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1.
Adv Exp Med Biol ; 1204: 1-30, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32152941

RESUMO

Most fungal species are harmless to humans and some exist as commensals on mucocutaneous surfaces. Yet many fungi are opportunistic pathogens, causing life-threatening invasive infections when the immune system becomes compromised. The fungal cell wall contains conserved pathogen-associated molecular patterns (PAMPs), which allow the immune system to distinguish between self (endogenous molecular patterns) and foreign material. Sensing of invasive microbial pathogens is achieved through recognition of PAMPs by pattern recognition receptors (PRRs). One of the predominant fungal-sensing PRRs is the C-type lectin receptor (CLR) family. These receptors bind to structures present on the fungal cell wall, eliciting various innate immune responses as well as shaping adaptive immunity. In this chapter, we specifically focus on the four major human fungal pathogens, Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans and Pneumocystis jirovecii, reviewing our current understanding of the CLRs that are involved in their recognition and protection of the host.


Assuntos
Fungos/imunologia , Imunidade Inata/imunologia , Lectinas Tipo C/imunologia , Aspergillus fumigatus/imunologia , Candida albicans/imunologia , Cryptococcus neoformans/imunologia , Humanos , Padrões Moleculares Associados a Patógenos/imunologia , Pneumocystis carinii/imunologia
2.
Infect Immun ; 88(2)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31767773

RESUMO

Aspergillus fumigatus is a ubiquitous fungal pathogen capable of causing multiple pulmonary diseases, including invasive aspergillosis, chronic necrotizing aspergillosis, fungal colonization, and allergic bronchopulmonary aspergillosis. Intact mucociliary barrier function and early airway neutrophil responses are critical for clearing fungal conidia from the host airways prior to establishing disease. Following inhalation, Aspergillus conidia deposit in the small airways, where they are likely to make their initial host encounter with epithelial cells. Challenges in airway infection models have limited the ability to explore early steps in the interactions between A. fumigatus and the human airway epithelium. Here, we use inverted air-liquid interface cultures to demonstrate that the human airway epithelium responds to apical stimulation by A. fumigatus to promote the transepithelial migration of neutrophils from the basolateral membrane surface to the apical airway surface. Promoting epithelial transmigration with Aspergillus required prolonged exposure with live resting conidia. Swollen conidia did not expedite epithelial transmigration. Using A. fumigatus strains containing deletions of genes for cell wall components, we identified that deletion of the hydrophobic rodlet layer or dihydroxynaphthalene-melanin in the conidial cell wall amplified the epithelial transmigration of neutrophils, using primary human airway epithelium. Ultimately, we show that an as-yet-unidentified nonsecreted cell wall protein is required to promote the early epithelial transmigration of human neutrophils into the airspace in response to A. fumigatus Together, these data provide critical insight into the initial epithelial host response to Aspergillus.


Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/imunologia , Parede Celular/imunologia , Células Epiteliais/imunologia , Neutrófilos/imunologia , Aspergilose/microbiologia , Linhagem Celular Tumoral , Células Epiteliais/microbiologia , Humanos , Pulmão/imunologia , Pulmão/microbiologia , Melaninas/imunologia , Naftóis/imunologia , Esporos Fúngicos/imunologia
3.
Zhonghua Nei Ke Za Zhi ; 58(11): 826-828, 2019 Nov 01.
Artigo em Chinês | MEDLINE | ID: mdl-31665859

RESUMO

This study aims to explore the diagnostic value of specific immunoglobulin E (sIgE) and specific immunoglobulin G (sIgG) of Aspergillus fumigatus in the diagnosis of allergic broncho-pulmonary aspergillosis (ABPA) and severe asthma with fungal sensitization (SAFS). A total of 17 ABPA patients and 14 SAFS patients were enrolled. The levels of sIgG [2 294.00 (1 527.00, 14 170.00) U/ml vs. 972.60 (650.90, 1 792.00) U/ml] and sIgE [8.77 (1.64, 16.85) kU/L vs. 1.04 (0.70, 2.05) kU/L] in ABPA patients were significantly higher than those in SAFS patients (P<0.05). Aspergillus fumigatus sIgG was strongly correlated with Aspergillus fumigatus sIgE (r(s)=0.797, P<0.001) in ABPA patients. When combined with Aspergillus fumigatus sIgG (>1 000.00 U/mL) and Aspergillus fumigatus sIgE (>1.00 kU/L), the sensitivity was 82.3% and specificity 78.6% for the differential diagnosis of ABPA and SAFS. It demonstrates the diagnostic value of Aspergillus fumigatus sIgG and sIgE.


Assuntos
Aspergilose Broncopulmonar Alérgica/diagnóstico , Aspergillus fumigatus/isolamento & purificação , Asma/complicações , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Anticorpos Antifúngicos/imunologia , Aspergilose Broncopulmonar Alérgica/sangue , Aspergilose Broncopulmonar Alérgica/imunologia , Aspergilose Broncopulmonar Alérgica/microbiologia , Aspergillus fumigatus/imunologia , Asma/sangue , Asma/diagnóstico , Humanos , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Índice de Gravidade de Doença
4.
Immunohorizons ; 3(8): 368-377, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31603851

RESUMO

The hallmark features of allergic asthma are type 2 (eosinophilic) inflammation and airways hyperresponsiveness (AHR). Although these features often comanifest in mouse lungs in vivo, we demonstrate in this study that the serine protease Alp1 from the ubiquitous mold and allergen, Aspergillus fumigatus, can induce AHR in mice unable to generate eosinophilic inflammation. Strikingly, Alp1 induced AHR in mice devoid of protease-activated receptor 2/F2 trypsin-like receptor 1 (PAR2/F2RL1), a receptor expressed in lung epithelium that is critical for allergic responses to protease-containing allergens. Instead, using precision-cut lung slices and human airway smooth muscle cells, we demonstrate that Alp1 directly increased contractile force. Taken together, these findings suggest that Alp1 induces bronchoconstriction through mechanisms that are largely independent of allergic inflammation and point to a new target for direct intervention of fungal-associated asthma.


Assuntos
Aspergillus fumigatus/imunologia , Asma/imunologia , Asma/microbiologia , Proteínas Fúngicas/imunologia , Serina Endopeptidases/imunologia , Alérgenos/imunologia , Animais , Aspergillus fumigatus/enzimologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Broncoconstrição/efeitos dos fármacos , Broncoconstrição/imunologia , Células Cultivadas , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Humanos , Inflamação/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/imunologia , Receptor PAR-2/genética , Receptor PAR-2/imunologia
5.
J Immunol Res ; 2019: 9164202, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482100

RESUMO

Collectin-12 (collectin placenta 1, CL-P1, or CL-12) is a newly identified pattern recognition molecule of the innate immune system. Recent evidences show that CL-12 plays important roles not only in innate immune protection against certain clinically important pathogens but also in scavenging of host molecules, leukocyte recruitment, and cancer metastasis. Furthermore, CL-12 has been shown to be associated with the pathogenesis of human diseases such as Alzheimer's disease and multiple sclerosis lesion development. Therefore, the functional consequence of CL-12 remains intriguing and awaits further elucidation. However, available protocols for the purification of recombinant CL-12 with high purity are laborious and inefficient and hamper further functional studies. Here, we report a simple, rapid, and efficient solution to obtain biologically active CL-12 with high purity. We established stable transfected Flp-In™-CHO cells expressing the recombinant CL-12 extracellular domain in high amounts. Recombinant CL-12 was purified from cell culture supernatants using a 3-step rapid purification procedure utilizing disposable affinity and ion exchange minicolumns. Purified recombinant CL-12 adopted an oligomeric structure with monomers, dimers, and trimers and retained its binding capacity towards the A. fumigatus strain that has been described before. Furthermore, we demonstrated the opsonic properties towards eight clinical isolates of A. fumigatus strains and diverse clinically important fungal pathogens. Purified recombinant CL-12 revealed a differential binding capacity towards selected fungal pathogens in vitro. In conclusion, we demonstrate a rapid and efficient purification solution for further biochemical and functional characterization of CL-12 and reveal opsonic properties of CL-12 towards diverse fungal pathogens.


Assuntos
Aspergillus fumigatus/imunologia , Colectinas/isolamento & purificação , Proteínas Opsonizantes/isolamento & purificação , Receptores Depuradores/isolamento & purificação , Animais , Aspergillus fumigatus/metabolismo , Células CHO , Colectinas/genética , Colectinas/metabolismo , Colectinas/farmacologia , Cricetulus , Humanos , Proteínas Opsonizantes/genética , Proteínas Opsonizantes/metabolismo , Proteínas Opsonizantes/farmacologia , Receptores Depuradores/genética , Receptores Depuradores/metabolismo
6.
Artigo em Inglês | MEDLINE | ID: mdl-31380292

RESUMO

Aspergillus fumigatus and A. flavus are the fungal pathogens responsible for most cases of invasive aspergillosis (IA). Early detection of the circulating antigen galactomannan (GM) in serum allows the prompt application of effective antifungal therapy, thus improving the survival rate of IA patients. However, the use of monoclonal antibodies (mAbs) for the diagnosis of IA is often associated with false positives due to cross-reaction with bacterial polysaccharides. More specific antibodies are therefore needed. Here we describe the characterization of the Aspergillus-specific mAb AP3 (IgG1κ), including the precise identification of its corresponding antigen. The antibody was generated using A. parasiticus cell wall fragments and was shown to bind several Aspergillus species. Immunofluorescence microscopy revealed that AP3 binds a cell wall antigen, but immunoprecipitation and enzyme-linked immunosorbent assays showed that the antigen is also secreted into the culture medium. The inability of AP3 to bind the A. fumigatus galactofuranose (Galf )-deficient mutant ΔglfA confirmed that Galf residues are part of the epitope. Several lines of evidence strongly indicated that AP3 recognizes the Galf residues of O-linked glycans on Aspergillus proteins. Glycoarray analysis revealed that AP3 recognizes oligo-[ß-D-Galf-1,5] sequences containing four or more residues with longer chains more efficiently. We also showed that AP3 captures GM in serum, suggesting it may be useful as a diagnostic tool for patients with IA.


Assuntos
Anticorpos Antifúngicos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Fungos/imunologia , Aspergilose/imunologia , Aspergillus/imunologia , Mananas/imunologia , Animais , Antígenos de Fungos/genética , Aspergillus/genética , Aspergillus flavus/genética , Aspergillus flavus/imunologia , Aspergillus fumigatus/imunologia , Parede Celular/química , Reações Cruzadas , Modelos Animais de Doenças , Epitopos/isolamento & purificação , Feminino , Testes Imunológicos , Mananas/genética , Camundongos , Camundongos Endogâmicos BALB C , Polissacarídeos Bacterianos/imunologia , Proteínas Recombinantes
7.
J Med Microbiol ; 68(9): 1341-1352, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31355743

RESUMO

Introduction. Timely detection of invasive aspergillosis (IA) caused by fungal pathogens, i.e. Aspergillus fumigatus and Aspergillus flavus, in immunocompromised patients is crucial in preventing high mortality.Aim. To develop a simple immunoassay for the detection of galactomannan (GM), an IA biomarker.Methodology. GM from A. fumigatus and A. flavus clinical strains was purified and characterized by X-ray diffraction, IR spectroscopy and 13C/1H nuclear magnetic resonance (NMR) for polyclonal antibody (pAb) production in rabbits. An enzyme-linked immunosorbent assay (ELISA) was standardized using concanavalin A to capture Aspergillus GM and pAbs to detect it. Gold nanoparticles (AuNPs) were synthesized and conjugated to pAbs for the development of a dot-blot immunoassay. The developed dot-blot was evaluated with 109 clinical serum and bronchoalveolar lavage samples.Results. Spectroscopy studies characterized the d-galactofuranosyl groups of GM responsible for the immune response and generation of pAbs. The ELISA employing pAbs showed a sensitivity of 1 ng ml-1 for Aspergillus GM. Furthermore, a sensitive, visual, rapid dot-blot assay developed by the conjugation of pAbs to AuNPs (~24±5 nm size, -36±2 mV zeta potential) had a detection limit of 1 pg ml-1 in serum. The pAbs interacted with Aspergillus spp. but did not cross-react with other fungal pathogen genera such as Penicillium and Candida. Evaluation of the dot-blot with 109 clinical samples showed high sensitivity (80 %) and specificity (93.2 %), with an overall assay accuracy of 89%.Conclusion. The developed nano-gold immunodiagnostic assay has immense potential for practical use in rapid, specific and sensitive on-site diagnosis of IA, even under resource-limited settings.


Assuntos
Aspergilose/diagnóstico , Ouro/química , Testes Imunológicos/métodos , Nanopartículas Metálicas/química , Animais , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/imunologia , Antígenos de Fungos/sangue , Antígenos de Fungos/imunologia , Aspergillus/imunologia , Aspergillus/isolamento & purificação , Aspergillus flavus/imunologia , Aspergillus flavus/isolamento & purificação , Aspergillus fumigatus/imunologia , Aspergillus fumigatus/isolamento & purificação , Humanos , Immunoblotting , Mananas/análise , Mananas/imunologia , Testes Imediatos , Coelhos , Sensibilidade e Especificidade
8.
mSphere ; 4(3)2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-31217305

RESUMO

Aspergillus fumigatus is a filamentous fungus which causes invasive pulmonary aspergillosis in immunocompromised individuals. In fungi, cell signaling and cell wall plasticity are crucial for maintaining physiologic processes. In this context, Msb2 is an important signaling mucin responsible for activation of a variety of mitogen-activated protein kinase (MAPK)-dependent signaling pathways that regulate cell growth in several organisms, such as the cell wall integrity (CWI) pathway. Here, we aimed to characterize the MSB2 homologue in A. fumigatus Our results showed that MsbA plays a role in the vegetative and reproductive development of the fungus, in stress adaptation, and in resistance to antifungal drugs by modulating the CWI pathway gene expression. Importantly, cell wall composition is also responsible for activation of diverse receptors of the host immune system, thus leading to a proper immune response. In a model of acute Aspergillus pulmonary infection, results demonstrate that the ΔmsbA mutant strain induced less inflammation with diminished cell influx into the lungs and lower cytokine production, culminating in increased lethality rate. These results characterize for the first time the role of the signaling mucin MsbA in the pathogen A. fumigatus, as a core sensor for cell wall morphogenesis and an important regulator of virulence.IMPORTANCE Aspergillus fumigatus is an opportunistic fungus with great medical importance. During infection, Aspergillus grows, forming hyphae that colonize the lung tissue and invade and spread over the mammal host, resulting in high mortality rates. The knowledge of the mechanisms responsible for regulation of fungal growth and virulence comprises an important point to better understand fungal physiology and host-pathogen interactions. Msb2 is a mucin that acts as a sensor and an upstream regulator of the MAPK pathway responsible for fungal development in Candida albicans and Aspergillus nidulans Here, we show the role of the signaling mucin MsbA in the pathogen A. fumigatus, as a core sensor for cell wall morphogenesis, fungal growth, and virulence. Moreover, we show that cell wall composition, controlled by MsbA, is detrimental for fungal recognition and clearance by immune cells. Our findings are important for the understanding of how fungal sensors modulate cell physiology.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Aspergillus fumigatus/genética , Proteínas de Bactérias/genética , Parede Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Mucinas/genética , Transportadores de Cassetes de Ligação de ATP/imunologia , Animais , Aspergilose/imunologia , Aspergillus fumigatus/imunologia , Proteínas de Bactérias/imunologia , Feminino , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucinas/imunologia , Transdução de Sinais , Virulência
9.
Artigo em Inglês | MEDLINE | ID: mdl-31192161

RESUMO

Dendritic cells (DCs) are antigen presenting cells which serve as a passage between the innate and the acquired immunity. Aspergillosis is a major lethal condition in immunocompromised patients caused by the adaptable saprophytic fungus Aspergillus fumigatus. The healthy human immune system is capable to ward off A. fumigatus infections however immune-deficient patients are highly vulnerable to invasive aspergillosis. A. fumigatus can persist during infection due to its ability to survive the immune response of human DCs. Therefore, the study of the metabolism specific to the context of infection may allow us to gain insight into the adaptation strategies of both the pathogen and the immune cells. We established a metabolic model of A. fumigatus central metabolism during infection of DCs and calculated the metabolic pathway (elementary modes; EMs). Transcriptome data were used to identify pathways activated when A. fumigatus is challenged with DCs. In particular, amino acid metabolic pathways, alternative carbon metabolic pathways and stress regulating enzymes were found to be active. Metabolic flux modeling identified further active enzymes such as alcohol dehydrogenase, inositol oxygenase and GTP cyclohydrolase participating in different stress responses in A. fumigatus. These were further validated by qRT-PCR from RNA extracted under these different conditions. For DCs, we outlined the activation of metabolic pathways in response to the confrontation with A. fumigatus. We found the fatty acid metabolism plays a crucial role, along with other metabolic changes. The gene expression data and their analysis illuminate additional regulatory pathways activated in the DCs apart from interleukin regulation. In particular, Toll-like receptor signaling, NOD-like receptor signaling and RIG-I-like receptor signaling were active pathways. Moreover, we identified subnetworks and several novel key regulators such as UBC, EGFR, and CUL3 of DCs to be activated in response to A. fumigatus. In conclusion, we analyze the metabolic and regulatory responses of A. fumigatus and DCs when confronted with each other.


Assuntos
Aspergillus fumigatus/imunologia , Aspergillus fumigatus/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Citocinas/metabolismo , Expressão Gênica , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Interleucinas/metabolismo , Redes e Vias Metabólicas , Proteínas NLR/metabolismo , Transdução de Sinais , Receptores Toll-Like/metabolismo , Transcriptoma
10.
J Med Microbiol ; 68(6): 924-929, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31090534

RESUMO

PURPOSE: The diagnosis of aspergillosis in cystic fibrosis (CF) patients remains a challenge due to overlapping features of both diseases. This is further complicated by inconsistent antibody reactivity to the currently used crude antigen, which has led a more focused evaluation of the efficacy of IgE response to a number of pure Aspergillus fumigatus recombinant proteins in patients with CF and asthma. In this study, we dissected the IgE and IgG responses to multiple A. fumigatus recombinant antigens in CF patients with different Aspergillus diseases. METHODOLOGY: Serum IgE and IgG antibodies were measured in 12 CF patients with allergic bronchopulmonary aspergillosis (ABPA), 12 with Aspergillus sensitization (AS) and 12 with Aspergillus bronchitis (AB) against recombinant antigens Asp f1, f2, f3, f4 and f6. RESULTS: The ABPA group showed significantly greater IgE reactivity to Asp f1, f2, f3 and f4 compared to patients with AS. Patients with AB expressed higher IgG positivity to Asp f1 and Asp f2 compared with those with ABPA. There were very low IgE antibody levels against all recombinant antigens in patients with AS. Aspf1 IgG reactivity in ABPA patients correlated with positive culture. CONCLUSION: The use of multiple recombinant antigens may improve the diagnostic accuracy in CF complicated with ABPA or AB. Asp f1 reactivity may relate to the presence of actively growing Aspergillus spp., which might be a useful marker for guiding antifungal therapy in ABPA.


Assuntos
Anticorpos Antifúngicos/sangue , Antígenos de Fungos/imunologia , Aspergilose Broncopulmonar Alérgica/diagnóstico , Aspergilose/diagnóstico , Aspergillus fumigatus/imunologia , Fibrose Cística/complicações , Adolescente , Adulto , Aspergilose/complicações , Aspergilose/microbiologia , Aspergilose Broncopulmonar Alérgica/complicações , Aspergilose Broncopulmonar Alérgica/microbiologia , Criança , Pré-Escolar , Feminino , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes , Estudos Retrospectivos , Adulto Jovem
11.
PLoS One ; 14(4): e0215535, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31022215

RESUMO

ß-(1→3)-D-Glucan is an essential component of the fungal cell wall. Mouse monoclonal antibodies (mAbs) against synthetic nona-ß-(1→3)-D-glucoside conjugated with bovine serum albumin (BSA) were generated using hybridoma technology. The affinity constants of two selected mAbs, 3G11 and 5H5, measured by a surface plasmon resonance biosensor assay using biotinylated nona-ß-(1→3)-D-glucan as the ligand, were approximately 11 nM and 1.9 nM, respectively. The glycoarray, which included a series of synthetic oligosaccharide derivatives representing ß-glucans with different lengths of oligo-ß-(1→3)-D-glucoside chains, demonstrated that linear tri-, penta- and nonaglucoside, as well as a ß-(1→6)-branched octasaccharide, were recognized by mAb 5H5. By contrast, only linear oligo-ß-(1→3)-D-glucoside chains that were not shorter than pentaglucosides (but not the branched octaglucoside) were ligands for mAb 3G11. Immunolabelling indicated that 3G11 and 5H5 interact with both yeasts and filamentous fungi, including species from Aspergillus, Candida, Penicillium genera and Saccharomyces cerevisiae, but not bacteria. Both mAbs could inhibit the germination of Aspergillus fumigatus conidia during the initial hours and demonstrated synergy with the antifungal fluconazole in killing C. albicans in vitro. In addition, mAbs 3G11 and 5H5 demonstrated protective activity in in vivo experiments, suggesting that these ß-glucan-specific mAbs could be useful in combinatorial antifungal therapy.


Assuntos
Anticorpos Monoclonais/farmacologia , Antifúngicos/farmacologia , Antígenos de Fungos/imunologia , Candidíase/tratamento farmacológico , beta-Glucanas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antifúngicos/imunologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/imunologia , Candida albicans/efeitos dos fármacos , Candida albicans/imunologia , Candidíase/imunologia , Candidíase/microbiologia , Parede Celular/efeitos dos fármacos , Parede Celular/imunologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Fluconazol/farmacologia , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Resultado do Tratamento
12.
Laryngoscope ; 129(10): 2230-2235, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30973971

RESUMO

OBJECTIVE: In the pathophysiology of chronic rhinosinusitis with nasal polyps (CRSwNP), Aspergillus fumigatus (A. fumigatus) can upregulate IL-33 from human sinonasal epithelial cells (SNECs), which then activates innate lymphoid cells causing release of IL-13, an important driver of allergic inflammation. However, the mechanism by which A. fumigatus mediates the induction of IL-33 expression remains to be elucidated. The objectives of this study were to determine the specific fungal component(s) and the receptor responsible for mediating the A. fumigatus induced increase in IL-33 expression in SNECs from patients with CRSwNP. METHODS: SNECs from CRSwNP patients were cultured and stimulated with various fungal components in the absence or presence of 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride, an irreversible serine protease inhibitor, or GB83, a reversible protease activated receptor 2 (PAR2) inhibitor. IL-33 expression was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). PAR2 expression was examined in inflamed mucosa from nonatopic control and CRSwNP patients. RESULTS: Elevation of IL-33 expression in primary SNECs was found in response to fungal protease but not fungal cell wall components. PAR2 expression was elevated in inflamed mucosa from CRSwNP patients in comparison to controls. The A. fumigatus fungal protease-mediated elevation in IL-33 expression by human SNECs was serine protease- and PAR2-dependent. CONCLUSION: These data suggest that serine protease activity of A. fumigatus is capable of inducing IL-33 expression in CRSwNP SNECs via PAR2, a potential therapeutic target in the treatment of CRSwNP. LEVEL OF EVIDENCE: NA Laryngoscope, 129:2230-2235, 2019.


Assuntos
Aspergillus fumigatus/imunologia , Interleucina-33/metabolismo , Pólipos Nasais/microbiologia , Receptores Acoplados a Proteínas-G/imunologia , Rinite/microbiologia , Sinusite/microbiologia , Células Cultivadas , Doença Crônica , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Feminino , Humanos , Imunidade Inata , Linfócitos/imunologia , Linfócitos/microbiologia , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/imunologia , Mucosa Nasal/microbiologia , Pólipos Nasais/imunologia , Seios Paranasais/imunologia , Seios Paranasais/microbiologia , Rinite/imunologia , Sinusite/imunologia
13.
Med Mycol J ; 60(1): 11-16, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30814465

RESUMO

Aspergillus fumigatus is a critical human fungal pathogen that infects the host via inhalation of airborne conidia. These conidia then germinate to form filamentous hyphae, which secrete various elements to survive in the host lung.Elements such as proteins secreted by A. fumigatus can act as virulence factors in host tissues. Among secreted proteins, we were interested in the thaumatin-like proteins of A. fumigatus. In our analysis of the function of thaumatin-like proteins, we found that, like CalA and CalB, CalC has a secreted form. Originally, CalC was predicted to be a GPI-anchored protein, as documented in the Aspergillus Genome Database. Here, we report on a novel secreted form of CalC. Furthermore, we established two novel hybridomas, C103 and C306, which recognized CalC. Monoclonal antibodies produced by these hybridomas responded to recombinant CalC produced by the mammalian cell line HEK293T and to the supernatant of cultured A. fumigatus.Taken together, our data suggest that calC can be spliced to give rise to a novel secretory form of CalC, which is present in the supernatant of cultured A. fumigatus. The hybridomas that we established will be helpful in understanding the biological role of A. fumigatus CalC.


Assuntos
Anticorpos Monoclonais , Aspergillus fumigatus/genética , Aspergillus fumigatus/imunologia , Proteínas Fúngicas , Aspergillus fumigatus/patogenicidade , Aspergillus fumigatus/fisiologia , Proteínas Fúngicas/metabolismo , Células HEK293/metabolismo , Humanos , Hifas/metabolismo , Pulmão/microbiologia , Virulência
14.
Med Mycol ; 57(Supplement_2): S118-S126, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30816976

RESUMO

Cystic fibrosis (CF), caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is the most common inherited life-limiting disease in North European people affecting 90,000 people worldwide. Progressive lung damage caused by recurrent infection and chronic airway inflammation is the major determinant of survival with a median age at death of 29 years. Approximately 60% of CF patients are infected with Aspergillus fumigatus, a ubiquitous environmental fungus, and its presence has been associated with accelerated lung function decline. Half of the patients infected with Aspergillus are <18 years of age. Yet time of acquisition of this fungus and determinants of CF-related Aspergillus disease severity and progression are not known. CFTR expression has been demonstrated in cells of the innate and adaptive immune system and has shown to be critical for normal function. Research delineating the role of CFTR-deficient phagocytes in Aspergillus persistence and infection in the CF lung, has only recently received attention. In this concise review we aim to present the current understanding with respect to when people with CF acquire infection with A. fumigatus and antifungal immune responses by CF immune cells.


Assuntos
Aspergillus fumigatus/imunologia , Fibrose Cística/complicações , Imunidade Inata , Leucócitos/imunologia , Aspergilose Pulmonar/imunologia , Aspergilose Pulmonar/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Humanos
15.
Exp Eye Res ; 182: 19-29, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30853520

RESUMO

Thymic stromal lymphopoietin (TSLP) is an interleukin 7 (IL-7)-like four helix bundle cytokine that plays diverse roles in the regulation of immune responses. In fungal infection, pattern recognition receptors (PRRs), including the cell surface Toll-like receptors (TLRs) and cytoplasmic NOD-like receptors, recognize pathogen-associated molecular patterns to initiate downstream signal cascades to active immune responses. Our previous studies reported that, in vitro human cornea epithelium cells represented a novel target of TSLP and that TSLP/TSLPR/STAT5 signaling played an important role in the response to Aspergillus fumigatus challenge. TSLP downstream signaling molecules upregulated TLR2 and MyD88/NF kappa B-p65 signaling. This phenomenon suggested that TSLP had an impact on PRRs in antifungal immunity. In mouse fungal keratitis induced by A. fumigatus, TSLP was mainly expressed in the epithelium as well as in some infiltrated immune cells in a time-dependent manner. Exogenous TSLP with Aspergillus led to severe keratitis and worse corneal recovery with higher levels of TLR2, TLR4, IL-6, and IL-8 as well as increased neutrophil infiltration. By contrast, when TSLP was suppressed by siRNA, fungal keratitis was mild with higher levels of antimicrobial peptides such as human beta-defensin (hBD9). Taken together, our data revealed an unreported function of TSLP in mediating an anti-fungal inflammatory response and serving as a target to control tissue injury and infection in A. fumigatus keratitis.


Assuntos
Aspergilose/genética , Citocinas/genética , Infecções Oculares Fúngicas/genética , Regulação da Expressão Gênica , Ceratite/genética , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Animais , Aspergilose/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/imunologia , Western Blotting , Células Cultivadas , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Infecções Oculares Fúngicas/metabolismo , Infecções Oculares Fúngicas/microbiologia , Imunidade Inata , Ceratite/metabolismo , Ceratite/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Células Estromais , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo
16.
Cell ; 176(6): 1340-1355.e15, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30799037

RESUMO

Th17 cells provide protection at barrier tissues but may also contribute to immune pathology. The relevance and induction mechanisms of pathologic Th17 responses in humans are poorly understood. Here, we identify the mucocutaneous pathobiont Candida albicans as the major direct inducer of human anti-fungal Th17 cells. Th17 cells directed against other fungi are induced by cross-reactivity to C. albicans. Intestinal inflammation expands total C. albicans and cross-reactive Th17 cells. Strikingly, Th17 cells cross-reactive to the airborne fungus Aspergillus fumigatus are selectively activated and expanded in patients with airway inflammation, especially during acute allergic bronchopulmonary aspergillosis. This indicates a direct link between protective intestinal Th17 responses against C. albicans and lung inflammation caused by airborne fungi. We identify heterologous immunity to a single, ubiquitous member of the microbiota as a central mechanism for systemic induction of human anti-fungal Th17 responses and as a potential risk factor for pulmonary inflammatory diseases.


Assuntos
Candida albicans/imunologia , Células Th17/imunologia , Células Th17/metabolismo , Aspergillus fumigatus/imunologia , Aspergillus fumigatus/patogenicidade , Candida albicans/patogenicidade , Reações Cruzadas/imunologia , Fibrose Cística/imunologia , Fibrose Cística/microbiologia , Humanos , Imunidade , Imunidade Heteróloga/imunologia , Células Th17/fisiologia
18.
Artigo em Inglês | MEDLINE | ID: mdl-30792969

RESUMO

Aspergillus fumigatus is an opportunistic fungal pathogen capable of causing severe infection in humans. One of the limitations in our understanding of how A. fumigatus causes infection concerns the initial stages of infection, notably the initial interaction between inhaled spores or conidia and the human airway. Using publicly-available datasets, we identified the Arp2/3 complex and the WAS-Interacting Protein Family Member 2 WIPF2 as being potentially responsible for internalization of conidia by airway epithelial cells. Using a cell culture model, we demonstrate that RNAi-mediated knockdown of WIPF2 significantly reduces internalization of conidia into airway epithelial cells. Furthermore, we demonstrate that inhibition of Arp2/3 by a small molecule inhibitor causes similar effects. Using super-resolution fluorescence microscopy, we demonstrate that WIPF2 is transiently localized to the site of bound conidia. Overall, we demonstrate the active role of the Arp2/3 complex and WIPF2 in mediating the internalization of A. fumigatus conidia into human airway epithelial cells.


Assuntos
Proteína 2 Relacionada a Actina/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Aspergillus fumigatus/imunologia , Proteínas de Transporte/metabolismo , Células Epiteliais/imunologia , Fagocitose , Linhagem Celular , Humanos , Proteínas dos Microfilamentos , Esporos Fúngicos/imunologia
19.
Front Immunol ; 10: 123, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30778357

RESUMO

ß2 integrin receptors consist of an alpha subunit (CD11a-CD11d) and CD18 as the common beta subunit, and are differentially expressed by leukocytes. ß2 integrins are required for cell-cell interaction, transendothelial migration, uptake of opsonized pathogens, and cell signaling processes. Functional loss of CD18-termed leukocyte-adhesion deficiency type 1 (LAD1)-results in an immunocompromised state characterized by frequent occurrence of severe infections. In immunosuppressed individuals Aspergillus fumigatus is a frequent cause of invasive pulmonary fungal infection, and often occurs in patients suffering from LAD1. Here, we asked for the importance of CD11b/CD18 also termed MAC-1 which is required for phagocytosis of opsonized A. fumigatus conidia by polymorphonuclear neutrophils (PMN) for control of pulmonary A. fumigatus infection. We show that CD11b-/- mice infected with A. fumigatus were unaffected in long term survival, similar to wild type (WT) mice. However, bronchoalveolar lavage (BAL) performed 1 day after infection revealed a higher lung infiltration of PMN in case of infected CD11b-/- mice than observed for WT mice. BAL derived from infected CD11b-/- mice also contained a higher amount of leukocyte-attracting CCL5 chemokine, but lower amounts of proinflammatory innate cytokines. In accordance, lung tissue of A. fumigatus infected CD11b-/- mice was characterized by lower cellular inflammation, and a higher fungal burden. In agreement, CD11b-/-PMN exerted lower phagocytic activity on serum-opsonized A. fumigatus conidia than WT PMN in vitro. Our study shows that MAC-1 is required for effective clearance of A. fumigatus by infiltrating PMN, and the establishment of an inflammatory microenvironment in infected lung. Enhanced infiltration of CD11b-/- PMN may serve to compensate impaired PMN function.


Assuntos
Aspergillus fumigatus/imunologia , Antígeno CD11b/metabolismo , Aspergilose Pulmonar Invasiva/imunologia , Aspergilose Pulmonar Invasiva/microbiologia , Antígeno de Macrófago 1/metabolismo , Infiltração de Neutrófilos/imunologia , Neutrófilos/metabolismo , Animais , Lavagem Broncoalveolar , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Inflamação/metabolismo , Estimativa de Kaplan-Meier , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose
20.
BMJ Case Rep ; 12(2)2019 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-30765445

RESUMO

Programmed cell death-1 (PD-1) inhibitors stimulate immune recognition of tumour cells in cancer patients, but have significant autoimmune side effects including pneumonitis. We report the case of a patient with asthma and mild eosinophilia who developed unusual pulmonary side effect of bronchiectasis, severe eosinophilia (absolute eosinophil count: 3200 c/mm3) and elevated IgE levels (7050 IU/mL; normal: <164 IU/mL) 4 months into therapy with the PD-1 inhibitor pembrolizumab. Aspergillus fumigatus IgG was elevated at 15.60 U/mL (normal: <12.01 U/mL). He responded to therapy with corticosteroids and voriconazole and was able to resume pembrolizumab thereafter with good clinical response.


Assuntos
Corticosteroides/uso terapêutico , Aspergilose Broncopulmonar Alérgica/diagnóstico por imagem , Aspergilose Broncopulmonar Alérgica/tratamento farmacológico , Aspergillus fumigatus/imunologia , Voriconazol/uso terapêutico , Anticorpos Antifúngicos/metabolismo , Anticorpos Monoclonais Humanizados/efeitos adversos , Aspergilose Broncopulmonar Alérgica/induzido quimicamente , Aspergilose Broncopulmonar Alérgica/imunologia , Eosinófilos/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios X
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