RESUMEN
Streptococcus pneumoniae is a major cause of pneumonia, wherein infection of respiratory mucosa drives a robust influx of neutrophils. We have previously shown that S. pneumoniae infection of the respiratory epithelium induces the production of the 12-lipoxygenase (12-LOX)-dependent lipid inflammatory mediator hepoxilin A3, which promotes recruitment of neutrophils into the airways, tissue damage, and lethal septicemia. Pneumolysin (PLY), a member of the cholesterol-dependent cytolysin (CDC) family, is a major S. pneumoniae virulence factor that generates â¼25-nm diameter pores in eukaryotic membranes and promotes acute inflammation, tissue damage, and bacteremia. We show that a PLY-deficient S. pneumoniae mutant was impaired in triggering human neutrophil transepithelial migration in vitro. Ectopic production of PLY endowed the nonpathogenic Bacillus subtilis with the ability to trigger neutrophil recruitment across human-cultured monolayers. Purified PLY, several other CDC family members, and the α-toxin of Clostridium septicum, which generates pores with cross-sectional areas nearly 300 times smaller than CDCs, reproduced this robust neutrophil transmigration. PLY non-pore-forming point mutants that are trapped at various stages of pore assembly did not recruit neutrophils. PLY triggered neutrophil recruitment in a 12-LOX-dependent manner in vitro. Instillation of wild-type PLY but not inactive derivatives into the lungs of mice induced robust 12-LOX-dependent neutrophil migration into the airways, although residual inflammation induced by PLY in 12-LOX-deficient mice indicates that 12-LOX-independent pathways also contribute to PLY-triggered pulmonary inflammation. These data indicate that PLY is an important factor in promoting hepoxilin A3-dependent neutrophil recruitment across pulmonary epithelium in a pore-dependent fashion.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Infiltración Neutrófila/inmunología , Streptococcus pneumoniae/patogenicidad , Estreptolisinas/metabolismo , Migración Transendotelial y Transepitelial/inmunología , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/inmunología , Animales , Bacillus subtilis/genética , Bacillus subtilis/patogenicidad , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Línea Celular , Membrana Celular/patología , Clostridium septicum/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/inmunología , Infecciones Neumocócicas/patología , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/inmunología , Estreptolisinas/genética , Factores de Virulencia/metabolismoRESUMEN
A feature shared by many inflammatory lung diseases is excessive neutrophilic infiltration. Neutrophil homing to airspaces involve multiple factors produced by several distinct cell types. Hepoxilin A(3) is a neutrophil chemoattractant produced by pathogen-infected epithelial cells that is hypothesized to facilitate neutrophil breach of mucosal barriers. Using a Transwell model of lung epithelial barriers infected with Pseudomonas aeruginosa, we explored the role of hepoxilin A(3) in neutrophil transepithelial migration. Pharmacological inhibitors of the enzymatic pathways necessary to generate hepoxilin A(3), including phospholipase A(2) and 12-lipoxygenase, potently interfere with P. aeruginosa-induced neutrophil transepithelial migration. Both transformed and primary human lung epithelial cells infected with P. aeruginosa generate hepoxilin A(3) precursor arachidonic acid. All four known lipoxygenase enzymes capable of synthesizing hepoxilin A(3) are expressed in lung epithelial cell lines, primary small airway epithelial cells, and human bronchial epithelial cells. Lung epithelial cells produce increased hepoxilin A(3) and lipid-derived neutrophil chemotactic activity in response to P. aeruginosa infection. Lipid-derived chemotactic activity is soluble epoxide hydrolase sensitive, consistent with hepoxilin A(3) serving a chemotactic role. Stable inhibitory structural analogs of hepoxilin A(3) are capable of impeding P. aeruginosa-induced neutrophil transepithelial migration. Finally, intranasal infection of mice with P. aeruginosa promotes enhanced cellular infiltrate into the airspace, as well as increased concentration of the 12-lipoxygenase metabolites hepoxilin A(3) and 12-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid. Data generated from multiple models in this study provide further evidence that hepoxilin A(3) is produced in response to lung pathogenic bacteria and functions to drive neutrophils across epithelial barriers.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Araquidonato 12-Lipooxigenasa/inmunología , Barrera Alveolocapilar/inmunología , Neutrófilos/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Migración Transendotelial y Transepitelial/inmunología , Ácido 8,11,14-Eicosatrienoico/inmunología , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Araquidonato 12-Lipooxigenasa/metabolismo , Barrera Alveolocapilar/metabolismo , Barrera Alveolocapilar/microbiología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Femenino , Humanos , Masculino , Ratones , Neutrófilos/metabolismo , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/metabolismo , Infecciones por Pseudomonas/metabolismo , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/microbiologíaRESUMEN
The widespread use of statins for hypercholesterolemia has uncovered pleiotropic anti-inflammatory properties that were unexpected based on the drugs' original design; yet, mechanisms for these protective actions remain uncertain. In this study lovastatin triggered biosynthesis of the anti-inflammatory and pro-resolving mediator 15-epi-lipoxin A(4) (15-epi-LXA(4)). During interactions between human neutrophils and airway epithelial cells, the statin-induced increase in 15-epi-LXA(4) was associated with increased 14,15-epoxyeicosatrienoic acid (14,15-EET) generation. When added to activated neutrophils, 14,15-EET enhanced 15-epi-LXA(4) biosynthesis. In a murine model of airway mucosal injury and inflammation, lovastatin increased 15-epi-LXA(4) formation in vivo and markedly decreased acute lung inflammation. Administration of 15-epi-LXA(4) also inhibited lung inflammation in an additive manner with lovastatin. Together, these results indicate that statin-triggered 15-epi-LXA(4) generation during human leukocyte-airway epithelial cell interactions is an endogenous mechanism for statin-mediated tissue protection at mucosal surfaces that may also be relevant in the statins' ability to stimulate the resolution of inflammation.
Asunto(s)
Anticolesterolemiantes/farmacología , Mediadores de Inflamación/inmunología , Lipoxinas/inmunología , Lovastatina/farmacología , Neumonía/inmunología , Mucosa Respiratoria/inmunología , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/inmunología , Ácido 8,11,14-Eicosatrienoico/metabolismo , Enfermedad Aguda , Animales , Línea Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Hipercolesterolemia/tratamiento farmacológico , Hipercolesterolemia/inmunología , Hipercolesterolemia/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Lipoxinas/metabolismo , Masculino , Ratones , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neumonía/tratamiento farmacológico , Neumonía/metabolismo , Mucosa Respiratoria/metabolismoRESUMEN
Neutrophil transmigration across mucosal surfaces contributes to dysfunction of epithelial barrier properties, a characteristic underlying many mucosal inflammatory diseases. Thus, insight into the directional movement of neutrophils across epithelial barriers will provide important information relating to the mechanisms of such inflammatory disorders. The eicosanoid hepoxilin A(3), an endogenous product of 12-lipoxygenase activity, is secreted from the apical surface of the epithelial barrier and establishes a chemotactic gradient to guide neutrophils from the submucosa across epithelia to the luminal site of an inflammatory stimulus, the final step in neutrophil recruitment. Currently, little is known regarding how hepoxilin A(3) is secreted from the intestinal epithelium during an inflammatory insult. In this study, we reveal that hepoxilin A(3) is a substrate for the apical efflux ATP-binding protein transporter multidrug resistance-associated protein 2 (MRP2). Moreover, using multiple in vitro and in vivo models, we show that induction of intestinal inflammation profoundly up-regulates apical expression of MRP2, and that interfering with hepoxilin A(3) synthesis and/or inhibition of MRP2 function results in a marked reduction in inflammation and severity of disease. Lastly, examination of inflamed intestinal epithelia in human biopsies revealed up-regulation of MRP2. Thus, blocking hepoxilin A(3) synthesis and/or inhibiting MRP2 may lead to the development of new therapeutic strategies for the treatment of epithelial-associated inflammatory conditions.