Effect of Vipera ammodytes ammodytes Snake Venom on the Human Cytokine Network

Local inflammation is a well-known symptom of envenomation by snakes of the family Viperidae, attributed primarily to the phospholipase A2s, metalloproteinases and L-amino acid oxidases contained in their venom. The inflammatory effect of snake venoms has been associated with a marked increase of the cytokines IL-1β, IL-6, IL-8, IL-10 and TNF-α. To determine the impact of Vipera ammodytes ammodytes snake venom on the expression of inflammation-related genes, we incubated human U937 monocyte cells with dilutions of snake venom. Gene expression was quantified for 28 different genes using a TaqMan® Array Human Cytokine Network 96-well Plate in a RT-qPCR system. Our results have demonstrated that 1.0 μg/mL Vipera ammodytes ammodytes venom solution induces a notable change in the expression of several cytokine network genes. Among the upregulated genes, there were several that encode interleukins, interferons, and tumor necrosis factors. We further report the downregulation of three interleukin-related genes. Our findings come as supportive information for the known complex effect of snake venoms on the human cytokine network. It also provides relevant new information regarding the expression of genes that have not been previously associated with the effect of snake venoms.


Introduction
The inflammatory process represents a defense mechanism of the body against harmful pathogens, damaged cells, or irritating substances. Inflammation can take an acute or chronic form. In its acute form, five typical signs of inflammation are usually present: heat, pain, redness, swelling, and loss of function of the affected tissues or organs. Chronic inflammatory processes are characterized by a species in tropical countries, these cases still represent a public health concern, mainly in the Balkan countries [27,28]. While there are several studies focused at isolating and characterizing proteins from the venom of V. ammodytes [25,[29][30][31][32], reports are scarce on the effects of unfractionated venom-a complex mixture of biologically active proteins-in humans.
Our study aimed to determine the effect of Vipera ammodytes ammodytes venom (VaaV) on the human cytokine network. Using a TaqMan ® Array Plate quantified by RT-qPCR, we assessed the expression of 28 cytokine-associated genes in monocytes treated with VaaV.

Effect of Treatment
Viability of cells treated with VaaV solution was assessed by microscopic evaluation. VaaV caused cell death at concentrations of 3.0, 10, 30, and 100 µg/mL. Monocyte cells treated with 1.0 µg/mL VaaV solution showed differentiation towards macrophage lineage as suggested by adherent polygonal cellular shape and growth arrest (Supplementary Figure S1). Cells incubated without treatment were assessed as viable and lacking signs of differentiation (Supplementary Figure S2). Total RNA was isolated from cell cultures deemed viable, namely those treated with 1.0 µg/mL VaaV and untreated cells.

Gene Expression in U937 Cells Treated with VaaV
Gene expression was assayed in triplicate using a TaqMan ® Array Human Cytokine Network Plate containing 28 genes coding inflammatory mediators and four endogenous control genes. The endogenous control genes (18S, GAPDH, HPRT1, GUSB) allowed for the correction of potential variations in RNA loading. Based on the obtained data, mean fold changes and standard errors were calculated if at least two relative quantification (RQ) values could be measured for one individual gene. The mean fold change of genes following treatment of cells with 1.0 µg/mL VaaV solution is presented in Figure 1 in a log2 RQ-based scale. A complete list of genes, obtained RQ values, calculated mean RQs, standard errors, and 90% confidence intervals are presented in Supplementary Table S1. Toxins 2018, 10, x FOR PEER REVIEW  3 of 10 countries [27,28]. While there are several studies focused at isolating and characterizing proteins from the venom of V. ammodytes [25,[29][30][31][32], reports are scarce on the effects of unfractionated venom-a complex mixture of biologically active proteins-in humans.
Our study aimed to determine the effect of Vipera ammodytes ammodytes venom (VaaV) on the human cytokine network. Using a TaqMan ® Array Plate quantified by RT-qPCR, we assessed the expression of 28 cytokine-associated genes in monocytes treated with VaaV.

Effect of Treatment
Viability of cells treated with VaaV solution was assessed by microscopic evaluation. VaaV caused cell death at concentrations of 3.0, 10, 30, and 100 μg/mL. Monocyte cells treated with 1.0 μg/mL VaaV solution showed differentiation towards macrophage lineage as suggested by adherent polygonal cellular shape and growth arrest (Supplementary Figure S1). Cells incubated without treatment were assessed as viable and lacking signs of differentiation (Supplementary Figure S2). Total RNA was isolated from cell cultures deemed viable, namely those treated with 1.0 μg/mL VaaV and untreated cells.

Gene Expression in U937 Cells Treated with VaaV
Gene expression was assayed in triplicate using a TaqMan ® Array Human Cytokine Network Plate containing 28 genes coding inflammatory mediators and four endogenous control genes. The endogenous control genes (18S, GAPDH, HPRT1, GUSB) allowed for the correction of potential variations in RNA loading. Based on the obtained data, mean fold changes and standard errors were calculated if at least two relative quantification (RQ) values could be measured for one individual gene. The mean fold change of genes following treatment of cells with 1.0 μg/mL VaaV solution is presented in Figure 1 in a log2 RQ-based scale. A complete list of genes, obtained RQ values, calculated mean RQs, standard errors, and 90% confidence intervals are presented in Supplementary  Table S1.

Upregulation of Interleukin-Related Genes
Our results showed that IL1A and IL1B genes presented a significant upregulation, with a mean fold change of 4.67 and 7.21, respectively. These genes encode two members of the IL-1 family: interleukin 1 alpha (IL-1α) and beta (IL-1β). The IL-1 family of cytokines has a major role in the initiation and regulation of inflammation. These cytokines possess a pronounced proinflammatory effect and are capable of inducing the expression of several other cytokines and chemokines, including IL-8 [33,34]. Our findings partially correlate with data available in the literature, as increased levels of IL-1β have frequently been reported following administration of snake venoms [35,36], svPLA 2 s [13,37], or SVMPs [12,20]. However, the expression of IL-1α following envenomation with snake venoms has not yet been the focus of research. Our finding that its gene is similarly upregulated as that of IL-1β suggests that IL-1α might have a pivotal role in the inflammatory process.
IL-10 is an immunosuppressive and anti-inflammatory cytokine that regulates and restrains the inflammatory response by limiting the production of cytokines and chemokines in macrophages and dendritic cells as well as by downregulating the expression of several chemokine receptors [34,38]. Increased IL-10 concentrations have been detected in human patients following envenomation with Daboia russelii venom [39] as well as in mice after administration of Crotalus durissus terrificus [40] and Bothrops spp. venom [41]. Furthermore, several studies have found that administration of SVMPs and svPLA 2 s isolated from Bothrops species leads to a marked increase in IL-10 expression [12,18,19]. The expression of IL-10 provides evidence that snake venoms are capable of modulating the expression of both pro-and anti-inflammatory cytokines. In accordance with these results, we report a significant upregulation of the IL10 gene (5.32-fold) following treatment with VaaV.
We found an approximately 1.4-fold increase in the expression of IL16, the gene encoding interleukin 16 (IL-16). IL-16 is a proinflammatory cytokine that functions as a chemoattractant for CD4 + and CD8 + T cells [34,42]. The expression level of IL16 in our study is a noteworthy finding, considering the increase of IL-16 levels has not been associated with the effects of snake venoms.

Downregulation of Interleukin-Related Genes
Among the studied genes, we observed the downregulation of two interleukin-encoding genes-IL18 and IL12A-responsible for the expression of interleukin 18 (IL-18) and interleukin 12 subunit alpha (IL-12α), respectively. Although statistically not significant, the results also show an indicative trend of downregulation for IL12B, the gene responsible for the expression of interleukin 12 subunit beta (IL-12β).
We identified a few cases of increased expression of IL-12 following administration of snake venoms or its components [43][44][45] but did not find any reports regarding the expression of IL-18. As both IL-12 and IL-18 induce the production of IFN-γ [34], the downregulation of the genes encoding these cytokines supports our findings regarding the lack of expression of IFNG, the gene responsible for encoding IFN-γ.

Upregulation of Chemokine-Related Genes
A marked upregulation following treatment of U937 cells with VaaV was observed for IL8 (6.97-fold increase), the gene encoding interleukin 8 (IL-8). The marked increase in IL8 expression suggests that IL-8 might be a significant mediator of inflammatory processes induced by snake venom. IL-8, or C-X-C motif chemokine ligand 8 (CXCL8), is a member of the CXC chemokine family. Its main function involves the recruitment of neutrophils to the site of injury or infection but also functions as a potent chemoattractant for other cell types, including basophils, eosinophils, NK cells, and T cells [34,46]. Release of IL-8 from neutrophils has been reported following in vitro treatment of human neutrophils with Bothrops bilineata venom [47] and Cr-LAAO, an L-amino acid oxidase isolated from Calloselasma rhodosthoma [22]. [48]. One of these mechanisms involves the direct activation of CD4 + and CD8 + T cells and dendritic cells and the subsequent release of various cytokines [49,50].

Interferon alpha (IFN-α) and interferon beta (IFN-β) are members of a highly related protein group called type I interferons (IFN-I). The main function of IFN-I consists of the induction of antiviral responses in cells through different mechanisms
Our results showed a marked increase in the interferon-related gene IFNB1 (2.7-fold), the gene responsible for the expression of IFN-β. Although statistically not significant, the results also show an indicative trend of upregulation for IFNA2 (6.57-fold), the gene responsible for the expression of a variant of IFN-α. To the best of our knowledge, we are the first to report the upregulation of interferon-related genes in connection with snake venoms that may support the antiviral activity of certain snake venom components, as suggested by previous reports in other contexts [51,52].

Upregulation of Tumor Necrosis Factor-Related Genes
The tumor necrosis factor superfamily represents a group of cytokines that play an important role in inflammatory processes, immunity and cell proliferation, differentiation and apoptosis, and the formation of secondary lymphoid organs. Tumor necrosis factor alpha (TNF-α) is secreted by macrophages in the acute phase of an inflammation, while lymphotoxin alpha (LT-α, TNF-β) is produced by activated type 1 T helper (Th1) lymphocytes. Both have a pronounced proinflammatory effect and have a significant role in cell apoptosis and tissue necrosis [53][54][55]. The latter is also involved in peripheral lymphoid organogenesis [56].

Limitations of the Study
The study design does not allow for the differentiation between the primary effect on gene expression induced directly by VaaV and the secondary effect on gene expression caused by the cytokines released following the action of VaaV treatment.
Furthermore, the use of unfractionated VaaV venom in the study does not allow the determination of the contribution to the overall observed effect by individual components contained in the venom. However, the current study design can be easily adapted to measure the effect of individual proteins on gene expression.

Conclusions
We report the influence of Vipera ammodytes ammodytes venom on the expression of a large number of inflammation-related genes in monocytes/macrophages. Various authors have reported the increased expression of IL-1β, IL-6, IL-8, IL-10, and TNF-α cytokines as a consequence of administration of snake venoms or the components thereof. We determined that the genes related to these cytokines, except the gene encoding IL-6, were markedly upregulated in our experiment. Thus, our findings come as supportive information for previous observations. Furthermore, we identified other upregulated genes, namely IL1A, IL16, IFNA2, and IFNB1. To the best of our knowledge, the cytokines encoded by these genes have not been previously associated with the effect of snake venoms or their components. Additionally, we report the downregulation of several interleukin-related genes, namely IL12A, IL12B, and IL18. Better understanding of the mechanisms and mediators involved in the inflammatory response following envenomation with snake venoms could be of potential use in the development of targeted venom antiserums.

Snake Venom
Lyophilized Vipera ammodytes ammodytes venom was obtained from the Institute of Immunology, Zagreb, Croatia. A stock solution with a concentration of 10 mg/mL was prepared by dissolving the lyophilized VaaV in phosphate buffered saline (PBS) (Lonza, Basel, Switzerland).

Treatment of Cells
U937 cell cultures were each treated with 1.0, 3.0, 10, 30, and 100 µg/mL VaaV solution for 48 h. Cells incubated without treatment were used as negative control. Following incubation, cell viability was assessed microscopically. Cells deemed viable were collected in RA1 lysis buffer (Macherey-Nagel GmbG, Düren, Germany) and stored at −80 • C until further analysis.

RNA Isolation and cDNA Construction
To determine the expression of genes associated with the human cytokine network, total RNA was isolated from cell cultures treated with 1.0 µg/mL VaaV solution and untreated cell cultures (negative control). Total RNA was isolated using a NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany) based on the manufacturer's recommended protocol. The obtained RNA concentrations were determined using a Nanodrop 2000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA). The isolated RNA was reverse transcribed to cDNA with a PikoReal 96 Real-Time PCR System (Thermo Scientific, Waltham, MA, USA) using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The PCR program used for cDNA synthesis consisted of sample incubation for 2 min at 50 • C and 10 min at 95 • C, followed by 40 cycles at 95 • C for 15 s and 60 • C for 60 s.

RT-qPCR
Gene expression was determined using a TaqMan ® Array Human Cytokine Network 96-well Plate (Part No. 4414255, Applied Biosystems, USA). The TaqMan ® Array Plate contains 28 assays for genes associated with pro-and anti-inflammatory cytokines and four assays for candidate endogenous control genes.
The quantitative real-time PCR amplification was performed on a 7500 Real-Time PCR System (Applied Biosystems, USA) in a 20 µL volume containing TaqMan Universal PCR Master Mix (Applied Biosystems, USA) and the cDNA samples. The PCR program used consisted of sample incubation for 2 min at 50 • C and 10 min at 95 • C, followed by 40 cycles at 95 • C for 15 s and 60 • C for 1 min. All assays were plated in triplicate. The obtained amplification data was evaluated using the 7500 Software v2.0.6 (Applied Biosystems, USA). Calculations and statistical analysis of the results was performed using Graphpad Prism software (version 6.0), GraphPad Software, La Jolla, CA, USA).

Supplementary Materials:
The following are available online at http://www.mdpi.com/2072-6651/10/7/259/s1, Figure S1: Monocyte cells treated with 1.0 µg/mL VaaV solution showing differentiation towards macrophage lineage as suggested by adherent polygonal cellular shape and growth arrest. Figure S2: Viable monocytes, lacking signs of differentiation following incubation without treatment, serving as negative control. Table S1: RQ values measured following treatment of U937 cells with 1.0 µg/mL VaaV. All assays were plated in triplicate.
Untreated cells served as reference (negative control). Mean RQ, standard error, and 90% confidence interval (using t-distribution for small set of samples) were calculated if at least two values were measured.
Author Contributions: E.S. and K.K. conceived and designed the experiments; F.B., K.B., and K.G. performed the experiments; A.C. analyzed the data; L.B. and F.B. wrote the manuscript; K.K. refined the manuscript for publication. All authors read and approved the final manuscript.