Lipidomic approaches to the study of the action of two secreted group IIA phospholipases from Bothrops asper snake venom on human monocytes: role of enzymatic activity

LEIGUEZ, ELBIO; RODRIGUEZ JUAN PABLO; GUIJAS, CARLOS; RUBIO, JULIO; ASTUDILLO, ALMA; LOMONTE, B.; GUTIERREZ, J. M.; TEIXEIRA, C.; BALSINDE, JESÚS

Guarujá

Congreso; XI Congress of the Pan-American Section of the International Society on Toxinology - XII Congress of the Brazilian Society of Toxinology; 2013

Introduciton: Bothrops asper (B.a.) is responsible for the vast majority of snakebite accidents in Central America. This venom causes an exacerbated local inflammation and pain, reactions that are poorly controlled by antivenoms. B.a. venom is constituted mainly by phospholipases A2 (PLA2s) that are considered responsible for triggering inflammation. In this work we have investigated the myotoxic action of MT- III (Asp-49) and/or MT-II (Lys-49), two PLA2s isolated from B.a. venom, on human peripheral blood monocytes using a lipidomic approach to characterize fatty acid (FA) release and metabolism induced by these toxins. Methods and Results: Gas chromatography and high performance liquid chromatography coupled to mass spectrometry analyses were used to detect and quantify lipid species released by PLA2 action on monocytes. The involvement of reacylation processes in lipid droplet (LD) biogenesis was evaluated by pharmacological intervention, and measured by confocal microscopy. Quantitative PCR was used to measure RNA levels of enzymes involved in FA metabolism. We observed large decreases in the content of saturated and polyunsaturated FA (60%) from phospholipids (PLs), in MT III-treated cells. Similarly, a significant diminution of the total cellular content of arachidonic acid (AA)- containing PL was observed, that depended on PL species and time of exposure. Large amounts of membrane lyso-PL and free FA such as oleic and AA were measured in monocytes stimulated by MT-III. MT-III promoted the appearance of high levels of triacylglycerol and cholesterol enriched in palmitoleic, stearic, and oleic acid (50-100%), along with an increase in LD synthesis. The latter process was abrogated by the acyl CoA synthetase inhibitor triacsin C. Additionally, we demonstrated that the increased availability of FA arising from the enzymatic hydrolysis of phospholipids activates the mitochondrial b-oxidation pathway and promotes eicosanoid synthesis. On the other hand, MT-II failed to produce any of the effects described above, which highlights the importance of the catalytic activity of the toxins. Conclusion: Our studies provide a complete lipid profile of the response of monocytes to venom PLA2, and reveals significant connections between LD biogenesis, cell signaling and biochemical pathways implicated, that contribute to initiate the innate immune response.