Cooper, Dawn Marie (2022) Investigating the effects of combined exposure to airborne house dust mite and particulate matter on bronchial epithelial cells. University of Southampton, Doctoral Thesis, 302pp.
Abstract
Background: Asthma is a chronic disease of the conducting airways affecting over 300 million people worldwide. Asthma exacerbations are associated with exposure to inhaled environmental factors, termed asthmagens, including airborne house dust mite (HDM) allergens and particulate matter (PM) air pollution. The bronchial epithelium is a critical part of the innate immune system and acts as the first barrier that encounters asthmagens. However , its physical, chemical, and immunological barrier functions are abnormal and dysfunctional. Understanding of the effects of asthmagens is generally based on exposures to single asthmagens in isolation neglecting more realistic simultaneous exposures. Furthermore, research examining the impact of co-exposure to asthmagens on the innate immune response of bronchial epithelial cells is also scarce. Aims: his project utilised an acute injury model to investigate the effects of co-exposure to HDM and PM (a common allergen and irritant known to exacerbate asthma) on bronchial epithelial cells, aiming to 1) assess time- and concentration-dependent effects of HDM and PM single and combined exposures, 2) ascertain intracellular mechanisms through which HDM and PM together exert effects, and 3) determine characteristics of HDM and PM associated with these effects. Methods: 16HBE14o- bronchial epithelial cell monolayer cultures were exposed to HDM extract and/or standard reference urban PM (SRM 1648a) for up to 72 hours. Cytotoxicity was determined by lactate dehydrogenase assay. Cytokine and chemokine release was measured by standard sandwich and multiplex enzyme-linked immunosorbent assay (ELISA). Gene expression was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Intracellular caspase-1 was measured by Western blotting. Elemental composition of whole, and water-soluble and -insoluble fractions of PM was determined by inductively-coupled plasma mass spectrometry (ICP-MS). PM-cell interactions were visualised by transmission electron microscopy with energy-dispersive x-ray (EDAX) spectroscopy. Mechanisms of action of PM and HDM were probed using metal chelation (desferrioxamine), antioxidant (N-acetylcysteine), inflammasome/caspase inhibition (MCC950, AC-YVAD-CMK), and HDM heat-treatment. Results: Sub-cytotoxic concentrations of HDM/PM induced additive increases in proinflammatory cytokine release. After 24 hours co-exposure to HDM (100 μg/mL) and PM (50 μg/mL), additive effects were observed for GM-CSF 48 pg/mL (IQR 31-66 pg/mL, p ≤ 0.0001), and TNF-α 167 pg/mL (IQR 22-1033, p ≤ 0.001). Interestingly, synergistic (significantly greater than additive) cytokine release was observed for IL-6 922 pg/mL (IQR 443-1765, p ≤ 0.01), IL- 1β 1 pg/mL (IQR 0.3-10, p ≤ 0.01) and IL-12p70 8 pg/mL (IQR 2-24 p ≤ 0.05). Synergy was mirrored at gene expression level after 24 hours for IL-6 (median fold change of 9). Coexposure also induced NLRP3 gene expression (median fold change of 4, p ≤ 0.01), implying a role for the NLRP3 inflammasome in the IL-1β response. However, the predominant form of caspase-1 was found to be the catalytically inactive δ-isoform. Nonetheless, caspase-1 inhibition diminished IL-1β release (by 7 fold, p ≤ 0.001). After 24 co-exposure, upregulation of the oxidative stress genes GCLM (median fold change of 3, p ≤ 0.001) and HMOX-1 (median fold change of 5, p ≤ 0.001) was observed. Upregulation of GCLM was also observed after 48 hours co-exposure (median fold change of 3, p ≤ 0.05) Effects of HDM were generally insensitive to heat-treatment. Iron chelation and antioxidant supplementation showed no effect. Notably, PM effects were predominantly associated with the insoluble PM fraction (HMOX-1 median fold change 2, p ≤ 0.01); this PM is transition metal-rich, especially iron (20483 ng/mg) and lead (3668 ng/mg), both almost entirely sequestered in the insoluble PM fraction. TEM showed the presence of cell-surface PM agglomerates and altered cell morphology while EDAX analysis indicated PM, copper, iron, and lead were present inside PM-exposed cells. Conclusion: Co-exposure to HDM and PM resulted in cytokine-specific additive or synergistic pro-inflammatory effects, with a possible role for the NLRP3 inflammasome and oxidative stress. The active component(s) in PM appear to be water-insoluble, with PM agglomerates attached to the apical cell surface, alongside evidence of increased intracellular copper, iron, and lead concentrations and altered cellular morphology. Synergistic effects on asthma associated signalling pathways, alongside pro-oxidant effects associated with airways dysfunction, suggest that more work to understand effects of asthmagen co-exposures is merited. This project highlights that the prevention of co-exposure to asthmagens could be an important focus for future work.
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