A C-type lectin induces NLRP3 inflammasome activation via TLR4 interaction in human peripheral blood mononuclear cells.

Lectins are a large group of proteins found in many snake venoms. BjcuL is a C-type lectin from Bothrops jararacussu snake venom that does not present cytotoxicity action on human peripheral blood mononuclear cells (PBMCs) at concentrations of 5 and 10 µg/mL. BjcuL demonstrates an immunomodulatory role in PBMCs with the production of pro- and anti-inflammatory cytokines (IL-2, IL-10, IFN-γ, IL-6, TNF-α, and IL-17) in addition to stimulate T cells to produce reactive oxygen species (ROS) that could play a role in the acute inflammatory reaction observed in the victims. Inflammasomes are an essential arm in cells of innate immunity to detect and sense a range of endogenous or exogenous, sterile, or infectious stimuli to elicit cellular responses and effector mechanisms. NLRP3 inflammasome is a significant target for this study, because the lectin is responsible for leukocyte activation stimulating the release of inflammatory mediators, which results in dynamic cellular responses to remove the detrimental process to the body in snakebites. Thus, this study aimed to investigate how isolated BjcuL from B. jararacussu venom affects NLRP3 inflammasome activation on PBMCs. For this, the cells were isolated by density gradient and incubated with BjcuL at different periods and concentrations for the evaluation of the activation of the NLRP3 inflammasome through gene and protein expressions of ASC, CASPASE-1, and NLRP3 by RT-qPCR, Western blot, and immunofluorescence, as well as the participation of Toll-like receptor 4 (TLR4) and ROS in the IL-1ß production, a product resultant of the NLRP3 inflammasome activation. Herein, BjcuL interacts with TLR4 as demonstrated by in vitro and in silico studies and induces cytokines release via NF-κB signaling. By genic and protein expression assays, BjcuL activates NLRP3 inflammasome, and the pharmacological modulation with LPS-RS, an antagonist of TLR4; LPS-SM, an agonist of TLR4; MCC950, a specific NLRP3 inhibitor, and rotenone, an inhibitor of mitochondrial ROS, confirmed the participation of TLR4 and ROS in the NLRP3 inflammasome activation and IL-1ß liberation. The effects of BjcuL on the regulation and activation of the NLRP3 inflammasome complex via TLR4 activation with ROS participation may be determinant for the development of the inflammatory local effects seen in snakebite victims. In addition, in silico together with in vitro studies provide information that may be useful in the rational design of TLR agonists as well as new adjuvants for immunomodulatory therapy.

Reactive oxygen species-dependent-NLRP3 inflammasome activation in human neutrophils induced by l-amino acid oxidase derived from Calloselasma rhodostoma venom.

l-Amino acid oxidase isolated from Calloselasma rhodostoma (Cr-LAAO) snake venom is a potent stimulus for neutrophil activation and production of inflammatory mediators, contributing to local inflammatory effects in victims of envenoming. Cr-LAAO triggered the activation of nicotinamide adenine dinucleotide phosphatase (NADPH) oxidase complex and protein kinase C (PKC)-α signaling protein for reactive oxygen species (ROS) production. This study aims to evaluate the ROS participation in the NLRP3 inflammasome complex activation in human neutrophil. Human neutrophils were isolated and stimulated for 1 or 2 h with RPMI (negative control), LPS (1 µg/mL, positive control) or Cr-LAAO (50 µg/mL). The neutrophil transcriptome was examined using the microarray technique, and RT-qPCR for confirmation of gene expression. Immunofluorescence assays for NLRP3, caspase-1, IL-1ß and GSDMD proteins was performed by Western blot in the presence and/or absence of Apocynin, an inhibitor of NADPH oxidase. IL-1ß release was also detected in the presence and/or absence of NLRP3, caspase-1 and NADPH oxidase inhibitors. Results showed that Cr-LAAO upregulated the expression of genes that participate in the NADPH oxidase complex formation and inflammasome assembly. NLRP3 was activated and accumulated in the cytosol forming punctas, indicating its activation. Gasdermin D was not cleaved but lactate dehydrogenase was released. Furthermore, ROS inhibition decreased the expression of NLRP3 inflammasome complex proteins, as observed by protein expression in the presence and/or absence of apocynin, an NADPH oxidase inhibitor. IL-1ß was also released, and pharmacological inhibition of NLRP3, caspase-1, and ROS reduced the amount of released cytokine. This is the first report demonstrating the activation of the NLRP3 inflammasome complex via ROS generation by Cr-LAAO, which may lead to the development of local inflammatory effects observed in snakebite victims.

Light Emitting Diode Photobiomodulation Enhances Oxidative Redox Capacity in Murine Macrophages Stimulated with Bothrops jararacussu Venom and Isolated PLA2s.

Photobiomodulation therapy associated with conventional antivenom treatment has been shown to be effective in reducing the local effects caused by bothropic venoms in preclinical studies. In this study, we analyzed the influence of photobiomodulation using light emitting diode (LED) on the oxidative stress produced by murine macrophages stimulated with Bothrops jararacussu venom and it isolated toxins BthTX-I and BthTX-II. Under LED treatment, we evaluated the activity of the antioxidant enzymes catalase, superoxide dismutase, and peroxidase as well as the release of hydrogen peroxide and the enzyme lactate dehydrogenase. To investigate whether NADPH oxidase complex activation and mitochondrial pathways could contribute to hydrogen peroxide production by macrophages, we tested the effect of two selective inhibitors, apocynin and CCCP3, respectively. Our results showed that LED therapy was able to decrease the production of hydrogen peroxide and the liberation of lactate dehydrogenase, indicating less cell damage. In addition, the antioxidant enzymes catalase, superoxide dismutase, and peroxidase increased in response to LED treatment. The effect of LED treatment on macrophages was inhibited by CCCP3, but not by apocynin. These findings show that LED photobiomodulation treatment protects macrophages, at least in part, by reducing oxidative stress caused B. jararacussu venom and toxins.

Bothrops jararacussu snake venom lectin induces mast cell activation and vascular permeability enhance in an animal model.

Bothrops species trigger an acute inflammatory response in victims, with activated leukocytes releasing several mediators that may contribute to local and systemic effects. The effects of BjcuL, a lectin isolated from B. jararacussu snake venom, on mast cells and vasopermeability were investigated in this study. BjcuL activates mast cells and increases vasopermeability through the involvement of histamine and platelet activating factor, which may play a role in the victims' acute inflammatory reaction.

Cytosolic phospholipase A2-α participates in lipid body formation and PGE2 release in human neutrophils stimulated with an L-amino acid oxidase from Calloselasma rhodostoma venom.

Cr-LAAO, an L-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, has been demonstrated as a potent stimulus for neutrophil activation and inflammatory mediator production. However, the mechanisms involved in Cr-LAAO induced neutrophil activation has not been well characterized. Here we investigated the mechanisms involved in Cr-LAAO-induced lipid body (also known as lipid droplet) biogenesis and eicosanoid formation in human neutrophils. Using microarray analysis, we show for the first time that Cr-LAAO plays a role in the up-regulation of the expression of genes involved in lipid signalling and metabolism. Those include different members of phospholipase A2, mostly cytosolic phospholipase A2-α (cPLA2-α); and enzymes involved in prostaglandin synthesis including cyclooxygenases 2 (COX-2), and prostaglandin E synthase (PTGES). In addition, genes involved in lipid droplet formation, including perilipin 2 and 3 (PLIN 2 and 3) and diacylglycerol acyltransferase 1 (DGAT1), were also upregulated. Furthermore, increased phosphorylation of cPLA2-α, lipid droplet biogenesis and PGE2 synthesis were observed in human neutrophils stimulated with Cr-LAAO. Treatment with cPLA2-α inhibitor (CAY10650) or DGAT-1 inhibitor (A922500) suppressed lipid droplets formation and PGE2 secretion. In conclusion, we demonstrate for the first time the effects of Cr-LAAO to regulate neutrophil lipid metabolism and signalling.

Light emitting diode (LED) photobiomodulation therapy on murine macrophage exposed to Bothropstoxin-I and Bothropstoxin-II myotoxins.

The light-emitting diode (LED) is considered a therapeutic tool due to its anti-inflammatory, analgesic, and wound-healing effects, which occur through angiogenesis, decrease in IL-1ß and IL-6 secretion, and acceleration of the cicatricial process. Snakebites are an important public health problem in tropical regions of the world. LED treatment is a therapeutic tool associated with serum therapy used to minimize the local effects of snakebites, including decrease in creatine kinase (CK) and lactate dehydrogenase (LDH) concentrations, myonecrosis, and inflammatory and haemorrhagic responses. In this study, we analysed the photobiomodulation effect of LED on the activation of murine macrophages induced by BthTX-I or BthTX-II isolated from Bothrops jararacussu venom. Photobiomodulation caused an increase in mitochondrial metabolism and a considerable decrease in cytotoxicity in murine macrophages. Moreover, it induced a decrease in reactive oxygen species and nitrogen liberation. However, photobiomodulation caused an increase in macrophage phagocytic capacity and lipid droplet formation. The results of this study corroborated with those of others in an unprecedented way and provide a better understanding of the mechanism of action of photobiomodulation, besides offering a coadjuvant action treatment for the local effects of snakebites, not achieved with serum therapy alone.

Role of l-amino acid oxidase isolated from Calloselasma rhodostoma venom on neutrophil NADPH oxidase complex activation.

The action of Cr-LAAO, an l-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on NADPH oxidase activation in isolated human neutrophil function was investigated. This enzyme has an intrinsic activity of hydrogen peroxide production. Cr-LAAO, in its native form, induces the ROS production in neutrophil and migration of cytosolic NADPH oxidase components p40phox, p47phox and p67phox to the membrane, and Rac, a GTPase protein member, with the involvement of intracellular signaling mediated by phospho PKC-α. In its inactive form, iCr-LAAO does not induce NADPH oxidase activation in neutrophil showing that the intrinsic enzymatic activity does not have a role in this process, suggesting that its primary structure is essential for the cell's stimulation. Accordingly, the data showed for the first time that the Cr-LAAO has a role in NADPH oxidase complex activation triggering relevant proinflammatory events in human neutrophils.

Effect of BjcuL, a lectin isolated from Bothrops jararacussu, on human peripheral blood mononuclear cells.

BjcuL is a C-type lectin with specificity for the binding of ß-d-galactose units isolated from Bothrops jararacussu venom. It triggers cellular infiltration in post capillary venules, increases edema and vascular permeability in murine models, contributes to in vitro neutrophil activation and modulates macrophage functional activation towards an M1 state. The purpose of this study was to investigate the effect of BjcuL on human peripheral blood mononuclear cells (PBMCs) activation with a focus on PBMCs proliferation and inflammatory mediators release. Results showed that BjcuL is not toxic to PBMCs, that BjcuL inhibits PBMCs proliferation and that it stimulates PBMCs to produce superoxide anion and hydrogen peroxide, primarily via lymphocyte stimulation, but does not stimulate the production of nitric oxide and PGE2. These results demonstrate that BjcuL has an immunomodulatory effect on PBMCs. Further studies are needed to confirm the immunomodulatory effect of BjcuL, to elucidate the molecular mechanisms of action responsible for its effects and to determine its potential application as an immunopharmacological and biotechnological tool.