Selected scorpion toxin exposures induce cytokine release in human peripheral blood mononuclear cells

Accepted Manuscript Selected scorpion toxin exposures induce cytokine release in human peripheral blood mononuclear cells Gerardo Corzo, Gerardo Pavel Espino-Solis PII:

S0041-0101(17)30018-1

DOI:

10.1016/j.toxicon.2017.01.007

Reference:

TOXCON 5540

To appear in:

Toxicon

Received Date: 23 August 2016 Revised Date:

1 December 2016

Accepted Date: 10 January 2017

Please cite this article as: Corzo, G., Espino-Solis, G.P., Selected scorpion toxin exposures induce cytokine release in human peripheral blood mononuclear cells, Toxicon (2017), doi: 10.1016/ j.toxicon.2017.01.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Selected scorpion toxin exposures induce cytokine release in human

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peripheral blood mononuclear cells

3 Gerardo Corzo2 and Gerardo Pavel Espino-Solis1,*

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Baylor Institute For Immunology Research, 3410 Worth Street, Suite 600. Dallas, TX 75246

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología,

Universidad Nacional Autónoma de México, UNAM. Apartado Postal 510-3, Cuernavaca

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Morelos, 61500, MEXICO.

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Institute For Immunology Research. Email: [email protected]

To whom correspondence should be addressed: Gerardo Pavel Espino-Solis, Baylor

15 Abstract

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A cytokine screening on human peripheral blood mononuclear cells (PBMCs) stimulated

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with selected scorpion toxins (ScTx’s) was performed in order to evaluate their effect on

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human immune cells. The ScTx’s chosen for this report were three typical buthid

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scorpion venom peptides, one with lethal effects on mammals Centruroides suffussus

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suffusus toxin II (CssII), another, with lethal effects on insects and crustaceans

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Centruroides noxius toxin 5 (Cn5), and one more without lethal effects Tityus discrepans

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toxin (Discrepin). A Luminex multiplex analysis was performed in order to determine the

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amounts chemokines and cytokines IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12-p40, IL-

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13, interferon alpha (IFN-α), interferon gamma (IFN-γ), tumor necrosis factor alpha

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TNF-α, and interferon-inducible protein-10 (IP-10) secreted from human PBMCs

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exposed to these toxins. Although, the ScTx Cn5 is not lethal for mammals, it was able to

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induce the secretion of cytokines IL-1β, IL-6, and TNF-α, IL-10 and IP-10 in comparison

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to the lethal CssII, which was able to induce only IP-10 secretion. Discrepin also was

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able to induce only IP-10. Interestingly, only low amounts of interferons α and β were

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induced in the presence of the ScTx’s assayed. In a synergic experiment, the combination

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of Discrepin and Cn5 displayed considerable reverse effects on induction of IL-1β, IL-6,

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IL-10 and TNF-α, but they had a slight synergic effect on IP-10 cytokine production in

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comparison with the single effect obtained with the Cn5 alone. Thus, the results obtained

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suggest that the profile of secreted cytokines promoted by ScTx Cn5 is highly related

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with a cytokine storm event, and also it suggests that the mammalian lethal neurotoxins

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are not solely responsible of the scorpion envenomation symptomatology.

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Introduction

Scorpions are venomous predatory arthropods which are members of the

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arachnidan class and order Scorpiones. They are living examples of 400 million years of

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successful evolution spread around the world, except in Antarctica (Ortiz et al. 2015).

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Scorpions are represented in 16 families with approximately 1,700 different species.

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Scorpions can produce potent venoms to kill or paralyze their preys and to ward off

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possible competitors and predators and their venom could be the key for they successful

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predominance (Chippaux and Goyffon 2008). The scorpion members of the buthidae

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family produce venom, which are highly toxic to mammals. In humans, the effects of a

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buthid scorpion sting can vary widely, from just local pain or inflammation to severe

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clinical complications, including death. Although, the severity of buthid scorpion

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envenomation is related to the presence of neurotoxins with pharmacological action on

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voltage-gated sodium channels (Nav) (Rodriguez de la Vega and Possani 2005), the

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scorpion venoms and their toxins from different species and areas of the world caused

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different immunological and toxicological manifestations (Hadaddezfuli et al. 2015).

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Such different toxicological manifestations may be related to cytokines, which are

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increased after envenomation (Magalhaes et al. 1999). Increases of pro-inflammatory and

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anti-inflammatory cytokines in human serum, cellular lines and mice has also been

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reported after scorpion envenomation or venom/toxins incubation (Petricevich 2010).

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Recent studies have been reported the effect of Hemiscorpius lepturus and Androctonus

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crassicauda scorpion venoms, which are able to induce the secretion of interleukin IL-12

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in human monocytes (Hadaddezfuli et al. 2015, Saadi et al. 2015). Studies performed

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with the Tityus serrulatus venom and/or its major toxins, showed an inmunomodulatory

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activity in macrophages. High levels of tumor necrosis factor TNF-α, interleukin IL-1β,

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IL- 6, IL-8 and IL-10 were observed in supernatants of macrophages from mice exposed

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to Tityus serrulatus venom and its major toxins (Petricevich et al. 2007, Fialho et al.

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2011). In the same way, increased levels of IL-1α, IL-1β, IL-6 and IL-10 were observed

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in sera from mice exposed to Centruroides noxius scorpion venom (Petricevich 2006).

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Depending on the concentrations used, toxins isolated from the venom of the scorpion

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Tityus serrulatus Toxin 5 (TsV), Toxin 1 (Ts1) and Toxin 6 (Ts6) stimulated the

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production of nitric oxide (NO), IL-6 and TNF-α in J774.1 cells. Toxin 2 (Ts2) alone

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stimulated the production of IL-10, suggesting an anti-inflammatory activity of Ts2.

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These findings are important for understanding the mechanisms involved in macrophage

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activation following scorpion envenomation (Zoccal et al. 2011), and these data is

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summarized in Table 1.

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Furthermore, there are clear examples of a cytokine cascade or cytokine storm

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when the presence of scorpion venom or toxins and the immune system goes out of the

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normal control and an inflammatory response also flares out of control. Although the

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general notion of an excessive or uncontrolled release of proinflammatory cytokines is

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well known, the concept of a cytokine storm and the biological consequences of cytokine

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overproduction are not clearly defined and the precise reason for this is not entirely

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understood, which may be caused by an exaggerated response when the immune system

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encounters a new and highly pathogenic invader (Tisoncik et al. 2012). It is known that

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the inflammation associated with a cytokine storm begins at a local site and spreads

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throughout the body via the systemic circulation. Redness, swelling or edema, heat,

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dolor, pain and loss of function are the hallmarks of acute inflammation. When localized

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in skin or other tissue, these responses increase blood flow, enable vascular leukocytes

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and plasma proteins to reach extravascular sites of injury, increase local temperatures

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(which is advantageous for host defense against bacterial infections), and generate pain,

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thereby warning the host of the local responses (Tisoncik et al. 2012). The pathology

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described above is highly related with a classic scorpion envenomation symptom.

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In order to evaluate what kind of scorpion peptide toxins are involved in cytokine

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induction on human immune cells, three typical buthid scorpion venom peptides were

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selected, one with lethal effects on mammals (CssII), another with lethal effects on

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insects and crustaceans (Cn5) and one more without lethal effects (Discrepin). Both CssII

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and Cn5 are 66 amino acid residue long peptides with four disulfide bridges isolated from

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the Mexican scorpions Centruroides suffussus suffusus and Centruroides noxius,

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respectively (Corzo et al. 2009, Saucedo et al. 2012), and Discrepin is a 38 residues

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short peptide with three disulfide bridges originated from the Venezuelan scorpion Tityus

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discrepans. CssII and Cn5 modifies the voltage-gated sodium channel currents, and

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Discrepin blocks preferentially the Ia currents of the voltage-gated potassium channels of

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rat cerebellum granular cells; however, it is no toxic to neither mammalians nor

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crustaceans (Prochnicka-Chalufour et al. 2006). Therefore, we performed a cytokine

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screening on human PBMCs stimulated with such scorpion toxins in order to evaluate

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their effect on human immune cells.

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2. Material and methods

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2.1. Peptide Toxins

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CssII and Cn5 were isolated from C. suffusus suffusus lyophilized crude venom

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(10 mg) according to Hernández-Salgado et al. (2009). Discrepin was chemically

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synthesized according to Prochnicka-Chalufour et al. (2006). Briefly, the crude venom

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was dissolved in 500 µL of 0.1% aqueous trifluoroacetic acid (TFA), and the insoluble

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material was removed by centrifugation at 14,000 g for 5 min. The soluble venom was

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used directly for high performance liquid chromatography (HPLC) fractionation using a

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reverse-phase semi-preparative C18 column (5C18MS, 10 x 250 mm Nacalai-Tesque,

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Japan) equilibrated in 0.1% trifluoroacetic acid (TFA), and eluted with a linear gradient

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of acetonitrile from 0 to 60% in 0.1%TFA, run for 60 min at a flow rate of 2 mL/min.

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Effluent absorbance was monitored at 280 nm. Fractions were collected in 1.5 mL tubes

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and dried out under vacuum. The HPLC fractions of interest were further purified by

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cation-exchange chromatography on TSK-gel sulfopropyl column (SP-5PW, 4.6 x 75

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mm, Tosoh, Japan). The fractions were diluted to 200 µL with 20 mM ammonium acetate

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in 1 M acetic acid pH 2.9, and they were further fractionated using a linear gradient of 2

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M ammonium acetate in 1 M acetic acid pH 5.9 from 0 to 50%, in 50 minutes (1

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mL/min). Eluted proteins were monitored by conductivity. If required, a final step

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purification, essentially for extra desalting, was performed in a C18 reverse-phase column

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(4.6 x 250 mm, Nacalai Tesque, Japan) equilibrated in 0.1% TFA, and eluted with a

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linear gradient of acetonitrile from 20 to 60% in 0.1% TFA, run for 60 min at a flow rate

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of 1 mL/min. Effluent absorbance was monitored at 230 nm. The identity of the scorpion

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peptides was confirmed by mass spectrometry as described (Zamudio et al. 1992).

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2.2. Peripheral blood mononuclear cells (PBMCs) isolation

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Human healthy PBMCs were isolated from whole blood by using the Ficoll-Paque

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method reported by the reagent provider (GE-Healthcare Bio-Sciences AB, Uppsala,

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Sweden). Fresh PBMCs were maintained in complete RPMI -1640 media (cRPMI),

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supplemented with 25 mM HEPES (GIBCO, Life Technologies, Co.), non essential

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Amino Acids (GIBCO, Life Technologies, Co.), Penicillin/streptomycin antibiotics

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(GIBCO, Life Technologies, Co.), 2 mM glutamine (GIBCO, Life Technologies, Co.), 1

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mM sodium pyruvate (GIBCO, Life Technologies, Co.), and 10 % AB serum. PBMCs

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were maintained in CO2 atmosphere at 37°C for incubation periods. The racial/ethnic

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distribution of the donors was as follows: Male Caucasian (28 years); Male African-

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American (30 years); Female African-American (40 years) and Male Asian (35 years).

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Donors ranged in age from 28 to 40 years with a mean age of 33 years.

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2.3. PBMCs stimulation with scorpion toxins The experiment was standardized for each treatment with 250,000 cells (PBMCs)

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in 200 µL volume, on cRPMI media for 12 h incubation; at 37°C in a CO2 atmosphere.

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Scorpion toxins were resuspended in sterile Dulbecco’s Phosphate Buffered Saline

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(DPBS) (Sigma-Aldrich, St. Louis, MO). All of them were assayed at 1 µg/200 µL

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reaction volume. As positive controls PMA at 50 ng/mL and Ionomycin at 1ng/mL were

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used. As negative controls PBMCs were maintained in cRMPI media without stimuli and

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arouse with sterile DPBS. All experiments were done in triplicate using PBMCs obtained

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from human healthy donors.

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2.4. Interleukins secretion monitoring by Luminex

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PBMCs were incubated in a 96 well plate for 12 h after which the incubation plate

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were centrifuged at 1,200 rpm for 5 min, and supernatants were obtained for further

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screening of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40, IL-13, IFN-γ, IFN-α, TNF-α

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and IP-10 by Luminex technology.

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2.5. Monocytic THP-1 cell line culture and stimulation with scorpion toxins THP-1 cell line were maintained in RMPI-1640 media (GIBCO, Life

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Technologies, Co.), Penicillin/streptomycin antibiotics (GIBCO, Life Technologies, Co.),

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2 mM glutamine (GIBCO, Life Technologies, Co.), the 10 % Fetal Bovine Serum (FBS),

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and maintained in CO2 atmosphere at 37°C for incubation periods. Scorpion toxin

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exposure was standardized for each treatment with 250,000 cells (PBMCs) in 200 µL

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volume, on RMPI media for 12 h incubation; at 37°C in a CO2 atmosphere. Scorpion

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toxins were resuspended in sterile Dulbecco’s Phosphate Buffered Saline (DPBS)

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(SIGMA-ALDRICH, St Louis, MO) all of them were assayed at 1 µg/200 µL reaction

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volume. As negative controls PBMCs were maintained in RMPI media without stimuli in

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sterile DPBS. All experiments were done in triplicate.

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2.6. Gene expression analysis by quantitative real time PCR (qPCR) RT-qPCR reactions were performed and reported according to MIQE guidelines

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(Bustin et al. 2009). Total RNA from THP-1 cells was obtained with the mirVana™

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miRNA Isolation Kit (Ambion–Life Technologies) in combination with DNase I

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treatment (Ambion) according to the manufacturer’s guidelines. Following extraction an

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Agilent 2100 Bioanalyzer (Agilent) was used to attain RNA Integrity Numbers (RIN) for

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each sample. All samples with RIN values > 5.5 were retained for further processing.

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RNA yields were attained using a Nanodrop 8000 (Nanodrop Technologies, Wilmington,

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DE). cDNA was synthesized from 500 ng RNA in a 20 µL reaction volume with

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Ambion® ArrayScript™ Reverse Transcriptase (10,000 units), random decamers (50

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µM) (Ambion) and dNTP Mix (2.5 mM) (Invitrogen), reaction was carried out at 42°C

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for 2 h. Enzyme was inactivated by incubation at 95°C for 5 min. All samples were

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diluted at a 1:25 ratio, with 1X TE Buffer, pH 8.0. Real time PCR reactions were

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performed in 384 reaction plates in a 10 µL final volume for each well, using 2 µL of

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cDNA diluted 1:25. TaqMan® Gene Expression Master Mix (Applied Biosystems®) was

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used for the reactions. Hydrolysis probes were purchased at Integrated DNA

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Technologies Company (www.idtda.com). Master Mix and TaqMan probe concentrations

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were used according to the manual provided in the reagent kit. qPCR reaction was

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performed in a real time instrument AB Applied Biosystems - ABI PRISM 7900 HT,

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using parameters: 1 cycle, 50°C per 2 min; holding 95 °C per 10 min; 40 Cycles: 95°C

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per 5s anneal/extend 60°C per 1 min. The genes actin beta (ACTB) and beta-2-

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microglobulin B2M were selected as reference genes standard curve in order to determine

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the hydrolysis probe efficiency (Supp. Material Figure 1).

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All data obtained were analyzed by using Bioplex Software (BIORAD, Hercules,

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CA). Three different methods were used to determine whether statistically significant

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differences occurred among the mean values obtained using the software package PRISM

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(GraphPad Software, Inc. La Jolla, CA). Dunnett's method was applied in analysis of

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variance (ANOVA) to create confidence intervals for differences between the mean of

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each factor level and the mean of a control group. Tukey's method was used in ANOVA

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to create confidence intervals for all pairwise differences between factor level means

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while controlling the family error rate to a level you specify. And Newman-Keuls to

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show significant difference between three or more sample means has been revealed

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by an ANOVA.

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3.0 Results

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In order to find the best time and toxin concentration to determine the amounts of

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secreted IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12-p40, IL-13, interferon alpha (IFN-α),

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interferon gamma (IFN-γ), tumor necrosis factor alpha TNF-

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protein-10 (IP-10), using a Luminex multiplex technology. A dose response experiment

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was performed, human PBMCs were incubated with the selected scorpion toxins

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(ScTx’s): CssII, Cn5 and Discrepin at three different concentrations (1, 0.33 and 0.11

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µg/mL), the readout of supernatants was carried out at 3 and 6 h after incubation at 37ºC

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under CO2 atmosphere conditions. The results obtained, suggest that lower concentration

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of scorpion toxins (0.11 µg/mL) and short time of incubation (3 h) was not sufficient to

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, and interferon-inducible

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induce cytokine production by PBMCs. However, PBMCs stimulated with the toxins at 1

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µg/mL, and incubated for 6 h, were able to secrete cytokines IL-1β, Il-6 and TNFα.

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Nevertheless, out of range bellow detection limit concentration results were obtained for

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the other panel of cytokines assayed such as: IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IFNα,

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IFNγ and IP-10. Based on these results, we decide to set up the next experiments using

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the scorpion toxins at 1 µg/mL and incubation time of 12 h. The results obtained from

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this experiment are shown on Supp. Material Figure 2. Cytokine profile data where the

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concentration was below the detection limit is not included.

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3.1. Cytokine profile induction by ScTx’s on human PBMCs (single toxin effect)

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Three scorpion toxins (ScTx’s) CssII, Cn5 and Discrepin were evaluated in their

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ability to modulate/induce cytokine secretion on human PBMCs. A Luminex multiplex

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analysis was performed in order to determine the amounts of secreted chemokines and

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cytokines. Human PBMCs isolated from two different healthy donors were incubated for

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12 h, at 37ºC under CO2 atmosphere conditions, with the different ScTx’s at 1 µg/mL,

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cells treated with sterile PBS or maintained on cRMPI without stimuli, were used as

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negative control. After incubation period, cells were centrifuged and supernatants were

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used to quantify/determine the analyte concentration. Cn5 induced the secretion of

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proinflammatory cytokines IL-1β, IL-6, and TNF-α (Figures 2A, E and K), anti-

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inflammatory cytokine IL-10 (Figure 2F), and chemokine IP-10 (Figure 2L) in

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comparison with CssII and Discrepin, which were able to induce only IP-10 secretion. A

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low amount of interferons α and β was induced by the ScTx’s assayed (Figure 2I-J). No

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induction of other cytokines was detected over levels seen in negative controls and these

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results are summarized in Figure 2. Positive controls of PBMCs stimulated with phorbol

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myristate acetate (PMA) or Ionomycin are show on Supp. Material Figure 3. The

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cytokine profile with this experiment, is consistent with the results obtained by other

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research groups were they report an increase in IL-1β, IL-6, IL-10 and TNF-α after the

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exposure of mice, rats and rabbits, or cells with scorpion venoms or purified toxins

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(Magalhaes et al. 1999, Fukuhara et al. 2003, Petricevich 2006, Adi-Bessalem et al. 2008,

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Liu et al. 2008, Abdoon and Fatani 2009, Zoccal et al. 2011, Zoccal et al. 2013,

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Hadaddezfuli et al. 2015, Saadi et al. 2015, Zoccal et al. 2016).

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3.2. Cytokine profile induction by ScTx’s on human PBMCs (synergistic effect screening)

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Since scorpion venoms are complex mixtures of peptides, proteins and small organic

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molecules and since such combination of molecules causes disturbance in the biological

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function of cells on target organisms, synergic experiments were performed in order to

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identify cytokine potentiation caused for the combination of ScTx’s. We decided to

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survey the possible effects of the toxins on the PBMCs by doing an experiment with

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same active concentration of selected toxins, the toxins were used at 1 µg/mL and

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incubation time of 12 h, these parameters were selected based on previous data, due the

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resolution obtained in cytokine quantification. The toxins were mixed and assayed in

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their capability to induce/modulate the cytokine secretion on human PBMCs. The

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combination of Discrepin and CssII did not show significant induction of IL-1β, IL-6, IL-

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10, TNF-α or IP-10 in comparison with the single effect obtained using the single toxins

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(Figures 3A-D). Combination of CssII and Cn5 also did not display synergic effects.

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However, the CssII abolishes the effect of Cn5 on their capability to induce IL-6, IL-10

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and TNF-α secretion (Figures 3B, C and D). Furthermore, no significant changes were

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observed on IL-1β or IP-10 (Figures 3A-E). Interestingly, the mixture of Discrepin and

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Cn5 shows considerable reverse effect on induction of IL-1β, IL-6, IL-10 and TNF-α

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(Figures 3A-D); and slight synergic effect on IP-10 induction (Figures 3E) in comparison

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with the single effect obtained with the Cn5 alone. A complete cytokine screening was

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performed and no effect of mixture was detected (Supp. Material, Figure 4). This

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preliminary screen uncovers some potentially interesting interactions that it will require

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further research.

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3.3. Cytokine profile induction by ScTx’s on THP-1 cell line (single toxin effect)

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CssII, Cn5 and Discrepin were evaluated in their ability to modulate/induce

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cytokine secretion from the monocytic cell line THP-1. A Luminex multiplex analysis

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was performed in order to determine the cytokine secretion and mRNA expression of IL-

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1β, TNF-

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atmosphere conditions, with the different ScTx’s at 1 µg/mL, cells treated with sterile

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PBS or maintained on cRMPI without stimuli were used as negative control. After

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incubation period, cells were centrifuged and used for RNA extraction and further gene

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expression analysis and the supernatants were used to quantify the cytokine

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concentration. The results obtained showed that Cn5 can induce the secretion of

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proinflammatory cytokines IL-1β and TNF-α (Figure 4A-B) and chemokine IP-10

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(Figure 4C). No significant amounts of cytokines were detected in cells treated with the

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ScTx’s CssII and Discrepin or negative controls. Relative gene expression analysis of IL-

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1β, TNF-

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consistent with the protein levels detected by Luminex assay, were Cn5 evoke the

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secretion and expression for these cytokines (Figure 4D-F), these results (protein

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secretion and gene expression) were evaluated in their association by a correlation

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analysis, were CssII and Cn5 displays a correlation (r) of 0.98 (p <0.09) and 1 (p<0.034),

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respectively, but lower values were obtained for Discrepin (r=0.22 and p<0.68) (Figures

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4G-I).

4.0 Discussion

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, and IP-10. THP-1 cells were incubated overnight at 37ºC under CO2

The ScTx’s assayed display an effect over the human blood cells causing

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disturbance and promoting the release of signaling molecules, these signals can mediate

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the recruiting of other cells, and promote or suppress inflammation (immune regulation).

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Our results directly indicate that encounter of scorpion toxin Cn5 with human peripheral

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blood mononuclear cells (PBMCs), triggers the secretion of pro-inflammatory cytokines

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IL-1β, IL-6 and TNF-α; anti-inflammatory cytokine IL-10 and interferon-inducible

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protein-10 (IP-10) (see Figures 2A, E, F, K and L). We found that the mixture of scorpion

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toxins Discrepin and CssII display a slight synergic effect promoting the secretion of IL-

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1β, IL-6, IL-10, TNF-α and IP-10 (Figure 3). When the combination of CssII with Cn5

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toxins was assayed, it was found that CssII abrogates the cytokine induction properties of

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Cn5 (Figure 3). Engrossing results were obtained with the mixture of Discrepin and Cn5;

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the data obtained with this treatment indicate that Discrepin may interfere/inhibit Cn5

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since the reduction of IL-1β, IL-6, IL-10 and TNF-α was observed (Figure 3A, B, C and

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D). Further dose–response experiments have to be performed in order to explain the

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mechanisms involved in the interference of effect observed between Discrepin and Cn5.

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One strategy to address this particular concern would be a dose-response experiment in

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transfected HEK cells with Kv4.3 channels, described as a Discrepin target (Picco et al.

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2014) and evaluate by patch clamp/electrophysiology approaches the effect of mix

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scorpion toxins, Cn5 and Discrepin. When the scorpion toxins were presented to the

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monocytic THP-1 cell line, the toxin Cn5 was able to induce the production of cytokines

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IL-1β, TNF-α and IP-10; therefore, the results are consistent with mRNA expression

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levels, data show in Figure 4. The broad repertoire of toxins within the scorpion venom,

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can impact in different ways the homeostasis of preys and predators – causing paralysis

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or death. The main targets of these toxins are the ion channels embedded on the

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membrane of many different cell lineages.

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The results obtained here are consistent with previous reports, where they show a

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pattern of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and the presence of anti-

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inflammatory cytokine IL-10, this is the most characteristic footprint of an organism or

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cell exposed to the whole venom or purified peptide toxins (Petricevich 2006, Petricevich

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et al. 2007, Fialho et al. 2011, Zoccal et al. 2011, Zoccal et al. 2013). Recently Zoccal et

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al. (2016) brought a new molecular concept of envenomation, which implicate the role of

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Tityus serrulatus scorpion venom regulating the inflammasome (Zoccal et al. 2016). In

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conclusion, an extensive screening of cytokines was conducted on human PBMCs

330

challenged with purified scorpion toxins, and assayed in their single or mixed (synergic)

331

effect. The profile of secreted cytokines (IL-1β, IL-6, IL-10, TNF-α and IP-10) promoted

332

by scorpion toxins are highly related with the cytokine storm event (D'Elia et al. 2013).

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PBMCs are not a homogenous cell population but are constituted by several

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immune cell types including, among others, B cells (~15 %), T cells (~70 %), monocytes

335

(~5 %), and natural killer (NK) cells (~10 %) (Corkum et al. 2015). Thus we expect that

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cells in PBMCs that respond may be a relatively minimum percentage of the total. The

337

results reported is this manuscript, in comparison to other reports is that cytokine

338

production was detected only at incubation times larger than 5 h after in vitro exposure to

339

purified toxins. Previous studies showing cytokine production at short times (2-5 h) used

340

enriched cell populations or plasma samples while our studies used total PBMCs where

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responding cells may be a minor subset of this population (Magalhaes et al. 1999, D'Suze

342

et al. 2003, Fukuhara et al. 2003, D'Suze et al. 2004, Petricevich 2006, Adi-Bessalem et

343

al. 2008, Liu et al. 2008, Abdoon and Fatani 2009, Zoccal et al. 2011, Zoccal et al. 2013,

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Ramirez-Bello et al. 2014, Hadaddezfuli et al. 2015, Saadi et al. 2015, Zoccal et al.

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2016).

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The venoms “hit early and strong” the host immune system causing imbalance

347

and triggering signals such as: inflammation, anti-inflammation, cell recruitment, etc.

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Given these points, the results support the theory of the synergic effect of venom

349

components, where there are some peptide toxins that target specific ion channels that

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causes death and others produces inflammation. The spectrum and degree of symptoms

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and signs of systemic envenoming are determined by several factors e.g. the scorpion

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species involved, body mass of the victim (children are more vulnerable than adults),

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amount of venom injected and the physical health of the sufferer.

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Further experimental analysis should be conducted in order to better

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understand the effect of whole venoms, single toxins and other venom components as

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mucoproteins, lipids, etc., on other mammal/human body systems such as: nervous

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system, immune system or reproductive system, with the horizon to find putative

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therapeutic targets to control neural disorders, immune suppressors or contraceptive

359

alternatives, respectively.

360 Acknowledgments

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This work received in part funding from the Direccion General de Asuntos del

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Personal Academico (DGAPA-UNAM) grant number IN204415 awarded to GC. The

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Baylor Scott and White Health Care System supported part of the work. The Baylor

365

Institute for Immunology Research BioBank and Project Management Core helped with

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blood samples. We would like to express our gratitude to Sandra Zurawski and Jerome

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Ellis for the technical help for the Luminex analysis. Gerard Zurawski is indebted for his

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support and editing of the manuscript.

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372 References

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toxin discrepin with Kv4.3 channels and A-type K(+) channels in cerebellum granular

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discrepin, a new K+-channel blocking peptide from the alpha-KTx15 subfamily."

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specific for Na+ channels and related peptides: biodiversity, structure-function

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toxin CssII from the venom of the scorpion Centruroides suffusus suffusus, and their

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effects on Nav1.5 sodium channels." Biochim Biophys Acta 1824(3): 478-487.

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(1992). "Amino acid sequence and immunological characterization with monoclonal

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antibodies of two toxins from the venom of the scorpion Centruroides noxius Hoffmann."

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Zoccal, K. F., S. Bitencourt Cda, A. Secatto, C. A. Sorgi, C. Bordon Kde, S. V. Sampaio,

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and Ts6 induce macrophage activation and production of immune mediators." Toxicon

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57(7-8): 1101-1108.

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Zoccal, K. F., S. Bitencourt Cda, C. A. Sorgi, C. Bordon Kde, S. V. Sampaio, E. C.

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Arantes and L. H. Faccioli (2013). "Ts6 and Ts2 from Tityus serrulatus venom induce

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inflammation by mechanisms dependent on lipid mediators and cytokine production."

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Toxicon 61: 1-10.

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Zoccal, K. F., C. A. Sorgi, J. I. Hori, F. W. Paula-Silva, E. C. Arantes, C. H. Serezani, D.

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S. Zamboni and L. H. Faccioli (2016). "Opposing roles of LTB4 and PGE2 in regulating

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the inflammasome-dependent scorpion venom-induced mortality." Nat Commun 7:

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10760.

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Tables

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Table 1. Pro and anti-inflammatory cytokines are induced by the effect of scorpion

476

venoms and toxins Scorpion/Toxin

Experimental

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Cytokines produced

References

IL-1β, IL-4, IL-6, IL-10 and TNF-α NO and paw edema IL-1β, IL-1a, IFN-γ, IL-6, IL-10 and TNF-α IL-6, IL-8, NO, and TNF-α IL-1β, IL-6, IL-8, IL-10, NO. TNF-α, IL-1α, IFN-γ and GM-CSF IL-1β, IL-6 and TNF-α IL-6 and TNF-α

(Adi-Bessalem et al. 2008) (Liu et al. 2008)

Androctonus australis hector Buthus martensi Karch

Rats Rats

Centruroides noxius

Mice Rabbits

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Leiurus quinquestriatus

Human and Rabbits

Tityus serrulatus

Human Rams Mice macrophages

Ts1, Ts2 and Ts6 toxins

Mice, cell lines

Androctonus crassicauda

479 480 481 482 483

NO, IL-6, IL-10 and TNF-α

IL-12

(Abdoon and Fatani 2009) (Magalhaes et al. 1999, Fukuhara et al. 2003) (D'Suze et al. 2003) (D'Suze et al. 2004) (Ramirez-Bello et al. 2014) (Zoccal et al. 2011, Zoccal et al. 2013) (Hadaddezfuli et al. 2015)

IL-12

(Saadi et al. 2015)

IL-1β, Inflammasome

(Zoccal et al. 2016)

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Human monocytes Human monocytes Mice

NO and TNF-α

(Petricevich 2006)

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Tityus discrepans Tityus discrepans Tityus discrepans inflammatory toxins

Hemiscorpius lepturus

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484 485 486 487

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488

Figure legends

489 Figure 1. Tridimentional structure of scorpion toxins CssII, PDB: 2LI7 (A), Cn5,

491

PDB: 2KJA (B) and Discrepin, PDB: 2AXK (C) and Multiple alignment of scorpion

492

toxins (D).

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Figure 2. Effect on cytokine secretion in human PBMCs by the scorpion toxins:

495

CssII, Cn5, and Discrepin; single effect experiment. A Luminex multiplex analysis

496

was performed to assess chemokine levels of interleukin (IL)-1β (A), IL-2 (B), IL-4 (C),

497

IL-5 (D), IL-6 (E), IL-10 (F), IL-12-p40 (G), IL-13 (H), interferon alpha (IFN-α) (I),

498

interferon gamma (IFN-γ) (J), tumor necrosis factor (TNF-

499

inducible protein-10 (IP-10) (L). Scorpion toxins were incubated at 1µg/200 µL for

500

12 h, at 37°C on CO2 atmosphere. Cytokine concentration is displayed as pg/mL in Y-

501

axis of the plot. A Dunnett’s multiple comparison tests were applied; the statistical tests

502

show significant differences (**p<0.001 ***p<0.0001).

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) (K) and interferon-

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Figure 3. Effect on cytokine secretion in human PBMCs by the scorpion toxins:

505

CssII, Cn5, and Discrepin; Single and synergic experiments (Major effect). A

506

Luminex multiplex analysis was performed to assess chemokine levels of interleukin

507

(IL)-1β (A), IL-6 (B), IL-10 (C), tumor necrosis factor (TNF-

508

inducible protein-10 (IP-10) (E). Scorpion toxins were incubated at 1µg/200 µL for

509

12 h, at 37°C on CO2 atmosphere. Cytokine concentration is displayed as pg/mL in Y-

510

axis of the plot. A Turkey’s multiple comparison tests were applied; the statistical tests

511

show significant differences (**p<0.001 ***p<0.0001).

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) (D) and interferon-

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Figure 4. Scorpion toxins, CssII, Cn5 and Discrepin differentially modulate the

514

cytokine secretion on THP-1 cell line. A-C, Luminex multiplex analysis was performed

515

to assess chemokine levels of interleukin (IL-1β) (A), interferon-inducible protein-10 (IP-

516

10) (B) and tumor necrosis factor (TNF-

517

specific TaqMan assays for genes IL-1β, IP-10 and TNF-α (D-F). A Newman-Keuls

) (C). Gene expression analysis by qPCR using

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518

multiple comparison tests were applied; the statistical tests show significant differences

519

(*p<0.01 **p<0.001). Correlation analysis of experiments assayed with scorpion toxins

520

(G-J). Scorpion toxins were incubated at 1µg/200 µL for 12 h, at 37°C on CO2

521

atmosphere. Cytokine concentration is displayed as pg/mL in Y-axis of the plot.

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522 Supplementary Material

524

Supp. Material Figure 1. THP-1 Standard Curve Plots: The standard curve was

526

generated on an ABI PRISM 7900 instrument by plotting the threshold cycle (Ct; see

527

Amplification Plot above) versus the log of the initial quantity of DNA for each reaction.

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Supp. Material Figure 2. Effect on cytokine production in human PBMCs by the

530

scorpion toxins: CssII, Cn5 and Discrepin; dose response experiment. A Luminex

531

multiplex analysis was performed to assess chemokine levels of IL-1β

532

D-F

and TNFα

-

, IL-6

G-I . A dose range (1 to 0.11 µg/mL) of each toxin was incubated

for 3 or 6 h, at 37 ° C on CO2 atmosphere. Cytokine concentration is displayed as

534

(pg/mL) in y-axis of the plot

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Supp. Material Figure 3. Positive controls, cytokine secretion on human PBMCs. A

537

Luminex multiplex analysis was performed to assess chemokine levels of interleukin

538

(IL)-1β (A), IL-2 (B), IL-4 (C), IL-5 (D), IL-6 (E), IL-10 (F), IL-12-p40 (G), IL-13 (H),

539

interferon alpha (IFN-α) (I), interferon gamma (IFN-γ) (J), tumor necrosis factor (TNF-

540

) (K) and interferon-inducible protein-10 (IP-10) (L). PBMCs isolated form healthy

541

donors were incubated in 200 µL volume, on cRMPI media for 12 h incubation; at 37°C

542

in a CO2 atmosphere and stimulated with PMA at 50 ng/mL and Ionomycin at 1 ng/mL.

543

As negative control PBMCs were maintained in cRMPI media.

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Supp. Material Figure 4. Effect on cytokine secretion in human PBMCs by the

546

scorpion toxins: CssII, Cn5, and Discrepin; synergic experiment.

547

multiplex analysis was performed to assess chemokine levels of interleukin (IL)-1β (A),

548

IL-2 (B), IL-4 (C), IL-5 (D), IL-6 (E), IL-10 (F), IL-12-p40 (G), IL-13 (H), interferon

A Luminex

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alpha (IFN-α) (I), interferon gamma (IFN-γ) (J), tumor necrosis factor (TNF-

550

interferon-inducible protein-10 (IP-10) (L). Scorpion toxins were incubated at 1µg/200

551

µL for 12 h at 37°C on CO2 atmosphere. Cytokine concentration is displayed as pg/mL in

552

Y-axis of the plot. A Dunnett’s multiple comparison tests were applied; the statistical

553

tests show significant differences (**p<0.001 ***p<0.0001). No significant statistical

554

data is not included.

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Highlights •

Cytokine screening on human peripheral blood mononuclear cells (PBMCs)

•

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stimulated with selected scorpion toxins

Scorpion toxins: Centruroides suffussus suffusus toxin II (CssII), Centruroides noxius toxin 5 (Cn5), and Tityus discrepans toxin (Discrepin).

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Luminex multiplex analysis of chemokines and cytokines

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