Phenotypic, functional and plasticity features of human PBMCs induced by venom secreted PLA2s.

Bothrops venom contains a high amount of secreted phospholipase A2 (sPLA2s) enzymes responsible for the inflammatory reaction and activation of leukocytes in cases of envenoming. PLA2s are proteins that have enzymatic activity and can hydrolyze phospholipids at the sn-2 position, thereby releasing fatty acids and lysophospholipids precursors of eicosanoids, which are significant mediators of inflammatory conditions. Whether these enzymes have a role in the activation and function of peripheral blood mononuclear cells (PBMCs) is not known. Here we show for the first time how two secreted PLA2s (BthTX-I and BthTX-II) isolated from the venom of Bothrops jararacussu affect the function and polarization of PBMCs. Neither BthTX-I nor BthTX-II exhibited significant cytotoxicity to isolated PBMCs compared with the control at any of the time points studied. RT-qPCR and enzyme-linked immunosorbent assays were used to determine changes in gene expression and the release of pro-inflammatory (TNF-α, IL-6, and IL-12) and anti-inflammatory (TGF-ß and IL-10) cytokines, respectively, during the cell differentiation process. Lipid droplets formation and phagocytosis were also investigated. Monocytes/macrophages were labeled with anti-CD14, -CD163, and -CD206 antibodies to assay cell polarization. Both toxins caused a heterogeneous morphology (M1 and M2) on days 1 and 7 based on immunofluorescence analysis, revealing the considerable flexibility of these cells even in the presence of typical polarization stimuli. Thus, these findings indicate that the two sPLA2s trigger both immune response profiles in PBMCs indicating a significant degree of cell plasticity, which may be crucial for understanding the consequences of snake envenoming.

Human neutrophils functionality under effect of an Asp49 phospholipase A2 isolated from Bothrops atrox venom.

Bothrops envenomation is associated with a cellular inflammatory response, characterized by pronounced neutrophil infiltration at the site of injury. Neutrophils act as the first line of defence, owing to their ability to migrate to the infected tissue, promoting an acute inflammatory response. At the site of inflammation, neutrophils perform defence functions such as phagocytosis, release of proteolytic enzymes, generation of reactive oxygen species (ROS), and synthesis of inflammatory mediators such as cytokines and lipid mediators. Neutrophils can also form neutrophil extracellular nets (NETs), webs composed of chromatin and granule proteins. This occurs after neutrophil activation and delivers high concentrations of anti-microbial molecules to the site of injury. This study evaluated the impact of BaTX-II, an Asp49 phospholipase A2 (PLA2) isolated from Bothrops atrox snake venom on human neutrophils in vitro. At non-toxic concentrations, BaTX-II induced hydrogen peroxide production by neutrophils, and this was reduced by wortmannin, a PI3K inhibitor. BaTX-II stimulated IL-1ß, IL-8, LTB4, myeloperoxidase (MPO), and DNA content release, consistent with NET formation. This is the first study to show the triggering of relevant pro-inflammatory events by PLA2 Asp49 isolated from secretory venom.

p38 MAPK is involved in human neutrophil chemotaxis induced by L-amino acid oxidase from Calloselasma rhodosthoma.

The action of LAAO, an L-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on isolated human neutrophil function was investigated. Cr-LAAO showed no toxicity on neutrophils. Cr-LAAO in its native form induced the neutrophil chemotaxis, suggesting that its primary structure is essential for stimulation the cell. p38 MAPK and PI3K have a role as signaling pathways of CR-LAAO induced chemotaxis. This toxin also induced the production of hydrogen peroxide and stimulated phagocytosis in neutrophils. Furthermore, Cr-LAAO was able to stimulate neutrophils to release IL-6, IL-8, MPO, LTB4 and PGE2. Together, the data showed that the Cr-LAAO triggers relevant proinflammatory events.

Activation of J77A.1 macrophages by three phospholipases A2 isolated from Bothrops atrox snake venom.

In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.