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Influence of CFTR on Lipid Metabolism Gene Expression in Marrow Derived Dendritic Cells infected with P. aeruginosa

ABSTRACT: Dysfunction of the cystic fibrosis transmembrane regulator (CFTR) in cystic fibrosis (CF) results in exaggerated and chronic inflammation as well as increased susceptibility to chronic pulmonary infections, in particular with Pseudomonas aeruginosa. Based on the concept that host immune responses do not seem to be adequate to eradicate P.aeruginosa from the lungs of CF patients and that dendritic cells (DC) play an important role in initiating and shaping adaptive immune responses, this study analyzed the role of CFTR in bone marrow-derived murine DC from CFTR knockout (CF) mice with and without exposure to P.aeruginosa. DC expressed CFTR mRNA and protein, although at much lower levels compared to whole lung. Microarray analysis of gene expression levels in DC generated from CF and wild type (WT) mice revealed significantly different expression of 16 genes in CF DC compared to WT DC. Among the genes with lower expression in CF DC was Caveolin-1, a membrane lipid raft protein. Messenger RNA and protein levels of Caveolin-1 were decreased in the CF DC compared to WT DC. Consistently, the active form of sterol-responsive element binding protein (SREBP), a negative regulator of Caveolin-1 expression, was increased in CF DC. Following exposure to P.aeruginosa, gene expression levels in CF and WT DC changed for 912 genes involved in inflammation, chemotaxis, signaling, cell cycling and apoptosis more than 1.5-fold. Among the genes that showed a different response between WT and CF DC infected with P.aeruginosa, were 3β-hydroxysterol-Δ7 reductase (Dhcr7) and stearoyl-CoA desaturase 2 (Scd2), two enzymes involved in the lipid metabolism that are also regulated by SREBP. These results suggest that CFTR dysfunction in non-epithelial cells results in changes in the expression of genes encoding factors involved in membrane structure and lipid-metabolism. These membrane alterations in immune cells may contribute to the abnormal inflammatory and immune response characteristic of CF. Keywords: CF control vs CF PAK vs WT control vs WT PAK

ORGANISM(S): Mus musculus

PROVIDER: GSE9488 | GEO | 2008/10/01

SECONDARY ACCESSION(S): PRJNA103271

REPOSITORIES: GEO

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Project description:Dysfunction of the cystic fibrosis transmembrane regulator (CFTR) in cystic fibrosis (CF) results in exaggerated and chronic inflammation as well as increased susceptibility to chronic pulmonary infections, in particular with Pseudomonas aeruginosa. Based on the concept that host immune responses do not seem to be adequate to eradicate P.aeruginosa from the lungs of CF patients and that dendritic cells (DC) play an important role in initiating and shaping adaptive immune responses, this study analyzed the role of CFTR in bone marrow-derived murine DC from CFTR knockout (CF) mice with and without exposure to P.aeruginosa. DC expressed CFTR mRNA and protein, although at much lower levels compared to whole lung. Microarray analysis of gene expression levels in DC generated from CF and wild type (WT) mice revealed significantly different expression of 16 genes in CF DC compared to WT DC. Among the genes with lower expression in CF DC was Caveolin-1, a membrane lipid raft protein. Messenger RNA and protein levels of Caveolin-1 were decreased in the CF DC compared to WT DC. Consistently, the active form of sterol-responsive element binding protein (SREBP), a negative regulator of Caveolin-1 expression, was increased in CF DC. Following exposure to P.aeruginosa, gene expression levels in CF and WT DC changed for 912 genes involved in inflammation, chemotaxis, signaling, cell cycling and apoptosis more than 1.5-fold. Among the genes that showed a different response between WT and CF DC infected with P.aeruginosa, were 3Î²-hydroxysterol-Î7 reductase (Dhcr7) and stearoyl-CoA desaturase 2 (Scd2), two enzymes involved in the lipid metabolism that are also regulated by SREBP. These results suggest that CFTR dysfunction in non-epithelial cells results in changes in the expression of genes encoding factors involved in membrane structure and lipid-metabolism. These membrane alterations in immune cells may contribute to the abnormal inflammatory and immune response characteristic of CF. Experiment Overall Design: comparison of gene expression in dendritic cells of cystic fibrosis mice without or with Pseudosmonas aeruginosa infection vs wild type dendritic cell controls

Project description:Cystic Fibrosis (CF) is a genetic disorder CF is caused by mutations of the gene encoding for the cystic fibrosis transmembrane conductance regulator protein (CFTR), a transmembrane anion channel expressed at the apical membrane of several organs, including the epithelial cells of the airway. CFTR mutations result in dysfunctional ion transport across the apical membrane at the surface of the epithelia, generating thickened and dehydrated secretions. In the lung, this leads to a decrease in the mucociliary clearance, favoring bacterial colonization and progressive obstruction of the duct. Although over 2000 CFTR variants have been identified so far, the most common mutation is a deletion of the phenylalanine in position 508 (F508del), which shows an allelic frequency of around 90% among CF patients. F508del-CFTR is incorrectly folded, causing its retention at the endoplasmic reticulum (ER) and subsequent proteasomal degradation. Among the several drugs available in CF pharmacological treatment, VX-809 (commercial name Lumacaftor) is the most used drug for patients carrying F508del-CFTR mutation. This drug corrects the aberrant folding of F508del-CFTR by favoring the correct intramolecular interactions, thus enabling a higher number of copies of the defective protein to reach the plasma membrane. Localization of Organelle Proteins by Isotope Tagging after Differential ultra-Centrifugation (LOPIT-DC, https://doi.org/10.1038/s41467-018-08191-w) workflow was used to study the spatial localization of proteins in the CFBE41o- cells and how it is impacted by CFTR rescue triggered by VX-809. LOPIT-DC is a powerful and well-established tool for the investigation of the spatial distribution of global proteome within cells, which combines subcellular fractionation based on differential centrifugation, quantitative mass spectrometry, and machine learning. Its application provides a global snapshot of the steady-state subcelluler distribution of proteins. The aim of this project is to identify proteins that change their cellular localization in association with the treatment, to uncover molecules that play a role in the trafficking of the defective CFTR to the plasma membrane.

Project description:Background: Cystic fibrosis (CF) is caused by mutations in the CFTR gene that impair function of this cAMP-regulated Cl- channel. In the small intestine, loss of CFTR function creates a dehydrated, acidic luminal environment which is believed to cause an accumulation of mucus, a phenotype characteristic of CF. CF mice have an innate immune response and impaired intestinal transit as well. We investigated whether lubiprostone, which activates the CLC2 Cl- channel, would improve the CF intestinal phenotype. Methods: Cftrtm1UNC (CF) and wildtype (WT) littermate mice on the C57BL/6 background were used. Lubiprostone (10ug/kg-day) was administered by gavage for two weeks. Mucus accumulation was estimated from crypt lumen widths in Carnoy's-fixed, PAS/AB stained sections. Luminal bacterial load was measured by qPCR for the bacterial 16S gene. Gastric emptying and small intestinal transit were assessed by gavage of rhodamine dextran. Gene expression was evaluated by Affymetrix Mouse430 2.0 microarray. Results: Crypt width in control CF mice was 700% that of WT mice (P<0.001). Lubiprostone did not affect WT crypt width but, unexpectedly, increased CF crypt width 22% (P=0.001). Lubiprostone increased bacterial load in WT mice to 490% of WT control levels (P=0.008). Conversely, lubiprostone decreased bacterial overgrowth in CF mice by 60% (P=0.005). Lubiprostone significantly increased gastric emptying at 20 min postgavage in both WT (P<0.001; control=57±3%, treated=81±4%) and CF mice (P<0.001; control=48±4%, treated=75±5%). After lubiprostone small intestinal transit was significantly enhanced in WT mice (P=0.024) but the effect was not significant in CF mice (P=0.377). Among other innate immune markers, expression of mast cell genes was elevated ~20-fold in the control CF intestine and lubiprostone decreased expression to WT control levels. Conclusions: These results indicate that lubiprostone has some benefits for the CF intestinal phenotype, especially on bacterial overgrowth and the innate immune response. The unexpected observation of increased mucus accumulation in the crypts of lubiprostone-treated CF mice suggests the possibility that lubiprostone increases mucus secretion. For each group (control wild type, lubiprostone-treated wild type, contol Cftr null, and lubiprostone-treated cftr null), equal amounts of total RNA extracted from the entire small intestine were pooled for analysis.

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Influence of CFTR on Lipid Metabolism Gene Expression in Marrow Derived Dendritic Cells infected with P. aeruginosa

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