RESUMEN
Leukotriene A4 hydrolase (LTA4H) is a bifunctional enzyme, with dual activities critical in defining the scale of tissue inflammation and pathology. LTA4H classically operates intracellularly, primarily within myeloid cells, to generate pro-inflammatory leukotriene B4. However, LTA4H also operates extracellularly to degrade the bioactive collagen fragment proline-glycine-proline to limit neutrophilic inflammation and pathological tissue remodeling. While the dichotomous functions of LTA4H are dictated by location, the cellular source of extracellular enzyme remains unknown. We demonstrate that airway extracellular LTA4H concentrations are governed by the level of pulmonary vascular permeability and influx of an abundant repository of blood-borne enzyme. In turn, blood LTA4H originates from liver hepatocytes, being released constitutively but further upregulated during an acute phase response. These findings have implications for our understanding of how inflammation and repair are regulated and how perturbations to the LTA4H axis may manifest in pathologies of chronic diseases.
RESUMEN
Acetylcholine (ACh) from neuronal and non-neuronal sources plays an important role in the regulation of immune responses and is associated with the development of several disease pathologies. We have previously demonstrated that group 2 innate lymphoid cell (ILC2)-derived ACh is required for optimal type 2 responses to parasitic infection and therefore sought to determine whether this also plays a role in allergic inflammation. RoraCre+ChatLoxP mice (in which ILC2s cannot synthesize ACh) were exposed to an allergenic extract of the fungus Alternaria alternata, and immune responses in the airways and lung tissues were analyzed. Airway neutrophilia and expression of the neutrophil chemoattractants CXCL1 and CXCL2 were enhanced 24 h after exposure, suggesting that ILC2-derived ACh plays a role in limiting excessive pulmonary neutrophilic inflammation. The effect of non-selective depletion of ACh was examined by intranasal administration of a stable parasite-secreted acetylcholinesterase. Depletion of airway ACh in this manner resulted in a more profound enhancement of neutrophilia and chemokine expression, suggesting multiple cellular sources for the release of ACh. In contrast, depletion of ACh inhibited Alternaria-induced activation of ILC2s, suppressing the expression of IL-5, IL-13, and subsequent eosinophilia. Depletion of ACh reduced macrophages with an alternatively activated M2 phenotype and an increase in M1 macrophage marker expression. These data suggest that ACh regulates allergic airway inflammation in several ways, enhancing ILC2-driven eosinophilia but suppressing neutrophilia through reduced chemokine expression.
Asunto(s)
Eosinofilia , Neumonía , Acetilcolina/farmacología , Acetilcolinesterasa/metabolismo , Animales , Inmunidad Innata , Inflamación/metabolismo , Interleucina-33/metabolismo , Pulmón , Linfocitos , RatonesRESUMEN
BACKGROUND: Proline-glycine-proline (PGP) is a bioactive fragment of collagen generated by the action of matrix metalloproteinase-9 (MMP-9) and prolylendopeptidase (PE), and capable of eliciting neutrophil chemotaxis and epithelial remodelling. PGP is normally then degraded by leukotriene A4 hydrolase (LTA4H) to limit inflammation and remodelling. This study hypothesized that early and persistent airway neutrophilia in Cystic Fibrosis (CF) may relate to abnormalities in the PGP pathway and sought to understand underlying mechanisms. METHODS: Broncho-alveolar lavage (BAL) fluid was obtained from 38 CF (9 newborns and 29 older children) and 24 non-CF children. BAL cell differentials and levels of PGP, MMP-9, PE and LTA4H were assessed. RESULTS: Whilst PGP was present in all but one of the older CF children tested, it was absent in non-CF controls and the vast majority of CF newborns. BAL levels of MMP-9 and PE were elevated in older children with CF relative to CF newborns and non-CF controls, correlating with airway neutrophilia and supportive of PGP generation. Furthermore, despite extracellular LTA4H commonly being greatly elevated concomitantly with inflammation to promote PGP degradation, this was not the case in CF children, potentially owing to degradation by neutrophil elastase. CONCLUSIONS: A striking imbalance between PGP-generating and -degrading enzymes enables PGP accumulation in CF children from early life and potentially supports airway neutrophilia.
Asunto(s)
Líquido del Lavado Bronquioalveolar/inmunología , Quimiotaxis de Leucocito/inmunología , Fibrosis Quística , Metaloproteinasa 9 de la Matriz/metabolismo , Neutrófilos , Oligopéptidos/metabolismo , Prolina/análogos & derivados , Prolil Oligopeptidasas/metabolismo , Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Broncoscopía/métodos , Niño , Fibrosis Quística/diagnóstico , Fibrosis Quística/inmunología , Fibrosis Quística/fisiopatología , Femenino , Humanos , Recién Nacido , Inflamación/metabolismo , Elastasa de Leucocito/metabolismo , Masculino , Neutrófilos/inmunología , Neutrófilos/patología , Prolina/metabolismo , Esputo/inmunologíaRESUMEN
Neutrophil mobilization, recruitment, and clearance must be tightly regulated as overexuberant neutrophilic inflammation is implicated in the pathology of chronic diseases, including asthma. Efforts to target neutrophils therapeutically have failed to consider their pleiotropic functions and the implications of disrupting fundamental regulatory pathways that govern their turnover during homeostasis and inflammation. Using the house dust mite (HDM) model of allergic airway disease, we demonstrate that neutrophil depletion unexpectedly resulted in exacerbated T helper 2 (TH2) inflammation, epithelial remodeling, and airway resistance. Mechanistically, this was attributable to a marked increase in systemic granulocyte colony-stimulating factor (G-CSF) concentrations, which are ordinarily negatively regulated in the periphery by transmigrated lung neutrophils. Intriguingly, we found that increased G-CSF augmented allergic sensitization in HDM-exposed animals by directly acting on airway type 2 innate lymphoid cells (ILC2s) to elicit cytokine production. Moreover, increased systemic G-CSF promoted expansion of bone marrow monocyte progenitor populations, which resulted in enhanced antigen presentation by an augmented peripheral monocyte-derived dendritic cell pool. By modeling the effects of neutrophil depletion, our studies have uncovered previously unappreciated roles for G-CSF in modulating ILC2 function and antigen presentation. More broadly, they highlight an unexpected regulatory role for neutrophils in limiting TH2 allergic airway inflammation.
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Presentación de Antígeno/inmunología , Células Dendríticas/inmunología , Hipersensibilidad/inmunología , Inmunidad Innata/inmunología , Inflamación/inmunología , Linfocitos/inmunología , Monocitos/inmunología , Neutrófilos/inmunología , Animales , Femenino , Humanos , Ratones , Ratones Endogámicos BALB CRESUMEN
We provide further evidence to support our assertion that PGP is a potent regulator of epithelial remodeling.
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Asma , Hipersensibilidad Respiratoria , Matriz Extracelular , HumanosRESUMEN
It is anticipated that bioactive fragments of the extracellular matrix (matrikines) can influence the development and progression of chronic diseases. The enzyme leukotriene A4 hydrolase (LTA4H) mediates opposing proinflammatory and anti-inflammatory activities, through the generation of leukotriene B4 (LTB4) and degradation of proneutrophilic matrikine Pro-Gly-Pro (PGP), respectively. We show that abrogation of LTB4 signaling ameliorated inflammation and airway hyperresponsiveness (AHR) in a murine asthma model, yet global loss of LTA4H exacerbated AHR, despite the absence of LTB4 This exacerbated AHR was attributable to a neutrophil-independent capacity of PGP to promote pathological airway epithelial remodeling. Thus, we demonstrate a disconnect between airway inflammation and AHR and the ability of a matrikine to promote an epithelial remodeling phenotype that negatively affects lung function. Subsequently, we show that substantial quantities of PGP are detectable in the sputum of moderate-severe asthmatics in two distinct cohorts of patients. These studies have implications for our understanding of remodeling phenotypes in asthma and may rationalize the failure of LTA4H inhibitors in the clinic.
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Remodelación de las Vías Aéreas (Respiratorias) , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Hipersensibilidad Respiratoria/fisiopatología , Resistencia de las Vías Respiratorias , Animales , Asma/complicaciones , Asma/inmunología , Asma/patología , Asma/fisiopatología , Bronquios/patología , Recuento de Células , Modelos Animales de Enfermedad , Epóxido Hidrolasas/deficiencia , Epóxido Hidrolasas/metabolismo , Humanos , Hipersensibilidad/complicaciones , Hipersensibilidad/inmunología , Hipersensibilidad/patología , Hipersensibilidad/fisiopatología , Inflamación/patología , Mediadores de Inflamación/metabolismo , Ratones Endogámicos C57BL , Moco/metabolismo , Neutrófilos/metabolismo , Oligopéptidos/metabolismo , Prolina/análogos & derivados , Prolina/metabolismo , Pyroglyphidae/fisiología , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/parasitología , Hipersensibilidad Respiratoria/patología , Esputo/metabolismo , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Matrikines are bioactive fragments of the extracellular matrix (ECM) that are fundamental in regulating a diverse array of physiological processes. The tripeptide Proline-Glycine-Proline (PGP) is a collagen-derived matrikine that has classically been described as a neutrophil chemoattractant. In this article, we describe our current understanding of the pathways that generate, degrade and modify PGP to dictate its bioavailability and stability. Additionally, we discuss our expanding appreciation of the capacity of PGP to regulate diverse cell types and biological processes, independent of its activity on neutrophils, including a putative role in wound repair. We argue that PGP functions as a primitive and conserved damage-associated molecular pattern, which is generated during infection or injury and subsequently acts to shape ensuing inflammatory and repair processes. As a fragment of the ECM that accumulates at the epicentre of the action, PGP is perfectly positioned to focus neutrophils to the exact site required and direct a localised repair response. However, it is essential that PGP is efficiently degraded, as if this matrikine is allowed to persist then pathology can ensue. Accordingly, we discuss how this pathway is subverted in chronic lung diseases giving rise to persistent inflammation and pathological tissue remodelling.
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Remodelación de las Vías Aéreas (Respiratorias) , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Enfermedades Pulmonares/metabolismo , Pulmón/metabolismo , Infiltración Neutrófila , Oligopéptidos/metabolismo , Fragmentos de Péptidos/metabolismo , Prolina/análogos & derivados , Animales , Humanos , Pulmón/fisiopatología , Enfermedades Pulmonares/fisiopatología , Prolina/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Alveolar macrophages are sentinels of the airways that must exhibit immune restraint to innocuous antigens but elicit a robust inflammatory response to pathogenic threats. How distinction between these dichotomous functions is controlled is poorly defined.Neutrophils are the first responders to infection, and we hypothesised that they may free alveolar macrophages from their hyporesponsive state, promoting their activation. Activation of the inflammasome and interleukin (IL)-1ß release is a key early inflammatory event that must be tightly regulated. Thus, the role of neutrophils in defining inflammasome activation in the alveolar macrophage was assessed. METHODS: Mice were infected with the X31 strain of influenza virus and the role of neutrophils in alveolar macrophage activation established through administration of a neutrophil-depleting (1A8) antibody. RESULTS: Influenza elicited a robust IL-1ß release that correlated (r=0.6849; p<0.001) with neutrophil infiltrate and was ablated by neutrophil depletion. Alveolar macrophages were shown to be the prominent source of IL-1ß during influenza infection, and virus triggered the expression of Nod-like receptor protein 3 (NLRP3) inflammasome and pro-IL-1ß in these cells. However, subsequent activation of the inflammasome complex and release of mature IL-1ß from alveolar macrophages were critically dependent on the provision of a secondary signal, in the form of antimicrobial peptide mCRAMP, from infiltrating neutrophils. CONCLUSIONS: Neutrophils are critical for the activation of the NLRP3 inflammasome in alveolar macrophages during respiratory viral infection. Accordingly, we rationalise that neutrophils are recruited to the lung to confront a viable pathogenic threat and subsequently commit alveolar macrophages to a pro-inflammatory phenotype to combat infection.
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Interleucina-1beta/inmunología , Macrófagos Alveolares/inmunología , Neutrófilos/inmunología , Infecciones del Sistema Respiratorio/inmunología , Virosis/inmunología , Animales , Femenino , Inflamasomas/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Infecciones del Sistema Respiratorio/virologíaRESUMEN
The neutrophil chemoattractant proline-glycine-proline (PGP) is generated from collagen by matrix metalloproteinase-8/9 (MMP-8/9) and prolyl endopeptidase (PE), and it is concomitantly degraded by extracellular leukotriene A4 hydrolase (LTA4H) to limit neutrophilia. Components of cigarette smoke can acetylate PGP, yielding a species (AcPGP) that is resistant to LTA4H-mediated degradation and can, thus, support a sustained neutrophilia. In this study, we sought to elucidate if an antiinflammatory system existed to degrade AcPGP that is analogous to the PGP-LTA4H axis. We demonstrate that AcPGP is degraded through a previously unidentified action of the enzyme angiotensin-converting enzyme (ACE). Pulmonary ACE is elevated during episodes of acute inflammation, as a consequence of enhanced vascular permeability, to ensure the efficient degradation of AcPGP. Conversely, we suggest that this pathway is aberrant in chronic obstructive pulmonary disease (COPD) enabling the accumulation of AcPGP. Consequently, we identify a potentially novel protective role for AcPGP in limiting pulmonary fibrosis and suggest the pathogenic function attributed to ACE in idiopathic pulmonary fibrosis (IPF) to be a consequence of overzealous AcPGP degradation. Thus, AcPGP seemingly has very divergent roles: it is pathogenic in its capacity to drive neutrophilic inflammation and matrix degradation in the context of COPD, but it is protective in its capacity to limit fibrosis in IPF.
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Inflamación/metabolismo , Peptidil-Dipeptidasa A/metabolismo , Fibrosis Pulmonar/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Femenino , Humanos , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Persona de Mediana Edad , Peptidil-Dipeptidasa A/sangre , Enfermedad Pulmonar Obstructiva Crónica/sangre , Fibrosis Pulmonar/patología , HumoRESUMEN
The pro-inflammatory mediator leukotriene B4 (LTB4) is implicated in the pathologies of an array of diseases and thus represents an attractive therapeutic target. The enzyme leukotriene A4 hydrolase (LTA4H) catalyses the distal step in LTB4 synthesis and hence inhibitors of this enzyme have been actively pursued. Despite potent LTA4H inhibitors entering clinical trials all have failed to show efficacy. We recently identified a secondary anti-inflammatory role for LTA4H in degrading the neutrophil chemoattractant Pro-Gly-Pro (PGP) and rationalized that the failure of conventional LTA4H inhibitors may be that they inadvertently prevented PGP degradation. We demonstrate that these inhibitors do indeed fail to discriminate between the dual activities of LTA4H, and enable PGP accumulation in mice. Accordingly, we have developed novel compounds that potently inhibit LTB4 generation whilst leaving PGP degradation unperturbed. These novel compounds could represent a safer and superior class of LTA4H inhibitors for translation into the clinic.
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Antiinflamatorios/síntesis química , Inhibidores Enzimáticos/síntesis química , Epóxido Hidrolasas/antagonistas & inhibidores , Leucotrieno B4/antagonistas & inhibidores , Neutrófilos/efectos de los fármacos , Secuencias de Aminoácidos , Animales , Antiinflamatorios/farmacología , Sitios de Unión , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Epóxido Hidrolasas/química , Epóxido Hidrolasas/genética , Epóxido Hidrolasas/metabolismo , Femenino , Expresión Génica , Humanos , Hidrólisis , Inflamación , Leucotrieno B4/biosíntesis , Ratones , Ratones Endogámicos BALB C , Simulación del Acoplamiento Molecular , Neutrófilos/citología , Neutrófilos/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Prolina/análogos & derivados , Prolina/química , Prolina/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , beta-Alanina/análogos & derivadosRESUMEN
Bioactive matrix fragments (matrikines) have been identified in a myriad of disorders, but their impact on the evolution of airway inflammation has not been demonstrated. We recently described a pathway where the matrikine and neutrophil chemoattractant proline-glycine-proline (PGP) could be degraded by the enzyme leukotriene A4 hydrolase (LTA4H). LTA4H classically functions in the generation of pro-inflammatory leukotriene B4, thus LTA4H exhibits opposing pro- and anti-inflammatory activities. The physiological significance of this secondary anti-inflammatory activity remains unknown. Here we show, using readily resolving pulmonary inflammation models, that loss of this secondary activity leads to more pronounced and sustained inflammation and illness owing to PGP accumulation. PGP elicits an exacerbated neutrophilic inflammation and protease imbalance that further degrades the extracellular matrix, generating fragments that perpetuate inflammation. This highlights a critical role for the secondary anti-inflammatory activity of LTA4H and thus has consequences for the generation of global LTA4H inhibitors currently being developed.