P038 Impact of cumulative low-level ozone on keratinocytes and implications for atopic eczema pathogenesis
Authors: Soha Tawfik, Bjorn Thomas, Matthew Caley, Eleri Jones, Ryan O’Shaughnessy, Edel O’Toole
Journal: British Journal of Dermatology (2024)

Abstract Ozone (O3) is a secondary air pollutant produced by photochemical reactions involving sunlight, volatile organic compounds and nitrogen oxides. In recent years, there has been a rising concentration of ground-level O3. Acute exposure to toxic O3 concentrations is known to result in skin eruptions mimicking atopic eczema (AE); however, the effect of daily low levels on the skin requires further research. A previous mathematical modelling study in British Bangladeshi patients with AE showed that long-term exposure to low-level O3 has the strongest association with eczema severity, as measured by Eczema Area and Severity Index. This study aimed to evaluate the effect of cumulative exposure to low levels of O3 on keratinocytes in two-dimensional (2D) and three-dimensional (3D) cultures. Subconfluent immortalized human keratinocytes were exposed to 70–80 ppb of O3 for 1 h daily, in a closed chamber for 5 days per week over 3 weeks. Protein and RNA were extracted after the fifth exposure weekly. Control cells were exposed to pure air (5–7 ppb O3) under similar conditions. Skin equivalent models were exposed similarly for 1 week. Oxidative stress was assessed by quantification of reactive oxygen species (ROS) and carbonylated protein (CP) expression level by immunoblotting. There was significant ROS generation following a single exposure in ozonated cells and increased CP expression during the first 2 weeks of O3 exposure. In total 88 genes were significantly changed by O3 exposure at a false discovery rate &amp;lt; 0.05, with a fold change of ≥ 1.25 or ≤ −1.25. Pathway enrichment analysis during the first 2 weeks of O3 exposure showed inflammatory pathways relevant to AE pathogenesis, such as P53 regulation, inflammasome, interleukin (IL)-10/IL-4/IL-13 signalling, and Janus kinase-signal transducer and activator of transcription (STAT). Longer exposure for 3 weeks activated DNA damage response pathways. At the protein expression level, Western blotting of lysates from three different experiments and live cell imaging of 2D culture showed increased activation of phospho-STAT3/total STAT3 and aryl hydrocarbon receptor by the end of the second week of exposure to O3. In 3D cultures, the same inflammatory changes were observed in addition to a proliferative phenotype and apoptosis in ozonated models compared with pure air. We suggest that chronic exposure to low O3 concentrations may be a proposed inflammatory mechanism in AE via increased oxidative stress, inflammatory changes, DNA damage and apoptosis. These mechanisms provide important therapeutic targets. Current pollution policy, such as reduction of traffic in cities focuses on reducing particulate matter pollution, can ironically be associated with a rise in O3. The overlooked rising O3 concentrations worldwide may be the cause of changing trends of increased prevalence and severity of AE, which require consideration of O3 pollution in future air quality control strategies.