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Identifying modulators of inflammation in an in vitro airway epithelial inflammation model using an arrayed CRISPRi screen

ABSTRACT: To study modulators of inflammatory airway disease, we developed an in vitro bronchial epithelial cell model system based on the BEAS-2B cell line exposed to lipopolysaccharide (LPS) and diesel exhaust particles (DEP). Combined LPS and DEP exposure triggered a molecular inflammatory phenotype, including elevated TSLP and IL-8 mRNA expression, similar to that observed in airway epithelial cells from asthma patients. In this model, we performed high-throughput perturbation of 24 mRNA and long non-coding (lncRNA) target genes through an arrayed CRISPR-interference screen, followed by shallow RNA-sequencing to identify modulators of inflammation. Perturbation of individual targets had a significant impact on the BEAS-2B DEP/LPS transcriptome. Some mRNA targets reversed the DEP/LPS gene expression signature upon knockdown and could serve as candidate therapeutic targets. Perturbation of other target genes however further amplified the pro-inflammatory signature and thus may have an anti-inflammatory role in these cells. In conclusion, we present a novel in vitro model system for airway inflammation that exhibited an inflammatory profile relevant for asthmatic patients, and demonstrate the feasibility to perform high-throughput target perturbation and molecular phenotyping by integrating CRISPR-interference and shallow RNA-sequencing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE271359 | GEO | 2024/09/15

REPOSITORIES: GEO

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Project description:Cigarette smoke (CS)-induced airway inflammation is an important pathologic feature of chronic obstructive pulmonary disease (COPD). Recent studies suggest a potential role of JunD in the regulation of inflammation, but its role in CS-induced airway inflammation has not been reported. This study aimed to determine its role in CS-induced airway inflammation through bioinformatics analysis and in vitro and in vivo experiments. Data from the Gene Expression Omnibus (GEO) database (GSE37147) were analyzed using weighted gene co-expression network analysis (WGCNA) and key driver analysis (KDA), and these analyses were validated using the GSE47460 dataset. The effect of CS on JunD expression was examined in lung tissues of COPD patients and CS-exposed mice and in CS extract (CSE)-exposed BEAS-2B cells. The effects of CSE on airway epithelial inflammatory injury after JunD knockdown or overexpression were also investigated. mRNA-seq and chromatin immunoprecipitation (ChIP)-seq were used to explore the mechanism of JunD-mediated CS-induced airway inflammation. Mice were injected with adeno-associated virus serotype 9 (AAV9)-JunD vector or control vector and then exposed to CS for 4 weeks, and lung tissue morphology and airway inflammation were evaluated. KDA of the lung function-related gene modules in GSE37147 revealed a potential role of JunD in COPD, which was validated in GSE47460. JunD was downregulated in lung tissues of COPD patients and CS-exposed mice and in BEAS-2B cells. JunD knockdown aggravated CSE-induced tumor necrosis factor (TNF)-α and interleukin (IL)-1β release by BEAS-2B cells, while JunD overexpression attenuated these effects. mRNA-seq and ChIP-seq identified several JunD-regulated genes, which are involved in the immune response and TNF signaling pathway and are commonly dysregulated in cell models of airway inflammation. In vivo, JunD overexpression attenuated the CS-induced inflammatory cell infiltration and inflammatory cytokine release in mouse lungs. Thus, JunD is involved in CS-induced airway inflammation and JunD-based therapy may be useful in CS-induced airway disorders.

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Identifying modulators of inflammation in an in vitro airway epithelial inflammation model using an arrayed CRISPRi screen

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