Electrophysiological characterization of the first Tityus serrulatus alpha-like toxin, Ts5: Evidence of a pro-inflammatory toxin on macrophages

Biochimie 115 (2015) 8e16

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Research paper

Electrophysiological characterization of the first Tityus serrulatus alpha-like toxin, Ts5: Evidence of a pro-inflammatory toxin on macrophages Manuela B. Pucca a, Steve Peigneur b, Camila T. Cologna a, Felipe A. Cerni a, Karina F. Zoccal c, Karla de C.F. Bordon a, Lucia H. Faccioli c, Jan Tytgat b, Eliane C. Arantes a, * ~o Preto, University of Sa ~o Paulo, Ribeira ~o Preto, SP, Brazil Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeira Toxicology and Pharmacology, University of Leuven, Leuven, Belgium c ~o Preto, University of Sa ~o Paulo, Ribeira ~o Preto, SP, Brazil Department of Clinical Analysis, School of Pharmaceutical Sciences of Ribeira a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 January 2015 Accepted 10 April 2015 Available online 20 April 2015

Tityus serrulatus (Ts) venom is composed of mainly neurotoxins specific for voltage-gated Kþ and Naþ channels, which are expressed in many cells such as macrophages. Macrophages are the first line of defense invasion and they participate in the inflammatory response of Ts envenoming. However, little is known about the effect of Ts toxins on macrophage activation. This study investigated the effect of Ts5 toxin on different sodium channels as well as its role on the macrophage immunomodulation. The electrophysiological assays showed that Ts5 inhibits the rapid inactivation of the mammalian sodium channels Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6 and Nav1.7. Interestingly, Ts5 also inhibits the inactivation of the insect Drosophila melanogaster sodium channel (DmNav1), and it is therefore classified as the first Ts a-like toxin. The immunological experiments on macrophages reveal that Ts5 is a proinflammatory toxin inducing the cytokine production of tumor necrosis factor (TNF)-a and interleukin (IL)-6. On the basis of recent literature, our study also stresses a possible mechanism responsible for venom-associated molecular patterns (VAMPs) internalization and macrophage activation and moreover we suggest two main pathways of VAMPs signaling: direct and indirect. This work provides useful insights for a better understanding of the involvement of VAMPs in macrophage modulation. © 2015 Published by Elsevier B.V.

Keywords: Macrophage modulation Sodium channel Ts5 Tityus serrulatus Toxin

1. Introduction The venom of Tityus serrulatus (Ts) is a complex mixture of molecules, mainly neurotoxins, enzymes such as proteases and hyaluronidase, nucleotides, low molecular mass peptides with

Abbreviations: Nav channel, voltage-gated sodium channel; Ts, Tityus serrulatus; IL, interleukin; TNF-alpha, tumor necrosis factor-alpha; PAMPs, pathogen-associated molecular patterns; DAMPs, damage-associated molecular patterns; PRRs, pattern recognition receptors; TLRs, toll-like receptors; VAMPs, venom-associated molecular patterns; NCX, Naþ/Ca2þ exchanger; RTK, receptor tyrosine kinase; PLC, phospholipase C. * Corresponding author. Department of Physics and Chemistry, School of Phar~o Preto, University of Sa ~o Paulo, Av. do Cafe , s/n, maceutical Sciences of Ribeira 14040-903, Ribeir~ ao Preto, SP, Brazil. Tel.: þ55 16 3315 4275; fax: þ55 16 3315 4880. E-mail address: [email protected] (E.C. Arantes). http://dx.doi.org/10.1016/j.biochi.2015.04.010 0300-9084/© 2015 Published by Elsevier B.V.

antimicrobial and anticancer activities, bradykinin-potentiating peptides and natriuretic peptide [1e4]. Neurotoxins are the most important compounds in Ts venom because of their capacity to interfere with the mechanisms of ionic permeability in excitable membrane cells and to interact specifically with voltage-gated Kþ and Naþ channels, leading to an intense depolarization and neurotransmitter release [5,6]. Potassium channel scorpion toxins are characterized by having 22e47 amino acid residues and generally act by inhibiting the channels [7,8]. Sodium channel scorpion toxins have 60e76 amino acids and can be classified according to their mode of action as a- and btoxins, depending on the specific binding site of the channel [9,10]. Additionally, the a-toxins can be divided into three pharmacological subgroups: classic a-toxins, insect a-toxins and a-like toxins. The classic a-toxins present high affinity for voltage-gated sodium channels of rat brains (mammal) and are almost non-toxic to

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insects. The insect a-toxins are potently toxic to insects and show low activity on mammalian brain channels, whereas a-like toxins act on both mammalian and insects channels [11]. Scorpion toxins that selectively interact with ion channels have been extensively used as pharmacological tools. These toxins can also be used as potential drugs for treating different diseases [4,8,12] as well as insecticides [13,14]. Moreover, many immunological properties (e.g. inflammation) have been attributed to scorpion venom and their isolated toxins [15e17]. For these reasons, Ts venom has been extensively studied and many of its toxins have been isolated through chromatography on a CM-52-cellulose column followed by additional chromatographic steps on a reversed-phase (RP) C18 column [4,18e22]. However, many studies are still required to understand the role of these toxins in envenoming by Ts. Among Ts toxins, Ts5 (also known as TsTX-V, Tityustoxin-5, Tityustoxin-V, alpha toxin TsTX-V, Ts V or Toxin V) is highly toxic (i.v. LD50 ¼ 94 ± 7 mg/kg in mice) and markedly delays the inactivation of Naþ channel, being classified as an a-toxin [23]. Moreover, studies have shown that Ts5 has also others features: i) it potentiates glucose-induced electrical activity and insulin secretion in rodent islet beta-cells [24]; ii) it reduces a reduction of 3H-GABA (gaminobutyric acid) and 3H-DA (dopamine) uptake in a Ca2þ dependent manner [25] and iii) it shows low effects on catecholamine release and blood pressure [26]. However, immunological and electrophysiological studies concerning Ts5 have not been performed so far. The aim of the present study was therefore to characterize electrophysiologically and immunologically the toxin Ts5. The activity of Ts5 was investigated on nine different Nav channels and its cytotoxicity and ability to stimulate cytokine and nitric oxide production in macrophage was examined. 2. Material and methods 2.1. T. serrulatus venom Ts venom was obtained by electrical stimulation [27] of scorpions maintained at the serpentarium of the Faculdade de Medicina ~o Preto da Universidade de Sa ~o Paulo (School of Medicine de Ribeira ~o Preto, University of Sa ~o Paulo, Brazil). of Ribeira 2.2. Ts5 isolation T. serrulatus desiccated venom (50 mg) was fractionated on a CM-cellulose-52 column connected to a Fast Protein Liquid Chromatography (FPLC) system, using the improved method described by Cerni et al. [22]. The fraction XIA eluted from CM-cellulose-52 was used to € obtain Ts5. RP-FPLC of the fraction XIA was performed in an Akta Purifier UPC-10 system (GE Healthcare, Uppsala, Sweden), using a 4.6 mm  250.0 mm C18 column (Shimadzu Corp., Kyoto, Japan) equilibrated with 0.1% (V/V) trifluoroacetic acid (TFA) at a flow rate of 0.8 mL/min. The samples were eluted with steps of concentration gradient from 0 to 100% of solution B (80% acetonitrile in 0.1% TFA), at a flow rate of 0.8 mL/min. Absorbance was monitored at 214 nm. Pure toxins were lyophilized and stored at 20  C. The determination of N-terminal amino acid residues of Ts5 was performed by Edman degradation [28], on a Protein Sequencer model PPSQ-33A (Shimadzu Co., Kyoto, Japan). 2.3. Sodium channel expression cRNA for all Navs (rNav1.2, rNav1.3, rNav1.4, hNav1.5, mNav1.6, rNav1.7, hNav1.8, Drosophila melanogaster sodium channel e

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DmNav and Bacillus halodurans sodium channel e NaChBac) were synthesized from linearized plasmids using large-scale T7 or SP6 mMESSAGEmMACHINE transcription kit. The harvesting of oocytes from anesthetized female Xenopus laevis frogs was performed as previously described [29]. Oocytes were injected with 30e50 nL of the different channels using a microinjector (Drummond Scientific, USA). ND-96 solution was used for the oocytes incubation (in mM e 96 NaCl, 2 KCl, 2 MgCl2, 1.8 Ca.Cl2, 5 HEPES, pH 7.4, supplemented with 50 mg/L gentamicin sulfate and 180 mg/L theophylline). The use of the frogs was in accordance with the license number LA1210239. 2.4. Electrophysiological measurements Sodium currents were recorded using the two-microelectrode voltage-clamp technique at room temperature (18e22  C). The recordings were processed by GeneClamp 500 amplifier (Axon Instruments, USA) controlled by a pClamp data acquisition system (Axon Instruments, USA). Whole-cell currents from oocytes were recorded 1e5 days after injection. Currents and voltage electrodes had resistances from 0.7 to 1.5 MU and were filled with 3 M KCl. Currents were sampled at 20 kHz and filtered at 2 kHz using a fourpole low-pass Bessel filter. Leak subtraction was performed using a P/4 protocol. For the assays, Ts5 diluted in ND-96 solution was added directly to the recording chamber to obtain the desired final concentration (1 mM). This concentration was previously used for electrophysiological characterization of other toxins [22,30]. Immediately after adding the toxin in the chamber containing the oocyte, the bath solution was mixed to obtain a homogenous final concentration within few seconds. For the activation protocols, 100 ms test depolarization, ranging from 90 mV to þ70 mV, were applied from a holding potential of 90 mV, in 5 mV increments at 5 s intervals. For the inactivation protocols, we employed double pulses, with a conditioning pulse applied from a holding potential of 100 mV to a range of potentials from 90 mV to 0 mV, in 5 mV increments for 50 ms, immediately followed by a test pulse to 0 mV (or 5 mV). Data were normalized to the maximal Nav current amplitude (Imax), plotted against the pre-pulse potential and fitted using the Boltzmann equation: INa/Imax ¼ 1/[1 þ exp((Vc  Vh)/kh)], where Vh was the voltage corresponding to half-maximal inactivation, Vc was the conditioning pre-pulse voltage, and kh was the slope factor. Each experiment was performed at least 3 times. 2.5. Cell line culture A murine peritoneal macrophage cell line (J774.1) was obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cells were cultured in RPMI (Roswell Park Memorial Institute Medium) e 1640 supplemented with 10% fetal bovine serum (RPMI-c) and 1% gentamicin. After the formation of a monolayer, cells were harvested with plastic cell scrapers and centrifuged at 400  g for 10 min at 10  C. The total number of cells was counted and the viability was determined in a Neubauer chamber (BOECO Germany, Hamburg, Germany) using Trypan blue (Gibco, Grand Island, NY). The cells were plated onto 96-well culture plates (Corning) at a concentration of 2.5  104 cells/well and maintained in RPMI-c overnight at 37  C in an incubator under a humidified atmosphere of 5% CO2 in air. The cells were then exposed for 24 h to Ts5 (12.5, 25 and 50 mg/mL). Nonstimulated cells were used as control. The culture supernatants were then harvested and stored at 20  C for subsequent cytokines and nitric oxide assay.

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2.6. Cytotoxicity assay

3. Results

Macrophage cell viability was evaluated using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (SigmaeAldrich, St. Louis, MO), as described by Mosmann (1983). Adherents cells were incubated for 24 h with Ts5 (12.5, 25 and 50 mg/mL). After this period, the cultures were incubated with 5% MTT in RPMI-c for 3 h. Subsequently, 50 mL of 20% sodium dodecyl sulfate in 0.01 M HCl was added to each well, and the plate was held at room temperature until the precipitate was completely solubilized. Absorbance was then measured at 570 nm with a spectrophotometer (mQuanti; Bio-Tek Instruments, Inc., Winooski, VT). The cytotoxicity of the Ts5 toxin was then expressed as a percentage of the viability observed in the unstimulated cells. For positive control, 20% dimethylsulfoxide (DMSO) was used.

3.1. Ts5 isolation

2.7. Cytokine and nitric oxide (NO) quantification Concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and interleukin (IL)-10 in culture supernatants were quantified by ELISA using purified biotinylated specific antibodies and cytokine standards, in accordance with the manufacturer instructions (R&D Systems, Minneapolis, MN). Cytokine concentrations were determined using a standard curve established with the appropriate recombinant cytokine (expressed in pg/mL). The optical densities were measured at 405 nm in a microplate reader (mQuant, Bio-Tek). Sensitivities were <10 pg/mL; limits of detection for the kits were 2000, 1000 and 2000 pg/mL for TNF-alpha, IL-6, and IL-10, respectively. The amount of nitrite ðNO 2 Þ present in the supernatants was measured as an indicator NO production by Griess method [31]. The amount of nitrite in the samples was obtained by a standard curve using serial NaNO2 dilutions. The Absorbance at 540 nm was recorded 10 min after addition of NaNO2. 2.8. Ts5 alignment The amino acid sequence of Ts5 and other Ts toxins used in the comparative analysis are deposited in the database Swiss-Prot. The alignment and percentage identity of the primary sequence of Ts5 with two other Ts toxins specifically acting on sodium channels for which electrophysiological and immunological data are available in the literature (Ts1 and Ts2). The sequence alignment figure was generated using ClustalW2 Multiple Sequence Alignment Tool (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

The Ts venom fractionation method used has already been described by Cerni et al. (2014) and the elution profile obtained on the present work is consistent with their results. As previously reported, the first fractionation step of Ts venom yielded 18 main fractions named IeXIII (some divided in A and B). Fraction XIA was used for further chromatographic step and purification of Ts5 [22]. Therefore Ts5 was obtained after a reversed-phase chromatography step of fraction XIA (Fig. 1). The purity of Ts5 was confirmed by mass spectrometry and its N-terminal primary sequence was verified by Edman degradation (data not shown). 3.2. Electrophysiological characterization Fig. 2 illustrates electrophysiological traces with the effect of 1 mM Ts5 on 9 different sodium channels on the voltage dependence of steady-state activation and inactivation curves. The results show that Ts5 inhibits rapid inactivation of Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7 and DmNav1 but does not affect Nav1.8 and NaChBac. The toxin exerts the biggest effect on DmNav1, followed by Nav1.6, Nav1.3 and Nav1.7. The presence of 1 mM Ts5 increased the INa peak amplitude by 35.4 ± 6.2% (n ¼ 3) for Nav1.3 and by 17.7 ± 2.3% (n ¼ 3) for Nav1.6. Application of the same concentration of toxin resulted in sustained non-inactivating currents of 71.3 ± 5.4% (n ¼ 3) of control for Nav1.3 and 58.9 ± 2.9% (n ¼ 3) of control for Nav1.6. Ts5 did not alter ion selectivity of the channels since no change in the reversal potential was observed. The toxin induced slowing of the fast inactivation but did not influence the voltageecurrent relationship (Fig. 2). Fig. 2 shows no shift in the voltage dependence of activation for all Nav isoforms tested, but for Nav1.2 / Nav1.7 and DmNav1 the steady-state inactivation curves became incomplete after toxin application. In terms of midpoint of the steady-state inactivation curve, only the Nav1.3 displayed a significant shift towards more hyperdepolarized potential values. The Vh shifted from 31.9 ± 0.5 mV to 42.3 ± 0.6 mV (p < 0.05, n  3). Ts5 affected the insect channel DmNav1 most profoundly. The INa peak amplitude increased slightly but significantly by

2.9. Statistical analysis The electrophysiological assays were tested for normality using a D'Agustino Pearson omnibus normality test. Data following a Gaussian distribution were analyzed for significance using one-way ANOVA, Bonferroni test. Non parametric data were analyzed for significance using the KruskaleWallis, Dunn's test. Data were analyzed using Clampfit 8.1 (Molecular Devices, Sunnyvale, CA), Excel 2010 (Microsoft Corp., Redmond, WA), and OriginPro 8.0 (OriginLab Corp., Northampton, MA). The macrophage cytotoxicity and cytokine and NO production induced by Ts5 were analyzed with GraphPad Prisma 5.0 software. The data from 2 independent set of experiments with n ¼ 3 each were analyzed using one-way ANOVA with the Tukey's Multiple Comparison test. The cytotoxicity is expressed as the mean ± standard error of the mean (SEM). The data of cytokine and NO production are expressed as the mean ± standard deviation (SD). Values of p  0.05 were considered significant.

Fig. 1. Reversed-phase FPLC of fraction XIA resulting from the Ts venom fractionation procedure. Ts5 was obtained from reversed-phase chromatography of the fraction XIA on a C18 column (4.6 mm  250 mm, 5 mm particles) equilibrated with 0.1% (V/V) of trifluoroacetic acid (TFA). Adsorbed proteins were eluted using a concentration gradient from 0% to 100% of solution B (80% acetonitrile in 0.1% TFA), represented by the dotted line. Flow: 0.8 mL/min. Absorbance was monitored at 214 nm, at 25  C.

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Fig. 2. Electrophysiological screening of the effect of Ts5 on 9 different cloned voltage-gated sodium channels. Left panels: representative whole-cell current traces under control conditions and after the addition of 1 mM Ts5 (*), n ¼ 3 cells. Right panels: effects of Ts5 on the voltage dependence of activation and steady-state inactivation curves under control conditions (closed symbols) and after the addition of 1 mM Ts5 (open symbols), n ¼ 3 cells ± SEM.

26.3 ± 4.2% (p < 0.05, n ¼ 5). However, the fast (“real time” kinetics) and steady-state inactivation were completely inhibited. 3.3. Immunological characterization To investigate the effects of Ts5 on the viability of J774.1 line, macrophages were exposed to Ts5 for 24 h. The concentrations (12.5e50 mg/mL) of Ts5 used in these experiments had no cytotoxic effects (see Fig. S1 in Supplementary material). Then our next step was to determine the capacity of Ts5 to induce NO, IL-10, IL-6 and TNF-alpha production in macrophages. As shown in Fig. 3A and B, Ts5 did not have, respectively, an effect on NO or IL-10 production after 24 h. On the other hand, Ts5 increased the concentrations of IL-6 (50 mg/mL) and TNF-alpha (at all toxin concentrations) in the cell supernatants when compared to non-stimulated cells (Figs. 3C, D). These results demonstrate that Ts5 selectively increased the production of certain cytokines. 4. Discussion The signs and symptoms of Ts envenoming involve intense activation of the immune system with the release of mainly proinflammatory cytokines such as TNF-alpha, IL-6, INF-alpha and other mediators such as leukotriene B4 and prostaglandin E2. The uncontrolled release of pro-inflammatory mediators by macrophages can induce a generalized inflammation that can lead to multiorgan failure [32e36]. Macrophages are crucial cells in the inflammation, being responsible for detecting pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) through their PRRs (pattern recognition receptors), such as toll-like receptors (TLRs) [37]. After pattern recognition by PRRs, macrophages activate an immune response that can result in the production of pro-inflammatory cytokines [38]. Recently, the term venom-associated molecular pattern (VAMP) was proposed to

refer to molecules from venoms that can be recognized by PRRs [16]. To date, distinguished reports have shown that Ts venom and toxins activate the macrophages and modulate the production of cytokines. Petricevich et al. showed an increase in vitro of IL-6 and INF-alpha production in the supernatants of peritoneal macrophages incubated with Ts venom [39]. Pessini et al. [33] reported that Ts venom as well as the major toxin Ts1 were able to induce the increase of IL-6, IL-1-alpha and TNF-alpha in mice. Lately, Zoccal et al. (2011) described the role of Ts1, Ts2 and Ts6 in the macrophage immunomodulation. Ts1 was able to increase the production of IL-6 and TNF-alpha; Ts2 stimulated the production of TNF-alpha, IL-10 and inhibited NO release; and Ts6 induced the production of IL-6 and NO, and decrease the production of TNFalpha [17]. In this study, Ts5 inhibits the rapid inactivation of Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7 and DmNav1 and therefore, it is considered the first alpha-like toxin from Ts venom described so far. The first described alpha-like toxins were Bom3 and Bom4 from Buthus occitanus mardochei and Lqh3 from Leiurus quinquestriatus hebraeus [40]. The flexible C-tail of alpha-like toxins enables that the toxin affects a broad range of Navs [41]. Ts5 (1 mM) exhibited the highest effect on DmNav1, followed by Nav1.6, Nav1.3 and Nav1.7. Similarly to Ts5, Lqh-3 (100 nM) slowed down the fast inactivation of hNav1.7, while rNav1.2 channels were almost insensitive to it [42]. Nav1.2 and Nav1.7 are mainly expressed in mammalian central and peripheral nervous systems, respectively (Chen et al., 2002). BmK-M1, an alpha-like neurotoxin from Buthus martensii Karsch, can be also considered as a cardiotoxin, since the toxin (EC50 ¼ 195 ± 15 nM) slowed the inactivation of the cardiac Naþ channel (hH1) functionally expressed in Xenopus oocytes [43]. BmK-M1 (EC50 ¼ 99.4 ± 20.1 nM) was also able to slow and partially inhibit the inactivation of rNav1.5 expressed in a mammalian HEK293t cell line [44]. On structural basis, the functional site of the recombinant BmK-M1 was investigated by site-directed mutagenesis [45e47].

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Fig. 3. Effect of Ts5 on NO and cytokine release. Adherent cells were stimulated with different concentrations of Ts5 (12.5, 25 and 50 mg/mL) for 24 h in 5% CO2 at 37  C. The supernatants were collected after 24 h. The amount of NO, IL-6 and TNF-a present in the supernatant was determined by ELISA. Values are expressed as means ± SD (n ¼ 4) (*p < 0.05, **p < 0.001 compared to control, non-stimulated cells).

BmK-M1 is constituted by a unique tertiary arrangement named site RC, where the residues 8e12 are connected to the C-terminal segment 58e64 by a disulfide bond (12e63) and a hydrogen bond network [48]. Most of the residues important for bioactivity are located within this site, such as the basic residues Lys62, His64 and Lys8 which may directly interact with the receptor site of Naþ channels through electrostatic interactions (Wang et al., 2003). Ts5 (34.4% of identity with BmK-M1) presents Ser, Arg and Glu at the positions corresponding to Lys62, His64 and Lys8, respectively (see Fig. S2 in Supplementary material). Guan et al. (2004) predicted that a substitution K8E (as observed in the primary structure of Ts5) or K8N could result in a misfolded protein, since the attempts to express BmK-M1 mutants K8E and K8N were unsuccessful [45]. On the other hand, replacing residue 8 in other scorpion toxins with Asp or Lys/Gln would result in a peptide bond 9e10 adopting a trans or cis form, respectively. The cis-to-trans isomerization appears to be sequence-dependent, since the mutation K8Q retains the wild-type cis conformation in the BmK-M1 mutant structure and may be related to the conversion of the scorpion alpha-toxins subgroups [45]. The residue Asn11 observed in Ts5, BmK-M1 and Lqh3 stabilizes the distinct conformation of the bioactive site, while His64 from BmK-M1 and Lqh3 (corresponding to Arg at Ts5) seems to be involved in the preference for phylogenetically distinct target sites (Wang et al., 2003). Most of the mutations in BmK-M1 similarly affected the anti-insect and antimammal activities of the toxin. However, the H10E mutation (as observed in Lqh3) decreased the anti-mammal activity of BmK-M1 without significantly affect the binding of the toxin to insect Naþ channels [47]. Similar reduction is expected with Ts5, since it presents an Asp at the position 10. Additionally, the neutralization H64A decreased 96-fold and 3.8-fold the binding affinity for cockroach and mice Naþ channels, respectively, showing that insect Naþ channels are much more sensitive to a histidine at position 64 than mammal Naþ channels [47]. Since Ts5 presents an

Arg at this position, which is a positively charged amino acid residue as histidine, no difference in the binding affinity is expected. All aromatic residues from BmK-M1 were individually substituted with Gly in association with a more conservative substitution of Phe, using site-directed mutagenesis (Sun et al., 2003). The results showed that the aromatic residues at the position 5, 35 and 47, which are also observed in Ts5, seem to be essential for the structure and function of the toxin BmK-M1, while Trp38 is involved in the pharmacological function [46]. The alpha-effect of Ts5 on a variety of mammal channels (total of six) also explains its high toxicity in mice, which presents an intravenous LD50 very similar to the most toxic Ts toxin, Ts1 (76 ± 9 and 94 ± 7 mg/kg for Ts1 and Ts5, respectively) [23,49]. Moreover, Ts5 presented electrophysiological results very similar to the Ts alpha-toxin Ts2 except on Nav1.4 and DmNav1 channels [30]. Indeed, Ts5 presents the highest identity with Ts2 compared with Ts1 (see Fig. S3 in Supplementary material). Regarding the immunological effects on macrophages, first we determined whether Ts5 could be considered as a cytotoxic stimulus or not. At the concentrations tested, Ts5 was not cytotoxic to macrophages and had no effect on NO and IL-10 release, but increased the production of IL-6 and TNF-alpha, although with different efficacies. However, Ts5 showed to be a pro-inflammatory toxin increasing the production of IL-6 (50 mg/mL) and TNF-alpha (12.5, 25 and 50 mg/mL). To facilitate the correlation of the concentrations used in this assay and in electrophysiological tests, each stimulated ELISA well containing macrophages received 1.7, 3.5 and 6.9 mM of Ts5 which correspond to 12.5, 25 and 50 mg/mL, respectively. To better understand the electrophysiological role of sodium channels on the macrophage production of inflammatory mediators, the results obtained in the presented work were compared with those concerning sodium channels Ts toxins previously described in the literature (Table 1).

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Previous studies have shown that voltage-gated sodium channels regulate macrophage phagocytosis [50e52]. However, Carrithers et al. (2007) were the first to show by RT-PCR that macrophages only express Nav1.5 and Nav1.6 channels. Furthermore, in contrast to other cell types (e.g. neurons and muscles), these channels are expressed only in intracellular structures: Nav1.5 is expressed in late endosome and regulates macrophage phagocytosis; and Nav1.6 is associated with cytoskeletal filaments and the endoplasmic reticulum. Nav1.5 localization on macrophage seems to facilitate the decrease of pH from the early stage (pH  6.8) to the lysosomal stage (pH  5.0). On the other hand, the neuronal channel Nav1.6 does not appear to facilitate phagocytosis, although it is believed to regulate cytoskeletal function [53]. Indeed, all Ts toxins specific to sodium channels that induced modulation of macrophages presented an effect on Nav1.5 and Nav1.6. Ts1 presents a beta-effect on Nav1.6 and a blockage on Nav1.5 [54], while Ts2 and Ts5 present an alpha-effect on both channels. Nevertheless, all of them produce different modulation of cytokine and NO, even Ts2 and Ts5, which modulate the inactivation of Nav1.5 and Nav1.6 channels in a very similar manner [30]. In this way, the different effects of Ts toxins on sodium channels (pure block, alpha- and/or beta-effect) are not sufficient to explain macrophage modulation. Based on this conclusion, we asked whether these toxins could be targeting other macrophage receptors. We also sought to understand how Ts toxins reach macrophage Nav1.5 and Nav1.6 located intracellularly. Recently, Zoccal et al. (2014) revealed that Ts venom and the major toxin Ts1 can induce the production of inflammatory mediators by interacting with TLR2 and CD14/TLR4, which confirmed that these toxins can target receptors different from sodium channels as well as a different pathway [16]. Macrophages play an important role in immunogenic challenges by producing reactive oxygen species, NO and pro-inflammatory cytokines that can aggravate the inflammation. These cells have receptors, such as, toll-like receptors (TLRs), the NLR family (nucleotide-binding oligomerization domain-like receptors), and the RLR family [RIG (retinoic acid-inducible gene)-I-like receptors] [55,56] that are recognized by pattern recognition receptors (PRRs), leading to the activation of intrinsic signaling pathways (e.g., myeloid differentiation factor 88; MyD88). Additionally, this recognition leads to the phosphorylation of NF-kB and AP-1 that induce the expression of pro-inflammatory cytokines and consequently, the production of various cytokines [16,55,57e59]. Another signaling pathway concerning toxins and macrophage activation was also described by Ramirez-Bello et al. (2014) using toxins from the Tityus discrepans venom, Inftx6 and Inftx7. In that work, the toxins have been shown to activate macrophages by interacting with the Naþ/Ca2þ exchanger (NCX) 1 or 3 in reverse mode and/or the Naþ/Hþ exchanger. This leads to an increase of the Naþ intracellular concentration which indirectly induces the activation of NCX3rv and the PLC-IP3 signaling. Moreover, in the same

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study the authors demonstrated that TTX prevents the action of Inftx6 on sodium channels [60,61]. Knowing that TTX is a sodium blocker which does not cross the membrane due to its insolubility in lipids [62], here we discuss the possible mechanism responsible for VAMPs internalization and macrophage activation. Furthermore, we suggest and propose two main pathways of VAMPs signaling: direct and indirect (Fig. 4). TLRs binding activate a variety of defense mechanisms on macrophages, including phagocytosis [63e65]. Therefore, VAMPs can be phagocytized by macrophages. Scorpion toxins that affect Naþ channels are basic proteins with 60e76 amino acid residues and connected by four disulphide bridges, knowing to be very stable [6]. In this way, we assume that VAMPs can resist degradation in the early and late endosome conditions but not on the phagolysosome, where the acid hydrolases are concentrated [66]. Indeed, there are studies with Anthrax lethal toxin (83 kDa) and edema factor (88.8 kDa) demonstrating that these toxins can reach the cytosol after endocytosis [67]. Before being digested by enzymes, VAMPs could bind to Nav1.5 channels in late endosomes and increase the cytosolic Naþ. This can be also supported by knockdown mice of the Nav1.5 channel, which reduce the initial calcium response following bacterial challenge and prevent the generation of prolonged and localized calcium oscillations during phagosolysosome maturation. Therefore, Nav1.5 alone could be the responsible to regulate phagocytosis and phagolysosome maturation in macrophages through spatial-temporal coordination of calcium signaling within a unique subcellular region [68]. Moreover, the cytosolic overload of Naþ induced by Nav1.5 could depolarize the macrophage membrane and activate the NCX1 and NCX3 channels in a reverse mode, increasing the intracellular Ca2þ [60]. Evidence that other receptors are also strongly involved with VAMPs signaling can be supported by the PLC pathway activation [60]. The increase of internal Ca2þ concentration from both pathways (NCX-reverse mode and PLC) results on macrophage modulation, formation of large vacuoles, modification of the round shape and spreading [69]. On the other hand, the involvement of Nav1.6 during VAMPs signaling is still obscure and until now, it remains speculative to assume that these toxins could reach the receptor in the cytosol or ER. In accordance with the described mechanism, the Ts2 and Ts5 toxins could activate macrophages using the direct and indirect pathway; however Ts1, a blocker of Nav1.5, possibly could only be able to activate the direct pathway. Ts1 could also impair the phagocytosis by blocking Nav1.5 and consequently the Naþ efflux from the late endosome. This could explain why a higher concentration of Ts1 (100 mg/mL) compared to Ts2 (25 mg/mL) and Ts5 (12.5 mg/mL) was necessary to increase the TNF-alpha production, since Ts1 only affects the direct pathway. Our results do not allow us to conclude which pathway is important for each proinflammatory mediator (IL-6, TNF-alpha and NO). Nevertheless,

Table 1 Ts toxins: mammalian sodium channel sensitivity and macrophage modulation. Ts toxin

Classification

Sensitive mammalian sodium channel

Macrophage modulation

Reference

Ts1

b-Toxin

Increases TNF-a, IL-6

[18,48]

Ts2

Classic a-toxin

Ts5

a-Like toxin

b-Effect on Nav1.2, Nav1.3, Nav1.4 and Nav1.6 Inhibits Nav1.5 a-Effect on Nav1.2, Nav1.5, Nav1.6 and Nav1.7 b-Effect on Nav1.3 a-Effect on Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6 and Nav1.7

a b c d

12.5 mg/mL. 25 mg/mL. 50 mg/mL. 100 mg/mL.

d

b

Increases TNF-a, IL-10 Decreases NOb Increases TNF-a,a IL-6c b

d

[18,43] Present article

14

M.B. Pucca et al. / Biochimie 115 (2015) 8e16

Fig. 4. Schematic diagram of VAMPs' signaling pathways implicated in macrophage modulation. Tityus serrulatus (Ts) toxins and other VAMPS (venom-associated molecular pattern), shown as colored stars, can activate macrophages by different pathways. Direct pathway: (1) TLR4/CD14/TLR2 can recognize VAMPS and (2) activate MYD88-dependent signaling via NF-kB or (3) MYD88-independent signaling via C-fos/Jun. (4) NF-kB and C-fos/Jun are transcription factors which activate the production of pro-inflammatory mediators [17]. Indirect pathway: (5) Activation of TLRs can activate VAMPs phagocytosis [54]. (6) In the late endosome, Nav1.5 activated by VAMPs toxins increases the movement of Naþ from the interior of the endosome to the cytoplasm [46,48]. (7) The increase of intracellular Naþ induces NCX1 and NCX3 activation in the reverse mode, allowing the entry of Ca2þ [48]. (8) Unknown receptors coupled to tyrosine kinase (RTK e receptor tyrosine kinase) or protein G (G protein-coupled receptor) could also recognize VAMPS and activate PLC pathway resulting in production of IP3. (9) IP3 diffuses to the endoplasmatic reticulum (ER), binds to its Ca2þ receptor (IP3R) which releases Ca2þ from the ER [48]. (10) The overload of intracellular Ca2þ induces macrophage activation, formation of large vacuoles, protein production (e.g. cytokines) and spreading [55]. (11) The activation of the direct and indirect pathways results in strongly pro-inflammatory mediators' production.

the model proposed here may be useful for understanding the involvement of Ts toxins in pro-inflammatory response. 5. Conclusion In conclusion, our results demonstrate that Ts5 is the first alphalike toxin described from Ts venom. Ts5 can exert a proinflammatory effect through its ability to stimulate the production of macrophage TNF-alpha and IL-6, two important cytokines with an important role in envenoming by Tityus species. Based on our results and recent literature reports, we suggest that VAMP signaling may involve direct and indirect pathways. Together, these findings provide a better understanding of the involvement of voltage-gated sodium channels in macrophage activation in general. 6. Financial support This work was supported by CNPq, Conselho Nacional de gico (Grant 402508/2012-2); Desenvolvimento Científico e Tecnolo ~o de Amparo a  Pesquisa do Estado de Sa ~o Paulo FAPESP, Fundaça (Grant 2009/07169-5, scholarship to MBP 2013/21329-0, scholarship to CTK 2013/26200-6, scholarship to KFZ 2014/03332-7 and scholarship to FAC 2013/21342-7); F.W.O. Vlaanderen (Grants G.0433.12 and G.A071.10N); Inter-University Attraction Poles Program Grant IUAP 7/10 from Belgian State and Belgian Science Policy; and KU Leuven Grant OT/12/081.

7. Citation of meeting abstracts This work was previously presented, in part, at the World Immune Regulation Meeting VIII, held in March 19e22, 2014, in Davos, Switzerland.

Conflict of interest The authors declare that there are no conflicts of interest.

Acknowledgements The authors thank John N. Wood (University College London, London, UK) for sharing rNav1.8; A.L. Goldin (University of California, Irvine, CA, USA) for sharing rNav1.2, rNav1.3, and mNav1.6; G. Mandel (State University of New York, Stony Brook, NY, USA) for sharing rNav1.4; R. G. Kallen (Roche Institute of Molecular Biology, Nutley, NJ, USA) for sharing hNav1.5; S.H. Heinemann (Friedrich€t Jena, Jena, Germany) for sharing the ratb1 Schiller-Universita subunit; S.C. Cannon (University of Texas Southwestern Medical Center, Dallas, TX, USA) for sharing the hb1 subunit and Martin S. Williamson (Rothhamsted Research, Harpenden, UK) for providing the Para and tipE clone. The Nav1.7 clone was kindly provided by ^ Cardoso Roche (Basel, Switzerland). The authors thank Iara Aime for technical assistance.

M.B. Pucca et al. / Biochimie 115 (2015) 8e16

Appendix A. Supplementary material Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biochi.2015.04.010.

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