The Effect Of The Human Dendritic Cells On Dengue Virus Infection

Dengue viruses (DV) are arthropod-borne flaviviruses that cause serious human diseases all over the world. There are four serotypes of DV (DV1,-2,-3 and -4),and infection by each of them may result in either a relatively benign febrile disease called Dengue fever (DF) or fatal outcomes,such as Dengue hemorrhagic fever (DHF) and Dengue Shock Syndrome (DSS).Although there are many proposed mechanisms,the pathogenesis of DHF and DSS is still far from understood where the adaptive immune response plays a significant role in determining the severity of clinical illness. Dendritic cells (DCs) are professional antigen-presenting cells and the uniqe stimulators of primary T-cells responses, and they produce an array of cytokines and chemokines. Thus, DCs are critical in the initiation of antimicrobial immunity, and they provide a crucial step in the development of adaptive immunity.Objective: To study dengue virus infection in human dendritic cells and cytokine productions by DV₂ infected human dendritic cells .To study the effect of cytokines on replication of DV₂ in human dendritic cells for a better understanding of the immuno-pathological mechanisms of DV₂ infection.Methods: The first part of study is to set up the in vitro cultural method of dendritic cells from human peripheral blood monocytes (PBMC): PBMC isolated from healthy human peripheral blood were incubated at 37℃, humidified 5%CO₂. After 6 hours, nonadherent cells were gently removed, and the adherent cells were cultured in medium with GM-CSF and IL-4 at 37℃, humidified 5%CO₂ for more than 7 days. DCs were then collected and identified by morphological features and underwent tests of surface antigen expression and lymphocytes stimulatory ability.In the second part of study, Dengue virus type â…¡ (DV₂) were infected with human dendritic cells in vitro, culture supernatants and cells were collected by different time postinfection (6h, 24h, 48h, 72h, 96h).Viral titers were evaluated by microplaque forming assay on C6/36 monolayer cells; DV antigen in human dendritic cells were demonstrated by an indirect immunofluorescent assay (IFA); Localization of DV in DC was observed under a transmission electron microscope.In the third part of study, Dengue virus type â…¡ were infected with human dendritic cells in vitro , culture supernatants were collected by different timepostinfection(6tu 241k 481k 72h> 96h), production of TNF-a > IL-6 and IFN-y in the culture supernatants were evaluated by ELISA.In the fourth part of study, a high, medium and low concentration of IL-6, TNF-a were incubated with DCs after DV2 absorption. Then culture supernatants were collected at various time postinfection (6h> 241k 481k 721k 96h) , virus titers were determined and the number of DCs at 48h postinfection were mensurated by MTT. Results: 1. A large amount of DCs could be obtained after 7 days, DCs displayed typical morphology with many bulbous protrusions and irregular in shape when observed by phase-contrast microscopy and electron microscope, the level of expression CD la was 80%⁹5% with immunohistochemical staining and indirect Immunofluorescent assay (IFA) and potently stimulated the proliferation of allogeneic lymphocytes.2. The virus titers were detected in the culture supernatants of infected DCs as early as 6h after infection; the highest viral titers were obtained at 48h, and then declined to very low titers at 96h. DV2 antigen was detected in infected DCs by IFA. After infection for 48h, DV particles were obvious in cystic vesicle, vacuoles.3. Dengue virus induces TNF-a and IL-6 secretion from infected DCs and does not induce IFN-y secretion.4. When cytokine was incubated with DCs after DV2 absorption, IL-6 at medium or low concentration increased DV2 replication in DCs, particular in medium concentration; while TNF-a can prohibited DV2 replication in DCs at high and mediumconcentration, particular in high concentration. Cytokines had no effect on the number of infected DCs.Conclusion: A large amount of DCs could be induced from peripheral blood monocytes in presence of GM-CSF and IL-4, the obtained DCs had typical morphology and antigen presenting ability.Human dendritic cells are targets of dengue virus infection. Dengue virus could efficiently infect DCs and produce virus particles.TNF-ou IL-6 production from DCs are increased with DV infection and the increased TNF-a, IL-6 can modulate virus replication. Dendritic cells may play an important role in DV2 pathogenicity and immunity.