Regulation of pro-inflammatory responses in a simple in vitro model of chronic oxidant exposure in COPD

Purpose: Chronic obstructive pulmonary disease (COPD) is caused by long-term exposure topollutants such as cigarette smoke (CS) that stimulates an inflammatory response andproduces oxidants that cause oxidative stress (OS) which is considered the main cause ofCOPD. Hydrogen peroxide (H2O2) is one of many oxidants identified to be elevated in thebreath condensates of COPD patients resulting in OS and it has been found to be associatedwith enhanced inflammation. Chronic inflammation is another key feature of COPD thatpersists long after smoking cessation and is driven by multiple pro-inflammatory cytokinesand OS. Inflammation and OS can lead to permanent epigenetic modulation patterns of genesthat are correlated with COPD. Protease-activated receptor 2 (PAR2) has been identified as acritical and key protease sensor and driver of pathology in several inflammatory diseasesincluding asthma, but the roles of this receptor in COPD are still unclear. This study aimed tounderstand how chronic exposure to H2O2 alone or in combination with other inflammatorymediators influences cell response relevant to COPD lung pathogenesis, and whether thesechallenges alter DNA methylation levels of the PAR2 promoter region, to reveal potentialimplications for, and mechanistic insights into, COPD development.Methods: H2O2 was used as a model oxidant to establish an in vitro chronic oxidative stresssystem. BEAS-2B cells were exposed chronically in a repeated exposure manner to H2O2 for 2h/day for 3 days at different concentrations, alone or in combination with inflammatorymediators (LPS, TGF-β and TNF-α; constant exposure for 48 h). Effects of pre-treatment witha selective IKK-2 inhibitor (TPCA-1) was investigated to assess the contribution of the NF-κBpathway. Cell viability was assessed by MTT assay. Cytokines were measured by ELISA,intracellular ROS production was detected by 2',7'-dichlorodihydrofluorescein diacetate (CMH2DCFDA)assay and mRNA transcript levels were assayed by qPCR. Model developmentexperiments were performed using primary human bronchial epithelial cells from healthy andCOPD donors (HBEC/COPD-HBEC). Methprimer software was used to predict the CpGislands/sites of the F2RL1 promoter region, and TFBS and JASPAR software were used toidentify potential motifs and their binding sites. DNA from treated and untreated cells wasisolated and bisulphite treated. Bisulphite-specific PCR was performed and PCR products werepurified and sequenced, prior to quantitative analysis (University of Glasgow Polyomics).Results: Cells tolerated a repeated exposure of H2O2 up to 15 μM in BEAS-2B, 25 μM in HBECand 50 μM in COPD-HBEC alone or in combination with 10 ng/ml of TGF-β and TNF-α or 500ng/ml of LPS with only moderate impact on viability (MTT), and no effect on visualappearance/morphology. Intracellular ROS were significantly elevated in the presence ofeither LPS or H2O2, with an additive effect of combined treatment. H2O2 stimulated modest orno release of IL-8 and IL-6 alone but synergistically enhanced LPS-induced IL-8 and IL-6.