RESUMEN
Recent investigations analyzed in depth the biochemical and biophysical properties of the endothelial glycocalyx. In comparison, this complex cell-covering structure is largely understudied in alveolar epithelial cells. To better characterize the alveolar glycocalyx ultrastructure, unaffected versus injured human lung tissue explants and mouse lungs were analyzed by transmission electron microscopy. Lung tissue was treated with either heparinase (HEP), known to shed glycocalyx components, or pneumolysin (PLY), the exotoxin of Streptococcus pneumoniae not investigated for structural glycocalyx effects so far. Cationic colloidal thorium dioxide (cThO2) particles were used for glycocalyx glycosaminoglycan visualization. The level of cThO2 particles orthogonal to apical cell membranes (â stained glycosaminoglycan height) of alveolar epithelial type I (AEI) and type II (AEII) cells was stereologically measured. In addition, cThO2 particle density was studied by dual-axis electron tomography (â stained glycosaminoglycan density in three dimensions). For untreated samples, the average cThO2 particle level was ≈ 18 nm for human AEI, ≈ 17 nm for mouse AEI, ≈ 44 nm for human AEII and ≈ 35 nm for mouse AEII. Both treatments, HEP and PLY, resulted in a significant reduction of cThO2 particle levels on human and mouse AEI and AEII. Moreover, a HEP- and PLY-associated reduction in cThO2 particle density was observed. The present study provides quantitative data on the differential glycocalyx distribution on AEI and AEII based on cThO2 and demonstrates alveolar glycocalyx shedding in response to HEP or PLY resulting in a structural reduction in both glycosaminoglycan height and density. Future studies should elucidate the underlying alveolar epithelial cell type-specific distribution of glycocalyx subcomponents for better functional understanding.
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Glicocálix , Dióxido de Torio , Ratones , Humanos , Animales , Liasa de Heparina , Electrones , GlicosaminoglicanosRESUMEN
BACKGROUND: Community-acquired pneumonia and associated sepsis cause high mortality despite antibiotic treatment. Uncontrolled inflammatory host responses contribute to the unfavorable outcome by driving lung and extrapulmonary organ failure. The complement fragment C5a holds significant proinflammatory functions and is associated with tissue damage in various inflammatory conditions. The authors hypothesized that C5a concentrations are increased in pneumonia and C5a neutralization promotes barrier stabilization in the lung and is protective in pneumococcal pulmonary sepsis. METHODS: The authors investigated regulation of C5a in pneumonia in a prospective patient cohort and in experimental pneumonia. Two complementary models of murine pneumococcal pneumonia were applied. Female mice were treated with NOX-D19, a C5a-neutralizing L-RNA-aptamer. Lung, liver, and kidney injury and the inflammatory response were assessed by measuring pulmonary permeability (primary outcome), pulmonary and blood leukocytes, cytokine concentrations in lung and blood, and bacterial load in lung, spleen, and blood, and performing histologic analyses of tissue damage, apoptosis, and fibrin deposition (n = 5 to 13). RESULTS: In hospitalized patients with pneumonia (n = 395), higher serum C5a concentrations were observed compared to healthy subjects (n = 24; 6.3 nmol/l [3.9 to 10.0] vs. 4.5 nmol/l [3.8 to 6.6], median [25 to 75% interquartile range]; difference: 1.4 [95% CI, 0.1 to 2.9]; P = 0.029). Neutralization of C5a in mice resulted in lower pulmonary permeability in pneumococcal pneumonia (1.38 ± 0.89 vs. 3.29 ± 2.34, mean ± SD; difference: 1.90 [95% CI, 0.15 to 3.66]; P = 0.035; n = 10 or 11) or combined severe pneumonia and mechanical ventilation (2.56 ± 1.17 vs. 7.31 ± 5.22; difference: 4.76 [95% CI, 1.22 to 8.30]; P = 0.011; n = 9 or 10). Further, C5a neutralization led to lower blood granulocyte colony-stimulating factor concentrations and protected against sepsis-associated liver injury. CONCLUSIONS: Systemic C5a is elevated in pneumonia patients. Neutralizing C5a protected against lung and liver injury in pneumococcal pneumonia in mice. Early neutralization of C5a might be a promising adjunctive treatment strategy to improve outcome in community-acquired pneumonia.
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Aptámeros de Nucleótidos/administración & dosificación , Complemento C5a/antagonistas & inhibidores , Neumonía Neumocócica/sangre , Neumonía Neumocócica/prevención & control , Sepsis/sangre , Sepsis/prevención & control , Animales , Anticuerpos Neutralizantes/administración & dosificación , Biomarcadores/sangre , Estudios de Cohortes , Complemento C5a/metabolismo , Femenino , Factores Inmunológicos/antagonistas & inhibidores , Factores Inmunológicos/sangre , Ratones , Ratones Endogámicos C57BL , Estudios ProspectivosRESUMEN
RATIONALE: During pneumonia, pathogen-host interaction evokes inflammation and lung barrier dysfunction. Tie2 activation by angiopoietin-1 reduces, whereas Tie2 blockade by angiopoietin-2 increases, inflammation and permeability during sepsis. The role of angiopoietin-1/-2 in pneumonia remains unidentified. OBJECTIVES: To investigate the prognostic and pathogenic impact of angiopoietins in regulating pulmonary vascular barrier function and inflammation in bacterial pneumonia. METHODS: Serum angiopoietin levels were quantified in pneumonia patients of two independent cohorts (n = 148, n = 395). Human postmortem lung tissue, pneumolysin- or angiopoietin-2-stimulated endothelial cells, isolated perfused and ventilated mouse lungs, and mice with pneumococcal pneumonia were investigated. MEASUREMENTS AND MAIN RESULTS: In patients with pneumonia, decreased serum angiopoietin-1 and increased angiopoietin-2 levels were observed as compared with healthy subjects. Higher angiopoietin-2 serum levels were found in patients with community-acquired pneumonia who died within 28 days of diagnosis compared with survivors. Receiver operating characteristic analysis revealed improved prognostic accuracy of CURB-65 for 28-day survival, intensive care treatment, and length of hospital stay if combined with angiopoietin-2 serum levels. In vitro, pneumolysin enhanced endothelial angiopoietin-2 release, angiopoietin-2 increased endothelial permeability, and angiopoietin-1 reduced pneumolysin-evoked endothelial permeability. Ventilated and perfused lungs of mice with angiopoietin-2 knockdown showed reduced permeability on pneumolysin stimulation. Increased pulmonary angiopoietin-2 and reduced angiopoietin-1 mRNA expression were observed in Streptococcus pneumoniae-infected mice. Finally, angiopoietin-1 therapy reduced inflammation and permeability in murine pneumonia. CONCLUSIONS: These data suggest a central role of angiopoietin-1/-2 in pneumonia-evoked inflammation and permeability. Increased angiopoietin-2 serum levels predicted mortality and length of hospital stay, and angiopoietin-1 may provide a therapeutic target for severe pneumonia.
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Angiopoyetina 1/uso terapéutico , Angiopoyetina 2/uso terapéutico , Células Endoteliales/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Inflamación/fisiopatología , Pulmón/efectos de los fármacos , Neumonía Neumocócica/tratamiento farmacológico , Neumonía Neumocócica/fisiopatología , Angiopoyetina 1/sangre , Angiopoyetina 2/sangre , Humanos , PronósticoRESUMEN
Descriptive histopathology of mouse models of pneumonia is essential in assessing the outcome of infections, molecular manipulations, or therapies in the context of whole lungs. Quantitative comparisons between experimental groups, however, have been limited to laborious stereology or ill-defined scoring systems that depend on the subjectivity of a more or less experienced observer. Here, we introduce self-learning digital image analyses that allow us to transform optical information from whole mouse lung sections into statistically testable data. A pattern-recognition-based software and a nuclear count algorithm were adopted to quantify user-defined pathologies from whole slide scans of lungs infected with Streptococcus pneumoniae or influenza A virus compared with PBS-challenged lungs. The readout parameters "relative area affected" and "nuclear counts per area" are proposed as relevant criteria for the quantification of lesions from hematoxylin and eosin-stained sections, also allowing for the generation of a heat map of, for example, immune cell infiltrates with anatomical assignments across entire lung sections. Moreover, when combined with immunohistochemical labeling of marker proteins, both approaches are useful for the identification and counting of, for example, immune cell populations, as validated here by direct comparisons with flow cytometry data. The solutions can easily and flexibly be adjusted to specificities of different models or pathogens. Automated digital analyses of whole mouse lung sections may set a new standard for the user-defined, high-throughput comparative quantification of histological and immunohistochemical images. Still, our algorithms established here are only a start, and need to be tested in additional studies and other applications in the future.
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Algoritmos , Técnicas Citológicas , Interpretación de Imagen Asistida por Computador/métodos , Pulmón/patología , Infecciones por Orthomyxoviridae/patología , Neumonía Neumocócica/patología , Neumonía Viral/patología , Enfermedad Aguda , Animales , Automatización de Laboratorios , Modelos Animales de Enfermedad , Virus de la Influenza A/patogenicidad , Pulmón/microbiología , Pulmón/virología , Ratones , Infecciones por Orthomyxoviridae/virología , Reconocimiento de Normas Patrones Automatizadas , Neumonía Neumocócica/microbiología , Neumonía Viral/virología , Valor Predictivo de las Pruebas , Programas Informáticos , Streptococcus pneumoniae/patogenicidadRESUMEN
OBJECTIVES: Severe pneumonia may evoke acute lung injury, and sphingosine-1-phosphate is involved in the regulation of vascular permeability and immune responses. However, the role of sphingosine-1-phosphate and the sphingosine-1-phosphate producing sphingosine kinase 1 in pneumonia remains elusive. We examined the role of the sphingosine-1-phosphate system in regulating pulmonary vascular barrier function in bacterial pneumonia. DESIGN: Controlled, in vitro, ex vivo, and in vivo laboratory study. SUBJECTS: Female wild-type and SphK1-deficient mice, 8-10 weeks old. Human postmortem lung tissue, human blood-derived macrophages, and pulmonary microvascular endothelial cells. INTERVENTIONS: Wild-type and SphK1-deficient mice were infected with Streptococcus pneumoniae. Pulmonary sphingosine-1-phosphate levels, messenger RNA expression, and permeability as well as lung morphology were analyzed. Human blood-derived macrophages and human pulmonary microvascular endothelial cells were infected with S. pneumoniae. Transcellular electrical resistance of human pulmonary microvascular endothelial cell monolayers was examined. Further, permeability of murine isolated perfused lungs was determined following exposition to sphingosine-1-phosphate and pneumolysin. MEASUREMENTS AND MAIN RESULTS: Following S. pneumoniae infection, murine pulmonary sphingosine-1-phosphate levels and sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 expression were increased. Pneumonia-induced lung hyperpermeability was reduced in SphK1 mice compared with wild-type mice. Expression of sphingosine kinase 1 in macrophages recruited to inflamed lung areas in pneumonia was observed in murine and human lungs. S. pneumoniae induced the sphingosine kinase 1/sphingosine-1-phosphate system in blood-derived macrophages and enhanced sphingosine-1-phosphate receptor 2 expression in human pulmonary microvascular endothelial cell in vitro. In isolated mouse lungs, pneumolysin-induced hyperpermeability was dose dependently and synergistically increased by sphingosine-1-phosphate. This sphingosine-1-phosphate-induced increase was reduced by inhibition of sphingosine-1-phosphate receptor 2 or its downstream effector Rho-kinase. CONCLUSIONS: Our data suggest that targeting the sphingosine kinase 1-/sphingosine-1-phosphate-/sphingosine-1-phosphate receptor 2-signaling pathway in the lung may provide a novel therapeutic perspective in pneumococcal pneumonia for prevention of acute lung injury.
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Lesión Pulmonar Aguda/metabolismo , Inflamación/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Neumonía Neumocócica/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Lesión Pulmonar Aguda/enzimología , Lesión Pulmonar Aguda/etiología , Animales , Femenino , Humanos , Inflamación/enzimología , Ratones , Ratones Endogámicos C57BL , Neumonía Neumocócica/complicaciones , Neumonía Neumocócica/enzimología , Receptores de Esfingosina-1-Fosfato , Streptococcus pneumoniaeRESUMEN
Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca(2+) mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxia-induced TRPC6 translocation to caveolae and Ca(2+) mobilization. Ca(2+) mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase- and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P2/4). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Hipertensión Pulmonar/metabolismo , Pulmón/patología , Vasoconstricción , Animales , Calcio/metabolismo , Señalización del Calcio , Ceramidas/química , Vasos Coronarios/metabolismo , Humanos , Hipoxia/patología , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CFTR , Miocitos del Músculo Liso/metabolismo , Oxígeno/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transporte de Proteínas , Arteria Pulmonar/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal , Esfingomielina Fosfodiesterasa/metabolismo , Canales Catiónicos TRPC/metabolismo , Canal Catiónico TRPC6 , Fosfolipasas de Tipo C/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
BACKGROUND: Community-acquired pneumonia (CAP) is a significant cause of morbidity and mortality worldwide. Despite effective antimicrobial therapy, CAP can induce pulmonary endothelial hyperpermeability resulting in life-threatening lung failure due to an exaggerated host-pathogen interaction. Treatment of acute lung injury is mainly supportive because key elements of inflammation-induced barrier disruption remain undetermined. Angiopoietin-1 (Ang-1)-mediated Tie2 activation reduces, and the Ang-1 antagonist Ang-2 increases, inflammation and endothelial permeability in sepsis. Vasculotide (VT) is a polyethylene glycol-clustered Tie2-binding peptide that mimics the actions of Ang-1. The aim of our study was to experimentally test whether VT is capable of diminishing pneumonia-induced lung injury. METHODS: VT binding and phosphorylation of Tie2 were analyzed using tryptophan fluorescence spectroscopy and phospho-Tie-2 enzyme-linked immunosorbent assay. Human and murine lung endothelial cells were investigated by immunofluorescence staining and electric cell-substrate impedance sensing. Pulmonary hyperpermeability was quantified in VT-pretreated, isolated, perfused, and ventilated mouse lungs stimulated with the pneumococcal exotoxin pneumolysin (PLY). Furthermore, Streptococcus pneumoniae-infected mice were therapeutically treated with VT. RESULTS: VT showed dose-dependent binding and phosphorylation of Tie2. Pretreatment with VT protected lung endothelial cell monolayers from PLY-induced disruption. In isolated mouse lungs, VT decreased PLY-induced pulmonary permeability. Likewise, therapeutic treatment with VT of S. pneumoniae-infected mice significantly reduced pneumonia-induced hyperpermeability. However, effects by VT on the pulmonary or systemic inflammatory response were not observed. CONCLUSIONS: VT promoted pulmonary endothelial stability and reduced lung permeability in different models of pneumococcal pneumonia. Thus, VT may provide a novel therapeutic perspective for reduction of permeability in pneumococcal pneumonia-induced lung injury.
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Permeabilidad Capilar/efectos de los fármacos , Pulmón/efectos de los fármacos , Fragmentos de Péptidos/farmacocinética , Animales , Infecciones Comunitarias Adquiridas/tratamiento farmacológico , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/uso terapéutico , Neumonía Neumocócica/tratamiento farmacológico , Espectrometría de Fluorescencia/métodos , Estadísticas no Paramétricas , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/patogenicidadRESUMEN
In patients with chronic obstructive pulmonary disease (COPD), Moraxella catarrhalis infection of the lower airways is associated with chronic colonization and inflammation during stable disease and acute exacerbations. Chronic smoke exposure induces chronic inflammation and impairs mucociliary clearance, thus contributing to bacterial colonization of the lower airways in COPD patients. The human-specific carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 5, expressed in human airways, has been shown to contribute to epithelial colonization of CEACAM-binding pathogens. To investigate the impact of CEACAM5 expression on pulmonary M. catarrhalis colonization, we infected mice transgenic for human CEACAM5 (hCEACAM5) and wild type mice intratracheally with M. catarrhalis with or without preceding smoke exposure and analyzed bacterial colonization and local and systemic inflammation. Our results show that airway infection with M. catarrhalis accelerated acute local but not systemic inflammation, albeit independent of hCEACAM5 expression. Long-term smoke exposure alone or prior to M. catarrhalis infection did not contribute to increased local or systemic inflammation. No difference was found in pulmonary clearance of M. catarrhalis in hCEACAM5-transgenic mice compared with wild-type mice. Smoke exposure neither altered time nor extent of persistence of M. catarrhalis in the lungs of both genotypes. In conclusion, M. catarrhalis induced a local acute immune response in murine airways. Neither hCEACAM5 expression nor chronic smoke exposure nor a combination of both was sufficient as prerequisites for the establishment of chronic M. catarrhalis colonization. Our results demonstrate the difficulties in mirroring conditions of chronic airways colonization of M. catarrhalis in a murine model.
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Antígeno Carcinoembrionario/metabolismo , Pulmón/metabolismo , Moraxella catarrhalis/inmunología , Infecciones por Moraxellaceae/inmunología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Animales , Antígeno Carcinoembrionario/genética , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Expresión Génica , Humanos , Pulmón/inmunología , Pulmón/microbiología , Ratones Endogámicos C57BL , Ratones Transgénicos , Infecciones por Moraxellaceae/metabolismo , Infecciones por Moraxellaceae/microbiología , Depuración Mucociliar , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Fumar/inmunología , Fumar/metabolismoRESUMEN
The cytosolic nucleotide oligomerization domain (NOD)-like receptors NOD1 and NOD2 are important contributors to the intracellular recognition of pathogens including Chlamydophila pneumoniae, but little is known about their influence on allergen-induced airway inflammation. In BALB/c mice, we observed that infection with C. pneumoniae before systemic sensitization with ovalbumin (OVA) and local OVA airway exposure diminished airway hyperresponsiveness (AHR). Thus, the impact of the NOD1 agonist FK156 and the NOD2 agonist muramyl dipeptide given 6 hours before each sensitization or airway challenge was evaluated regarding AHR, OVA-specific plasma immunoglobulins, bronchoalveolar lavage fluid differentials, and cytokines. Spleen dendritic cells of FK156-treated mice were isolated and cocultured with OVA-specific T cells isolated from DO11.10 mice, and T-cell proliferation was quantified after OVA restimulation. T-cell proliferation was investigated in vivo in lungs and lymph nodes of FK156-treated and OVA-exposed DO11.10 mice. FK156, but not muramyl dipeptide, reduced AHR and pulmonary eosinophilic infiltration if given before OVA sensitization or challenge, whereas T-helper (Th)2 cytokines were not diminished. Dendritic cells from FK156-treated mice evoked less OVA-specific T-cell proliferation as compared with solvent-treated controls. Similarly, antigen-specific T-cell activation in lung tissue was diminished after FK156 treatment. We conclude that NOD1 activation reduced AHR in allergen-induced lung inflammation, which was accompanied by a reduction of allergen-specific T-cell proliferation.
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Alérgenos/inmunología , Hiperreactividad Bronquial/inmunología , Proteína Adaptadora de Señalización NOD1/inmunología , Células Th2/inmunología , Animales , Proliferación Celular , Chlamydophila pneumoniae/inmunología , Citocinas/inmunología , Células Dendríticas/inmunología , Femenino , Inmunoglobulinas/inmunología , Inflamación/inmunología , Ligadura , Pulmón/inmunología , Ganglios Linfáticos/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Proteína Adaptadora de Señalización NOD2/inmunología , Ovalbúmina/inmunología , Sistema Respiratorio/inmunologíaRESUMEN
Surfactant protein A has been shown to enhance opsonization and clearance of Staphylococcus aureus in vitro. Here, the phagocytosis of alveolar S. aureus was investigated in vivo using intravital microscopy. Fluorescence labelled S. aureus Newman cells were intratracheally administered to anesthetized mice and the alveolar surface was observed for fifteen minutes. Confirming previously reported in vitro data, surfactant protein A-deficient mice showed a significantly reduced uptake of bacteria compared to wild-type mice.
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Pulmón/metabolismo , Macrófagos Alveolares/metabolismo , Fagocitosis/fisiología , Proteína A Asociada a Surfactante Pulmonar/fisiología , Staphylococcus aureus/metabolismo , Animales , Pulmón/química , Pulmón/microbiología , Macrófagos Alveolares/química , Macrófagos Alveolares/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Staphylococcus aureus/químicaRESUMEN
Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. In this study, we examine an innate immune recognition pathway that senses pneumococcal infection, triggers type I IFN production, and regulates RANTES production. We found that human and murine alveolar macrophages as well as murine bone marrow macrophages, but not alveolar epithelial cells, produced type I IFNs upon infection with S. pneumoniae. This response was dependent on the pore-forming toxin pneumolysin and appeared to be mediated by a cytosolic DNA-sensing pathway involving the adapter molecule STING and the transcription factor IFN regulatory factor 3. Indeed, DNA was present in the cytosol during pneumococcal infection as indicated by the activation of the AIM2 inflammasome, which is known to sense microbial DNA. Type I IFNs produced by S. pneumoniae-infected macrophages positively regulated gene expression and RANTES production in macrophages and cocultured alveolar epithelial cells in vitro. Moreover, type I IFNs controlled RANTES production during pneumococcal pneumonia in vivo. In conclusion, we identified an immune sensing pathway detecting S. pneumoniae that triggers a type I IFN response and positively regulates RANTES production.
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Quimiocina CCL5/biosíntesis , Factor 3 Regulador del Interferón/fisiología , Interferón Tipo I/biosíntesis , Macrófagos Alveolares/inmunología , Proteínas de la Membrana/fisiología , Mucosa Respiratoria/inmunología , Streptococcus pneumoniae/inmunología , Animales , Comunicación Autocrina/inmunología , Proteínas Bacterianas/fisiología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Técnicas de Cocultivo , Citosol/inmunología , Citosol/metabolismo , ADN Bacteriano/inmunología , ADN Bacteriano/metabolismo , Modelos Animales de Enfermedad , Humanos , Inmunidad Innata , Interferón Tipo I/fisiología , Pulmón/citología , Pulmón/inmunología , Pulmón/metabolismo , Macrófagos Alveolares/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Comunicación Paracrina/inmunología , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Estreptolisinas/fisiologíaRESUMEN
Activin A strongly influences immune responses; yet, few studies have examined its role in infectious diseases. We measured serum activin A levels in two independent tuberculosis (TB) patient cohorts and in patients with pneumonia and sarcoidosis. Serum activin A levels were increased in TB patients compared to healthy controls, including those with positive tuberculin skin tests, and paralleled severity of disease, assessed by X-ray scores. In pneumonia patients, serum activin A levels were also raised, but in sarcoidosis patients, levels were lower. To determine whether blockade of the activin A signaling axis could play a functional role in TB, we harnessed a soluble activin type IIB receptor fused to human IgG1 Fc, ActRIIB-Fc, as a ligand trap in a murine TB model. The administration of ActRIIB-Fc to Mycobacterium tuberculosis-infected mice resulted in decreased bacterial loads and increased numbers of CD4 effector T cells and tissue-resident memory T cells in the lung. Increased frequencies of tissue-resident memory T cells corresponded with downregulated T-bet expression in lung CD4 and CD8 T cells. Altogether, the results suggest a disease-exacerbating role of ActRIIB signaling pathways. Serum activin A may be useful as a biomarker for diagnostic triage of active TB or monitoring of anti-tuberculosis therapy. IMPORTANCE: Tuberculosis remains the leading cause of death by a bacterial pathogen. The etiologic agent of tuberculosis, Mycobacterium tuberculosis, can remain dormant in the infected host for years before causing disease. Significant effort has been made to identify biomarkers that can discriminate between latently infected and actively diseased individuals. We found that serum levels of the cytokine activin A were associated with increased lung pathology and could discriminate between active tuberculosis and tuberculin skin-test-positive healthy controls. Activin A signals through the ActRIIB receptor, which can be blocked by administration of the ligand trap ActRIIB-Fc, a soluble activin type IIB receptor fused to human IgG1 Fc. In a murine model of tuberculosis, we found that ActRIIB-Fc treatment reduced mycobacterial loads. Strikingly, ActRIIB-Fc treatment significantly increased the number of tissue-resident memory T cells. These results suggest a role for ActRIIB signaling pathways in host responses to Mycobacterium tuberculosis and activin A as a biomarker of ongoing disease.
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Mycobacterium tuberculosis , Neumonía , Sarcoidosis , Tuberculosis , Humanos , Ratones , Animales , Ligandos , Tuberculina , Activinas , Inmunoglobulina G , BiomarcadoresRESUMEN
Hospital-acquired pneumonia (HAP) is associated with high mortality and costs, and frequently caused by multidrug-resistant (MDR) bacteria. Although prior antimicrobial therapy is a major risk factor for HAP, the underlying mechanism remains incompletely understood. Here, we demonstrate that antibiotic therapy in hospitalized patients is associated with decreased diversity of the gut microbiome and depletion of short-chain fatty acid (SCFA) producers. Infection experiments with mice transplanted with patient fecal material reveal that these antibiotic-induced microbiota perturbations impair pulmonary defense against MDR Klebsiella pneumoniae. This is dependent on inflammatory monocytes (IMs), whose fatty acid receptor (FFAR)2/3-controlled and phagolysosome-dependent antibacterial activity is compromized in mice transplanted with antibiotic-associated patient microbiota. Collectively, we characterize how clinically relevant antibiotics affect antimicrobial defense in the context of human microbiota, and reveal a critical impairment of IM´s antimicrobial activity. Our study provides additional arguments for the rational use of antibiotics and offers mechanistic insights for the development of novel prophylactic strategies to protect high-risk patients from HAP.
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Antibacterianos , Antiinfecciosos , Humanos , Ratones , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Monocitos , Antiinfecciosos/farmacología , Klebsiella pneumoniae , PulmónRESUMEN
OBJECTIVES: Pneumonia is associated with a high morbidity and mortality worldwide. Streptococcus pneumoniae remains the most common cause of pneumonia, and pneumococcal antibiotic resistance is increasing. The purified bacteriophage endolysin Cpl-1 rapidly and specifically kills pneumococci. We tested the hypothesis that a single dose of recombinant aerosolized Cpl-1 would rescue mice with severe pneumococcal pneumonia. METHODS: Female C57Bl/6 mice (aged 8-12 weeks) were transnasally infected with pneumococci. When severe pneumonia was established 24 h after infection, mice were treated with 25 µL of aerosolized Cpl-1. Survival was monitored for 10 days and the pulmonary and systemic bacterial burdens were assessed. Furthermore, cytokines were quantified in bronchoalveolar lavage fluid, and lung morphology was analysed histologically. RESULTS: The endolysin efficiently reduced pulmonary bacterial counts and averted bacteraemia. Although concentrations of inflammatory cytokines were increased shortly after Cpl-1 inhalation, mice recovered rapidly, as shown by increasing body weight, and inflammatory infiltrates resolved in the lungs, leading to a reduction in mortality of 80%. CONCLUSIONS: Administration of Cpl-1 by inhalation may offer a new therapeutic perspective for the treatment of pneumococcal lung infection.
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Antibacterianos/administración & dosificación , Bacteriófagos/enzimología , Productos Biológicos/administración & dosificación , Endopeptidasas/administración & dosificación , Neumonía Neumocócica/tratamiento farmacológico , Streptococcus pneumoniae/efectos de los fármacos , Administración por Inhalación , Animales , Antibacterianos/aislamiento & purificación , Carga Bacteriana , Productos Biológicos/aislamiento & purificación , Modelos Animales de Enfermedad , Endopeptidasas/aislamiento & purificación , Femenino , Ratones , Ratones Endogámicos C57BL , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and sepsis. Pneumococci can be divided into >90 serotypes that show differences in the pathogenicity and invasiveness. We tested the hypotheses that the innate immune inflammasome pathway is involved in fighting pneumococcal pneumonia and that some invasive pneumococcal types are not recognized by this pathway. We show that human and murine mononuclear cells responded to S. pneumoniae expressing hemolytic pneumolysin by producing IL-1ß. This IL-1ß production depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Some serotype 1, serotype 8, and serotype 7F bacteria, which have previously been associated with increased invasiveness and with production of toxins with reduced hemolytic activity, or bacterial mutants lacking pneumolysin did not stimulate notable IL-1ß production. We further found that NLRP3 was beneficial for mice during pneumonia caused by pneumococci expressing hemolytic pneumolysin and was involved in cytokine production and maintenance of the pulmonary microvascular barrier. Overall, the inflammasome pathway is protective in pneumonia caused by pneumococci expressing hemolytic toxin but is not activated by clinically important pneumococcal sequence types causing invasive disease. The study indicates that a virulence factor polymorphism may substantially affect the recognition of bacteria by the innate immune system.
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Proteínas Portadoras/fisiología , Variación Genética/inmunología , Inflamasomas/metabolismo , Neumonía Neumocócica/inmunología , Neumonía Neumocócica/patología , Estreptolisinas/genética , Animales , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Humanos , Inmunidad Innata/genética , Inflamasomas/fisiología , Interleucina-18/fisiología , Interleucina-1beta/biosíntesis , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/genética , Proteína con Dominio Pirina 3 de la Familia NLR , Proteína Adaptadora de Señalización NOD2/fisiología , Neumonía Neumocócica/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Estreptolisinas/biosíntesis , Estreptolisinas/deficiencia , Receptor Toll-Like 2/deficiencia , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genética , Receptor Toll-Like 9/fisiologíaRESUMEN
Dysfunction of endothelial cells (ECs) lining the inner surface of blood vessels are causative for a number of diseases. Hence, the ability to therapeutically modulate gene expression within ECs is of high therapeutic value in treating diseases such as those associated with lung edema. mRNAs formulated with lipid nanoparticles (LNPs) have emerged as a new drug modality to induce transient protein expression for modulating disease-relevant signal transduction pathways. In the study presented here, we tested the effect of a novel synthetic, nucleoside-modified mRNA encoding COMP-Ang1 (mRNA-76) formulated into a cationic LNP on attenuating inflammation-induced vascular leakage. After intravenous injection, the respective mRNA was found to be delivered almost exclusively to the ECs of the lung, while sparing other vascular beds and bypassing the liver. The mode of action of mRNA-76, such as its activation of the Tie2 signal transduction pathway, was tested by pharmacological studies in vitro and in vivo in respective mouse models. mRNA-76 was found to prevent lung vascular leakage/lung edema as well as neutrophil infiltration in a lipopolysaccharide-challenging model.
RESUMEN
The majority of cases of community-acquired pneumonia are caused by Streptococcus pneumoniae and most studies on pneumococcal host interaction are based on cell culture or animal experiments. Thus, little is known about infections in human lung tissue. Cyclooxygenase-2 and its metabolites play an important regulatory role in lung inflammation. Therefore, we established a pneumococcal infection model on human lung tissue demonstrating mitogen-activated protein kinase (MAPK)-dependent induction of cyclooxygenase-2 and its related metabolites. In addition to alveolar macrophages and the vascular endothelium, cyclooxygenase-2 was upregulated in alveolar type II but not type I epithelial cells, which was confirmed in lungs of patients suffering from acute pneumonia. Moreover, we demonstrated the expression profile of all four E prostanoid receptors at the mRNA level and showed functionality of the E prostanoid(4) receptor by cyclic adenosine monophosphate production. Additionally, in comparison to previous studies, cyclooxygenase-2/prostaglandin E(2) related pro- and anti-inflammatory mediator regulation was partly confirmed in human lung tissue after pneumococcal infection. Overall, cell type-specific and MAPK-dependent cyclooxygenase-2 expression and prostaglandin E(2) formation in human lung tissue may play an important role in the early phase of pneumococcal infections.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Pulmón/enzimología , Pulmón/microbiología , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/metabolismo , Ensayo de Unidades Formadoras de Colonias , Dinoprostona/metabolismo , Células Epiteliales/microbiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica/métodos , Inflamación , Sistema de Señalización de MAP Quinasas , Microscopía Fluorescente/métodos , Infecciones Neumocócicas/enzimología , Prostaglandinas/metabolismo , Alveolos Pulmonares/microbiologíaRESUMEN
Community acquired pneumonia, mainly caused by Streptococcus pneumoniae (S.pn.), is a common cause of death worldwide. Despite adequate antibiotic therapy, pneumococcal pneumonia can induce pulmonary endothelial hyperpermeability leading to acute lung injury, which often requires mechanical ventilation (MV) causing ventilator-induced lung injury (VILI). Endothelial stabilization is mediated by angiopoietin-1 induced Tie2 activation. PEGylated (polyethylene glycol) Tie2-agonist Vasculotide (VT) mimics Angiopietin-1 effects. Recently, VT has been shown to reduce pulmonary hyperpermeability in murine pneumococcal pneumonia. The aim of this study was to determine whether VT reduces lung damage in S.pn. infected and mechanically ventilated mice. Pulmonary hyperpermeability, immune response and bacterial load were quantified in S.pn. infected mice treated with Ampicillin + /-VT and undergoing six hours of MV 24 h post infection. Histopathological lung changes, Tie2-expression and -phosphorylation were evaluated. VT did not alter immune response or bacterial burden, but interestingly combination treatment with ampicillin significantly reduced pulmonary hyperpermeability, histological lung damage and edema formation. Tie2-mRNA expression was reduced by S.pn. infection and/or MV but not restored by VT. Moreover, Tie2 phosphorylation was not affected by VT. These findings indicate that VT may be a promising adjunctive treatment option for prevention of VILI in severe pneumococcal pneumonia.
Asunto(s)
Neumonía Neumocócica , Receptor TIE-2/agonistas , Lesión Pulmonar Inducida por Ventilación Mecánica , Ampicilina/farmacología , Angiopoyetina 1/farmacología , Animales , Antibacterianos/farmacología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos , Permeabilidad , Neumonía Neumocócica/tratamiento farmacológico , Polietilenglicoles/farmacología , ARN Mensajero/farmacología , Respiración Artificial , Streptococcus pneumoniae , Lesión Pulmonar Inducida por Ventilación Mecánica/prevención & controlRESUMEN
Introduction: Inflammation is a major pathological feature of pulmonary arterial hypertension (PAH), particularly in the context of inflammatory conditions such as systemic sclerosis (SSc). The endothelin system and anti-endothelin A receptor (ETA) autoantibodies have been implicated in the pathogenesis of PAH, and endothelin receptor antagonists are routinely used treatments for PAH. However, immunological functions of the endothelin B receptor (ETB) remain obscure. Methods: Serum levels of anti-ETB receptor autoantibodies were quantified in healthy donors and SSc patients with or without PAH. Age-dependent effects of overexpression of prepro-endothelin-1 or ETB deficiency on pulmonary inflammation and the cardiovascular system were studied in mice. Rescued ETB-deficient mice (ETB-/-) were used to prevent congenital Hirschsprung disease. The effects of pulmonary T-helper type 2 (Th2) inflammation on PAH-associated pathologies were analyzed in ETB-/- mice. Pulmonary vascular hemodynamics were investigated in isolated perfused mouse lungs. Hearts were assessed for right ventricular hypertrophy. Pulmonary inflammation and collagen deposition were assessed via lung microscopy and bronchoalveolar lavage fluid analyses. Results: Anti-ETB autoantibody levels were elevated in patients with PAH secondary to SSc. Both overexpression of prepro-endothelin-1 and rescued ETB deficiency led to pulmonary hypertension, pulmonary vascular hyperresponsiveness, and right ventricular hypertrophy with accompanying lymphocytic alveolitis. Marked perivascular lymphocytic infiltrates were exclusively found in ETB-/- mice. Following induction of pulmonary Th2 inflammation, PAH-associated pathologies and perivascular collagen deposition were aggravated in ETB-/- mice. Conclusion: This study provides evidence for an anti-inflammatory role of ETB. ETB seems to have protective effects on Th2-evoked pathologies of the cardiovascular system. Anti-ETB autoantibodies may modulate ETB-mediated immune homeostasis.
Asunto(s)
Hipertensión Arterial Pulmonar , Receptor de Endotelina B , Animales , Autoanticuerpos/inmunología , Endotelina-1/inmunología , Hipertensión Pulmonar Primaria Familiar/inmunología , Humanos , Hipertrofia Ventricular Derecha/inmunología , Inflamación/inmunología , Ratones , Hipertensión Arterial Pulmonar/inmunología , Receptor de Endotelina B/inmunología , Esclerodermia Sistémica/inmunologíaRESUMEN
Pneumonia is the most common cause of the acute respiratory distress syndrome (ARDS). Here, we identified loss of endothelial cystic fibrosis transmembrane conductance regulator (CFTR) as an important pathomechanism leading to lung barrier failure in pneumonia-induced ARDS. CFTR was down-regulated after Streptococcus pneumoniae infection ex vivo or in vivo in human or murine lung tissue, respectively. Analysis of isolated perfused rat lungs revealed that CFTR inhibition increased endothelial permeability in parallel with intracellular chloride ion and calcium ion concentrations ([Cl-]i and [Ca2+]i). Inhibition of the chloride ion-sensitive with-no-lysine kinase 1 (WNK1) protein with tyrphostin 47 or WNK463 replicated the effect of CFTR inhibition on endothelial permeability and endothelial [Ca2+]i, whereas WNK1 activation by temozolomide attenuated it. Endothelial [Ca2+]i transients and permeability in response to inhibition of either CFTR or WNK1 were prevented by inhibition of the cation channel transient receptor potential vanilloid 4 (TRPV4). Mice deficient in Trpv4 (Trpv4-/-) developed less lung edema and protein leak than their wild-type littermates after infection with S. pneumoniae. The CFTR potentiator ivacaftor prevented lung CFTR loss, edema, and protein leak after S. pneumoniae infection in wild-type mice. In conclusion, lung infection caused loss of CFTR that promoted lung edema formation through intracellular chloride ion accumulation, inhibition of WNK1, and subsequent disinhibition of TRPV4, resulting in endothelial calcium ion influx and vascular barrier failure. Ivacaftor prevented CFTR loss in the lungs of mice with pneumonia and may, therefore, represent a possible therapeutic strategy in people suffering from ARDS due to severe pneumonia.