Effect of NSR infection on intracellular and extracellular CD83 levels.

<p>(A) The levels of soluble CD83 in supernatants from cells harvested 24 h after stimulation with LPS, infection with NSR, or from cells mock infected with NSRmock were determined by ELISA. Bars represent average CD83 concentrations ±SD. Results from one of two independently performed experiments with similar results are shown. (B) Detection of CD83 in cell lysates by Western blot at 24 hpi. The different treatments are shown above the top panel and the probed proteins are depicted at the right. The positions of molecular weight standard proteins are shown at the left. The top blot was stripped and re-probed with antibodies against GAPDH and GFP, which served as loading control and control to confirm NSR infection, respectively. Results from one of two independent experiments with cells from two donors are shown.</p

Surface expression of CD40, CD80, CD83, CD86, MHC-I and MHC-II on DCs at 24 h after NSR infection as measured by flow cytometry.

<p>Immature DCs were infected with NSR, mock-infected with NSRmock, or stimulated with LPS (left and middle panels). Alternatively, cells were infected with NSR or mock-infected with NSRmock in the presence of LPS (right panels). The left panel shows representative histograms of surface marker measurements on cells stimulated with LPS, mock infected cells (NSRmock), cells infected with NSR (GFP+) and uninfected bystander DCs (GFP-). Expression of markers in untreated cells and an irrelevant isotype control are depicted. The middle and right panels represent average data from 4 independent experiments performed with cells from 3 donors. The box plots depict MFI of the different markers relative to untreated cells. A black asterisk indicates upregulation compared to the control and a red asterisk indicates downregulation.</p

Analysis of CD83 and CD80 surface expression in time.

<p>DCs were treated with NSRmock, NSR, LPS, LPS+NSRmock or LPS+NSR and were harvested at 0, 4, 8,12, 16, 24 and 48 h post treatment. Surface expression of CD83 (upper panels) and CD80 (lower panels) were measured by flow cytometry. Left panels show histograms from one representative experiment. Time points are depicted with different colors and the color code is shown at the right. IC–isotype control. Right panels illustrate average data from three independent experiments with cells from three different donors. Bars represent means ±SD of the fold change of MFI relative to untreated cells.</p

Infection of DCs by NSR.

<p>(A) DCs were infected with NSR for 24 h and evaluated for expression of GFP, using an EVOS fluorescence microscope. (B) Infection efficiency under optimal conditions as determined by flow cytometry. (C) Viability and percentage of infected cells at different time points after infection. Cells were infected with NSR or mock-infected with NSRmock, harvested at the indicated time points, stained with 7AAD and analysed by flow cytometry. The percentage of GFP expressing cells (bars) and the viability after NSR or NSRmock infections (lines) is depicted. Viability of the cells was calculated relative to the viability at 8 hpi, which was set at 100%. The data depict average values from two experiments with cells from two different donors ±SD. (D) Morphology of DCs stimulated with the indicated stimuli at 24 h post treatment.</p

Visualization of GAPDH, CD80 and CD83 mRNAs in infected cells using FISH.

<p>DCs were stimulated for 24 h with LPS, infected with NSR or left untreated and then fixed and subjected to FISH. Shown are (A) representative cells of each treatment condition from three independently performed experiments with cells from three different donors and (B) average ±SD spot counts of cells probed for GAPDH, CD80 and CD83. Relevant statistical significance is indicated with an asterisk.</p

Analysis of CD83, CD80, GAPDH and PPIA mRNA levels.

<p>DC RNA samples, prepared 24 h after treatment (as indicated), were analysed by qRT-PCR. Bars represent average Ct values from triplicates ±SD with cells from one donor. The experiment is a representative of 3 independently performed experiments with cells from 3 different donors. Statistical significance is indicated with an asterisk.</p

Cytokine secretion by NSR-infected DCs.

<p>Supernatants of infected or control-treated DCs were harvested at 24 hpi and analysed with a luminex-based cytokine assay. Bars represent the mean cytokine concentrations ± SD of triplicates with cells from one donor. Statistical significance between infected (NSR) and mock-infected (NSRmock) conditions is indicated.</p

Expression of CD83 after inhibition of cellular protein degradation routes.

<p>(A) Flow cytometry analysis of CD83 surface expression after inhibition of the proteasome. DCs were stimulated with NSRmock, LPS+NSRmock or LPS+NSR for 8 h and then clasto Lactacystin β-lactone (CLBL) was added at two concentrations, as indicated. Control cells were left untreated or were treated with DMSO. Cells were analysed at 24 hpi for CD83 expression. Bars represent average MFI ±SD from two experiments with cells from one donor (B) Detection of total amounts of CD83 in cell lysates by Western blot. Cells were stimulated as described under point “A” and treatments are indicated above the image. (C) Inhibition of endocytosis. DCs were stimulated with LPS+NSRmock, LPS+NSR or left unstimulated. Cytochalasin D (Cyt D) or the solvent DMSO were subsequently added at different time points. The moments of adding Cyt D/DMSO and harvesting of cells are indicated above each graph. Bars represent average fold change of the MFI relative to unstimulated cells treated with DMSO ±SD. Average values of three experiments with cells from one donor are depicted. Relevant statistical significances are shown.</p