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Inactivation of CFTR by CRISPR/Cas9 alters transcriptional regulation of inflammatory pathways and other networks

ABSTRACT: Individuals with cystic fibrosis (CF) experience elevated inflammation in multiple organs, but whether this reflects an inherent feature of CF cells or is a consequence of a pro-inflammatory environment is not clear. Using CRISPR/Cas9-mediated mutagenesis of CFTR, 17 subclonal cell lines were generated from Caco-2 cells. Clonal lines with functional CFTR (CFTR+) were compared to those without (CFTR-) to directly address the role of CFTR in inflammatory gene regulation. All lines maintained CFTR mRNA production and formation of tight junctions. CFTR+ lines displayed short circuit currents in response to forskolin, while the CFTR- lines did not. Baseline expression of both cytokines was not different between the lines regardless of CFTR genotype. All lines responded to TNFa and IL1b by increasing IL6 and CXCL8 (IL8) mRNA levels, but the CFTR- lines produced more CXCL8 mRNA than the CFTR+ lines. Transcriptomes of 6 CFTR- and 6 CFTR+ lines, before and after stimulation by TNFa, were compared for differential expression as a function of CFTR genotype. While some genes appeared to be differentially expressed simply because of CFTR’s absence, others required stimulation for differences to be apparent. Together, these data suggest cells respond to CFTR’s absence by modulating transcriptional networks, some of which are only apparent when cells are exposed to different environmental contexts, such as inflammation. With regards to inflammation, these data suggest a model in which CFTR’s absence leads to a poised, pro-inflammatory state of cells that is only revealed by stimulation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE130226 | GEO | 2019/05/02

REPOSITORIES: GEO

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Project description:Background: CFTR modulators will decrease some etiologies of inflammation in the CF airway; however, current data indicate that non-resolving airway infection and inflammation will persist in individuals with CF and chronic bacterial infections. Thus, identification of therapies that diminish CF airway inflammation without allowing unrestrained bacterial growth remains a critical research goal. Novel strategies for combatting deleterious airway inflammation require a better understanding of the cellular contributions to chronic CF airway disease, and how cells change after initiation of CFTR modulator therapy. Peripheral blood monocytes, which traffic to the CF airway, can develop both pro-inflammatory and inflammation-resolving phenotypes, and represent intriguing targets for focused therapies to dampen CF airway inflammation. Material and Methods: In order to characterize the inflammatory phenotype of CF blood monocytes, and how these cells change after initiation of CFTR modulator therapy, we studied adults (n=10) with CF, chronic airway infections, and the CFTR-R117H mutations before and 7 days after initiation of ivacaftor. Transcriptomes of freshly isolated blood monocytes were interrogated by RNA-sequencing (RNA-seq). Plasma concentrations of cytokines and chemokines were evaluated by multiplex ELISA. Results: RNAseq identified approximately 50 monocyte genes for which basal expression was significantly changed in all 10 subjects after 7 days of ivacaftor. Of these, the majority were increased in expression post ivacaftor, including many genes traditionally associated with enhanced inflammation and immune responses. Pathway analyses confirmed that transcriptional pathways were overwhelmingly up-regulated in monocytes after 7 days of ivacaftor, including transcriptional modules associated with immunity, cell cycle, oxidative phosphorylation, and the unfolded protein response. Ivacaftor increased plasma concentrations of CXCL2, a neutrophil chemokine secreted by monocytes and macrophages, and CCL2, a monocyte chemokine. These results demonstrate that ivacaftor causes acute changes in blood monocytes and plasma chemokines, and suggest that increased monocyte inflammatory signals and potentially improved trafficking to the lung contribute to changes in airway inflammation in people taking CFTR modulators. To our knowledge, this is the first report investigating the transcriptomic response of isolated blood monocytes in CF subjects.

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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Inactivation of CFTR by CRISPR/Cas9 alters transcriptional regulation of inflammatory pathways and other networks

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