RESUMEN
Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia. Protease-activated receptor 2 (PAR2) is expressed by different cell types in the lungs and can mediate inflammatory responses. We sought to determine the role of PAR2 during pneumococcal pneumonia. Pneumococcal pneumonia or sepsis was induced in wild-type and PAR2 knock-out (Par2-/-) mice by infection with viable S. pneumoniae. Par2-/- mice demonstrated improved host defense, a largely preserved lung barrier integrity, and reduced mortality during pneumococcal pneumonia. PAR2 deficiency did not influence bacterial growth after intravenous infection. Inhibition of the endogenous PAR2 activating proteases tissue factor/factor VIIa or tryptase did not impact on bacterial burdens during pneumonia. In a PAR2 reporter cell line it was demonstrated that S. pneumoniae-derived proteases are able to cleave PAR2. These results show that S. pneumoniae is able to cleave and exploit PAR2 to disseminate systemically from the airways.
Asunto(s)
Neumonía Neumocócica/microbiología , Receptor PAR-2 , Streptococcus pneumoniae/fisiología , Animales , Carga Bacteriana , Coagulación Sanguínea , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Proteínas del Helminto/farmacología , Humanos , Inflamación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neumonía Neumocócica/patología , Organismos Libres de Patógenos EspecíficosRESUMEN
Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia (CAP). Granzyme A (GzmA) is a serine protease produced by a variety of cell types involved in the immune response. We sought to determine the role of GzmA on the host response during pneumococcal pneumonia. GzmA was measured in bronchoalveolar lavage fluid (BALF) harvested from CAP patients from the infected and contralateral uninfected side and in lung tissue slides from CAP patients and controls. In CAP patients, GzmA levels were increased in BALF obtained from the infected lung. Human lungs showed constitutive GzmA expression by both parenchymal and nonparenchymal cells. In an experimental setting, pneumonia was induced in wild-type (WT) and GzmA-deficient (GzmA(-/-)) mice by intranasal inoculation of S. pneumoniae In separate experiments, WT and GzmA(-/-) mice were treated with natural killer (NK) cell depleting antibodies. Upon infection with S. pneumoniae, GzmA(-/-) mice showed a better survival and lower bacterial counts in BALF and distant body sites compared with WT mice. Although NK cells showed strong GzmA expression, NK cell depletion did not influence bacterial loads in either WT or GzmA(-/-) mice. These results implicate that GzmA plays an unfavorable role in host defense during pneumococcal pneumonia by a mechanism that does not depend on NK cells.
Asunto(s)
Granzimas/fisiología , Neumonía Neumocócica/enzimología , Streptococcus pneumoniae/inmunología , Animales , Líquido del Lavado Bronquioalveolar , Estudios de Casos y Controles , Femenino , Humanos , Inmunidad Celular , Células Asesinas Naturales/fisiología , Pulmón/enzimología , Pulmón/inmunología , Pulmón/microbiología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Infiltración Neutrófila , Neumonía Neumocócica/inmunología , Neumonía Neumocócica/microbiologíaRESUMEN
OBJECTIVES: Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia. In patients, thrombocytopenia is correlated with an adverse outcome of pneumonia. Platelets can modulate the host response to infection in several ways, that is, by facilitating clot formation, production of antimicrobial proteins, and interaction with neutrophils. We studied the effect of thrombocytopenia during murine pneumococcal pneumonia. DESIGN: Animal study. SETTING: University research laboratory. SUBJECTS: Mice. INTERVENTIONS: Pneumonia was induced by intranasal inoculation of S. pneumoniae. Platelets were depleted by anti-mouse thrombocyte serum; controls received nonimmunogenic serum. In separate studies, mice were treated with the platelet P2Y12 receptor inhibitor clopidogrel or placebo. MEASUREMENTS AND MAIN RESULTS: Thrombocytopenic mice (platelet counts < 1% of uninfected controls) showed a reduced survival during pneumococcal pneumonia (27% vs 75% among controls; p = 0.003), which was associated with higher bacterial loads in lungs, spleen, and blood. Thrombocytopenic mice showed enhanced coagulation activation (thrombin-antithrombin complexes) in plasma. Proinflammatory cytokine levels were higher in plasma but not in lungs of thrombocytopenic mice. Although clopidogrel treatment strongly prolonged the bleeding time, it did not impact on bacterial loads during pneumococcal pneumonia. CONCLUSIONS: Platelets play a protective role during pneumococcal pneumonia independent of their aggregation.
Asunto(s)
Neumonía Neumocócica/inmunología , Streptococcus pneumoniae , Trombocitopenia/inmunología , Animales , Antitrombina III/metabolismo , Clopidogrel , Citocinas/metabolismo , Modelos Animales de Enfermedad , Ratones , Péptido Hidrolasas/metabolismo , Neumonía Neumocócica/epidemiología , Neumonía Neumocócica/patología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Trombocitopenia/epidemiología , Trombocitopenia/patología , Ticlopidina/análogos & derivados , Ticlopidina/farmacologíaRESUMEN
BACKGROUND: Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia. Mast cells (MCs) are located mainly at the host-environment interface where they function as sentinels. OBJECTIVE: Our goal was to study the role of MCs during pneumonia caused by S. pneumoniae. METHODS: Lung tissue of patients who had died from pneumococcal pneumonia or a nonpulmonary cause was stained for MCs and tryptase. Wild-type (WT) and MC-deficient (Kit(W-sh/W-sh)) mice were observed or sacrificed after induction of pneumonia by intranasal inoculation of S. pneumoniae. In separate experiments, WT mice were treated with doxantrazole or cromoglycate, which are MC stabilizing agents. RESULTS: The constitutive presence of tryptase-positive MCs was reduced in affected lungs from pneumonia patients. Kit(W-sh/W-sh) mice showed a prolonged survival during the first few days after median lethal dose (LD)100 and LD50 infection, while overall mortality did not differ from that in WT mice. Relative to WT mice, Kit(W-sh/W-sh) mice showed reduced bacterial counts with less bacterial dissemination to distant organs and less inflammation. Neither doxantrazole nor cromoglycate influenced antibacterial defense or inflammatory responses after airway infection with S. pneumoniae. CONCLUSIONS: MCs exhibit an unfavorable role in host defense during pneumococcal pneumonia by a mechanism independent of degranulation.
Asunto(s)
Mastocitos/fisiología , Neumonía Neumocócica/inmunología , Streptococcus pneumoniae/inmunología , Animales , Carga Bacteriana , Femenino , Interacciones Huésped-Patógeno/inmunología , Humanos , Pulmón/microbiología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Neumonía Neumocócica/microbiología , Neumonía Neumocócica/patologíaRESUMEN
BACKGROUND: Recombinant human activated protein C (APC) improves survival of patients with severe sepsis; this beneficial effect is especially apparent in patients with pneumococcal pneumonia. The aim of this study was to determine the effect of APC treatment initiated after induction of pneumococcal pneumonia on pulmonary coagulation, inflammation, and survival, with or without concurrent antibiotic therapy. METHODS: Mice were infected intranasally with viable Streptococcus pneumoniae and were treated intraperitoneally after 24 h of infection with vehicle, recombinant mouse (rm) APC (125 µg), ceftriaxone (500 µg), or rm-APC plus ceftriaxone. Treatment with rm-APC or vehicle was repeated every 8 h for a maximum of 96 h. Animals were either killed 48 h after infection or were monitored in a survival study (with an extra dose of ceftriaxone given after 72 h). RESULTS: Rm-APC treatment inhibited pulmonary activation of coagulation, as reflected by lower levels of thrombin-antithrombin complexes and D-dimer. Rm-APC did not affect the pulmonary levels of 55 inflammatory mediators in the context of antibiotic therapy. Rm-APC added to ceftriaxone markedly improved survival, compared with ceftriaxone treatment alone. CONCLUSIONS: Rm-APC inhibits pulmonary activation of coagulation and, when added to antibiotic therapy, improves survival in murine pneumococcal pneumonia.
Asunto(s)
Trastornos de las Proteínas de Coagulación/tratamiento farmacológico , Trastornos de las Proteínas de Coagulación/microbiología , Neumonía Neumocócica/tratamiento farmacológico , Proteína C/uso terapéutico , Animales , Antibacterianos/uso terapéutico , Factores de Coagulación Sanguínea/antagonistas & inhibidores , Ceftriaxona/administración & dosificación , Ceftriaxona/uso terapéutico , Evaluación Preclínica de Medicamentos , Quimioterapia Combinada , Humanos , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos C57BL , Neumonía Neumocócica/complicaciones , Proteína C/administración & dosificación , Proteína C/farmacología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéuticoRESUMEN
BACKGROUND: Critically ill patients are at a constant risk of direct (e.g., by pneumonia) or indirect lung injury (e.g., by sepsis). Excessive alveolar fibrin deposition is a prominent feature of lung injury, undermining pulmonary integrity and function. METHODS: We examined the effect of local administration of recombinant human tissue factor pathway inhibitor (rh-TFPI), a natural anticoagulant, in two well-established models of lung injury in rats. Rats received intratracheal instillation of Pseudomonas aeruginosa, causing direct lung injury, or they received an intravenous injection of Escherichia coli lipopolysaccharide (LPS), causing indirect lung injury. Rats were randomized to local treatment with rh-TFPI or placebo through repeated nebulization. RESULTS: Challenge with P. aeruginosa or LPS was associated with increased coagulation and decreased fibrinolysis in bronchoalveolar lavage fluid (BALF) and plasma. Rh-TFPI levels in BALF increased after nebulization, whereas plasma rh-TFPI levels remained low and systemic TFPI activity was not affected. Nebulization of rh-TFPI attenuated pulmonary and systemic coagulation in both models, without affecting fibrinolysis. Nebulization of rh-TFPI modestly reduced the inflammatory response and bacterial growth of P. aeruginosa in the alveolar compartment. CONCLUSIONS: Local treatment with rh-TFPI does not alter systemic TFPI activity; however, it attenuates both pulmonary and systemic coagulopathy. Furthermore, nebulized rh-TFPI modestly reduces the pulmonary inflammatory response and allows increased bacterial clearance in rats with direct lung injury caused by P. aeruginosa.
Asunto(s)
Antiinflamatorios/farmacología , Anticoagulantes/farmacología , Lipoproteínas/farmacología , Lesión Pulmonar/tratamiento farmacológico , Animales , Antiinflamatorios/administración & dosificación , Anticoagulantes/administración & dosificación , Coagulación Sanguínea/efectos de los fármacos , Líquido del Lavado Bronquioalveolar , Modelos Animales de Enfermedad , Fibrinólisis/efectos de los fármacos , Humanos , Inflamación/tratamiento farmacológico , Lipopolisacáridos/toxicidad , Lipoproteínas/administración & dosificación , Lesión Pulmonar/patología , Masculino , Pseudomonas aeruginosa , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Proteínas RecombinantesRESUMEN
Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. Coagulation and inflammation interact in the host response to infection. Tissue factor pathway inhibitor (TFPI) is a natural anticoagulant protein that inhibits tissue factor (TF), the main activator of inflammation-induced coagulation. It was the objective of this study to investigate the effect of endogenous TFPI levels on coagulation, inflammation and bacterial growth during S. pneumoniae pneumonia in mice. The effect of low endogenous TFPI levels was studied by administration of a neutralising anti-TFPI antibody to wild-type mice, and by using genetically modified mice expressing low levels of TFPI, due to a genetic deletion of the first Kunitz domain of TFPI (TFPIK1(-/-)) rescued with a human TFPI transgene. Pneumonia was induced by intranasal inoculation with S. pneumoniae and samples were obtained at 6, 24 and 48 hours after infection. Anti-TFPI reduced TFPI activity by ~50 %. Homozygous lowTFPI mice and heterozygous controls had ~10 % and ~50 % of normal TFPI activity, respectively. TFPI levels did not influence bacterial growth or dissemination. Whereas lung pathology was unaffected in all groups, mice with ~10 % (but not with ~50 %) of TFPI levels displayed elevated lung cytokine and chemokine concentrations 24 hours after infection. None of the groups with low TFPI levels showed an altered procoagulant response in lungs or plasma during pneumonia. These data argue against an important role for endogenous TFPI in the antibacterial, inflammatory and procoagulant response during pneumococcal pneumonia.
Asunto(s)
Coagulación Sanguínea , Lipoproteínas/metabolismo , Pulmón/microbiología , Neumonía Neumocócica/microbiología , Streptococcus pneumoniae/patogenicidad , Animales , Carga Bacteriana , Citocinas/sangre , Modelos Animales de Enfermedad , Genotipo , Interacciones Huésped-Patógeno , Humanos , Mediadores de Inflamación/sangre , Lipoproteínas/sangre , Lipoproteínas/deficiencia , Lipoproteínas/genética , Pulmón/inmunología , Pulmón/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Neumonía Neumocócica/sangre , Neumonía Neumocócica/genética , Neumonía Neumocócica/inmunología , Streptococcus pneumoniae/crecimiento & desarrollo , Streptococcus pneumoniae/inmunología , Factores de TiempoRESUMEN
Pulmonary coagulopathy is intrinsic to pulmonary injury including pneumonia. Anticoagulant strategies could benefit patients with pneumonia, but systemic administration of anticoagulant agents may lead to suboptimal local levels and may cause systemic hemorrhage. We hypothesized nebulization to provide a safer and more effective route for local administration of anticoagulants. Therefore, we aimed to examine feasibility and safety of nebulization of recombinant human tissue factor pathway inhibitor (rh-TFPI) in a well-established rat model of Streptococcus (S.) pneumoniae pneumonia. Thirty minutes before and every 6 hours after intratracheal instillation of S. pneumonia causing pneumonia, rats were subjected to local treatment with rh-TFPI or placebo, and sacrificed after 42 hours. Pneumonia was associated with local as well as systemic activation of coagulation. Nebulization of rh-TFPI resulted in high levels of rh-TFPI in bronchoalveolar lavage fluid, which was accompanied by an attenuation of pulmonary coagulation. Systemic rh-TFPI levels remained undetectable, and systemic TFPI activity and systemic coagulation were not affected. Histopathology revealed no bleeding in the lungs. We conclude that nebulization of rh-TFPI seems feasible and safe; local anticoagulant treatment with rh-TFPI attenuates pulmonary coagulation, while not affecting systemic coagulation in a rat model of S. pneumoniae pneumonia.
Asunto(s)
Anticoagulantes/administración & dosificación , Coagulación Sanguínea/efectos de los fármacos , Líquido del Lavado Bronquioalveolar/química , Lipoproteínas/administración & dosificación , Neumonía Neumocócica/tratamiento farmacológico , Administración Intranasal , Animales , Anticoagulantes/efectos adversos , Modelos Animales de Enfermedad , Estudios de Factibilidad , Humanos , Lipoproteínas/efectos adversos , Masculino , Nebulizadores y Vaporizadores , Neumonía Neumocócica/sangre , Neumonía Neumocócica/microbiología , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Mast cells are implicated in allergic and innate immune responses in asthma, although their role in models using an allergen relevant for human disease is incompletely understood. House dust mite (HDM) allergy is common in asthma patients. Our aim was to investigate the role of mast cells in HDM-induced allergic lung inflammation. METHODS: Wild-type (Wt) and mast cell-deficient Kit(w-sh) mice on a C57BL/6 background were repetitively exposed to HDM via the airways. RESULTS: HDM challenge resulted in a rise in tryptase activity in bronchoalveolar lavage fluid (BALF) of Wt mice, indicative of mast cell activation. Kit(w-sh) mice showed a strongly attenuated HDM- induced recruitment of eosinophils in BALF and lung tissue, accompanied by reduced pulmonary levels of the eosinophil chemoattractant eotaxin. Remarkably, Kit(w-sh) mice demonstrated an unaltered capacity to develop lung pathology and increased mucus production in response to HDM. The increased plasma IgE in response to HDM in Wt mice was absent in Kit(w-sh) mice. CONCLUSION: These data contrast with previous reports on the role of mast cells in models using ovalbumin as allergen in that C57BL/6 Kit(w-sh) mice display a selective impairment of eosinophil recruitment without differences in other features of allergic inflammation.