A three-dimensional lung cell model to leptospira virulence investigations

Leptospirosis is a worldwide zoonosis and a serious public health threat in tropical and subtropical areas. The etiologic agents of leptospirosis are pathogenic spirochetes from the genus Leptospira. In severe cases, patients develop a pulmonary hemorrhage that is associated with high fatality rates. Several animal models were established for leptospirosis studies, such as rodents, dogs, and monkeys. Although useful to study the relationship among Leptospira and its hosts, the animal models still exhibit economic and ethical limitation reasons and do not fully represent the human infection. As an attempt to bridge the gap between animal studies and clinical information from patients, we established a three-dimensional (3-D) human lung cell culture for Leptospira infection. We show that Leptospira is able to efficiently infect the cell lung spheroids and also to infiltrate in deeper areas of the cell aggregates. The ability to infect the 3-D lung cell aggregates was time-dependent. The 3-D spheroids infection occurred up to 120 h in studies with two serovars, Canicola and Copenhageni. We standardized the number of bacteria in the initial inoculum for infection of the spheroids and we also propose two alternative culture media conditions. This new approach was validated by assessing the expression of three genes of Leptospira related to virulence and motility. The transcripts of these genes increased in both culture conditions, however, in higher rates and earlier times in the 3-D culture. We also assessed the production of chemokines by the 3-D spheroids before and after Leptospira infection, confirming induction of two of them, mainly in the 3-D spheroids. Chemokine CCL2 was expressed only in the 3-D cell culture. Increasing of this chemokine was observed previously in infected animal models. This new approach provides an opportunity to study the interaction of Leptospira with the human lung epithelium in vitro.

Crotoxin Modulates Events Involved in Epithelial-Mesenchymal Transition in 3D Spheroid Model.

Epithelial-mesenchymal transition (EMT) occurs in the early stages of embryonic development and plays a significant role in the migration and the differentiation of cells into various types of tissues of an organism. However, tumor cells, with altered form and function, use the EMT process to migrate and invade other tissues in the body. Several experimental (in vivo and in vitro) and clinical trial studies have shown the antitumor activity of crotoxin (CTX), a heterodimeric phospholipase A2 present in the Crotalus durissus terrificus venom. In this study, we show that CTX modulates the microenvironment of tumor cells. We have also evaluated the effect of CTX on the EMT process in the spheroid model. The invasion of type I collagen gels by heterospheroids (mix of MRC-5 and A549 cells constitutively prepared with 12.5 nM CTX), expression of EMT markers, and secretion of MMPs were analyzed. Western blotting analysis shows that CTX inhibits the expression of the mesenchymal markers, N-cadherin, α-SMA, and αv. This study provides evidence of CTX as a key modulator of the EMT process, and its antitumor action can be explored further for novel drug designing against metastatic cancer.

Crotoxin Inhibits Endothelial Cell Functions in Two- and Three-dimensional Tumor Microenvironment.

Antitumor property of Crotoxin (CTX), the major toxin from Crotalus durissus terrificus snake venom, has been demonstrated in experimental animal models and clinical trials. However, the direct action of this toxin on the significant events involved in neovascularization, which are essential for tumor growth and survival, has not been confirmed. This study investigated the effects of CTX on the key parameters of neovascularization in two- and three-dimensional culture models. Murine endothelial cell lines derived from thymus hemangioma (t.End.1) were treated at different concentrations of CTX (6.25-200 nM). Endothelial cell proliferation, cell adhesion, and actin cytoskeletal dynamics on laminin (10 µg/ml), type I collagen (10 µg/ml), and fibronectin (3 µg/ml) were evaluated along with the endothelial cell migration and formation of capillary-like tubes in 3D Matrigel. CTX concentration of 50 nM inhibited tube formation on 3D Matrigel and impaired cell adhesion, proliferation, and migration under both culture medium and tumor-conditioned medium. These actions were not accountable for the loss of cell viability. Inhibition of cell adhesion to different extracellular matrix components was related to the reduction of αv and α2 integrin distribution and cytoskeletal actin polymerization (F-actin), accompanied by inhibition of focal adhesion kinase (FAK), Rac1 (GTPase) signaling proteins, and actin-related protein 2/3 (Arp 2/3) complex. This study proved that CTX inhibits the major events involved in angiogenesis, particularly against tumor stimuli, highlighting the importance of the anti-angiogenic action of CTX in inhibition of tumor progression.

Crotoxin modulates events involved in epithelial–mesenchymal transition in 3D spheroid model

Epithelial–mesenchymal transition (EMT) occurs in the early stages of embryonic development and plays a significant role in the migration and the differentiation of cells into various types of tissues of an organism. However, tumor cells, with altered form and function, use the EMT process to migrate and invade other tissues in the body. Several experimental (in vivo and in vitro) and clinical trial studies have shown the antitumor activity of crotoxin (CTX), a heterodimeric phospholipase A2 present in the Crotalus durissus terrificus venom. In this study, we show that CTX modulates the microenvironment of tumor cells. We have also evaluated the effect of CTX on the EMT process in the spheroid model. The invasion of type I collagen gels by heterospheroids (mix of MRC-5 and A549 cells constitutively prepared with 12.5 nM CTX), expression of EMT markers, and secretion of MMPs were analyzed. Western blotting analysis shows that CTX inhibits the expression of the mesenchymal markers, N-cadherin, α-SMA, and αv. This study provides evidence of CTX as a key modulator of the EMT process, and its antitumor action can be explored further for novel drug designing against metastatic cancer.

Crotoxin inhibits endothelial cell functions in two- and three-dimensional tumor microenvironment

Antitumor property of Crotoxin (CTX), the major toxin from Crotalus durissus terrificus snake venom, has been demonstrated in experimental animal models and clinical trials. However, the direct action of this toxin on the significant events involved in neovascularization, which are essential for tumor growth and survival, has not been confirmed. This study investigated the effects of CTX on the key parameters of neovascularization in two- and three-dimensional culture models. Murine endothelial cell lines derived from thymus hemangioma (t.End.1) were treated at different concentrations of CTX (6.25–200 nM). Endothelial cell proliferation, cell adhesion, and actin cytoskeletal dynamics on laminin (10 µg/ml), type I collagen (10 µg/ml), and fibronectin (3 µg/ml) were evaluated along with the endothelial cell migration and formation of capillary-like tubes in 3D Matrigel. CTX concentration of 50 nM inhibited tube formation on 3D Matrigel and impaired cell adhesion, proliferation, and migration under both culture medium and tumor-conditioned medium. These actions were not accountable for the loss of cell viability. Inhibition of cell adhesion to different extracellular matrix components was related to the reduction of αv and α2 integrin distribution and cytoskeletal actin polymerization (F-actin), accompanied by inhibition of focal adhesion kinase (FAK), Rac1 (GTPase) signaling proteins, and actin-related protein 2/3 (Arp 2/3) complex. This study proved that CTX inhibits the major events involved in angiogenesis, particularly against tumor stimuli, highlighting the importance of the anti-angiogenic action of CTX in inhibition of tumor progression.

Elevated plasma levels of hepatocyte growth factor in rats experimentally envenomated with Bothrops jararaca venom: role of snake venom metalloproteases

The hepatocyte growth factor (HGF)/c-met pathway, which mainly consists of HGF activator (HGFA) and its substrate HGF, protects various types of cells via anti-apoptotic and anti-inflammatory signals. Thrombin is the main physiological activator of such plasmatic pathway, and increased plasma concentrations of HGF have been considered as a molecular marker for some pathological conditions, such as disseminated intravascular coagulation. Since thrombin generation is often linked to tissue injury, and these events are common during snake venom-induced consumption coagulopathies (VICC), our goals were to examine whether Bothrops jararaca venom (Bjv), which induces VICC in vivo: (i) activates the HGF/c-met pathway in vivo and (ii) cleaves zymogen forms of HGFA and HGF (proHGFA and proHGF, respectively) in vitro. Two experimental groups (n = 6, each) of male adult Wistar rats were subcutaneously injected with 500?µL of 0.9% NaCl solution (control) or sub-lethal doses (1.6 mg/kg) of Bjv. Three hours after envenomation, whole blood samples were collected from the carotid arteries to evaluate relevant coagulation parameters using rotational thromboelastometry and fibrinogen level (colorimetric assay). Additionally, the plasma concentration of HGF was assayed (ELISA). Thromboelastometric assays showed that blood clotting and fibrin polymerization were severely impaired 3 h after Bjv injection. Total plasma HGF concentrations were almost 6-fold higher in the Bjv-injected group (410.0 ± 91) compared with control values (68 ± 18 pg/mL, p < 0.05). Western blotting assay showed that Bjv processed proHGFA and proHGF, generating bands resembling those generated by thrombin and kallikrein, respectively. In contrast to the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), the metalloprotease inhibitor ethylenediaminetetraacetic acid disodium salt (Na2-EDTA) strongly reduced the ability of Bjv to process proHGFA and generated one active band similar to that of thrombin. Since Bjv contains prothrombin and factor X activators, increased intravascular thrombin formation might partly explain the increased HGF levels after bothropic envenomation. In conclusion, these findings suggest that snake venom metalloproteases may be determinant for elevation of plasma levels of HGF in rats experimentally envenomated with Bjv.

Elevated plasma levels of hepatocyte growth factor in rats experimentally envenomated with Bothrops jararaca venom: role of snake venom metalloproteases

The hepatocyte growth factor (HGF)/c-met pathway, which mainly consists of HGF activator (HGFA) and its substrate HGF, protects various types of cells via anti-apoptotic and anti-inflammatory signals. Thrombin is the main physiological activator of such plasmatic pathway, and increased plasma concentrations of HGF have been considered as a molecular marker for some pathological conditions, such as disseminated intravascular coagulation. Since thrombin generation is often linked to tissue injury, and these events are common during snake venom-induced consumption coagulopathies (VICC), our goals were to examine whether Bothrops jararaca venom (Bjv), which induces VICC in vivo: (i) activates the HGF/c-met pathway in vivo and (ii) cleaves zymogen forms of HGFA and HGF (proHGFA and proHGF, respectively) in vitro. Two experimental groups (n = 6, each) of male adult Wistar rats were subcutaneously injected with 500?µL of 0.9% NaCl solution (control) or sub-lethal doses (1.6 mg/kg) of Bjv. Three hours after envenomation, whole blood samples were collected from the carotid arteries to evaluate relevant coagulation parameters using rotational thromboelastometry and fibrinogen level (colorimetric assay). Additionally, the plasma concentration of HGF was assayed (ELISA). Thromboelastometric assays showed that blood clotting and fibrin polymerization were severely impaired 3 h after Bjv injection. Total plasma HGF concentrations were almost 6-fold higher in the Bjv-injected group (410.0 ± 91) compared with control values (68 ± 18 pg/mL, p < 0.05). Western blotting assay showed that Bjv processed proHGFA and proHGF, generating bands resembling those generated by thrombin and kallikrein, respectively. In contrast to the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), the metalloprotease inhibitor ethylenediaminetetraacetic acid disodium salt (Na2-EDTA) strongly reduced the ability of Bjv to process proHGFA and generated one active band similar to that of thrombin. Since Bjv contains prothrombin and factor X activators, increased intravascular thrombin formation might partly explain the increased HGF levels after bothropic envenomation. In conclusion, these findings suggest that snake venom metalloproteases may be determinant for elevation of plasma levels of HGF in rats experimentally envenomated with Bjv.

Interação tumor-estroma durante a transição epitéliomesenquimal in vitro: envolvimento da integrina a11b1 e modulação da Crotoxina sobre este processo / Tumor-stroma interaction during in vitro epithelialmesenchymal transition: involvement of integrin a11 B1 and modulatory effect of Crotoxin on this process

During tumor progression, tumor cells are regulated by the microenvironment and acquire highly proliferative behavior within the epithelial compartment, having as characteristic the high motility and invasiveness to the adjacent stroma! tissue. This behavior is a result of the phenotypic alteration of epithelial tumor cells, due to a biological process called epithelial-rnesenchymal transition (EMT). ln this process, fibroblasts are pointed out as fundamental elements in the EMT induction, performing their functions via integrins. Recently, a11 has been shown to be a type I collagen receptor of mesenchyme-derived cells, particularly fibroblasts, which promote cell adhesion and migration to this substrate and may also contribute to the invasion of tumor cells, making it thus, an important target of study. Components obtained from snake venoms modulate integrin-mediated functions, resulting in modulation of cell signaling and consequently regulation of events involved with tumor progressions, such as proliferation, migration, and apoptosis. The aim of this study was 1) to evaluate in vitro the role of a11 integrin on EMT and its importance in the phenotype stabilization; 2) to evaluate the possible inhibitory effect of CTX on the function of this integrin in fibroblasts in spheroid model and; 3) to evaluate the modulatory effect of CTX on key markers in EMT in spheroid model. Firstly, epithelial cell lines (NMuMG and A549) treated with EMT-inducing factors were used to evaluate the expression of epithelial and mesenchymal markers. Secondly, the experimental model used was in vitro interaction between cancer-associated fibroblasts (CAFs-mock or CAF-a11) or normal fibroblasts (MRC-5) and tumor cell lines (A549 or Calu-3), evaluating: a) tumor cells proliferation inside the spheroids; b) invasion and migratory behavior of composite spheroids in type I collagen gel; c) secretion of TGF-ß1 by CAFs; d) expression of EMT markers: E-cadherin, N-cadherin, vimentin and a-SMA, by immunofluorescence assay and Western blotting; e) quantification of MMPs (9 and 13) by ELISA. Taken together, the data obtained allow us to assert that a11 integrin is modulated by growth factors involved with EMT and that its participation was fundamental for the stabilization of the myofibroblastic phenotype. CTX interfered with CAFs adhesion, tumor cells proliferation and its inhibitory effect during migration and cell invasion is more significant when the tumor-stroma interaction occurs in the spheroid model, accompanied by inhibition of the secretion of active TGF-ß1. Furthermore, CTX inhibited differentiation of MRC-5 cells in the tumor microenvironment and inhibited the expression of N-cadherin, a-SMA and av mesenchymal markers, evaluated in 2D or 3D matrices, by Western blot and confocal microscopy. This study shows CTX as an important modulatory molecule of the EMT process and highlights the mechanisms involved in the antitumor action described for CTX in experimental studies and clinical trials.

Durante a progressão tumoral, as células tumorais são reguladas pelo microambiente e adquirem comportamento altamente proliferativo dentro do compartimento epitelial, tendo como característica a alta motilidade e invasividade ao tecido estremai adjacente. Este comportamento é resultado da alteração fenotípica das células turnarais epiteliais, decorrente de um processo biológico denominado transição epitélio-mesenquimal (EMT). Neste processo, os fibroblastes são apontados como elementos fundamentais na indução da EMT, desempenhando suas funções via integrinas. Muito recentemente, foi demonstrado que a a11 é um receptor para colágeno tipo I de células derivadas do mesênquima, particularmente de fibroblastes, que promovem a adesão e migração celular a este substrato podendo, ainda, contribuir para a invasão das células turnarais, tornando-a assim, um importante alvo de estudo. Componentes obtidos de venenos de serpentes modulam funções mediadas por integrinas, resultando na modulação da sinalização celular e consequentemente, sobre a regulação de eventos envolvidos com a· progressão tumoral, tais como a proliferação, migração e apoptose. Portanto, este estudo tem como objetivos: 1) avaliar in vitro a participação da integri na a11 sobre a EMT e sua importância na estabilização do fenótipo; 2) avaliar o possível efeito inibitório da CTX sobre a função desta integrina em fibroblastes no modelo de esferóide e; 3) avaliar o efeito modulatório da CTX sobre os marcadores-chave na EMT, em modelo de esferóide. Para tanto, primeiramente, foi utilizado linhagens de células epiteliais (NMuMG e A549) tratadas com fatores indutores da EMT para avaliação da expressão dos marcadores epiteliais e mesenquimais. Para o segundo delineamento experimental, foi empregado a interação in vitro entre as linhagens de fibroblastos associados ao câncer (CAFs-mock ou CAF-a11) ou fibroblastos normais (MRC-5) e as linhagens de célula tumoral (A549 ou Calu-3) no modelo de esferóide, avaliando-se: a) a proliferação de células turnarais dentro dos esferóides; b) a invasão e o comportamento migratório dos esferóides compostos no gel de colágeno tipo I; c) a secreção de TGF-ß1 pelas CAFs; d) a expressão das moléculas-chave da EMT: E-caderina, N-caderina, vimentina e a-SMA, por meio do ensaio de imunofluorescência e de Western blotting; e) quantificação de MMPs (9 e 13) por ELISA. Em conjunto, os dados obtidos nos permite afirmar que a integrina a11 é modulada por fatores de crescimentos envolvidos com a EMT e que sua participação foi fundamental para estabilização do fenótipo miofibroblástico. CTX interferiu com a adesão de CAFs e proliferação das células tumorais. O efeito inibitório da CTX durante a migração e invasão celular foi mais significativo quando ocorreu a interação tumor-estroma no modelo de esferóide, acompanhado da inibição da secreção de TGF-ß1 ativo. Ainda, a CTX inibiu a diferenciação de células MRC-5 no microambiente tumoral e inibiu a expressão dos marcadores mesenquimais Ncaderina, a-SMA e av, avaliados em matrizes 20 ou 30, por Western blot e microscopia confocal. Este estudo mostra a CTX como importante molécula moduladora do processo EMT e evidencia os mecanismos envolvidos na ação antitumoral descrita para a CTX em estudos experimentais e ensaios clínicos.

Interação tumor-estroma durante a transição epitéliomesenquimal in vitro: envolvimento da integrina a11b1 e modulação da Crotoxina sobre este processo. / Tumor-stroma interaction during in vitro epithelialmesenchymal transition: involvement of integrin a11 B1 and modulatory effect of Crotoxin on this process.

During tumor progression, tumor cells are regulated by the microenvironment and acquire highly proliferative behavior within the epithelial compartment, having as characteristic the high motility and invasiveness to the adjacent stroma! tissue. This behavior is a result of the phenotypic alteration of epithelial tumor cells, due to a biological process called epithelial-rnesenchymal transition (EMT). ln this process, fibroblasts are pointed out as fundamental elements in the EMT induction, performing their functions via integrins. Recently, a11 has been shown to be a type I collagen receptor of mesenchyme-derived cells, particularly fibroblasts, which promote cell adhesion and migration to this substrate and may also contribute to the invasion of tumor cells, making it thus, an important target of study. Components obtained from snake venoms modulate integrin-mediated functions, resulting in modulation of cell signaling and consequently regulation of events involved with tumor progressions, such as proliferation, migration, and apoptosis. The aim of this study was 1) to evaluate in vitro the role of a11 integrin on EMT and its importance in the phenotype stabilization; 2) to evaluate the possible inhibitory effect of CTX on the function of this integrin in fibroblasts in spheroid model and; 3) to evaluate the modulatory effect of CTX on key markers in EMT in spheroid model. Firstly, epithelial cell lines (NMuMG and A549) treated with EMT-inducing factors were used to evaluate the expression of epithelial and mesenchymal markers. Secondly, the experimental model used was in vitro interaction between cancer-associated fibroblasts (CAFs-mock or CAF-a11) or normal fibroblasts (MRC-5) and tumor cell lines (A549 or Calu-3), evaluating: a) tumor cells proliferation inside the spheroids; b) invasion and migratory behavior of composite spheroids in type I collagen gel; c) secretion of TGF-ß1 by CAFs; d) expression of EMT markers: E-cadherin, N-cadherin, vimentin and a-SMA, by immunofluorescence assay and Western blotting; e) quantification of MMPs (9 and 13) by ELISA. Taken together, the data obtained allow us to assert that a11 integrin is modulated by growth factors involved with EMT and that its participation was fundamental for the stabilization of the myofibroblastic phenotype. CTX interfered with CAFs adhesion, tumor cells proliferation and its inhibitory effect during migration and cell invasion is more significant when the tumor-stroma interaction occurs in the spheroid model, accompanied by inhibition of the secretion of active TGF-ß1. Furthermore, CTX inhibited differentiation of MRC-5 cells in the tumor microenvironment and inhibited the expression of N-cadherin, a-SMA and av mesenchymal markers, evaluated in 2D or 3D matrices, by Western blot and confocal microscopy. This study shows CTX as an important modulatory molecule of the EMT process and highlights the mechanisms involved in the antitumor action described for CTX in experimental studies and clinical trials.

Durante a progressão tumoral, as células tumorais são reguladas pelo microambiente e adquirem comportamento altamente proliferativo dentro do compartimento epitelial, tendo como característica a alta motilidade e invasividade ao tecido estremai adjacente. Este comportamento é resultado da alteração fenotípica das células turnarais epiteliais, decorrente de um processo biológico denominado transição epitélio-mesenquimal (EMT). Neste processo, os fibroblastes são apontados como elementos fundamentais na indução da EMT, desempenhando suas funções via integrinas. Muito recentemente, foi demonstrado que a a11 é um receptor para colágeno tipo I de células derivadas do mesênquima, particularmente de fibroblastes, que promovem a adesão e migração celular a este substrato podendo, ainda, contribuir para a invasão das células turnarais, tornando-a assim, um importante alvo de estudo. Componentes obtidos de venenos de serpentes modulam funções mediadas por integrinas, resultando na modulação da sinalização celular e consequentemente, sobre a regulação de eventos envolvidos com a· progressão tumoral, tais como a proliferação, migração e apoptose. Portanto, este estudo tem como objetivos: 1) avaliar in vitro a participação da integri na a11 sobre a EMT e sua importância na estabilização do fenótipo; 2) avaliar o possível efeito inibitório da CTX sobre a função desta integrina em fibroblastes no modelo de esferóide e; 3) avaliar o efeito modulatório da CTX sobre os marcadores-chave na EMT, em modelo de esferóide. Para tanto, primeiramente, foi utilizado linhagens de células epiteliais (NMuMG e A549) tratadas com fatores indutores da EMT para avaliação da expressão dos marcadores epiteliais e mesenquimais. Para o segundo delineamento experimental, foi empregado a interação in vitro entre as linhagens de fibroblastos associados ao câncer (CAFs-mock ou CAF-a11) ou fibroblastos normais (MRC-5) e as linhagens de célula tumoral (A549 ou Calu-3) no modelo de esferóide, avaliando-se: a) a proliferação de células turnarais dentro dos esferóides; b) a invasão e o comportamento migratório dos esferóides compostos no gel de colágeno tipo I; c) a secreção de TGF-ß1 pelas CAFs; d) a expressão das moléculas-chave da EMT: E-caderina, N-caderina, vimentina e a-SMA, por meio do ensaio de imunofluorescência e de Western blotting; e) quantificação de MMPs (9 e 13) por ELISA. Em conjunto, os dados obtidos nos permite afirmar que a integrina a11 é modulada por fatores de crescimentos envolvidos com a EMT e que sua participação foi fundamental para estabilização do fenótipo miofibroblástico. CTX interferiu com a adesão de CAFs e proliferação das células tumorais. O efeito inibitório da CTX durante a migração e invasão celular foi mais significativo quando ocorreu a interação tumor-estroma no modelo de esferóide, acompanhado da inibição da secreção de TGF-ß1 ativo. Ainda, a CTX inibiu a diferenciação de células MRC-5 no microambiente tumoral e inibiu a expressão dos marcadores mesenquimais Ncaderina, a-SMA e av, avaliados em matrizes 20 ou 30, por Western blot e microscopia confocal. Este estudo mostra a CTX como importante molécula moduladora do processo EMT e evidencia os mecanismos envolvidos na ação antitumoral descrita para a CTX em estudos experimentais e ensaios clínicos.

Crotoxin-treated macrophages stimulate ROS production and killing activity in co-cultured neutrophils

Crotoxin (CTX), the predominant toxin in Crotalus durissus terrificus snake venom (CdtV), has anti-inflammatory and immunomodulatory effects. Despite its inhibitory action on neutrophil migration and phagocytosis, CTX does not directly affect the production of reactive oxygen species (ROS) by the neutrophils. In contrast, it enhances the generation of reactive oxygen and nitrogen intermediates by macrophages. Given the importance of macrophage-neutrophil interactions in innate antimicrobial defense, the aim of this study was to investigate the effect of CTX on neutrophil ROS production and killing activity, either through CTX-treated macrophage co-culture or conditioned medium of CTX-treated macrophages. The results showed an important modulatory action of CTX on the neutrophil function as well as neutrophil-macrophage interactions, as demonstrated by the increased production of hydrogen peroxide, hypochlorous acid, nitric oxide and TNF- a , along with the increased fungicidal activity of neutrophils.

Crotoxin-Treated Macrophages Stimulate ROS Production and Killing Activity in co-cultured Neutrophils

Crotoxin (CTX), the predominant toxin in Crotalus durissus terrificus snake venom (CdtV), has anti-inflammatory and immunomodulatory effects. Despite its inhibitory action on neutrophil migration and phagocytosis, CTX does not directly affect the production of reactive oxygen species (ROS) by the neutrophils. In contrast, it enhances the generation of reactive oxygen and nitrogen intermediates by macrophages. Given the importance of macrophage-neutrophil interactions in innate antimicrobial defense, the aim of this study was to investigate the effect of CTX on neutrophil ROS production and killing activity, either through CTX-treated macrophage co-culture or conditioned medium of CTX-treated macrophages. The results showed an important modulatory action of CTX on the neutrophil function as well as neutrophil-macrophage interactions, as demonstrated by the increased production of hydrogen peroxide, hypochlorous acid, nitric oxide and TNF- a , along with the increased fungicidal activity of neutrophils.