Monte Carlo studies of two-dimensional polymer–solvent systems

<div><p>Allergic bronchopulmonary aspergillosis (ABPA) in asthma is a severe, life-affecting disease that potentially affects over 4.8 million people globally. In the UK, ABPA is predominantly caused by the fungus <i>Aspergillus fumigatus</i>. Phagocytosis is important in clearance of this fungus, and Early Endosome Antigen 1 (<i>EEA1</i>) has been demonstrated to be involved in phagocytosis of fungi. We sought to investigate the role of <i>EEA1</i> mutations and phagocytosis in ABPA. We used exome sequencing to identify variants in <i>EEA1</i> associated with ABPA. We then cultured monocyte-derived macrophages (MDMs) from 17 ABPA subjects with <i>A</i>. <i>fumigatus</i> conidia, and analyzed phagocytosis and phagolysosome acidification in relation to the presence of these variants. We found that variants in <i>EEA1</i> were associated with ABPA and with the rate of phagocytosis of <i>A</i>. <i>fumigatus</i> conidia and the acidification of phagolysosomes. MDMs from ABPA subjects carrying the disease associated genotype showed increased acidification and phagocytosis compared to those from ABPA subjects carrying the non-associated genotypes or healthy controls.The identification of ABPA-associated variants in EEA that have functional effects on MDM phagocytosis and phagolysosome acidification of <i>A</i>. <i>fumigatus</i> conidia revolutionizes our understanding of susceptibility to this disease, which may in future benefit patients by earlier identification or improved treatments. We suggest that the increased phagocytosis and acidification observed demonstrates an over-active MDM profile in these patients, resulting in an exaggerated cellular response to the presence of <i>A</i>. <i>fumigatus</i> in the airways.</p></div

MOESM1 of Genetic susceptibility to allergic bronchopulmonary aspergillosis in asthma: a genetic association study

Additional file 1. Supplementary tables and figures

Additional file 1 of Development of a novel mycobiome diagnostic for fungal infection

Additional file 2: Supplementary_figures.pd

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

<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

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>EEA1</i> mutations associated with ABPA.

<p><i>EEA1</i> mutations associated with ABPA.</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>EEA1</i> expression, phagocytosis and phagolysosome acidification after siRNA treatment.

<p>Mean and SEM shown. Groups compared by t-test. A) siRNA treatment. B) Phagocytosis. C) Phagolysosome acidification.</p