RESUMEN
Excessive neutrophil infiltration of the lungs is a common contributor to immune-related pathology in many pulmonary disease states. In response to pathogenic infection, airway epithelial cells produce hepoxilin A3 (HXA3), initiating neutrophil transepithelial migration. Migrated neutrophils amplify this recruitment by producing a secondary gradient of leukotriene B4 (LTB4). We sought to determine whether this two-step eicosanoid chemoattractant mechanism could be exploited by the pathogen Pseudomonas aeruginosa. ExoU, a P. aeruginosa cytotoxin, exhibits phospholipase A2 (PLA2) activity in eukaryotic hosts, an enzyme critical for generation of certain eicosanoids. Using in vitro and in vivo models of neutrophil transepithelial migration, we evaluated the impact of ExoU expression on eicosanoid generation and function. We conclude that ExoU, by virtue of its PLA2 activity, augments and compensates for endogenous host neutrophil cPLA2α function, leading to enhanced transepithelial migration. This suggests that ExoU expression in P. aeruginosa can circumvent immune regulation at key signaling checkpoints in the neutrophil, resulting in exacerbated neutrophil recruitment.
Asunto(s)
Proteínas Bacterianas/inmunología , Leucotrieno B4/inmunología , Infiltración Neutrófila/inmunología , Infecciones por Pseudomonas/inmunología , Migración Transendotelial y Transepitelial/inmunología , Animales , Western Blotting , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Pseudomonas aeruginosa/patogenicidad , Virulencia/inmunologíaRESUMEN
Eicosanoids are a group of bioactive lipids that are shown to be important mediators of neutrophilic inflammation; selective targeting of their function confers therapeutic benefit in a number of diseases. Neutrophilic airway diseases, including cystic fibrosis, are characterized by excessive neutrophil infiltration into the airspace. Understanding the role of eicosanoids in this process may reveal novel therapeutic targets. The eicosanoid hepoxilin A3 is a pathogen-elicited epithelial-produced neutrophil chemoattractant that directs transepithelial migration in response to infection. Following hepoxilin A3-driven transepithelial migration, neutrophil chemotaxis is amplified through neutrophil production of a second eicosanoid, leukotriene B4 (LTB4). The rate-limiting step of eicosanoid generation is the liberation of arachidonic acid by phospholipase A2, and the cytosolic phospholipase A2 (cPLA2)α isoform has been specifically shown to direct LTB4 synthesis in certain contexts. Whether cPLA2α is directly responsible for neutrophil synthesis of LTB4 in the context of Pseudomonas aeruginosa-induced neutrophil transepithelial migration has not been explored. Human and mouse neutrophil-epithelial cocultures were used to evaluate the role of neutrophil-derived cPLA2α in infection-induced transepithelial signaling by pharmacological and genetic approaches. Primary human airway basal stem cell-derived epithelial cultures and micro-optical coherence tomography, a new imaging modality that captures two- and three-dimensional real-time dynamics of neutrophil transepithelial migration, were applied. Evidence from these studies suggests that cPLA2α expressed by neutrophils, but not epithelial cells, plays a significant role in infection-induced neutrophil transepithelial migration by mediating LTB4 synthesis during migration, which serves to amplify the magnitude of neutrophil recruitment in response to epithelial infection.
Asunto(s)
Antígenos de Plaqueta Humana/metabolismo , Fibrosis Quística/inmunología , Neutrófilos/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Mucosa Respiratoria/inmunología , Migración Transendotelial y Transepitelial , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Comunicación Celular , Línea Celular , Quimiotaxis , Técnicas de Cocultivo , Citosol/metabolismo , Humanos , Leucotrieno B4/metabolismo , Ratones , Neutrófilos/microbiología , Mucosa Respiratoria/microbiología , Mucosa Respiratoria/patología , Tomografía de Coherencia ÓpticaRESUMEN
Neutrophilic infiltration is a leading contributor to pathology in a number of pulmonary disease states, including cystic fibrosis. Hepoxilin A3 (HXA3) is a chemotactic eicosanoid shown to mediate the transepithelial passage of neutrophils in response to infection in several model systems and at multiple mucosal surfaces. Another well-known eicosanoid mediating general neutrophil chemotaxis is leukotriene B4 (LTB4). We sought to distinguish the roles of each eicosanoid in the context of infection of lung epithelial monolayers by Pseudomonas aeruginosa. Using human and mouse in vitro transwell model systems, we used a combination of biosynthetic inhibitors, receptor antagonists, as well as mutant sources of neutrophils to assess the contribution of each chemoattractant in driving neutrophil transepithelial migration. We found that following chemotaxis to epithelial-derived HXA3 signals, neutrophil-derived LTB4 is required to amplify the magnitude of neutrophil migration. LTB4 signaling is not required for migration to HXA3 signals, but LTB4 generation by migrated neutrophils plays a significant role in augmenting the initial HXA3-mediated migration. We conclude that HXA3 and LTB4 serve independent roles to collectively coordinate an effective neutrophilic transepithelial migratory response.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Bacterias/inmunología , Leucotrieno B4/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Migración Transendotelial y Transepitelial/inmunología , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Infecciones Bacterianas/inmunología , Señalización del Calcio , Línea Celular , Quimiotaxis de Leucocito/inmunología , Modelos Animales de Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Infiltración Neutrófila/inmunología , Pseudomonas aeruginosa/inmunología , Receptores de Leucotrieno B4/antagonistas & inhibidores , Receptores de Leucotrieno B4/genética , Receptores de Leucotrieno B4/metabolismo , Migración Transendotelial y Transepitelial/genéticaRESUMEN
Intestinal helminth infections elicit Th2-type immunity, which influences host immune responses to additional threats, such as allergens, metabolic disease, and other pathogens. Th2 immunity involves a shift of the CD4+ T-cell population from type-0 to type-2 (Th2) with increased abundance of interleukin (IL)-4 and IL-13. This study sought to investigate if existing gut-restricted intestinal helminth infections impact bacterial-induced acute airway neutrophil recruitment. C57BL/6 mice were divided into four groups: uninfected; helminth-Heligmosomoides polygyrus infected; Pseudomonas aeruginosa infected; and coinfected. Mice infected with H. polygyrus were incubated for 2 weeks, followed by P. aeruginosa intranasal inoculation. Bronchial alveolar lavage, blood, and lung samples were analyzed. Interestingly, infection with gut-restricted helminths resulted in immunological and structural changes in the lung. These changes include increased lung CD4+ T cells, increased Th2 cytokine expression, and airway goblet cell hyperplasia. Furthermore, coinfected mice exhibited significantly more airspace neutrophil infiltration at 6 hours following P. aeruginosa infection and exhibited an improved rate of survival compared with bacterial infected alone. These results suggest that chronic helminth infection of the intestines can influence and enhance acute airway neutrophil responses to P. aeruginosa infection.
Asunto(s)
Helmintiasis/patología , Parasitosis Intestinales/patología , Pulmón/microbiología , Nematospiroides dubius/aislamiento & purificación , Neutrófilos/inmunología , Pseudomonas aeruginosa/metabolismo , Animales , Helmintiasis/inmunología , Helmintiasis/microbiología , Mediadores de Inflamación/metabolismo , Parasitosis Intestinales/inmunología , Parasitosis Intestinales/microbiología , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Nematospiroides dubius/patogenicidad , Células Th2/inmunologíaRESUMEN
Salmonella spp. and Shigella spp. are responsible for millions of cases of enteric disease each year worldwide. While these pathogens have evolved distinct strategies for interacting with the human intestinal epithelium, they both induce significant proinflammatory responses that result in massive transepithelial migration of neutrophils across the intestinal mucosa. It has previously been shown with Salmonella enterica serotype Typhimurium that the process of neutrophil transmigration is mediated in part by the secretion of hepoxilin A(3) (HXA(3); 8-hydroxy-11,12-epoxy-eicosatetraenoic acid), a potent neutrophil chemoattractant, from the apical surface of infected model intestinal epithelium. This study confirms that HXA(3) is also secreted in response to infection by Shigella flexneri, that it is produced by a pathway involving 12/15-lipoxygenase (12/15-LOX), and that S. enterica serovar Typhimurium and S. flexneri share certain elements in the mechanism(s) that underlies the otherwise separate signal transduction pathways that are engaged to induce polymorphonuclear leukocyte (PMN) transepithelial migration (protein kinase C and extracellular signal-regulated kinases 1 and 2, respectively). PMN transepithelial migration in response to infection with S. flexneri was dependent on 12/15-LOX activity, the enzyme responsible for the initial metabolism of arachidonic acid to HXA(3). Probing further into this pathway, we also found that S. enterica serovar Typhimurium and S. flexneri activate different subtypes of phospholipase A(2), a critical enzyme involved in the liberation of arachidonic acid from cellular membranes. Thus, although S. enterica serovar Typhimurium and S. flexneri utilize different mechanisms for triggering the induction of PMN transepithelial migration, we found that their reliance on 12/15-LOX is conserved, suggesting that enteric pathogens may ultimately stimulate similar pathways for the synthesis and release of HXA(3).
Asunto(s)
Movimiento Celular/inmunología , Neutrófilos/inmunología , Fosfolipasas A2/química , Fosfolipasas A2/inmunología , Salmonella typhimurium/enzimología , Salmonella typhimurium/inmunología , Shigella flexneri/enzimología , Shigella flexneri/inmunología , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/metabolismo , Línea Celular , Ensayos de Migración de Leucocitos , Humanos , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , IsoenzimasRESUMEN
Neutrophil breach of the mucosal surface is a common pathological consequence of infection. We present an advanced co-culture model to explore neutrophil transepithelial migration utilizing airway mucosal barriers differentiated from primary human airway basal cells and examined by advanced imaging. Human airway basal cells were differentiated and cultured at air-liquid interface (ALI) on the underside of 3 µm pore-sized transwells, compatible with the study of transmigrating neutrophils. Inverted ALIs exhibit beating cilia and mucus production, consistent with conventional ALIs, as visualized by micro-optical coherence tomography (µOCT). µOCT is a recently developed imaging modality with the capacity for real time two- and three-dimensional analysis of cellular events in marked detail, including neutrophil transmigratory dynamics. Further, the newly devised and imaged primary co-culture model recapitulates key molecular mechanisms that underlie bacteria-induced neutrophil transepithelial migration previously characterized using cell line-based models. Neutrophils respond to imposed chemotactic gradients, and migrate in response to Pseudomonas aeruginosa infection of primary ALI barriers through a hepoxilin A3-directed mechanism. This primary cell-based co-culture system combined with µOCT imaging offers significant opportunity to probe, in great detail, micro-anatomical and mechanistic features of bacteria-induced neutrophil transepithelial migration and other important immunological and physiological processes at the mucosal surface.
Asunto(s)
Técnicas de Cultivo de Célula , Técnicas de Cocultivo , Inflamación/metabolismo , Inflamación/patología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Línea Celular , Movimiento Celular/inmunología , Polaridad Celular , Quimiotaxis de Leucocito/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Técnica del Anticuerpo Fluorescente , Humanos , Inflamación/inmunología , Inflamación/microbiología , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/microbiologíaRESUMEN
Gut commensal bacteria contribute to the pathogenesis of inflammatory bowel disease, in part by activating the inflammasome and inducing secretion of interleukin-1ß (IL-1ß). Although much has been learned about inflammasome activation by bacterial pathogens, little is known about how commensals carry out this process. Accordingly, we investigated the mechanism of inflammasome activation by representative commensal bacteria, the Gram-positive Bifidobacterium longum subspecies infantis and the Gram-negative Bacteroides fragilis. B. infantis and B. fragilis induced IL-1ß secretion by primary mouse bone marrow-derived macrophages after overnight incubation. IL-1ß secretion also occurred in response to heat-killed bacteria and was only partly reduced when phagocytosis was inhibited with cytochalasin D. Similar results were obtained with a wild-type immortalized mouse macrophage cell line but neither B. infantis nor B. fragilis induced IL-1ß secretion in a mouse macrophage line lacking the nucleotide-binding/leucine-rich repeat pyrin domain containing 3 (NLRP3) inflammasome. IL-1ß secretion in response to B. infantis and B. fragilis was significantly reduced when the wild-type macrophage line was treated with inhibitors of potassium efflux, either increased extracellular potassium concentrations or the channel blocker ruthenium red. Both live and heat-killed B. infantis and B. fragilis also induced IL-1ß secretion by human macrophages (differentiated THP-1 cells or primary monocyte-derived macrophages) after 4 hours of infection, and the secretion was inhibited by raised extracellular potassium and ruthenium red but not by cytochalasin D. Taken together, our findings indicate that the commensal bacteria B. infantis and B. fragilis activate the NLRP3 inflammasome in both mouse and human macrophages by a mechanism that involves potassium efflux and that does not require bacterial viability or phagocytosis.
Asunto(s)
Bifidobacterium longum/fisiología , Inflamasomas/metabolismo , Macrófagos/microbiología , Viabilidad Microbiana , Fagocitosis , Potasio/metabolismo , Animales , Transporte Biológico , Células de la Médula Ósea/citología , Caspasa 1/metabolismo , Línea Celular , Humanos , Interleucina-1beta/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
Mucosal surfaces serve as protective barriers against pathogenic organisms. Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils. This process has the potential to be destructive to tissues if excessive or held in an unresolved state. Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration. This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil. Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface. The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1). Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000). The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls. This in vitro model system can be manipulated and applied to other mucosal surfaces. Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections. A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.
Asunto(s)
Técnicas de Cocultivo/métodos , Neutrófilos/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Migración Transendotelial y Transepitelial/inmunología , Línea Celular , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Células Epiteliales/patología , Humanos , Pulmón/citología , Pulmón/inmunología , Pulmón/microbiología , Neutrófilos/microbiología , Neutrófilos/patología , Infecciones por Pseudomonas/patologíaRESUMEN
Pregnancy is a leading risk factor for severe complications during an influenza virus infection. Women infected during their second and third trimesters are at increased risk for severe cardiopulmonary complications, premature delivery, and death. Here, we establish a murine model of aerosolized influenza infection during pregnancy. We find significantly altered innate antiviral responses in pregnant mice, including decreased levels of IFN-ß, IL-1α, and IFN-γ at early time points of infection. We also find reduced cytotoxic T cell activity and delayed viral clearance. We further demonstrate that pregnancy levels of the estrogen 17-ß-estradiol are able to induce key anti-inflammatory phenotypes in immune responses to the virus independently of other hormones or pregnancy-related stressors. We conclude that elevated estrogen levels result in an attenuated anti-viral immune response, and that pregnancy-associated morbidities occur in the context of this anti-inflammatory phenotype.
Asunto(s)
Inmunidad Adaptativa , Estrógenos/metabolismo , Inmunidad Innata , Infecciones por Orthomyxoviridae/inmunología , Orthomyxoviridae/inmunología , Inmunidad Adaptativa/efectos de los fármacos , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/inmunología , Citocinas/biosíntesis , Citocinas/inmunología , Estrógenos/administración & dosificación , Estrógenos/sangre , Femenino , Inmunidad Innata/efectos de los fármacos , Leucocitos/inmunología , Leucocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Embarazo , Transducción de SeñalRESUMEN
In human intestinal disease induced by Salmonella enterica serotype Typhimurium (S. typhimurium) transepithelial migration of polymorphonuclear leukocytes (PMNs) rapidly follows attachment of the bacteria to the epithelial apical membrane. Previously, we have shown that the S. typhimurium effector protein, SipA, plays a pivotal role in signalling epithelial cell responses that lead to the transepithelial migration of PMNs. Thus, the objective of this study was to determine the functional domain of SipA that regulates this signalling event. SipA was divided into two fragments: the SipAb C-terminal fragment(426-684) (259 AA), which binds actin, and the SipAa fragment(2-425) (424 AA), which a role has yet to be described. In both in vitro and in vivo models of S. typhimurium-induced intestinal inflammation the SipAa fragment exhibited a profound ability to induce PMN transmigration, whereas the SipAb actin-binding domain failed to induce PMN transmigration. Subsequent mapping of the SipAa domain identified a 131-amino-acid region (SipAa3(294-424)) responsible for modulating PMN transepithelial migration. Interestingly, neither intracellular translocation nor actin association of SipA was necessary for its ability to induce PMN transepithelial migration. As these results indicate SipA has at least two separate functional domains, we speculate that during infection S. typhimurium requires delivery of SipA to both extracellular and intracellular spaces to maximize pro-inflammatory responses and mechanisms of bacterial invasion.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Mucosa Intestinal , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Infiltración Neutrófila , Estructura Terciaria de Proteína , Infecciones por Salmonella/metabolismo , Salmonella typhimurium , Animales , Proteínas Bacterianas/genética , Movimiento Celular/fisiología , Colon/microbiología , Colon/patología , Femenino , Humanos , Interleucina-8/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidadRESUMEN
T cell activation by dendritic cells (DCs) is critical to the initiation of adaptive immune responses and protection against pathogens. Here, we demonstrate that a specialized DC subset in Peyer's patches (PPs) mediates the rapid activation of pathogen specific T cells. This DC subset is characterized by the expression of the chemokine receptor CCR6 and is found only in PPs. CCR6(+) DCs were recruited into the dome regions of PPs upon invasion of the follicle associated epithelium (FAE) by an enteric pathogen and were responsible for the rapid local activation of pathogen-specific T cells. CCR6-deficient DCs were unable to respond to bacterial invasion of PPs and failed to initiate T cell activation, resulting in reduced defense against oral infection. Thus, CCR6-dependent regulation of DCs is responsible for localized T cell dependent defense against entero-invasive pathogens.