RESUMEN
Aspirin, with its active compound acetylsalicylic acid (ASA), shows antiviral activity against rhino- and influenza viruses at high concentrations. We sought to investigate whether ASA and its metabolite salicylic acid (SA) inhibit SARS-CoV-2 since it might use similar pathways to influenza viruses. The compound-treated cells were infected with SARS-CoV-2. Viral replication was analysed by RTqPCR. The compounds suppressed SARS-CoV-2 replication in cell culture cells and a patient-near replication system using human precision-cut lung slices by two orders of magnitude. While the compounds did not interfere with viral entry, it led to lower viral RNA expression after 24 h, indicating that post-entry pathways were inhibited by the compounds.
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Chronic respiratory infections with the gram-negative bacterium Pseudomonas aeruginosa are an important co-morbidity for the quality of life and prognosis of people with cystic fibrosis (CF). Such long-term colonization, sometimes lasting up to several decades, represents a unique opportunity to investigate pathogen adaptation processes to the host. Our studies aimed to resolve if and to what extent the bacterial adaptation to the CF airways influences the fitness of the pathogen to grow and to persist in the lungs. Marker-free competitive fitness experiments of serial P. aeruginosa isolates differentiated by strain-specific SNPs, were performed with murine and human precision cut lung slices (PCLS). Serial P. aeruginosa isolates were selected from six mild and six severe CF patient courses, respectively. MPCLS or hPCLS were inoculated with a mixture of equal numbers of the serial isolates of one course. The temporal change of the composition of the bacterial community during competitive growth was quantified by multi-marker amplicon sequencing. Both ex vivo models displayed a strong separation of fitness traits between mild and severe courses. Whereas the earlier isolates dominated the competition in the severe courses, intermediate and late isolates commonly won the competition in the mild courses. The status of the CF lung disease rather than the bacterial genotype drives the adaptation of P. aeruginosa during chronic CF lung infection. This implies that the disease status of the lung habitat governed the adaptation of P. aeruginosa more strongly than the underlying bacterial clone-type and its genetic repertoire.
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Fibrosis Quística , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Animales , Fibrosis Quística/complicaciones , Fibrosis Quística/microbiología , Humanos , Pulmón/microbiología , Ratones , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genéticaRESUMEN
Human rhinovirus (RV) is a major risk factor for chronic obstructive pulmonary disease (COPD) and asthma exacerbations. The exploration of RV pathogenesis has been hampered by a lack of disease-relevant model systems. We performed a detailed characterization of host responses to RV infection in human lung tissue ex vivo and investigated whether these responses are disease relevant for patients with COPD and asthma. In addition, impact of the viral replication inhibitor rupintrivir was evaluated. Human precision-cut lung slices (PCLS) were infected with RV1B with or without rupintrivir. At Days 1 and 3 after infection, RV tissue localization, tissue viability, and viral load were determined. To characterize host responses to infection, mediator and whole genome analyses were performed. RV successfully replicated in PCLS airway epithelial cells and induced both antiviral and proinflammatory cytokines such as IFNα2a, CXCL10, CXCL11, IFN-γ, TNFα, and CCL5. Genomic analyses revealed that RV not only induced antiviral immune responses but also triggered changes in epithelial cell-associated pathways. Strikingly, the RV response in PCLS was reflective of gene expression changes described in patients with COPD and asthma. Although RV-induced host immune responses were abrogated by rupintrivir, RV-triggered epithelial processes were largely refractory to antiviral treatment. Detailed analysis of RV-infected human PCLS and comparison with gene signatures of patients with COPD and asthma revealed that the human RV PCLS model represents disease-relevant biological mechanisms that can be partially inhibited by a well-known antiviral compound and provide an outstanding opportunity to evaluate novel therapeutics.
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Asma/genética , Interacciones Huésped-Patógeno/genética , Pulmón/virología , Infecciones por Picornaviridae/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Anciano , Antivirales/farmacología , Asma/patología , Bronquios/patología , Bronquios/fisiología , Células Epiteliales/patología , Células Epiteliales/virología , Femenino , Perfilación de la Expresión Génica , Genoma Humano , Humanos , Isoxazoles/farmacología , Pulmón/fisiología , Masculino , Persona de Mediana Edad , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Infecciones por Picornaviridae/tratamiento farmacológico , Infecciones por Picornaviridae/patología , Enfermedad Pulmonar Obstructiva Crónica/patología , Pirrolidinonas/farmacología , Rhinovirus/patogenicidad , Valina/análogos & derivados , Valina/farmacologíaRESUMEN
Smokers with apparently "healthy" lungs suffer from more severe and frequent viral respiratory infections, but the mechanisms underlying this observation are still unclear. Epithelial cells and dendritic cells (DC) form the first line of defense against inhaled noxes such as smoke or viruses. We therefore aimed to obtain insight into how cigarette smoke affects DCs and epithelial cells and how this influences the response to viral infection. Female C57BL/6J mice were exposed to cigarette smoke (CS) for 1 h daily for 24 days and then challenged i.n. with the viral mimic and Toll-like receptor 3 (TLR3) ligand poly (I:C) after the last exposure. DC subpopulations were analyzed 24 h later in whole lung homogenates by flow cytometry. Calu-3 cells or human precision-cut lung slices (PCLS) cultured at air-liquid interface were exposed to CS or air and subsequently inoculated with influenza H1N1. At 48 h post infection cytokines were analyzed by multiplex technology. Cytotoxic effects were measured by release of lactate dehydrogenase (LDH) and confocal imaging. In Calu-3 cells the trans-epithelial electrical resistance (TEER) was assessed. Smoke exposure of mice increased numbers of inflammatory and plasmacytoid DCs in lung tissue. Additional poly (I:C) challenge further increased the population of inflammatory DCs and conventional DCs, especially CD11b+ cDCs. Smoke exposure led to a loss of the barrier function in Calu-3 cells, which was further exaggerated by additional influenza H1N1 infection. Influenza H1N1-induced secretion of antiviral cytokines (IFN-α2a, IFN-λ, interferon-γ-induced protein 10 [IP-10]), pro-inflammatory cytokine IL-6, as well as T cell-associated cytokines (e.g., I-TAC) were completely suppressed in both Calu-3 cells and human PCLS after smoke exposure. In summary, cigarette smoke exposure increased the number of inflammatory DCs in the lung and disrupted epithelial barrier functions, both of which was further enhanced by viral stimulation. Additionally, the antiviral immune response to influenza H1N1 was strongly suppressed by smoke. These data suggest that smoke impairs protective innate mechanisms in the lung, which could be responsible for the increased susceptibility to viral infections in "healthy" smokers.
RESUMEN
The peptide sequence KKIRVRLSA was synthesized in a dimeric structure (SET-M33DIM) and evaluated as a candidate drug for infections due to multidrug-resistant (MDR) Gram-negative pathogens. SET-M33DIM showed significant antibacterial activity against MDR strains of Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli (Minimal Inhibitory Concentration [MICs], 1.5-11 µM), and less activity against Pseudomonas aeruginosa (MICs, 11-22 µM). It showed very low toxicity in vitro, ex vivo, and in vivo; in cytotoxicity tests, its EC50 was as much as 22 times better than that of SET-M33, a peptide with the same amino-acid sequence, but synthesized in tetra-branched form (638 vs 28 µM). In in vivo and ex vivo experiments, SET-M33DIM cleared P. aeruginosa infection, significantly reducing signs of sepsis in animals, and restoring cell viability in lung tissue after bacterial challenge. It also quelled inflammation triggered by LPS and live bacterial cells, inhibiting expression of inflammatory mediators in lung tissue, cultured macrophages, and bronchial cells from a cystic fibrosis patient.
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Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Péptidos/síntesis química , Péptidos/farmacología , Neumonía Bacteriana/tratamiento farmacológico , Infecciones por Pseudomonas/tratamiento farmacológico , Animales , Antibacterianos/síntesis química , Farmacorresistencia Bacteriana Múltiple , Femenino , Huésped Inmunocomprometido , Lipopolisacáridos , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Neumonía Bacteriana/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa , Células RAW 264.7 , Pruebas de ToxicidadRESUMEN
BACKGROUND: Antiviral drugs such as rupintrivir may have an immune-modulatory effect in experimentally induced allergic asthma with subsequent RV infection. We infected lung slices of house-dust mite (HDM)-sensitized asthmatic mice ex vivo with human rhinovirus (RV) and investigated the effect of the antiviral drug rupintrivir on RV-induced cytokine response in lung tissue of HDM-sensitized mice ex vivo. METHODS: Mice were sensitized with HDM. Precision-cut lung slices (PCLS) were prepared from HDM-sensitized or non-sensitized mice. Lung slices were infected ex vivo with RV or RV together with rupintrivir. Modulation of immune responses was evaluated by cytokine secretion 48 h post infection. RESULTS: In vivo HDM sensitization resulted in a TH-2/TH-17-dominated cytokine response that persisted in PCLS ex vivo. RV infection of PCLS from non-sensitized mice resulted in the induction of an antiviral and pro-inflammatory immune response, as indicated by the secretion of IFN-α, IFN-ß, IFN-γ, TNF-α, MCP-1, IP-10, IL-10, and IL-17A. In contrast, PCLS from HDM-sensitized mice showed an attenuated antiviral response, but exaggerated IL-4, IL-6, and IL-10 secretion upon infection. Rupintrivir inhibited exaggerated pro-inflammatory cytokine IL-6 and TH-2 cytokine IL-4 in HDM-sensitized mice. CONCLUSIONS: In summary, this study demonstrates that treatment with rupintrivir influences virus-induced IL-4 and IL-6 cytokine release under experimental conditions ex vivo.
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Interleucina-4/inmunología , Isoxazoles/farmacología , Pulmón/inmunología , Pyroglyphidae/inmunología , Pirrolidinonas/farmacología , Rhinovirus , Células Th2/inmunología , Animales , Antivirales/farmacología , Citocinas/antagonistas & inhibidores , Citocinas/inmunología , Femenino , Interleucina-4/antagonistas & inhibidores , Pulmón/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Técnicas de Cultivo de Órganos , Fenilalanina/análogos & derivados , Células Th2/efectos de los fármacos , Valina/análogos & derivadosRESUMEN
Rhinovirus (RV) is the predominant virus causing respiratory tract infections. Bronchobini® is a low dose multi component, multi target preparation used to treat inflammatory respiratory diseases such as the common cold, described to ease severity of symptoms such as cough and viscous mucus production. The aim of the study was to assess the efficacy of Bronchobini® in RV infection and to elucidate its mode of action. Therefore, Bronchobini®'s ingredients (BRO) were assessed in an ex vivo model of RV infection using mouse precision-cut lung slices, an organotypic tissue capable to reflect the host immune response to RV infection. Cytokine profiles were assessed using enzyme-linked immunosorbent assay (ELISA) and mesoscale discovery (MSD). Gene expression analysis was performed using Affymetrix microarrays and ingenuity pathway analysis. BRO treatment resulted in the significant suppression of RV-induced antiviral and pro-inflammatory cytokine release. Transcriptome analysis revealed a multifactorial mode of action of BRO, with a strong inhibition of the RV-induced pro-inflammatory and antiviral host response mediated by nuclear factor kappa B (NFkB) and interferon signaling pathways. Interestingly, this was due to priming of these pathways in the absence of virus. Overall, BRO exerted its beneficial anti-inflammatory effect by priming the antiviral host response resulting in a reduced inflammatory response to RV infection, thereby balancing an otherwise excessive inflammatory response.
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Antivirales/farmacología , Inductores de Interferón/farmacología , Interferones/metabolismo , Pulmón/efectos de los fármacos , Infecciones por Picornaviridae/tratamiento farmacológico , Extractos Vegetales/farmacología , Transcriptoma , Animales , Antivirales/uso terapéutico , Femenino , Inductores de Interferón/uso terapéutico , Pulmón/metabolismo , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Infecciones por Picornaviridae/inmunología , Infecciones por Picornaviridae/virología , Extractos Vegetales/uso terapéutico , Rhinovirus/efectos de los fármacos , Rhinovirus/patogenicidad , Transducción de SeñalRESUMEN
The increasing number of severe infections with multi-drug-resistant pathogens worldwide highlights the need for alternative treatment options. Given the pivotal role of phagocytes and especially alveolar macrophages in pulmonary immunity, we introduce a new, cell-based treatment strategy to target bacterial airway infections. Here we show that the mass production of therapeutic phagocytes from induced pluripotent stem cells (iPSC) in industry-compatible, stirred-tank bioreactors is feasible. Bioreactor-derived iPSC-macrophages (iPSC-Mac) represent a highly pure population of CD45+CD11b+CD14+CD163+ cells, and share important phenotypic, functional and transcriptional hallmarks with professional phagocytes, however with a distinct transcriptome signature similar to primitive macrophages. Most importantly, bioreactor-derived iPSC-Mac rescue mice from Pseudomonas aeruginosa-mediated acute infections of the lower respiratory tract within 4-8 h post intra-pulmonary transplantation and reduce bacterial load. Generation of specific immune-cells from iPSC-sources in scalable stirred-tank bioreactors can extend the field of immunotherapy towards bacterial infections, and may allow for further innovative cell-based treatment strategies.
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Infecciones Bacterianas/prevención & control , Reactores Biológicos , Inmunoterapia/métodos , Células Madre Pluripotentes Inducidas/citología , Macrófagos/citología , Infecciones del Sistema Respiratorio/prevención & control , Animales , Infecciones Bacterianas/inmunología , Técnicas de Cultivo de Célula , Humanos , Macrófagos/fisiología , Ratones , Microscopía Electrónica de Rastreo , Pseudomonas aeruginosa/patogenicidad , Infecciones del Sistema Respiratorio/inmunologíaRESUMEN
Objectives: In the context of cystic fibrosis, Pseudomonas aeruginosa biofilms often develop in the vicinity of airway mucus, which acts as a protective physical barrier to inhaled matter. However, mucus can also adsorb small drug molecules administered as aerosols, including antibiotics, thereby reducing their bioavailability. The efficacy of antibiotics is typically assessed by determining the MIC using in vitro assays. This widespread technique, however, does not consider either bacterial biofilm formation or the influence of mucus, both of which may act as diffusion barriers, potentially limiting antibiotic efficacy. Methods: We grew P. aeruginosa biofilms in the presence or absence of human tracheal mucus and tested their susceptibility to tobramycin and colistin. Results: A significant reduction of tobramycin efficacy was observed when P. aeruginosa biofilms were grown in the presence of mucus compared with those grown in the absence of mucus. Diffusion of tobramycin through mucus was reduced; however, this reduction was more pronounced in biofilm/mucus mixtures, suggesting that biofilms in the presence of mucus respond differently to antibiotic treatment. In contrast, the influence of mucus on colistin efficacy was almost negligible and no differences in mucus permeability were observed. Conclusions: These findings underline the important role of mucus in the efficacy of anti-infective drugs.
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Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Colistina/farmacología , Moco/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Tobramicina/farmacología , Biopelículas/crecimiento & desarrollo , Humanos , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/crecimiento & desarrollo , Tráquea/metabolismoRESUMEN
BACKGROUND: Respiratory viruses such as respiratory syncytial virus (RSV) or rhinovirus are one of the major causes for respiratory tract infections causing common cold disease. Respiratory viral infections range from mild symptoms in adults to serious illness especially in the very young or elderly as well as patients suffering from lung diseases or being immunocompromised due to other reasons. Engystol (EGY-2) is a multicomponent, multitarget preparation consisting of Vincetoxicum hirundinaria and Sulfur in various dilutions. The study objective was to test the effect of EGY-2 on the innate immune response during the early onset of respiratory viral infection in vivo as exemplified in a mouse model of RSV-induced respiratory inflammation. METHODS: Naïve BALB/c mice were infected with 1x106 infectious units RSV A2 intranasally to cause a mild respiratory infection. EGY-2 was administered daily per oral gavage starting seven days prior to RSV infection at doses of 0.4 to 5.1 tablets/kg. Control groups received placebo treatment. Animals were sacrificed 1 to 3 days post infection (p.i.) to analyse the infection and induced immune response in the lung. Viral load in bronchoalveolar lavage fluid (BALF) and lung homogenate was determined by TCID50 assay as well as immunofluorescence staining of BALF cells using anti-RSV antibody and microscopic analysis. The RSV induced immune response was assessed by evaluation of BALF differential cell count, BALF cytokine secretion and analysis of the phagocytic capacity of alveolar macrophages. RESULTS: EGY-2 significantly reduced the RSV induced neutrophil and early lymphocyte influx on day 1 p.i. in BALF. EGY-2 treatment significantly diminished the RSV induced secretion of pro-inflammatory cytokines such as IFN-γ, IL-1ß, IL-6, KC and TNF-α at day 1. EGY-2 treatment was not protective for RSV infection per se, as no alteration in the viral load in lung and BALF was detected. Enhanced numbers of phagocytic-active macrophages were observed in EGY-2 treated animals on day 1 and this macrophage population showed strongly enhanced phagocytic activity on day 1 and day 3. CONCLUSION: The data suggest a beneficial immunomodulatory effect of EGY-2 during early onset of respiratory viral infection in vivo, mediated by stimulation of macrophage phagocytosis, resulting in a reduced innate inflammatory response in terms of neutrophil and early lymphocyte infiltration as well as reduced inflammatory cytokine secretion.
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Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Extractos Vegetales/farmacología , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/inmunología , Animales , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Ratones , Infiltración Neutrófila/efectos de los fármacos , Infiltración Neutrófila/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Infecciones por Virus Sincitial Respiratorio/metabolismo , Carga ViralRESUMEN
RATIONALE: The pathogenesis of respiratory syncytial virus (RSV) bronchiolitis in infants remains poorly understood. Mouse models implicate pulmonary T cells in the development of RSV disease. T cell responses are initiated by dendritic cells (DCs), which accumulate in lungs of RSV-infected mice. In infants with RSV bronchiolitis, previous reports have shown that DCs are mobilised to the nasal mucosa, but data on lower airway DC responses are lacking. OBJECTIVE: To determine the presence and phenotype of DCs and associated immune cells in bronchoalveolar lavage (BAL) and peripheral blood samples from infants with RSV bronchiolitis. METHODS: Infants intubated and ventilated due to severe RSV bronchiolitis or for planned surgery (controls with healthy lungs) underwent non-bronchoscopic BAL. Immune cells in BAL and blood samples were characterised by flow cytometry and cytokines measured by Human V-Plex Pro-inflammatory Panel 1 MSD kit. MEASUREMENTS AND MAIN RESULTS: In RSV cases, BAL conventional DCs (cDCs), NK T cells, NK cells and pro-inflammatory cytokines accumulated, plasmacytoid DCs (pDCs) and T cells were present, and blood cDCs increased activation marker expression. When stratifying RSV cases by risk group, preterm and older (≥4â months) infants had fewer BAL pDCs than term born and younger (<4â months) infants, respectively. CONCLUSIONS: cDCs accumulate in the lower airways during RSV bronchiolitis, are activated systemically and may, through activation of T cells, NK T cells and NK cells, contribute to RSV-induced inflammation and disease. In addition, the small population of airway pDCs in preterm and older infants may reveal a distinct endotype of RSV bronchiolitis with weak antiviral pDC responses.
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Bronquiolitis Viral/inmunología , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/análisis , Células Dendríticas/inmunología , Infecciones por Virus Sincitial Respiratorio/complicaciones , Factores de Edad , Antígenos CD/sangre , Bronquiolitis Viral/sangre , Bronquiolitis Viral/virología , Líquido del Lavado Bronquioalveolar/química , Linfocitos T CD4-Positivos , Antígenos CD40/sangre , Linfocitos T CD8-positivos , Estudios de Casos y Controles , Recuento de Células , Citocinas/sangre , Femenino , Humanos , Inmunoglobulinas/sangre , Lactante , Recién Nacido , Células Asesinas Naturales , Macrófagos , Masculino , Glicoproteínas de Membrana/sangre , Monocitos , Células T Asesinas Naturales , Fenotipo , Nacimiento Prematuro/inmunología , Nacimiento a Término/inmunología , Antígeno CD83RESUMEN
OBJECTIVES: Our previous data demonstrated that allergic airway inflammation induces migration of dendritic cells (DC) into airway sensory jugular and nodose ganglia (jugular-nodose ganglion complex; JNC). Here we investigated the effects of steroid treatment regarding the expression and migration of DC and calcitonin gene-related peptide (CGRP)-immunoreactive neurons of vagal sensory ganglia during allergic airway inflammation. METHODS: A house dust mite (HDM) model for allergic airway inflammation was used. The mice received 0.3 mg fluticasone propionate per kilogram of body weight in the last 9 days. JNC slices were analyzed on MHC II, the neuronal marker PGP9.5, and the neuropeptide CGRP. RESULTS: Allergic airway inflammation increased the numbers of DC and CGRP-expressing neurons in the JNC significantly in comparison to the controls (DC/neurons: HDM 44.58 ± 1.6% vs. saline 33.29 ± 1.6%, p < 0.05; CGRP-positive neurons/total neurons: HDM 30.65 ± 1.9% vs. saline 19.49 ± 2.3%, p < 0.05). Steroid treatment did not have any effect on the numbers of DC and CGRP-expressing neurons in the JNC compared to HDM-treated mice. CONCLUSIONS: The present findings indicate an important role of DC and CGRP-containing neurons in the pathogenesis of allergic airway inflammation. However, steroid treatment did not have an effect on the population of DC and neurons displaying CGRP in the JNC, whereas steroid treatment was found to suppress allergic airway inflammation.