Immunopathogenesis of Dengue Virus-Induced Redundant Cell Death: Apoptosis and Pyroptosis

Abstract

Dengue virus infection is a self-limited condition, which is of particular importance in tropical and subtropical regions and for which no specific treatment or effective vaccine is available. There are several hypotheses explaining dengue pathogenesis. These usually refer to host immune responses, including antibody-dependent enhancement, cytokine expression, and dengue virus particles including NS1 protein, which lead to cell death by both apoptosis and pyroptosis. A clear understanding of the pathogenesis should facilitate the development of vaccines and therapies. This review focuses on the immunopathogenesis in relation to clinical manifestations and patterns of cell death, focusing on the pathogenesis of severe dengue.

Introduction

Dengue virus infection is one of the most common health problems in tropical and subtropical regions and the leading cause of dengue hemorrhagic fever (DHF) (38). This virus is endemic in over 125 countries, containing approximately 50% of the world's population (13,34). Around 100 million cases of dengue virus infection are annually reported (6). More than 500,000 cases of this infection develop into DHF, leading to 22,000 fatal cases annually. This virus is icosahedral in shape and is a positive-sense single-stranded RNA virus, which belongs to the Flaviviridae family and the Flavivirus genus.

The dengue virus consists of three structural proteins, namely C (capsid), PrM/M (pre/membrane), and E (envelope), and seven nonstructural (NS) proteins, namely NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. All these proteins are responsible for the formation of dengue virus particles. Dengue viruses include four specific serotypes: DENV-1, DENV-2, DENV-3, and DENV-4, which are distributed in different regions (6,18,32,45,46,50). The virus can infect and replicate in multiple cell types, which is the cause of the disease pathogenesis (37). Moreover, secondary infection by a dengue virus with a different serotype can increase the severity of the disease, influenced by an antibody-dependent enhancement (ADE) (15).

Currently, supportive treatment is the only valuable approach for dengue-infected patients (43). Now, a dengue vaccine is available, named Dengvaxia^®, which can reduce the likelihood of contracting the dengue virus to only 60%; unfortunately, this vaccine is not supported worldwide (11,17). Therefore, in 2011, the World Health Organization developed a system of dengue diagnosis by classifying the severity of dengue as follows: the first category is DF, which usually presents as high fever, retro-orbital pain, muscle and/or joint pain, severe headache, nausea, vomiting, and rash. The second category includes four grades of DHF (DHF grades I–IV), which involve the clinical symptoms of DF plus rapid breathing, severe abdominal pain, restlessness, fatigue, low blood pressure, and also plasma leakage from endothelial cell damage. In particularly severe cases, profound shock can occur (27,31).

There are several competing hypotheses about the pathogenesis of dengue virus infection (21). An interesting pathogenesis study demonstrated that the cell death rate in fatal cases of dengue infection was greater than in nonfatal cases, especially in dendritic cells. This finding indicated that the cell death rate is relevant to the pathogenesis of dengue virus infection (51). Thus, study of the pathogenesis of dengue virus infection in relation to cell death might be valuable for developing therapies that could reduce the mortality rate of dengue infection.

Mechanism of Dengue Infection, Virus Replication, and Dengue Pathogenesis

During blood feeding of dengue virus-infected mosquitoes, the proboscis penetrates the epidermis and releases the dengue virus residing in the mosquito's salivary glands to target cells (8,16). The virus then begins to replicate using ligand–receptor interaction between the host plasma membrane and viral envelope proteins. Next, endocytosis of these interaction complexes is induced by the host target cell (22). The forming of endosome with acidic condition (59), virion uncoating the envelop into nucleocapsid form. Nucleocapsid viral particles induce a further translation process in the endoplasmic reticulum (ER) and start to assemble using the ER membrane under conditions of pH 7.2. Then, viral maturation and modification occur in the Golgi apparatus. The precursor membrane (prM) protein is cleaved by furin protease to generate the mature virion. Subsequently, virions leave the host target cell to infect other host cells for further replication (50).

The pathogenesis of dengue virus is multifactorial and complex in both host and viral factor; some hypotheses are still unclear (30,56). After the dengue virus invade skin barrier, the first target cell is dendritic cell, the infection enhanced via nonspecific receptor dendritic cell specific ICAM3 grabbing nonintegrin (DC-SIGN), then, dendritic cell migrate to lymph node for present antigen to T cell, beginning the host immune responses (18). The host factor and viral factor act as key of clinical presentation and severity level, and the age and immune status are important for severity determination of host factor (42).

During primary infection in children usually subclinical infection, the secondary infection by heterotypic dengue virus, the disease severity such as plasma leakage or tend to dengue shock syndrome are commonly occur. This age-dependent also could be occurring in adult leading to severe bleeding tendency, and will be more severe in abnormal immune status such as dengue virus infection accompanied with asthma or another chronic diseases, can be life-threatening (18,20). Secondary heterotypic infection enhanced the severity of disease could be explained by ADE phenomenon (23), owing to dengue virus could be infection and replication in various cell type as well as in peripheral blood mononuclear cell, the important hypothesis that the monocyte and macrophages has a crucial role for ADE phenomenon that occur via the immune complexity of dengue virus and non-neutralizing antibodies from primary heterotypic infection support the dengue virus infection to monocyte and macrophages through Fc receptor (18).

The other pathogenesis mostly occurs through the immune cell infection, leading to cytokine production and complement activation that might contribute to endothelial cell damage and lack of homeostasis, correlating with disease severity (26). Moreover, the NS1 viral factor also the important in pathogenesis of vascular damage induce via TLR-4 and also play role in cross-reactivity antibody with platelet and endothelial cells (33).

Immunopathogenesis of Dengue Virus Infection

Despite several key factors being included in the hypotheses regarding dengue pathogenesis, the interesting ones in the context of this study involve the immune system (52). Four key features of this study are as follows: (i) viral load effect related to NS1 protein, (ii) secondary infection with a virus with different serotypes, (iii) rapid viral load reduction, and (iv) cytokine bombardment (50).

Viral Load Effect Related to NS1 Protein

NS1 is a dengue viral protein that has been described as a soluble complement-fixing antigen (7,47). A previous study reported that antibodies of NS1 can undergo cross-reactions with self-antigens on endothelial cells and platelets. This is interesting for clarifying the vascular endothelial cell damage in severe dengue cases. Interested in this finding, the NS1 protein can be called a viral toxin due to NS1 antigen consequence with toll-like receptor (TLR) 4, inducing the proinflammatory cytokine expression that might be triggering the cytokine storm phenomena caused by vascular leakage and severe dengue clinical presentation. An in vivo study supported this hypothesis by using TLR4 antagonist and TLR4 antibody and presenting their effects at reducing vascular permeability and maintaining endothelial integrity; this could provide therapeutic options in the future and supports the inclusion of NS1 in efforts to develop dengue vaccines (4,33).

NS1 protein is essential for dengue virus replication in both mammalian and mosquito cells. A study by Fan et al. found that alanine mutations in the NS1 protein, such as Asn-130, Ans-207, and Glu-173, lead to impairment and/or delay of dengue 2 virus replication (12). Another study by Paranavitane et al. confirmed the role of NS1 in dengue pathogenesis, describing that positivity for the NS1 antigen, especially positivity beyond day 5 of illness, is associated with a high risk of severe dengue (40). In addition, it was shown that an NS1 antigen and antibody combination test can increase the efficiency of dengue virus diagnosis (28). The specificity of an NS1-based dengue test was reported to be between 86.1% and 100%, with few false-positive results (39).

Secondary Infection with a Virus with a Different Serotype

An alternative hypothesis has proposed that dengue severity is related to secondary infection with dengue virus of a different serotype. This phenomenon is supported by ADE, which is a critical risk factor for DHF grade III and IV in patients previously exposed to the dengue virus. The mechanism behind this is facilitated by preexisting antibodies from the primary infection, which is predominantly associated with Fc-receptor-positive monocytic lineage cells, especially macrophages (38,49). Despite previous infection, the antibodies cannot neutralize the virus, which are known as nonneutralizing serum antibodies. Unfortunately, such antibodies form a dengue–Fc complex. Then, the dengue virus is taken up by Fc-receptor-bearing cells (53).

An important study by Chan et al. found that leukocyte immunoglobulin-like receptor B1 (LILR-B1) is critical for the ADE phenomenon. The subneutralization antibody of dengue virus recognized by LILR-B1 on host target cell membranes activates signals through tyrosine-based immunoreceptors to inhibit the expression of interferon-stimulated genes, which is the important sign for ADE phenomenon. This suggests that LILR-B1 inhibition should be considered for the design of vaccines and therapies (9). The downstream effects of the ADE phenomenon are an increase in viral load, which increases the number of infected cells, and an increase in disease severity; this phenomenon is driven by two pathways: (i) Extrinsic ADE pathway: This pathway plays an important role in increasing the number of infected cells, which is influenced by antibody-mediated cell binding and the entry of both mature and immature dengue particles into target cells. (ii) Intrinsic ADE pathway: This pathway suppresses the innate antiviral response, leading to the enhancement of viral production per infected cell, predominantly by TLR suppression (15).

Furthermore, an in vivo model first developed by Ng et al. demonstrated that mice born from a mother infected with dengue serotype 1 and then infected with dengue serotype 2 themselves exhibited increased severity accompanied by plasma leakage, when compared with mice just infected with dengue serotype 2 (36).

Rapid Viral Load Reduction

During dengue virus infection, the clinical course involves three distinct phases: febrile phase, critical phase, and recovery phase. The phenomenon of rapid decrease of viremia after the febrile phase is a consequence of severe form of disease in the critical phase usually associated with shock, bleeding, and organ impairment (50,58). This indicates that, in the febrile phase, the host immune response is initiated, including new antibodies and/or preexisting antibodies inducing innate immune cells, complement activation, the adaptive immune system (T and B cells), autoantibodies, and also cytokines and other soluble mediators such as interleukin (IL)-6, IL-8, MCP-1, TNF-α, and IFNγ. These act against the virus and cause the viral titer to decrease rapidly. After this strong immune response, other factors become involved, including cross-reactions of autoantibodies for NS1 protein, leading to thrombocytopenia, and cross-reactions with endothelial cells, leading to endothelial cell dysfunction by disrupting tight junctions; these processes involve mediators such as MMPs, HMGB1, IL-10, and TGF-β (19). These processes strongly support the immune response and are important for combating virus infection in the febrile phase, mainly by viral clearance, which causes the viral load to decrease rapidly. After this rapid decrease, residual soluble immune mediators such as cytokines and antibodies have some critical effects on dengue pathogenesis.

Cytokine Bombardment

Cytokines are important mediators that play a major role in severe dengue disease progression. With increased capillary permeability, cytokines including TNF-α, IFNγ, and IL-1 cooperate to induce vascular leakage, causing severe clinical manifestations. Cytokine bombardment usually occurs in patients with severe dengue, which is generally believed to be associated with a high level of circulating proinflammatory cytokines, leading to the activation of vascular leakage and shock (16). The levels of TNF-α, IFNγ, and IL-10 found in severely dengue-infected serum samples in a previous study by Rathakrishnan et al. support this hypothesis. In addition, a study of the cytokines in febrile patients found elevations of MCP-2, IP-10, and TRAIL (44) and a meta-analysis also showed the elevations of IL-6, IL-8, TNF-α, IFNγ, VEGF-A, and VCAM-1 in severe dengue patients. Among cytokines associated with dengue shock syndrome in children, elevations of IFNγ and VCAM-1 were also identified. However, at present, the roles of some cytokines/mediators in dengue pathogenesis remain unclear (29). However, an in vivo study found that a lack of CCR2 could decrease cytokine bombardment and tissue damage (16).

Dengue Virus-Induced Redundant Cell Death

During the replication of dengue virus in host target cells, programmed cell death is promoted, which is crucial for the control of infection alongside host immune system activities. Previous studies demonstrated that two types of cell death occur after dengue infection, namely, apoptosis (31,55) and pyroptosis (54,57).

Apoptosis upon Dengue Infection

Apoptosis is a form of type I programmed cell death, which occurs by a controlled mechanism that is triggered under normal physiological conditions, during development and aging, as well as in response to various stresses and pathologies, including infections by viruses and some bacteria. Apoptosis involves two main pathways that are interconnected: the extrinsic pathway and the intrinsic pathway (5,41).

With regard to dengue infection, a study by Jan et al. (25) found that, after dengue viral protein accumulation in ER membranes, rather than virus release, ER stress may be induced, thereby activating the apoptotic pathway, which is found in mouse neuroblastoma cells (25). In addition, Nagila et al. found that NF-κB is activated in dengue virus-infected hepatocytes and induces the expression of CD137. Furthermore, anti-CD137 antibody binding to CD137 may activate caspase cascades, triggering the apoptosis of hepatocytes, as confirmed in a study by Netsawang et al. (35).

Currently, Torrentes-Carvalho et al. (55) described that the dengue infected monocyte by the CD14-TLR-2 coreceptors, the recognition-mediated activation transcription factors that involved in viral clearance. The NF-κB was activated and translocation to the nucleus for activation pro-inflammatory cytokine expression, including TNF-α, this cytokine interact with its transmembrane receptors is tumor necrosis factor receptor (TNFR), leading to initially caspase-8 activation within a sequence of caspase-8 induces effector caspase-3 activation. During infection, viral RNA induces Fas receptor expression, which interacts with Fas ligand that is present on other immune cells, triggering apoptotic signaling, the same as in the TNF/TNFR pathway. Mitochondria also play an important role in this context. Indeed, mitochondrial alteration promotes caspase-9 activation, which then activates caspase-3, leading to DNA cleavage and apoptosis (Fig. 1, left) (55).

FIG. 1.

(left) The mechanism of apoptotic cell death upon dengue virus infection. The virus is recognized by plasma membrane receptors in a monocyte cell model presenting coreceptors of CD14 and TLR-2 (3). Activation of NFkB leads to transcription and release TNF-α, which is then recognized by the TNF receptor. The stimulation of caspase-8 by the TNF receptor induces caspase-3 to undertake DNA cleavage. Viral RNA also induces the release of cytochrome C from mitochondria, which cooperate with caspase-9, and enhances caspase-3 activation, supporting the DNA cleavage mechanism. At the same time, the mitochondria also produce ROS to induce apoptosis. Moreover, viral RNA stimulates the Fas receptor on the inner cell membrane, leading to recognition of the Fas ligand and eventually apoptosis (48). (right) Dengue viral particles are recognized by CLEC5A receptor on a macrophage model. This leads to Syk activation by the phosphorylation of DAP12, inducing caspase-1. Caspase-1 damages DNA and induces pore formation in the plasma membrane, producing an ionic gradient and water influx, leading to osmotic lysis of the cell. Inflammasome activation acts in cooperation with proinflammatory cytokine (IL-1β and IL-18) production. After osmotic lysis of the cell, the proinflammatory cytokines are released (24,57). ROS, reactive oxygen species.

Pyroptosis Pathway Associated with Dengue Pathogenesis

Pyroptosis is distinct from other types of cell death, both morphologically and mechanistically (5). The caspase 1 is a key feature of pyroptosis pathway, which downstream to secretion the pro-inflammatory cytokines such as IL-1β and IL-18, contributed the effect to plasma membrane rapidly loss of integrity (1,2). This type of cell death is influenced by caspase-1, which induces inflammasome activation followed by proinflammatory cytokine production. Caspase-1 also has an important role in inducing membrane pore formation and DNA cleavage. Then, the ionic gradient of cell will be occur and increase the osmotic pressure leading to water influx, the cell will be swelling and consequence to osmotic lysis. After cell osmotic lysis, inflammatory cytokines are released outside the cell (14,48). The destruction of actin cytoskeleton has also been observed in the process of pyroptosis, but the mechanism and importance of this remain unclear (5).

Recently, a dengue virus study by Wu et al. (57) focusing on pyroptosis demonstrated that C-type lectin domain family 5 member A (CELC5A), originally identified as a DAP-12-associated molecule, recognized dengue virus particles in a human macrophage model and induced the DAP-12 phosphorylation to Syk activation downstream to induction of NALP3 inflammasome and release of inflammatory cytokines IL-1β and IL-18. The activation of caspase-1 during the downstream cascade then induced pyroptosis. In addition, a study by Chen et al. also found a significant relationship between CLEC5A and lethal dengue disease; furthermore, blockage of CLEC5A by anti-CLEC5A monoclonal antibodies increased the survival rate in a patients suffering from DHF and dengue shock syndrome (10,57). A previous study also found that caspase-1 induction of IL-1β secretion by dengue-infected monocytes was related to the severity of dengue-related disease (Fig. 1, right) (54).

Concluding Remarks

There are currently a range of hypotheses about dengue immunopathogenesis, which predominantly focus on cell death (apoptosis and pyroptosis) and dysfunction. The pathogenesis of dengue virus infection is strongly related to the host immune response against the virus, as well as certain factors from viral particles. NS1 protein is one component of dengue virus that plays a critical role in recognition, with the TLR4 cascade causing the alteration of vascular endothelial cells accompanied by plasma leakage, which is associated with the severity of dengue-related disease. The host immune response is hypothesized to be one of the most important factors, especially the ADE phenomenon by secondary infection with dengue virus of a different serotype. Furthermore, autoantibodies of NS1 protein that can cross-react with platelets and endothelial cells could lead to the hallmarks of severe dengue-related disease, namely, thrombocytopenia and endothelial cell damage. However, cytokines also play an important role in the pathogenesis of the disease, particularly TNF-α, IFNγ, and IL-10, which synergistically play a crucial role in increasing endothelial cell permeability.

In terms of the redundant cell death caused by dengue virus infection, most studies found two patterns of cell death: apoptosis and pyroptosis. Dengue virus infection may induce the apoptosis of infected cells directly during its replication process and may induce apoptosis in uninfected cells. Apoptosis may lead to a cascade that impairs the effects of immune responses to a high viral load and cytokine bombardment, which usually occur in severe forms of dengue-related disease. Meanwhile, pyroptosis in dengue virus infection involves a cascade from viral recognition by the CLEC5A cascade to activated caspase-1, which induces apoptosis. Both these types of cell death might have an impact on pathogenesis and the severity determination of dengue virus infection, such as the endothelial cell death effect on vascular damage, endothelial permeability, and also overall homeostasis. Clarifying the mechanisms of cell death caused by dengue virus infection would significantly increase our understanding of this disease and should lead to the development of therapies and vaccines, and antiviral strategies.

Footnotes

Acknowledgments

Scholarship from The Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0001/2557) to S.S. and research grant from Mahidol University to N.L. are gratefully acknowledged. The authors thank Pantira Singkum and Laddawan Bangsai for help in generating the schematic diagram.

Author Disclosure Statement

The authors declare that they have no conflicts of interest.

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