Several cytokines and chemokines have been reported to be induced in response to DENV infection in vitro and in vivo. It has been suggested that the short-lived vascular leakage observed in DENV infections in vivo is most likely mediated by transiently released/produced soluble mediator(s) [Avirutnan et al., 1998
]. The effect of chemokines is not restricted to the regulation of local trafficking of leukocytes, but also would have an effect on systemic targets [Farber, 1997
]. A combination of different chemokines signaling through different receptors in different cells or tissues, rather than an individual molecule, determines the final outcome. Hence, it is believed that the over-expression of different chemokines and cytokines would probably contribute to the DENV-induced immune-mediated pathology, including endothelial cell dysfunction.
In this study, gene expression analysis was used to identify additional cytokines and chemokines that could be involved in the cellular response to DENV infection in vitro and in vivo. Gene expression analysis showed three genes with cytokine/chemokine activity (MCP-2/CCL8, IP-10/CXCL10 and TRAIL/TNFSF10) up-regulated more than 10-fold in human primary immune cells infected with DENV in vitro; one gene (RANTES) was up-regulated 5-fold and one gene (MIP-1β/CCL4) was marginally up-regulated. MCP-2/CCL8 has not been previously described in DENV infections in vitro or in patients. IP-10/CXCL10 has been shown to be induced in vitro and recently was reported to be increased in serum of DENV-infected patients [Fink et al., 2007
]. TRAIL/TNFSF10 expression has been reported in human primary cells in response to DENV infection in vitro [Warke et al., 2007
]. MIP-1β mRNA expression has been found in PBMC from DENV-infected patients [Spain-Santana et al., 2001
] and serum levels were recently reported [Bozza et al., 2008
As noted above, MCP-2 has not been previously associated with DENV infections. Previous studies have shown higher levels of MCP-1 in patients with DHF and the authors hypothesized that the mechanism of vascular leakage in their in vitro model using DENV2-infected HUVECs was partially dependent on MCP-1 [Lee et al., 2006
]. MCP-1 and MCP-2 are co-expressed, although MCP-1 is produced in higher quantities and lower concentrations of MCP-1 are required to induce chemotaxis of monocytes and activated T lymphocytes [Proost et al., 1996
]. However, MCP-1 binds only to CCR2 while MCP-2 is able to bind to CCR1, CCR2 and CCR5. Therefore, MCP-1 and MCP-2 could have independent effects during DENV infection. Interestingly, a recent report showed that the MCP-2 – CCR2 interaction counteracts the activation signaling normally generated by other chemokines [O’Boyle et al., 2007
]. MCP-2 might contribute to the immune response to DENV infection by engaging with different receptors in various cell types and/or suppressing the effect of other chemokines.
IP-10 was among the highest up-regulated genes in DC and was also up-regulated in other primary cells in response to DENV infection in vitro. Increased levels of IP-10 were also found in DENV-infected patients. Interestingly, a tendency of higher levels of IP-10 in DENV-infected patients with hemorrhagic manifestations was found, but no differences in IP-10 levels in serum from patients with primary and secondary DENV infections. Recently, higher serum levels of IP-10 in DENV-infected patients were also reported by others [Fink et al., 2007
]. The data presented here confirms the data published by Fink et al in a different cohort of patients (Venezuela vs. Singapore). IP-10 is induced by IFN-γ and IFN-α/β and is chemoattractant to activated T lymphocytes and NK cells [Farber, 1997
]. IP-10 has been found to be induced in various viral infections in vivo and in vitro [Chen et al., 2006
; Diago et al., 2006
; Roe et al., 2007
; Warke et al., 2007
]. It has been suggested that IP-10 has a role in the protective immune response against DENV, competing with the virus for the binding to heparan sulfate, and blocking entry and replication [Chen et al., 2006
] [Hsieh et al., 2006
]. On the other hand, high serum levels of IP-10 have been found in patients with chronic inflammatory conditions [Laine et al., 2007
]. Thus, IP-10 could have a protective role against DENV, but could also be associated with the potentially damaging inflammatory response if produced in high amounts and in an uncontrolled manner.
Gene expression analysis showed a slight increase of MIP-1β expression in various primary human cells infected with DENV. MIP-1β levels were slightly increased in serum from DENV-infected patients during the febrile period of the disease. Interestingly, MIP-1β levels were higher in patients with primary infections during the post-febrile period of the disease. Higher levels of MIP-1β in primary infections may suggest a protective role of MIP-1β in dengue infections. Recently, Bozza et al [Bozza et al., 2008
] reported elevated levels of MIP-1β associated with mild dengue disease. MIP-1β is induced in response to LPS, TNF-α, IFN-γ, and viral infections [Maurer and von Stebut, 2004
] and induces chemotaxis of monocytes, T lymphocytes, NK cells and immature DC. MIP-1β expression was induced in K562 cells in response to DENV infection in vitro and also was found in PBMC isolated from DENV-infected patients [Spain-Santana et al., 2001
]. The induction of MIP-1β in response to DENV might be limited to certain cell types or restricted to specialized areas, and hence systemic levels or cell-specific levels might underestimate the role of MIP-1β in vivo.
TRAIL levels were increased in serum from DENV-infected patients during the febrile period of the disease. Interestingly, higher levels of TRAIL were found in patients with primary infections. Recently, a potent antiviral effect of TRAIL in DENV infection in vitro was reported [Warke et al., 2007
]. TRAIL pre-treatment of DENV-infected DC in vitro also suppressed the production of pro-inflammatory chemokines and cytokines. These results could suggest a protective role of TRAIL in DENV infections in vivo. TRAIL is a member of the TNF family of proteins, originally identified as a promoter of apoptosis in tumor cell lines and some primary tumors [Ashkenazi et al., 1999
]. Recent studies have shown that TRAIL can induce proliferation of T lymphocytes and negatively regulate inflammation and the innate immune response through an apoptosis-independent mechanism [Diehl et al., 2004
; Secchiero et al., 2005
; Song et al., 2000
; Vassina et al., 2005
]. The mechanism of action of TRAIL to reduce the production of cytokines and chemokines is not known. The effect of TRAIL as an antiviral could be responsible for the suppression of DENV-induced chemokines and cytokines, as lower levels of virus might account for lower induction of these soluble mediators, but we cannot rule out a direct anti-inflammatory role of TRAIL. Further investigation should be conducted to elucidate the mechanisms involved in TRAIL-mediated control of the virus and the inflammatory response.
The clinical events that characterize DHF occur around the time of defervescence [Kalayanarooj et al., 1997
]. Higher levels of MCP-2 and IP-10, as well as other mediators, around the time of defervescence could have a role promoting and increasing the potentially damaging inflammatory response and thereby contribute to the events that lead to endothelial cell dysfunction, vascular leakage and altered coagulation. The specific up-regulation of TRAIL in DENV-infected patients and the previous observation that TRAIL has an antiviral effect in DENV-infections in vitro, suggests a role of TRAIL in the control of virus infection. The tendency of higher serum TRAIL levels in primary infections could suggest a protective role, limiting virus replication and consequently the transition to a severe disease and a potential therapeutic use for TRAIL in limiting the damaging inflammatory response triggered by DENV in severe cases. Further investigation, using a larger cohort of patients and patients with different grades of severity should be conducted to confirm these observations.