RESUMEN
Neutrophil elastase (NE) is taken up by macrophages, retains intracellular protease activity, and induces a pro-inflammatory phenotype. However, the mechanism of NE-induced pro-inflammatory polarization of macrophages is not well understood. We hypothesized that intracellular NE degrades histone deacetylases (HDAC) and Sirtuins, disrupting the balance of lysine acetylation and deacetylation and resulting in nuclear to cytoplasmic translocation of a major alarmin, High Mobility Group Box 1 (HMGB1), a pro-inflammatory response in macrophages. Human blood monocytes were obtained from healthy donors or from subjects with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). Monocytes were differentiated into blood monocyte derived macrophages (BMDMs) in vitro. Human BMDMs were exposed to NE or control vehicle, and the abundance of HDACs and Sirtuins was determined by Western blotting of total cell lysates or nuclear extracts or determined by ELISA. HDAC, Sirtuin, and Histone acetyltransferase (HAT) activities were measured. NE degraded most HDACs and Sirtuin (Sirt)1, resulting in decreased HDAC and sirtuin activities, with minimal change in HAT activity. We then evaluated whether the NE-induced loss of Sirt activity or loss of HDAC activities would alter the cellular localization of HMGB1. NE treatment or treatment with Trichostatin A (TSA), a global HDAC inhibitor, both increased HMGB1 translocation from the nucleus to the cytoplasm, consistent with HMGB1 activation. NE significantly degraded Class I and II HDAC family members and Sirt 1, which shifted BMDMs to a pro-inflammatory phenotype.
Asunto(s)
Proteína HMGB1 , Histona Desacetilasas , Elastasa de Leucocito , Macrófagos , Sirtuina 1 , Humanos , Acetilación , Células Cultivadas , Fibrosis Quística/metabolismo , Histona Acetiltransferasas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Proteína HMGB1/metabolismo , Ácidos Hidroxámicos , Elastasa de Leucocito/metabolismo , Macrófagos/metabolismo , Monocitos/metabolismo , Proteolisis , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Sirtuina 1/metabolismoRESUMEN
Neutrophil elastase (NE), a major inflammatory mediator in chronic obstructive pulmonary disease (COPD) airways, impairs macrophage function, contributing to persistence of airway inflammation. We hypothesized that NE activates a novel mechanism of macrophage-induced inflammation: release of macrophage extracellular traps (METs). The METs are composed of extracellular DNA decorated with granule proteinases and oxidants and may trigger persistent airway inflammation in COPD. To test the hypothesis, human blood monocytes were isolated from whole blood of subjects with COPD recruited following informed written consent. Patient demographics and clinical data were collected. Cells were cultured in media with GM-CSF to differentiate into blood monocyte derived macrophages (BMDMs). The BMDMs were treated with FITC-NE and unlabeled NE to determine intracellular localization by confocal microscopy and intracellular proteinase activity by DQ-Elastin assay. After NE exposure, released extracellular traps were quantified by abundance of extracellular DNA in conditioned media using the Pico Green assay. BMDM cell lysates were analyzed by Western analysis for proteolytic degradation of histone H3 or H4 or upregulation of peptidyl arginine deiminase (PAD) 2 and 4, two potential mechanisms to mediate extracellular trap DNA release. We observed that NE was taken up by COPD BMDM, localized to the cytosol and nucleus, and retained proteinase activity in the cell. NE induced MET release at doses as low as 50 nM. NE treatment caused histone H3 clipping but no effect on histone H4 nor PAD 2 or 4 abundance or activity. In summary, NE activated COPD MET release by clipping histone H3, a prerequisite for chromatin decondensation.
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Trampas Extracelulares , Elastasa de Leucocito , Enfermedad Pulmonar Obstructiva Crónica , Humanos , ADN , Trampas Extracelulares/metabolismo , Histonas/metabolismo , Histonas/farmacología , Inflamación/metabolismo , Elastasa de Leucocito/genética , Elastasa de Leucocito/metabolismo , Elastasa de Leucocito/farmacología , Macrófagos/metabolismo , Neutrófilos , Enfermedad Pulmonar Obstructiva Crónica/metabolismoRESUMEN
Patients with cystic fibrosis (CF) have decreased severity of severe acute respiratory syndrome-like coronavirus-2 (SARS-CoV-2) infections, but the underlying cause is unknown. Patients with CF have high levels of neutrophil elastase (NE) in the airway. We examined whether respiratory epithelial angiotensin-converting enzyme 2 (ACE-2), the receptor for the SARS-CoV-2 spike protein, is a proteolytic target of NE. Soluble ACE-2 levels were quantified by ELISA in airway secretions and serum from patients with and without CF, the association between soluble ACE-2 and NE activity levels was evaluated in CF sputum. We determined that NE activity was directly correlated with increased ACE-2 in CF sputum. Additionally, primary human bronchial epithelial (HBE) cells, exposed to NE or control vehicle, were evaluated by Western analysis for the release of cleaved ACE-2 ectodomain fragment into conditioned media, flow cytometry for the loss of cell surface ACE-2, its impact on SARS-CoV-2 spike protein binding. We found that NE treatment released ACE-2 ectodomain fragment from HBE and decreased spike protein binding to HBE. Furthermore, we performed NE treatment of recombinant ACE-2-Fc-tagged protein in vitro to assess whether NE was sufficient to cleave recombinant ACE-2-Fc protein. Proteomic analysis identified specific NE cleavage sites in the ACE-2 ectodomain that would result in loss of the putative N-terminal spike-binding domain. Collectively, data support that NE plays a disruptive role in SARS-CoV-2 infection by catalyzing ACE-2 ectodomain shedding from the airway epithelia. This mechanism may reduce SARS-CoV-2 virus binding to respiratory epithelial cells and decrease the severity of COVID19 infection.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , Fibrosis Quística , Elastasa de Leucocito , Humanos , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/metabolismo , Fibrosis Quística/metabolismo , Elastasa de Leucocito/metabolismo , Unión Proteica , Proteómica , Mucosa Respiratoria/metabolismo , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
RATIONALE: Identifying neonatal and post-discharge exposures among extremely low gestational age newborns (ELGANs) that drive increased pulmonary morbidity and abnormal lung function at 1 year of age proves challenging. OBJECTIVE: The NIH-sponsored Prematurity and Respiratory Outcomes Program (PROP), evaluated infant pulmonary function tests (iPFTs) at 1 year corrected age to determine which demographic and clinical factors are associated with abnormal lung function. METHODS: iPFTs were performed on a PROP subcohort of 135 participants following Institutional Review Board (IRB)-approved written consent. Demographic data, Neonatal Intensive Care Unit (NICU) clinical care, and post-NICU exposures were analyzed for association with iPFTs. MAIN RESULTS: A significant decrease in forced expiratory volume at 0.5 s (FEV0.5 ) and/or forced expiratory flows at 75% of forced vital capacity (FEF75 ), were associated with male sex and African American race. Clinical factors including longer duration of ventilatory support, exposure to systemic steroids, and weight less than the 10th percentile at 36 weeks postmenstrual age were also associated with airflow obstruction, whereas supplemental oxygen requirement and bronchopulmonary dysplasia were not. Additionally, the need for respiratory medications, technology, or hospitalizations during the first year, ascertained by a quarterly survey, were the only post-NICU factors associated with decreased FEV0.5 and FEF75 . Only 7% of infants had reversible airflow obstruction. CONCLUSIONS: Neonatal demographic factors, respiratory support in the NICU, and a history of greater post-NICU medical utilization for respiratory disease had the strongest association with lower lung function at 1 year in ELGANs.
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Cuidados Posteriores , Displasia Broncopulmonar , Displasia Broncopulmonar/complicaciones , Edad Gestacional , Humanos , Lactante , Recién Nacido , Masculino , Alta del Paciente , Pruebas de Función RespiratoriaRESUMEN
Neutrophil extracellular traps increase cystic fibrosis (CF) airway inflammation. We hypothesized that macrophage exposure to neutrophil elastase (NE) would trigger the release of macrophage extracellular traps (METs), a novel mechanism to augment NE-induced airway inflammation in CF. Experiments were performed using human blood monocyte derived macrophages (hBMDM) from patients with and without CF to test specific mechanisms associated with MET release, and MET release by NE was confirmed in alveolar macrophages from Cftr-null and wild-type littermate mice exposed to intratracheal NE in vivo. Human BMDM were exposed to FITC-NE, and intracellular FITC-NE was localized to cytoplasmic and nuclear domains. Intracellular NE was proteolytically active as indicated by DQ-Elastin substrate cleavage. NE (100 to 500 nM) significantly increased extracellular PicoGreen fluorescence consistent with DNA release/ MET release from hBMDM in the absence of cell death. MET release was further confirmed by confocal microscopy in hBMDM treated with NE, and in alveolar macrophages from Cftr-null and wild-type littermate mice that had been exposed to intratracheal NE. NE-triggered MET release was associated with H3 citrullination detected by immunofluorescence assays and with partial cleavage of histone H3 but not H4. Exposure to NE caused release of METs from both CF and non-CF hBMDM in vitro and murine alveolar macrophages in vivo. MET release was associated with NE-activated H3 clipping, a mechanism associated with chromatin decondensation, a prerequisite for METs.
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Fibrosis Quística/metabolismo , Trampas Extracelulares/metabolismo , Elastasa de Leucocito/metabolismo , Macrófagos/metabolismo , Adulto , Animales , Lavado Broncoalveolar , Citrulinación , Fibrosis Quística/patología , ADN/metabolismo , Femenino , Histonas/metabolismo , Humanos , Masculino , Ratones , Persona de Mediana Edad , Proteolisis , Adulto JovenRESUMEN
Neutrophil elastase (NE) is a major inflammatory protease released by neutrophils and is present in the airways of patients with cystic fibrosis (CF), chronic obstructive pulmonary disease, non-CF bronchiectasis, and bronchopulmonary dysplasia. Although NE facilitates leukocyte transmigration to the site of infection and is required for clearance of Gram-negative bacteria, it also activates inflammation when released into the airway milieu in chronic inflammatory airway diseases. NE exposure induces airway remodeling with increased mucin expression and secretion and impaired ciliary motility. NE interrupts epithelial repair by promoting cellular apoptosis and senescence and it activates inflammation directly by increasing cytokine expression and release, and indirectly by triggering extracellular trap release and exosome release, which magnify protease activity and inflammation in the airway. NE inhibits innate immune function by digesting opsonins and opsonin receptors, degrading innate immune proteins such as lactoferrin, and inhibiting macrophage phagocytosis. Importantly, NE-directed therapies have not yet been effective in preventing the pathologic sequelae of NE exposure, but new therapies are being developed that offer both direct antiprotease activity and multifunctional anti-inflammatory properties.
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Enfermedad Crónica , Elastasa de Leucocito/fisiología , Enfermedades Pulmonares/metabolismo , Pulmón , Neutrófilos/metabolismo , Animales , Humanos , Pulmón/metabolismo , Pulmón/patología , Neutrófilos/citologíaRESUMEN
Patients with cystic fibrosis (CF) have defective macrophage phagocytosis and efferocytosis. Several reports demonstrate that neutrophil elastase (NE), a major inflammatory protease in the CF airway, impairs macrophage phagocytic function. To date, NE-impaired macrophage phagocytic function has been attributed to cleavage of cell surface receptors or opsonins. We applied an unbiased proteomic approach to identify other potential macrophage targets of NE protease activity that may regulate phagocytic function. Using the murine macrophage cell line, RAW 264.7, human blood monocyte-derived macrophages, and primary alveolar macrophages from Cftr-null and wild-type littermate mice, we demonstrated that NE exposure blocked phagocytosis of Escherichia coli bio-particles. We performed liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteomic analysis of the conditioned media from RAW264.7 treated either with active NE or inactive (boiled) NE as a control. Out of 840 proteins identified in the conditioned media, active NE upregulated 142 proteins and downregulated 211 proteins. NE released not only cell surface proteins into the media but also cytoskeletal, mitochondrial, cytosolic, and nuclear proteins that were detected in the conditioned media. At least 32 proteins were associated with the process of phagocytosis including 11 phagocytic receptors [including lipoprotein receptor-related protein 1 (LRP1)], 7 proteins associated with phagocytic cup formation, and 14 proteins involved in phagocytic maturation (including calpain-2) and phagolysosome formation. NE had a broad effect on the proteome required for regulation of all stages of phagocytosis and phagolysosome formation. Furthermore, the NE sheddome/secretome included proteins from other macrophage cellular domains, suggesting that NE may globally regulate macrophage structure and function.
Asunto(s)
Elastasa de Leucocito/metabolismo , Lisosomas/metabolismo , Macrófagos/metabolismo , Fagocitosis , Fagosomas/metabolismo , Adolescente , Adulto , Animales , Niño , Preescolar , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Femenino , Humanos , Elastasa de Leucocito/genética , Lisosomas/genética , Lisosomas/patología , Macrófagos/fisiología , Masculino , Ratones , Ratones Mutantes , Fagosomas/genética , Fagosomas/patología , Células RAW 264.7RESUMEN
Cystic fibrosis (CF) lung disease is marked by high concentrations of neutrophil elastase (NE) and DNA polymers; both factors contribute to airway disease. Although inhaled recombinant human dornase alfa reduces the frequency of CF pulmonary exacerbations, it also increases free NE activity in the sputum. There are no approved anti-NE therapies for patients with CF. We investigated whether synthetic, low-molecular weight polysulfated hyaluronan GlycoMira-1111 (GM-1111) would be effective as an anti-NE drug using ex vivo CF sputum. Anti-NE activity of GM-1111 was tested in CF sputum in the presence or absence of dornase alfa and/or hypertonic saline using a spectrophotometric assay specific for human NE and was compared with unfractionated heparin. We tested whether GM-1111 disaggregated DNA from CF sputum (using gel electrophoresis analysis) or modified CF sputum viscoelastic properties (using a dynamic rheometer). GM-1111 and unfractionated heparin had near equivalent anti-NE activity in CF sputum in the presence of dornase alfa. Both GM-1111 and unfractionated heparin retained anti-NE activity in hypertonic saline but with decreased activity. GM-1111 increased the release of soluble DNA in CF sputum, resulting in improved depolymerization efficacy of dornase alfa. GM-1111 decreased CF sputum elasticity. GM-1111 inhibited NE activity, enhanced DNA depolymerization by deoxyribonuclease, and decreased viscoelastic properties of CF sputum, similar to effects reported previously for unfractionated heparin. Unlike heparins, GM-1111 is synthetic, with minimal anticoagulant activity, and is not derived from animal products. These key attributes provide advantages over unfractionated heparin as a potential therapeutic for CF.
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Fibrosis Quística/tratamiento farmacológico , Ácido Hialurónico/uso terapéutico , Elastasa de Leucocito/metabolismo , Esputo/efectos de los fármacos , Esputo/metabolismo , Adulto , Antiinflamatorios/uso terapéutico , Fibrosis Quística/metabolismo , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Femenino , Heparina/uso terapéutico , Humanos , Masculino , Proteínas Recombinantes/metabolismo , ReologíaRESUMEN
Neutrophil elastase (NE) is a major protease in the airways of patients with cystic fibrosis (CF) that activates airway inflammation by several mechanisms. NE stimulates epithelial toll like receptors (TLR) resulting in cytokine upregulation and release, upregulates MUC5AC, a major airway mucin, degrades both phagocytic receptors and opsonins resulting in both neutrophil and macrophage phagocytic failure, generates oxidative stress via extracellular generation and uptake of heme free iron, and activates other proteases. Altogether, these mechanisms create a significant inflammatory challenge that impairs innate immune function and results in airway remodeling. Currently, a major gap in our therapeutic approach to CF lung disease is the lack of an effective therapeutic strategy targeting active NE and its downstream pro-inflammatory sequelae. Polysulfated glycosaminoglycans (GAGs) are potent anti-elastase drugs that have additional anti-inflammatory properties. Heparin is a prototype of a glycosaminoglycan with both anti-elastase and anti-inflammatory properties. Heparin inhibits NE in an allosteric manner with high potency. Heparin also inhibits cathepsin G, blocks P-selectin and L-selectin, hinders ligand binding to the receptor for advanced glycation endproducts, and impedes histone acetyltransferase activity which dampens cytokine transcription and High Mobility Group Box 1 release. Furthermore, nebulized heparin treatment improves outcomes for patients with chronic obstructive pulmonary disease (COPD), asthma, acute lung injury and smoke inhalation. However, the anticoagulant activity of heparin is a potential contraindication for this therapy to be developed for CF lung disease. Therefore, modified heparins and other GAGs are being developed that retain the anti-elastase and anti-inflammatory qualities of heparin with minimal to no anticoagulant activity. The modified heparin, 2-O, 3-O desulfated heparin (ODSH), maintains anti-elastase and anti-inflammatory activities in vitro and in vivo, and has little residual anticoagulant activity. Heparan sulfate with O-sulfate residues but not N-sulfate residues blocks allergic asthmatic inflammation in a murine model. Polysulfated hyaluronic acid abrogates allergen- triggered rhinosinusitis in a murine model. Finally, nonsaccharide glycosaminoglycan mimetics with specific sulfate modifications can be designed to inhibit NE activity. Altogether, these novel GAGs or GAG mimetics hold significant promise to address the unmet need for inhaled anti-elastase and anti-inflammatory therapy for patients with CF.
RESUMEN
RATIONALE: Bronchopulmonary dysplasia (BPD) is associated with post-prematurity respiratory disease (PRD) in survivors of extreme preterm birth. Identifying early biomarkers that correlate with later development of BPD and PRD may provide insights for intervention. In a preterm baboon model, elevated gastrin-releasing peptide (GRP) is associated with BPD, and GRP inhibition mitigates BPD occurrence. OBJECTIVE: We performed a prospective cohort study to investigate whether urine GRP levels obtained in the first postnatal week were associated with BPD, PRD, and other urinary biomarkers of oxidative stress. METHODS: Extremely low gestational age infants (23-28 completed weeks) were enrolled in a US multicenter observational study, The Prematurity and Respiratory Outcomes Program (http://clinicaltrials.gov/ct2/show/NCT01435187). We used multivariable logistic regression to examine the association between urine GRP in the first postnatal week and multiple respiratory outcomes: BPD, defined as supplemental oxygen use at 36 + 0 weeks postmenstrual age, and post-PRD, defined by positive quarterly surveys for increased medical utilization over the first year (PRD score). RESULTS: A total of 109 of 257 (42%) infants had BPD, and 120 of 217 (55%) had PRD. On adjusted analysis, GRP level more than 80 was associated with BPD (adjusted odds ratio [aOR], 1.83; 95% confidence interval [CI], 1.03-3.25) and positive PRD score (aOR, 2.46; 95% CI, 1.35-4.48). Urine GRP levels correlated with duration of NICU ventilatory and oxygen support and with biomarkers of oxidative stress: allantoin and 8-hydroxydeoxyguanosine. CONCLUSIONS: Urine GRP in the first postnatal week was associated with concurrent urine biomarkers of oxidative stress and with later diagnoses of BPD and PRD.
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Displasia Broncopulmonar/orina , Péptido Liberador de Gastrina/orina , Recien Nacido Extremadamente Prematuro , Enfermedades del Prematuro/orina , Enfermedades Respiratorias/orina , Biomarcadores/orina , Displasia Broncopulmonar/diagnóstico , Femenino , Humanos , Recién Nacido , Enfermedades del Prematuro/diagnóstico , Modelos Logísticos , Masculino , Estudios Prospectivos , Trastornos Respiratorios , Enfermedades Respiratorias/diagnósticoRESUMEN
Cystic fibrosis (CF) is a disease of dysregulated salt and fluid homeostasis that results in the massive accumulation of neutrophil elastase, resulting in lung degradation and death. The current CF therapy relies on inhaled deoxyribonuclease and hypertonic saline but does not address the elastolytic degradation of the lung. We reasoned that allosteric agents targeting the heparin-binding site of neutrophil elastase would offer a therapeutic paradigm. Screening a library of 60 nonsaccharide glycosaminoglycan mimetics (NSGMs) led to the discovery of 23 hits against neutrophil elastase. To identify a lead NSGM that works in sync with the current CF-relieving agents, we developed a rigorous protocol based on fundamental computational, biochemical, mechanistic, and adverse effect studies. The lead NSGM so identified neutralized neutrophil elastase present in the sputum of CF patients in the presence of deoxyribonuclease and high-salt conditions. Our work presents the process for discovering potent, small, synthetic, allosteric, anti-CF agents, while also identifying a novel lead for further studies in animal models of CF.
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Fibrosis Quística/tratamiento farmacológico , Descubrimiento de Drogas , Heparina/metabolismo , Elastasa de Leucocito/metabolismo , Terapia Molecular Dirigida , Esputo/efectos de los fármacos , Esputo/metabolismo , Sitios de Unión/efectos de los fármacos , Humanos , Relación Estructura-ActividadRESUMEN
Cystic fibrosis (CF) is a multifactorial disease in which dysfunction of protease-antiprotease balance plays a key role. The current CF therapy relies on dornase α, hypertonic saline, and antibiotics and does not address the high neutrophil elastase (NE) activity observed in the lung and sputum of CF patients. Our hypothesis is that variants of heparin, which potently inhibit NE but are not anticoagulant, would help restore the protease-antiprotease balance in CF. To realize this concept, we studied molecular principles governing the effectiveness of different heparins, especially 2-O,3-O-desulfated heparin (ODSH), in the presence of sputum components and therapeutic agents. Using sputa from CF patients and an NE activity assay, we found that heparins are ineffective if used in the absence of dornase. This is true even when mucolytics, such as DTT or N-acetylcysteine, were used. Computational modeling suggested that ODSH and DNA compete for binding to an overlapping allosteric site on NE, which reduces the anti-NE potential of ODSH. NE inhibition of both DNA and ODSH is chain length-dependent, but ODSH chains exhibit higher potency per unit residue length. Likewise, ODSH chains exhibit higher NE inhibition potential compared with DNA chains in the presence of saline. These studies suggest fundamental differences in DNA and ODSH recognition and inhibition of NE despite engaging overlapping sites and offer unique insights into molecular principles that could be used in developing antiprotease agents in the presence of current treatments, such as dornase and hypertonic saline.
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Fibrosis Quística/fisiopatología , Heparina/análogos & derivados , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/metabolismo , Oligosacáridos/farmacología , Inhibidores de Proteasas/farmacología , Esputo/enzimología , Simulación por Computador , Heparina/farmacología , HumanosRESUMEN
OBJECTIVE: Vascular endothelial growth factor (VEGF) and sphingolipid metabolites, sphingosine 1-phosphate (S1P), and ceramides are important to lung development and repair. We hypothesized specific sphingolipid and VEGF alterations would be associated with BPD development and aimed to investigate the early tracheal aspirate (TA) VEGF and S1P relationship with later diagnosis of preterm infant bronchopulmonary dysplasia, BPD. DESIGN: TA VEGF and lipidomics were measured in TA from Infants <32 weeks gestational age at birth with and without later BPD. BPD was defined using the NICHD severity BPD definition. Clinical demographics and medical course were identified with statistical analysis performed with JMP, Statistical Analysis Software. RESULTS: The analysis included 25 infants (9 NoBPD and 16 BPD) with mean gestational age of 27.8 ± 2.5 SD weeks and 25.1 ± 1.9 SD weeks respectively, P < 0.01. Later development of BPD was associated with elevated mean TA VEGF 604.3 ± 150.2 SE pg/mL versus NoBPD 120 ± 34.3 SE pg/mL, elevated S1P, 11.5 ± 2.3 SE pmol/mL versus NoBPD 4.8 ± 0.6 SE pmol/mL, and elevated selected ceramides during the first week of life. CONCLUSIONS: Airway VEGF and sphingolipid metabolites were distinctly elevated within the first days of postnatal life in preterm infants with later BPD progression. These biomarkers may be useful as indicators of lung injury development or as targets to decrease BPD risk.
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Displasia Broncopulmonar/metabolismo , Esfingolípidos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Biomarcadores/metabolismo , Femenino , Edad Gestacional , Humanos , Lactante , Recién Nacido , Recien Nacido Prematuro , Masculino , Tráquea/metabolismoRESUMEN
INTRODUCTION: Sphingolipids are associated with the regulation of pulmonary inflammation. Although sphingolipids have been investigated in the context of cystic fibrosis (CF), the focus has been on loss of CF transmembrane conductance regulator (CFTR) function in mice, and in CF human lung epithelial cell lines. The sphingolipid content of CF sputum and the potential link between ceramide and airway inflammation in CF remain relatively unexplored. METHODS: Fifteen patients with CF provided two spontaneously expectorated sputum samples, one collected during a hospitalization for an acute pulmonary exacerbation and one from an outpatient visit at a time of clinical stability. Sputum was processed, and the supernatant assessed for active neutrophil elastase (NE) using a chromogenic microplate assay and sphingolipid content using reverse phase high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Relevant demographic data including age, sex, CF genotype, FEV1 % predicted, and sputum bacteriology were assessed as possible modifying factors that could influence the correlation between NE and sputum sphingolipids. Data were analyzed for linear correlation, with statistical significance pre-defined as P < 0.05. RESULTS: There was a significant association between the concentration of active NE and ceramide, sphingomyelin, and monohexosylceramide moieties as well as sphingosine-1-phosphate. The presence of Methicillin-resistant Staphylococcus aureus (MRSA), FEV1 % predicted, and female gender further strengthened the association of NE and sphingolipids, but Pseudomonas aeruginosa had no effect on the association between NE and sphingolipids. CONCLUSIONS: These data suggest that NE may increase pro-inflammatory sphingolipid signaling, and the association is strengthened in female patients and patients with MRSA.
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Fibrosis Quística/metabolismo , Elastasa de Leucocito/metabolismo , Esfingolípidos/metabolismo , Esputo/metabolismo , Adolescente , Adulto , Animales , Niño , Fibrosis Quística/microbiología , Fibrosis Quística/fisiopatología , Femenino , Volumen Espiratorio Forzado , Humanos , Pulmón/metabolismo , Pulmón/microbiología , Pulmón/fisiopatología , Masculino , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Ratones , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/fisiopatología , Pseudomonas aeruginosa/aislamiento & purificación , Esputo/microbiología , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/fisiopatología , Adulto JovenRESUMEN
BACKGROUND: Cystic fibrosis (CF) airway secretions are abnormal, contributing to decreased clearance and a cycle of infection and inflammation. CF sputum properties may predict disease progression. We hypothesized that sputum viscoelasticity and clearance abnormalities would inversely correlate with pulmonary function during exacerbation and that sputum properties would return to baseline after therapy. METHODS: We collected sputa longitudinally from 13 subjects with CF with moderate to severe lung disease during both clinical stability and exacerbation. Dynamic rheology was analyzed, using a cone-and-plate rheometer. Mucociliary clearability was measured on mucus-depleted frog palate, cough clearability in a simulated cough machine, and sputum hydration as percent solids was measured following lyophilization. RESULTS: Elastic modulus G' and viscous modulus G'' increased during exacerbation and returned to baseline levels with recovery (P < .05 for both). Solid content did not change. Sputum mucociliary clearability decreased during exacerbations (P < .01) but not cough clearance. FEV1 % predicted was inversely correlated with G' and G'' (P < .01 for both). The regression slope of the natural log-transformed G' and G'' vs FEV1 % predicted was statistically homogeneous among subjects (estimated common slope m = -3.84, P < .001 and m = -8.53, P < .0001, respectively). CONCLUSIONS: Among these subjects with CF, there is a striking identity of the slope defining the relationship between ln G' or ln G'' and FEV1. There are dramatic increases in dynamic viscosity and elasticity during a pulmonary exacerbation with return to baseline at recovery. This suggests that sputum viscoelastic properties are tightly associated with lung function and disease status.
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Fibrosis Quística/metabolismo , Fibrosis Quística/fisiopatología , Reología/métodos , Esputo/metabolismo , Tos , Módulo de Elasticidad , Femenino , Humanos , Estudios Longitudinales , Masculino , Depuración Mucociliar , Pruebas de Función Respiratoria , Adulto JovenRESUMEN
The respiratory epithelium is lined by mucus, a gel consisting of water, ions, proteins, and macromolecules. The major macromolecular components of mucus are the mucin glycoproteins, which are critical for local defense of the airway. There are three classes of mucins in the airways: those that are secreted but do not polymerize (MUC7), those that are secreted and polymerize to form gels (MUC5AC, MUC5B), and those that have transmembrane domains and are cell surface associated (MUC1, MUC4, MUC16, MUC20). The mucins are regulated at the transcriptional, posttranscriptional, and epigenetic levels, and posttranslational modifications play an important role in mucin binding and clearance of microbes and pollutants. The development of mice deficient in specific mucins, and the cystic fibrosis pig, has greatly advanced our understanding of the role of mucins as innate immune mediators and how mucins and mucus contribute to lung disease. These observations suggest new strategies to ameliorate mucus obstruction by targeting mucociliary clearance and mucin hyperconcentration. Furthermore, a polymorphism in the promoter of MUC5B is strongly associated with risk of developing pulmonary fibrosis, supporting a novel function for MUC5B to influence interstitial lung disease. Exciting new data support the concept not only that mucins and mucus are important for lung homeostasis and protection from environmental threats but also that goblet cells play an important role as regulators of innate immune function. These insights into the innate immune properties of mucins and goblet cells support a shift from the current paradigm of repressing increased mucin expression to targeting regulation of specific mucins and the abnormal airway milieu.
Asunto(s)
Fibrosis Quística , Células Caliciformes/metabolismo , Mucinas/metabolismo , Moco/metabolismo , Remodelación de las Vías Aéreas (Respiratorias) , Animales , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Fibrosis Quística/fisiopatología , Células Caliciformes/patología , Humanos , Depuración MucociliarRESUMEN
Altered sphingolipid metabolism is associated with increased inflammation; however, the impact of inflammatory mediators, including neutrophil elastase (NE), on airway sphingolipid homeostasis remains unknown. Using a well-characterized mouse model of NE oropharyngeal aspiration, we investigated a potential link between NE-induced airway inflammation and increased synthesis of various classes of sphingolipids, including ceramide species. Sphingolipids in bronchoalveolar lavage fluids (BAL) were identified and quantified using reverse-phase high-performance liquid chromatography/electrospray ionization tandem mass spectrometry analysis. BAL total and differential cell counts, CXCL1/keratinocyte chemoattractant (KC) protein levels, and high-mobility group box 1 (HMGB1) protein levels were determined. NE exposure increased BAL long-chain ceramides, total cell and neutrophil counts, and upregulated KC and HMGB1. The mRNA and protein levels of serine palmitoyltransferase (SPT) long-chain subunits 1 and 2, the multimeric enzyme responsible for the first, rate-limiting step of de novo ceramide generation, were determined by qRT-PCR and Western analyses, respectively. NE increased lung SPT long-chain subunit 2 (SPTLC2) protein levels but not SPTLC1 and had no effect on mRNA for either subunit. To assess whether de novo ceramide synthesis was required for NE-induced inflammation, myriocin, a SPT inhibitor, or a vehicle control was administered intraperitoneally 2 h before NE administration. Myriocin decreased BAL d18:1/22:0 and d18:1/24:1 ceramide, KC, and HMGB1 induced by NE exposure. These results support a feed-forward cycle of NE-generated ceramide and ceramide-driven cytokine signaling that may be a potential target for intervention in lung disease typified by chronic neutrophilic inflammation.