Bothrops moojeni snake venom induces an inflammatory response in preadipocytes: Insights into a new aspect of envenomation.

Bothrops envenomation is a public health problem in Brazil. Despite the advances in the knowledge of the pathogenesis of systemic and local effects induced by Bothrops venom, the target tissues to this venom are not completely characterised. As preadipocytes are important cells of the adipose tissue and synthesize inflammatory mediators, we investigated the ability of B. moojeni snake venom (Bmv) to stimulate an inflammatory response in 3T3-L1 preadipocytes in vitro, focusing on (1) the release of PGE2, IL-6, TNF-α, MCP-1, KC, leptin and adiponectin; (2) the mechanisms involved in PGE2 release and (3) differentiation of these cells. Cytotoxicity of Bmv was determined by MTT assay. The concentrations of PGE2, cytokines and adipokines were quantified by EIA. Participation of the COX-1 and COX-2 enzymes, NF-κB and PGE2 receptors (EP1-4) was assessed using a pharmacological approach, and protein expression of the COX enzymes and P-NF-κB was analysed by western blotting. Preadipocyte differentiation was quantified by Oil Red O staining. Bmv (1 µg/mL) induced release of PGE2, IL-6 and KC and increased expression of COX-2 in preadipocytes. Basal levels of TNF-α, MCP-1, leptin and adiponectin were not modified. Treatment of cells with SC560 (COX-1 inhibitor) and NS398 (COX-2 inhibitor) inhibited Bmv-induced PGE2 release. Bmv induced phosphorylation of NF-κB, and treatment of the cells with TPCK and SN50, which inhibit distinct NF-κB domains, significantly reduced Bmv-induced PGE2 release, as did the treatment with an antagonist of PGE2 receptor EP1, unlike treatment with antagonists of EP2, EP3 or EP4. Bmv also induced lipid accumulation in differentiating cells. These results demonstrate that Bmv can activate an inflammatory response in preadipocytes by inducing the release of inflammatory mediators; that PGE2 production is mediated by the COX-1, COX-2 and NF-κB pathways; and that engagement of EP1 potentiates PGE2 synthesis via a positive feedback mechanism. Our findings highlight the role of the adipose tissue as another target for Bmv and suggest that it contributes to Bothrops envenomation by producing inflammatory mediators.

Bothrops moojeni snake venom induces an inflammatory response in preadipocytes: insights into a new aspect of envenomation

Bothrops envenomation is a public health problem in Brazil. Despite the advances in the knowledge of the pathogenesis of systemic and local effects induced by Bothrops venom, the target tissues to this venom are not completely characterised. As preadipocytes are important cells of the adipose tissue and synthesize inflammatory mediators, we investigated the ability of B. moojeni snake venom (Bmv) to stimulate an inflammatory response in 3T3-L1 preadipocytes in vitro, focusing on (1) the release of PGE2, IL-6, TNF-α, MCP-1, KC, leptin and adiponectin; (2) the mechanisms involved in PGE2 release and (3) differentiation of these cells. Cytotoxicity of Bmv was determined by MTT assay. The concentrations of PGE2, cytokines and adipokines were quantified by EIA. Participation of the COX-1 and COX-2 enzymes, NF-κB and PGE2 receptors (EP1-4) was assessed using a pharmacological approach, and protein expression of the COX enzymes and P-NF-κB was analysed by western blotting. Preadipocyte differentiation was quantified by Oil Red O staining. Bmv (1 μg/mL) induced release of PGE2, IL-6 and KC and increased expression of COX-2 in preadipocytes. Basal levels of TNF-α, MCP-1, leptin and adiponectin were not modified. Treatment of cells with SC560 (COX-1 inhibitor) and NS398 (COX-2 inhibitor) inhibited Bmv-induced PGE2 release. Bmv induced phosphorylation of NF-κB, and treatment of the cells with TPCK and SN50, which inhibit distinct NF-κB domains, significantly reduced Bmv-induced PGE2 release, as did the treatment with an antagonist of PGE2 receptor EP1, unlike treatment with antagonists of EP2, EP3 or EP4. Bmv also induced lipid accumulation in differentiating cells. These results demonstrate that Bmv can activate an inflammatory response in preadipocytes by inducing the release of inflammatory mediators; that PGE2 production is mediated by the COX-1, COX-2 and NF-κB pathways; and that engagement of EP1 potentiates PGE2 synthesis via a positive feedback mechanism. Our findings highlight the role of the adipose tissue as another target for Bmv and suggest that it contributes to Bothrops envenomation by producing inflammatory mediators.

Inflammatory Effects of Bothrops Phospholipases A2: Mechanisms Involved in Biosynthesis of Lipid Mediators and Lipid Accumulation.

Phospholipases A2s (PLA2s) constitute one of the major protein groups present in the venoms of viperid and crotalid snakes. Snake venom PLA2s (svPLA2s) exhibit a remarkable functional diversity, as they have been described to induce a myriad of toxic effects. Local inflammation is an important characteristic of snakebite envenomation inflicted by viperid and crotalid species and diverse svPLA2s have been studied for their proinflammatory properties. Moreover, based on their molecular, structural, and functional properties, the viperid svPLA2s are classified into the group IIA secreted PLA2s, which encompasses mammalian inflammatory sPLA2s. Thus, research on svPLA2s has attained paramount importance for better understanding the role of this class of enzymes in snake envenomation and the participation of GIIA sPLA2s in pathophysiological conditions and for the development of new therapeutic agents. In this review, we highlight studies that have identified the inflammatory activities of svPLA2s, in particular, those from Bothrops genus snakes, which are major medically important snakes in Latin America, and we describe recent advances in our collective understanding of the mechanisms underlying their inflammatory effects. We also discuss studies that dissect the action of these venom enzymes in inflammatory cells focusing on molecular mechanisms and signaling pathways involved in the biosynthesis of lipid mediators and lipid accumulation in immunocompetent cells.

A Metalloproteinase Induces an Inflammatory Response in Preadipocytes with the Activation of COX Signalling Pathways and Participation of Endogenous Phospholipases A2.

Matrix metalloproteinases (MMPs) are proteolytic enzymes that have been associated with the pathogenesis of inflammatory diseases and obesity. Adipose tissue in turn is an active endocrine organ capable of secreting a range of proinflammatory mediators with autocrine and paracrine properties, which contribute to the inflammation of adipose tissue and adjacent tissues. However, the potential inflammatory effects of MMPs in adipose tissue cells are still unknown. This study investigates the effects of BmooMPα-I, a single-domain snake venom metalloproteinase (SVMP), in activating an inflammatory response by 3T3-L1 preadipocytes in culture, focusing on prostaglandins (PGs), cytokines, and adipocytokines biosynthesis and mechanisms involved in prostaglandin E2 (PGE2) release. The results show that BmooMPα-I induced the release of PGE2, prostaglandin I2 (PGI2), monocyte chemoattractant protein-1 (MCP-1), and adiponectin by preadipocytes. BmooMPα-I-induced PGE2 biosynthesis was dependent on group-IIA-secreted phospholipase A2 (sPLA2-IIA), cytosolic phospholipase A2-α (cPLA2-α), and cyclooxygenase (COX)-1 and -2 pathways. Moreover, BmooMPα-I upregulated COX-2 protein expression but not microsomal prostaglandin E synthase-1 (mPGES-1) expression. In addition, we demonstrate that the enzymatic activity of BmooMPα-I is essential for the activation of prostanoid synthesis pathways in preadipocytes. These data highlight preadipocytes as important targets for metalloproteinases and provide new insights into the contribution of these enzymes to the inflammation of adipose tissue and tissues adjacent to it.

Inflammatory effects of Bothrops phospholipases A2: mechanisms involved in biosynthesis of lipid mediators and lipid accumulation

Phospholipases A2s (PLA2s) constitute one of the major protein groups present in the venoms of viperid and crotalid snakes. Snake venom PLA2s (svPLA2s) exhibit a remarkable functional diversity, as they have been described to induce a myriad of toxic effects. Local inflammation is an important characteristic of snakebite envenomation inflicted by viperid and crotalid species and diverse svPLA2s have been studied for their proinflammatory properties. Moreover, based on their molecular, structural, and functional properties, the viperid svPLA2s are classified into the group IIA secreted PLA2s, which encompasses mammalian inflammatory sPLA2s. Thus, research on svPLA2s has attained paramount importance for better understanding the role of this class of enzymes in snake envenomation and the participation of GIIA sPLA2s in pathophysiological conditions and for the development of new therapeutic agents. In this review, we highlight studies that have identified the inflammatory activities of svPLA2s, in particular, those from Bothrops genus snakes, which are major medically important snakes in Latin America, and we describe recent advances in our collective understanding of the mechanisms underlying their inflammatory effects. We also discuss studies that dissect the action of these venom enzymes in inflammatory cells focusing on molecular mechanisms and signaling pathways involved in the biosynthesis of lipid mediators and lipid accumulation in immunocompetent cells.

A metalloproteinase induces an inflammatory response in preadipocytes with the activation of COX signalling pathways and participation of endogenous phospholipases A2

Matrix metalloproteinases (MMPs) are proteolytic enzymes that have been associated with the pathogenesis of inflammatory diseases and obesity. Adipose tissue in turn is an active endocrine organ capable of secreting a range of proinflammatory mediators with autocrine and paracrine properties, which contribute to the inflammation of adipose tissue and adjacent tissues. However, the potential inflammatory effects of MMPs in adipose tissue cells are still unknown. This study investigates the effects of BmooMPα-I, a single-domain snake venom metalloproteinase (SVMP), in activating an inflammatory response by 3T3-L1 preadipocytes in culture, focusing on prostaglandins (PGs), cytokines, and adipocytokines biosynthesis and mechanisms involved in prostaglandin E2 (PGE2) release. The results show that BmooMPα-I induced the release of PGE2, prostaglandin I2 (PGI2), monocyte chemoattractant protein-1 (MCP-1), and adiponectin by preadipocytes. BmooMPα-I-induced PGE2 biosynthesis was dependent on group-IIA-secreted phospholipase A2 (sPLA2-IIA), cytosolic phospholipase A2-α (cPLA2-α), and cyclooxygenase (COX)-1 and -2 pathways. Moreover, BmooMPα-I upregulated COX-2 protein expression but not microsomal prostaglandin E synthase-1 (mPGES-1) expression. In addition, we demonstrate that the enzymatic activity of BmooMPα-I is essential for the activation of prostanoid synthesis pathways in preadipocytes. These data highlight preadipocytes as important targets for metalloproteinases and provide new insights into the contribution of these enzymes to the inflammation of adipose tissue and tissues adjacent to it.

A Representative GIIA Phospholipase A2 Activates Preadipocytes to Produce Inflammatory Mediators Implicated in Obesity Development.

Adipose tissue secretes proinflammatory mediators which promote systemic and adipose tissue inflammation seen in obesity. Group IIA (GIIA)-secreted phospholipase A2 (sPLA2) enzymes are found to be elevated in plasma and adipose tissue from obese patients and are active during inflammation, generating proinflammatory mediators, including prostaglandin E2 (PGE2). PGE2 exerts anti-lipolytic actions and increases triacylglycerol levels in adipose tissue. However, the inflammatory actions of GIIA sPLA2s in adipose tissue cells and mechanisms leading to increased PGE2 levels in these cells are unclear. This study investigates the ability of a representative GIIA sPLA2, MT-III, to activate proinflammatory responses in preadipocytes, focusing on the biosynthesis of prostaglandins, adipocytokines and mechanisms involved in these effects. Our results showed that MT-III induced biosynthesis of PGE2, PGI2, MCP-1, IL-6 and gene expression of leptin and adiponectin in preadipocytes. The MT-III-induced PGE2 biosynthesis was dependent on cytosolic PLA2 (cPLA2)-α, cyclooxygenases (COX)-1 and COX-2 pathways and regulated by a positive loop via the EP4 receptor. Moreover, MT-III upregulated COX-2 and microsomal prostaglandin synthase (mPGES)-1 protein expression. MCP-1 biosynthesis induced by MT-III was dependent on the EP4 receptor, while IL-6 biosynthesis was dependent on EP3 receptor engagement by PGE2. These data highlight preadipocytes as targets for GIIA sPLA2s and provide insight into the roles played by this group of sPLA2s in obesity.

A Lipidomic Perspective of the Action of Group IIA Secreted Phospholipase A2 on Human Monocytes: Lipid Droplet Biogenesis and Activation of Cytosolic Phospholipase A2α.

Phospholipase A2s constitute a wide group of lipid-modifying enzymes which display a variety of functions in innate immune responses. In this work, we utilized mass spectrometry-based lipidomic approaches to investigate the action of Asp-49 Ca2+-dependent secreted phospholipase A2 (sPLA2) (MT-III) and Lys-49 sPLA2 (MT-II), two group IIA phospholipase A2s isolated from the venom of the snake Bothrops asper, on human peripheral blood monocytes. MT-III is catalytically active, whereas MT-II lacks enzyme activity. A large decrease in the fatty acid content of membrane phospholipids was detected in MT III-treated monocytes. The significant diminution of the cellular content of phospholipid-bound arachidonic acid seemed to be mediated, in part, by the activation of the endogenous group IVA cytosolic phospholipase A2α. MT-III triggered the formation of triacylglycerol and cholesterol enriched in palmitic, stearic, and oleic acids, but not arachidonic acid, along with an increase in lipid droplet synthesis. Additionally, it was shown that the increased availability of arachidonic acid arising from phospholipid hydrolysis promoted abundant eicosanoid synthesis. The inactive form, MT-II, failed to produce any of the effects described above. These studies provide a complete lipidomic characterization of the monocyte response to snake venom group IIA phospholipase A2, and reveal significant connections among lipid droplet biogenesis, cell signaling and biochemical pathways that contribute to initiating the inflammatory response.

A representative metalloprotease induces PGE2 synthesis in fibroblast-like synoviocytes via the NF-κB/COX-2 pathway with amplification by IL-1ß and the EP4 receptor.

Inflammatory joint conditions are characterized by synovial inflammation, which involves activation of fibroblast-like synoviocytes (FLSs) and production of inflammatory mediators and matrix metalloproteases (MMPs) in joints. This study showed that the snake venom metalloprotease (SVMP) BaP1 activates FLSs to produce PGE2 by a mechanism dependent on COX-2, mPGES-1 and iPLA2s. BaP1 also induces IL-1ß release, which up-regulates the production of PGE2 at a late stage of the stimulation. Expression of COX-2 and mPGES-1 are induced by BaP1 via activation of NF-κB pathway. While NF-κB p50 and p65 subunits are involved in up-regulation of COX-2 expression, only p65 is involved in BaP1-induced mPGES-1 expression. In addition, BaP1 up-regulates EP4 receptor expression. Engagement of this receptor by PGE2 triggers a positive feedback loop for its production by up-regulating expression of key components of the PGE2 biosynthetic cascade (COX-2, mPGES-1 and the EP4 receptor), thus contributing to amplification of BaP1-induced effects in FLSs. These data highlight the importance of FLS as a target for metalloproteases in joint inflammation and provide new insights into the roles of MMPs in inflammatory joint diseases. Moreover, our results may give insights into the importance of the catalytic domain, of MMPs for the inflammatory activity of these enzymes.

A Lipidomic perspective of the action of group IIA secreted phospholipase A2 on human monocytes: lipid droplet biogenesis and activation of cytosolic phospholipase A2a

Phospholipase A2s constitute a wide group of lipid-modifying enzymes which display a variety of functions in innate immune responses. In this work, we utilized mass spectrometry-based lipidomic approaches to investigate the action of Asp-49 Ca2+-dependent secreted phospholipase A2 (sPLA2) (MT-III) and Lys-49 sPLA2 (MT-II), two group IIA phospholipase A2s isolated from the venom of the snake Bothrops asper, on human peripheral blood monocytes. MT-III is catalytically active, whereas MT-II lacks enzyme activity. A large decrease in the fatty acid content of membrane phospholipids was detected in MT III-treated monocytes. The significant diminution of the cellular content of phospholipid-bound arachidonic acid seemed to be mediated, in part, by the activation of the endogenous group IVA cytosolic phospholipase A2a. MT-III triggered the formation of triacylglycerol and cholesterol enriched in palmitic, stearic, and oleic acids, but not arachidonic acid, along with an increase in lipid droplet synthesis. Additionally, it was shown that the increased availability of arachidonic acid arising from phospholipid hydrolysis promoted abundant eicosanoid synthesis. The inactive form, MT-II, failed to produce any of the effects described above. These studies provide a complete lipidomic characterization of the monocyte response to snake venom group IIA phospholipase A2, and reveal significant connections among lipid droplet biogenesis, cell signaling and biochemical pathways that contribute to initiating the inflammatory response.

A representative metalloprotease induces PGE2 synthesis in fibroblast-like synoviocytes via the NF-capaB/COX-2 pathway with amplification by IL-1ß and the EP4 receptor

Inflammatory joint conditions are characterized by synovial inflammation, which involves activation of fibroblast-like synoviocytes (FLSs) and production of inflammatory mediators and matrix metalloproteases (MMPs) in joints. This study showed that the snake venom metalloprotease (SVMP) BaP1 activates FLSs to produce PGE2 by a mechanism dependent on COX-2, mPGES-1 and iPLA2s. BaP1 also induces IL-1ß release, which up-regulates the production of PGE2 at a late stage of the stimulation. Expression of COX-2 and mPGES-1 are induced by BaP1 via activation of NF-capaB pathway. While NF-capaB p50 and p65 subunits are involved in up-regulation of COX-2 expression, only p65 is involved in BaP1-induced mPGES-1 expression. In addition, BaP1 up-regulates EP4 receptor expression. Engagement of this receptor by PGE2 triggers a positive feedback loop for its production by up-regulating expression of key components of the PGE2 biosynthetic cascade (COX-2, mPGES-1 and the EP4 receptor), thus contributing to amplification of BaP1-induced effects in FLSs. These data highlight the importance of FLS as a target for metalloproteases in joint inflammation and provide new insights into the roles of MMPs in inflammatory joint diseases. Moreover, our results may give insights into the importance of the catalytic domain, of MMPs for the inflammatory activity of these enzymes.

A Lipidomic perspective of the action of group IIA secreted phospholipase A2 on human monocytes: lipid droplet biogenesis and activation of cytosolic phospholipase A2a

Phospholipase A2s constitute a wide group of lipid-modifying enzymes which display a variety of functions in innate immune responses. In this work, we utilized mass spectrometry-based lipidomic approaches to investigate the action of Asp-49 Ca2+-dependent secreted phospholipase A2 (sPLA2) (MT-III) and Lys-49 sPLA2 (MT-II), two group IIA phospholipase A2s isolated from the venom of the snake Bothrops asper, on human peripheral blood monocytes. MT-III is catalytically active, whereas MT-II lacks enzyme activity. A large decrease in the fatty acid content of membrane phospholipids was detected in MT III-treated monocytes. The significant diminution of the cellular content of phospholipid-bound arachidonic acid seemed to be mediated, in part, by the activation of the endogenous group IVA cytosolic phospholipase A2a. MT-III triggered the formation of triacylglycerol and cholesterol enriched in palmitic, stearic, and oleic acids, but not arachidonic acid, along with an increase in lipid droplet synthesis. Additionally, it was shown that the increased availability of arachidonic acid arising from phospholipid hydrolysis promoted abundant eicosanoid synthesis. The inactive form, MT-II, failed to produce any of the effects described above. These studies provide a complete lipidomic characterization of the monocyte response to snake venom group IIA phospholipase A2, and reveal significant connections among lipid droplet biogenesis, cell signaling and biochemical pathways that contribute to initiating the inflammatory response.

A representative metalloprotease induces PGE2 synthesis in fibroblast-like synoviocytes via the NF-capaB/COX-2 pathway with amplification by IL-1ß and the EP4 receptor

Inflammatory joint conditions are characterized by synovial inflammation, which involves activation of fibroblast-like synoviocytes (FLSs) and production of inflammatory mediators and matrix metalloproteases (MMPs) in joints. This study showed that the snake venom metalloprotease (SVMP) BaP1 activates FLSs to produce PGE2 by a mechanism dependent on COX-2, mPGES-1 and iPLA2s. BaP1 also induces IL-1ß release, which up-regulates the production of PGE2 at a late stage of the stimulation. Expression of COX-2 and mPGES-1 are induced by BaP1 via activation of NF-capaB pathway. While NF-capaB p50 and p65 subunits are involved in up-regulation of COX-2 expression, only p65 is involved in BaP1-induced mPGES-1 expression. In addition, BaP1 up-regulates EP4 receptor expression. Engagement of this receptor by PGE2 triggers a positive feedback loop for its production by up-regulating expression of key components of the PGE2 biosynthetic cascade (COX-2, mPGES-1 and the EP4 receptor), thus contributing to amplification of BaP1-induced effects in FLSs. These data highlight the importance of FLS as a target for metalloproteases in joint inflammation and provide new insights into the roles of MMPs in inflammatory joint diseases. Moreover, our results may give insights into the importance of the catalytic domain, of MMPs for the inflammatory activity of these enzymes.

Inflammation Induced by Platelet-Activating Viperid Snake Venoms: Perspectives on Thromboinflammation.

Envenomation by viperid snakes is characterized by systemic thrombotic syndrome and prominent local inflammation. To date, the mechanisms underlying inflammation and blood coagulation induced by Viperidae venoms have been viewed as distinct processes. However, studies on the mechanisms involved in these processes have revealed several factors and signaling molecules that simultaneously act in both the innate immune and hemostatic systems, suggesting an overlap between both systems during viper envenomation. Moreover, distinct classes of venom toxins involved in these effects have also been identified. However, the interplay between inflammation and hemostatic alterations, referred as to thromboinflammation, has never been addressed in the investigation of viper envenomation. Considering that platelets are important targets of viper snake venoms and are critical for the process of thromboinflammation, in this review, we summarize the inflammatory effects and mechanisms induced by viper snake venoms, particularly from the Bothrops genus, which strongly activate platelet functions and highlight selected venom components (metalloproteases and C-type lectins) that both stimulate platelet functions and exhibit pro-inflammatory activities, thus providing insights into the possible role(s) of thromboinflammation in viper envenomation.

A Secreted Phospholipase A2 Induces Formation of Smooth Muscle Foam Cells Which Transdifferentiate to Macrophage-Like State.

Vascular smooth muscle cells (VSMCs) loaded with lipid droplets (LDs) are markers of atherosclerosis. In this disease, inflammatory Group IIA-secreted phospholipase A2s (GIIA sPLA2s) are highly expressed in VSMCs, but their actions in these cells are unknown. Here, we investigated the ability of myotoxin III (MT-III), an ophidian GIIA sPLA2 sharing structural and functional features with mammalian GIIA sPLA2s, to induce LD formation and lipid metabolism factors involved in this effect. Modulation of VSMC phenotypes by this sPLA2 was also evaluated. Incubation of VSMCs with MT-III significantly increased the number of LDs. MT-III upregulated scavenger receptor type 1 (SR-A1) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein expression and enhanced acetylated-low density lipoprotein (acLDL) uptake by VSMCs, revealing the ability of a GIIA PLA2 to modulate scavenger receptor activities. MT-III induced translocation and protein expression of PPAR-γ and -ß/δ. Inhibition of peroxisome proliferator-activated receptors (PPARs) and diacylglycerol O-acyltransferase (DGAT) and acyl-CoA:cholesterolacyltransferase (ACAT) enzymes abrogated MT-III-induced LD formation. Moreover, in response to MT-III, VSMCs acquired phagocytic activity and expressed macrophage markers CD68 and MAC-2. In conclusion, MT-III is able to stimulate VSMCs and recruit factors involved in lipid uptake and metabolism, leading to the formation of VSMC-derived foam cells with acquisition of macrophage-like markers and functions.

Crotalus durissus ruruima snake venom and a Phospholipase A2 isolated from this venom elicit macrophages to form lipid droplets and synthesize inflammatory lipid mediators

Viper snake Crotalus durissus ruruima (Cdr) is a subspecies found in northern area of Brazil. Among the snakes of Crotalus genus subspecies, the venom of Cdr presents highest level of crotoxin, which is the major component of Crotalus snake venoms, formed by two subunits (crotapotin and a phospholipase A2 named CBr) and presents potent neurotoxic activity. Curiously, the venom of C. d. ruruima (CdrV) is better neutralized by antibothropic than by anticrotalic serum, strongly suggesting that this venom has similarities with venom of Bothrops genus snakes with regard to the ability to induce inflammation. Macrophages are cells with a central role in inflammatory and immunological responses. Upon inflammatory stimuli, these cells exhibit increased numbers of lipid droplets, which are key organelles in the synthesis and release of inflammatory mediators. However, the effects of CdrV and CBr in macrophage functions are unknown. We herein investigated the ability of CdrV and CBr to activate macrophages with focus on the formation of lipid droplets (LDs), synthesis of lipid mediators, and mechanisms involved in these effects. The involvement of LDs in PGE2 biosynthesis was also assessed. Stimulation of murine macrophages with CdrV and CBr induced an increased number of LDs and release of prostanoids (PGE2, PGD2, and TXB2). Neither CdrV nor CBr induced the expression of COX-1 and COX-2 by macrophages. LDs induced by both CdrV and CBr are associated to PLIN2 recruitment and expression and were shown to be dependent on COX-1, but not COX-2 activity. Moreover, PGE2 colocalized to CdrV- and CBr-induced LDs, revealing the role of these organelles as sites for the synthesis of prostanoids. These results evidence, for the first time, the ability of a whole snake venom to induce formation of LDs and the potential role of these organelles for the production of inflammatory mediators during envenomation by Crotalus snakes.

A secreted phospholipase A2 induces formation of smooth muscle foam cells which transdifferentiate to macrophage-like state

Vascular smooth muscle cells (VSMCs) loaded with lipid droplets (LDs) are markers of atherosclerosis. In this disease, inflammatory Group IIA-secreted phospholipase A2s (GIIA sPLA2s) are highly expressed in VSMCs, but their actions in these cells are unknown. Here, we investigated the ability of myotoxin III (MT-III), an ophidian GIIA sPLA2 sharing structural and functional features with mammalian GIIA sPLA2s, to induce LD formation and lipid metabolism factors involved in this effect. Modulation of VSMC phenotypes by this sPLA2 was also evaluated. Incubation of VSMCs with MT-III significantly increased the number of LDs. MT-III upregulated scavenger receptor type 1 (SR-A1) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein expression and enhanced acetylated-low density lipoprotein (acLDL) uptake by VSMCs, revealing the ability of a GIIA PLA2 to modulate scavenger receptor activities. MT-III induced translocation and protein expression of PPAR-gama and -ß/d. Inhibition of peroxisome proliferator-activated receptors (PPARs) and diacylglycerol O-acyltransferase (DGAT) and acyl-CoA:cholesterolacyltransferase (ACAT) enzymes abrogated MT-III-induced LD formation. Moreover, in response to MT-III, VSMCs acquired phagocytic activity and expressed macrophage markers CD68 and MAC-2. In conclusion, MT-III is able to stimulate VSMCs and recruit factors involved in lipid uptake and metabolism, leading to the formation of VSMC-derived foam cells with acquisition of macrophage-like markers and functions.

Inflammation induced by platelet-activating viperid snake venoms: perspectives on thromboinflammation

Envenomation by viperid snakes is characterized by systemic thrombotic syndrome and prominent local inflammation. To date, the mechanisms underlying inflammation and blood coagulation induced by Viperidae venoms have been viewed as distinct processes. However, studies on the mechanisms involved in these processes have revealed several factors and signaling molecules that simultaneously act in both the innate immune and hemostatic systems, suggesting an overlap between both systems during viper envenomation. Moreover, distinct classes of venom toxins involved in these effects have also been identified. However, the interplay between inflammation and hemostatic alterations, referred as to thromboinflammation, has never been addressed in the investigation of viper envenomation. Considering that platelets are important targets of viper snake venoms and are critical for the process of thromboinflammation, in this review, we summarize the inflammatory effects and mechanisms induced by viper snake venoms, particularly from the Bothrops genus, which strongly activate platelet functions and highlight selected venom components (metalloproteases and C-type lectins) that both stimulate platelet functions and exhibit pro-inflammatory activities, thus providing insights into the possible role(s) of thromboinflammation in viper envenomation.

Crotalus durissus ruruima snake venom and a Phospholipase A2 isolated from this venom elicit macrophages to form lipid droplets and synthesize inflammatory lipid mediators

Viper snake Crotalus durissus ruruima (Cdr) is a subspecies found in northern area of Brazil. Among the snakes of Crotalus genus subspecies, the venom of Cdr presents highest level of crotoxin, which is the major component of Crotalus snake venoms, formed by two subunits (crotapotin and a phospholipase A2 named CBr) and presents potent neurotoxic activity. Curiously, the venom of C. d. ruruima (CdrV) is better neutralized by antibothropic than by anticrotalic serum, strongly suggesting that this venom has similarities with venom of Bothrops genus snakes with regard to the ability to induce inflammation. Macrophages are cells with a central role in inflammatory and immunological responses. Upon inflammatory stimuli, these cells exhibit increased numbers of lipid droplets, which are key organelles in the synthesis and release of inflammatory mediators. However, the effects of CdrV and CBr in macrophage functions are unknown. We herein investigated the ability of CdrV and CBr to activate macrophages with focus on the formation of lipid droplets (LDs), synthesis of lipid mediators, and mechanisms involved in these effects. The involvement of LDs in PGE2 biosynthesis was also assessed. Stimulation of murine macrophages with CdrV and CBr induced an increased number of LDs and release of prostanoids (PGE2, PGD2, and TXB2). Neither CdrV nor CBr induced the expression of COX-1 and COX-2 by macrophages. LDs induced by both CdrV and CBr are associated to PLIN2 recruitment and expression and were shown to be dependent on COX-1, but not COX-2 activity. Moreover, PGE2 colocalized to CdrV- and CBr-induced LDs, revealing the role of these organelles as sites for the synthesis of prostanoids. These results evidence, for the first time, the ability of a whole snake venom to induce formation of LDs and the potential role of these organelles for the production of inflammatory mediators during envenomation by Crotalus snakes.

A secreted phospholipase A2 induces formation of smooth muscle foam cells which transdifferentiate to macrophage-like state

Vascular smooth muscle cells (VSMCs) loaded with lipid droplets (LDs) are markers of atherosclerosis. In this disease, inflammatory Group IIA-secreted phospholipase A2s (GIIA sPLA2s) are highly expressed in VSMCs, but their actions in these cells are unknown. Here, we investigated the ability of myotoxin III (MT-III), an ophidian GIIA sPLA2 sharing structural and functional features with mammalian GIIA sPLA2s, to induce LD formation and lipid metabolism factors involved in this effect. Modulation of VSMC phenotypes by this sPLA2 was also evaluated. Incubation of VSMCs with MT-III significantly increased the number of LDs. MT-III upregulated scavenger receptor type 1 (SR-A1) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein expression and enhanced acetylated-low density lipoprotein (acLDL) uptake by VSMCs, revealing the ability of a GIIA PLA2 to modulate scavenger receptor activities. MT-III induced translocation and protein expression of PPAR-gama and -ß/d. Inhibition of peroxisome proliferator-activated receptors (PPARs) and diacylglycerol O-acyltransferase (DGAT) and acyl-CoA:cholesterolacyltransferase (ACAT) enzymes abrogated MT-III-induced LD formation. Moreover, in response to MT-III, VSMCs acquired phagocytic activity and expressed macrophage markers CD68 and MAC-2. In conclusion, MT-III is able to stimulate VSMCs and recruit factors involved in lipid uptake and metabolism, leading to the formation of VSMC-derived foam cells with acquisition of macrophage-like markers and functions.