Fungus-treated MDDCs enhance HIV-1 endocytosis and alter viral intracellular sequestration.

<p>(A) Enhanced HIV-1 endocytosis in Fungus-treated MDDCs. Immature MDDCs were treated with heat-killed fungi as described above. HIV-1 VLPs (40 ng amounts of p24^gag) were added for 2 h incubation, and some samples were prior-blocked with anti-DC-SIGN antibodies, and trypsin treatment for 5 min at room temperature was used to remove surface-bound virus. Gag-GFP level was detected by flow cytometry, and the positive percentages and the calculated MFI values from one representative out of four experiments are denoted. (B) Internalized HIV-1 VLPs are sequestrated in CD81⁺ DC-SIGN^- compartments in fungus-stimulated MDDCs. Immature MDDCs were treated with heat-killed fungi and pulsed with HIV-1 VLPs as described for panel A, MDDCs were fixed and immunostained for DC-SIGN or CD81, and cells were observed by confocal microscopy. Scale bars, 2 µm.</p

Fungal stimulation promotes MDDC-mediated HIV-1 transmission to CD4⁺ T cells.

<p>Immature MDDCs were treated with heat-killed fungi species as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027609#pone-0027609-g002" target="_blank">Figure 2</a>. After pulsing with 40 ng p24^gag amounts of HIV-1 VLPs (A), or with 5 ng p24^gag amounts of pseudotyped HIV-Luc/NL4-3 or HIV-Luc/JRFL(B and C), CD4⁺ T target cells were added for co-culture, HIV-1 VLP transfer was detected either by flow cytometry after 30 min (A), or by measuring HIV-1 infection after 3 days co-culture (B and C). (A) Enhanced viral association with Hut/CCR5 cells mediated by fungus-stimulated MDDCs. Hut/CCR5 cells (CD11c^-) were gated from co-culture, and viral association was measured by detecting Gag-GFP level by flow cytometry. The positive percentages and the values of MFI from one representative out of three experiments are shown; (B, C) Increased HIV-1 <i>trans</i> infection mediated by fungus-stimulated MDDCs, either with Hut/CCR5 cells (B) or PHA-activated autologous primary CD4⁺ T cells as target cells (C). Asterisks indicate significantly enhanced HIV-1 <i>trans</i> infection mediated by fungus-treated MDDCs compared with that of medium-treated cells (**<i>P</i> <0.01, ***<i>P</i> <0.001, paired <i>t</i> test); Results of one representative experiment out of four are shown. All data are means ± SD. cps, counts per second.</p

Fungal stimulation induces MDDCs activation.

<p>(A) <i>Ex vivo</i> culture and characterizations of fungi species associated with HIV-1 infection. <i>P. Marneffei</i> was isolated and identified from a skin lesion from an AIDS patient. Sub-cultured <i>P. Marneffei</i> showed dimorphisms, with a yeast form at 37°C growth (PMy) and a mycelial form at 25°C (PMm-i); the production of red pigments at 25°C is indicated (PMm-ii). <i>C. Albicans</i> was isolated from the tongue of an AIDS patient (CA-i), and <i>identified with sub-inoculation</i> in CHROMagar Candida displaying the green color (CA-ii), and in Corn Tween agar showing the formation of sporulation (CA-iii), <i>C. albicans</i> was sub-cultured <i>ex vivo</i> (CA-iv). (B) Activation of MDDCs was monitored by flow cytometry. Immature MDDCs were treated with heat-killed <i>C. albicans</i>, PMy and PMm at a ratio of 1∶10 for 48 h, and medium treatment was used as a control. MDDCs were gated as a CD11c⁺ population, and fungus-stimulated MDDCs showed increased expression of HLA-DR and the co-stimulatory molecules CD83 and CD86, with decreased expression of DC-SIGN, compared with the medium-treated controls. The positive percentages and values of MFI from one representative out of six experiments are indicated.</p

Fungus-stimulated MDDCs increase surface expression of ICAM-1 and facilitate the formation of virological synapses between MDDCs and CD4⁺ T target cells.

<p>(A) Increased ICAM-1 expression on fungus-stimulated MDDCs. Immature MDDCs were treated separately with heat-inactivated <i>C. albicans</i>, PMy and PMm or medium. The surface level of ICAM-1 was detected by flow cytometry. The MFI values are shown. (B, C) Enhanced viral concentration on the contact sites between MDDCs and T cells. Immature MDDCs were treated with fungi as above, HIV-1 VLPs (40 ng p24^gag) were added for 2 h incubation, and Hut/CCR5 or PHA-activated autologous primary CD4⁺ T cells were added as target cells for 30 min co-culture. Cells were fixed and observed by confocal microscopy. β-actin (B) or tetraspanin CD81 (C) were stained and shown in red. Nuclei were stained by DAPI. DIC, differential interference contrast. Scale bars, 5 µm.</p

HIV-1 infection is blocked in MDDCs after fungal stimulation.

<p>Immature MDDCs were treated separately with heat-killed fungus species <i>C. albicans</i>, PMy and PMm or control medium for 48 h, and then were incubated with single-cycle luciferase reporter virus HIV-Luc/JRFL (5 ng of p24^gag) for 2 h. After washing, HIV-1-pulsed MDDCs were incubated and harvested at the indicated times, and HIV-1 infection was detected by measuring the luciferase activity in cell lysates. Results of one representative experiment out of three are shown. All data are mean ± standard deviation (SD).cps, counts per second.</p