16 research outputs found
Mutations that permit residual CFTR function delay acquisition of multiple respiratory pathogens in CF patients
<p>Abstract</p> <p>Background</p> <p>Lung infection by various organisms is a characteristic feature of cystic fibrosis (CF). <it>CFTR </it>genotype effects acquisition of <it>Pseudomonas aeruginosa (Pa)</it>, however the effect on acquisition of other infectious organisms that frequently precede <it>Pa </it>is relatively unknown. Understanding the role of CFTR in the acquisition of organisms first detected in patients may help guide symptomatic and molecular-based treatment for CF.</p> <p>Methods</p> <p>Lung infection, defined as a single positive respiratory tract culture, was assessed for 13 organisms in 1,381 individuals with CF. Subjects were divided by predicted CFTR function: 'Residual': carrying at least one partial function <it>CFTR </it>mutation (class IV or V) and 'Minimal' those who do not carry a partial function mutation. Kaplan-Meier estimates were created to assess <it>CFTR </it>effect on age of acquisition for each organism. Cox proportional hazard models were performed to control for possible cofactors. A separate Cox regression was used to determine whether defining infection with <it>Pa</it>, mucoid <it>Pa </it>or <it>Aspergillus (Asp) </it>using alternative criteria affected the results. The influence of severity of lung disease at the time of acquisition was evaluated using stratified Cox regression methods by lung disease categories.</p> <p>Results</p> <p>Subjects with 'Minimal' CFTR function had a higher hazard than patients with 'Residual' function for acquisition of 9 of 13 organisms studied (HR ranging from 1.7 to 3.78 based on the organism studied). Subjects with minimal CFTR function acquired infection at a younger age than those with residual function for 12 of 13 organisms (p-values ranging: < 0.001 to 0.017). Minimal CFTR function also associated with younger age of infection when 3 alternative definitions of infection with <it>Pa</it>, mucoid <it>Pa </it>or <it>Asp </it>were employed. Risk of infection is correlated with CFTR function for 8 of 9 organisms in patients with good lung function (>90%ile) but only 1 of 9 organisms in those with poorer lung function (<50%ile).</p> <p>Conclusions</p> <p>Residual CFTR function correlates with later onset of respiratory tract infection by a wide spectrum of organisms frequently cultured from CF patients. The protective effect conferred by residual CFTR function is diminished in CF patients with more advanced lung disease.</p
Identification of metabolic pathways influenced by the G-protein coupled receptors GprB and GprD in Aspergillus nidulans
Heterotrimeric G-protein-mediated signaling pathways play a pivotal role in transmembrane signaling in eukaryotes. Our main aim was to identify signaling pathways regulated by A. nidulans GprB and GprD G-protein coupled receptors (GPCRs). When these two null mutant strains were compared to the wild-type strain, the DeltagprB mutant showed an increased protein kinase A (PKA) activity while growing in glucose 1% and during starvation. In contrast, the DeltagprD has a much lower PKA activity upon starvation. Transcriptomics and (1)H NMR-based metabolomics were performed on two single null mutants grown on glucose. We noted modulation in the expression of 11 secondary metabolism gene clusters when the DeltagprB and DeltagprD mutant strains were grown in 1% glucose. Several members of the sterigmatocystin-aflatoxin gene cluster presented down-regulation in both mutant strains. The genes of the NR-PKS monodictyphenone biosynthesis cluster had overall increased mRNA accumulation in DeltagprB, while in the DeltagprD mutant strain the genes had decreased mRNA accumulation. Principal component analysis of the metabolomic data demonstrated that there was a significant metabolite shift in the DeltagprD strain. The (1)H NMR analysis revealed significant expression of essential amino acids with elevated levels in the DeltagprD strain, compared to the wild-type and DeltagprB strains. With the results, we demonstrated the differential expression of a variety of genes related mainly to secondary metabolism, sexual development, stress signaling, and amino acid metabolism. We propose that the absence of GPCRs triggered stress responses at the genetic level. The data suggested an intimate relationship among different G-protein coupled receptors, fine-tune regulation of secondary and amino acid metabolisms, and fungal development
Proinflammatory Phenotype and Increased Caveolin-1 in Alveolar Macrophages with Silenced CFTR mRNA
The inflammatory milieu in the respiratory tract in cystic fibrosis (CF) has been linked to the defective expression of the cystic transmembrane regulator (CFTR) in epithelial cells. Alveolar macrophages (AM), important contibutors to inflammatory responses in the lung, also express CFTR. The present study analyzes the phenotype of human AM with silenced CFTR. Expression of CFTR mRNA and the immature form of the CFTR protein decreased 100-fold and 5.2-fold, respectively, in AM transfected with a CFTR specific siRNA (CFTR-siRNA) compared to controls. Reduction of CFTR expression in AM resulted in increased secretion of IL-8, increased phosphorylation of NF-κB, a positive regulator of IL-8 expression, and decreased expression of IκB-α, the inhibitory protein of NF-κB activation. AM with silenced CFTR expression also showed increased apoptosis. We hypothesized that caveolin-1 (Cav1), a membrane protein that is co-localized with CFTR in lipid rafts and that is related to inflammation and apoptosis in macrophages, may be affected by decreased CFTR expression. Messenger RNA and protein levels of Cav1 were increased in AM with silenced CFTR. Expression and transcriptional activity of sterol regulatory element binding protein (SREBP), a negative transcriptional regulator of Cav1, was decreased in AM with silenced CFTR, but total and free cholesterol mass did not change. These findings indicate that silencing of CFTR in human AM results in an inflammatory phenotype and apoptosis, which is associated to SREBP-mediated regulation of Cav1
Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells
The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2α) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2α. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-α. This was concomitant with increased IL-8 synthesis and cPLA2α activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-β-cyclodextrin induced further cPLA2α activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-α-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2α and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-α-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis