NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) modulation by intracellular Cl concentration.

The impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) activity induces intracellular chloride (Cl ) accumulation. The anion Cl , acting as a second messenger, stimulates the secretion of interleukin-1β (IL-1β), which starts an autocrine positive feedback loop. Here, we show that NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) are indirectly modulated by the intracellular Cl concentration, showing maximal expression and activity at 75 mM Cl , in the presence of the ionophores nigericin and tributyltin. The expression of PYD and CARD domain containing (PYCARD/ASC) remained constant from 0 to 125 mM Cl . The CASP1 inhibitor VX-765 and the NLRP3 inflammasome inhibitor MCC950 completely blocked the Cl -stimulated IL-1β mRNA expression and partially the IL-1β secretion. DCF fluorescence (cellular reactive oxygen species, cROS) and MitoSOX fluorescence (mitochondrial ROS, mtROS) also showed maximal ROS levels at 75 mM Cl , a response strongly inhibited by the ROS scavenger N-acetyl-L-cysteine (NAC) or the NADPH oxidase (NOX) inhibitor GKT137831. These inhibitors also affected CASP1 and NLRP3 mRNA and protein expression. More importantly, the serum/glucocorticoid regulated kinase 1 (SGK1) inhibitor GSK650394, or its shRNAs, completely abrogated the IL-1β mRNA response to Cl and the IL-1β secretion, interrupting the autocrine IL-1β loop. The results suggest that Cl effects are mediated by SGK1, in which under Cl modulation stimulates the secretion of mature IL-1β, in turn, responsible for the upregulation of ROS, CASP1, NLRP3 and IL-1β itself, through autocrine signalling.