An in vitro model for dengue virus infection that exhibits human monocyte infection, multiple cytokine production and dexamethasone immunomodulation

Abstract

An important cytokine role in dengue fever pathogenesis has been described. These molecules can be associated with haemorrhagic manifestations, coagulation disorders, hypotension and shock, all symptoms implicated in vascular permeability and disease worsening conditions. Several immunological diseases have been treated by cytokine modulation and dexamethasone is utilized clinically to treat pathologies with inflammatory and autoimmune ethiologies. We established an in vitro model with human monocytes infected by dengue virus-2 for evaluating immunomodulatory and antiviral activities of potential pharmaceutical products. Flow cytometry analysis demonstrated significant dengue antigen detection in target cells two days after infection. TNF-α, IFN-α, IL-6 and IL-10 are produced by in vitro infected monocytes and are significantly detected in cell culture supernatants by multiplex microbead immunoassay. Dexamethasone action was tested for the first time for its modulation in dengue infection, presenting optimistic results in both decreasing cell infection rates and inhibiting TNF-α, IFN-α and IL-10 production. This model is proposed for novel drug trials yet to be applyed for dengue fever.Key words: dengue -dexamethasone -monocytes -cytokines -therapeutics Dengue fever (DF) is an acute infectious viral disease that presents a broad severity spectrum from asymptomatic or oligosymptomatic to extremely severe clinical manifestations. It is believed that immunological mechanisms play a key role in pathogenesis (WHO 1997, Gubler 2002. Proinflammatory cytokines have been associated with haemodynamic and coagulation disorders that may lead to increased vascular permeability and leakage and to hypovolemic shock resulting eventually in death (Green & Rothman 2006). No specific treatment exists for dengue and research has been unsuccessful in finding improved conditions with steroids, antivirals or substances that decrease capillary permeability (Sumarmo et al. 1982, Tassniyom et al. 1993, Pea et al. 2001, Gibbons & Vaughn 2002, Ligon 2005. In patients, fluid-replacement therapy should be administrated according to the severity and paracetamol/metamizole has been prescribed for fever and analgesia.Glucocorticoids are widely adopted as anti-inflammatory drugs for several inflammatory diseases and are known to suppress of inflammatory mediators expression (Barnes & Adcock 1995, Joyce et al. 1997. Dexamethasone inhibits cytokine gene expression such as TNF-α by blocking transcription factors NF-κB and activator factor-1 (Steer et al. 2000). IL-1β, iNOS, ciclooxigenase-2 and monocyte chemoattractant protein-1 have also been described to destabilize mRNAs during this treatment (Amano et al. 1993, Ristimaki et al. 1996, Poon et al. 1999, Lasa et al. 2001, Korhonen et al. 2002.Dexamethasone is applied in the clinics to treat pathologies with inflammatory and autoimmune origins, such as rheumatoid arthritis, multiple melanomas and bacterial meningitis (Jimenez-Zepeda & DominguezMartinez 2006, van de Beek & de Gans 2006, Schutt et a...

Dengue-2 and yellow fever 17DD viruses infect human dendritic cells, resulting in an induction of activation markers, cytokines and chemokines and secretion of different TNF-α and IFN-α profiles

“…In our in vitro infection model, we observed markedly higher TNF-α production after infection with pathogenic virus (DENV2) as compared to the non-pathogenic viruses (YF17DD and YF17D/DENV2), providing further evidence that this cytokine plays a role in flavivirus immunopathogenesis. Other authors have also observed TNF-α production in DC or monocyte cultures in the presence of DENV2 and some authors even associate it with the infected cell (Hacker et al 1998, Querec et al 2006, Deauvieau et al 2007, Reis et al 2007, Ahmad et al 2008, Nightingale et al 2008, Levy et al 2009. A moderate level of TNF-α production may be beneficial to mature cells that may become good antigen presenters.…”

“…Indeed, DCs have a crucial role in initiating host defence mechanisms; through antigen presentation and cytokine production, they can define the fate of the immunological response (Blanco et al 2008). Both DENV and YF vaccine viruses are known to infect DCs and monocytes in vitro (Wu et al 2000, Barba-Spaeth et al 2005, Reis et al 2007). We aimed to characterise the similarities and differences between flavivirus infections with distinct virulence patterns that elicit long lasting in vivo immunity (Monath 2001, Halstead 2007.…”

“…Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors' buffy coats by centrifugation on a density gradient (400 g for 30 min in Ficoll-Paque Plus, Amersham Biosciences Corp), as described previously (Reis et al 2007). Cells were resuspended at 10 6 cells/mL in RPMI 1640 supplemented with 200 mM glutamine, 100 U/mL penicillin and 10 mg/mL streptomycin (Gibco, Invitrogen) and incubated at 37ºC under a humid atmosphere with 5% CO2.…”

“…Cytokine detection in cell culture supernatant by multiplex microbead immunoassay -A multiplex biometric immunoassay, which contains fluorescent microspheres conjugated with a monoclonal antibody specific for a target protein, was employed for cytokine and chemokine measurement according to the manufacturer's instructions (Upstate, Millipore) as described previously (Reis et al 2007). Measured cytokines including the following: IFN-α, IL-6, IL-1Ra, IL-10, MCP-1/CCL-2, MIP-1β/ CCL-4, IP-10/CXCL-10 and RANTES/CCL-5.…”

Dengue-2 infection and the induction of apoptosis in human primary monocytes

“…Monocytes and T lymphocytes are activated during human infection, producing several pro-inflammatory mediators and may have amplified activation during secondary infection to become sources of intense cytokine production. Several investigations have followed patients during acute infection to monitor their plasma cytokine content and found that TNF-α is among the first detected cytokines in patients as well as in peripheral blood mononuclear cell (PBMC) cultures (Bethell et al 1998, Braga et al 2001, Reis et al 2007). In general, these mediators may promote platelet and endothelial activation, culminating in thrombocytopenia, vascular permeability, hypotension and shock, as observed in severe cases of dengue (Fink et al 2006).…”