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The pathogenic mechanism of the severe form of dengue is complicated. Recent reports indicate that apoptotic death of various tissues or organs may be associated with vascular leakage, and ultimately leads to the death of DENV-infected patients. In the present study, we provide additional evidence supporting the detrimental role of apoptosis in DENV infection. A comparison of the rate of apoptosis in PBMCs isolated from patients suffering DF, a mild form of the disease, and the rate in patients with DHF, a life-threatening disease, revealed that PBMCs from DHF patients underwent apoptosis at a significantly higher rate than those suffering from DF alone. This suggests that the severity of natural DENV infection correlates with PBMC apoptosis. In addition, this cell death was induced not only by DENV itself, but also by the apoptotic activities of pro-inflammatory cytokines, such as TNF-α, and IL-1β, that were upregulated in DHF patients. The death of these mononuclear cells that function in an innate immune system may explain the higher viral load in DHF patients than in DF patients. Interestingly, a gene expression profile pattern elucidated that apoptosis occurring during natural DENV infection involved mainly the extrinsic apoptosis pathway, which is mediated via both caspase-dependent and caspase-independent mechanisms. In conclusion, our data highlight the adverse effect of apoptosis induced by DENV and by pro-inflammatory cytokines during natural DENV infection.
Dengue fever and DHF/DSS, the world's most significant arthropod-borne viral diseases, are caused by DENV. DENV, a member of the Flavivirus genus, has caused life-threatening epidemics in more than 100 countries in tropical and subtropical areas (1). Each year, 50–100 million people are estimated to be infected by DENV (2–4). While the majority of infected patients experience uncomplicated DF, an acute febrile illness typically lasting 3–7 days, on less frequent occasions the infection can cause DHF or DSS, a potentially fatal plasma leakage syndrome (2, 5, 6). Although it has been shown that the severity of the disease depends upon both virus and host factors, the determinants that predispose infected patients to develop mild or severe forms of the disease are still uncertain (7–9).
Apoptosis, a programmed cell death that regulates homeostasis in multicellular organisms, is mediated via at least two signaling pathways: extrinsic and intrinsic pathways. The extrinsic pathway is induced by the binding of death ligands to death receptors, which then initiates death inducing signaling complex (DISC) formation. This results in a stimulation of the death execution pathway via a regulation of caspase function. However, the intrinsic pathway, a receptor-independent pathway, is triggered intracellularly by irregular homeostasis (10).
Apoptosis is commonly detected in a broad range of viral infections, and has been shown to play both protective and destructive roles, depending on the cell type and viral system. For example, apoptosis is a mechanism of CD4+ T lymphocyte depletion during HIV infection, of neuronal death in poliomyelitis in a mouse model, and of immunological anergy during measles virus infection; whereas it is responsible for an attenuation of rabies virus infection (11).
DENV is one of the viruses that induces apoptosis in several cell types, such as monocytes, dendritic cells, endothelial cells and hepatocytes; this is important to its pathogenesis (12–15). A recent study has shown that DENV can replicate in megakaryocytes, and drives these cells to undergo apoptosis. This effect could be an important event in the etiology of thrombocytopenia in DENV infection (16). In addition, apoptosis has been found in liver, brain and intestinal tissues, as well as in pulmonary microvascular endothelial cells, in fatal DSS cases (17). This evidence indicates that apoptosis can be induced by DENV infection, and contributes to its degree of severity.
As the most recognized target cells of DENV in humans are mononuclear phagocytes, which constitute a major part of the population of innate immune cells, any alteration in this population of cells during DENV infection might contribute significantly to the clinical outcome (18). To investigate this hypothesis, PBMCs from DF and DHF patients were examined for both cellular and genetic modifications. By using cDNA microarray analysis, we found that PBMCs from DENV-infected patients underwent apoptosis mainly through the extrinsic pathway, according to gene expression profiling. The percentages of PBMC apoptosis between DF and DHF patients were compared to determine whether the death of these PBMCs contributes to the disease severity. Indeed, we were able to show a direct correlation between the level of PBMC apoptosis and the severity of the disease. Moreover, we also determined that the death of these innate immune cells was partly induced by the pro-inflammatory cytokines that were upregulated in DHF serum.
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- MATERIALS AND METHODS
To efficiently terminate a viral infection, the cooperation of innate and adaptive immune responses is required. Modification or dysfunction of one of these immune arms generally results in an increased disease severity (24, 25). One function of apoptosis, a fundamental regulatory mechanism of various systems in humans, is to serve as an innate immune response to eliminate viral-infected cells (26). However, apoptosis is also used by several viruses to perturb the balance of subsets of immune cells for the viruses’ own benefit, which, in turn, is detrimental to the host (27, 28). DENV, for instance, was previously reported to induce apoptosis in the pathogenesis of DHF via CD8+ T-cell depletion and original antigenic sin (5, 29).
By investigating apoptosis in PBMC from dengue patients, we demonstrated that PBMC from patients who developed DF, a self-limiting form of the disease, underwent apoptosis at a lower percentage than PBMC from patients who developed DHF, a more serious form of dengue. As PBMC are one of the critical components of the immune system, greater damage to this population of cells may, at least in part, explain the higher viral load in DHF than in DF patients, resulting in increased severity of the disease. However, whether the percentage of PBMC apoptosis can be used as an indicator of DHF progression will require further investigation.
A number of studies have indicated that DENV-induced apoptosis is mediated via both extrinsic and intrinsic pathways (30–32). In particular, an extrinsic pathway via TNF-α was found to be critical for DENV-induced hemorrhage in a mouse model (33). To obtain insight into the mechanism of DENV-mediated PBMC apoptosis, cDNA microarray was used to screen for the apoptotic gene expression profile of DENV-infected PBMC. This analysis revealed that most of the upregulated apoptotic genes in DHF PBMC from the acute phase involve an extrinsic apoptosis pathway, including TNFR-I/TNF-α and Fas/FasL. Support for a receptor-mediated pathway was demonstrated by an upregulation of caspase-8, which is a downstream molecule of this cascade, and its substrate, Rho-GDP dissociation inhibitor beta (ARHGDIB), in the same array screening. The reduction in the number of apoptotic cells after adding the TNF-α antagonist, etanercept, implicates the role of TNFR-I/TNF-α in DENV-induced PBMC apoptosis. However, PBMC apoptosis in DHF patients is not determined exclusively by TNF-α, but rather by multiple factors, as the TNF-α antagonist only partially inhibited apoptosis. In addition, the expression of TNF-α at the proteomic level, as determined by ELISA, paralleled that of the genomic level. To further support array analysis, the amount of TNF-α protein detected in acute-phase DHF plasma was significantly higher than that found in DF plasma, suggesting a detrimental role of TNF-α in DENV infection. These findings agree with previous reports in which TNF-α and its family members are important apoptosis mediators during DENV infection (30, 31, 34).
The upregulation of IL-1β which was observed in the screening suggests another possible extrinsic pathway, which signals through JNK and nuclear factor-kappa B (NF-κB) (35–37). As the activation of NF-κB can promote the expression of the sIER3 transcript, which is known to trigger apoptosis via caspase-3 and Bax, the fact that sIER3 was also upregulated in DHF PBMC may imply that DENV uses both IL-1β-mediated and TNFR-I/TNF-α-mediated apoptosis pathways (21, 38, 39). Furthermore, ribosomal protein S3 (RPS3), which is part of caspase-independent TNF-α/FasL-mediated apoptosis, and directly causes DNA degradation after translocation from the cytosol into the nucleus, was also found to be upregulated, suggesting another alternative apoptosis pathway induced by DENV (40). Collectively, these data demonstrate that natural DENV infection is able to induce PBMC apoptosis by various pathways, but mainly through the extrinsic pathway via both caspase-dependent and caspase-independent mechanisms. Interestingly, the present study did not detect upregulation of any anti-apoptotic gene, but was able to find downregulation of one anti-apoptotic gene, BAG1.
The severity of the progression of the disease is associated with the degree of viral burden, which, in turn, depends largely on the ability of the virus to evade the immune response. Our data suggest that natural DENV uses multiple pathways to induce apoptosis in the immune cells, PBMC in particular, leading to exacerbation of the disease. Whether interfering with any of these key elements can prevent disease progression is worth further examination.