory disorders with elevated cytokine levels are not accompanied by increased vascular permeability as seen in severe dengue. Thus, the challenge is to identify the key elements of the host immune response that are causally linked to papillary permeability from those that constitute the normal host immune response.