Abstract 15736: The Sphk1/s1p Signaling Axis Regulates Pulmonary Vascular Inflammation by Activating Nf-kb and Nlrp3 Inflammasome

Abstract

Rationale: Pulmonary vascular inflammation is activated during the development of pulmonary arterial hypertension (PAH). Sphingosine kinase 1 (Sphk1) catalyzes the formation of sphingosine-1-phosphate (S1P), which has been shown to promote inflammation by activating nuclear factor kappa B (NF-κB) and the nucleotide oligomerization domain-like receptor 3 (NLRP3) inflammasome. However, the impact of S1P on NLRP3 inflammasome activation in PAH is unknown. Here, we tested whether Sphk1/S1P regulates pulmonary vascular inflammation via activating NF-κB and the NLRP3 inflammasome. Methods: Human pulmonary arterial smooth muscle cells (PASMCs) and endothelial cells (PAECs) were treated with S1P or were exposed to hypoxia. Sphk1 plasmid or siRNA was used to increase and decrease Sphk1/S1P levels, respectively. Activation of NF-κB signaling and the NLRP3 inflammasome (increased NLRP3, cleaved caspase-1, IL-1β and IL-18) were detected by western blots or real-time PCR. ELISA was used to measure secretion of IL-1β. Results: S1P treatment led to a biphasic phosphorylation and degradation of IκBα accompanied by increased phosphorylation and expression of NF-κB in PASMCs. S1P treatment also resulted in increased expression of inflammasome mediators, NLRP3 and cleaved caspase-1, enhanced intracellular expression of IL-1b and IL-18, and higher levels of secreted IL-1β compared to controls. Notably, over expression of Sphk1 in PASMCs led to increased NF-κB and NLRP3 expression, whereas suppression of Sphk1 expression reduced NLRP3, cleavage of IL-1β and IL-18 expression and phosphorylation of NF-κB. Similar phenomena with respect to inflammasome activation and IL-1β secretion was observed in PASMCs exposed to hypoxia. Increased NF-κB and NLRP3 activation, cleavage of caspase-1 were also observed in S1P treated PAECs, however no changes in IL-1β secretion levels were detected. Conclusion: These findings suggest that S1P induces pulmonary vascular pro-inflammatory signaling via activation of NF-κB and the NLRP3 inflammasome, and that the S1P/Sphk1 signaling axis may be involved in this context. Activation of the inflammasome may be an important mechanism underlying PAH pathogenesis, targeting of which may be exploited for the treatment of PAH.