Dendritic cells (DCs) are potent antigen presenting cells whose role in eliciting immune responses in the context of human immunodeficiency virus type-1 (HIV-1) infection remains unclear. The objective of this project was to determine if HIV-1 modulates DC function by downregulating the production of IL-23 and IL-27, cytokines important in the generation of cellular immune responses. Dendritic cells were isolated from the epidermal and dermal skin layers resulting from elective abdominoplasties by CD1a and CD1c microbeads. Circulating DCs were isolated from peripheral blood by CD1c microbeads. As an experimental model, monocyte-derived DCs (MDDCs) were generated from freshly isolated monocytes which were cultured for 6 days with IL-4 and GM-CSF. The phenotypes of these 4 DC populations were compared. To determine the effects of an endogenous source of HIV-1 Tat on cytokine expression, MDDCs were infected with a pLXIN (ptat) construct containing the HIV-1 tat wt gene or the empty vector for 24h, before a 4h LPS (1mug/mL) stimulation. To determine the effects of an exogenous source of Tat, which is known to be secreted by infected cells, DCs were pre-treated with recombinant Tat protein (rTat) for 1h before LPS stimulation. Alternatively, DCs were cultured in the presence of HIV-1 dual-tropic strain 92HT593 for 24h before LPS stimulation. IL-23p19 and IL-27EBI3 and p28 mRNA expression were evaluated by quantitative real-time RT-PCR (qRT-PCR) and reported as relative expression levels. DCs are readily infected with the ptat, as shown by RT-PCR. The presence of endogenous Tat resulted in a decrease in IL-23p19, IL-27EBI3 and p28 mRNA expression. Incubation of DCs with rTat similarly decreased IL-23p19, IL-27EBI3 and p28 mRNA expression. When cultured in the presence of HIV-192HT593, a similar downregulation in IL-23p19 and IL-27EBI3 was observed, with no significant effect on p28 expression. In conclusion, the presence of HIV-1 Tat protein whether from an endogenous or exogenous source significantly downregulated the expression of IL-23p19 and IL-27EBI3 and p28 mRNAs. HIV-1 similarly downregulated the gene expression of IL-23p19 and IL-27EBI3, but appeared to have no effect on IL-27p28 gene expression. HIV-1 disregulation of IL-27 subunits EBI3 and p28 and IL-23p19 may be a mechanism by which HIV-1 evades the infection-clearing immune response. Understanding the function of cytokines expressed and secreted by DCs to initiate T cell polarization may lead to better understanding of HIV-1 pathogenesis and the development of novel therapies for HIV infection.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/27966 |
| Date | January 2008 |
| Creators | Chenier, Andreane |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 89 p. |
Page generated in 0.011 seconds