Molecular Aspects Involved in the Mechanisms of Bothrops jararaca Venom-Induced Hyperalgesia: Participation of NK1 Receptor and Glial Cells.

Accidents caused by Bothrops jararaca (Bj) snakes result in several local and systemic manifestations, with pain being a fundamental characteristic. The inflammatory process responsible for hyperalgesia induced by Bj venom (Bjv) has been studied; however, the specific roles played by the peripheral and central nervous systems in this phenomenon remain unclear. To clarify this, we induced hyperalgesia in rats using Bjv and collected tissues from dorsal root ganglia (DRGs) and spinal cord (SC) at 2 and 4 h post-induction. Samples were labeled for Iba-1 (macrophage and microglia), GFAP (satellite cells and astrocytes), EGR1 (neurons), and NK1 receptors. Additionally, we investigated the impact of minocycline, an inhibitor of microglia, and GR82334 antagonist on Bjv-induced hyperalgesia. Our findings reveal an increase in Iba1 in DRG at 2 h and EGR1 at 4 h. In the SC, markers for microglia, astrocytes, neurons, and NK1 receptors exhibited increased expression after 2 h, with EGR1 continuing to rise at 4 h. Minocycline and GR82334 inhibited venom-induced hyperalgesia, highlighting the crucial roles of microglia and NK1 receptors in this phenomenon. Our results suggest that the hyperalgesic effects of Bjv involve the participation of microglial and astrocytic cells, in addition to the activation of NK1 receptors.

Molecular aspects involved in the mechanisms of Bothrops jararaca venom-induced Hyperalgesia: participation of NK1 Receptor and glial cells

Accidents caused by Bothrops jararaca (Bj) snakes result in several local and systemic manifestations, with pain being a fundamental characteristic. The inflammatory process responsible for hyperalgesia induced by Bj venom (Bjv) has been studied; however, the specific roles played by the peripheral and central nervous systems in this phenomenon remain unclear. To clarify this, we induced hyperalgesia in rats using Bjv and collected tissues from dorsal root ganglia (DRGs) and spinal cord (SC) at 2 and 4 h post-induction. Samples were labeled for Iba-1 (macrophage and microglia), GFAP (satellite cells and astrocytes), EGR1 (neurons), and NK1 receptors. Additionally, we investigated the impact of minocycline, an inhibitor of microglia, and GR82334 antagonist on Bjv-induced hyperalgesia. Our findings reveal an increase in Iba1 in DRG at 2 h and EGR1 at 4 h. In the SC, markers for microglia, astrocytes, neurons, and NK1 receptors exhibited increased expression after 2 h, with EGR1 continuing to rise at 4 h. Minocycline and GR82334 inhibited venom-induced hyperalgesia, highlighting the crucial roles of microglia and NK1 receptors in this phenomenon. Our results suggest that the hyperalgesic effects of Bjv involve the participation of microglial and astrocytic cells, in addition to the activation of NK1 receptors.

Crotoxin Modulates Macrophage Phenotypic Reprogramming.

Macrophage plasticity is a fundamental feature of the immune response since it favors the rapid and adequate change of the functional phenotype in response to the pathogen or the microenvironment. Several studies have shown that Crotoxin (CTX), the major toxin of the Crotalus durissus terrificus snake venom, has a long-lasting antitumor effect both in experimental models and in clinical trials. In this study, we show the CTX effect on the phenotypic reprogramming of macrophages in the mesenchymal tumor microenvironment or those obtained from the peritoneal cavity of healthy animals. CTX (0.9 or 5 µg/animal subcutaneously) administered concomitantly with intraperitoneal inoculation of tumor cells (1 × 107/0.5 mL, injected intraperitoneally) of Ehrlich Ascitic Tumor (EAT) modulated the macrophages phenotype (M1), accompanied by increased NO• production by cells from ascites, and was evaluated after 13 days. On the other hand, in healthy animals, the phenotypic profile of macrophages was modulated in a dose-dependent way at 0.9 µg/animal: M1 and at 5.0 µg/animal: M2; this was accompanied by increased NO• production by peritoneal macrophages only for the dose of 0.9 µg/animal of CTX. This study shows that a single administration of CTX interferes with the phenotypic reprogramming of macrophages, as well as with the secretory state of cells from ascites, influencing events involved with mesenchymal tumor progression. These findings may favor the selection of new therapeutic targets to correct compromised immunity in different systems.

Importância da galectina-3 na ação moduladora da crotoxina sobre função de monócito/macrófago

Crotoxin (CTX) has biological effects both in experimental models and in clinical trials. Our group showed that the antitumor activity of CTX is associated with the modulating actions of the toxin on the secretory activity of inflammatory mediators and on the energy metabolism of macrophages. Furthermore, we demonstrated that CTX, in vivo, interferes with the phenotypic reprogramming of resident peritoneal macrophages and the tumor microenvironment, suggesting a reprogramming or prevalence of the M1 phenotype of macrophages. Among the mechanisms involved with the phenotypic reprogramming of monocytes/macrophages, galectin-3 has been described as an immunoregulatory protein, which may favor the classic pro-inflammatory condition (macrophage-M1) or alternative (macrophage-M2, anti-inflammatory), depending on the pathophysiological condition, with involvement of the PI3K/Akt signaling pathway. Therefore, the objective of this project was to investigate whether CTX is capable of interfering with the expression and secretion of galectin-3 by monocytes and the participation of this lectin in the modulating activity of CTX on the phenotypic condition of these cells by different stimuli. For this purpose, in vitro assays were carried out using cells of the human monocytic lineage (THP-1), pre-incubated in the absence (control) or presence of CTX (0.3; 0.6 and 1.2 μg/mL), by 2 hours and maintained in culture for 72 hours under different microenvironmental stimuli: a) only in fresh RPMI 1640 culture medium (to evaluate the per se activity of CTX on the investigated proteins); b) with culture medium containing 800 μg/mL of LPS; or c) and conditioned medium (CM) from MDA-MB-231 tumor cells. The Western Blotting assay was performed to mark the proteins: PI3Kp110a, Akt (types 1 and 2) and galectin-3. Functional/phenotypic parameters, such as H2O2 release, secretion of pro-(TNF-α) and anti-inflammatory cytokines (IL-10/TGF-β1) and Gal-3, and phenotypic markers (M1- CD80/CD86 and M2-CD163/CD206) was determined in order to associate possible changes in the functional/phenotypic behavior (M1/M2) of these cells and with the expression of signaling proteins involved in the reprogramming of monocytes/macrophages, in the face of different stimuli. The results indicate that CTX modulates the Akt signaling pathway and with the expression and secretion of Gal-3, as well as with the release of H2O2 and against different signals in the microenvironment. Furthermore, a pro-inflammatory and pro-tumor role is attributed to Gal-3 in inflammatory and tumoral conditions and here, the results show the participation of this lectin in the immunomodulatory effect of CTX, both in the action per se of the toxin and in the inflammatory and tumoral microenvironments. Furthermore, particularly the concentration of CTX 0.6 and 1.2 μg/ml seems to promote a THP-1 mixed monocyte/macrophage profile or switch phenotype. This study contributes to knowledge about the immunomodulatory actions of CTX, showing for the first time the participation of Gal-3 in this process.

A Crotoxina (CTX) apresenta efeitos biológicos tanto em modelos experimentais como em estudos clínicos. Nosso grupo mostrou que a atividade antitumoral da CTX está associada às ações moduladoras da toxina sobre a atividade secretora de mediadores inflamatórios e sobre o metabolismo energético de macrófagos. Ainda, demonstramos que a CTX, in vivo, interfere com a reprogramação fenotípica de macrófagos peritoneais residentes e do microambiente tumoral, sugerindo uma reprogramação ou prevalência do fenótipo M1 de macrófagos. Dentre os mecanismos envolvidos com a reprogramação fenotípica de monócitos/macrófagos, a galectina-3 vem sendo descrita como uma proteína imunorreguladora, podendo favorecer a condição pró- inflamatória clássica (macrófago-M1) ou alternativa (macrófago-M2, anti-inflamatório), conforme a condição fisiopatológica, com envolvimento da via de sinalização PI3K/Akt. Portanto, o objetivo do presente projeto foi investigar se a CTX é capaz de interferir com a expressão e secreção de galectina-3 por monócitos e a participação desta lectina na atividade moduladora da CTX sobre a condição fenotípica dessas células, frente a diferentes estímulos. Para tanto, ensaios in vitro foram realizados utilizando células da linhagem monocítica humana (THP-1), pré-incubadas na ausência (controle) ou presença de CTX (0,3; 0,6 e 1,2 μg/mL), por 2 horas e mantidas em cultura por 72 horas em diferentes estímulos microambientais: a) apenas em meio de cultura RPMI 1640 fresco (para avaliar a atividade per se da CTX sobre as proteínas investigadas); b) com meio de cultura contendo 800 μg/mL de LPS; ou c) e meio condicionado de células tumorais MDA-MB-231. O ensaio de Western Blotting foi realizado para marcação das proteínas: PI3Kp110a, Akt (tipos 1 e 2) e galectina-3. Parâmetros funcionais/fenotípicos, como a liberação de H2O2, secreção de citocinas pró- (TNF-α) e anti-inflamatórias (IL-10/TGF-β1) e Gal-3 e marcadores fenotípicos (M1-CD80/CD86 e M2-CD163/CD206) foi determinado visando associar possíveis alterações sobre o comportamento funcional/fenotípico (M1/M2) dessas células e com a expressão das proteínas sinalizadoras envolvidas com a reprogramação dos monócitos/macrófagos, frente aos diferentes estímulos. Os resultados indicam que a CTX modula a via de sinalização Akt e com a expressão e secreção de Gal-3, bem como, com a liberação de H2O2 e frente a diferentes sinais no microambiente. Ainda, é atribuída a Gal-3 papel pró-inflamatória e pró-tumoral em condições inflamatória e tumoral e aqui, os resultados mostram a participação dessa lectina no efeito imunomodulador da CTX, tanto na ação per se da toxina quanto nos microambientes inflamatório e tumoral. Além disso, particularmente as concentrações de CTX 0,6 e 1,2 μg/mL parece promover um perfil misto de monócito/macrófago THP-1 ou fenótipo switch. Este estudo contribui com o conhecimento sobre as ações imunomoduladoras da CTX, evidenciando pela primeira vez a participação da Gal-3 nesse processo.

Crotoxin modulates macrophage phenotypic reprogramming

Macrophage plasticity is a fundamental feature of the immune response since it favors the rapid and adequate change of the functional phenotype in response to the pathogen or the microenvironment. Several studies have shown that Crotoxin (CTX), the major toxin of the Crotalus durissus terrificus snake venom, has a long-lasting antitumor effect both in experimental models and in clinical trials. In this study, we show the CTX effect on the phenotypic reprogramming of macrophages in the mesenchymal tumor microenvironment or those obtained from the peritoneal cavity of healthy animals. CTX (0.9 or 5 μg/animal subcutaneously) administered concomitantly with intraperitoneal inoculation of tumor cells (1 × 107/0.5 mL, injected intraperitoneally) of Ehrlich Ascitic Tumor (EAT) modulated the macrophages phenotype (M1), accompanied by increased NO• production by cells from ascites, and was evaluated after 13 days. On the other hand, in healthy animals, the phenotypic profile of macrophages was modulated in a dose-dependent way at 0.9 μg/animal: M1 and at 5.0 μg/animal: M2; this was accompanied by increased NO• production by peritoneal macrophages only for the dose of 0.9 μg/animal of CTX. This study shows that a single administration of CTX interferes with the phenotypic reprogramming of macrophages, as well as with the secretory state of cells from ascites, influencing events involved with mesenchymal tumor progression. These findings may favor the selection of new therapeutic targets to correct compromised immunity in different systems.

Effects of methylene blue-mediated photodynamic therapy on a mouse model of squamous cell carcinoma and normal skin.

BACKGROUND: Photodynamic therapy is used to treat a variety of cancers and skin diseases by inducing apoptosis, necrosis, immune system activation, and/or vascular damage. Here, we describe the effects of a single photodynamic therapy session using methylene blue on a mouse model of squamous cell carcinoma and normal skin. METHODS: The photodynamic therapy protocol comprised application of a 1% methylene blue solution, followed by irradiation with a diode laser for 15 min at 74 mW/cm2, for a total dose of 100 J/cm2. Morphological changes, cell proliferation, apoptosis, collagen quantity, immune system activity, and blood vessel number were analyzed 24 h and 15 days after photodynamic therapy. RESULTS: In the squamous cell carcinoma group, photodynamic therapy reduced tumor size and cell proliferation and raised cytokine levels. In normal skin, it decreased cell proliferation and collagen quantity and increased apoptosis and blood vessel numbers. CONCLUSIONS: The effects of photodynamic therapy were greater on normal skin than squamous cell carcinoma tissues. The reduced epithelial thickness and keratinization of the former are factors that contribute to the efficacy of this treatment. Adjustments to the treatment protocol are necessary to potentiate the effects for squamous cell carcinoma therapy.

Efeito da crotoxina sobre a reprogramação fenotípica dos macrófagos estimulados pelo microambiente tumoral / Crotoxin effect on the phenotypic reprogramming of tumor microenvironment-stimulated macrophages

Several studies have shown that Crotoxin (CTX), the major toxin of the Crotalus durissus terrificus snake venom, has an antitumor effect both in experimental models and in clinical trials. Our group has been demonstrating that the antitumor activity of the toxin is also associated with the CTX modulatory actions on the secretory activity of inflammatory mediators (cytokines, reactive oxygen and nitrogen species) and on the energetic metabolism (glycolytic and glutaminolytic pathways) of macrophages. Despite this evidence, it has not been demonstrated to date whether the metabolic alterations induced by CTX establish a phenotypic profile of the resident macrophages and influence the reprogramming of macrophages induced by the tumor microenvironment. Therefore, the present study investigated the effect of CTX on the phenotypic reprogramming of macrophages in the tumor microenvironment. Balb/c mice received administration of different CTX concentrations (0.9 or 5 μg/animal subcutaneously in sterile saline) at the 1st, 6th or 1st and 6th day of inoculation of tumor cells (1x107 /0.5 mL, injected intraperitoneally) of Ehrlich Ascitic Tumor (EAT). After 6 or 13 days of EAT induction, we analyzed: a) immunophenotyping of macrophages; b) total volume of ascitic fluid; total and differential count of cells (tumor and leukocytes) in the ascitic fluid; determination of nitric oxide (NO) production and secretion of cytokines IL-1β, IL-10 and release of TNF-. Functional and phenotypic parameters were also established in macrophages obtained from the peritoneal cavity of healthy animals to evaluate the action per se of the toxin. The different doses of CTX, administered on day 1, modulated the macrophages phenotype (M1), accompanied by increased NO production. In healthy animals, the phenotypic profile of macrophages was dependent on the administered dose (0.9 μg/animal: M1 and 5 μg/animal: M2), accompanied by increased NO production when administered at a dose of 0.9 μg/animal. When the parameters were evaluated on the 6th day after the treatment concomitant to the induction of EAT, there was a significant decrease in ascitic volume, accompanied by the decrease of tumor cells and inhibition of IL-10 production. In healthy animals, both doses induced increased leukocytes in the peritoneal cavity. When the animals were treated with CTX (particularly at the dose of 0.9 μg/animal), on the 6th day of EAT induction, a decrease in the number of tumor cells and inhibition of TNF-α release was again observed. In addition, treatment with CTX induced an increase in the number of leukocytes in the peritoneal cavity of healthy animals. In another treatment protocol, where the animals were treated with two doses given on the 1st and 6th days of EAT induction, the ascitic volume decreased again, accompanied by a significant increase in TNF-α release. Therefore, the data obtained in the present study indicate that CTX interferes with the phenotypic reprogramming of macrophages, as well as with the secretory state of these cells, influencing events involved with tumor progression.

Diferentes estudos demonstraram que a Crotoxina (CTX), toxina majoritária do veneno de serpente Crotalus durissus terrificus apresenta efeito antitumoral, tanto em modelos experimentais como em ensaios clínicos. Nosso grupo vem demonstrando que a atividade antitumoral da toxina também está associada às ações moduladoras da CTX sobre a atividade secretória de mediadores inflamatórios (citocinas, espécies reativas intermediárias do oxigênio e do nitrogênio e lipoxinas) e sobre o metabolismo energético (vias glicolítica e glutaminolítica) de macrófagos. Apesar dessas evidências, não foi demonstrado até o momento se as alterações metabólicas induzidas pela CTX estabelecem um perfil fenotípico dos macrófagos residentes e se influenciam a reprogramação de macrófagos induzida pelo microambiente tumoral. Portanto, o presente estudo investigou o efeito da CTX sobre a reprogramação fenotípica de macrófagos no microambiente tumoral. Camundongos Balb/c receberam a administração de diferentes concentrações CTX (0,9 ou 5 μg/animal, via subcutânea, em salina estéril), no 1o, 6o ou 1o e 6o dias da inoculação de células tumorais (1x107 /0,5 mL, i.p) de Tumor Ascítico de Ehrlich (TAE). Após 6 ou 13 dias da indução do TAE, foram analisados: a) imunofenotipagem dos macrófagos; b) volume total do líquido ascítico; contagem total e diferencial de células (tumorais e leucócitos) presentes no líquido ascítico; determinação da produção de óxido nítrico (NO) e secreção das citocinas IL-1β, IL-10 e liberação de TNF-. Os parâmetros funcionais e fenotípicos também foram estabelecidos em macrófagos obtidos da cavidade peritoneal de animais saudáveis, para avaliar a ação per se da toxina. As diferentes doses de CTX, administrada no 1o dia, modularam o fenótipo dos macrófagos (M1), acompanhado pelo aumento da produção de NO. Em animais saudáveis, o perfil fenotípico dos macrófagos foi dependente da dose administrada (0,9 μg/animal: M1 e 5 μg/animal: M2), acompanhado pelo aumento da produção de NO, quando administrada a dose de 0,9 μg/animal. Quando os parâmetros foram avaliados no 6o dia após o tratamento concomitante à indução da TAE, houve diminuição significativa do volume ascítico, acompanhado pela diminuição de células tumorais e inibição da produção de IL-10. Em animais saudáveis, ambas as doses induziram aumento de leucócitos na cavidade peritoneal. Quando os animais foram tratados com CTX (particularmente na dose de 0,9 μg/animal) no 6o dia da indução da TAE, observou-se novamente diminuição do número de células tumorais e inibição da liberação de TNF-. Ainda, o tratamento com a CTX induziu aumento do número de leucócitos na cavidade peritoneal de animais saudáveis. Em outro protocolo de tratamento, onde os animais foram tratados com duas doses, administradas no 1o e no 6o dia da indução da TAE, observou-se novamente a diminuição do volume ascítico, acompanhado por significativo aumento da liberação de TNF-. Portanto, os dados obtidos no presente estudo indicam que a CTX interfere com a reprogramação fenotípica de macrófagos, bem como com o estado secretório dessas células, influenciando eventos envolvidos com a progressão tumoral.

Crotoxin, a rattlesnake toxin, down-modulates functions of bone marrow neutrophils and impairs the Syk-GTPase pathway

Neutrophils have a critical role in the innate immune response; these cells represent the primary line of defense against invading pathogens or tissue injury. Crotoxin (CTX), the major toxin of the South American rattlesnake (Crotalus durissus terrificus) venom, presents longstanding anti-inflammatory properties, inhibiting neutrophil migration and phagocytosis by peritoneal neutrophils for 14 days. Herein, to elucidate these sustained inhibitory effects induced by CTX, we performed in vitro and in vivo studies evaluating the functionality of bone marrow neutrophils and possible molecular mechanisms associated with these effects. CTX inhibited the processes of chemotaxis, adhesion to fibronectin, and phagocytosis of opsonized particles; however, it did not affect ROS production or degranulation in bone marrow neutrophils. To understand the molecular mechanisms that orchestrate this effect, we investigated the expression of CR3 on the neutrophil surface and the total expression and activity of signaling proteins from the Syk-GTPase pathway, which is involved in actin polymerization. CTX down-regulated both subunits of CR3, as well as, the activity of Syk, Vav1, Cdc42, Rac1 and RhoA, and the expression of the subunit 1B from Arp2/3. Together, our findings demonstrated that CTX inhibits the functionally of bone marrow neutrophils and that this effect may be associated with an impairment of the Syk-GTPase pathway. This study demonstrates, for the first time, that the sustained down-modulatory effect of CTX on circulating and peritoneal neutrophils is associated with functional modifications of neutrophils still in the bone marrow, and it also contributes to a better understanding of the anti-inflammatory effect of CTX.

Efeito da Cotoxina na Sinalização Intracelular Envolvida na Fagositose por Neutrófilos

A Crotoxina (CTX), principal constituinte do veneno de Crotalus durissus terrificus (VCdt), tem ação anti-inflamatória prolongada, modulando funções de macrófagos e neutrófilos. Estados anteriores demonstraram que uma única administração dessa toxina inibe, por até 14 dias, a atividade fagocítica de neutrófilos obtidos no modelo de peritonite induzido por carragenina. Considerando a rápida metaboização da CTX e a vida média curta dos neutrófilos, é dificil explicar a ação prolongada dessa toxina. Nesse sentido, o objetivo do estudo é avaliar o efeito da CTX in vidro e in vivo sobre a funcionalidade de neutrófilos da medula óssea de camundongos, bem como sobre os mecanismos moleculares envolvidos nas funções avaliadas. Para os ensaios in vitro, os netrófilos foram incubados com a CTx (0,08 ug/mL), por 1 dia ou 24 horas. Para os ensaios in vivo, os animais foram pré-tratados com uma única administração de CTX (4mg/kg), 1 dia antes do isolamento das células. Após os tratamentos acima, os seguintes parâmetros funcionais foram avaliados: a) fagocitose de partículas de zimosan opsonizadas com C3bi e b) expressão das molécuilas sinalizadoras envolvidas no processo de fagocitose. Os resultados aqui apresentados mostraram que a CTX, in vitro e in vivo, inibiu o processo de fagocitose de partículas opsonizadas, bem como, a atividade de Syk, Vav1, Cdc42, Racl e RhoA e a expressão da subunidade 1B do complexo Arp 2/3. Em conjunto, os resultados obtidos mostraram que a CTX inibe a atividade funcional dos neutrófilos da medula óssea e essa ação está associada a inibição da atividade de proteínas sinalizadoras de Syk e de suas proteínas dowstream o que resulta da redução da formação de filamentos de F-actina. Dessa forma, esse estudo comprova a hipótese de que a ação inibitória prolongada da CTX sobre os neutrófilos circulantes e peritoneais está associada as alterações funcionais dos neutrófilos da medula. Esses dados contribuem para explicar o efeito inibitório da CTX sobre a respota inflamatória, particularmente sobre os eventos celulares dessa resposta.