RESUMO
Chronic intestinal inflammation is a poorly understood manifestation of cystic fibrosis (CF), which may be refractory to ion channel CF transmembrane conductance regulator (CFTR) modulator therapy. People with CF exhibit intestinal dysbiosis, which has the potential for stimulating intestinal and systemic inflammation. CFTR is expressed in organ epithelia, leukocytes, and other tissues. Here, we investigate the contribution of intestinal epithelium-specific loss of Cftr [iCftr knockout (KO)] to dysbiosis and inflammation in mice treated with either of two antiobstructive dietary regimens necessary to maintain CF mouse models [polyethylene glycol (PEG) laxative or a liquid diet (LiqD)]. Feces collected from iCftr KO mice and their wild-type (WT) sex-matched littermates were used to measure fecal calprotectin to evaluate inflammation and to perform 16S rRNA sequencing to characterize the gut microbiome. Fecal calprotectin was elevated in iCftr KO relative to WT mice that consumed either PEG or LiqD. PEG iCftr KO mice did not show a change in α diversity versus WT mice but demonstrated a significant difference in microbial composition (ß diversity) with included increases in the phylum Proteobacteria, the family Peptostreptococcaceae, four genera of Clostridia including C. innocuum, and the mucolytic genus Akkermansia. Fecal microbiome analysis of LiqD-fed iCftr KO mice showed both decreased α diversity and differences in microbial composition with increases in the Proteobacteria family Enterobacteriaceae, Firmicutes families Clostridiaceae and Peptostreptococcaceae, and enrichment of Clostridium perfringens, C. innocuum, C. difficile, mucolytic Ruminococcus gnavus, and reduction of Akkermansia. It was concluded that epithelium-specific loss of Cftr is a major driver of CF intestinal dysbiosis and inflammation with significant similarities to previous studies of pan Cftr KO mice.NEW & NOTEWORTHY Chronic intestinal inflammation is a manifestation of cystic fibrosis (CF), a disease caused by loss of the anion channel CF transmembrane conductance regulator (CFTR) that is expressed in many tissues. This study shows that intestinal epithelial cell-specific loss of CFTR [inducible Cftr knockout (KO)] in mice is sufficient to induce intestinal dysbiosis and inflammation. Experiments were performed on mice consuming two dietary regimens routinely used to prevent obstruction in CF mice.
Assuntos
Clostridioides difficile , Fibrose Cística , Obstrução Intestinal , Animais , Humanos , Camundongos , Clostridioides difficile/genética , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Disbiose/microbiologia , Expectorantes/uso terapêutico , Fezes , Inflamação , Complexo Antígeno L1 Leucocitário/uso terapêutico , Camundongos Endogâmicos CFTR , Camundongos Knockout , RNA Ribossômico 16SRESUMO
BACKGROUND: Mucus stasis, a hallmark of muco-obstructive disease, results from impaired mucociliary transport and leads to lung function decline and chronic infection. Although therapeutics that target mucus stasis in the airway, such as hypertonic saline or rhDNAse, show some therapeutic benefit, they do not address the underlying electrostatic defect apparent in mucins in CF and related conditions. We have previously shown poly (acetyl, arginyl) glucosamine (PAAG, developed as SNSP113), a soluble, cationic polymer, significantly improves mucociliary transport in a rat model of CF by normalizing the charge defects of CF mucin. Here, we report efficacy in the CFTR-sufficient, ENaC hyperactive, Scnn1b-Tg mouse model that develops airway muco-obstruction due to sodium hyperabsorption and airway dehydration. METHODS: Scnn1b-Tg mice were treated with either 250 µg/mL SNSP113 or vehicle control (1.38% glycerol in PBS) via nebulization once daily for 7 days and then euthanized for analysis. Micro-Optical Coherence Tomography-based evaluation of excised mouse trachea was used to determine the effect on the functional microanatomy. Tissue analysis was performed by routine histopathology. RESULTS: Nebulized treatment of SNSP113 significantly improved mucociliary transport in the airways of Scnn1b-Tg mice, without altering the airway surface or periciliary liquid layer. In addition, SNSP113 significantly reversed epithelial hypertrophy and goblet cell metaplasia. Finally, SNSP113 significantly ameliorated eosinophilic crystalline pneumonia and lung consolidation in addition to inflammatory macrophage influx in this model. CONCLUSION: Overall, this study extends the efficacy of SNSP113 as a potential therapeutic to alleviate mucus stasis in muco-obstructive diseases in CF and potentially in related conditions.
Assuntos
Obstrução das Vias Respiratórias , Fibrose Cística , alfa 2-Macroglobulinas Associadas à Gravidez , Feminino , Gravidez , Camundongos , Animais , Ratos , Depuração Mucociliar , Camundongos Transgênicos , Modelos Animais de Doenças , Camundongos Endogâmicos CFTR , Pulmão , Canais Epiteliais de Sódio/genéticaRESUMO
Mutations in the CFTR chloride channel result in intestinal obstructive episodes in cystic fibrosis (CF) patients and in CF animal models. In this study, we explored the possibility of reducing the frequency of obstructive episodes in cftr-/- mice through the oral application of a gut-selective NHE3 inhibitor tenapanor and searched for the underlying mechanisms involved. Sex- and age-matched cftr+/+ and cftr-/- mice were orally gavaged twice daily with 30 mg kg-1 tenapanor or vehicle for a period of 21 days. Body weight and stool water content was assessed daily and gastrointestinal transit time (GTT) once weekly. The mice were sacrificed when an intestinal obstruction was suspected or after 21 days, and stool and tissues were collected for further analysis. Twenty-one day tenapanor application resulted in a significant increase in stool water content and stool alkalinity and a significant decrease in GTT in cftr+/+ and cftr-/- mice. Tenapanor significantly reduced obstructive episodes to 8% compared to 46% in vehicle-treated cftr-/- mice and prevented mucosal inflammation. A decrease in cryptal hyperproliferation, mucus accumulation, and mucosal mast cell number was also observed in tenapanor- compared to vehicle-treated, unobstructed cftr-/- mice. Overall, oral tenapanor application prevented obstructive episodes in CFTR-deficient mice and was safe in cftr+/+ and cftr-/- mice. These results suggest that tenapanor may be a safe and affordable adjunctive therapy in cystic fibrosis patients to alleviate constipation and prevent recurrent DIOS.
Assuntos
Fibrose Cística , Obstrução Intestinal , Animais , Fibrose Cística/complicações , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Isoquinolinas , Camundongos , Camundongos Endogâmicos CFTR , Trocador 3 de Sódio-Hidrogênio , Sulfonamidas , ÁguaRESUMO
Macrophages represent prominent immune orchestrators of cystic fibrosis (CF) inflammation and, as such, are an ever-increasing focus of CF research with several reports of intrinsic immune dysfunction related to loss of CFTR activity in macrophages themselves. Animal models of CF have contributed, in no small part, to a deepening of our understanding of the pathophysiology of the disease and towards therapeutic development. A commonly-used animal model in CF research is the Cftrtm1Unc Tg(FABP-hCFTR) mouse, which displays gut-specific expression of a human CFTR transgene in order to rescue the high rate of early mortality in Cftr-null mice associated with severe intestinal obstruction. We find significant variation in the response to inflammatory challenge of patient macrophages and cells derived from the Cftrtm1Unc Tg(FABP-hCFTR) mouse and show that macrophages derived from this mouse exhibit aberrant expression of human CFTR. This may contribute to the absence of inflammatory changes in this model.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Intestinos/metabolismo , Animais , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Modelos Animais de Doenças , Humanos , Macrófagos , Camundongos , Camundongos Endogâmicos CFTR , FenótipoRESUMO
Cystic fibrosis (CF) is an autosomal recessive gene disorder that affects tens of thousands of patients worldwide. Individuals with CF often succumb to progressive lung disease and respiratory failure following recurrent infections with bacteria. Viral infections can also damage the lungs and heighten the CF patient's susceptibility to bacterial infections and long-term sequelae. Vitamin A is a key nutrient important for immune health and epithelial cell integrity, but there is currently no consensus as to whether vitamin A should be monitored in CF patients. Here we evaluate previous literature and present results from a CF mouse model, showing that oral vitamin A supplements significantly reduce lung lesions that would otherwise persist for 5-6 weeks post-virus exposure. Based on these results, we encourage continued research and suggest that programs for the routine monitoring and regulation of vitamin A levels may help reduce virus-induced lung pathology in CF patients.
Assuntos
Fibrose Cística/metabolismo , Pulmão/patologia , Infecções por Respirovirus/metabolismo , Vírus Sendai/fisiologia , Vitamina A/metabolismo , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Suplementos Nutricionais , Modelos Animais de Doenças , Proteínas de Ligação a Ácido Graxo/genética , Humanos , Pulmão/virologia , Camundongos , Camundongos Endogâmicos CFTR , Camundongos Transgênicos , Regiões Promotoras Genéticas , Vitamina A/administração & dosagemRESUMO
BACKGROUND: Destruction of blood-brain barrier (BBB) ââis one of the main mechanisms of secondary brain injury following intracerebral hemorrhage (ICH). Frizzled-7 is a key protein expressed on the surface of endothelial cells that controls vascular permeability through the Wnt-canonical pathway involving WNT1-inducible signaling pathway protein 1 (WISPI). This study aimed to investigate the role of Frizzled-7 signaling in BBB preservation after ICH in mice. METHODS: Adult CD1 mice were subjected to sham surgery or collagenase-induced ICH. Frizzled-7 activation or knockdown was performed by administration of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) by intracerebroventricular injection at 48 h before ICH induction. WISP1 activation or WISP1 knockdown was performed to evaluate the underlying signaling pathway. Post-ICH assessments included neurobehavior, brain edema, BBB permeability, hemoglobin level, western blot and immunofluorescence. RESULTS: The brain expressions of Frizzled-7 and WISP1 significantly increased post-ICH. Frizzled-7 was expressed in endothelial cells, astrocytes, and neurons after ICH. Activation of Frizzled-7 significantly improved neurological function, reduced brain water content and attenuated BBB permeability to large molecular weight substances after ICH. Whereas, knockdown of Frizzled-7 worsened neurological function and brain edema after ICH. Activation of Frizzled-7 significantly increased the expressions of Dvl, ß-Catenin, WISP1, VE-Cadherin, Claudin-5, ZO-1 and reduced the expression of phospho-ß-Catenin. WISP1 knockdown abolished the effects of Frizzled-7 activation on the expressions of VE-Cadherin, Claudin-5 and ZO-1 at 24 h after ICH. CONCLUSIONS: Frizzled-7 activation potentially attenuated BBB permeability and improved neurological deficits after ICH through Dvlââ/ß-Catenin/WISP1 pathway. Frizzled-7 may be a potential target for the development of ICH therapeutic drugs.
Assuntos
Barreira Hematoencefálica , Proteínas de Sinalização Intercelular CCN/metabolismo , Hemorragia Cerebral , Proteínas Desgrenhadas/metabolismo , Receptores Frizzled/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , beta Catenina/metabolismo , Animais , Comportamento Animal , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/fisiopatologia , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/fisiopatologia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos CFTR , Transdução de Sinais/fisiologiaRESUMO
Vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide with potent anti-inflammatory, bronchodilatory and immunomodulatory functions, is secreted by intrinsic neurons innervating all exocrine glands, including the pancreas, in which it exerts a regulatory function in the secretion of insulin and glucagon. Cystic fibrosis-related diabetes (CFRD) is the most common co-morbidity associated with cystic fibrosis (CF), impacting approximately 50% of adult patients. We recently demonstrated a 50% reduction of VIP abundance in the lungs, duodenum and sweat glands of C57Bl/6 CF mice homozygous for the F508del-CFTR disease-causing mutation. VIP deficiency resulted from a reduction in VIPergic and cholinergic innervation, starting before signs of CF disease were observed. As VIP functions as a neuromodulator with insulinotropic effect on pancreatic beta cells, we sought to study changes in VIP in the pancreas of CF mice. Our goal was to examine VIP content and VIPergic innervation in the pancreas of 8- and 17-week-old F508del-CFTR homozygous mice and to determine whether changes in VIP levels would contribute to CFRD development. Our data showed that a decreased amount of VIP and reduced innervation are found in CF mice pancreas, and that these mice also exhibited reduced insulin secretion, up-regulation of glucagon production and high random blood glucose levels compared to same-age wild-type mice. We propose that low level of VIP, due to reduced innervation of the CF pancreas and starting at an early disease stage, contributes to changes in insulin and glucagon secretion that can lead to CFRD development.
Assuntos
Fibrose Cística/metabolismo , Complicações do Diabetes/etiologia , Complicações do Diabetes/metabolismo , Pâncreas/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Glicemia/metabolismo , Glucagon/metabolismo , Homozigoto , Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CFTRRESUMO
BACKGROUND: Cystic fibrosis transmembrane conductance regulator (CFTR) has been associated with vascular tone and blood pressure (BP), however, its role in the genesis of hypertension remains elusive. In the present study, we investigated the regulating effect of CFTR on angiotensin II (Ang II) -induced hypertension and defined the molecular role of CFTR in vasoconstriction. RESULTS: We found that CFTR mRNA and protein expression were markedly down-regulated in the arteries from Ang II induced hypertensive animals. During the development of hypertension, BP of Cftr-â£/- mice was significantly higher than that of Cftr+â£/+ mice. Arteries from Cftr-â£/- mice or pre-incubated with CFTR specific inhibitor CFTR(inh)-172 exhibited a greater contractile response to Ang II. In vascular smooth muscle cells (VSMCs), the phosphorylation of myosin light chain (MLC), which is the core of VSMCs contraction, was negatively modulated by CFTR. Furthermore, intracellular Ca2+ concentration ([Ca2+]i) rise in response to Ang II was negatively modulated by CFTR, while no alteration was observed in resting VSMCs. Ras homolog family member A/Rho-associated protein kinase (RhoA/Rock) mediated phosphorylation of myosin phosphatase target subunit 1 (MYPT1), a regulator of MLC phosphorylation, was negatively modulated by CFTR in both resting and Ang II-stimulated VSMCs. CONCLUSIONS: This study demonstrates that CFTR is a negative regulator of vasoconstriction and hypertension, and the underlying mechanism contains two possible pathways: (1) in resting VSMCs, CFTR altered MLC phosphorylation through RhoA/Rock pathway; (2) in Ang II stimulated VSMCs, the regulating effect was mediated by both Ca2+ influx and RhoA/Rock mediated pathway.
Assuntos
Angiotensina II , Cálcio , Hipertensão , Vasoconstrição , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Hipertensão/genética , Hipertensão/metabolismo , Camundongos , Camundongos Endogâmicos CFTR , Camundongos Knockout , Músculo Liso Vascular/citologia , Fosforilação , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
AIM: Cystic fibrosis patients have an increased risk of developing metabolic alkalosis presumably as a result of altered renal HCO3- handling. In this study, we directly assess the kidneys' ability to compensate for a chronic base-load in the absence of functional CFTR. METHODS: Comprehensive urine and blood acid-base analyses were done in anaesthetized WT mice or mice lacking either CFTR or pendrin, with or without 7 days of oral NaHCO3 loading. The in vivo experiments were complemented by a combination of immunoblotting and experiments with perfused isolated mouse cortical collecting ducts (CCD). RESULTS: Base-loaded WT mice maintained acid-base homeostasis by elevating urinary pH and HCO3- excretion and decreasing urinary net acid excretion. In contrast, pendrin KO mice and CFTR KO mice were unable to increase urinary pH and HCO3- excretion and unable to decrease urinary net acid excretion sufficiently and thus developed metabolic alkalosis in response to the same base-load. The expression of pendrin was increased in response to the base-load in WT mice with a paralleled increased pendrin function in the perfused CCD. In CFTR KO mice, 7 days of base-loading did not upregulate pendrin expression and apical Cl- /HCO3- exchange function was strongly blunted in the CCD. CONCLUSION: CFTR KO mice develop metabolic alkalosis during a chronic base-load because they are unable to sufficiently elevate renal HCO3- excretion. This can be explained by markedly reduced pendrin function in the absence of CFTR.
Assuntos
Alcalose , Regulador de Condutância Transmembrana em Fibrose Cística , Animais , Bicarbonatos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Rim/metabolismo , Camundongos , Camundongos Endogâmicos CFTRRESUMO
Cystic fibrosis (CF) is the most common genetic disease in Caucasians. CF is manifested by abnormal accumulation of mucus in the lungs, which serves as fertile ground for the growth of microorganisms leading to recurrent infections and ultimately, lung failure. Mucus in CF patients consists of DNA from dead neutrophils as well as mucins produced by goblet cells. MUC5AC mucin leads to pathological plugging of the airways whereas MUC5B has a protective role against bacterial infection. Therefore, decreasing the level of MUC5AC while maintaining MUC5B intact would in principle be a desirable mucoregulatory treatment outcome. Fenretinide prevented the lipopolysaccharide-induced increase of MUC5AC gene expression, without affecting the level of MUC5B, in a lung goblet cell line. Additionally, fenretinide treatment reversed the pro-inflammatory imbalance of fatty acids by increasing docosahexaenoic acid and decreasing the levels of arachidonic acid in a lung epithelial cell line and primary leukocytes derived from CF patients. Furthermore, for the first time we also demonstrate the effect of fenretinide on multiple unsaturated fatty acids, as well as differential effects on the levels of long- compared to very-long-chain saturated fatty acids which are important substrates of complex phospholipids. Finally, we demonstrate that pre-treating mice with fenretinide in a chronic model of P. aeruginosa lung infection efficiently decreases the accumulation of mucus. These findings suggest that fenretinide may offer a new approach to therapeutic modulation of pathological mucus production in CF.
Assuntos
Fibrose Cística/complicações , Fenretinida/administração & dosagem , Pulmão/efeitos dos fármacos , Pneumonia/prevenção & controle , Infecções por Pseudomonas/prevenção & controle , Administração Oral , Animais , Ácido Araquidônico/metabolismo , Linhagem Celular , Fibrose Cística/genética , Fibrose Cística/patologia , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/metabolismo , Humanos , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos CFTR , Mucina-5AC/metabolismo , Mucina-5B/metabolismo , Muco/metabolismo , Fosfolipídeos/metabolismo , Pneumonia/microbiologia , Pneumonia/patologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/patogenicidade , Ratos , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismoRESUMO
BACKGROUND & AIMS: The bile acid (BA)-activated farnesoid X receptor (FXR) controls hepatic BA synthesis and cell proliferation via the intestinal hormone fibroblast growth factor 19. Because cystic fibrosis (CF) is associated with intestinal dysbiosis, anomalous BA handling, and biliary cirrhosis, we investigated FXR signaling in CF. METHODS: Intestinal and hepatic expression of FXR target genes and inflammation markers was assessed in Cftr null mice and controls. Localization of the apical sodium-dependent BA transporter was assessed, and BAs in gastrointestinal tissues were analyzed. The CF microbiota was characterized and FXR signaling was investigated in intestinal tissue and organoids. RESULTS: Ileal murine fibroblast growth factor 19 ortholog (Fgf15) expression was strongly reduced in CF mice, compared with controls. Luminal BA levels and localization of apical sodium-dependent BA transporter was not affected, and BAs induced Fgf15 up to normal levels in CF ileum, ex vivo, and CF organoids. CF mice showed a dysbiosis that was associated with a marked up-regulation of genes involved in host-microbe interactions, including those involved in mucin glycosylation, antimicrobial defense, and Toll-like receptor signaling. Antibiotic treatment reversed the up-regulation of inflammatory markers and restored intestinal FXR signaling in CF mice. Conversely, FXR-dependent gene induction in ileal tissue and organoids was repressed by bacterial lipopolysaccharide and proinflammatory cytokines, respectively. Loss of intestinal FXR activity was associated with a markedly blunted hepatic trophic response to oral BA supplementation, and with impaired repression of Cyp7a1, the gene encoding the rate-limiting enzyme in BA synthesis. CONCLUSIONS: In CF mice, the gut microbiota represses intestinal FXR activity, and, consequently, FXR-dependent hepatic cell proliferation and feedback control of BA synthesis.
Assuntos
Fibrose Cística/imunologia , Disbiose/imunologia , Fatores de Crescimento de Fibroblastos/metabolismo , Íleo/patologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Ácidos e Sais Biliares/biossíntese , Ácidos e Sais Biliares/imunologia , Proliferação de Células , Colesterol 7-alfa-Hidroxilase/metabolismo , Fibrose Cística/complicações , Fibrose Cística/patologia , Modelos Animais de Doenças , Regulação para Baixo , Disbiose/microbiologia , Disbiose/patologia , Retroalimentação Fisiológica , Feminino , Microbioma Gastrointestinal/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Íleo/imunologia , Íleo/microbiologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Fígado/citologia , Fígado/imunologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos CFTR , Regulação para CimaRESUMO
High energy expenditure is reported in cystic fibrosis (CF) animal models and patients. Alterations in skeletal muscle oxidative capacity, fuel utilization, and the creatine kinase-phosphocreatine system suggest mitochondrial dysfunction. Studies were performed on congenic C57BL/6J and F508del (Cftrtm1kth) mice. Indirect calorimetry was used to measure gas exchange to evaluate aerobic capacity during treadmill exercise. The bioenergetic function of skeletal muscle subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) was evaluated using an integrated approach combining measurement of the rate of oxidative phosphorylation by polarography and of electron transport chain activities by spectrophotometry. CF mice have reduced maximal aerobic capacity. In SSM of these mice, oxidative phosphorylation was impaired in the presence of complex I, II, III, and IV substrates except when glutamate was used as substrate. This impairment appeared to be caused by a defect in complex V activity, whereas the oxidative system of the electron transport chain was unchanged. In IFM, oxidative phosphorylation and electron transport chain activities were preserved, whereas complex V activity was reduced, in CF. Furthermore, creatine kinase activity was reduced in both SSM and IFM of CF skeletal muscle. The decreased complex V activity in SSM resulted in reduced oxidative phosphorylation, which could explain the reduced skeletal muscle response to exercise in CF mice. The decrease in mitochondrial creatine kinase activity also contributed to this poor exercise response.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Fibrose Cística/metabolismo , Metabolismo Energético/genética , Músculo Esquelético/metabolismo , Animais , Fibrose Cística/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CFTR , Camundongos Transgênicos , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/patologia , Fosforilação Oxidativa , Estresse Oxidativo/genética , Condicionamento Físico Animal/fisiologia , Deleção de SequênciaRESUMO
MicroRNAs (miRNAs) have recently been shown to be important for spermatogenesis; both DROSHA and Dicer1 KO mice exhibit infertility due to abnormal miRNA expression. However, the roles of individual miRNAs in spermatogenesis remain elusive. Here we demonstrated that miR-15b, a member of the miR-15/16 family, is primarily expressed in testis. A miR-15b transgenic mouse model was constructed to investigate the role of miR-15b in spermatogenesis. Impaired spermatogenesis was observed in miR-15b transgenic mice, suggesting that appropriate expression of miR-15b is vital for spermatogenesis. Furthermore, we demonstrated that overexpression of miR-15b reduced CDC25A gene post-transcriptional activity by targeting the 3'-UTR region of CDC25A, thus regulating spermatogenesis. In vitro results further demonstrated that a mutation in CFTR could affect the interaction between Ago2 with Dicer1 and that Dicer1 activity regulates miR-15b expression. We extended our study to azoospermia patients and found that infertile patients have a significantly higher level of miR-15b in semen and plasma samples. Taken together, we propose that CFTR regulation of miR-15b could be involved in the post-transcriptional regulation of CDC25A in mammalian testis and that miR-15b is important for spermatogenesis.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , MicroRNAs/genética , Espermatogênese/genética , Fosfatases cdc25/genética , Animais , Feminino , Regulação da Expressão Gênica , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CFTR , Camundongos Transgênicos , Mutação , Processamento Pós-Transcricional do RNA/genética , Fosfatases cdc25/metabolismoRESUMO
Cystic fibrosis (CF) is characterized by increased mucus viscosity and delayed mucociliary clearance that contributes to progressive decline of lung function. Mucus in the respiratory and GI tract is excessively adhesive in the presence of airway dehydration and excess extracellular Ca2+ upon mucin release, promoting hyperviscous, densely packed mucins characteristic of CF. Therapies that target mucins directly through ionic interactions remain unexploited. Here we show that poly (acetyl, arginyl) glucosamine (PAAG), a polycationic biopolymer suitable for human use, interacts directly with mucins in a Ca2+-sensitive manner to reduce CF mucus viscoelasticity and improve its transport. Notably, PAAG induced a linear structure of purified MUC5B and altered its sedimentation profile and viscosity, indicative of proper mucin expansion. In vivo, PAAG nebulization improved mucociliary transport in CF rats with delayed mucus clearance, and cleared mucus plugging in CF ferrets. This study demonstrates the potential use of a synthetic glycopolymer PAAG as a molecular agent that could benefit patients with a broad array of mucus diseases.
Assuntos
Fibrose Cística/tratamento farmacológico , Glucosamina/análogos & derivados , Mucina-5B/metabolismo , Depuração Mucociliar/efeitos dos fármacos , Muco/efeitos dos fármacos , Polímeros/farmacologia , Animais , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Modelos Animais de Doenças , Furões , Glucosamina/farmacologia , Glucosamina/uso terapêutico , Humanos , Camundongos , Camundongos Endogâmicos CFTR , Mucina-5B/química , Muco/metabolismo , Polímeros/uso terapêutico , Estrutura Quaternária de Proteína/efeitos dos fármacos , Ratos , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/patologia , Viscosidade/efeitos dos fármacosRESUMO
Hyperproliferation occurs in a variety of tissues and organs during cystic fibrosis (CF). However, the associated molecular mechanisms remain elusive. We investigated the molecular link between cystic fibrosis transmembrane conductance regulator (CFTR) defects and hyperproliferation, and showed that the length of the entire gastrointestinal tract was longer and the intestinal crypts were deeper in CF mice compared to those in wild-type animals. PCNA expression increased in CF mouse intestines and CFTR-knockdown cells. Villin1, an intestinal differentiation marker, was downregulated in CF mice. Ihh and Gli1 were significantly downregulated, whereas TCF4 was activated in CF mouse intestines and CFTR-knockdown Caco2 cells. Importantly, ß-catenin activators rescued Gli1 suppression, suggesting that hedgehog signaling might be mediated by the Wnt/ß-catenin pathway in the absence of functional CFTR. Moreover, PCNA positivity in the crypts of CF mice was alleviated by LiCl, which activates Wnt/ß-catenin signaling. Further, a strong positive correlation was observed between the expression of CFTR and Ihh in intestines. Our study revealed a previously unidentified role of CFTR in regulating hedgehog signaling through ß-catenin, providing novel insights into the physiological function of CFTR and CF-related diseases.
Assuntos
Proliferação de Células , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Proteínas Hedgehog/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Via de Sinalização Wnt , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Células CACO-2 , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Células HCT116 , Células HT29 , Proteínas Hedgehog/genética , Humanos , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Masculino , Camundongos Endogâmicos CFTR , Mutação , Fenótipo , Ratos , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
BACKGROUND: Cystic fibrosis (CF) is a genetic disease characterized by chronic inflammation of the lungs that is ineffective at clearing pathogens. B-cell activating factor (BAFF), a cytokine involved in the development of B-cells, is known to be elevated in CF patients with subclinical infections. We postulate that the elevated BAFF levels in CF patients might be triggered by Pseudomonas aeruginosa infection and it might play a protective role in the regulation of lung responses to infection. METHODS: To address this hypothesis, we used a well characterized model of CFTR.KO mice infected with a clinical strain of P. aeruginosa (PA508). We quantified cell types with flow cytometry, concentration of cytokines by ELISA tests, bacterial load by colony counting and lung physiology by metacholine-induced lung resistance. RESULTS: Our data demonstrates that BAFF is not elevated in uninfected CF mice, and infection with Pseudomonas leads to significant induction of this regulatory cytokine. We also demonstrate that the maintenance of BAFF levels and its induction during the infection is important for clearance of Pseudomonas infection as its depletion during the course of infection leads to decrease in the resolution of infection both in WT and CFTR-KO mice. Interestingly, the depletion of BAFF not only results in a depletion of B cells numbers but also to a significant decrease in the number of regulatory T cells in the non-infected lungs. CONCLUSIONS: Overall, our data demonstrate for the first time that BAFF is an important regulatory molecule helping to maintain the immunological response to infection and clearance of lung infection.
Assuntos
Fator Ativador de Células B/metabolismo , Fibrose Cística , Infecções por Pseudomonas/imunologia , Infecções Respiratórias/imunologia , Animais , Fibrose Cística/imunologia , Fibrose Cística/microbiologia , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos CFTR , Depuração Mucociliar/fisiologia , Pseudomonas aeruginosa/fisiologiaRESUMO
Ivacaftor is the first drug to target directly defects in the cystic fibrosis transmembrane conductance regulator (CFTR), which causes cystic fibrosis (CF). To understand better how ivacaftor potentiates CFTR channel gating, here we investigated the effects of temperature on its action. As a control, we studied the benzimidazolone UCCF-853, which potentiates CFTR by a different mechanism. Using the patch-clamp technique and cells expressing recombinant CFTR, we studied the single-channel behavior of wild-type and F508del-CFTR, the most common CF mutation. Raising the temperature of the intracellular solution from 23 to 37°C increased the frequency but reduced the duration of wild-type and F508del-CFTR channel openings. Although the open probability ( Po) of wild-type CFTR increased progressively as temperature was elevated, the relationship between Po and temperature for F508del-CFTR was bell-shaped with a maximum Po at ~30°C. For wild-type CFTR and to a greatly reduced extent F508del-CFTR, the temperature dependence of channel gating was asymmetric with the opening rate demonstrating greater temperature sensitivity than the closing rate. At all temperatures tested, ivacaftor and UCCF-853 potentiated wild-type and F508del-CFTR. Strikingly, ivacaftor but not UCCF-853 abolished the asymmetric temperature dependence of CFTR channel gating. At all temperatures tested, Po values of wild-type CFTR in the presence of ivacaftor were approximately double those of F508del-CFTR, which were equivalent to or greater than those of wild-type CFTR at 37°C in the absence of the drug. We conclude that the principal effect of ivacaftor is to promote channel opening to abolish the temperature dependence of CFTR channel gating.
Assuntos
Aminofenóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos Endogâmicos CFTR/metabolismo , Quinolonas/farmacologia , Animais , Benzodioxóis/farmacologia , Linhagem Celular , Cricetinae , Fibrose Cística/metabolismo , Humanos , Transporte de Íons/efeitos dos fármacos , Camundongos , Mutação/efeitos dos fármacos , TemperaturaRESUMO
Low linoleic acid concentration is a common finding in patients with cystic fibrosis and associated with severe clinical phenotype. Low docosahexaenoic and arachidonic acids are more inconsistently found in patients, but arachidonic/docosahexaenoic ratio is usually high. In animal models with cftr mutations or KO animals for the cftr gene, linoleic acid deficiency has not been consistently reported and some report docosahexaenoic deficiency as the major fatty acid abnormality. We hereby describe fatty acid profile in a severe clinical cystic fibrosis phenotype in mice with a duplication of exon 3 generated in the cystic fibrosis gene of C57B1/6J mice ( cftrm1Bay allele). In 43/50 animals, plasma phospholipid fatty acids were repeatedly analyzed (mean three times/animal) covering ages between 7 and 235 days. Linoleic acid concentrations were significantly lower in cftr-/- mice compared to heterozygotes ( P = 0.03) and wild type mice ( P < 0.001). Females had significantly lower linoleic acid than males, not related to age. Arachidonic acid did not differ but docosahexaenoic acid was higher in cftr-/- than in wild type mice ( P < 0.001). The arachidonic/docosahexaenoic acid ratio did not differ but arachidonic/linoleic acid ratio was higher in cftr-/- mice compared to wild type mice ( P = 0.007). Similar to clinical studies, type of mutation is important for lipid abnormality with low linoleic acid most consistently found in the animals. Rodents differ in metabolism by synthesizing docosahexaenoic acid more efficiently comparing to humans, suggesting greater influence by diet. Precaution seems important when comparing animal and humans. Impact statement In translational research, animal models are important to investigate the effect of genetic mutations in specific diseases and their metabolism. Special attention has to be given to differences in physiology and metabolism between species and humans, which otherwise can hazard the conclusions. Our work illustrates that the different synthesis capacity in mice and humans for DHA would explain different results in different models for cystic fibrosis and different influences of diets. To avoid disappointing clinical results, these facts have to be considered before extensive clinical studies are started based on results from single animal studies.
Assuntos
Ácido Araquidônico/sangue , Fibrose Cística/sangue , Fibrose Cística/patologia , Ácidos Docosa-Hexaenoicos/sangue , Ácido Linoleico/sangue , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CFTR , Camundongos KnockoutRESUMO
Purpose: The role of cystic fibrosis transmembrane conductance regulator (CFTR) in lacrimal gland (LG) function has only recently received some attention, mainly from our group. In the present study, we investigated the potential changes of LG pathology, tear secretion, ocular surface integrity, and fluid secretion in isolated LG ducts from CFTR knockout (KO) mice. Methods: Tear production and ocular surface integrity were investigated in anesthetized wild-type (WT) and KO mice using cotton threads and fluorescein staining, respectively. Immunofluorescence was used to localize CFTR protein in the LGs. Ductal fluid secretions evoked by forskolin (10 µM); cell-permeable cAMP analogue (8-bromo cAMP, 100 µM); or carbachol (100 µM) were measured in isolated LG ducts using video-microscopy. Intracellular Ca2+ homeostasis underlying carbachol stimulation was investigated with microfluorometry. Results: Significant decrease in tear secretion and impaired ocular surface integrity were observed in KO mice. Immunofluorescence demonstrated the predominant presence of CFTR protein in the apical membranes of the duct cells from WT mice. Continuous fluid secretion was evoked by forskolin and 8-bromo cAMP in LG ducts from WT mice, while no secretory response was observed in ducts from KO mice. Carbachol caused similar secretory responses in ducts from WT and KO animals without significant differences in cytosolic Ca2+ signaling. Conclusions: Our results suggest the important role of CFTR in LG ductal secretion and in the maintenance of ocular surface integrity, suggesting that CFTR may be a promising target of novel therapeutic approaches in the treatment of dry eye.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Síndromes do Olho Seco/metabolismo , Aparelho Lacrimal/metabolismo , Lágrimas/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Síndromes do Olho Seco/patologia , Aparelho Lacrimal/patologia , Camundongos , Camundongos Endogâmicos CFTRRESUMO
BACKGROUND: Dysfunction in cystic fibrosis transmembrane conductance regulator (CFTR) can be elicited by cigarette smoke and is observed in patients with chronic bronchitis. We have previously demonstrated in human airway epithelial cell monolayers that roflumilast, a clinically approved phosphodiesterase 4 inhibitor that reduces the risk of exacerbations in chronic obstructive pulmonary disease patients with chronic bronchitis and a history of exacerbations, activates CFTR-dependent chloride secretion via a cAMP-mediated pathway, partially restores the detrimental effects of cigarette smoke on CFTR-mediated ion transport, and increases CFTR-dependent gastrointestinal fluid secretion in isolated murine intestine segments. Based on these findings, we hypothesized that roflumilast could improve CFTR-mediated chloride transport and induce secretory diarrhea in mice exhibiting cigarette smoke-induced CFTR dysfunction. METHODS: A/J mice expressing wild type CFTR (+/+) were exposed to cigarette smoke or air with or without roflumilast and the effect of treatment on CFTR-dependent chloride transport was quantified using nasal potential difference (NPD) measurements in vivo and short-circuit current (Isc) analysis of trachea ex vivo. Stool specimen were collected and the wet/dry ratio measured to assess the effect of roflumilast on secretory diarrhea. RESULTS: Acute roflumilast treatment increased CFTR-dependent chloride transport in both smoke- and air-exposed mice (smoke, -2.0 ± 0.4 mV, 131.3 ± 29.3 µA/cm2, P < 0.01 and air, 3.9 ± 0.8 mV, 147.7 ± 38.0 µA/cm2, P < 0.01 vs. vehicle -0.3 ± 0.7 mV, 10.4 ± 7.0 µA/cm2). Oral administration of roflumilast over five weeks completely reversed the deleterious effects of cigarette smoke on CFTR function in smoke-exposed animals, in which CFTR-dependent chloride transport was 64% that of air controls (roflumilast, -15.22 ± 2.7 mV vs. air, -14.45 ± 1.4 mV, P < 0.05). Smoke exposure increased the wet/dry ratio of stool specimen to a level beyond which roflumilast had little additional effect. CONCLUSIONS: Roflumilast effectively rescues CFTR-mediated chloride transport in vivo, further implicating CFTR activation as a mechanism through which roflumilast benefits patients with bronchitis.