Neutrophil infiltration and extracellular DNA are closely associated with enhanced sDectin-1 staining.

<p>Vaginal swab samples with <i>C</i>. <i>albicans</i> were scored by level of neutrophil infiltration, then stained and imaged as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201436#pone.0201436.g001" target="_blank">Fig 1</a>. (A) Samples with high levels of neutrophil infiltration had sDectin-1-positive filaments, while those with low levels of infiltration had none. (B) Samples with high infiltration had a significantly higher level of extracellular DNA (eDNA), as imaged using Sytox Green. (C) Samples with high levels of eDNA also had high percentages of sDectin-1-positive cells. Samples with eDNA in every field were categorized as High eDNA, samples for which <100% of fields had eDNA were categorized as Low eDNA. (D) Representative field of a sample (#SP-14314) with no fields containing eDNA, no hyphae, and no sDectin-1-positive cells. Black arrowheads indicate nuclei from epithelial cells in the lavage. Image is maximum projection of 5 z-slices, created by ImageJ. (E) Representative field of a sample (#SP-12522) with high levels of eDNA, hyphae, and sDectin-1 positive cells. Black arrowheads indicate epithelial nuclei and white arrowheads indicate areas of diffuse Sytox Green staining of eDNA. Image is maximum projection of 10 z-slices, created by ImageJ. Scalebar = 20 μm. Statistics used in (A-C) were Mann-Whitney non-parametric tests. Significance throughout the figure is indicated with * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Enhanced Dectin-1 recognition is filament-specific and co-localizes with enhanced chitin deposition.

<p>Vaginal swab samples were stained with sDectin-1-Fc (sDectin-1; red), Calcofluor white (CFW; blue; outline of fungi for morphology and for identification of sites of increased chitin deposition), and Sytox Green (green; stains only extracellular DNA and DNA inside cells with compromised membranes). The EGFP-expressing strain KAH3-EGFP was spiked in before staining as a positive control. Several 40x fields (8–16 per sample) were imaged and scored for fungal morphology, sites of sDectin-1 staining, co-localization of sDectin-1 staining and increased CFW staining, and presence of extracellular DNA. (A) In samples from <i>C</i>. <i>albicans</i>-infected patients, only filaments were found with enhanced sDectin-1 staining. (B) In samples from patients infected with non-<i>albicans Candida</i> or a mix of <i>C</i>. <i>albicans</i> and non-<i>albicans Candida</i>, only filaments were stained with sDectin-1. The only sample with any sDectin-1+ cells was a mix of <i>C</i>. <i>albicans</i> and <i>C</i>. <i>norvegensis</i> (Sample #SP-97366). The difference in frequency of staining is not significantly different between filaments and yeast (p >0.9999), because there was virtually no staining of either morphology. (C) Representative field from <i>C</i>. <i>albicans</i>-infection sample (#SP-66117). A single filament segment that is swollen (purple arrow) has high levels of sDectin-1 and CFW staining. The top image is a three-color overlay and the images below separate the sDectin-1 and CFW. (D) Representative field from a non-<i>albicans Candida</i> infection sample (#SP-12622). Most of the fungi are yeast from the infected patient without high levels of sDectin-1 or CFW staining (white arrows). In the upper right is a cluster of cells from the spiked-in control KAH3-EGFP (white arrowheads) with enhanced sDectin-1 and CFW staining. The CFW is overexposed to visualize the weakly-staining <i>C</i>. <i>krusei</i> cells from the patient, but the cell outlines can be clearly seen in the sDectin-1 staining image in red. (E) Schematic to illustrate that the majority of sites (58%) with enhanced sDectin-1 staining also had increased chitin deposition. Images are maximum projections of 6 slices (J) or 5 slices (K). Scalebar = 20 μm throughout. Statistics used for (H) and (I) were Mann-Whitney non-parametric tests. Significance throughout the figure is indicated with: n.s. p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.</p

<i>Lactobacilli</i> are found in most samples and are not associated with neutrophil infiltration or lack of sDectin-1+ cells.

<p><i>Lactobacilli</i> are found in most samples and are not associated with neutrophil infiltration or lack of sDectin-1+ cells.</p

Staining with sDectin-1 on hyphae is associated with neutrophil infiltration and extracellular DNA.

<p>Staining with sDectin-1 on hyphae is associated with neutrophil infiltration and extracellular DNA.</p

Primer sequences used for quantitative RT-PCR.

<p>Primers were designed with the help of Beacon Designer software (Bio-Rad, Hercules, CA, USA) and provided by Invitrogen.</p

TNF-α and IL-6 production by PM and PMN stimulated with human and/or mouse CDRs and mouse V_HCDR3 uptake by different cell populations.

<p>PM (<b>A</b>) or PM and PMN (<b>B</b>) (both 5×10⁶/ml) were cultured in the presence or absence (NS) of human and/or mouse CDRs, LPS, or NC (all 10 µg/ml) for 18 h. After incubation, TNF-α and IL-6 levels were evaluated in culture supernatants by specific ELISA assays. *, <i>P</i><0.05 (treated <i>vs</i> untreated cells, n = 7). DC, PM, PMN, and T cells (all 1×10⁶/ml) were incubated for 1 h in the presence or absence (NS) of b-V_HCDR3 or b-NC (both 10 µg/ml). After incubation, permeabilized cells were reacted with FITC-labelled mAb to biotin and analyzed by FACScan flow cytometry. Data are reported as the percentage of positive cells (<b>C</b>). *, <i>P</i><0.05 (b-V_HCDR3 treated <i>vs</i> untreated cells, n = 5). Error bars, s.e.m.</p

Kinetic of V_HCDR3 uptake by PM.

<p>PM (1×10⁶/ml) were incubated for different times with b-V_HCDR3 or b-NC (all 10 µg/ml). After incubation, permeabilized cells were reacted with FITC-labelled mAb to biotin and analyzed by FACScan flow cytometry. Data are reported as the mean fluorescence intensity (MFI) (upper panel) and percentage of positive cells (lower panel) (<b>A</b>). *, <i>P</i><0.05 (b-V_HCDR3-treated <i>vs</i> untreated cells, n = 7). Error bars, s.e.m. In selected experiments cells were incubated for 1 h as above described, reacted with FITC-labelled mAb to biotin in the presence of Evans' Blue as a counter stain, and subsequently examined under fluorescent light microscopy. Note the green fluorescence of b-V_HCDR3 treated cells. Original magnification 20×(<b>B</b>). Images shown are from one representative experiment out of five with similar results.</p

Segregation of CD45 GalXM-induced on BW5147 cells.

<p>Fluorescence microscopy analysis of BW5147 and BW5147 (T200⁻) cells incubated for 2 h in the presence or absence (NS) of GalXM (<b>A</b>) or GalXM-FLUOS (green) (<b>B</b>) (both 10 µg/ml). After incubation, cells were labelled with RPE mAb to CD45 (red) and then examined under fluorescent light microscopy in the presence of DAPI (blue). Note the CD45 segregation in BW5147 cells treated with GalXM (<b>A</b>). The colocalization of GalXM with CD45 and the receptor segregation on BW5147 cells was demonstrated in the merged image of panel B (<b>B</b>). Original magnification 100x.</p

CDRs sequences.

<p>CDRs sequences.</p

GalXM-induced apoptosis of BW5147 cells.

<p>(<b>A</b>) BW5147 and BW5147 (T200⁻) cells (both 1×10⁶/ml) were incubated for 18 h in the presence or absence (NS) of PHA (10 µg/ml) or GalXM (10 µg/ml). After incubation, cells were collected by cytospin and stained by Hemacolor. GalXM-treated cells exhibited altered morphology, surface blubs and nuclear fragmentation (arrows). N = cell nucleus; original magnification 40x. In selected experiments, cells were incubated for 18 h in the presence or absence (NS) of PHA (10 µg/ml), mAb to CD3 (1 µg/ml) or GalXM (10 µg/ml). After incubation, the percentage of cells undergoing apoptosis was evaluated by PI staining and analyzed using FACScan flow cytometry. Data are expressed as fold increase of percentage of apoptotic cells (<b>B</b>), or shown as FACScan histograms from one representative experiment out of seven with similar results (<b>C</b>). *, <i>p</i><0.05 (treated <i>vs</i> untreated, n = 7). Error bars denote s.e.m.</p