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

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

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

<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

Microarray experiments on DCs challenged with fungi and single agonists.

<p>Microarray experiments on DCs challenged with fungi and single agonists.</p

Immune response (IR) genes activated by different cell wall components and <i>Saccharomyces cerevisiae</i> in DCs.

<p>Transcriptional analysis was performed on DCs after 4 hours of stimulation with Curdlan, Zymosan, Mannan and <i>S. cerevisiae</i> yeast or without any stimuli. (A) Venn diagram of upregulated DEGs. (B) IR differentially regulated genes (p≤0.05) commonly expressed among the four stimulation conditions. (C) IR differentially regulated genes (p≤0.05) commonly expressed among Zymosan and <i>S. cerevisiae</i> yeast stimulation condition and not affected upon single cell wall components stimulation. (D) Cytokine detection assays were performed for IL-1β, TNFα, IL-6 and IL-12p70 measurement on supernatants of DCs stimulated for 18 hours with Sc cells or zymosan; **p<0.01.</p

Immunoblot analyses of Dectin-1 and Syk upon fungal recognition.

<p>DCs were stimulated with live <i>S. cerevisiae</i> (Sc) cells, <i>Candida</i> (A) or different concentration of Curdlan (µg/ml, B, C) for the time indicated. Cell lysates were isolated and immunoblotted with the specific primary antibodies and reprobed with anti-β-tubulin antibody to control for the equal loading of cell lysates. Representative blots are shown. Quantitative analysis of protein expression is summarized in bar graphs, presented as mean integrated intensity of specific bands normalized to β-tubulin ± SD of 3–5 independent experiments.</p