Immune response to different <i>Aspergillus</i> color mutants.

<p>Different mutant strains elicit diverse inflammatory responses and prime peculiar adaptive T_H response. (A) DCs were cultured with UV-killed conidia or hyphae for 24 hours or without any stimuli (unstimulated, us) and supernatants were used for TNFα, IL-10, IL-1β, IL-6, IL-12p70 and IL-23 measurements. Data are represented as mean+SD (N = 6), *p≤0.05, **p≤0.01, conidial mutant strains <i>vs</i> conidial WT strain; ^§p≤0.05, ^§§p≤0.01, hyphal mutant strains vs hyphal WT strain. Complete statistically significant p-values are collected in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056651#pone.0056651.s002" target="_blank">Table S2</a>. (B,C) Healthy PBMCs were cultured with live conidia or without any stimulus, and cytokine protein and transcript levels were measured. Data are represented as mean+SD (N = 6), *p≤0.05, **p≤0.01, mutants strain <i>vs</i> WT strain.</p

Effect of melanin content in the immune reactivity of <i>A. fumigatus</i> clinical isolates.

<p>Ability of DC to discriminate among different melanin color mutants and clinical isolates of the <i>Aspergillus</i> species was tested as differential cytokine production. DCs were cultured with live conidia of wt CEA10 strain, different knock out (KO) mutants, Aspergillosis isolates or without any stimulus (unstimulated, us) for 24 hours and supernatants used for TNFα, IL-10, IL-1β, IL-6, IL-12p70 and IL-23 measurements. *p≤0.05, **p≤0.01 and ***p≤0.001, ****p≤0.0001, mutants strains <i>vs</i> CEA10 strain, isolates <i>vs</i> CEA10 strain.</p

Immune response to different WT <i>Aspergillus</i> strains.

<p>Different WT strains elicit diverse inflammatory responses and prime peculiar adaptive Th response. (A) DCs were cultured with UV-killed conidia or hyphae of WT strains for 24 hours or without any stimuli (unstimulated, us) and supernatants were used for TNFα, IL-10, IL-1β, IL-6, IL-12p70 and IL-23 measurements. Data are represented as mean+SD (N = 6), *p≤0.05, **p≤0.01, INF⁻ conidia <i>vs</i> INF⁺conidia; ^§p≤0.05, ^§§p≤0.01, INF⁻ hyphae <i>vs</i> INF⁺ hyphae. Complete statistically significant p-values are collected in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056651#pone.0056651.s002" target="_blank">Table S2</a>. (B,C) Healthy PBMCs were cultured with live conidia or without any stimulus, and cytokine protein (A) and transcript (B) levels were measured. Data are represented as mean+SD (N = 6), *p≤0.05, **p≤0.01, INF⁻<i>vs</i> INF⁺ strains.</p

Immune response to different <i>Aspergillus</i> mutant strains.

<p>Different mutant strains elicit diverse inflammatory responses and prime peculiar adaptive T_H response. (A) DCs were cultured with UV-killed conidia or hyphae for 24 hours or without any stimuli (unstimulated, us) and supernatants were used for TNFα, IL-10, IL-1β, IL-6, IL-12p70 and IL-23 measurements. Data are represented as mean+SD (N = 6), *p≤0.05, **p≤0.01, conidial mutant strains <i>vs</i> the conidial WT strain; ^§p≤0.05, ^§§p≤0.01, hyphal mutant strains vs hyphal WT strain. Complete statistically significant p-values are collected in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056651#pone.0056651.s002" target="_blank">Table S2</a>. (B,C) Healthy PBMCs were cultured with live conidia or without any stimulus, and cytokine protein and transcript levels were measured. Data are represented as mean+SD (N = 6), *p≤0.05, **p≤0.01, mutants strain <i>vs</i> WT strain. (D) C57BL/6 mice were infected intranasally with different strains of <i>A. fumigatus</i> (6 mice/group). Survival (%), fungal growth (mean log₁₀ CFU ± SE, N = 3) in the lungs and brains of infected mice were assessed at different days post-infection (dpi). The CFUs between wild-type and the corresponding mutant strains were statistically significant (p-values ranging from ≤0.01 to ≤0.001). Lung histology (PAS staining) and BAL morphometry [%, mean ± SD, of mononuclear (MNC) or polymorphonuclear (PMN) cells] were done at 4 dpi. Representative images of two independent experiments were depicted; bars indicate magnifications. Total lung RNA was extracted at 4 dpi and the relative expression of <i>Il1β</i>, <i>Cxcl1</i> and <i>Cxcl2</i> genes was assessed by RT-PCR. Lung homogenates at 4 dpi were tested for levels of IL-17A, IFN-γ, and IL-10 by specific ELISA (mean values ± SD, N = 3). *p≤0.05, **p≤0.01 and ***p≤0.001, wild-type strains <i>vs</i> uninfected mice or mutants strains <i>vs</i> the wild-type strain.</p

<i>In vivo</i> immune response to <i>A. fumigatus</i> mutant strains.

<p>Different mutant strains elicit diverse inflammatory responses and prime peculiar adaptive T_H response. (A) DCs were cultured with UV-killed conidia or hyphae for 24 hours or without any stimuli (unstimulated, us) and supernatants were used for TNFα, IL-10, IL-1β, IL-6, IL-12p70 and IL-23 measurements. Data are represented as mean+SD (N = 6), *p≤0.05, **p≤0.01, conidial mutant strains <i>vs</i> the conidial WT strain; ^§p≤0.05, ^§§p≤0.01, hyphal mutant strains vs hyphal WT strain. Complete statistically significant p-values are collected in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056651#pone.0056651.s002" target="_blank">Table S2</a>. (B,C) Healthy PBMCs were cultured with live conidia or without any stimulus, and cytokine protein and transcript levels were measured. Data are represented as mean+SD (N = 6), *p≤0.05, **p≤0.01, mutants strain <i>vs</i> WT strain. (D) C57BL/6 mice were infected intranasally with different strains of <i>A. fumigatus</i> (6 mice/group). Survival (%), fungal growth (mean log₁₀ CFU ± SE, N = 3) in the lungs and brains of infected mice were assessed at different days post-infection (dpi). The CFUs between wild-type and the corresponding mutant strains were statistically significant (p values ranging from ≤0.01 to ≤0.001). Lung histology (PAS staining) and BAL morphometry [%, mean ± SD, of mononuclear (MNC) or polymorphonuclear (PMN) cells] were done at 4 dpi. Representative images of two independent experiments were depicted; bars indicate magnifications. Total lung RNA was extracted at 4 dpi and the relative expression of <i>Il1β</i>, <i>Cxcl1</i> and <i>Cxcl2</i> genes was assessed by RT-PCR. Lung homogenates at 4 dpi were tested for levels of IL-17A, IFN-γ, and IL-10 by specific ELISA (mean values ± SD, N = 3). *p≤0.05, **≤0.01 and ***p≤0.001, wild-type strains <i>vs</i> uninfected mice or mutants strains <i>vs</i> the wild-type strain.</p

<i>In vivo</i> immune response to <i>A. fumigatus</i> color mutants.

<p>C57BL/6 mice were infected intranasally with different strains of <i>A. fumigatus</i> (6 mice/group). Survival (%), fungal growth (mean log₁₀ CFU ± SE, N = 3) in the lungs and brains of infected mice were assessed at different days post-infection (dpi). The CFUs between wild-type and the corresponding mutant strains were statistically significant (p values ranging from ≤0.01 to ≤0.001). Lung histology (PAS staining) and BAL morphometry [%, mean ± SD, of mononuclear (MNC) or polymorphonuclear (PMN) cells] were done at 4 dpi. Representative images of two independent experiments were depicted; bars indicate magnifications. Total lung RNA was extracted at 4 dpi and the relative expression of <i>Il1β</i>, <i>Cxcl1</i> and <i>Cxcl2</i> genes was assessed by RT-PCR. Lung homogenates at 4 dpi were tested for levels of IL-17A, IFN-γ, and IL-10 by specific ELISA (mean values ± SD, N = 3). *p≤0.05, **p≤0.01 and ***p≤0.001, wild-type strains <i>vs</i> uninfected mice or mutants strains <i>vs</i> the corresponding wild-type strain.</p

<i>In vivo</i> immune reactivity to <i>A. fumigatus</i> WT strains.

<p>C57BL/6 mice were infected intranasally with different strains of <i>A. fumigatus</i> (6 mice/group). Survival (%), fungal growth (mean log₁₀ CFU ± SE, N = 3) in the lungs and brains of infected mice were assessed at different days post-infection (dpi). The CFUs between wild-type and the corresponding mutant strains were statistically significant (p values ranging from ≤0.01 to ≤0.001). Lung histology (PAS staining) and BAL morphometry [%, mean ± SD, of mononuclear (MNC) or polymorphonuclear (PMN) cells] were done at 4 dpi. Representative images of two independent experiments were depicted; bars indicate magnifications. Total lung RNA was extracted at 4 dpi and the relative expression of <i>Il1β</i>, <i>Cxcl1</i> and <i>Cxcl2</i> genes was assessed by RT-PCR. Lung homogenates at 4 dpi were tested for levels of IL-17A, IFN-γ, and IL-10 by specific ELISA (mean values ± SD, N = 3). *p≤0.05, **p≤0.01 and ***p≤0.001, wild-type strains <i>vs</i> uninfected mice.</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

Vaginal candidiasis in IL-22- or IDO1-deficient mice.

<p>C57BL/6, IL-22- or IDO1-deficient mice (<i>n</i> = 6) were intravaginally inoculated with 5×10⁶<i>C. albicans</i> blastoconidia. (<b>A</b>) Periodic acid-Schiff-staining of vaginal sections and inflammatory cell recruitment in vaginal fluids (May–Grünwald Giemsa staining in the insets) at different days post-infection (dpi). Representative images of histology sections and vaginal fluids were acquired with a 40× and 100× objective respectively. Scale bars, 100 µm. (<b>B</b>) Polymorphonuclear cells (PMNs) quantification in the vaginal fluids. PMNs were identified by nuclear morphology and enumerated per field at ×100 magnification. Each point represents an individual mouse, and horizontal bar indicates the means. **<i>P</i><0.01 and ***<i>P</i><0.001, IDO1- or IL-22-deficient <i>vs.</i> wild-type mice at the indicated days. (<b>C</b>) <i>S100a8</i> and <i>S100a9</i> mRNA expression in vaginal tissue by real-time RT-PCR. The mRNA-normalized data were expressed as relative mRNA in IDO1- or IL-22-deficient <i>vs.</i> wild-type mice. *<i>P</i><0.05 and **<i>P</i><0.01, IDO1- or IL-22-deficient <i>vs.</i> wild-type mice at the indicated days. (<b>D</b>) Levels of calprotectin during vaginal candidiasis. **<i>P</i><0.01, IDO1- or IL-22-deficient <i>vs.</i> wild-type mice. (<b>E</b>) Vaginal fungal burden in mice at different dpi. CFU were quantified by culturing serial dilutions of vaginal fluids (VF) from each mouse and expressed as Log₁₀ CFU/100 µl VF ± s.e.m. *<i>P</i><0.05, IDO1- or IL-22-deficient <i>vs.</i> wild-type mice at the days indicated. Data are pooled or representative (histology) from four independent experiments. (<b>F</b>) Cytokine levels (pg/mg, cytokine/total proteins for each sample) in the vaginal fluids (at 3 dpi for IL-17F and IL-17A). Results represent mean cytokine levels (± s.e.m.) from samples pooled from three experiments (<i>n</i> = 4–6 total samples per group). *<i>P</i><0.05, IDO1- or IL-22-deficient <i>vs.</i> wild-type mice. (<b>G</b>) Levels of IL-22 (pg/mg, cytokine/total proteins) and (<b>H</b>) fungal growth (at 3 dpi) in mice treated with 300 µg of mAb neutralizing IL-22 or isotype control mAb (None) given intraperitoneally the day of and 1 day after the primary infection. (<b>I</b>) Fungal growth (at 3 dpi) in mice treated intravaginally with rIL-22 or PBS (None) the day of and 1 and 2 days after the infection. Pooled data from two experiments (<i>n</i> = 6). *<i>P</i><0.05 and ***<i>P</i><0.001, treated <i>vs.</i> untreated mice. N.S., not significant.</p

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