Simvastatin Inhibits Allergen‐induced IL‐33 Secretion in Human Bronchial Epithelial Cells by Blocking ATP Release and Ca2+ Uptake

Abstract

Previous studies have demonstrated that simvastatin possesses immunomodulatory effects in airways, but the precise mechanisms and therapeutic potential are poorly understood. The objective of the present study was to investigate the inhibitory effects of simvastatin and b‐methyl‐cyclodextrin (β‐MCD) on the innate immune response of the airway epithelium induced by Alternaria alternata (100 μg/ml) and house dust mite (200 μg/ml, HDM) allergens. Experiments were performed with cultured human bronchial epithelial cells transfected with the human IL‐33 gene to produce a robust and reproducible cell model for the study of IL‐33 secretion. Cells were pretreated with varying concentrations of simvastatin ranging from 0.1 to 10 μM for 24 hours prior to allergen exposure. IL‐33 release was quantified by ELISA (Quantikine®) following the procedure provided by the manufacturer. Simvastatin produced a concentration‐dependent inhibition of IL‐33 release that was nearly abolished at 10 μM (IC50; 1.0 μM). In contrast, simvastatin was ineffective at blocking the effects of HDM on IL‐33 release (IC50; >10 μM). Similarly, pretreatment with β‐MCD (5 mM) for 30 minutes also inhibited the Alternaria response, suggesting that depletion of cholesterol from the plasma membrane was responsible for the effects of simvastatin and β‐MCD. Subsequent experiments explored the effects of simvastatin and β‐MCD on Alternaria‐evoked Ca2+ uptake, which is necessary for activating IL‐33 release. Simvastatin (10 μM, 24 hrs) and β‐MCD (5 mM; 30 minutes) both abolished the increase in [Ca2+]i induced by Alternaria. We next examined the possibility that abolition of Ca2+ uptake resulted from inhibition of ATP release, since ATP activates P2X7 receptors that mediate Ca2+ influx in response to Alternaria exposure. ATP release was measured in real time using a luciferin/luciferase‐based photon counting assay. Both simvastatin and β‐MCD blocked ATP release. Moreover, addition of 100 μM cholesterol to the extracellular media rescued the effects of simvastatin. These results demonstrated that inhibition of IL‐33 secretion by simvastatin and β‐MCD following Alternaria exposure involved suppression of ATP release that was dependent on membrane cholesterol content. Thus, membrane cholesterol likely plays a pivotal role in innate immune function of airway epithelial cells.Support or Funding InformationThis study was supported by NIH R01 AI128729 to HK and SMO.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.