Abstract 15143: The Sphk1/S1P/S1PR2 Axis Regulates Pulmonary Vascular Remodeling by Activating a Mitochondrial ROS and NLRP3 Inflammasome Regulatory Loop

Abstract

Introduction: Sphk1/S1P/S1PR2 axis regulated NLRP3 inflammasome and promoted pyroptosis by activating NF-κB in human PASMC. Abnormal or inappropriate ROS mediates the development of PAH modulating inflammation, proliferation, and energy metabolism. Mitochondrial ROS promotes deubiquitylation of NLRP3 and activate the inflammasome. However, the function and regulation of S1P axis mediated mitochondrial ROS and NLRP3 inflammasome in PAH remain unknown. Hypothesis: We hypothesized that S1P axis regulated pulmonary vascular remodeling by activated mitochondrial ROS and NLRP3 inflammasome. Methods: NLRP3 inflammasome and pyroptosis activation were detected in PASMC from patients with PAH. JTE013, PF543, MCC950 and MitoTEMPO were used to modulate S1P, NLRP3 inflammasome and mitochondrial ROS signaling, respectively. Activation of inflammasome and pyroptosis were detected by western blots (WB), real-time PCR or immunofluorescence (IF). IL-1β secretion was detected by ELISA. ROS were measured by Mitosox Red and DCFDA. Mitochondrial membrane potential was determined by JC-1 dye assay. PASMCs which were isolated from Sphk1 fl/fl Tagln Cre (+) and Sphk1 fl/fl mice were used to detect the role of S1P axis in vivo. Results: NLRP3 Inflammasome and pyroptosis were activated in PASMCs from patients with PAH. JTE013 or PF543 treatment inhibited NLRP3, ASC, N-GSDMD, HMGB1, cleaved IL-1β and IL-18 expression, and suppressed mitochondrial ROS, as well as mitochondrial hyperpolarization induced by S1P. Treatment with MitoTEMPO decreased S1P induced-NLRP3, ASC, N-GSDMD, HMGB1, cleaved IL-1β and IL-18 expression and inhibited IL-1β secretion. MCC950 controlled the activation of mitochondrial ROS, as well as mitochondrial hyperpolarization which were activated by S1P. NLRP3, cleaved IL-1β, ASC, HMGB1 and N-GSDMD expression were decreased in mPASMCs from Sphk1 fl/fl Tagln Cre (+) mice. Conclusions: S1P induces activation of mitochondrial ROS and NLRP3 inflammasome, and that the Sphk1/S1P/S1PR2 signaling axis may be involved in this context. Activation of the mitochondrial ROS and NLRP3 inflammasome regulatory loop may be an important mechanism underlying PAH pathogenesis, targeting of which may be exploited for the treatment of PAH.