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.

Estudo dos efeitos desencadeados pelo veneno de Bothrops moojeni na síntese de mediadores inflamatórios em pré- adipócitos e os mecanismos envolvidos

Snakebites constitute an important public health problem, recognized by World Health Organization. In Brazil, Bothrops genus snakes are responsible for the major number of registered envenomation cases. Among this genus, the species B. moojeni is known for the severity of local inflammatory response. However, systemic repercussions of bothropic envenomation are still not well stablished. In this context, adipose tissue is a metabolically active organ, known for producing many proinflammatory mediators. Preadipocytes and adipocytes are important cell types that constitute this tissue and possess the capacity of synthetize components that drive to inflammation. Therefore, this study aims to investigate the effects of B. moojeni whole venom (BmV), in cultured preadipocytes, with focus on: i) release of cytokines and chemokines (TNF-α, IL-6, IL-10, MCP-1 and KC); ii) release of prostaglandin E2 (PGE2); iii) protein expression of cyclooxygenase-1 and -2 (COX-1 and -2); iv) participation of COX-1, -2, and PGE2 receptors (EP1-4) on PGE2 release; v) activation of transcription factor NF-κB; vi) participation of NF-κB on PGE2 release and COX-2 protein expression; and vii) preadipocytes differentiation into mature adipocytes. Moreover, the venom effects in adipocytes were investigated, focusing on: i) PGE2 release and ii) COX-2 protein expression. In order to this, 3T3-L1 preadipocytes were cultured in DMEM medium, with 10% BFS, 1% L-glutamine and antibiotics until confluence. Next, they were incubated with DMEM only, or TNF-α (20 ng/mL), or BmV (1 μg/mL) for different experimental periods of time. TNF-α, IL-6, IL- 10, MCP-1, KC and PGE2 concentration, in supernatants, was evaluated by ELISA. COX-1, -2, and P-NF-κB protein expression was determined by Western blotting analysis. COX-1, -2, EP1-4 receptors and NF-κB participation on PGE2 release were evaluated utilizing pharmacological treatments with specific inhibitors and/or antagonists. BmV effect on preadipocytes differentiation into mature adipocytes was evaluated according to protocol previously described in literature. Taken together, results obtained show that BmV activates proinflammatory pathways in preadipocytes and mature adipocytes. This venom acts on these cells stimulating the synthesis of cytokines that regulate inflammation (IL-6 and IL-10), or that possess chemotactic activity (KC), besides PGE2. Moreover, BmV was capable of activating mature adipocytes to PGE2 synthesis. In preadipocytes, COX-1 and -2 and EP1 receptor are important factors of venom-induced mechanisms involved in PGE2 release, with COX-2 protein expression increased in this process. It was also demonstrated NF-κB participation in PGE2 synthesis by preadipocytes. Finally, BmV is able to enhance preadipocytes differentiation into mature adipocytes. Taken together, this work indicates that adipose tissue cells are targets for BmV and must collaborate to the biosynthesis of inflammatory mediators involved in bothropic envenoming pathophysiology.

Os acidentes ofídicos constituem um importante problema de saúde pública, reconhecido pela Organização Mundial da Saúde. No Brasil, as serpentes do gênero Bothrops são responsáveis pelo maior número de envenenamentos registrados. Dentro deste gênero, a espécie B. moojeni é conhecida pela gravidade da resposta inflamatória exacerbada, no local da picada. No entanto, as repercussões sistêmicas do envenenamento botrópico, em diferentes tecidos, ainda não são bem estabelecidas. Neste contexto, o tecido adiposo é um órgão metabolicamente ativo, conhecido por produzir diversos mediadores pró-inflamatórios. Os pré-adipócitos e adipócitos são importantes tipos celulares que constituem este tecido e possuem a capacidade de sintetizar componentes que conduzem à inflamação. Sendo assim, o objetivo do presente estudo foi investigar os efeitos do veneno bruto de B. moojeni (VBm), em pré-adipócitos, em cultura, quanto a: i) liberação de citocinas e quimiocinas (TNF-α, IL-6, IL-10, MCP-1 e KC); ii) liberação de prostaglandina E2 (PGE2); iii) expressão proteica de ciclo-oxigenases-1 e -2 (COX-1 e -2); iv) participação das COX-1 e -2, e dos receptores de PGE2 (EP1-4) na liberação de PGE2; v) ativação do fator de transcrição NF-κB; vi) participação do NF-κB na liberação de PGE2 e na expressão proteica de COX-2; e vii) diferenciação de pré- adipócitos em adipócitos maduros. Ainda, o efeito do veneno em adipócitos foi investigado quando à: i) liberação de PGE2 e ii) expressão proteica de COX-2. Pré- adipócitos da linhagem 3T3-L1 foram cultivados em meio DMEM, contendo 10% de SFB, 1% de L-glutamina e antibiótico até a confluência. Em seguida, foram incubados com apenas DMEM, ou TNF-α (20 ng/mL), ou VBm (1 μg/mL) por diferentes períodos de tempo experimentais. A concentração de TNF-α, IL-6, IL-10, MCP-1 e KC, nos sobrenadantes, foi avaliada por ELISA. A expressão proteica de COX-1 e -2 e P-NF-κB foi determinada por Western blotting. A participação das COX-1 e -2, dos receptores EP1-4 e do NF-κB na liberação de PGE2 foi avaliada utilizando-se inibidores e/ou antagonistas específicos. O efeito do VBm na diferenciação de pré-adipócitos em adipócitos maduros foi avaliada seguindo protocolo descrito na literatura. Em conjunto, os resultados obtidos evidenciaram que o VBm ativa vias pró-inflamatórias em pré-adipócitos e adipócitos maduros. Este veneno atua sobre estas células estimulando a síntese de citocinas, que regulam a inflamação (IL-6 e IL-10), ou possuem atividade quimiotáxica (KC), além de PGE2. Além disso, o VBm foi capaz de ativar adipócitos maduros para a síntese de PGE2. Em pré-adipócitos, as COX-1 e -2 e o receptor EP1 são fatores importantes nos mecanismos desencadeados pelo veneno, que acarretam a liberação de PGE2, sendo que a COX-2 tem sua expressão proteica aumentada neste processo. Também foi demonstrada a participação do NF-κB na síntese de PGE2 em pré- adipócitos. Por fim, o VBm é capaz de aumentar a diferenciação de PAds em adipócitos maduros, provavelmente através dos mecanismos antilipolíticos da PGE2. Em conclusão, este trabalho aponta que as células do tecido adiposo são alvos do VBm e devem colaborar para a biossíntese de mediadores inflamatórios, envolvidos na fisiopatologia do envenenamento botrópico.

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 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.