INTRACELLULAR CL- MODULATION OF IL-1ß SECRETION AND THE NLRP3 INFLAMMASOME EXPRESSION/ACTIVITY REQUIRE SGK1

MARIÁNGELES CLAUZURE; MARÍA MACARENA MASSIP COPIZ; TOMÁS SANTA COLOMA; CONSUELO MORI; CRISTIAN J. A. ASENSIO; ÁNGEL G. VALDIVIESO; VERÓNICA SOTOMAYOR

Mar del Plata

Congreso; REUNIÓN ANUAL DE SOCIEDADES DE BIOCIENCIA 2019; 2019

The impairment of the CFTR activityinduces intracellular chloride [Cl-]i accumulation andconsequently, as a second messenger, stimulates the secretionof interleukin-1ß (IL-1ß). We have previously described that thissecretion starts an autocrine positive feedback loop. Moreover,the expression of two subunits of the inflammasome complex:NLR family pyrin domain containing 3 (NLRP3) and caspase-1(CASP1), that are involved in the IL-1ß maturation, are indirectlymodulated by the [Cl-]i. On the other hand, cellular andmitochondrial ROS (reactive oxygen species) also are regulatedby [Cl-]i. Recently, other authors found that differences in [Cl-]imodulates SGK1 (serum-glucocorticoid kinase 1) phosphorylationand subsequently regulates NF-kB activation in airway epithelialcells. Therefore, we decided to study the effects of SGK1 on IL-1ß expression at different [Cl-]i. In this study we used IB3-1 cells(a bronchial cell line derived from a cystic fibrosis patient with a DF508/W1282X CFTR genotype) and Caco-2 cells (transfectedwith CFTR-shRNA). The cells were incubated for 1 h at 5 or 75mM Cl-, in presence of ionophores tributyltin (10 μM) andnigericin (5 μM) to equilibrate [Cl-]e and [Cl-]i. To explore if SGK1was also involved in the IL-1ß response to [Cl-]i, we used theSGK1 inhibitor GSK650394 at 0, 0.1, 1 and 10 μM. After, wedetermine IL-1ß expression by quantitative real-time RT-PCR andELISA quantification in culture media. To analyze the ROSresponse, we determined DCF fluorescence and MitoSOXfluorescence by microplate reader and/or flow cytometry. Theresults showed that SGK1 inhibitor diminished the response ofIL-1ß mRNA to changes in the [Cl-]i from 5 to 75 mM;GSK650394, at 10 μM, completely abrogated the IL-1ß mRNAresponse to Cl- 75 mM (p