Fungal allergen-induced IL-33 secretion involves cholesterol-dependent, VDAC-1-mediated ATP release from the airway epithelium

Key points: Alternaria aeroallergens induce the release of ATP from human bronchial epithelial (HBE) cells by activating a conductive pathway involving voltage-dependent anion channel-1 (VDAC-1) and by exocytosis of ATP localized within membrane vesicles. Inhibition of VDAC-1 blocked Alternaria-evoked Ca²⁺ uptake across the plasma membrane of HBE cells and interleukin (IL)-33 release into the extracellular media. Reducing cholesterol content with a cholesterol scavenger (β-methylcyclodextrin) or statin compound (simvastatin) blocked ATP and IL-33 release by lowering the expression of VDAC-1 in the plasma membrane. Pretreatment with simvastatin for 24 h also inhibited the increase in tight junction macromolecule permeability that occurs following Alternaria exposure. These results establish a novel role for VDAC-1 as a mechanism underlying ATP release induced by fungal allergens and suggests a possible therapeutic use for cholesterol lowering compounds in reducing Alternaria-stimulated allergic inflammation. Abstract: Human bronchial epithelial (HBE) cells exposed to allergens derived from the common saprophytic fungus, Alternaria alternata release ATP, which in turn stimulates P2X₇ receptor-mediated Ca²⁺ uptake across the plasma membrane. The subsequent increase in intracellular calcium concentration induces proteolytic processing and secretion of interleukin (IL)-33, a critical cytokine involved in the initiation of allergic airway inflammation. A major objective of the present study was to identify the mechanism responsible for conductive ATP release. The results show that pretreatment of HBE cells with inhibitors of the voltage-dependent anion channel-1 (VDAC-1) or treatment with a VDAC-1 selective blocking antibody or silencing mRNA expression of the channel by RNA interference, inhibit Alternaria-evoked ATP release. Moreover, inhibition of VDAC-1 channel activity or reducing protein expression blocked the secretion of IL-33. Similarly, reducing the cholesterol content of HBE cells with simvastatin or the cholesterol scavenger β-methylcyclodextrin also blocked ATP release and IL-33 secretion by decreasing the level of VDAC-1 expression in the plasma membrane. In addition, simvastatin inhibited the increase in tight junction macromolecule permeability that was previously observed after Alternaria exposure. These results demonstrate a novel function for VDAC-1 as the conductive mechanism responsible for Alternaria-induced ATP release, an essential early step in the processing, mobilization and secretion of IL-33 by the airway epithelium. Furthermore, the simvastatin-evoked reduction of VDAC-1 expression in the plasma membrane, suggests the possibility that cholesterol lowering compounds may be beneficial in alleviating allergic airway inflammation induced by fungal allergens.