7 research outputs found
<i>In vivo</i> infection of resident and migratory DCs in the skin-draining lymph node of IFNAR<sup>−/−</sup> mice and their migratory behavior.
<p>(A) Gating strategy to identify lymph node (LN)-resident and migratory DCs in the skin-draining LN of IFNAR<sup>−/−</sup> mice. Resident DC subsets are either CD8<sup>+</sup> (1), CD11b<sup>−</sup> (2) or CD11b<sup>+</sup> (3), while migratory DC subsets incorporate those found in the skin: CD103<sup>+</sup> (4), CD11b<sup>−</sup> (5), CD11b<sup>+</sup> (6) and Langerhans Cells (LCs, 7). (B) Mice were inoculated i.d. with 1×10<sup>6</sup> pfu of DENV-2 D2Y98P/ear and skin-draining lymph nodes (LN) were harvested 2 and 4 days post infection (dpi). Presence of DENV E protein was established by flow cytometry in both LN-resident and migratory DC subsets. (C) Summary of data from (B), 4 to 5 mice (average of two LN/mouse) per group pooled from two independent experiments, mean ± SEM, analyzed with one-way ANOVA followed by Tukey's multiple comparison test, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; non-significant differences are not indicated. (D) Quantification of DCs isolated from LNs at 2 and 4 dpi to address infiltration and migration of different cell subsets. Same conditions as in (C).</p
<i>In vivo</i> infection of migratory DCs in the skin of IFNAR−/− mice.
<p>(A) Mice were inoculated intra-dermally (i.d) with 1×10<sup>6</sup> pfu of DENV-2 D2Y98P/ear, and ears were harvested 2 and 4 days post infection. DENV E protein expression was detected by flow cytometry of skin-resident DC subsets and infiltrating monocytes (Gating strategy to identify DC subsets/monocytes shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004548#ppat.1004548.s002" target="_blank">Figure S2</a>) (B) Summary data of (A), 4 to 5 mice (taking the average of two ears/mouse) per group pooled from two independent experiments, mean ± SEM, analyzed with one-way ANOVA followed by Tukey's multiple comparison test, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; non-significant differences are not indicated (C) Quantification of DCs isolated from ears at 2 and 4 dpi to address infiltration and migration of different cell subsets. Same conditions as in (B).</p
Infection characteristics of DENV in DC subsets from human skin.
<p>(A) Cells from collagenase-treated healthy human skin were exposed to DENV-2 at MOI 2 for the indicated times. Presence of DENV E protein in DC subsets was established by flow cytometry. (B) Amount of live virus in supernatants of cells from (A) was quantified by plaque-forming assay and used to calculate titer in plaque-forming units per ml (pfu/ml), n = 3 donors, mean ± SEM from three independent experiments. (C) Sorted DC subsets were infected with DENV-2 at MOI 2 and viral RNA was measured in the supernatant by quantitative real time PCR at 0, 24 and 48 hpi. n = 3, mean ± SEM from three independent experiments. (D) Analysis of skin DC subsets and macrophages by flow cytometry in whole human skin. Each dot represents one donor, mean ± SEM. (E) Results from (C) and (D) were used to calculate the relative contribution of each DC subset to the total viral load at 24 and 48 hpi. (F) Infected DC subsets from skin were stained with Annexin V and labeled for DENV E protein after 24 h of exposure to virus to determine the extent of apoptosis (one representative donor of three), mean % per quadrant ± SEM.</p
T cell stimulation capacity of DENV-exposed DC subsets and their migratory response towards the chemokine CCL19.
<p>(A) Co-culture of sorted infected DCs with allogeneic, CFSE labeled CD3<sup>+</sup> T cells (ratio 1∶10). Proliferation was measured after 5 days by flow cytometry, one representative result for CD14<sup>+</sup> dermal DCs is shown. (B) Summary data from all three susceptible DC subsets are shown, each line represents one donor. Statistical analysis was performed using paired two-sided t-test, *p<0.05; ns, not significant, p-values are indicated for each DC subset (C) CCR7 expression on non-treated and DENV-2-exposed DCs at 24 hpi. One representative experiment of three is shown. (D) Skin cell migration was assessed using a 5 µm pore-sized membrane (see Methods Section) with either medium alone or CCL19 (20 ng/ml) in the bottom chamber. Cells were allowed to migrate for 2 h at 37°C before CellTiter Glo activity was measured (RLU, relative light units). Composite data of 4–6 donors is shown, mean ± SEM from 4 independent experiments. (E) Whole skin cells were analyzed by flow cytometry before and after migration towards CCL19. Migrated cells (in the lower well of the chemotaxis plate) were enriched in HLA-DR<sup>+</sup> cells compared to input cells (left graph). Migrated HLA-DR<sup>+</sup> cells were enriched in CD1c<sup>+</sup> DCs and LCs, but not CD14<sup>+</sup> DCs (right graph; non-infected cells are illustrated; similar results were obtained for infected cells). n = 3 donors, mean ± SEM from three individual experiments.</p
Identification of DENV susceptible cells from healthy human skin samples.
<p>(A) Flow cytometry of collagenase-treated healthy human skin incubated with medium (mock) or DENV-2 (multiplicity of infection, MOI 2) for 48 h (left). All cells (grey) were overlaid with infected cells (red) to determine expression of CD45 and HLA-DR. (B) Summary data of infected cells from (A) according to their expression of CD45 and HLA-DR of n = 7 donors, mean ± SEM from 7 independent experiments. (C) Mock, UV-inactivated and LIVE DENV-2 treated HLA-DR<sup>+</sup> cells from (A) were subjected to surface molecule expression analysis to identify four different subsets of dendritic cells (DCs) (gating strategy shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004548#ppat.1004548.s001" target="_blank">Figure S1</a>): CD14<sup>+</sup> dermal DCs, epidermal Langerhans cells (LCs), CD1c<sup>+</sup> DCs and CD141<sup>+</sup> DCs. Infection was quantified by flow cytometry according to detection of DENV E protein. (D) Summary data of (C), each dot represents one donor, mean ± SEM. (E) Cells were exposed to DENV-1, -3 and -4 (MOI 10) for 48 h and presence of DENV E protein detected by flow cytometry, n = 3 donors, mean ± SEM from three independent experiments.</p
Infection of primary skin DCs is independent of DC-SIGN.
<p>(A) DEPC-inactivated, fluorescently-labeled DENV-3 was added to skin cells for 2 h at 4°C or 37°C (left) before flow cytometric analysis of viral uptake. Representative results of one donor are shown (right) and summary data are depicted as mean fluorescence intensity (MFI) at 37°C minus MFI at 4°C (bottom), each dot represents one donor, mean ± SEM. (B) APC subsets were incubated with DC-SIGN blocking antibody for 1 h before infection for 24. n = 3 donors, Mean ± SEM from two independent experiments. (C) Total skin cells were treated with IFN-β for 24 h before infection for 24 h. Infected APC subsets were analyzed by flow cytometry. n = 4, mean ± SEM from three independent experiments.</p
Model for skin dengue infection.
<p>(A) The mosquito searches for blood vessels in the dermis and thereby releases saliva that contains virus. The proboscis bypasses the epidermis. The question mark indicates that it is not clear whether during the process of probing virus is also released into the epidermis. (B) Virus in the dermis infects CD1c<sup>+</sup> and CD14<sup>+</sup> dermal DCs and macrophages (MP), but not CD141<sup>+</sup> DCs. Infected CD1c<sup>+</sup> DCs and possibly LCs migrate to draining LNs to initiate the adaptive immune response. Based on their non-migratory behavior <i>ex vivo</i> (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004548#ppat-1004548-g003" target="_blank">Fig. 3E</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004548#ppat.1004548-McGovern1" target="_blank">[12]</a>) CD14<sup>+</sup> DCs are not expected to migrate to draining LNs. Based on mouse studies, monocyte-derived cells that infiltrate into the skin are infected efficiently (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004548#ppat-1004548-g006" target="_blank">Fig. 6</a>) and contribute to the local and systemic immune response. (C) Table summarizing the role of skin APCs during infection with DENV using single cell suspensions.</p