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

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

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

One hundred forty pathogens detected by SeptiFast (SF) and/or blood culture (BC) in 1009 patients.

1<p><i>Streptococcus</i> spp. group includes <i>S. agalactiae, S. pyogenes, S. anginosus</i>, <i>S. bovis</i>, <i>S. constellatus</i>, <i>S. cristatus</i>, <i>S. gordonii</i>, <i>S. intermedius, S. milleri, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. salivarius, S. sanguinis, S. thermophilus, S. vestibularis, S. viridans</i>.</p>2<p>Coagulase-negative staphylococci group includes <i>S. epidermidis, S. haemolyticus, S. hominis, S. pasteuri, S. warneri, S. cohnii, S. lugdunensis, S. capitis, S. caprae, S. saprophyticus</i>, and <i>S. xylosus</i>.</p>3<p>ND, not included in the SF master list <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053279#pone.0053279-Lehmann1" target="_blank">[6]</a>.</p

ROC curves for SeptiFast and blood cultures results according to different cut-off of PCT.

<p>SeptiFast: Area Under the Curve (AUC) = 0.927; 95% confidence interval (CI): 0.899–0.955, p<0.0001 (panel A). Blood culture: AUC = 0.820; 95% CI: 0.769–0.870, p>0.0001 (panel B).</p

Sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio of different cut-off values of PCT on SeptiFast results.

<p>PPV = positive predictive value.</p><p>NPV = negative predictive value.</p><p>LR+ = positive likelihood ratio.</p><p>LR− = negative likelihood ratio.</p

IL-22 and IDO1 Affect Immunity and Tolerance to Murine and Human Vaginal Candidiasis

<div><p>The ability to tolerate <i>Candida albicans</i>, a human commensal of the gastrointestinal tract and vagina, implicates that host defense mechanisms of resistance and tolerance cooperate to limit fungal burden and inflammation at the different body sites. We evaluated resistance and tolerance to the fungus in experimental and human vulvovaginal candidiasis (VVC) as well as in recurrent VVC (RVVC). Resistance and tolerance mechanisms were both activated in murine VVC, involving IL-22 and IL-10-producing regulatory T cells, respectively, with a major contribution by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 was responsible for the production of tolerogenic kynurenines, such that replacement therapy with kynurenines restored immunoprotection to VVC. In humans, two functional genetic variants in <i>IL22</i> and <i>IDO1</i> genes were found to be associated with heightened resistance to RVVC, and they correlated with increased local expression of IL-22, IDO1 and kynurenines. Thus, IL-22 and IDO1 are crucial in balancing resistance with tolerance to <i>Candida</i>, their deficiencies are risk factors for RVVC, and targeting tolerance via therapeutic kynurenines may benefit patients with RVVC.</p></div

IDO1 and kynurenines mediate tolerance in murine VVC.

<p>(<b>A</b>) IDO1 protein and (<b>B</b>) gene expression in the vagina of C57BL/6 mice (<i>n</i> = 4) intravaginally infected with <i>C. albicans</i>. Proteins in vaginal cell lysates (3 dpi) were visualized by western blotting with rabbit polyclonal IDO1 specific antibody. Scanning densitometry was done on a Scion Image apparatus. Western blots out of 2 independent experiments and corresponding pixel density ratio normalized against β-tubulin. <i>Ido1</i> mRNA expression [normalized to mRNA of naïve (dpi 0) mice] in vaginal tissue (RT-PCR) at different dpi. (<b>C</b>) Relative concentrations of kynurenines (Kyn) and (<b>D</b>) kynurenine-to-tryptophan (Kyn/Trp) ratio in vaginal fluids at different dpi. Pooled results from 3 different experiments. *<i>P</i><0.05, IDO1-deficient <i>vs.</i> C57BL/6 mice at the days indicated. N.S., not significant. (<b>E</b>) Vaginal fungal growth (Log₁₀ CFU/100 µl VF ± s.e.m.) at different dpi in mice (<i>n</i> = 6) treated intraperitoneally with a mixture of l-kynurenine, 3-hydroxykynurenine and 3-hydroxyanthranilic acid or PBS (None). Pooled data from 3 different experiments.*<i>P</i><0.05, treated <i>vs.</i> untreated mice at the days indicated. N.S., not significant. (<b>F</b>) Periodic acid-Schiff-stained vaginal sections and inflammatory cell recruitment in vaginal fluids (May–Grünwald Giemsa staining in the insets) acquired with a 40× and 100× objective, respectively, at 21 dpi. Scale bars, 100 µm. Representative image from 3 experiments. (<b>G</b>) Cytokine levels (pg/mg, cytokine/total proteins for each sample, at 21 dpi) in the vaginal fluids of mice treated as above. Pooled data from 3 different experiments. *<i>P</i><0.05, treated <i>vs.</i> untreated (None) mice. (<b>H</b>) Vaginal immunohistochemistry of naïve or infected mice at 3 days after re-challenge. Double staining was done with anti-IL-10-FITC and polyclonal rabbit to FoxP3 followed by anti-rabbit TRITC. Cell nuclei were stained with DAPI (blue). Representative pictures (out of 2 experiments) were taken with a 20× objective. Scale bars, 50 µm.</p

Treg cells control the Th1/Th17 cell balance in murine VVC.

<p>(<b>A</b>) IFN-γ and IL-17A production (pg/mg, cytokine/total proteins for each sample) in the vagina fluids of mice (<i>n</i> = 6) intravaginally inoculated with 5×10⁶<i>C. albicans</i> blastoconidia and re-infected 3 weeks later. Assays were done at 3 days after the primary infection (infected) or re-challenge. Pooled data from 5 experiments. (<b>B</b>) Vaginal immunohistochemistry of re-infected mice. Staining was done with anti-IFN-γ-PE and anti-IL-17A-PE antibody. Cell nuclei were stained with DAPI (blue). Representative pictures (out of 2 experiments) were taken with a 20× objective. Scale bars, 200 µm. (<b>C</b>) Expression (RT-PCR) of transcript factor genes in purified CD4⁺ T cells from the draining lymph nodes. Assays were done at 3 days after the primary infection (infected) or re-challenge. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001, knockout <i>vs.</i> C57BL/6 mice at 3 days after re-challenge and re-challenged <i>vs.</i> infected mice. Control immunostaining performed on the vaginas from naïve mice revealed an increased expression of IL-17A in IL-17RA- and IL-10-deficient mice and of IFN-γ in IL-10-deficient mice. (<b>D</b>) Fungal growth in C57BL/6 or IL-10-deficient mice (<i>n</i> = 6) inoculated with 5×10⁶<i>C. albicans</i> blastospores. Fungal growth was quantified in the vaginal fluids at different days post-infection (dpi) and expressed as Log₁₀ CFU/100 µl VF ± s.e.m. (<b>E</b>) Polymorphonuclear cells (PMNs) quantification in the vaginal fluids at different dpi. PMNs were identified by nuclear morphology and enumerated per field at ×100 magnification. Pooled data from 3 experiments. *<i>P</i><0.05 and **<i>P</i><0.01, 3 or 21 <i>vs.</i> 0 dpi.</p