Patients with cystic fibrosis have a lesion in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which is associated with abnormal regulation of other ion channels, abnormal glycosylation of secreted and cell surface molecules, and vulnerability to bacterial infection and inflammation in the lung usually leading to the death of these patients. The exact mechanism(s) by which mutation in CFTR leads to lung infection and inflammation is not clear. Mice bearing different mutations in the murine homolog to CFTR (Cftr) (R117H, S489X, Y122X, and DeltaF508, all backcrossed to the C57BL/6J background) were compared with respect to growth and in their ability to respond to lung infection elicited with Pseudomonas aeruginosa-laden agarose beads. Body weights of mice bearing mutations in Cftr were significantly smaller than wild-type mice at most ages. The inflammatory responses to P. aeruginosa-laden agarose beads were comparable in mice of all four Cftr mutant genotypes with respect to absolute and relative cell counts in bronchoalveolar lavage fluid, and cytokine levels (TNF-alpha, IL-1beta, IL-6, macrophage inflammatory protein-2, and keratinocyte chemoattractant) and eicosanoid levels (PGE2 and LTB4) in epithelial lining fluid: the few small differences observed occurred only between cystic fibrosis mice bearing the S489X mutation and those bearing the knockout mutation Y122X. Thus we cannot implicate either misprocessing of CFTR or failure of CFTR to reach the plasma membrane in the genesis of the excess inflammatory response of CF mice. Therefore, it appears that any functional defect in CFTR produces comparable inflammatory responses to lung infections with P. aeruginosa.
"STOCK Cftrtm1Unc-TgN(FABPCFTR)#Jaw were bred, housed and used as in our previous studies [46,47]. Male mice (9 per group) 6-8 weeks of age, body weight at least 16 g, were used in these experiments and bred and housed under standard laboratory conditions. "
[Show abstract][Hide abstract] ABSTRACT: 17α-Ethynyl-5-androsten-3β, 7β, 17β-triol (HE3286) is a synthetic derivative of an endogenous steroid androstenetriol (β-AET), a metabolite of the abundant adrenal steroid deyhdroepiandrosterone (DHEA), with broad anti-inflammatory activities. We tested the ability of this novel synthetic steroid with improved pharmacological properties to limit non-productive lung inflammation in rodents and attempted to gauge its immunological impact.
In mice, oral treatment with HE3286 (40 mg/kg) significantly (p < 0.05) decreased neutrophil counts and exudate volumes (~50%) in carrageenan-induced pleurisy, and myeloperoxidase in lipopolysaccharide-induced lung injury. HE3286 (40 mg/kg) was not found to be profoundly immune suppressive in any of the classical animal models of immune function, including those used to evaluate antigen specific immune responses in vivo (ovalbumin immunization). When mice treated for two weeks with HE3286 were challenged with K. pneumoniae, nearly identical survival kinetics were observed in vehicle-treated, HE3286-treated and untreated groups.
HE3286 represents a novel, first-in-class anti-inflammatory agent that may translate certain benefits of β-AET observed in rodents into treatments for chronic inflammatory pulmonary disease.
Journal of Inflammation 10/2010; 7(1):52. DOI:10.1186/1476-9255-7-52 · 2.02 Impact Factor
"Mice homozygous for the ΔF508 CFTR mutation were described previously , as were mice carrying the R117H CFTR mutation . Mice heterozygous for CFTR expression (Cftrtm1Unc) were obtained from Jackson Laboratories (Bar Harbor, MA). "
[Show abstract][Hide abstract] ABSTRACT: Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis.
Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age.
Membrane cholesterol measurements are elevated in both R117H and DeltaF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTRinh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility.
The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content.
Respiratory research 05/2010; 11(1):61. DOI:10.1186/1465-9921-11-61 · 3.09 Impact Factor
"We compared 8–12 week old B6.129S6- Cftrtm2Mrc mice to wild type (WT) littermates. The B6.129S6- Cftrtm2Mrc mice are R117H murine Cftr mutants that have been back-crossed into the C57BL6j background . Age and sex matched CF and normal littermate mice were euthanized by CO2. "
[Show abstract][Hide abstract] ABSTRACT: Cystic fibrosis is characterized by recurring pulmonary exacerbations that lead to the deterioration of lung function and eventual lung failure. Excessive inflammatory responses by airway epithelia have been linked to the overproduction of the inflammatory cytokine IL-6 and IL-8. The mechanism by which this occurs is not fully understood, but normal IL-1beta mediated activation of the production of these cytokines occurs via H2O2 dependent signaling. Therefore, we speculated that CFTR dysfunction causes alterations in the regulation of steady state H2O2. We found significantly elevated levels of H2O2 in three cultured epithelial cell models of CF, one primary and two immortalized. Increases in H2O2 heavily contributed to the excessive IL-6 and IL-8 production in CF epithelia. Proteomic analysis of three in vitro and two in vivo models revealed a decrease in antioxidant proteins that regulate H2O2 processing, by > or =2 fold in CF vs. matched normal controls. When cells are stimulated, differential expression in CF versus normal is enhanced; corresponding to an increase in H2O2 mediated production of IL-6 and IL-8. The cause of this redox imbalance is a decrease by approximately 70% in CF cells versus normal in the expression and activity of the transcription factor Nrf-2. Inhibition of CFTR function in normal cells produced this phenotype, while N-acetyl cysteine, selenium, an activator of Nrf-2, and the overexpression of Nrf-2 all normalized H2O2 processing and decreased IL-6 and IL-8 to normal levels, in CF cells. We conclude that a paradoxical decrease in Nrf-2 driven antioxidant responses in CF epithelia results in an increase in steady state H2O2, which in turn contributes to the overproduction of the pro-inflammatory cytokines IL-6 and IL-8. Treatment with antioxidants can ameliorate exaggerated cytokine production without affecting normal responses.
PLoS ONE 10/2008; 3(10):e3367. DOI:10.1371/journal.pone.0003367 · 3.23 Impact Factor
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