Immunomodulatory actions and epigenetic alterations induced by proteases from Bothrops snake venoms in human immune cells

Toxicology in Vitro 61 (2019) 104586

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Immunomodulatory actions and epigenetic alterations induced by proteases from Bothrops snake venoms in human immune cells

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Danilo L. Menaldoa, , Tássia R. Costaa, Diego L. Ribeirob, Fabiana A. Zambuzia, ⁎ Lusânia M.G. Antunesa, Fabíola A. Castroa, Fabiani G. Frantza, Suely V. Sampaioa, a Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil b Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Snake venoms Bothrops Metalloprotease Serine protease Immunomodulation Epigenetics

The aim of this study was to evaluate the immunomodulatory effects of two toxins from Bothrops snake venoms (the P–I metalloprotease Batroxase and the thrombin-like serine protease Moojase) on human peripheral blood mononuclear cells (PBMC), also investigating changes in the expression of genes related to epigenetic alterations and their immunotherapeutic potential. After 24 h of PBMC stimulation, Batroxase (2 μg/mL) and Moojase (4 μg/ mL) increased some cytokine levels (including IL-6 and IL-10), but did not promote cell death processes (apoptosis/necrosis) or alterations in the global DNA methylation levels. Gene expression experiments (RTqPCR) showed that most of the genes with altered transcript levels encode enzymes that act on histones, such as acetyltransferases (HAT1), deacetylases (HDACs), methyltransferases (DOT1L) or demethylases (KDM5B), indicating that these toxins may alter gene regulation through epigenetic changes mainly related to histones and to methyl-CpG binding proteins (MECP2). Subsequently, the immunotherapeutic potential of these toxins was evaluated using in vitro cytotoxicity assays with NK cells and K562 leukemic cells. Both toxins were able to potentiate the NK cell cytotoxic effects against K562 tumor cells, and the effect of Batroxase was dependent on the concomitant stimulus with IL-2, whereas Moojase increased the NK cytotoxicity independently of IL-2. Thus, Batroxase and Moojase presented interesting immunomodulatory effects that could be explored for the development of new strategies in anticancer immunotherapies.

1. Introduction Envenomations by Bothrops snakes are characterized by the proteolytic, coagulant and hemorrhagic actions of venom toxins, including metalloproteases and serine proteases, which lead to local manifestations such as inflammation, pain, edema, cutaneous lesions, bleeding and tissue necrosis (Matsui et al., 2000; Kini, 2005; Cruz et al., 2009). Although most of these events are directly related to the action of certain toxins on specific molecules or tissues, numerous effects of the envenomations occur due to their actions on components of the immune system that lead to inflammatory processes, with involvement of leukocytes (neutrophils, monocytes and lymphocytes) and chemical mediators (cytokines and eicosanoids) (Teixeira et al., 2009). With that in mind, several recent studies have been investigating the immunomodulatory properties of different toxins from snake venoms (Menaldo et al., 2013, 2017; Costa et al., 2017; Burin et al., 2018; Cedro

et al., 2018; Sartim et al., 2018), aiming not only to better understand the physiological processes induced by these toxins in envenomations, but also to discover potential new agents for diverse applications, such as in anticancer therapy. In the last few years, clinical and scientific studies have been searching for ways to use our own immune system to fight diseases, which led to new discoveries and treatment strategies, known as immunotherapy, with excellent results against cancer (Latteyer et al., 2016; Abou-Shousha et al., 2016). The current immunotherapy used in the treatment of cancer has been developed in two main areas: the first is the use of antibodies capable of specifically recognizing tumor cells and redirecting an immune response against them, and the second is the use of immunomodulatory cytokines (Kontermann, 2012). The proinflammatory cytokine IL-2 (Proleukin®), for example, is currently used in the treatment of metastatic melanoma and renal cell carcinoma, inducing apoptosis of tumor cells by the activation of cytotoxic immune

⁎ Corresponding authors at: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida do Café, s/n, Monte Alegre, CEP 14040903, Ribeirão Preto, São Paulo, Brazil. E-mail addresses: [email protected] (D.L. Menaldo), [email protected] (S.V. Sampaio).

https://doi.org/10.1016/j.tiv.2019.06.020 Received 20 May 2019; Received in revised form 28 June 2019; Accepted 29 June 2019 Available online 02 July 2019 0887-2333/ © 2019 Elsevier Ltd. All rights reserved.

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mononuclear cells (PBMC), also investigating changes in the expression of genes related to epigenetic alterations. In addition, cytotoxicity assays using NK cells (effector cells) and K562 tumor cells (target cells) were performed to examine the potential of these toxins as possible molecules of interest in future anticancer immunotherapies.

cells, such as macrophages and natural killers (NK) (Vazquez-Lombardi et al., 2013). NK cells are large granular lymphocytes with effector functions in the innate immune system, acting as the first line of defense against tumor cells and intracellular pathogens, such as viruses and bacteria, without the need for prior recognition of a specific antigen (Lodoen and Lanier, 2006; Zwirner and Domaica, 2010; O'sullivan and Sun, 2015). In recent years, NK cells have emerged as key components of the immune response against tumor cells and thus became interesting targets for immunological intervention and immunotherapeutic approaches (Zwirner and Domaica, 2010). Considering that some Bothrops toxins have already been described as capable of modulating the action of different cells from the immune system, such as neutrophils, macrophages and mast cells (Menaldo et al., 2017), it is plausible to conceive that these toxins would also act on NK cells, which could result in positive immunomodulatory effects of interest in anticancer immunotherapy. In the last decades, the literature has shown that cancer, neurodegenerative, autoimmune and infectious diseases may arise or be aggravated due to epigenetic alterations, either by increasing the incidence of certain enzymes or by increasing the enzymatic activities (Portela and Esteller, 2010). Thus, the involvement of epigenetic mechanisms in the regulation of events that control the phenotypic characteristics of cells of the immune system, as well as their contribution to the immunopathology of several diseases, have been increasingly explored. In general, the term epigenetic refers to the mechanisms involved in the gene expression control without modifying the primary DNA nucleotide sequence of organisms and which are potentially inheritable throughout cell generations. Epigenetic mechanisms include chemical modifications in DNA and/or their associated histones, resulting in changes in the physical accessibility of DNA to transcriptional factors (Portela and Esteller, 2010; Carson et al., 2011). Such modifications can be grouped into three main categories: DNA methylation, histone modifications and nucleosome positioning (Portela and Esteller, 2010). In summary, the mechanisms involved in the epigenetic regulation of genes can be generalized as transcriptional activators or gene silencers. Although it is known that the methylation pattern of DNA can be influenced by environmental factors such as diet, drugs and toxins (Abdolmaleky et al., 2008; Machado et al., 2019), the evaluation of epigenetic alterations promoted by snake venom toxins is yet to be determined. In the present study, we used two known proteases from Bothrops snake venoms. The first one, Batroxase, is a P–I hemorrhagic metalloprotease from Bothrops atrox venom that presents molecular mass of 22.9 kDa and neutral pI (Cintra et al., 2012; Menaldo et al., 2015). This enzyme showed proteolytic activity on extracellular membrane constituents (such as type IV collagen and fibronectin), fibrin and fibrinogen, and pronounced thrombolytic activity both in vitro and in vivo (Cintra et al., 2012; Jacob-Ferreira et al., 2016). Batroxase also induced inflammatory processes in vivo, contributing to the formation of edema and hyperalgesia (De Toni et al., 2015), as well as stimulating the recruitment of leukocytes and the production of cytokines and eicosanoids (Menaldo et al., 2017). Furthermore, this metalloprotease was able to act on the human complement system in vitro, modulating its activation pathways, degrading components and generating anaphylatoxins (Menaldo et al., 2016). The second toxin, Moojase, was recently described by our research group (Amorim et al., 2018) as an isoform of Batroxobin, a thrombin-like serine protease from Bothrops moojeni venom described in the 1930s and widely used as a defibrinogenating agent (Serrano, 2013). Moojase is a glycoprotein with molecular mass of 30.3 kDa and acidic character, capable of coagulating platelet-poor plasma and fibrinogen solutions, besides promoting aggregation of washed platelets and fibrin(ogen)olytic effects (Amorim et al., 2018). In this context, our study aimed to evaluate the immunomodulatory effects of Batroxase and Moojase on human peripheral blood

2. Materials and methods 2.1. Venoms and toxins Bothrops atrox venom was acquired from the Center for Extraction of Animal Toxins (CETA, Morungaba, SP, Brazil), and Bothrops moojeni was provided by the Center for the Study of Venoms and Venomous Animals (CEVAP, Botucatu, SP, Brazil). Batroxase was isolated from B. atrox venom and Moojase from B. moojeni venom according to previously described methodologies (Menaldo et al., 2015; Amorim et al., 2018). After isolation, protein concentrations were determined using Bradford's reagent (Sigma-Aldrich) or BCA kit (Thermo Fischer Scientific), according to the manufacturers' recommendations. The purity levels of the toxins were confirmed by the electrophoretic profile on 12% polyacrylamide gel under denaturing and reducing conditions (Laemmli, 1970), and by the chromatographic profile on a C18 reverse phase column, as previously described (Menaldo et al., 2015; Amorim et al., 2018). 2.2. Human blood The peripheral blood used to obtain leukocytes was collected from 4 to 5 healthy volunteers between 20 and 40 years old, of both sexes, and who did not use medications for a minimum period of 10 days prior to collection. The blood was collected aseptically in Vacutainer tubes with heparin (BD) by venipuncture. All experimental protocols involving humans were properly approved by the Research Ethics Committee of the School of Pharmaceutical Sciences of Ribeirão Preto - University of São Paulo (CEP/FCFRP-USP protocol #404 – CAAE n° 55170816.0.0000.5403). 2.3. Isolation of mononuclear cells Peripheral blood mononuclear cells (PBMC) were isolated from the blood collected from the volunteers by using Histopaque-1077 separation medium (Sigma-Aldrich), following the manufacturer's recommendations. After centrifugation at 400 xg for 30 min and room temperature, the white cell layer (PBMC) was harvested and washed twice with phosphate buffered saline (PBS, pH 7.2). The cells were then resuspended in RPMI 1640 complete culture medium (supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin antibiotics and 0.024% NaHCO3) and counted in a Countess II FL automated cell counter (Invitrogen) before use in the experiments described next. 2.4. Production of mediators and cell death evaluation 2.4.1. PBMC culture To assess toxin-induced cytokine production and cell death, initially 5 × 105 mononuclear cells were added to each well of 24-well culture plates and incubated at 37 °C for 24 h in a 5% CO2 incubator. Then, cells were stimulated with PBS (negative control), cisplatin at 100 μg/mL (Incel, Darrow, positive control), or the toxins Batroxase or Moojase (1, 2 or 4 μg/mL) for another 24 h at 37 °C. After the incubation period, the culture plate was centrifuged at 400 xg for 10 min and the supernatants were removed and stored at −20 °C for subsequent cytokine dosage. Cells retained on the plate wells were then prepared for analysis of cell death by flow cytometry methodology. 2.4.2. Dosage of cytokines The cytokines IL-6, IL-1β, IL-10 and TNF-α were quantified in PBMC 2

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that encode enzymes that induce repression or activation of gene transcription through processes such as methylation/demethylation, acetylation/deacetylation of DNA and histones, among others. qPCR reactions were performed using 50 ng of cDNA for each gene analyzed and an equal volume of TaqMan® Fast Universal PCR Mastermix 2× (Applied Biosystems, cat. 4352042), in a StepOnePlus™ Real-Time PCR system (Applied Biosystems), according to manufacturer's instructions. The results were analyzed using the DataAssist v3.01 software (Applied Biosystems), and the limit value of the threshold cycle (Ct) was set at 40. Normalization was done by subtracting the Ct value of the gene of interest from the mean Ct value of the two housekeeping genes (HPRT1 and GUSB). The values obtained for the negative control (PBMC stimulated with PBS) were used as reference for comparison. The relative expression of each gene was calculated by fold change (2−ΔΔCt) (Pfaffl et al., 2002). Differences in gene expression were assessed using Student's t-test and the criteria for statistical significance were set at p < 0.05 and fold change ≥1.5 or ≤ 0.5 (Costa et al., 2018).

supernatants by enzyme-linked immunosorbent assay (ELISA), using specific biotinylated antibodies and cytokine standards, according to manufacturer's instructions (R&D Systems). Absorbance readings were done at 405 nm on a microplate reader (Spectramax190, Molecular Devices) and the concentration of cytokines was calculated from standard curves, expressing the results in pg/mL. 2.4.3. Cell death by apoptosis/necrosis PBMC were detached from culture plate wells by using Accutase (Sigma-Aldrich) for 5 min at 37 °C, transferred to FACS tubes and then kept on ice. Cell death induced by toxins was assessed with an apoptosis/necrosis kit (Invitrogen, cat. V13241), containing annexin V (AV) and propidium iodide (PI), following the manufacturer's recommendations. Analysis was done by flow cytometry, acquiring 30,000 events for each sample on a FACS Canto II (BD Biosciences) cytometer and analyzing the data by dot-plot using the FACSDiva v.6.1.3 software (BD Biosciences). Labeling of cells with AV and/or PI allowed to classify them as viable cells (AV−PI−), cells in apoptosis (AV+PI−), cells in necrosis (AV−PI+) or cells in late apoptosis (AV +PI+).

2.6. Functional evaluation of NK cells by cytotoxicity assays

2.5. Evaluation of epigenetic alterations

2.6.1. Isolation of NK cells from PBMC by magnetic separation Purification of NK cells from PBMC of 5 subjects (item 2.3.) was standardized for negative magnetic separation by using the NK Cell Isolation Kit, Human (Miltenyi Biotec, cat. 130-092-657) and MACS LS separation columns (Miltenyi Biotec, Cat. 130-042-401), according to the manufacturer's recommendations. Briefly, after cell counting, the PBMC suspension was centrifuged at 400 ×g, 4 °C for 10 min, the supernatant was discarded and the mononuclear cells resuspended in cold beads buffer (PBS, pH 7.2, containing 2 mM EDTA and 5% bovine serum albumin - BSA) in the proportion of 40 μL to every 107 cells. Then, 10 μL of human NK Cell Biotin-Antibody Cocktail solution was added for each 107 cells, followed by homogenization and incubation for 5 min in the refrigerator (2–8 °C). Then, 30 μL of cold beads buffer and 20 μL of NK Cell Microbead Cocktail were added to every 107 cells, incubating for another 10 min in the refrigerator (2–8 °C). After that period, 500 μL of cold beads buffer was added to the cell suspension, which was then transferred to the MACS LS column, collecting the fraction eluting first and containing the unlabeled cells (NK cells). The NK cell suspension was then centrifuged at 400 ×g, 4 °C for 10 min, the supernatant was discarded, and the NK cells were resuspended in RPMI 1640 complete culture medium and counted in a Countess II FL automated cell counter (Invitrogen).

2.5.1. Global DNA methylation analysis To assess the global DNA methylation levels of toxin-stimulated PBMC, 1 × 106 cells were added to wells of 12-well culture plates and incubated at 37 °C for 24 h in a 5% CO2 incubator. After this period, cells were stimulated with PBS (negative control) or the toxins Batroxase (2 μg/mL) or Moojase (4 μg/mL) for further 24 h at 37 °C. Then, the genomic DNA was extracted from cells by using the PureLink® Genomic DNA Mini Kit (Invitrogen, cat. K1820-01), following the manufacturer's recommendations. DNA methylation levels of control and toxin-stimulated groups were determined with the Imprint® Methylated DNA quantification kit (Sigma-Aldrich, cat. MDQ1), following the manufacturer's specifications. Absorbance reading was done at 450 nm on a microplate reader (Spectramax190, Molecular Devices) and the relative percentage of global methylation levels of samples was calculated using a methylated DNA standard provided by the kit, which quantifies 5-methyl-cytidine (5mC) content. 2.5.2. Gene expression analysis (RT-qPCR) To evaluate the expression of genes related to epigenetic changes in toxin-stimulated PBMC, 2 × 106 cells/well were grown in 12-well culture plates at 37 °C for 24 h in a 5% CO2 incubator. After this, cells were stimulated with PBS (negative control) or the toxins Batroxase (2 μg/ mL) or Moojase (4 μg/mL) for further 24 h at 37 °C. Then, the total RNA was extracted from cells by using the PureLink® RNA Mini Kit (Invitrogen, cat. 12183018A), according to the manufacturer's specifications. The amount and quality of the extracted RNA were determined by spectrophotometry (NanoDrop 2000, Thermo Scientific), considering the A260/280 and A260/230 absorbance ratios between 1.8 and 2.2. To eliminate contaminations with genomic DNA, RNA samples were treated with DNase I, amplification grade (1 U/μL; Invitrogen, cat. 18068015), according to the manufacturer's instructions. cDNA was then synthesized from 3 μg of the total RNA extracted by using the High-Capacity cDNA Reserve kit Transcription (Applied Biosystems, cat. 4368814), according to the manufacturer's specifications. Three distinct reactions of cDNA synthesis were performed for each sample, combining the resulting products to obtain a single mixture as described by Pfaffl et al. (2004) and Bustin and Mueller (2005). The resulting cDNAs were then used for gene quantification analysis by realtime polymerase chain reaction (RT-qPCR). In order to identify the gene expression profile of cells stimulated with the toxins, RT2 Profiler PCR Array plates (Applied Biosystems) were customized with constitutive genes, used as endogenous controls (housekeepings), and genes related to epigenetic alterations, i.e. genes

2.6.2. K562 cell culture K562 is a human tumor cell line from chronic myelogenous leukemia (CML) in blastic phase and was obtained from the American Type Culture Collection (ATCC CCL-243). Five days prior to the cytotoxicity assays, K562 cells were thawed, resuspended in RPMI 1640 complete medium and maintained in 75 cm2 cell culture flasks at approximately 1 × 105 cells/mL. 2.6.3. Cytotoxicity assay of NK cells in co-culture with K562 tumor cells Initially, purified NK cells were plated (1 × 105 cells in 150 μL of RPMI 1640 complete culture medium per well) in a 96-well U-bottom plate. In certain wells, 1 μL of IL-2 (Miltenyi Biotec, cat. 130-097-743) at 10 μg/mL was added to obtain a final concentration per well that was previously standardized to stimulate NK cells without causing significant cytotoxicity. Then, this plate was incubated for 48 h at 37 °C in a 5% CO2 incubator. After this period, toxins diluted in 50 μL of RPMI 1640 complete culture medium were added at the predefined concentrations (Batroxase at 2 μg/mL or Moojase at 4 μg/mL). Again, the plate with the samples was incubated for 24 h at 37 °C in a 5% CO2 incubator. Near the end of this incubation period, K562 cells were labeled with CFSE (carboxyfluorescein diacetate succinimidyl ester, BD Biosciences), 3

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a fluorescent cell marker which allows the detection of cells by flow cytometry. For each 1 × 106 K562 cells in 100 μL of PBS, 100 μL of a 4 μM CFSE solution (diluted in PBS) was added, followed by incubation at 37 °C for 10 min and protected from light. Then, 1.8 mL of RPMI 1640 complete medium was added and another incubation took place in an ice bath for 10 min. Thereafter, the labeled cells were washed and resuspended in RPMI complete medium for co-cultivation with the NK cells. For the cytotoxicity assay, NK cells were used as effector cells and the K562 tumor cells as target cells, using the NK:K562 ratio of 2:1 (this ratio was initially standardized to suit the purposes of the present study). Thus, 5 × 104 CFSE-labeled K562 cells were added to each well containing 1 × 105 NK cells, and these cells remained in direct coculture for 4 h at 37 °C in a 5% CO2 incubator. After this period, the different cell groups were transferred to distinct FACS tubes, adding 2 μL of 100 μg/mL PI solution (BD Biosciences) to each tube immediately prior to flow cytometry analysis. The events were acquired on a FACS Canto II flow cytometer (BD Biosciences), using the FACSDiva software v.6.1.3 (BD Biosciences) to record the data. In addition to the test groups containing NK and K562 cells with the toxins, in the presence or absence of IL-2, control groups were also evaluated, such as only NK cells with toxins or only K562 cells with the toxins, and both in the presence or absence of IL-2. Each group was assessed in duplicate for each of the 5 experiments conducted, with each experiment using PBMC from a different subject. According to this methodology, dead K562 target cells were labeled as CFSE+PI+, and the percentage of cytotoxicity was determined by relating the number of CFSE+PI+ cells to the total number of CFSE labeled cells. The cytotoxicity to NK cells was also determined by using gates and considering the relation between CFSE-PI+ cells and the total number of cells not labeled with CFSE.

In the presence of Moojase, the behavior of PBMC was different: there was an increase in cytokine levels at increasing concentrations of this toxin. In addition, only levels of IL-6 and IL-10 showed significant increases in relation to the controls of cells not stimulated with the toxin, although the results also showed a tendency of increase in IL-1β and TNF-α levels when higher concentrations of Moojase (4 μg/mL) were used (Fig. 1B). Regarding the cell viability of the monocytes and lymphocytes after stimulation with this toxin, no significant changes were observed (Fig. 2B), indicating that Moojase was not cytotoxic to PBMC at the concentrations evaluated. Thus, the concentration of 4 μg/ mL was chosen for the next experiments with Moojase. The increase in cytokine production by the toxins Batroxase and Moojase indicates that both have immunomodulatory effects on blood mononuclear cells, including monocytes and lymphocytes. This behavior of Batroxase on human leukocytes is consistent with what we had previously described using mice leukocytes (Menaldo et al., 2017). The effects observed for Moojase had not yet been investigated neither in vivo or in vitro, and although less expressive, indicate that this toxin is also related to the modulation of the action of immune cells, inducing increased levels of cytokines that promote recruitment of various types of leukocytes and production of other mediators, possibly amplifying the inflammatory responses. Cytokines are a large family of soluble proteins and glycoproteins that function as key modulators of the immune system, being responsible for the communication between cells and the regulation of all phases of the inflammatory response (Arend and Gabay, 2004). Activated leukocytes release a broad spectrum of cytokines, including: IL-6 and TNF-α, which increase leukocyte influx by stimulating maturation of polymorphonuclear cells and expression of adhesion molecules (Fasshauer et al., 2003; Moreira et al., 2012); IL-1β, which can stimulate lymphocytes and macrophages, besides regulating the synthesis and release of prostaglandins and chemokines (Rucavado et al., 2002; Cunha et al., 2008); and IL-10, which has regulatory effects related to the control of the inflammatory process, inhibiting the expression of pro-inflammatory cytokines (e.g. TNF-α and IL-1β), prostaglandins and nitric oxide synthase (Howard and O’Garra, 1992; Fukuhara et al., 2003; Hernández-Cruz et al., 2008). In the present study, the induction of cell death mechanisms (apoptosis and necrosis) was investigated to determine safe working concentrations to use the toxins Batroxase and Moojase as immunomodulatory agents without causing cytotoxicity on mononuclear cells. Toxins classified as proteases are not usually evaluated as possible cytotoxic agents because they usually present particular effects related to specific protein targets, such as coagulation cascade factors (for metallo and serine proteases) or basement membrane components of capillaries (for metalloproteases) (Serrano and Maroun, 2005; Escalante et al., 2006). However, it is worth remembering that exposure of cells to every toxin may trigger cell death processes depending on the concentration and duration of the stimulus. Thus, our data displayed that stimulation of PBMC with Batroxase at 2 μg/mL or Moojase at 4 μg/mL for 24 h were safe conditions to be used in the following experiments.

2.7. Statistical analysis Data were expressed as mean values ± SEM. Statistical variations were analyzed using one-way ANOVA and Tukey's post-test or Student's t-test, and were calculated by the GraphPad Prism 5 software or the DataAssist v3.01 software (Applied Biosystems). Values of p < 0.05 were considered as statistically significant. 3. Results and discussion 3.1. Cytokine production and evaluation of cell death mechanisms After isolation of the toxins Batroxase and Moojase, our first step was to determine concentrations of each of the toxins capable of inducing immunomodulatory effects without causing cytotoxicity. For this, PBMC from 4 individuals were stimulated for 24 h at 37 °C with 3 concentrations of each toxin: 1, 2 and 4 μg/mL. After incubation, cell supernatants were used to determine the concentration of pro- or antiinflammatory cytokines (Fig. 1), while cell viability was investigated using cell death markers (annexin V and PI), which indicate mechanisms of death by apoptosis and/or necrosis (Fig. 2). The results presented in Fig. 1A showed that Batroxase, at all concentrations evaluated, was able to stimulate the mononuclear cells to produce different cytokines (IL-6, IL-1β, IL-10 and TNF-α), in general with higher levels of cytokines produced in the presence of lower concentrations of the toxin (1 and 2 μg/mL). This inversely proportional relationship between toxin concentration and cytokine levels can be explained by that observed in Fig. 2A, which shows that Batroxase, at the highest concentration evaluated (4 μg/mL), induced a significant reduction in the cell viability of monocytes, with a consequent increase in the number of cells in apoptosis. Thus, the concentration of 2 μg/mL of Batroxase was chosen for the subsequent assays of the present study, since this concentration was able to modulate the activity of the mononuclear cells without causing significant cytotoxicity.

3.2. Gene expression and epigenetic alterations Considering that DNA methylation is one of the main epigenetic mechanisms for regulating the gene expression (Portela and Esteller, 2010), we initiated our studies to evaluate epigenetic modifications investigating variations in global methylation levels in the genomic DNA of mononuclear cells stimulated with the toxins Batroxase or Moojase (Fig. 3). As in the previous assays, PBMC from 4 individuals were stimulated for 24 h with the toxins at the selected concentrations (Batroxase at 2 μg/mL and Moojase at 4 μg/mL), and then the methylation percentages of the extracted DNAs were determined by ELISA, using a methylation control as reference. As shown in Fig. 3, there were no statistical differences between the groups, which indicates that the stimulation of mononuclear cells with these toxins for 24 h did not 4

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Fig. 1. Dosage of cytokines by ELISA. The concentrations of the cytokines IL-6, IL-1β, IL-10 and TNF-α were determined in supernatants of PBMC stimulated with Batroxase (A) or Moojase (B) for 24 h, by using specific ELISA kits (R&D Systems). Data expressed as mean ± SEM (n = 4). Statistical differences (ANOVA followed by Tukey's post-test) in relation to the control cells (shown as 0 on the x-axis) were indicated by *p < 0.05, **p < 0.01 or ***p < 0.001.

comparison to untreated cells (negative controls normalized to the fold change value of 1.0). GUSB and HPRT1 housekeeping genes were evaluated as endogenous controls and showed levels of expression in the toxin-stimulated cells very close to those obtained for the negative control cells (fold change values close to 1.0), thus validating these gene expression experiments. The transcriptional levels of a gene within a cell can be altered by several conditions, such as the cell cycle phase or exposure to stimuli such as drugs, cytokines or toxins (Moura et al., 2014). The results in Table 1 showed that the expression of various genes in PBMC stimulated with Batroxase or Moojase was altered in comparison to the control group. We could also observe a large variation between samples of different individuals in the expression of some genes, which highlighted the importance of establishing criteria to determine the significance of these results. Thus, according to the criteria described in Section 2.5.2., 14 of the 44 evaluated genes had their expression levels

promote any significant changes in the global DNA methylation levels under the conditions evaluated. Although Batroxase and Moojase at the concentrations evaluated did not significantly alter the global DNA methylation levels of mononuclear cells, this does not necessary mean that they cannot induce methylation/demethylation of specific genes. In addition, there are other epigenetic mechanisms in which they may be involved, such as histone modifications. To evaluate these possibilities, our next goal was to evaluate changes in the expression of genes related to epigenetic alterations in PBMC of 4 individuals stimulated with Batroxase (2 μg/ mL) or Moojase (4 μg/mL) for the same period of 24 h. Using the qPCR array technique and custom plates, 44 genes encoding enzymes with functions related to the activation or repression of gene transcription, such as methyltransferases, acetyltransferases, demethylases, deacetylases, among others, were evaluated. Table 1 lists all the assessed genes and the fold change obtained for cells stimulated with each toxin in

Fig. 2. Evaluation of cell death mechanisms by flow cytometry. PBMC were stimulated for 24 h with PBS (NC, negative control), cisplatin (100 μg/mL, PC, positive control) or 3 concentrations of each toxin: Batroxase and Moojase. After that, cells were labeled with annexin V (AV) and propidium iodide (PI) and evaluated by flow cytometry, using gates for monocytes (A) and lymphocytes (B). According to the labeling, cells were classified as viable (AV−PI−), in apoptosis (AV+PI−), in necrosis (AV−PI+) or in late apoptosis (AV+PI+). Data expressed as mean ± SEM (n = 4). Statistical differences (ANOVA followed by Tukey's post-test) in relation to the negative control (NC) were indicated by *p < 0.05, **p < 0.01 or ***p < 0.001. 5

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Table 1 Relative expression of genes associated with epigenetic changes in PBMC after stimulation with Batroxase or Moojase for 24 h.

Fig. 3. Evaluation of global DNA methylation levels by ELISA. PBMC were stimulated for 24 h with PBS (NC, negative control), Batroxase (2 μg/mL) or Moojase (4 μg/mL). After that, the genomic DNA of cells was extracted and the DNA methylation levels were quantified by using an ELISA kit (Sigma-Aldrich). The methylation percentages of each sample were calculated in relation to a control of methylated DNA provided by the kit (PC, positive control), considered as 100%. Data expressed as mean ± SEM (n = 4). No statistical differences (ANOVA followed by Tukey's post-test) were observed in relation to the negative control (NC).

significantly increased or repressed after 24 h of stimulation of PBMC with the toxins. Batroxase promoted increased expression of DOT1L, EHMT2, HAT1, KAT5, KDM5B, HDAC2, HDAC5, MECP2, SAP18, SAP30, SIN3A and TRDMT1 genes, and down regulated the gene MBD3, whereas Moojase up regulated the expression of DOT1L, HAT1, KDM5B, HDAC3 and MECP2 genes. These results demonstrate that both toxins, especially Batroxase, can influence the expression of genes related to epigenetic alterations, including genes encoding enzymes responsible for the activation or repression of gene transcription. DNA methylation is one of the main mechanisms of transcriptional activation or gene silencing. The main functional consequence of DNA methylation is a modification in the compaction degree of chromatin, which hinders the access of the cell transcription machinery to the DNA, inhibiting the gene expression (Orphanides and Reinberg, 2002). Chromatin compaction requires the combined activities of several classes of enzymes, including (I) DNA methyltransferases (DNMTs), which specifically methylate DNA cytosines to produce 5-methylcytosines; (II) histone acetyltransferases (HATs) and histone deacetylases (HDACs), which acetylate and deacetylate, respectively, lysine residues in histones; (III) histone methyltransferases (HMTs), which methylate lysine or arginine residues in histones; and (IV) histone demethylases (KDMs), which operate by hydroxylation of a methyl group, followed by dissociation of a formaldehyde. All these classes of enzymes have crucial functions in the establishment and maintenance of DNA methylation patterns and in the epigenetic status (Vaquero et al., 2003; Bombail et al., 2004; Klose and Zhang, 2007). Methyl-CpG binding proteins (MBDs) also have important functions in gene silencing, specifically recognizing and binding to methylated regions of DNA, and then forming complexes with other proteins, such as histone deacetylases, to direct the chromatin remodeling (Klose and Bird, 2006). Genes that presented altered transcriptional levels after PBMC stimulation with Batroxase or Moojase encode enzymes that can be divided into classes according to the functions they exert on DNA or histones. Table 2 shows that most of the genes that had their expression influenced by the toxins encode enzymes that promote histone modifications, such as acetyltransferases, deacetylases, methyltransferases or demethylases. These results are in agreement with those concerning global DNA methylation and the absence of significant differences between the control cells and the toxin-stimulated cells (Fig. 3). In fact, the only gene that had its expression enhanced by Batroxase and encodes an enzyme that promotes DNA methylation was TRDMT1, which is not a DNA methyltransferase itself. Initially called DNMT2, this enzyme had its name changed to TRDMT1 (tRNA aspartic acid

Batroxase (2 μg/mL)

Moojase (4 μg/mL)

Genes

FC

p-value

FC

p-value

Activation of gene transcription ASH1L (Hs00218516_m1) ATF2 (Hs01095345_m1) CARM1 (Hs00406354_m1) DOT1L (Hs01579928_m1) EHMT2 (Hs00198710_m1) HAT1 (Hs00186320_m1) KAT2A (Hs00904943_gH) KAT2B (Hs00187332_m1) KAT5 (Hs00197310_m1) KDM1A (Hs01002741_m1) KDM5B (Hs00981910_m1) KDM6B (Hs00996325_g1) KMT2C (Hs01005521_m1) KMT2E (Hs01096121_m1) PRMT5 (Hs01047345_g1) SETD1B (Hs00324585_m1) SMYD3 (Hs01585866_m1) SUV39H1 (Hs00957892_m1)

1.6883 1.6539 1.4939 1.7805 2.0587 1.8315 1.6987 1.6301 1.9052 1.6576 1.9553 1.7244 1.6912 1.6345 1.6760 1.6518 1.6955 1.2010

0.0606 0.0627 0.2471 0.0300 0.0129 0.0490 0.0573 0.0748 0.0344 0.0611 0.0352 0.0561 0.0557 0.0805 0.0637 0.0676 0.0518 0.4423

1.5095 1.3104 1.1939 1.7861 1.7657 2.0348 1.3638 1.4513 1.4764 1.3640 1.8590 1.5144 1.3133 1.4012 1.3448 1.3245 1.2753 0.9456

0.1895 0.3220 0.6097 0.0264 0.1772 0.0469 0.2690 0.1662 0.1943 0.2699 0.0402 0.1441 0.3515 0.2478 0.3033 0.3507 0.3166 0.7878

Repression of gene transcription CHD4 (Hs00172349_m1) DNMT1 (Hs00945875_m1) DNMT3A (Hs01027162_m1) DNMT3B (Hs00171876_m1) HDAC1;A2M (Hs02621185_s1) HDAC2 (Hs00231032_m1) HDAC3 (Hs00187320_m1) HDAC4 (Hs01041648_m1) HDAC5 (Hs00608351_m1) HDAC6 (Hs00997427_m1) HDAC7 (Hs01045864_m1) HDAC8 (Hs00954353_g1) HDAC9 (Hs01081558_m1) HDAC10 (Hs00368899_m1) HDAC11 (Hs00978031_g1) HMBS (Hs00609296_g1) MBD2 (Hs00969366_m1) MBD3 (Hs00922219_m1) MECP2 (Hs04187588_m1) RBBP4 (Hs01568507_g1) RBBP7 (Hs00171476_m1) RPLP0 (Hs00420895_gH) SAP18 (Hs00705532_s1) SAP30 (Hs01009153_g1) SIN3A (Hs00411592_m1) TRDMT1 (Hs00189402_m1)

1.3900 1.2668 1.1218 1.1769 1.4910 1.5033 1.4437 1.6668 1.7610 1.9937 1.4207 1.2924 0.8674 1.5432 1.7560 1.0796 0.6862 0.2659 5.8419 1.7445 1.6506 1.4108 1.6966 1.5974 1.6762 2.3512

0.0083 0.1334 0.5727 0.7022 0.1045 0.0433 0.1913 0.0630 0.0461 0.0644 0.0301 0.3848 0.5219 0.1704 0.2815 0.5662 0.0991 0.0113 0.0142 0.0550 0.0657 0.4373 0.0150 0.0002 0.0036 0.0283

1.4294 1.7953 0.9306 1.0197 1.3373 0.9177 1.6561 1.2299 1.6859 1.3367 1.0890 1.4722 0.7732 1.4188 1.5128 0.8063 0.7497 0.7594 3.0925 1.4927 1.4364 0.9610 1.4352 1.6710 1.7979 1.9527

0.0879 0.1019 0.7337 0.9552 0.1608 0.6783 0.0152 0.3899 0.2795 0.2942 0.6582 0.1319 0.2833 0.2710 0.4300 0.3159 0.2831 0.5968 0.0191 0.2538 0.2937 0.9179 0.2670 0.1285 0.0860 0.1979

Fold change (FC) values were obtained by 2−ΔΔCt method and the results normalized using GUSB and HPRT1 housekeeping genes. The relative expression of each gene was analyzed in PBMC from 4 individuals (n = 4). Bold highlighted values fit the established criteria for statistical significance: t-test p < 0.05 and FC ≥1.5 (up regulation) or ≤ 0.5 (down regulation).

methyltransferase 1) since, despite its high similarity with 5-methylcytosine methyltransferases, it only had residual DNA methyltransferase activity, preferentially acting on aspartic acid transfer RNAs (Goll et al., 2006). Therefore, we can suggest that none of the evaluated genes encoding enzymes that induce DNA methylation had altered expression after PBMC stimulation with Batroxase or Moojase. On the other hand, both proteases induced increased expression of at least one representative gene from each of the enzyme classes that act on histones (Table 2), which indicates that these two toxins can alter gene regulation through histone-related epigenetic changes. In addition to increasing the expression of histone methyltransferases, acetyltransferases, deacetylases and demethylases, the highest fold change values after PBMC stimulation with Batroxase or Moojase were observed for the MECP2 gene (Table 1), which encodes a

6

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Table 2 Classification of genes with altered expression after PBMC stimulation with Batroxase or Moojase for 24 h. Class DNA methyltransferases Histone acetyltransferases Histone methyltransferases Histone deacetylases

Histone demethylases Methyl-CpG binding proteins

a b

Symbol a

TRDMT1 HAT1a,b KAT5a DOT1La,b EHMT2a HDAC2a HDAC3b HDAC5a SAP18a SAP30a SIN3Aa KDM5Ba,b MECP2a,b MBD3a

Full name

Transcriptional effects

tRNA aspartic acid methyltransferase 1 histone acetyltransferase 1 lysine acetyltransferase 5 DOT1 like histone lysine methyltransferase euchromatic histone lysine methyltransferase 2 histone deacetylase 2 histone deacetylase 3 histone deacetylase 5 Sin3A associated protein 18 Sin3A associated protein 30 SIN3 transcription regulator family member A lysine demethylase 5B methyl-CpG binding protein 2 methyl-CpG binding domain protein 3

Repression Activation Activation Repression

Repression Repression

Transcript levels altered by Batroxase. Transcript levels altered by Moojase.

stimulating cytokine). After this period, CFSE-labeled K562 tumor cells were co-cultured with NK cells in the ratio 2:1 (NK:K562) for 4 h at 37 °C. Subsequently, the dead cells were labeled by the addition of PI and the analyses were then performed by flow cytometry. In vitro cytotoxicity assays using NK and K562 cells are based on the cytotoxic properties of NK cells (used as effector cells) on tumor cells (used as target cells) (Zamai et al., 1998). NK cells can lyse a variety of tumor cells by exocytosis of perforins and granzymes, which lead to the formation of lytic pores in the target cell membranes. Some tumor cells are less susceptible to the cell death mediated by NK cells because they prevent the binding of perforins to their cell membranes, and therefore, such cytotoxicity assays often include the activation of NK cells by stimulatory cytokines, such as IL-2 and IL-12 (Lehmann et al., 2000, 2001). In the present study, we used stimuli with IL-2 and/or the toxins to compare the effects and infer possible mechanisms of cytotoxicity. Fig. 4A shows that the death percentage of K562 cells in the presence of only toxins and/or IL-2, at the conditions assessed, is low (~3–4%). After incubation of K562 cells with NK cells, in the absence of IL-2 or toxins, there was a significant increase in cytotoxicity (~28%), as expected (Fig. 4A). NK cells previously stimulated with Moojase had a greater cytotoxic effect on tumor cells (~39%), a significant difference compared to the NK + K562 group without toxins, while the same could not observed for Batroxase, which induced about 30% cytotoxicity on K562 cells (Fig. 4A). These results indicate that Moojase by itself, at the concentration of 4 μg/mL, potentiated the cytotoxic effect of NK cells. Next, we evaluated the cytotoxicity of NK cells after stimulation with IL-2, alone or together with each of the toxins. Cytotoxicity for the K562 + NK + IL-2 group (~45%) was significantly higher in comparison to that of the K562 + NK group (~28%) (Fig. 4A). NK cells stimulated concomitantly with IL-2 and Batroxase (K562 + NK + IL2 + Batroxase) showed even higher cytotoxicity on K562 cells (~58%) and significantly superior than that of groups K562 + NK (~28%), K562 + NK + Batroxase (~30%) and K562 + NK + IL-2 (~45%), whereas stimulation with IL-2 and Moojase (K562 + NK + IL2 + Moojase) resulted in significantly higher cytotoxicity (~51%) compared to that of groups K562 + NK (~28%) and K562 + NK + Moojase (~39%) (Fig. 4A). As a secondary evaluation, we also determined the death percentage of the NK cells themselves (Fig. 4B), in order to verify the viability of these effector cells during the period of the experiment (> 72 h) and in the presence of different variables (IL-2, toxins). The percentage of dead NK cells in the presence of only toxins and/or IL-2 overall ranged from 2 to 5%. After the co-cultivation period with K562 cells, with or without toxins, this same range of cytotoxicity was maintained, and in the presence of IL-2 (K562 + NK + IL-2 group without toxins), only a small

protein of the same name that is part of the family of methyl-CpG binding proteins (MBDs). These proteins can recognize methylated DNA and mediate gene silencing through the recruitment of chromatin remodeling complexes and histone deacetylases (Klose and Bird, 2006). MECP2 has two domains, a methyl-binding (MBD) and a transcriptional repression (TRD) domain. The MBD domain binds to the methylated CpG sites in the DNA strands, and then the TRD domain reacts with SIN3A to recruit histone deacetylases (HDACs) (Wakefield et al., 1999; Dhasarathy and Wade, 2008). Considering that Batroxase induced increased expression not only of the MECP2 gene, but also of the genes HDAC2, HDAC5 and SIN3A, it is possible that the proteins encoded by these genes could play important roles in silencing certain genes after PBMC stimulation with this toxin. Batroxase also changed the transcriptional levels of another gene encoding a MBD family protein: MBD3, which was down regulated in relation to the negative control. MBD3 is the only member of the family that does not specifically bind to methylated DNAs but can bind to unmethylated or 5-hydroxymethylated DNAs due to the alteration of two amino acid residues in its MBD domain (Saito and Ishikawa, 2002). This protein is an essential subunit of the nucleosome remodeling deacetylase complex (NuRD) involved in chromatin remodeling (Saito and Ishikawa, 2002; Le Guezennec et al., 2006; Du et al., 2015). The fact that the PBMC stimulation with Batroxase promoted inverse effects on the expression of the genes MECP2 and MBD3, increasing the transcriptional levels of the first and decreasing the expression of the second, is an unusual result since they belong to the same protein family. However, considering the differences between MECP2 and MBD3 discussed above, it is possible that the stimulus with this toxin could somehow decrease the availability of MBD3 to prevent the formation of NuRD complexes that would repress important genes for the cells. There is also the possibility that this decreased expression is related to deleterious effects on the cells, since there are reports that decreased MBD3 levels are related to the formation of gastric neoplasias (Pontes et al., 2014).

3.3. NK cytotoxicity against K562 tumor cells After determining immunomodulatory and non-cytotoxic concentrations of Batroxase and Moojase and verifying that they promote changes in the gene expression of mononuclear cells, we investigated the immunotherapeutic potential of these toxins by evaluating the cytotoxicity of toxin-stimulated NK cells on K562 tumor cells (Fig. 4). Initially, NK cells were isolated from PBMC of 5 individuals. Then, NK cells were stimulated for 24 h with the toxins, at the same concentrations used in previous assays (Batroxase at 2 μg/mL and Moojase at 4 μg/mL), in the presence or absence of IL-2 (used as a NK cell 7

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Fig. 4. Evaluation of NK cytotoxicity against K562 tumor cells by flow cytometry. NK cells (effector cells) were stimulated for 24 h with Batroxase (2 μg/mL) or Moojase (4 μg/mL) in the presence or absence of IL-2 (0.07 μg/mL). Control cells (without toxins) were incubated for the same period only in RPMI 1640 culture medium. Then, CFSE-labeled K562 cells (target cells) were added in the ratio 2:1 (NK:K562), followed by incubation for 4 h at 37 °C. The analysis was done by flow cytometry after addition of propidium iodide (PI). The percentage of cell death was evaluated for both K562 tumor cells (A) and NK cells (B). Data expressed as mean ± SEM (n = 5). Statistical differences (ANOVA followed by Tukey's post-test) indicated by *p < 0.05 or ***p < 0.001 in relation to the group K562 + NK without toxins; ###p < 0.001 in relation to the group K562 + NK with Batroxase; &p < 0.05 in relation to the group K562 + NK + IL-2 without toxins; or £ p < 0.05 in relation to the group K562 + NK with Moojase.

elucidate the mechanisms for these increased cytotoxic effects.

non-significant increase (~8%) was observed (Fig. 4B). As mentioned in the methodology, the concentration of IL-2 used in these assays was previously standardized to achieve this non-cytotoxic behavior, since, despite being an important cytokine for the activation and proliferation of NK cells, IL-2 can cause apoptosis in these cells depending on the exposure conditions (Ross and Caligiuri, 1997). Interestingly, concomitant stimulation with IL-2 and Moojase (K562 + NK + IL2 + Moojase group) resulted in a significant increase in the percentage of dead NK cells (~17%) (Fig. 4B). As we evaluated all the variables separately, we could observe that this cytotoxic effect is exclusively related to the combined action of IL-2 and Moojase on NK cells. This result indicates that the combined stimulation of NK cells with IL-2 and Moojase results in an increase not only in their cytotoxicity on K562 cells, but also on NK cells themselves. Taken together, our results showed that both Bothrops toxins were able to stimulate NK cells, increasing their cytotoxicity against K562 leukemic cells. The mechanisms by which the toxins amplify the cytotoxic effects of NK cells still need to be better studied, but considering that both Batroxase and Moojase are proteases, it is possible to suggest that they have similar effects to granzymes, which are the serine proteases present in NK cell granules. As previously mentioned, the cytotoxic effects of NK cells are related to the release of perforins and granzymes. Initially, the pore-forming protein perforin binds to the target cell membrane in the presence of calcium, leading to loss of osmotic stability and subsequent influx of granzymes, which are capable of inducing apoptosis by caspase-dependent or caspase-independent mechanisms (Lehmann et al., 2000; Pardo et al., 2002; Ida et al., 2005). Thus, it is possible that the perforins released by NK cells enable the entrance of the toxins into tumor cells along with granzymes, consequently enhancing toxicity and leading to cell death processes, such as apoptosis. In this sense, the concomitant stimulation with IL-2 would be important to activate NK cells, which would release more of their granules containing perforins and granzymes, thus facilitating the action of the toxins on tumor cells. However, we must also consider that toxins (mainly Moojase, according to the results) may have direct stimulatory effects on NK cells, such as those promoted by IL-2. It is also possible that the observed differences between the effects of Batroxase and Moojase are due to the different classes of proteases to which they belong, the first being a metalloprotease (therefore with lower proteolytic specificity) and the second a serine protease (like the granzymes). Further studies should be conducted in the future to better

4. Conclusions The proteases Batroxase and Moojase, at non-cytotoxic concentrations, showed immunomodulatory effects on human peripheral blood mononuclear cells in vitro, inducing the production of pro-inflammatory and regulatory cytokines. Both toxins also promoted alterations in the expression (transcripts) of genes encoding enzymes that act on histones (acetyltransferases, deacetylases, methyltransferases or demethylases), or methyl-CpG binding proteins, indicating the induction of epigenetic changes that may lead to transcriptional activation or repression in the stimulated cells. Furthermore, cytotoxicity assays have shown that both Batroxase and Moojase stimulated the cytotoxic effects of IL-2-activated NK cells on K562 tumor cells, suggesting an interesting immunotherapeutic potential that can be exploited in future anticancer therapies. Thus, the results obtained in this study should contribute to a better understanding of the unexplored effects of animal toxins on epigenetic processes that culminate in alterations in the protein profile of cells stimulated with these molecules, allowing the determination of how these changes can influence the cellular phenotypes and functions. These data should contribute to better elucidate the systemic targets in which snake venom toxins act and thus provide basic scientific tools for the development of new drugs that have targeted action. Conflict of interests The authors declare no conflict of interests. Acknowledgements This work was supported by the São Paulo Research Foundation (FAPESP, grant #2011/23236-4 to S.V.S.) and by the Coordination for the Improvement of Higher Education Personnel (CAPES Brazil, financing code 001 to D.L.M.). The authors thank the Center for the Study of Venoms and Venomous Animals (CEVAP) from São Paulo State University (Botucatu, SP, Brazil) for providing the B. moojeni venom used in this study. Thanks are also due to Fabiana Rossetto de Morais (FCFRP-USP) for performing the flow cytometry analyses, and Thiago Abrahao Silva and Luciana Ambrósio (FCFRP-USP) for providing 8

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technical assistance.

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