Dynamics of action of a Lys-49 and an Asp-49 PLA2s on inflammasome NLRP3 activation in murine macrophages.

Phospholipases A2 (PLA2s) are proteins found in snake venoms with hemolytic, anticoagulant, myotoxic, edematogenic, bactericidal and inflammatory actions. In Bothrops jararacussu snake venom were isolated a Lys49-PLA2 (BthTX-I) and an Asp49-PLA2 (BthTX-II) with myotoxic and inflammatory actions. Both PLA2s can activate the NLRP3 inflammasome, an intracytoplasmic platform that recognizes molecules released when tissue is damaged liberating IL-1ß that contributes to the inflammatory response observed in envenoming. The dynamic of action of BthTX-I and BthTX-II in both thioglycollate (TG)-elicited macrophages and C2C12 myoblasts and the involvement of EP1 and EP2 receptors, and PGE2 in NLRP3 inflammasome activation were evaluated. Both toxins induced PGE2 liberation and inflammasome components (NLRP3, Caspase-1, ASC, IL-1ß, and IL18), IL-6, P2X7, COX-1, COX-2, EP2 and EP4 gene expression in TG-elicited macrophages but not in C2C12 myoblasts. EP2 (PF04418948) and EP4 (GW627368X) inhibitors abolished this effect. Both PLA2s also induced NLRP3 inflammasome protein expression that was abolished with the inhibitors used. Immunofluorescence and IL-1ß assays confirmed the NLRP3 activation in TG-elicited macrophages with the participation of both EP2 and EP4 receptors confirming their involvement in this effect. All in all, BthTX-I and BthTX-II activate macrophages and induce the NLRP3 inflammasome complex activation with the participation of the PGE2 via COX pathway and EP2 and EP4, both PGE2 receptors, contributing to the local inflammatory effects observed in envenoming.

Inflammasome NLRP3 activation induced by Convulxin, a C-type lectin-like isolated from Crotalus durissus terrificus snake venom.

Convulxin (CVX), a C-type lectin-like protein isolated from the venom of the snake species, Crotalus durissus terrificus, stimulates platelet aggregation by acting as a collagen receptor agonist for glycoprotein VI found in the platelets. The effect of CVX on platelets has been studied, but its effect on human peripheral blood mononuclear cells (PBMCs) remains unclear. Given the significance of PBMCs in inflammation, this study explored the effect of CVX on PBMCs, specifically regarding NLRP3 inflammasome activation by assessing cell viability, ability to induce cell proliferation, reactive oxygen species (ROS) and nitric oxide production, interleukin (IL)-2 and IL-10 secretion, NLRP3 complex activation, and the role of C-type lectin-like receptors (CTLRs) in these. CVX was not toxic to PBMCs at the investigated concentrations and did not increase PBMC growth or IL-2 release; however, CVX induced IL-10 release and ROS generation via monocyte activation. It also activated the NLRP3 complex, resulting in IL-1ß induction. Furthermore, the interaction between CVX and Dectin-2, a CTLR, induced IL-10 production. CVX interaction with CTLR has been demonstrated by laminarin therapy. Because of the involvement of residues near the Dectin-2 carbohydrate-recognition site, the generation of ROS resulted in inflammasome activation and IL-1ß secretion. Overall, this work helps elucidate the function of CVX in immune system cells.

NLRP3 inflammasome activation in human peripheral blood mononuclear cells induced by venoms secreted PLA2s.

Bothropic venoms contains high amount of secreted phospholipases A2 (sPLA2s) that play a significant role in leukocyte activation and inflammation. Monocytes and lymphocytes are highly functional immune system cells that mediate and provide efficient responses during the inflammation. NLRP3 inflammasome is a multiprotein complex found in immune system cells that is triggered by pathogen- and damage-associated molecular patterns, PAMPs and DAMPs, respectively. PLA2s' effect on human peripheral blood mononuclear cells (PBMCs) is still incompletely understood. PBMCs were isolated by density gradient and incubated with RPMI (control), LPS, BthTX-I (PLA2-Lys49) or BthTX-II (PLA2-Asp49) isolated from Bothrops jararacussu venom, to evaluate viability, and the results showed that there was no cell death. RT-qPCR and immunoblot were used to assess the gene and protein expression of NLRP3 components. Results indicated that there was substantial amplification of ASC, Caspase-1, IL-6, and IL-1ß in 1 h and NLRP3 in 2 h. Protein expression was measured, and the results revealed substantial expression of the NLRP3 inflammasome complex after 4 h. IL-1ß and LDH was quantified in the supernatant of the cells. Taken together, the findings demonstrate that BthTX-I and BthTX-II activate the NLRP3 inflammasome complex in human PBMCs and contribute to the inflammatory response seen in envenoming.

Phospholipase A2 Inhibitor from Crotalus durissus terrificus rattlesnake: Effects on human peripheral blood mononuclear cells and human neutrophils cells.

Crotalus Neutralizing Factor (CNF) is an inhibitor of phospholipase A2 (PLA2), present in the blood plasma of Crotalus durissus terrificus snake. This inhibitor neutralizes the lethal and enzymatic activity of crotoxin, the main neurotoxin from this venom. In this study, we investigated the effects of CNF on the functionality of human peripheral blood mononuclear cells (PBMCs) and human neutrophils. The following parameters were evaluated: viability and proliferation, chemotaxis, cytokines and LTB4 production, cytosolic PLA2s activity, myeloperoxidase (MPO) and superoxide anion (O2-) production. CNF showed no toxicity on PBMCs or neutrophils, and acts by stimulating the release of TNF-α and LTB4, but neither stimulates IL-10 and IL-2 nor affects PBMCs proliferation and O2- release. In neutrophils, CNF induces chemotaxis but does not induce the release of both MPO and O2-. However, it induces LTB4 and IL-8 production. These data show the influence of CNF on PBMCs' function by inducing TNF-α and LTB4 production, and on neutrophils, by stimulating chemotaxis and LTB4 production, via cytosolic PLA2 activity, and IL-8 release. The inflammatory profile produced by CNF is shown for the first time. Our present results suggest that CNF has a role in activation of leukocytes and exert proinflammatory effects on these cell.

The effect of 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene lupane compound isolated from Combretum leprosum Mart. on peripheral blood mononuclear cells.

BACKGROUND: The Combretum leprosum Mart. plant, popularly known as mofumbo, is used in folk medicine for inflammation, pain and treatment of wounds. From this species, it is possible to isolate three triterpenes: (3ß, 6ß, 16ß-trihydroxylup-20(29)-ene) called lupane, arjunolic acid and molic acid. In this study, through preclinical tests, the effect of lupane was evaluated on the cytotoxicity and on the ability to activate cellular function by the production of TNF-α, an inflammatory cytokine, and IL-10, an immuno regulatory cytokine was assessed. The effect of lupane on the enzymes topoisomerase I and II was also evaluated. METHODS: For this reason, peripheral blood mononuclear cells (PBMCs) were obtained and cytotoxicity was assessed by the MTT method at three different times (1, 15 and 24 h), and different concentrations of lupane (0.3, 0.7, 1.5, 6, 3 and 12 µg/mL). The cell function was assessed by the production of TNF-α and IL-10 by PBMCs quantified by specific enzyme immunoassay (ELISA). The activity of topoisomerases was assayed by in vitro biological assays and in silico molecular docking. RESULTS: The results obtained showed that lupane at concentrations below 1.5 µg/mL was not toxic to the cells. Moreover, lupane was not able to activate cellular functions and did not alter the production of IL-10 and TNF-α. Furthermore, the data showed that lupane has neither interfered in the action of topoisomerase I nor in the action of topoisomerase II. CONCLUSION: Based on preclinical results obtained in this study, we highlight that the compound studied (lupane) has moderate cytotoxicity, does not induce the production of TNF-α and IL-10, and does not act on human topoisomerases. Based on the results of this study and taking into consideration the reports about the anti-inflammatory and leishmanicidal activity of 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene, we suggest that this compound may serve as a biotechnological tool for the treatment of leishmaniasis in the future.

Effect of L-amino acid oxidase from Calloselasma rhodosthoma snake venom on human neutrophils.

The in vitro effects of LAAO, an l-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on isolated human neutrophil function were investigated. LAAO showed no toxicity on neutrophils. At non-cytotoxic concentrations, LAAO induced the superoxide anion production by isolated human neutrophil. This toxin, in its native form, is also able to stimulate the production of hydrogen peroxide in neutrophils, suggesting that its primary structure is essential for stimulation the cell. Moreover, the incubation of LAAO and phenol red medium did not induce the production of hydrogen peroxide. Furthermore, LAAO was able to stimulate neutrophils to release proinflammatory mediators such as IL-8 and TNF-α as well as NETs liberation. Together, the data showed that the LAAO triggers relevant proinflammatory events. Particular regions of the molecule distinct from the LAAO catalytic site may be involved in the onset of inflammatory events.