Fumigatus Extract Research Articles

Dual inhibition of airway inflammation and fibrosis by common β cytokine receptor blockade

BackgroundSevere asthmatics can present with eosinophilic type 2 (T2), neutrophilic or mixed inflammation that drives airway remodeling and exacerbations and represents a major treatment challenge. The common beta (βc) receptor signals for three cytokines GM-CSF, IL-5 and IL-3, that collectively mediate T2 and neutrophilic inflammation. ObjectivesTo determine the pathogenesis of βc receptor mediated inflammation and remodeling in severe asthma, and to investigate βc antagonism as a therapeutic strategy for mixed granulocytic airway disease. Methodsβc gene expression was analysed in bronchial biopsies from mild/moderate and severe asthmatics. House dust mite extract (HDM) and Aspergillus fumigatus extract (ASP) models were used to establish asthma-like pathology and airway remodeling in human βc transgenic mice. Lung tissue gene expression was analysed by RNA-sequencing. The monoclonal antibody CSL311 targeting the shared cytokine binding site of βc was used to block βc signaling. Resultsβc gene expression was increased in severe asthmatics. CSL311 potently reduced lung neutrophils, eosinophils and interstitial macrophages, and improved airway pathology and lung function in the acute steroid resistant HDM model. Chronic intranasal ASP exposure induced airway inflammation and fibrosis and impaired lung function that was inhibited by CSL311. CSL311 normalised the ASP-induced fibrosis-associated extracellular matrix gene expression network and strongly reduced signatures of cellular inflammation in the lung. Conclusionsβc cytokines drive steroid-resistant mixed myeloid cell airway inflammation and fibrosis. The anti-βc antibody CSL311 effectively inhibits mixed T2/neutrophilic inflammation and severe asthma-like pathology and reverses fibrosis gene signatures induced by exposure to commonly encountered environmental allergens.

Abnormal adenosine metabolism of neutrophils inhibits airway inflammation and remodeling in asthma model induced by Aspergillus fumigatus

BackgroundNeutrophils consume a large amount of energy when performing their functions. Compared with other white blood cells, neutrophils contain few mitochondria and mainly rely on glycolysis and gluconeogenesis to produce ATP. The inflammatory site is hypoxic and nutrient poor. Our aim is to study the role of abnormal adenosine metabolism of neutrophils in the asthmatic airway inflammation microenvironment.MethodIn this study, an asthma model was established by intratracheal instillation of Aspergillus fumigatus extract in Ecto-5'-Nucleotidase (CD73) gene–knockout and wild-type mice. Multiple analyses from bronchoalveolar lavage fluid (BALF) were used to determine the levels of cytokines and chemokines. Immunohistochemistry was used to detect subcutaneous fibrosis and inflammatory cell infiltration. Finally, adenosine 5’-(α, β-methylene) diphosphate (APCP), a CD73 inhibitor, was pumped subcutaneously before Aspergillus attack to observe the infiltration of inflammatory cells and subcutaneous fibrosis to clarify its therapeutic effect.ResultPAS staining showed that CD73 knockout inhibited pulmonary epithelial cell proliferation and bronchial fibrosis induced by Aspergillus extract. The genetic knockdownof CD73 significantly reduced the production of Th2 cytokines, interleukin (IL)-4, IL-6, IL-13, chemokine (C–C motif) ligand 5 (CCL5), eosinophil chemokine, neutrophil IL-17, and granulocyte colony-stimulating factor (G-CSF). In addition, exogenous adenosine supplementation increased airway inflammation. Finally, the CD73 inhibitor APCP was administered to reduce inflammation and subcutaneous fibrosis.ConclusionElevated adenosine metabolism plays an inflammatory role in asthma, and CD73 could be a potential therapeutic target for asthma.

Bimetallic Assembled Silver Nanoparticles Impregnated in Aspergillus fumigatus Extract Damage the Bacterial Membrane Surface and Release Cellular Contents

The bactericidal effects of nanomaterials play an essential role in cytoplasmic leakage, leading to bacterial cell death. In this study, silver nanoparticles (AgNPs) were synthesized using a fungal extract of Aspergillus fumigatus (A. fumigatus). The physicochemical properties of the bare and myco-synthesized AgNPs (MS-AgNPs) were examined by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, ultraviolet–visible (UV–vis) spectroscopy, scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM). XRD revealed the crystalline structure of the prepared NPs. The FTIR spectrum of the MS-AgNPs revealed the presence of the stretching vibrations of hydroxyl (−OH) and carbonyl groups (C=O). The UV results showed absorption from 450 nm to 590 nm, confirming the synthesis of the AgNPs. SEM and TEM showed rough cubic shapes (spheres), 20–60 nm in size, while EDX confirmed the presence of 60% Ag in the sample. The MS-AgNPs revealed the highest antibacterial activity against Staphylococcus aureus, with a zone of inhibition of 18.21 ± 2.1 mm, followed by Shigella dysenteriae and Salmonella typhi. The bimetallic-AgNPs played a vital role in cell membrane damage and the release of cellular contents, specifically nucleic acids and proteins. These results suggest that MS-AgNPs have promising antimicrobial capabilities and might be beneficial for an extensive array of biological applications.

Biosynthesis of Antimicrobial Silver Nanoparticles against Gram-Negative and Gram-positive Bacteria by the Entophytic Fungus Aspergillus Fumigatus

Multi drug resistance is increasing day by day due to misuse of antibiotics. Several potent metabolites are produced by fungi. Synthesis of silver nanoparticle (AgNPs) Due to its simple, harmless, time-saving, and cost-effective characteristics, it has acquired great popularity in recent years. A variety of analytical techniques were used to synthesize AgNPs from Aspergillus fumigatus extracts, including X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM). The effect of synthesis AgNPs and crude extract noted against different bacterial pathogens. Maximum antibacterial activity were noticed against tested bacteria by both fungal crude extract and (AgNPs) Maximum antibacterial activity of Ethyl acetate crude extract at 50µl concentration (12mg/1ml DMSO) showed (15mm) zone of inhibition against E.coli. While minimum antibacterial activity of Ethyl crude extract at 50µl concentration was observed against S.typhi (12mm). Highest antibacterial activity of Ethyl acetate crude extract at 100µl was noted against E.coli which showed (20mm) zone of inhibition. While (17mm) zone of inhibition was observed against S.typhi at 100µl concentration Ethyl acetate crude extract and AgNPs (25mm) zone of inhibition was observed against E.coli at 100µl concentration Ethyl acetate crude extract and AgNPs respectively. During UV-visible spectroscopy, surface Plasmon Resonance (SPR) was observed at 432 nm, which confirmed the synthesis of AgNPs. The SEM micrograph demonstrated the spherical shape of AgNPs. The results of FTIR research revealed that phenolic, carboxyl, and hydroxyl groups played a crucial role in the reduction of Ag+ ions into AgNPs, while amide linkage and amino acids stabilized AgNPs. AgNPs synthesized were XRD peaks that revealed phase purity, size, internal crystalline structure, and nature. In the pharmaceutical and medical fields, AgNPs synthesized from Aspergillus fumigatus extract could be of great importance. While, the combination of AgNPs and crude extract Aspergillus fumigatus enhances their antimicrobial effect which increase their importance in future studies Keywords: Fungal secondary metabolites, silver nanoparticles, crude extract, MDR pathogen bacteria , SEM, TEM, UV, FTIR, XRD.

Resveratrol attenuates inflammation and apoptosis through alleviating endoplasmic reticulum stress via Akt/mTOR pathway in fungus-induced allergic airways inflammation

BackgroundResveratrol has shown pleiotropic effects against inflammation and oxidative response. The present study aimed to investigate the effects and mechanisms of resveratrol on fungus-induced allergic airway inflammation. MethodsFemale BALB/c mice were injected intraperitoneally with Aspergillus fumigatus (Af) extract emulsified with aluminum on day 0 and 7 and intranasally challenged with Af extracts on day 14 and 15. Resveratrol or dexamethasone or a vehicle was injected intraperitoneally 1 h before each challenge. Mice were sacrificed for serum, bronchoalveolar lavage fluid (BALF), and lungs 24 h after the last challenge. The control group was administered with saline. BEAS-2B was used for the experiments in vitro that Af-exposed airway epithelial cells. ResultsResveratrol and dexamethasone attenuated the airway inflammation and eosinophilia, and reduced not only the production of IL-4, IL-5, and IL-13 in the BALF and lung tissues but also the mRNA levels of lung IL-6, TNF-α, and TGF-β induced by Af challenge (P < 0.05). Furthermore, Af-induced lung endoplasmic reticulum (ER) stress-related proteins PERK, CHOP, and GRP78 and the apoptosis markers including cleaved caspase-3 and cleaved caspase-7 were both suppressed significantly by resveratrol (P < 0.05). In vitro, activation of ER stress and the Akt/mTOR pathway in Af-exposed BEAS-2B cells were effectively ameliorated by resveratrol. Inhibition of the Akt/mTOR pathway using LY294002 suppressed the ER stress while ER stress inhibitor 4-PBA decreased the apoptosis in Af-exposed BEAS-2B cells. ConclusionsOur findings collectively revealed that resveratrol alleviated the Af-exposed allergic inflammation and apoptosis through inhibiting ER stress via Akt/mTOR pathway, exerting therapeutic effects on the fungus-induced allergic lung disorder.

Fibrin(ogen) Mechanistically Contributes to Atopic Dermatitis Pathogenesis and Allergic Sensitization

Adults with atopic dermatitis (AD) have an increased risk for thromboembolic events. While coagulation is critical for almost all healing responses, dysregulated clotting and fibrin(ogen) deposition lead to inflammation that exacerbates tissue damage and impairs tissue repair. While plasma fibrin(ogen) has been associated with established asthma, fibrin(ogen)'s mechanistic role in AD pathogenesis and allergic sensitization has not been investigated. Here we show that fibrinogen plays a critical role in a murine model atopic dermatitis pathogenesis by markedly attenuating disease severity, barrier dysfunction, and allergic sensitization.To determine if fibrinogen impacts AD pathogenesis, we used mice with complete fibrinogen deficiency (Fib -/-), partial deficiency (Fib +/-), and wildtype controls (Fib WT) in our established model of AD. This model uses repeated cutaneous allergen sensitization with heat-killed Aspergillus fumigatus (Asp) extract. Notably, Fib +/- mice produce approximately half as much fibrinogen as Fib WT mice. After each patch and at sacrifice each mouse underwent an objective disease severity assessment as well as transepidermal water loss (TEWL) measurements to assess skin barrier function. Our results show that complete and partial fibrinogen deficiency abrogated AD development. We found that Fib -/- and Fib +/- mice had markedly attenuated TEWL (P-value <0.0001) and disease severity (P-value <0.0001) compared with controls (Figure 1). In addition, we found that the TEWL and disease severity in Fib -/- and Fib +/- were comparable to unchallenged controls. There was also no difference in either outcome between Fib -/- and Fib +/- mice within the experimental group. Lastly, none of the Fib -/- and Fib +/- mice developed spontaneous bleeding.Next, to elucidate the impact of fibrinogen on allergic sensitization we measured plasma allergen specific immunoglobulin E (sIgE) to Asp in all mice in our AD model at sacrifice. We found that Fib -/- and Fib +/- mice had significantly lower Asp sIgE compared with controls (p-value <0.0001 and 0.0002, respectively; Figure 2). In addition, we found that the amount of sIgE to Asp produced was comparable between Fib -/- and Fib +/- mice as well as with unchallenged controls.Thus, our findings suggest that fibrin(ogen) plays a critical mechanistic role in driving AD development and allergic sensitization. While fibrinogen's role in established allergic disease has been shown, these novel findings suggest a critical role for fibrinogen in allergic disease pathogenesis which may allow for additional therapeutic targets to treat atopic dermatitis and other allergic diseases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Improved saccharification of Chlorella vulgaris biomass by fungal secreted enzymes for bioethanol production

Microalgal carbohydrates can serve as a potential source of bioethanol after saccharification and fermentation. This study aimed to assess the utility of fungal crude enzyme extracts isolated from Penicillium chrysogenum, Aspergillus tubingensis, Aspergillus fumigatus, and Thermomyces lanuginosus in the degradation of intact or lipid-extracted Chlorella vulgaris biomass. All fungal extracts were able to release higher concentrations of sugars from lipid-extracted biomass than intact biomass. Notably, A. fumigatus crude enzyme extract produced the most efficient saccharification reaction at 37 °C, which released 67% and 94% of C. vulgaris carbohydrates from intact and lipid-extracted biomass, respectively. Moreover, the A. fumigatus extract could provide complete saccharification from intact biomass at 50 °C. Proteomic characterisation of the A. fumigatus secretome identified enzymes that were up-regulated or uniquely expressed in the presence of C. vulgaris biomass and included cellulases, glucanases, hemicellulases, chitinases, pectinases, amylases, lipases and proteases. These are therefore candidates for the degradation of complex carbohydrates, lipids and proteins in the microalgae biomass. However, the expression of much of the secretome was independent of carbon source as found by a comparison between microalgae biomass and glucose, indicating constitutive expression of key enzymes. These findings should facilitate rational design of enzyme cocktails for bioethanol production from microalgae.

TSLP rather than IL-33 signaling drives intestinal mucosal mast accumulation and allergen-induced diarrhea in mice following epicutaneous sensitization to food allergen.

Abstract Background: A major route of sensitization to food allergen is through an impaired skin barrier. IL33 and TSLP have both been implicated in epicutaneous sensitization and development of food allergy. Method: We assessed the respective contributions of TSLP and IL33 to the development of food allergy in TSLP and IL33 receptor deficient mice following epicutaneous food allergen sensitization, where mice were exposed to thrice weekly skin patches of either saline, OVA or a combination of OVA and Aspergillus fumigatus extract (ASP). Results: ASP+OVA-patched but not OVA-patched mice developed an atopic dermatitis (AD)-like skin phenotype, while sensitization to OVA occurred in both OVA and OVA+ASP patched mice. OVA-specific IgE levels were significantly lower in OVA(±ASP)-patched TSLPR−/− mice compared to wild type mice and ST2−/− mice. Repeated intragastric challenges with 50mg of OVA, induced intestinal accumulation of mast cells but not ILC2s in OVA(±ASP)-patched WT and ST2−/− mice, and to a much lesser degree in TSLPR−/− mice. While OVA-induced mast cell degranulation assessed by measuring MCPT1 blood levels and the development of food allergy (diarrhea occurrences) was observed in almost all WT mice and two thirds of OVA+ASP patched ST2−/− mice, all TSLPR−/− mice were protected from developing food allergy and had significantly lower MCPT1 levels. Conclusion: Epicutaneous sensitization to food allergen and subsequent development of food allergy does not require atopic dermatitis skin lesions and is dependent on TSLP, suggesting that prophylactic targeting of TSLP may be useful in mitigating the development of food allergy early in life.

Antimicrobial activity and antioxidant properties of Brucea mollis Wall. ex Kurz: a medicinal plant of Northeast India

Brucea mollis Wall. ex Kurz is a potent medicinal plant occurring on hill slopes of Karbi Anglong district of Assam. The species is popularly known to have antimalarial activity in traditional medicine by a few ethnic communities of Northeastern regions. In the present study, an attempt was made to study the antimicrobial and antioxidant activity of B. mollis. The antibacterial and antifungal activity of plant extracts was screened by the disc diffusion method of Kirby-Bauer sensitivity test against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Serratia marcescens, Aspergillus fumigatus and Aspergillus niger. Among all the extracts (leaf ethanol, leaf methanol, callus ethanol, and callus methanol), the callus ethanol extracts was found to exhibit better antimicrobial activity against all the bacterial strains at 20 mg/ml concentration. Callus ethanol extracts exhibited maximum inhibition 17.33 ± 0.33 mm against B. subtilis. In case of antifungal activity, leaf methanol extracts exhibited 17.66 ± 0.33 mm against A. fumigatus and leaf ethanol extracts exhibited 18.33 ± 0.33 mm inhibition against A. niger at 20 mg/ml. The minimum inhibitory concentration (MIC) of the both leaves and callus against the bacterial and fungal strains exhibited lowest 0.156 mg/ml MIC against (A) niger from leaf methanol extract; against (B) subtilis, S. aureus and A. fumigatus from callus ethanol extract and against S. marcescens from callus methanol extract. Moreover, DPPH, Hydrogen peroxide (H2O2) radical scavenging activity, reducing power assay, total phenolic, and flavonoid content were investigated. Both the extracts showed antioxidant activity in a dose-dependent manner and ethanolic extract exhibited better antioxidant properties than methanolic extract.

Priming with Toll-like receptor 3 agonist or interferon-gamma enhances the therapeutic effects of human mesenchymal stem cells in a murine model of atopic dermatitis

BackgroundAtopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease. Great efforts have been recently made to treat AD using mesenchymal stem cells (MSCs), which have immunomodulatory functions. However, the immunomodulatory effects of MSCs need to be enhanced for clinical application in the treatment of AD.ObjectivesTo evaluate and characterise the therapeutic effects of human Wharton’s jelly-derived MSCs (WJ-MSCs) primed with the Toll-like receptor 3 agonist poly I:C or interferon-γ (IFN-γ) in a murine model of AD.MethodsMice were treated with Aspergillus fumigatus extract to induce AD and then subcutaneously injected with non-primed, poly I:C-primed or IFN-γ-primed WJ-MSCs. Clinical symptom scores, transepidermal water loss (TEWL), histological characteristics and cytokine levels were determined. Transcriptome profiling and pathway analyses of primed WJ-MSCs were conducted.ResultsThe clinical symptom score and TEWL in skin lesions were reduced in mice administered non-primed and primed WJ-MSCs. Epidermal thickness and inflammatory cell infiltration in skin lesions were reduced more in mice administered primed WJ-MSCs than in mice administered non-primed WJ-MSCs. Secretion of interleukin-17 was significantly reduced in skin draining lymph nodes of mice administered primed WJ-MSCs. Genomics and bioinformatics analyses demonstrated the enrichment of certain pathways specifically in WJ-MSCs primed with poly I:C or IFN-γ.ConclusionsPriming with poly I:C- or IFN-γ improved the therapeutic effects of WJ-MSCs in a murine model of AD. This study suggests that priming with poly I:C or IFN-γ enhances the immunomodulatory functions of WJ-MSCs and can be used as a novel therapeutic approach for AD.

Human IgE mAbs define variability in commercial Aspergillus extract allergen composition.

Sensitization to Aspergillus species is associated with allergic respiratory diseases. Allergen immunotherapy with nonstandardized Aspergillus extracts is commonly used as therapy in these patients. Unfortunately, no method exists to measure the relevant allergen protein content in diagnostic and therapeutic extracts. Thus, there is a critical need for Aspergillus extract standardization. We hypothesized that development of Aspergillus-specific human IgE mAbs would allow for the characterization of the relevant human allergenic epitopes among currently available commercial Aspergillus fumigatus extracts. Patients with allergic bronchopulmonary mycosis were recruited from Vanderbilt University Medical Center. IgE antibody-secreting B cells were grown and immortalized using human hybridoma techniques first described here. Twenty-six human Aspergillus-reactive IgE mAbs were used as capture and detection reagents to characterize the Aspergillus allergen content of commercial extracts. We found extreme variability in the specificity and quantity of their protein targets. Just 4 mAbs reacted with all available extracts, and only 1 of 4 extracts contained the major allergen Asp f 1. This degree of variability will almost certainly affect the efficacy of these reagents when used in diagnosis and treatment. Human IgE mAbs represent an innovative tool for the evaluation of relevant human allergenic epitopes, which may assist in future development and long-term standardization of mold extracts.

A synthetic glycosaminoglycan reduces sinonasal inflammation in a murine model of chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is characterized by sustained mucosal inflammation, impaired mucociliary clearance, loss of cilia and epithelial barrier breakdown, and tissue remodeling. Certain glycosaminoglycans inhibit various inflammatory mediators, suppress bacterial growth, and provide important functions in mucosal tissue repair and mucociliary clearance. Herein, we evaluated the effects of a synthetic glycosaminoglycan, GM-1111, on the clinical signs and inflammatory tissue changes associated with CRS in mice. CRS was generated by repeated intranasal applications of Aspergillus fumigatus (A. fumigatus) extracts over 4 weeks. Mice were then intranasally administered GM-1111 (600 μg per dose, 5 times a week) or vehicle (phosphate buffered saline, PBS) for an additional 4 weeks while still being given A. fumigatus extracts to maintain a chronic inflammatory environment with acute exacerbations. Clinical signs indicative of sinonasal inflammation were recorded throughout the study. After 9 weeks, whole blood and sinonasal tissues were harvested for hematological, histological, and biochemical examination. The clinical signs, white blood cell counts, tissue markers of sinonasal inflammation, and histological changes caused by A. fumigatus extract administration were compared to the healthy (PBS vehicle) and GM-1111-treated groups (n = 12 per treatment group). Compared to vehicle-treated animals, animals treated with GM-1111 demonstrated significant reductions in clinical signs (p<0.05), degenerative tissue changes, goblet cell hyperplasia, inflammatory cell infiltration (p<0.01), innate immunity- (tlr2, tlr4, myd88, il1b, tnfa, il6, and il12) and adaptive immunity-associated (ccl11, ccl24, ccl5, il4, il5, and il13) cytokine gene expression (p<0.05 to p<0.0001) in sinonasal tissues, and serum IgE levels (p<0.01). Our data suggest that GM-1111 significantly reduces local and systemic effects of CRS-associated sinonasal inflammation.

Autoclave-assisted green synthesis of silver nanoparticles using A. fumigatus mycelia extract and the evaluation of their physico-chemical properties and antibacterial activity

AbstractSilver nanoparticles (AgNPs) were synthesized usingAspergillus fumigatus(A. fumigatus) mycelia extract via the hydrothermal method. The main reducing and stabilizing groups and components ofA. fumigatusextract, such as amine, hydroxyl, amid, protein, enzymes, and cell saccharide compounds, were identified by Fourier transform infrared (FT-IR). Central composition design was used to plan the experiments, and response surface methodology was applied to evaluate of the effects of independent variables, including the amount of the prepared extract (5–7 ml) and heating time (10–20 min) at 121°C and 1.5 bar), on the particle size of the synthesized AgNPs, as manifested in broad emission peak (λmax). More stable and spherical monodispersed AgNPs, with mean particle size, polydispersity index (PDI) value, and maximum ζ potential value of 23 nm, 0.270, and +35.3 mV, respectively, were obtained at the optimal synthesis conditions using 7 ml ofA. fumigatusextract and heating time of 20 min. The synthesized AgNPs indicated high antibacterial activity against both Gram-positive and Gram-negative bacteria.

C3a receptor antagonism as a novel therapeutic target for chronic rhinosinusitis.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory disease with an unknown etiology. Recent studies have implicated the complement system as a potential modulator of disease immunopathology. We preformed proteomic pathway enrichment analysis of differentially increased proteins, and found an enrichment of complement cascade pathways in the nasal mucus of individuals with CRSwNP as compared to control subjects. Sinonasal mucus levels of C3 correlated with worse subjective disease severity, whereas no significant difference in systemic C3 levels could be determined in plasma samples. Given that human sinonasal epithelial cells were the predominate sinonasal source of C3 and C3a staining, we focused on their role in in-vitro studies. Baseline intracellular C3 levels were higher in CRSwNP cells, and following exposure to Aspergillus fumigatus (Af) extract they released significantly more C3 and C3a. Inhibition of C3aR signaling led to a decrease in Af-induced C3 and C3a release, both in-vitro and in-vivo. Finally, we found in-vivo that C3aR deficiency or inhibition significantly reduced inflammation and CRS development in a mouse model of Af induced CRS. These findings demonstrate that local sinonasal complement activation correlates with subjective disease severity, and that local C3aR antagonism significantly ameliorates Af induced CRS in a rodent model.

Etv5 Regulates IL-10 Production in Th Cells.

IL-10 is an immunoregulatory cytokine that has broad effects across the immune system. In Th cell subsets, Th2 cells produce considerable amounts of IL-10. The transcription factors that regulate IL-10 production in Th2 cells are still incompletely described. In this study, we demonstrate that the ETS family transcription factor ETS variant (Etv)5 regulates IL-10 production in Th2 cells. T cell-specific Etv5-deficient and littermate control mice demonstrated that IL-10 production and gene expression were significantly decreased in the absence of Etv5. In an Aspergillus fumigatus extract-induced inflammation model, IL-10-producing CD4+ T cells in bronchoalveolar lavage and lung were significantly decreased in mice that lacked Etv5 in T cells, compared with control mice. We showed that Etv5 directly binds to the Il10 locus conserved noncoding sequence 3 site and that it activates gene expression in a luciferase reporter assay and following retroviral transduction. Etv5 deficiency did not affect the expression of other transcription factors known to be important for expression of IL-10, including Jun family members, GATA3, E4BP4, and IFN regulatory factor 4. However, in the absence of Etv5, binding of these transcription factors to the Il10 locus was dramatically reduced. Ectopic Etv5 expression in Th2 cells that lack Etv5 restored IL-10 production and the binding of IL-10-inducing transcription factors including E4BP4, IFN regulatory factor 4, and GATA3. Taken together, we conclude that Etv5 plays a crucial role in regulating IL-10 production in Th2 cells by facilitating the binding of IL-10-inducing transcription factors at the Il10 locus.

Relative Allergen Content of Commercial Aspergillus Fumigatus Extracts Determined by Human Monoclonal IgE

Relative Allergen Content of Commercial Aspergillus Fumigatus Extracts Determined by Human Monoclonal IgE

Etv5 promotes IL-10 production in Th2 cells

Abstract The anti-inflammatory cytokine, IL-10, suppresses inflammatory responses at mucosal surfaces. Among T helper cell subsets, Th2 cells produce the highest concentrations. However, the transcription factors that regulate Il10 gene expression have not been completely defined. Etv5 is a member of the ETS transcription factor family that promotes production of IL-17 in Th17 cells and IL-9 in Th9 cells. To define the effects of Etv5 deficiency on cytokine expression in Th2 cells, T cells from CD4 specific Etv5 knockout (Etv5fl/flCD4-Cre+, termed Etv5 KO below) and littermate control (Etv5fl/fl CD4-Cre-, termed WT below) mice were differentiated under Th2 conditions. Etv5 was required for maximal IL-10 production and modestly repressed the Th2 cytokines IL-4, IL-5 and IL-13. In mice sensitized with Aspergilus fumigatus extract, IL-10 production in both BAL and lung was significantly reduced in Etv5 KO mice, consistent with in vitro data. Using ChIP and reporter assays, we showed that Etv5 directly binds to the Il10 locus, specifically at the CNS3 region. Etv5 promotes IL-10 production by recruiting IL-10 inducing transcription factors including Jun family members, IRF4 and E4BP4, and the binding of these factors to the Il10 locus, but not the expression of these factors, was decreased in the absence of Etv5. Ectopic GATA3 and E4BP4 expression recovers IL-10 production to wild type amounts, suggesting that Etv5 enhances the efficiency of function of these factors. Importantly, ectopic Etv5 expression in Etv5 KO Th2 cells restored binding of the factors to the Il10 locus. Thus, Etv5 promotes IL-10 production by enhancing the recruitment of IL-10 inducing transcription factors.

Characterisation of Alternaria alternata manganese-dependent superoxide dismutase, a cross-reactive allergen homologue to Asp f 6

It is well known that Alternaria alternata presents a significant level of allergenic cross-reactivity with several other phylogenetically related and non-related allergenic moulds. To improve the molecular diagnosis, the identification and characterisation of all clinically relevant allergens, including both species-specific and cross-reacting proteins, is required. In this study we report the molecular and immunological characterisation of the A. alternata manganese-dependent superoxide dismutase (Alt a MnSOD) and its cross-reactivity with Asp f 6, a diagnostic marker allergen in allergic bronchopulmonary aspergillosis (ABPA). The cDNA coding for Alt a MnSOD sequence was isolated by RACE and PCR. Alt a MnSOD is a protein of 191 amino acids that presented significant homology and potential cross-reactive epitopes with Asp f 6. The recombinant protein was produced in Escherichia coli and the immunoreactivity was evaluated in patient sera. Immunoblotting analyses showed that seven of sixty-one A. alternata-sensitised patient sera and two ABPA patient sera reacted with the recombinant Alt a MnSOD. The native counterpart contained in both A. alternata and Aspergillus fumigatus extracts inhibited IgE binding to the recombinant molecule. The allergen was named Alt a 14 by the official Allergen nomenclature subcommittee. Thus, Alt a 14 is a relevant allergen in A. alternata sensitisation that may be used to improve diagnostic procedures. Evidence of cross-reactivity between Asp f 6 and Alt a 14-recognition by ABPA patient sera suggest the existence of an Alt a 14-mediated mechanism that, similar to Asp f 6, may be related to the pathogenesis of ABPA.

SERPINB3/B4 Contributes to Early Inflammation and Barrier Dysfunction in an Experimental Murine Model of Atopic Dermatitis

Serine proteases are critical for epidermal barrier homeostasis and their aberrant expression and/or activity is associated with chronic skin diseases. Elevated levels of the serine protease inhibitors SERPINB3 and SERPINB4 are seen in patients with atopic dermatitis and psoriasis. However their mechanistic role in the skin is unknown. To evaluate the contribution of Serpinb3a (mouse homolog of SERPINB3 and SERPINB4) in atopic dermatitis, we examined the effect of topical Aspergillus fumigatus extract exposure in wild-type and Serpinb3a null mice on transepidermal water loss (TEWL), sensitization and inflammation. Allergen exposure induced Serpinb3a expression in the skin, along with increased TEWL, epidermal thickness, and skin inflammation, all of which were attenuated in the absence of Serpinb3a. Attenuated TEWL correlated with decreased expression of the pro-inflammatory marker S100A8. Silencing of SERPINB3/B4 in human keratinocytes decreased S100A8 expression supporting a role for SERPINB3/B4 in initiation of the acute inflammatory response. RNA-Seq analysis following allergen exposure identified a network of pro-inflammatory genes induced in the wild type mice that was absent in the Serpinb3a null mice. In conclusion, Serpinb3a deficiency attenuates barrier dysfunction and the early inflammatory response following cutaneous allergen exposure, supporting a role for Serpinb3a (mice) and SERPINB3/B4 (humans) early in atopic dermatitis.

Aspergillus Fumigatus Extract Suppresses the mRNA Expression of the IP‐10 in Human Bronchial Epithelial Cells

Objectives: Immunologic reactions to antigens released by Aspergillus fumigatus can cause infections such as allergic bronchopulmonary aspergillosis (ABPA). The mucosal surface of the respiratory tract provides host defense mechanism. The aim of this study was to investigate whether respiratory epithelial cells recognize A. fumigatus extract (AE) and initiate an immune response. We report inhibition of Th1 biased epithelial inflammation by an Aspergillus extract. Methods: BEAS-2B epithelial cells were cultured in a 37°C incubator in media supplemented with 5% FBS. Cells were treated in duplicate with AE for 1 hour and were stimulated with the TLR3 activator dsRNA for 6 hours. RNA was isolated and was reverse transcribed to cDNA. Realtime polymerase chain reaction analysis was performed in the presence of specific primers and fluorescently labeled probes. Results: IFN-β and dsRNA both induced IP-10 mRNA expression in BEAS-2B cells (IFN-β 1081-fold; n = 3; P &lt; .001) (dsRNA 3044-fold; n = 3; P &lt; .001). When BEAS-2B cells were pretreated with AE for 1 hour and then stimulated with IFN-β or dsRNA for 6 hours, induction of IP-10 mRNA was suppressed relative to levels produced by IFN-β and dsRNA alone. Suppressive effects increased with increasing AE concentration. IP-10 protein was induced by IFN-β (control [undetectable] vs IFN-β treated [16.6 ± 9.5 pg/mL] at 6 hours) and by dsRNA (control [undetectable] vs dsRNA [25.3 ± 6.9 pg/mL], at 6 hours, n = 3). We observed significant suppression of IP-10 protein when cells were treated with 1:320 W/V AE concentration (16.6 ± 9.5 pg/mL control vs 2.4 ± 0.25 pg/mL AE treated: P = .005, n = 3). Conclusions: We found that AE inhibits the IP-10 mRNA and protein secretion in bronchial epithelial cells.