Activation of the Innate Immune Response against DENV in Normal Non-Transformed Human Fibroblasts

In this work, we demonstrate that that both human whole skin and freshly isolated skin fibroblasts are productively infected with Dengue virus (DENV). In addition, primary skin fibroblast cultures were established and subsequently infected with DENV-2; we showed in these cells the presence of the viral antigen NS3, and we found productive viral infection by a conventional plaque assay. Of note, the infectivity rate was almost the same in all the primary cultures analyzed from different donors. The skin fibroblasts infected with DENV-2 underwent signaling through both TLR3 and RIG-1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5) and β defensin 2 (HβD2). In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3), but not interferon regulatory factor 7 IRF7, when compared with mock-infected fibroblasts. Our data suggest that fibroblasts might even participate producing mediators involved in innate immunity that activate and contribute to the orchestration of the local innate responses. This work is the first evaluating primary skin fibroblast cultures obtained from different humans, assessing both their susceptibility to DENV infection as well as their ability to produce molecules crucial for innate immunity

dsRNA weakly colocalizes with lipid raft markers in DENV-infected HMEC-1.

<p>(A) HMEC-1 cells, mock infected or infected with DENV-2, were fixed 48 h after infection, permeabilized, and analyzed by immunofluorescence. Cells were double stained with mouse monoclonal antibodies against dsRNA (red always) and NS2B (green) (a); dsRNA and NS3 (green) (b); dsRNA and NS5 (green) (c);(B) dsRNA and Cav-1 (green) (a); or dsRNA and flotillin (green) (b). Colocalization of dsRNA was observed at different times after infection. (C)HMEC-1cells were infected with DENV-2 and double stained at different times after infection (18 h and 24 h) dsRNA, red, Cav-1, green; and mock infected, not shown. Nuclear DNA was counterstained with DAPI, and the merged image is shown at higher magnification.</p

Depletion of cholesterol by MβCD affects DENV-2 infection in HMEC-1.

<p>HMEC-1cells were infected at 10 MOI. At 120 min after adsorption, the inoculated virus was washed off, and the cells were cultured in fresh medium. At 2 or 6 h post infection, the cells were mock treated (A) or treated with 10 mM MβCD (B) for 2 h. The drug was then washed off, and the cells were cultured in fresh serum-free medium for 48 h. Detergent-resistant membrane fractions obtained from sucrose gradient ultracentrifugation were collected, and Cav-1 and NS3 were analyzed. The recovered fractions were numbered from 1 (top fraction) to 11 (bottom fraction). (C) The culture supernatants were harvested to determine the viral titration by plaque-forming assays. (D) Disaggregation of lipid rafts after10 mM MβCD treatment.</p

DENV-2 NS3 with Cav-1 colocalize at early stages after infection.

<p>(A) HMEC-1cells were infected with DENV-2 and double stained at different times after infection (12, 24 and 48hpi) or were mock infected (not shown). In all cases, cells were double stained with mouse monoclonal antibodies against NS3 (green) and the lipid raft marker Cav-1 (red). Nuclear DNA was stained with DAPI. (B) Slides were analyzed by confocal laser scanning microscopy (Leica SP5 Objective HCXPLAPO63 63Oil). The graph shows the colocalization index. (C) HMEC-1 cells were transfected with (a) NS2BNS3pro-GFP, (b) NS3pro-GFP, and,(c) NS2B-GFP. After 24 h, they were stained with the lipid raft marker Cav-1 (red), and colocalization was analyzed.</p

Colocalizationanalysis of DENV NS2B, NS3, and NS5 with caveolin and flotillin in infected cells.

<p>HMEC-1 cells were mock infected or infected with DENV-2, fixed 36 h after infection with 4% paraformaldehyde, permeabilized, and immunolabeled. Cells were double stained with mouse monoclonal antibodies against NS2B (A), NS3 (B), or NS5 (C); the lipid raft markers Cav-1 (upper panel) and Flt-1 (middle panel); and the non-raft marker TfR (bottom panel). DAPI was used for nuclear DNA staining. Slides were analyzed by confocal laser scanning microscopy. (D) The degree of overlap between the green and red signals for each viral protein was statistically analyzed and expressed as a Pearson's coefficient by the microscopy software.</p

NS3 and NS2B are present in detergent-resistant membrane fractions.

<p>HMEC-1cells were infected with DENV-2 at 10 MOI for 48 h and lysed using 1% Brij in TNEV buffer. Lysates were analyzed by sucrose gradient ultracentrifugation (A). The floating bands from sucrose gradient ultracentrifugation were collected and analyzed by dot blot with an anti-Flt-1 antibody (a lipid resident protein). (B) The samples from each fraction were then analyzed by western blotting using specific antibodies against NS3, NS2B, Flt-1, Cav-1 and TfR as a marker of non-raft fractions.</p

dsRNA weakly colocalizes with lipid raft markers in DENV-infected HMEC-1.

<p>(A) HMEC-1 cells, mock infected or infected with DENV-2, were fixed 48 h after infection, permeabilized, and analyzed by immunofluorescence. Cells were double stained with mouse monoclonal antibodies against dsRNA (red always) and NS2B (green) (a); dsRNA and NS3 (green) (b); dsRNA and NS5 (green) (c);(B) dsRNA and Cav-1 (green) (a); or dsRNA and flotillin (green) (b). Colocalization of dsRNA was observed at different times after infection. (C)HMEC-1cells were infected with DENV-2 and double stained at different times after infection (18 h and 24 h) dsRNA, red, Cav-1, green; and mock infected, not shown. Nuclear DNA was counterstained with DAPI, and the merged image is shown at higher magnification.</p

Localization of lipid raft resident proteins and viral DENV proteins.

<p>(A) HMEC-1cells were stained with specific antibodies against Cav-1 and Flt-1, and immunofluorescence were analyzed by confocal laser scanning microscopy. (B) HMEC-1 cells were mock infected or infected with DENV-2 and then double-stained with specific antibodies (developed in our laboratory) targeting NS2B (green) and NS3 (red) or NS3 (red) and NS5 (green).</p

The Immune Response in Adipocytes and Their Susceptibility to Infection: A Possible Relationship with Infectobesity

The current obesity pandemic has been expanding in both developing and developed countries. This suggests that the factors contributing to this condition need to be reconsidered since some new factors are arising as etiological causes of this disease. Moreover, recent clinical and experimental findings have shown an association between the progress of obesity and some infections, and the functions of adipose tissues, which involve cell metabolism and adipokine release, among others. Furthermore, it has recently been reported that adipocytes could either be reservoirs for these pathogens or play an active role in this process. In addition, there is abundant evidence indicating that during obesity, the immune system is exacerbated, suggesting an increased susceptibility of the patient to the development of several forms of illness or death. Thus, there could be a relationship between infection as a trigger for an increase in adipose cells and the impact on the metabolism that contributes to the development of obesity. In this review, we describe the findings concerning the role of adipose tissue as a mediator in the immune response as well as the possible role of adipocytes as infection targets, with both roles constituting a possible cause of obesity