Infection of human monocyte-derived dendritic cells. Part 2
The hemorrhagic viruses, including the members of the Bunyaviridae as well as dengue viruses, target endothelial cells and immune cells, mainly monocyte-derived cells such as the professional antigen-presenting cells, Dendritic cells (DCs). DCs activation triggers their maturation and trans-endothelial migration occurring during wound healing or inflammation. These processes require extracellular matrix remodeling and involve changes in endothelial permeability regulated by the production of matrix metalloproteases (gMMPs) or vascular endothelial growth factor (VEGF). However, in excess, these soluble factors can have deleterious effects on endothelial cell integrity. Data from different reports show that endothelial cells infected by dengue virus trigger secretion of soluble factors such as VEGF and the decrease of VEGF-R2 receptor. We have recently reported in vitro and in vivo showing that soluble factors secreted from DV-infected DCs enhance endothelial permeability and down-regulate expression of endothelial junction proteins, Pecam-1 and VE-cadherin in a gMMP-9-dependent manner. More recently, complementary and convergent studies, to our own previous data on dengue, have reported that Hantavirus-infected endothelial cells enhances the permeability via the reduction of VE-cadherin expression due to its dissociation with VEGF-receptor2 (VEGF-R2) which, in turn, become associated with VEGF. An accurate understanding of Hantavirus pathogenesis is pivotal to design de novo therapeutic or vaccine approaches that are still lacking against this hemorrhagic viral infection. In this study, we show that ANDV-infected DC are quickly activated and rapidly progress to an intermediate maturation and pro-inflammatory state that contributes to the increase of soluble factors in their supernatant able to trigger the enhancement of endothelial permeability.
Methods
Virus and cells
The primary isolate, ANDV strain CHI-7913 was propagated in the epithelial Vero-E6 cell line (ATCC CRL 1586). Titrated supernatants of these cells were used to infect, at a MOI of 1 for 2 h, human iDCs derived from peripheral blood monocytes (PBMC), as previously described. In these experiments, UV (λ: 250 nm; 15 min)-irradiated ANDV was used as the negative control. Four days post-DC infection, ANDV N-protein was detected by indirect immunofluorescence (IFA) using a well characterized anti-ANDV N monoclonal antibody (MAb). Total RNA was extracted using the High Pure viral nucleic acid kit (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacture’s protocol and 1 μl of total RNA was amplified in a one step RT-PCR (SuperScript III One-Step RT-PCR with Platinum Taq, Invitrogen) using primers that recognize the nucleocapsid coding region (forward primer: 5′ ACA CGA ACA ACA GCT CGT GAC ‘3 and reverse primer: 5′ AGG CTC AAG CCC TGT TGG ATC ‘3). To assess the viral infectivity, from ANDV-positive DCs, their supernatants were used to infect Vero-E6 cells.