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1.
Am J Physiol Lung Cell Mol Physiol ; 323(5): L525-L535, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36041220

RESUMO

E-cigarette vaping is a major aspect of nicotine consumption, especially for children and young adults. Although it is branded as a safer alternative to cigarette smoking, murine and rat models of subacute and chronic e-cigarette vaping exposure have shown many proinflammatory changes in the respiratory tract. An acute vaping exposure paradigm has not been demonstrated in the golden Syrian hamster, and the hamster is a readily available small animal model that has the unique benefit of becoming infected with and transmitting respiratory viruses, including SARS-CoV-2, without genetic alteration of the animal or virus. Using a 2-day, whole body vaping exposure protocol in male golden Syrian hamsters, we evaluated serum cotinine, bronchoalveolar lavage cells, lung, and nasal histopathology, and gene expression in the nasopharynx and lung through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Depending on the presence of nonnormality or outliers, statistical analysis was performed by ANOVA or Kruskal-Wallis tests. For tests that were statistically significant (P < 0.05), post hoc Tukey-Kramer and Dunn's tests, respectively, were performed to make pairwise comparisons between groups. In nasal tissue, RT-qPCR analysis revealed nicotine-dependent increases in gene expression associated with type 1 inflammation (CCL-5 and CXCL-10), fibrosis [transforming growth factor-ß (TGF-ß)], nicotine-independent increase oxidative stress response (SOD-2), and a nicotine-independent decrease in vasculogenesis/angiogenesis (VEGF-A). In the lung, nicotine-dependent increases in the expression of genes involved in the renin-angiotensin pathway [angiotensin-converting enzyme (ACE), ACE2], coagulation (tissue factor, Serpine-1), extracellular matrix remodeling (MMP-2, MMP-9), type 1 inflammation (IL-1ß, TNF-α, and CXCL-10), fibrosis (TGF-ß and Serpine-1), oxidative stress response (SOD-2), neutrophil extracellular traps release (ELANE), and vasculogenesis and angiogenesis (VEGF-A) were identified. To our knowledge, this is the first demonstration that the Syrian hamster is a viable model of e-cigarette vaping. In addition, this is the first report that e-cigarette vaping with nicotine can increase tissue factor gene expression in the lung. Our results show that even an acute exposure to e-cigarette vaping causes significant upregulation of mRNAs in the respiratory tract from pathways involving the renin-angiotensin system, coagulation, extracellular matrix remodeling, type 1 inflammation, fibrosis, oxidative stress response, neutrophil extracellular trap release (NETosis), vasculogenesis, and angiogenesis.


Assuntos
Sistemas Eletrônicos de Liberação de Nicotina , Transcriptoma , Vaping , Animais , Cricetinae , Masculino , Enzima de Conversão de Angiotensina 2 , Angiotensinas , Cotinina , Fibrose , Inflamação/patologia , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Mesocricetus , Nicotina/farmacologia , Renina , Superóxido Dismutase , Tromboplastina , Fator de Crescimento Transformador beta , Fator de Necrose Tumoral alfa , Vaping/efeitos adversos , Fator A de Crescimento do Endotélio Vascular
2.
JCI Insight ; 7(13)2022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35608904

RESUMO

Chronic type 2 (T2) inflammatory diseases of the respiratory tract are characterized by mucus overproduction and disordered mucociliary function, which are largely attributed to the effects of IL-13 on common epithelial cell types (mucus secretory and ciliated cells). The role of rare cells in airway T2 inflammation is less clear, though tuft cells have been shown to be critical in the initiation of T2 immunity in the intestine. Using bulk and single-cell RNA sequencing of airway epithelium and mouse modeling, we found that IL-13 expanded and programmed airway tuft cells toward eicosanoid metabolism and that tuft cell deficiency led to a reduction in airway prostaglandin E2 (PGE2) concentration. Allergic airway epithelia bore a signature of PGE2 activation, and PGE2 activation led to cystic fibrosis transmembrane receptor-dependent ion and fluid secretion and accelerated mucociliary transport. These data reveal a role for tuft cells in regulating epithelial mucociliary function in the allergic airway.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Animais , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dinoprostona , Interleucina-13/metabolismo , Camundongos , Sistema Respiratório
3.
J Cyst Fibros ; 21(4): 637-643, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35248469

RESUMO

BACKGROUND: A decrease in the lumacaftor-mediated increase in F508del-CFTR function and expression upon prolonged exposure to ivacaftor (VX-770) has previously been described. However, the efficacy observed with ivacaftor-containing CFTR modulator therapies in vivo is in conflict with these reports. We hypothesized that a portion of the apparent decrease in CFTR function observed after prolonged ivacaftor exposure in vitro was due to an increase in constitutive CFTR-mediated ion transport. METHODS: Human nasal epithelial (HNE) cells were obtained by brushings from three CF individuals homozygous for the F508del CFTR mutation. Differentiated epithelia were pre-treated with prolonged (24 h) exposure to either lumacaftor (VX-809; 3 µM), tezacaftor (VX-661; 3 µM), elexacaftor (VX-445; 3 µM), and/or ivacaftor (0.1-6.4 µM) or DMSO (vehicle control), and CFTR function was assayed by Ussing chamber electrophysiology. RESULTS: In cells treated with lumacaftor, constitutive CFTR activity was not increased at any concentration of co-treatment with ivacaftor. Constitutive CFTR activity was also unchanged in cells treated with the combination of tezacaftor and elexacaftor. An increase in constitutive CFTR activity above the DMSO controls was only observed in cells treated with the combination of tezacaftor and elexacaftor and co-treated with at least 0.1 µM ivacaftor. CONCLUSIONS: These results demonstrate that ivacaftor is a critical component in the triple combination therapy along with tezacaftor and elexacaftor to increase constitutive CFTR function. This work further elucidates the mechanism of action of the effective triple combination therapeutic that is now the primary clinical tool in treating CF.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Aminofenóis , Benzodioxóis , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dimetil Sulfóxido/uso terapêutico , Combinação de Medicamentos , Humanos , Indóis , Mutação , Pirazóis , Piridinas , Pirrolidinas , Quinolonas
4.
Am J Physiol Lung Cell Mol Physiol ; 322(3): L305-L314, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35020527

RESUMO

Cystic fibrosis (CF) is a genetic disease caused by mutations of the gene encoding a cAMP-activated Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR modulator therapies consist of small-molecule drugs that rescue mutant CFTR. Regimens of single or combinations of CFTR modulators still rely on endogenous levels of cAMP to regulate CFTR activity. We investigated CFTR activation by the natural mediator prostaglandin E2 (PGE2) and lubiprostone (a Food and Drug Administration-approved drug known to target prostaglandin receptors) and tested the hypothesis that receptor-mediated CFTR activators can be used in combination with currently available CFTR modulators to increase function of mutant CFTR. Primary-cultured airway epithelia were assayed in Ussing chambers. Experimental CFTR activators and established CFTR modulators were applied for 24 h and/or acutely and analyzed for their effect on CFTR activity as measured by changes in short-circuit current (ISC). In non-CF airway epithelia, acute application of lubiprostone and PGE2 activated CFTR to the levels comparable to forskolin (Fsk). Pretreatment (24 h) with antagonists to prostaglandin receptors EP2 and EP4 abolished the ability of lubiprostone to acutely activate CFTR. In F508del homozygous airway epithelia pretreated with the triple combination of elexacaftor, tezacaftor, and ivacaftor (ELEXA/TEZ/IVA; i.e., Trikafta), acute application of lubiprostone was able to maximally activate CFTR. Prolonged (24 h) cotreatment of F508del homozygous epithelia with ELEXA/TEZ/IVA and lubiprostone increased acute CFTR activation by ∼60% compared with the treatment with ELEXA/TEZ/IVA alone. This work establishes the feasibility of targeting prostaglandin receptors to activate CFTR on the airway epithelia and demonstrates that cotreatment with lubiprostone can further restore modulator-rescued CFTR.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Aminofenóis/farmacologia , Aminofenóis/uso terapêutico , Benzodioxóis/uso terapêutico , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dinoprostona/farmacologia , Humanos , Lubiprostona/farmacologia , Lubiprostona/uso terapêutico , Mutação , Prostaglandinas , Receptores de Prostaglandina E Subtipo EP2 , Transdução de Sinais
5.
Sci Rep ; 11(1): 22616, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34799640

RESUMO

Quantitation of CFTR function in vitro is commonly performed by acutely stimulating then inhibiting ion transport through CFTR and measuring the resulting changes in transepithelial voltage (Vte) and current (ISC). While this technique is suitable for measuring the maximum functional capacity of CFTR, it may not provide an accurate estimate of in vivo CFTR activity. To test if CFTR-mediated ion transport could be measured in the absence of acute CFTR stimulation, primary airway epithelia were analyzed in an Ussing chamber with treatment of amiloride followed by CFTR(inh)-172 without acute activation of CFTR. Non-CF epithelia demonstrated a decrease in Vte and ISC following exposure to CFTR(inh)-172 and in the absence of forskolin/IBMX (F/I); this decrease is interpreted as a measure of spontaneous CFTR activity present in these epithelia. In F508del/F508del CFTR epithelia, F/I-induced changes in Vte and ISC were ~ fourfold increased after treatment with VX-809/VX-770, while the magnitude of spontaneous CFTR activities were only ~ 1.6-fold increased after VX-809/VX-770 treatment. Method-dependent discrepancies in the responses of other CF epithelia to modulator treatments were observed. These results serve as a proof of concept for the analysis of CFTR modulator responses in vitro in the absence of acute CFTR activation. Future studies will determine the usefulness of this approach in the development of novel CFTR modulator therapies.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Fibrose Cística/terapia , Células Epiteliais/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Aminofenóis , Aminopiridinas/farmacologia , Animais , Benzodioxóis/farmacologia , Produtos Biológicos , Células Cultivadas , Colforsina/farmacologia , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Eletrofisiologia/métodos , Epitélio/metabolismo , Genótipo , Humanos , Camundongos , Células NIH 3T3 , Quinolonas
6.
Sci Rep ; 11(1): 19810, 2021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34615919

RESUMO

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), which lead to early death due to progressive lung disease. The development of small-molecule modulators that directly interact with CFTR to aid in protein folding ("correctors") and/or increase channel function ("potentiators") have proven to be highly effective in the therapeutic treatment of CF. Notably, incorporation of the next-generation CFTR corrector, elexacaftor, into a triple combination therapeutic (marketed as Trikafta) has shown tremendous clinical promise in treating CF caused by F508del-CFTR. Here, we report on a newly-described role of elexacaftor as a CFTR potentiator. We explore the acute and chronic actions, pharmacology, and efficacy of elexacaftor as a CFTR potentiator in restoring function to multiple classes of CFTR mutations. We demonstrate that the potentiating action of elexacaftor exhibits multiplicative synergy with the established CFTR potentiator ivacaftor in rescuing multiple CFTR class defects, indicating that a new combination therapeutic of ivacaftor and elexacaftor could have broad impact on CF therapies.


Assuntos
Aminofenóis/farmacologia , Benzodioxóis/farmacologia , Agonistas dos Canais de Cloreto/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística , Indóis/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Pirrolidinas/farmacologia , Quinolinas/farmacologia , Células Cultivadas , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Combinação de Medicamentos , Humanos
8.
BMJ Open Respir Res ; 8(1)2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33622672

RESUMO

INTRODUCTION: The incubation of airway epithelia cells at low temperatures is a common in vitro experimental approach used in the field of cystic fibrosis (CF) research to thermo-stabilise F508del-CFTR and increase its functional expression. Given that the airway epithelium includes numerous ion transporters other than CFTR, we hypothesised that there was an impact of low temperature incubation on CFTR-independent ionoregulatory mechanisms in airway epithelia derived from individuals with and without CF. METHODS: After differentiation at the air-liquid interface, nasal epithelia were incubated at either 37°C or 29°C (low temperature) for 48 hours prior to analysis in an Ussing chamber. RESULTS: While F508del-CFTR activity was increased after low temperature incubation, activity of CFTR in non-CF epithelia was unchanged. Importantly, cultures incubated at 29°C demonstrated decreased transepithelial potential difference (TEPD) and short-circuit currents (Isc) at baseline. The predominant factor contributing to the reduced baseline TEPD and Isc in 29°C cultures was the reduced activity of the epithelial sodium channel (ENaC), evidenced by a reduced responsiveness to amiloride. This effect was observed in cells derived from both non-CF and CF donors. DISCUSSION: Significant transcriptional downregulation of ENaC subunits ß and γ were observed, which may partially explain the decreased ENaC activity. We speculate that low temperature incubation may be a useful experimental paradigm to reduce ENaC activity in in vitro epithelial cultures.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Canais Epiteliais de Sódio , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação para Baixo , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Epitélio/metabolismo , Humanos , Temperatura
9.
Physiol Rep ; 8(19): e14603, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33038073

RESUMO

INTRODUCTION: One method for assessing the in vitro response to CFTR-modulating compounds is by analysis of epithelial monolayers in an Ussing chamber, where the apical and basolateral surfaces are isolated and the potential difference, short-circuit current, and transepithelial resistance can be monitored. The effect of a chloride ion gradient across airway epithelia on transepithelial chloride transport and the magnitude of CFTR modulator efficacy were examined. METHODS: CFTR-mediated changes in the potential difference and transepithelial currents of primary human nasal epithelial cell cultures were quantified in Ussing chambers with either symmetrical solutions or reduced chloride solutions in the apical chamber. CFTR activity in homozygous F508del CFTR epithelia was rescued by treatment with VX-661, C4/C18, 4-phenylbutyrate (4-PBA) for 24 hr at 37°C or by incubation at 29°C for 48 hr. RESULTS: Imposing a chloride gradient increased CFTR-mediated and CaCC-mediated ion transport. Treatment of F508del CFTR homozygous cells with CFTR modulating compounds increased CFTR activity, which was significantly more evident in the presence of a chloride gradient. This observation was recapitulated with temperature-mediated F508del CFTR correction. CONCLUSIONS: Imposing a chloride gradient during Ussing chamber measurements resulted in increased CFTR-mediated ion transport in expanded non-CF and F508del CFTR homozygous epithelia. In F508del CFTR homozygous epithelia, the magnitude of response to CFTR modulating compounds or low temperature was greater when assayed with a chloride gradient compared to symmetrical chloride, resulting in an apparent increase in measured efficacy. Future work may direct which methodologies utilized to quantify CFTR modulator response in vitro are most appropriate for the estimation of in vivo efficacy.


Assuntos
Benzodioxóis/farmacologia , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/tratamento farmacológico , Indóis/farmacologia , Adulto , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Células Epiteliais/metabolismo , Epitélio/metabolismo , Feminino , Humanos , Transporte de Íons/efeitos dos fármacos , Masculino , Mucosa Nasal/metabolismo
10.
Nat Commun ; 11(1): 2485, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427931

RESUMO

Cigarette smoke first interacts with the lung through the cellularly diverse airway epithelium and goes on to drive development of most chronic lung diseases. Here, through single cell RNA-sequencing analysis of the tracheal epithelium from smokers and non-smokers, we generate a comprehensive atlas of epithelial cell types and states, connect these into lineages, and define cell-specific responses to smoking. Our analysis infers multi-state lineages that develop into surface mucus secretory and ciliated cells and then contrasts these to the unique specification of submucosal gland (SMG) cells. Accompanying knockout studies reveal that tuft-like cells are the likely progenitor of both pulmonary neuroendocrine cells and CFTR-rich ionocytes. Our smoking analysis finds that all cell types, including protected stem and SMG populations, are affected by smoking through both pan-epithelial smoking response networks and hundreds of cell-specific response genes, redefining the penetrance and cellular specificity of smoking effects on the human airway epithelium.


Assuntos
Células Epiteliais/metabolismo , Perfilação da Expressão Gênica/métodos , Pulmão/metabolismo , Mucosa Respiratória/metabolismo , Fumar/genética , Traqueia/metabolismo , Animais , Células Cultivadas , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Humanos , Pulmão/citologia , Camundongos , Células NIH 3T3 , não Fumantes/estatística & dados numéricos , Mucosa Respiratória/citologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Fumantes/estatística & dados numéricos , Traqueia/citologia
11.
Free Radic Biol Med ; 146: 324-332, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31740228

RESUMO

A hallmark of cystic fibrosis (CF) lung pathology is an increased susceptibility to pulmonary infections. Thiocyanate (-SCN) is an endogenous component of the innate immunity's peroxidase system that converts -SCN to the antimicrobial agent hypothiocyanite (HOSCN). We have previously shown that the host thioredoxin reductase (TrxR), but not the pathogen's TrxR, can selectively detoxify HOSCN thereby decreasing inflammation and oxidative stress. We tested whether the -SCN analog selenocyanate (-SeCN) shares these properties against several clinical CF bacterial isolates. We examined oxidant production from a lactoperoxidase (LPO) system using -SeCN as a potential substrate. The LPO system generated an oxidant similar in nature to HOSCN and consistent with being HOSeCN. The rate of oxidant generation using -SeCN was significantly less than seen for -SCN. An LPO system was used to generate HOSCN or HOSeCN and compared for antimicrobial activity during in situ exposure of clinical CF isolates of P. aeruginosa (PA), B. cepacia complex (BCC), and methicillin-resistant S. aureus (MRSA) obtained from CF sputum samples. Bacterial viability was assessed by colony forming units. Selective detoxification of HOSeCN was determined by comparing its metabolism by mammalian thioredoxin reductase (TrxR) to bacterial TrxR following the consumption of NADPH. We also assessed potential toxicity of equivalent HOSeCN generation, which demonstrated in situ antimicrobial activity, in human bronchial epithelial cells with a cell viability assay. The -SeCN/HOSeCN system was much more potent than -SCN/HOSCN system at killing PA, BCC and MRSA isolates. The -SeCN/HOSeCN system was more effective at killing -SCN/HOSCN resistant isolates. Mammalian TrxR selectively detoxified HOSeCN whereas the bacterial TrxR enzyme showed little activity. Human bronchial epithelial cells exposed to equivalent flux of HOSeCN that killed several CF pathogens showed no decrease in viability. -SeCN may be an effective therapeutic for the treatment of CF lung pathogens that are difficult to treat with current antibiotics.


Assuntos
Anti-Infecciosos , Staphylococcus aureus Resistente à Meticilina , Pró-Fármacos , Animais , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Cianatos , Humanos , Compostos de Selênio , Tiocianatos
12.
Am J Respir Cell Mol Biol ; 61(5): 560-566, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30958968

RESUMO

Chronic obstructive pulmonary disease (COPD) is a major cause of mortality worldwide and is characterized by an excessive airway neutrophilic response. The neutrophil chemoattractant proline-glycine-proline (PGP) and its more potent acetylated form (acPGP) have been found to be elevated in patients with COPD and act via CXCR2. Here, we investigated the impact of neutralizing PGP peptides in a murine model for emphysema. The PGP-neutralizing peptide l-arginine-threonine-arginine (RTR) was used first in a 6-week model of cigarette smoke exposure, where it attenuated lung inflammation. Then, in a model of chronic smoke exposure, mice were exposed to cigarette smoke and RTR treatment was initiated after 10 weeks of smoke exposure. This treatment was continued together with smoke exposure for another 13 weeks, for a total of 23 weeks of smoke exposure. RTR significantly inhibited neutrophil and macrophage influx into the lungs in the 6-week model of exposure. RTR also attenuated the development of emphysema, normalized lung volumes, and reduced right ventricular hypertrophy in the chronic exposure model. Murine epithelia expressed CXCR2, and this expression was increased after smoke exposure. In vitro, human bronchial epithelial cells also demonstrated robust expression of CXCR2, and stimulation of primary human bronchial epithelial cells with acPGP led to increased release of MMP-9 and IL-8. Overall, these results provide evidence that acPGP plays a critical role during the development of emphysema in cigarette smoke-induced injury, and highlight a new epithelial mechanism by which acPGP augments neutrophilic inflammation.


Assuntos
Inflamação/metabolismo , Neutrófilos/metabolismo , Enfisema Pulmonar/etiologia , Animais , Células Cultivadas , Humanos , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Oligopeptídeos/metabolismo , Prolina/análogos & derivados , Prolina/metabolismo , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Enfisema Pulmonar/metabolismo , Fumaça/efeitos adversos
13.
Cell ; 176(1-2): 113-126.e15, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30633902

RESUMO

Here, we describe a novel pathogenic entity, the activated PMN (polymorphonuclear leukocyte, i.e., neutrophil)-derived exosome. These CD63+/CD66b+ nanovesicles acquire surface-bound neutrophil elastase (NE) during PMN degranulation, NE being oriented in a configuration resistant to α1-antitrypsin (α1AT). These exosomes bind and degrade extracellular matrix (ECM) via the integrin Mac-1 and NE, respectively, causing the hallmarks of chronic obstructive pulmonary disease (COPD). Due to both ECM targeting and α1AT resistance, exosomal NE is far more potent than free NE. Importantly, such PMN-derived exosomes exist in clinical specimens from subjects with COPD but not healthy controls and are capable of transferring a COPD-like phenotype from humans to mice in an NE-driven manner. Similar findings were observed for another neutrophil-driven disease of ECM remodeling (bronchopulmonary dysplasia [BPD]). These findings reveal an unappreciated role for exosomes in the pathogenesis of disorders of ECM homeostasis such as COPD and BPD, providing a critical mechanism for proteolytic damage.


Assuntos
Exossomos/fisiologia , Neutrófilos/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/citologia , Células Cultivadas , Matriz Extracelular/metabolismo , Feminino , Humanos , Inflamação , Integrinas , Elastase de Leucócito/metabolismo , Pulmão/metabolismo , Pulmão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , alfa 1-Antitripsina/metabolismo
15.
PLoS One ; 13(12): e0209026, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30540818

RESUMO

Cystic fibrosis (CF) is the most common life-shortening genetic disease and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Several current therapies aim at improving availability and/or function of the mutant CFTR proteins. The combination therapeutic lumacaftor/ivacaftor (Orkambi, luma/iva) partially corrects folding and potentiates CFTR function impaired by the F508del mutation. Despite the potential for clinical benefit, a substantial number of patients discontinue treatment due to intolerable adverse effects. The aim of the present study is to identify differences between individuals who continued treatment and those who discontinued due to adverse respiratory effects to potentially inform treatment decisions. Clinical data from the year prior to treatment initiation were analyzed from 82 patients homozygous for the F508del mutation treated at the Colorado Adult CF Program. Blood samples were collected from 30 of these subjects before initiation of treatment to examine expression of circulating leukocyte surface antigens and cytokines. Clinical and demographic characteristics were analyzed along with inflammatory markers to determine biomarkers of drug discontinuation. The use of oral prednisone and/or nasal budesonide in the year prior to luma/iva initiation was more prevalent in CF subjects who did not tolerate luma/iva (82% vs. 43%). Increased age, but not gender or initial lung function, was associated with higher probability of discontinuing treatment due to side effects overall. Worse lung function (lower ppFEV1, ppFEF25-75 ≤ 60%) was associated with higher incidence of discontinuing treatment due to pulmonary adverse effects. In a nested cohort of patients, increased surface levels of CXCR2 on CD14+CD16- monocytes were associated with discontinuation. Overall, the patients who tolerated luma/iva were distinguishable from those who did not tolerate the drug based on clinical and cellular markers obtained prior to treatment initiation.


Assuntos
Corticosteroides/uso terapêutico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Leucócitos/metabolismo , Receptores de Interleucina-8B/metabolismo , Adulto , Aminofenóis/efeitos adversos , Aminofenóis/uso terapêutico , Aminopiridinas/efeitos adversos , Aminopiridinas/uso terapêutico , Benzodioxóis/efeitos adversos , Benzodioxóis/uso terapêutico , Fibrose Cística/genética , Combinação de Medicamentos , Feminino , Volume Expiratório Forçado , Deleção de Genes , Homozigoto , Humanos , Modelos Logísticos , Masculino , Adesão à Medicação , Quinolonas/efeitos adversos , Quinolonas/uso terapêutico , Tetraspanina 30/metabolismo
16.
Eur Respir J ; 49(4)2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28381428

RESUMO

Matrix metalloprotease-9 (MMP-9) plays a role in progression of cystic fibrosis, and doxycycline can reduce MMP-9 in vitro Here, we explore the effect of doxycycline during cystic fibrosis exacerbation treatment on MMP-9 related readouts and clinical end-points.This randomised, double-blind, placebo-controlled study enrolled hospitalised patients with cystic fibrosis undergoing exacerbation. In total, 20 participants were given doxycycline and 19 participants were given placebo over an 8-day period during hospitalisation. Biospecimens were collected at the beginning and the end of the study period. Primary end-points were total MMP-9 levels in the sputum and safety/tolerability. Secondary end-points included change in lung function, time to next exacerbation, and markers of MMP-9-related protease activity (active MMP-9 and TIMP-1). Nonparametric testing was used for within-group and between-group analyses.Doxycycline was well tolerated, with no treatment discontinuations or serious adverse events. Doxycycline reduced total sputum MMP-9 levels by 63.2% (p<0.05), and was also associated with a 56.5% reduction in active MMP-9 levels (p<0.05), a 1.6-fold increase in sputum TIMP-1 (p<0.05), improvement in forced expiratory volume in 1 s (p<0.05), and an increase in time to next exacerbation (p<0.01).Adjunctive use of doxycycline improved dysregulated MMP-9 levels in sputum, along with biomarkers consistent with a reduced proteolytic pulmonary environment. Improvement in clinical outcome measures suggests an important therapeutic benefit of doxycycline for individuals with cystic fibrosis.


Assuntos
Fibrose Cística/tratamento farmacológico , Doxiciclina/uso terapêutico , Metaloproteinase 9 da Matriz/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Adolescente , Adulto , Alabama , Método Duplo-Cego , Feminino , Volume Expiratório Forçado , Humanos , Estimativa de Kaplan-Meier , Modelos Lineares , Pulmão/fisiopatologia , Masculino , Escarro/química , Adulto Jovem
17.
Glia ; 65(6): 945-963, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28300326

RESUMO

In neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), chronic activation of microglia contributes to disease progression. Activated microglia produce cytokines, chemokines, and other factors that normally serve to clear infection or damaged tissue either directly or through the recruitment of other immune cells. The molecular program driving this phenotype is classically linked to the transcription factor NF-κB and characterized by the upregulation of proinflammatory factors such as IL-1ß, TNF-α, and IL-6. Here, we investigated the role of HuR, an RNA-binding protein that regulates gene expression through posttranscriptional pathways, on the molecular and cellular phenotypes of activated microglia. We performed RNA sequencing of HuR-silenced microglia and found significant attenuation of lipopolysaccharide-induced IL-1ß and TNF-α inflammatory pathways and other factors that promote microglial migration and invasion. RNA kinetics and luciferase reporter studies suggested that the attenuation was related to altered promoter activity rather than a change in RNA stability. HuR-silenced microglia showed reduced migration, invasion, and chemotactic properties but maintained viability. MMP-12, a target exquisitely sensitive to HuR knockdown, participates in the migration/invasion phenotype. HuR is abundantly detected in the cytoplasmic compartment of activated microglia from ALS spinal cords consistent with its increased activity. Microglia from ALS-associated mutant SOD1 mice demonstrated higher migration/invasion properties which can be blocked with HuR inhibition. These findings underscore an important role for HuR in sculpting the molecular signature and phenotype of activated microglia, and as a possible therapeutic target in ALS and other neurodegenerative diseases.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Microglia/metabolismo , Adulto , Idoso , Esclerose Lateral Amiotrófica/patologia , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Proteína Semelhante a ELAV 1/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Metaloproteinase 12 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/patologia , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia
18.
J Cyst Fibros ; 16(3): 358-366, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28025037

RESUMO

BACKGROUND: Concomitant use of oral azithromycin and inhaled tobramycin occurs in approximately half of US cystic fibrosis (CF) patients. Recent data suggest that this combination may be antagonistic. METHODS: Test the hypothesis that azithromycin reduces the clinical benefits of tobramycin by analyses of clinical trial data, in vitro modeling of P. aeruginosa antibiotic killing, and regulation of the MexXY efflux pump. RESULTS: Ongoing administration of azithromycin associates with reduced ability of inhaled tobramycin, as compared with aztreonam, to improve lung function and quality of life in a completed clinical trial. In users of azithromycin FEV1 (L) increased 0.8% during a 4-week period of inhaled tobramycin and an additional 6.4% during a subsequent 4-week period of inhaled aztreonam (P<0.005). CFQ-R respiratory symptom score decreased 1.8 points during inhaled tobramycin and increased 8.3 points during subsequent inhaled aztreonam (P<0.001). A smaller number of trial participants not using azithromycin had similar improvement in lung function and quality of life scores during inhaled tobramycin and inhaled aztreonam. In vitro, azithromycin selectively reduced the bactericidal effects tobramycin in cultures of clinical strains of P. aeruginosa, while up regulating antibiotic resistance through MexXY efflux. CONCLUSIONS: Azithromycin appears capable of reducing the antimicrobial benefits of tobramycin by inducing adaptive bacterial stress responses in P. aeruginosa, suggesting that these medications together may not be optimal chronic therapy for at least some patients.


Assuntos
Azitromicina , Aztreonam , Fibrose Cística , Infecções por Pseudomonas , Pseudomonas aeruginosa/efeitos dos fármacos , Tobramicina , Antibacterianos/administração & dosagem , Antibacterianos/farmacocinética , Azitromicina/administração & dosagem , Azitromicina/farmacocinética , Aztreonam/administração & dosagem , Aztreonam/farmacocinética , Fibrose Cística/complicações , Fibrose Cística/microbiologia , Fibrose Cística/psicologia , Vias de Administração de Medicamentos , Interações Medicamentosas , Monitoramento de Medicamentos/métodos , Feminino , Humanos , Masculino , Infecções por Pseudomonas/diagnóstico , Infecções por Pseudomonas/tratamento farmacológico , Testes de Função Respiratória/métodos , Tobramicina/administração & dosagem , Tobramicina/farmacocinética , Resultado do Tratamento , Adulto Jovem
19.
J Cyst Fibros ; 15(1): 67-73, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25769931

RESUMO

BACKGROUND: Ivacaftor improves clinical outcome by potentiation of mutant G551D CFTR. Due to the presence of CFTR in monocytes and polymorphonuclear neutrophils (PMNs), we hypothesized that ivacaftor may impact leukocyte activation. METHODS: We examined blood leukocytes from G551D CF subjects prior to and at one and six months after receiving ivacaftor. Blood leukocytes from ivacaftor-naïve G551D, F508del, and healthy controls were also treated with ivacaftor ex vivo to assess mutation-specific effects. RESULTS: Compared to healthy controls, G551D CF subjects had significantly higher expression of active CD11b on PMNs and of CD63 on monocytes, which were normalized by in vivo ivacaftor treatment. Ex vivo exposure to ivacaftor of blood cells from G551D, but not F508del and healthy subjects, resulted in changes in CXCR2 and CD16 expression on PMNs. CONCLUSIONS: In vivo and ex vivo exposure of G551D CF leukocytes to ivacaftor resulted in an altered activation profile, suggesting mutation-specific leukocyte modulation.


Assuntos
Aminofenóis/administração & dosagem , Antígeno CD11b/análise , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística , Monócitos , Neutrófilos , Quinolonas/administração & dosagem , Tetraspanina 30/análise , Adulto , Agonistas dos Canais de Cloreto/administração & dosagem , Fibrose Cística/sangue , Fibrose Cística/tratamento farmacológico , Feminino , Humanos , Masculino , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Mutação , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Estatística como Assunto
20.
Am J Respir Cell Mol Biol ; 54(3): 359-69, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26222144

RESUMO

Proteases are important regulators of pulmonary remodeling and airway inflammation. Recently, we have characterized the enzyme prolyl endopeptidase (PE), a serine peptidase, as a critical protease in the generation of the neutrophil chemoattractant tripeptide Pro-Gly-Pro (PGP) from collagen. However, PE has been characterized as a cytosolic enzyme, and the mechanism mediating PE release extracellularly remains unknown. We examined the role of exosomes derived from airway epithelia as a mechanism for PE release and the potential extracellular signals that regulate the release of these exosomes. We demonstrate a specific regulatory pathway of exosome release from airway epithelia and identify PE as novel exosome cargo. LPS stimulation of airway epithelial cells induces release of PE-containing exosomes, which is significantly attenuated by small interfering RNA depletion of Toll-like receptor 4 (TLR4). These differences were recapitulated upon intratracheal LPS administration in mice competent versus deficient for TLR4 signaling. Finally, sputum samples from subjects with cystic fibrosis colonized with Pseudomonas aeruginosa demonstrate elevated exosome content and increased PE levels. This TLR4-based mechanism highlights the first report of nonstochastic release of exosomes in the lung and couples TLR4 activation with matrikine generation. The increased quantity of these proteolytic exosomes in the airways of subjects with chronic lung disease highlights a new mechanism of injury and inflammation in the pathogenesis of pulmonary disorders.


Assuntos
Brônquios/enzimologia , Fibrose Cística/enzimologia , Células Epiteliais/enzimologia , Exossomos/enzimologia , Proteínas Mitocondriais/metabolismo , Serina Endopeptidases/metabolismo , Receptor 4 Toll-Like/metabolismo , Adulto , Animais , Brônquios/efeitos dos fármacos , Brônquios/microbiologia , Estudos de Casos e Controles , Linhagem Celular , Fibrose Cística/genética , Fibrose Cística/microbiologia , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Exossomos/efeitos dos fármacos , Exossomos/microbiologia , Feminino , Humanos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Endogâmicos C3H , Camundongos Knockout , Prolil Oligopeptidases , Pseudomonas aeruginosa/isolamento & purificação , Interferência de RNA , Transdução de Sinais , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/genética , Transfecção , Adulto Jovem
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