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Gene expression profile at single cell level of human bronchial epithelium cells cultured at the air-liquid interface (ALI) treated with CdCl2 (10µM) or DMSO (0,1%)

ABSTRACT: We use scRNA-seq to identify the signature of CdCl2 expsosure of ALI culture. CdCl2 was dissolved in DMSO (0,1%) and the effect of DMSO is also reported.

ORGANISM(S): Homo sapiens

PROVIDER: GSE316799 | GEO | 2026/02/23

REPOSITORIES: GEO

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Project description:Chronic obstructive pulmonary disease (COPD) has the highest increased risk due to household air pollution arising from biomass fuel burning. However, knowledge on COPD patho-mechanisms is mainly limited to tobacco smoke exposure. In this study, a repeated direct wood smoke (WS) exposure was performed using normal- (bro-ALI) and chronic bronchitis-like bronchial (bro-ALI-CB), and alveolar (alv-ALI) lung mucosa models at air-liquid interface (ALI) to assess broad toxicological end points. The bro-ALI and bro-ALI-CB models were developed using human primary bronchial epithelial cells and the alv-ALI model was developed using a representative type-II pneumocyte cell line. The lung models were exposed to WS (10 minutes/exposure; 5-exposures over 3-days; n=6-7 independent experiments). Sham exposed samples served as control. WS composition was analyzed following passive sampling. Cytotoxicity, total cellular reactive oxygen species (ROS) and stress responsive NFkB were assessed by flow cytometry. WS exposure induced changes in gene expression were evaluated by RNA-seq (p≤0.01) followed by pathway enrichment analysis. Secreted levels of proinflammatory cytokines were assessed in the basal media. Non-parametric statistical analysis was performed. 147 unique compounds were annotated in WS of which 42 compounds have inhalation toxicity (9 very high). WS exposure resulted in significantly increased ROS in bro-ALI (11.2%) and bro-ALI-CB (25.7%) along with correspondingly increased NFkB levels (bro-ALI: 35.6%; bro-ALI-CB: 18.1%). A total of 1262 (817-up and 445-down), 329 (141-up and 188-down), and 102 (33-up and 69-down) genes were differentially regulated in the WS-exposed bro-ALI, bro-ALI-CB, and alv-ALI models respectively. The enriched pathways included the terms acute phase response, mitochondrial dysfunction, inflammation, oxidative stress, NFkB, ROS, xenobiotic metabolism of AHR, and chronic respiratory disorder. The enrichment of the ‘cilium’ related genes was predominant in the WS-exposed bro-ALI (180-up and 7-down). The pathways primary ciliary dyskinesia, ciliopathy, and ciliary movement were enriched in both WS-exposed bro-ALI and bro-ALI-CB. Interleukin-6 and tumor necrosis factor-α were reduced (p<0.05) in WS-exposed bro-ALI and bro-ALI-CB.

Project description:Idiopathic Pulmonary Fibrosis (IPF) is a devastating form of respiratory disease with a life expectancy of 3-4 years. Inflammation, epithelial injury and myofibroblast proliferation have been implicated in disease initiation and, recently, epithelial-fibroblastic crosstalk has been identified as a central driver. However, the ability to interrogate this crosstalk is limited due to the absence of in vitro models that mimic physiological conditions. To investigate IPF dysregulated cross-talk, primary normal human bronchial epithelial (NHBE) cells and primary normal human lung fibroblasts (NHLF) or diseased human lung fibroblasts (DHLF) from IPF patients, were co-cultured in direct contact at the air-liquid interface (ALI). Intercellular crosstalk was assessed by comparing cellular phenotypes of co-cultures to respective monocultures, through optical, biomolecular and electrical methods. A co-culture-dependent decrease in epithelium thickness, basal cell mRNA (P63, KRT5) and an increase in transepithelial electrical resistance (TEER) was observed. This effect was significantly enhanced in DHLF co-cultures and lead to the induction of epithelial to mesenchymal transition (EMT) and increased mRNA expression of TGFβ-2, ZO-1 and DN12. When stimulated with exogenous TGFβ, NHBE and NHLF monocultures showed a significant upregulation of EMT (COL1A1, FN1, VIM, ASMA) and senescence (P21) markers, respectively. In contrast, direct NHLF/NHBE co-culture indicated a protective role of epithelial-fibroblastic cross-talk against TGFβ-induced EMT, fibroblast-to-myofibroblast transition (FMT) and inflammatory cytokine release (IL-6, IL-8, IL-13, IL-1β, TNF-α). DHLF co-cultures showed no significant phenotypic transition upon stimulation, likely due to the constitutively high expression of TGFβ isoforms prior to any exogenous stimulation. The model developed provides an alternative method to generate IPF-related bronchial epithelial phenotypes in vitro, through the direct co-culture of human lung fibroblasts with NHBEs. These findings highlight the importance of fibroblast TGFβ signaling in EMT but that monocultures give rise to differential responses compared to co-cultures, when exposed to this pro-inflammatory stimulus. This holds implications for any translation conclusions drawn from monoculture studies and is an important step in development of more biomimetic models of IPF. In summary, we believe this in vitro system to study fibroblast-epithelial crosstalk, within the context of IPF, provides a platform which will aid in the identification and validation of novel targets.

Dataset Information

Gene expression profile at single cell level of human bronchial epithelium cells cultured at the air-liquid interface (ALI) treated with CdCl2 (10µM) or DMSO (0,1%)

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