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Human CFTR deficient iPSC-macrophages reveal impaired functional and transcriptomic response upon Pseudomonas aeruginosa infection

ABSTRACT: Cystic fibrosis (CF) is a hereditary autosomal recessive disease driven by deleterious variants of the CFTR gene, leading, among other symptoms, to increased lung infection susceptibility. Mucus accumulation in the CF lung is, as of yet, considered as one important factor contributing to its colonization by opportunistic pathogens such as Pseudomonas aeruginosa. However, in recent years evidence was provided that alveolar macrophages, which form the first line of defense against airborne pathogens, seem to be intrinsically defective with regard to bactericidal functionality in the CF lung. To assess the impact of CFTR deficiency in human macrophages only insufficient systems are available. To address this problem and to evaluate the role of CFTR in human macrophages, we successfully differentiated human induced pluripotent stem cells (iPSC) from a CF p.Phe508del homozygous individual and a healthy donor into primitive macrophages (iMacΔF508 and iMacWT), respectively, and compared the bactericidal functionality in the relevant cell type. iMacΔF508 showed impaired P. aeruginosa clearance and intracellular killing capacity in comparison to iMacWT. Furthermore, iMacΔF508 exhibited a less acidic lysosomal pH, and upon P. aeruginosa infection, there were signs of mitochondrial fragmentation and autophagosome formation together with a hyperinflammatory phenotype and deficient type I interferon response. In summary, we present a defective phenotype in iMacΔF508 upon P. aeruginosa infection, which will constitute an ideal platform to further study the role of macrophages in the context of CF.

ORGANISM(S): Homo sapiens

PROVIDER: GSE276816 | GEO | 2024/09/27

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

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Human CFTR deficient iPSC-macrophages reveal impaired functional and transcriptomic response upon Pseudomonas aeruginosa infection

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