Disruption of lipid rafts blunts T cell response <i>to C</i>. <i>albicans</i>.

<p>Monocytes, treated or not with DAmb or MBCD before incubation with HK <i>C</i>. <i>albicans</i> (Ca) or with MP65, were co-cultured with autologous memory CD4⁺ T lymphocytes for 6 days. At day 6 cell proliferation was measured by thymidine incorporation, expressed as count per minutes (cpm), and culture supernatants were harvested for cytokine production analysis by ELISA. <b>A</b>. Part of the monocytes treated with drugs and pulsed with <i>C</i>. <i>albicans</i> were extensively washed or not before incubation with T cells (+/- washing). Drugs removal only partially restored T cell response supporting a major effect on monocytes. <b>B</b>. T cell response to MP65, which does not require phagocytosis for its uptake and processing, was left unmodified by the presence of drugs excluding a non specific effect on antigen processing and/or presentation by monocytes. Asterisks indicate values significantly different from non-treated monocytes: (*p<0.05; **p<0.001). One experiment out of two is shown as means ± SD of triplicate wells.</p

<i>Candida albicans</i> Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses

<div><p>Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-β-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen <i>Candida albicans</i> by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes <i>Candida albicans</i> cell wall-associated β-glucan, is recruited to lipid rafts upon <i>Candida albicans</i> uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to <i>Candida albicans</i> but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to <i>Candida albicans</i> in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.</p></div

Disruption of lipid rafts does not modify cytokine production by monocytes in response to <i>C</i>. <i>albicans</i>.

<p>Monocytes, pretreated or not with DAmb or MBCD, were incubated with or without <i>C</i>. <i>albicans</i>. Supernatants were collected and cytokine levels were evaluated by ELISA. Histograms indicate mean values ± SE of three independent experiments. The asterisk indicates values (<i>p<</i>0.05) significantly different from untreated monocytes. <i>ns</i>: not significant.</p

Disruption of lipid rafts inhibits <i>C</i>. <i>albicans</i> phagocytosis by human monocytes.

<p><b>A.</b> Phagocytosis inhibition assessed by flow cytometry. Human monocytes were pretreated with MBCD, DAmB or left untreated (none), and then incubated with HK FITC-conjugated <i>C. albicans</i> or <i>Staphylococcus aureus</i> (<i>St. aureus</i>). The data highlight that drugs specifically impair fungal phagocytosis. Markers were set up to exclude background staining of cells incubated with the microorganisms at 0°C. Numbers indicate the percentage of monocytes with ingested microorganisms. Data shown are from one experiment representative of four. <b>B.</b> Phagocytosis analysis by confocal microscopy. Phagocytosis was performed as in A. Samples were acquired in 3D stacks by DIC and three-color fluorescent images. The triple staining allows to clearly establish if a FITC-conjugated <i>C</i>. <i>albicans</i> (green) was internalized by a monocyte labelled by anti-CD14 (red). DNA was counterstained with DAPI (blue). The first column (DIC overlay) shows the combined signals from the DIC and confocal fluorescent channels of a single xy slice. In the second column (3D rendering), the 3D reconstructions of the 22 slices highlighting the reciprocal spatial information of fungal and human cells are displayed. The third column (orthogonal views) shows the xy, xz and yz single planes at the indicated orthogonal xyz axes (white lines). Scale bar for all panels is 3 μ.</p

Effects of lipid raft perturbation on Dectin-1 dynamics during phagocytosis.

<p>Confocal time lapse analysis of Dectin-1 (red) and lipid rafts (CTB, magenta) dynamics in stimulated human monocytes after MBCD or DAmb treatment. Merge panels show the combined confocal fluorescent signals. The DIC images showing cell morphology are merged with the fluorescent channels in the last column. Scale bars are 5 μm. First lane are control cell not treated with drugs. MBCD or DAmb were added to the monocytes before fungi stimulation. Drugs treatment caused a complete disaggregation of lipid rafts (CTB channel). Dectin-1 polarization at the sites of <i>C</i>. <i>albicans</i> contact was strongly impaired. The resulting effect of these events was a severely decreased phagocytosis. See also Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142531#pone.0142531.s002" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142531#pone.0142531.s003" target="_blank">S3</a> Movies.</p

Involvement of lipid rafts in Dectin-1-mediated phagocytosis of <i>C</i>. <i>albicans</i>.

<p>Confocal time lapse analysis of Dectin-1 (red) and lipid rafts (CTB, magenta) dynamics in control, unstimulated human monocytes (<b>A</b>) and in FITC-<i>C</i>. <i>albicans</i> (green) stimulated human monocytes (<b>B</b>). Merge panels show the combined confocal fluorescent signals. The DIC images showing cell morphology are merged with the fluorescent channels in the last column. <b>A</b>. In absence of external stimuli, Dectin-1 receptor is scattered on the plasma membrane and no signal could be associated with the lipid rafts compartments. Scale bar is 5 μm. <b>B</b>. The timescale on the left indicates recording time. Soon after <i>C</i>. <i>albicans</i> stimulus, CTB signal starts to aggregate, indicating lipid rafts assembly in larger structures. With phagocytosis progression Dectin-1 association with lipid raft domains became stronger and clustering in correspondence of the point of monocyte-fungal cell contact increased and persisted during uptake and internalization. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142531#pone.0142531.s001" target="_blank">S1 Movie</a>. Scale bars are 5 μm.</p

Dectin-1 is the predominant PRR involved in phagocytosis of <i>C</i>. <i>albicans</i> by human monocytes.

<p><b>A</b>. Flow cytometry analysis of monocyte surface expression of <i>C</i>. <i>albicans</i>-specific PRRs. Circulating human monocytes express Dectin-1 and CR3 receptors but not MR and DC-SIGN. The appropriate isotype control for each mAb is reported as histogram with dotted lines. One experiment representative of five is shown. <b>B</b>. Inhibition of <i>C</i>. <i>albicans</i> phagocytosis by blocking <i>C</i>. <i>albicans-</i>specific PRRs. Monocytes were pre-treated with laminarin, mannan, acetylglucosamine (GlcNAc) to block Dectin-1, Dectin-2 or CR3, respectively or with a specific antibody anti-CR3, before incubation with FITC-conjugated <i>C</i>. <i>albicans</i>. Only laminarin impairs fungus phagocytosis, supporting the relevant role of Dectin-1 on <i>C</i>. <i>albicans</i> uptake. The asterisk indicates values significantly different from untreated monocytes (<i>p</i><0.05). Results are the mean ± standard error (SE) of three independent experiments performed. <b>C.</b> Inhibition of the uptake of the specific Dectin-1 ligand β-glucan following monocyte lipid rafts disruption. Monocytes were pre-treated or not with DAmb or MBCD and then incubated with HK FITC-conjugated <i>C</i>. <i>albicans</i> or FITC-conjugated β-glucan. The extent of phagocytosis was assessed by flow cytometry. The uptake reduction was similar for both β-glucan and <i>C</i>. <i>albicans</i> suggesting the involvement of lipid rafts in Dectin-1 mediated phagocytosis. The asterisk indicates values significantly different (<i>p<</i>0.05) from untreated monocytes. Results are expressed as mean ± SE of three independent experiments.</p

MSU crystals enhance Ca²⁺ dependent ROS production.

<p>(<b>A</b>) dTHP-1 cells were incubated with 3 μM Fluo-3/AM at 37°C for 1 hour in the dark and were stimulated with 5 μg/ml MSU. After stimulation, fluorescence emission was continuously monitored for 30 minutes and expressed as to determine relative alteration in intensity. (<b>B</b>) dTHP-1 cells were incubated for 1 hour at the dark with 10 μM DCF, or with 3 μM Fluo-3/AM (inset of the figure), and were stimulated with 5 μg/ml MSU. Ca^<b>2+</b> dependence ROS generation was assessed by adding 20 μM BAPTA-AM or 3 mM EGTA 30 minutes and 15 minutes before MSU addition, respectively. Fluorescence emission was monitored at 20 minutes after stimulation. Results are expressed as mean ± SD of arbitrary fluorescence units performed in triplicate and are representative of three separate experiments. * p < 0.01 in comparison with non-stimulated control cells</p

MSU crystals enhance antimycobacterial activity.

<p>(<b>A</b>) Differentiated THP-1 (dTHP1) cells were infected with BCG at the MOI of 1 and then stimulated or not with 0.5, 5, 50 μg/ml of MSU for 3 and 5 days. The results are expressed as means ± Standard Deviation (SD) of CFU values performed in triplicate and are representative of three independent experiments. * p ≤ 0.001 in comparison with non-stimulated control cells. (<b>B</b>) Stimulation of human macrophages with MSU enhances phagocytosis of BCG. Differentiated THP-1 cells were exposed to BCG at the MOI of 1 for 3 hour in the presence or not of 0.05, 0.5, 5 μg/ml MSU. Results are expressed as mean ± SD of CFU values performed in triplicate and are representative of two independent experiments. * p < 0.05 in comparison with non-stimulated control cells.</p

MSU crystals promote maturation of phagosomes containing BCG.

<p>(<b>A</b>) dTHP-1 cells were infected with NHS labelled BCG at the MOI of 1 and then stimulated overnight with 5 μg/ml of MSU, in the presence or absence 10 μM Chloroquine. Results are expressed in terms of mean ± SD of arbitrary fluorescence units of triplicate values and are representative of two independent experiments. * p < 0.001 in comparison with non-stimulated control cells, ° p < 0.001 in comparison with MSU stimulated cells. (<b>B</b>) dTHP-1 cells were infected with BCG, stimulated overnight with 5 μg/ml of MSU, in the presence or absence of 10 μM Chloroquine (Cq), and then labelled with 1 μM Lysosensor green DND 189. Results are expressed as mean ± SD of pH values from cultures performed in triplicate and are representative of two independent experiments. * p < 0.001 in comparison with non-stimulated control cells, ° p < 0.001 in comparison with BCG infected MSU stimulated cells (<b>C</b>) Protease activity of BCG infected dTHP-1 cells stimulated overnight with 5 μg/ml of MSU was analysed by loading of 10 μg/ml DQ red BSA for 2h at 37°C. Results are expressed as mean ± SD of triplicate values and are representative of three independent experiments. * p ≤ 0.01 in comparison with non-stimulated control cells. (<b>D</b>) Confocal microscopy representative images out of 10 per condition showing the increase of Auramine-stained BCG (green) residing in LAMP-3 positive vacuoles (red) after stimulation with 5 μg/ml of MSU. One representative experiment out of three is shown. (<b>E</b>) Summary of the mean percentage ± standard deviation (SD) of BCG co-localizing in LAMP-3-positive vacuoles determined by acquiring at least 10 images per condition and by counting ≥ 50 dTHP-1 cells per sample, after stimulation or not with MSU. Three different experiments were assessed. * p < 0.05 in comparison with BCG-infected cells (data were analyzed using the unpaired Student’s <i>t</i>-test).</p