Comprehensive study of resveratrol associated to sulfadiazine in lymphocytes infected with Toxoplasma gondii: involvement of purinergic signaling in immune response
Nathieli Bianchin Bottari, Priscila Marquezan Copetti, Bianca Fagan Bissacotti, Taís Vidal, Anielen Dutra Da Silva, Mateus Fracasso, Karine Paula Reichert, Jelson Nauderer, Vera Maria Melchiors Morsch, Cinthia Melazzo, Aleksandro Schafer Da Silva, Maria Rosa Chitolina Schetinger
TL;DR
This study explores how resveratrol affects immune responses in lymphocytes infected with Toxoplasma gondii, suggesting it could be a potential treatment for toxoplasmosis.
Contribution
The study identifies resveratrol's role in modulating purinergic signaling and immune responses during Toxoplasma gondii infection in lymphocytes.
Findings
Resveratrol increased ATP and ADP hydrolysis in infected lymphocytes.
Resveratrol upregulated P1 and P2 purinergic receptors in T. gondii-infected lymphocytes.
Resveratrol showed anti-inflammatory and antioxidant effects through adenosine receptors.
Abstract
Toxoplasma gondii is a highly successful intracellular pathogen. Its success is largely achieved by the parasite’s ability to avoid the host immune response. During T. gondii infection lymphocytes play an active role in host defense and purinergic signaling has been shown to contribute to parasite control. Several studies have demonstrated the importance of ATP (adenosine triphosphate) signaling via purinergic receptor, as a component of the inflammatory response against T. gondii. Here, we hypothesized whether RSV, a natural polyphenol, could be involved in T. gondii control triggered by purinergic signaling, during acute infection in lymphocytes. Thus, the outcomes of this study were lymphocyte viability, modulation of ectonucleotidase activity, purinergic receptor expression and inflammatory mediators. T. gondii infection diminished lymphocytes viability 24h after RH-tachyzoites…
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Taxonomy
TopicsAdenosine and Purinergic Signaling · Toxoplasma gondii Research Studies · Sirtuins and Resveratrol in Medicine
Introdução
In the last decade, several studies have been highlighted the mechanisms of immune evasion by Toxoplasma gondii [1, 2]. This opportunistic protozoan invades almost any nucleated cell and drives different strategies to successfully manipulate the host immune system to establish a long-term latent infection maintaining an optimal replicative niche [3]. During these processes, T. gondii regulates the host cell by modulating morphological, physiological, immunological, genetic, and cellular biological aspects of the host cell [4, 5].
Upon infection, host cells are modulated to keep a delicate balance between facilitating and eliminating the infection [6]. A key part of this balance involves recognition of T. gondii by innate and adaptative immune cells, such as lymphocytes, the primary cell type that controls the parasite in the mammalian adaptive immune response [7, 8]. However, the extension of these studies to human cells remain not understood.
During the early stages of infection, T. gondii-specific antigens are rapidly recognized by dendritic cells (CD), macrophages and neutrophils. Once activated, they release cytokines such as IL-12 and IFN-γ that bias the host response toward a Th1 inflammatory environment which leads to the development of immune protection mediated primarily by memory CD8 + T cells and require CD4 + T cell help for priming. [8, 9]. In addition, replicating parasites lyse host cells, disseminate, and lead to extensive host tissue destruction and enhanced inflammation [2]. Generation of T lymphocytes possessing parasite-specific cytolytic activity is a characteristic of both human and murine infection.
Nucleotide-mediated signaling is also considered a common pathway for host immune response against intracellular pathogens, such as T. gondii [10]. Once established the T. gondii infection, adenosine 5´-triphosphate (ATP) is release from apoptotic and activated immune cells to extracellular medium [11]. Extracellularly, ATP acts as a Danger Associated Molecular Patterns (DAMPs) and initiate signaling cascades to induce an inflammatory response [12]. In the extracellular compartment, ATP can be sensed by P2 purinergic receptors that are activated solely by ATP to mediate the influx or efflux of various cations (Na^+^, Ca^2+^, K^+^) in cells [13]. P2X and P2Y receptors are present on a variety of immune cells, including lymphocytes. The P2X_7_ receptor has been suggested to play an important role in ATP-induced inflammation mainly in T. gondii infections [14–16].
The pool of ATP is controlled by ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1 or CD39) enzyme, which dephosphorylated ATP or ADP into AMP. Subsequently 5’-ectonucleotidase (or CD73) enzyme hydrolyze AMP into adenosine (Ado), which is desamined by ADA enzyme. Members of this enzyme family are expressed on the surface of lymphoid cell [17]. Different of ATP, adenosine (Ado) exhibits mostly immune suppressive effects. Ado inhibits Th1 and Th2 differentiation by decreasing T cell proliferation and IL-2 production. Many of these effects are regulated by the A2 receptors [18].
One of the most important cellular response upon stimulation of the P2X_7_ is associated to the NLRP3 inflammasome activation in infections mediated by intracellular parasites, such as T. gondii [19]. P2X_7_-mediated canonical activation of NLRP3 and IL1-β secretion induced T. gondii control in macrophages by a mechanism dependent on IL-1β-induced mitochondrial ROS production [20, 21]. ATP release also induces reactive species production in various pathologies, including intracellular pathogen infections [22].
Despite all mechanisms reported, no current drug treatments that trigger hypersensitivity reactions are available, making the discovery of new therapeutic approaches highly important research priority [23]. In this way, elucidation of the molecular mechanisms underlying regulation of the protective immune response will provide important clues for understanding the basis of successful long-term interaction between parasites and host.
Previous studies conducted by our research group has been investigate the effect of resveratrol (RSV), a natural polyphenol found in grapes as a candidate to treatment of toxoplasmosis [24, 25]. RSV showed benefic effects against T. gondii and also modulate purinergic signaling in infected mice. Chen et al. [26], successfully showed that RSV inhibit the growth of RH tachyzoites presumably through the deprivation of redox homeostasis in infected macrophages culture. Moreover, RSV has been related to a non-selective adenosine receptor agonist [27]. Thus, this study aimed to evaluate the effects of resveratrol (RSV) on the viability and oxidative stress of T. gondii-infected lymphocytes, focusing on the role of purinergic signaling in driving the host immune response during toxoplasmosis.
Material and Methods
Parasite Maintenance and Culture
The type I virulent RH strain of T. gondii (RH) was stored in phosphate-buffered saline (PBS) solution containing 20% glycerin in liquid nitrogen until analysis. The RH strain was removed from liquid nitrogen and recovered in a water bath of 37 °C for 30 min and then maintained in vitro by serial passage on human foreskin fibroblasts (HFF). Cells were grown in complete growth medium RPMI (Gibco, USA) supplemented with 10% fetal bovine serum (FBS). When over 90% of the infected cells consisted lysed, the parasites were obtained and followed by centrifugation at 100 × g for 10 min at 4 °C, the supernatants were collected, centrifuged at 2.000 × g for 10 min to remove the supernatants, and resuspended RPMI 1640 medium supplemented with 5% fetal bovine serum at 37 °C in 5% CO_2_ (for treatment). Tachyzoites sediment were counted in a Neubauer chamber before distribution and were added at 3:1 parasite-to-cell basead to Ehmen and Lüder [28].
Isolation of Polymorphonuclear Cells (PMNs)
Peripheral blood mononuclear cells (PBMC) were obtained from wisely healthy volunteers sample according the approval of the Research Ethics Committee of the Human of Federal University of Santa Maria (CAAE:13192719.0.0000.5346). Each independent experiment corresponded to cells obtained from a different donor.
Peripheral venous blood was collected in an EDTA-containing tube. Polymorphonuclear cells (PMNs) were isolated by Ficoll-Hypaque density gradient separation. Briefly, 30 ml (ml) of blood was diluted with saline 0.9% buffer and layered on an equal amount of Histopaque-1077 (Sigma-Aldrich, St. Louis, Missouri, USA). Blood components were separated by 30-min centrifugation at 2.000 rpm, 25 °C without brake plasma and PBMC-rich interphase were removed. Contaminating erythrocytes were removed from PMN-rich supernatant by hypotonic lysis using sterile water, after which the PMNs were resuspended in medium. Purity of cells was determined by differential staining with Giemsa stain, and the viability of cells was assessed by trypan blue exclusion. The cells were plated for culture maintenance at a density of 1 × 10^5^cells/cm^2^ and incubated 24, 48 and 72 h at 37 °C, in 5% CO_2_, and the medium with parasite and treatments.
Treatments
Resveratrol (RSV, C_14_H_12_O_3_; molecular weight 228.25 g/mol; purity > 98%, Sigma Aldrich) were diluted in DMSO 0.1% and added to the growth medium at final concentrations of 1 to 75 µM. Sulfamethoxazole-trimethoprim (Bactrim®) were dissolved in DSMO 0.1% and RPMI medium (Invitrogen-Gibco) at final concentration of 25 μg/mL according to the methodology described by Sanfelic et al. [29]. Classical in vitro studies have shown that sulfonamides are effective against T. gondii within the low-to-mid µg/mL range, allowing parasite control while preserving host cell viability [30]. The treatments were incubed for 24hs after T. gondii infection.
Lymphocytes Morphology and Proliferation Assay
Approximately 90% of the cells obtained from PBMCs displayed typical lymphocyte morphology, characterized by small cell size, a high nucleus-to-cytoplasm ratio, round and dense nuclei, and scant cytoplasm, and were therefore considered lymphocytes. Lymphocytes were further confirmed by flow cytometry scatter properties (FSC-A vs.SSC-A) using singlet gatting and confirmed by FL1-H (BD FACSCalibur; BD Biosciences, La Jolla, CA).
After confirmation, lymphocytes were plated in culture flasks at an initial density of 1 × 10^5^ cells/cm^2^ in the presence or absence of RSV (1, 10, 25, 50, or 75 µM) and incubated 24 to 72 h. Images of viable lymphocytes under control conditions and after addition of RSV or SMX-TMP were recorded after 24 h using an inverted microscope (Nikon) coupled to a digital camera. Five micrograph images were taken per experimental condition.
Lymphocyte Viability by MTT Assay
The effects of the RSV on the viability of T. gondii-infected lymphocytes was evaluated based on mitochondrial oxidation using the colorimetric MTT (3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MI, USA) assay [31]. The experiments were performed in 96-well plates (1 × 10^4^ cells/well/ 200 μl) at intervals 24 h after T. gondii and/or RSV exposition. Control and treated cells were incubated with MTT at a final concentration of 1 mg/mL for 3 h. Then the cells were centrifuged, the medium was removed, and 50 µL isopropanol was added, with agitation for 1 min. The optical density of each sample was measured at 570 nm using a spectrophotometer microplate reader. The data are given as percentages of viable treated cells, relative to untreated control cells.
Enzymes Activities
For enzymatic assay, lymphocyte cells were washed three times with phosphate-free incubation medium in the absence of substrate after 24 h T*. gondii* or RSV exposition. Twenty microliters of cell suspension (0.8–1.0 mg/mL protein according to [32]) were added to the reaction mixture of E-NTPDase by method of Lunkes et al. [33] or E − 5′-nucleotidase by the method of Heymann et al. [34]. The samples were preincubated for 10 min at 37 °C in a final volume of 200 μL. The release of inorganic phosphate (Pi) was measured by the malachite green method. All assays were performed in triplicate. Activity was expressed as µmol Pi released/min/mg of protein.
ADA activity assays were performed as method previously described by Giusti and Galanti [35] as the measurement of ammonia produced when adenosine deaminase acts in excess of adenosine (Ado) substrate. For the assay, 50 μL of cell suspension was reacted for 60 min with 21 mmol/L of adenosine, pH 6.5, at 37 °C. The reaction was stopped with 106.2 mM phenol and 167.8 mM sodium nitroprusside and a hypochlorite solution. The amount of ammonia produced was measured by absorption at 620 nm, and results were expressed in units per liter (U ADO/mg protein).
Flow Cytometric Antibody Labeling Analysis
After 24 h treatments lymphocytes were dissociated and fixed for 20 min in ice-cold 1% PFA in PBS, washed with PBS supplemented with 2% FBS, and incubated for 30 min with primary antibodies against P2X_7_ (1:500 Santa Cruz), P2Y_12_ (1:500, Santa cruz), A1 (1:1000, Santa Cruz) receptors; CD39 (1:500 Santa Cruz) and ADA1 (1:500 Santa Cruz) enzymes. Alexa Fluor 555, 488 or 647-conjugated secondary antibodies (1:500) (Life Technologies, Carlsbad, CA) were added into cells and analyzed by flow cytometry (BD FACS Calibur; BD Biosciences, La Jolla, CA).
Lymphocytes were identified from total events by first excluding doublets (FSC-H vs FSC-A) and selecting viable cells. The lymphocyte population was then gated based on FSC-H vs FL1-H (Fig. 2D). Subsequent analysis of lymphocyte subpopulations was performed for CD39 + and ADA1 + cells**.** Thirty thousand events were acquired per sample. Data were analyzed by using the FlowJo V10 software (FlowJo, BD Biosciences) and plotted in dot plot format.
Western Blot Analysis
Protein density was performed by Western blot analysis of the P2X_7_, P2Y_11_ and A_2A_ receptors, and mature IL-1β and IL-10 cytokines. In brief, cells were initially homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer added with 1 mM phosphatase and protease inhibitors and centrifuged at 12.000 rpm at 4 °C for 10 min. Protein content was then determined using the Lowry method (Sigma-Aldrich). Subsequently, samples were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immun-Blot® PVDF membranes (Bio-Rad Laboratories). After blocking, membranes were incubated overnight at 4 °C with the primary antibodies: P2X_7_ (dilution 1: 500), P2Y_11_ (dilution 1: 500), A_2A_ (dilution 1: 800), and cytokines (dilution 1: 500), all obtained from Santa Cruz Biotechnology. After this step, membranes were washed with Tris-buffered saline (pH 7.6) with 0.1% Tween 20 (TBST) and further incubated with anti-rabbit or anti-mouse secondary antibodies (dilution 1: 10.000, Santa Cruz Biotechnology) at room temperature for 90 min. The membranes were washed again, incubated with an enhanced chemifluorescent substrate (Immobilon® Forte Western HRP Substrate, Merck), and analyzed with a ChemiDoc Imaging System (Bio-Rad Laboratories). The membranes were reprobed and tested for β-actin immunoreactivity as a control for the protein concentration. The band intensity was quantified via ImageJ 1.6.0 version to determine the relative protein density.
Quantification of ROS and Nitrite
T. gondii-infected lymphocytes in RPMI buffer were seeded into each well of a 24-well cell culture plate, followed by addition of 1 to 75 µM resveratrol, 30 µM H_2_O_2_, and DMSO for 12 h. DCFH-DA (2′,7′-dichlorofluorescein diacetate; 10 µM; Sigma Aldrich) was then added to the culture medium, and the tachyzoites were incubated for an extra 60 min before collection by centrifugation at 1.300 × g for 10 min and washing with sterile PBS. Pellets were resuspended in PBS and detected by use of the FITC channel of a flow cytometer (BD FACS Calibur; BD Biosciences, La Jolla, CA). NOx levels were measured according to Miranda et al. (2001), which indirectly measure quantities of nitrite/nitrate levels, and the results were expressed as μmol nitrite/mg protein.
Statistical Analysis
All of the data are expressed as means ± standard error of the mean (SEM) from at least three independent tests with three replicate wells per condition reported with 95% confidence intervals. Data were assessed for normality and homogeneity of variances were analyzed by two-way analysis of variance (ANOVA) with Tukey´s as post hoc test for multiples comparison using GraphPad Prism (version 5) software. Significant results were considered when p < 0.05.
Results
RSV Preserves Cell Viability of Lymphocytes Infected by T. gondii
Primary human lymphocytes were isolated from peripheral blood samples drawn from healthy donors and separated through a density gradient. The isolated cell population was analyzed by microscopy and was characterized by small cell size, a high nucleus-to-cytoplasm ratio, round and dense nuclei, and scant cytoplasm. Analysis of the cells by microscopy confirmed that the population consisted predominantly of lymphocytes due to the characteristic (Fig. 1A). The lymphocytes were incubated with T. gondii RH strain and 24 h after infection exposure to RSV (1–75 µM) or SMX-TMP (25 μg/mL) treatments for 24, 48 and 72 h. The cell viability was lower in T. gondii-infected cells 24 h when compared to uninfected. The SMX-TMP reduce cell viability in uninfected and infected cells in 24, 48 and 72 h compared to negative control. The RSV treatment did not alter lymphocytes viability in 24 h, beside even though increase cell viability 48 and 72 h in comparison to negative control. However, in T. gondii infected cells RSV treatment 1 up to 50 µM increase the percentage of viable lymphocytes analyzed by MTT after 24 (Fig. 1B) and 72 h (Fig. 1D) of T. gondii infection compared to positive control (infected group) (p < 0.001).
Fig. 1. Effect of RSV on lymphocytes viability exposed to* T. gondii*.** A**) Representative microscopic lymphocytes characterized by small size, a high nucleus-to-cytoplasm ratio, round and dense nuclei, and scant cytoplasm. Images 24 h after T. gondii-infection and RSV exposition. Scale bar indicate 40 µm** B**) Cell viability after 24h exposition;** C**) Cell viability after 48h exposition;** D**) Cell viability after 72h exposition. The experiments were performed in triplicate. Data are expressed as means ± S.E.M from representative three independent experiments performed by two-way ANOVA followed by Tukey´s post test. Significant values with p < 0.001. *compared to uninfected (control group) versus treatments; # compared to positive control (infected group) versus treatments
Effects of RSV on Ectonucleotidase and ADA Enzymes are Critical to Regulation of T. gondii Infection
The capacity of the peripheral blood lymphocytes to hydrolyze extracellular ATP and ADP by E-NTPDase and adenosine deamination by E-ADA was evaluated in this study (Fig. 2). RSV and SMX-TMP treatment did not alter ectonucleotidase and E-ADA in lymphocytes when compared to control group (p > 0.05). An increase of NTPDase were observed when ATP (30%) and ADP (17.5%) were used as substrate, as also E-ADA (55%) augmented in infected cells when compared to control (p < 0.05). However, ATP and ADP hydrolysis were lower when RSV free or combinate to SMX-TMP were added into medium. The correspondent nucleoside adenosine was high in infected and treated cells (Fig. 2D).
Fig. 2RSV modulate ectonucleotidases in* T. gondii*-infected lymphocytes.** A**- NTPDase activity using ATP as substrate;** B**- NTPDase activity using ADP as substrate;** C**- E- ADA;** D**- 3D dot plot of CD39 on using R-Phycoerythrin (R-PE)-conjugated mouse anti-human monoclonal antibody against CD39;** E**- Percentage of CD39+ expression cell;** F**- ADA1+ expression analyzed by flowcytometry. The experiments were performed in triplicate. Data are expressed as means ± S.E.M from representative three independent experiments performed by two-way ANOVA followed by Tukey´s post test. Significant values with p < 0.05. ^*^ compared to uninfected (control group) versus treatments; # compared to positive control (infected group) versus treatments
The presence of CD39 was confirmed on the gate of lymphocytes infected with T. gondii and/or RSV incubation (Fig. 2D) by flowcytometry. The RSV treatment and SMX-TMP diminished the percentage of CD39^+^ lymphocytes when compared to negative control (p < 0.001) (Fig. 2E). Infected lymphocytes also presented a decrease on CD39^+^ cells when compared to negative control (p < 0.05). No significant differences on CD39^+^ treated cells were observed when compared to positive control (Fig. 2E) (p > 0.05).
In order to screen whether adenosine deaminase-1 (ADA-1), indispensable for T. gondii immune response the expression was analyzed (Fig. 2F). T. gondii-infected lymphocytes significantly decreased ADA-1 expression when compared to negative control (p < 0.05). However, the addition of RSV (50 μM) or SMX-TMP on infected cell culture was associated with higher frequency of rescued ADA-1^+^.
Resveratrol Upmodulate Purinergic Receptors in T. gondii-Infected Lymphocytes
After 24 h of T. gondii infection, lymphocytes were treated with RSV and/or SMX-TMP and purine receptors by cytometry and/or western blotting analysis were evaluated (Fig. 3). P2X_7_ receptor are augmented in infected lymphocytes when compared to negative control in both assays. RSV 10 and 50 μM reduce P2X_7_ receptor in T. gondii- infected cells when compared to positive control (infected). Similarly results can be observed when RSV 50 μM were associated to SMX-TMP (Fig. 3A, D).
Fig. 3. Role of resveratrol in purine receptors of* T. gondii*-infected lymphocytes.** A**- Percentage of P2X7+ cells;** B**- Percentage of P2Y12+ cells;** C**- Percentage of A1+ cells analyzed by Flowcytometry.** D**- P2X7 density confirmed by western blotting;** E**- P2Y11 density;** F**- A2A density. β-actin was used as a loading control to normalize protein levels. The experiments were performed in triplicate. Data are expressed as means ± S.E.M from representative three independent experiments performed by two-way ANOVA followed by Tukey´s post test. Significant values with *p < 0.05, **p<0.01. *compared to uninfected (control group) versus treatments; # compared to positive control (infected group) versus treatments. Representative original Western blot images in Figure S1
In order to established the role of nucleotide signaling mediated by P2 receptors, P2Y12 and P2Y11, involved in cell proliferation and chemotaxis, were analyzed (Fig. 3B, F). No significant differences were observed in marked P2Y12R^+^ lymphocytes RSV-treated in comparison to control. T. gondii-infected lymphocytes augmented P2Y12R^+^ when compared to negative control (p < 0.05). RSV 50 μM overexpress P2Y12R^+^ in infected cells, while RSV 50 μM plus SMX-TMP down express P2Y12R^+^ cells (Fig. 3B). The P2Y11R density was higher when RSV 10 and 50 μM were exposed to uninfected lymphocytes when compared to control (Fig. 3F). SMX-TMP reduce P2Y11R density in healthy lymphocytes (p < 0.05). T. gondii-infected cells promotes a high P2Y11 protein density when compared to negative control (p < 0.05). The treatments with RSV (10 and 50 μM) and RSV (50 μM + SMX-TMP) diminished P2Y11 density in T. gondii-infected lymphocytes compared to positive control (Fig. 3F).
For instance, adenosine signaling through the A1 and A_2A_ receptors were investigated by cytometry and western blotting respectively (Fig. 3C, E) once promotes chemotaxis to sites of infection and their adhesion to inflamed endothelium. A1R^+^ lymphocytes were augmented when exposed to RSV (10 and 50 μM) when compared to control in uninfected cells (Fig. 3C). The T. gondii-infection reduced the percentage of A1R^+^ lymphocyte compared to negative control (p < 0.05). In contrast, RSV treatments (10 and 50 μM) isolated and in combination with SMX-TMP (RSV 50 μM + SMX-TMP) over express A1 receptor (Fig. 3C) compared to positive control. On the other hand, T. gondii-infection augmented A_2A_ density in infected cells when compared to control (Fig. 3E). In contrast, RSV reduce A_2A_ density receptor when compared to control isolated or in combination to SMX-TMP (Fig. 3E) (p < 0.05).
RSV Regulate Inflammatory Mediators During T. gondii Infection
Until here, we demonstrate that during an inflammatory response against T. gondii, there are a close participation of purine enzymes and receptors. ATP from damaged cells, undergoing oxidative stress is released into the extracellular space. This extracellular ATP could amplify the inflammatory immune response through the recruitment and activation of immune cells such as T cells, neutrophils, and macrophages. Thus, oxidative parameters and cytokines were measured (Fig. 4). The data revels an increase Nox (Fig. 4B) in infected lymphocytes when compared to control. No significant results were observed when cells were treated with RSV (10 and 50 μM) for ROS levels. However, when the lymphocytes were outdared by T. gondii RSV treatment increase Nox levels possible stimulating inflammation and at the same time acts as a cell damage signal to promote cell migration to sites of tissue injury.
Fig. 4. Antioxidant and immunomodulator effect of RSV in* T. gondii*- infected lymphocytes.** A**- ROS levels analyzed by flowcytometry;** B**- NOx levels;** C**- Mature IL-1b density by western blotting;** D**- IL-10 density by western blotting. β-actin was used as a loading control to normalize protein levels. The experiments were performed in triplicate. Data are expressed as means ± S.E.M from representative three independent experiments performed by two-way ANOVA followed by Tukey´s post test.Significant values with ^^ p < 0.05. ^^compared to uninfected (control group) versus treatments; # compared to positive control (infected group) versus treatments. Representative original Western blot images in Figure S1
The pro-inflammatory cytokine IL-1β and the anti-inflammatory cytokine IL-10 were also determined (Fig. 4C, D) by western blotting analysis. The results indicate an increase of IL-1β (Fig. 4C) in infected lymphocytes in contrast to a decrease of IL-10 (Fig. 4D) levels when compared to control. RSV treatments diminished proinflammatory (IL-1β) and augmented anti-inflammatory cytokines levels in the supernatant medium containing infected lymphocytes exhibiting this biological anti-inflammatory effect.
Discussion
T. gondii elicits a robust innate immune response during infection through the signaling pathways that result in modulation of cell adhesion and migration, secretion of immunoregulatory cytokines, production of microbicidal molecules, and cellular apoptosis. Several studies considered new approaches and strategies for toxoplasmosis treatment including protein pathways regulation, drugs and vaccines [36]. Here, we demonstrate a molecular mechanism governing human immune evasion by T. gondii through purinergic signaling providing new insights into potential therapeutic targets that could contribute to minimize disease and improved outcomes for human health. Furthermore, we showed that RSV free and combinate to SMX-TMP (therapeutically drug choice for toxoplamosis) plays a protective role in host defense against T. gondii parasite, recommending this association.
First of all, different concentrations of RSV isolated and combinate to SMX-TMP were tested in RH strain of T. gondii-infected lymphocytes at 24, 48 and 72 h. As expected, 24 h after T. gondii exposition, viable lymphocytes diminished when compared to control (Fig. 1A). T. gondii tachyzoites invade host cells and replicate rapidly before inducing a potent immune response that controls parasite numbers through direct cellular killing. Along the time tested (48 and 72 h) no alterations in infected lymphocytes number and cell viability were observed, probably in response to reduce tachyzoites proliferation during the infection. Tonin et al., [37] have showed lymphocytes showed an increase of their blood levels when compared to the control group and diminished along the curse of infection.
RSV at high tested concentrations (10 to 50 μM) improve number of viable cells analyzed by MTT in normal and infected cells. The maximum concentration analyzed (75 μM) diminished cell viability. Thus, 10 and 50 μM concentration were used for sub sequentially analysis.
The effects of this biologically active compound on the immune system are associated with immune-regulatory property on immune cells mediated by sirtuin-1 (Sirt1) [38]. Its effect as an immunomodulator has been demonstrated in various animal models and in different cell lines as describe by Meng et al., [39]. Previous studies by our research group have demonstrated by histological analysis in liver and brain that RSV clearly diminished infiltrate lymphocytes in resveratrol-treated mice compared with the control mice [25, 40].
Enzymes activities were measured at 24 h after tachyzoites exposition. The results revel an increase of ATP (Fig. 2A) and ADP (Fig. 2B) hydrolysis by NTPDase in infected lymphocytes when compared to control. The function of ATP in the immune system is complex and may be concentration dependent, but in general ATP is involved in pro-inflammatory functions as stimulation of lymphocyte proliferation and cytokine release [41]. Our findings are in accordance with Tonin and colleagues [37] that showed E-NTPDase increased activities observed on lymphocytes of infected mice.
RSV-treated cells reduce ATP and ADP hydrolysis by NTPDase in infected cells (Fig. 2A, B). Resveratrol has immunomodulatory effects [39]. In a study conducted by Huang et al., [42] RSV produced anti-inflammatory effects by inhibiting canonical and non-canonical inflammasomes in macrophages suppressing ATP-induced transcription.
It well established that CD39 enzyme hydrolyses extracellular ATP (eATP) and ADP into AMP, while another ectonucleotidase, CD73, dephosphorylates AMP to adenosine [17]. Cells incubated with RSV demonstrated stable to mildly decreased levels of CD39 activity in infected group (Fig. 2E). The finding that CD39 is down-regulated in infected lymphocytes suggesting that CD39 may function as an immunomodulant via ATPase activity.
Adenosine, in contrast, is a labile molecule that binds to the adenosine receptor expressed on lymphocytes and dampens cell proliferation and inflammatory cytokine secretion [18]. Adenosine concentrations are further regulated by adenosine deaminase (ADA), which catalyzes the deamination of adenosine generating inosine and ammonia [12]. During T. gondii-infection, E-ADA activity was lower in the infected lymphocytes (Fig. 2C). The same can be observed when ADA-1 expression were determined by flowcytometry (Fig. 2F). However, our results are in contrast to Tonin et al. [37] which showed ADA activity is increased in rat T cells infected by RH strain of T. gondii compared to non-infected rat. In contrast, our data supporting the idea that adenosine favors T. gondii persistence as already reported by Mahamed et al. [43, 44].
Human ADA1 binds CD26 on T-cells, favoring adenosine turn-over [45]. Therefore, the interplay of CD39 with CD73 and CD26 regulates the levels of eATP, ADP, AMP, and adenosine, with major consequences for the purinergic control of inflammation and adaptative immune responses in parasites [46]. RSV treatment (50 μM) of infected lymphocytes increased the percentage of ADA1^+^ cells (Fig. 2F), thus our findings suggest that molecular action of RSV create an opportunity to improve anti-inflammatory effect of adenosine.
We next investigated whether eATP activates P2 purinergic receptors expressed by infected lymphocytes, triggering a range of proinflammatory responses (Fig. 3). In both methodologies used the P2X_7_ receptor is overexpressed in T. gondii-infected lymphocytes when compared to control group. Several publications demonstrated an important role of the P2X_7_R in the determination of resistance or susceptibility to acquired T. gondii [14–16, 47]. eATP binds to P2X_7_R triggered the elimination of the parasite in a P2X_7_R dependent manner [14]. Therefore, high concentrations of eATP activate the P2X_7_R and can induce the apoptosis of infected cells leading to the death of the parasite. However, RSV treatment diminished P2X_7_R halting pro-inflammatory activity and apoptosis probably by inhibiting inflammasome as reported by Huang et al. [42].
P2Y receptors are expressed in virtually all cells including immune cells and were also investigated in the present study (Fig. 3B, E), due to important function in immune cell differentiation and maturation, migration, and cell apoptosis [48]. P2Y_11_ couple to Gq proteins and activate phospholipase C for signal transmission, while P2Y_12_ receptor couple to Gαi/o proteins o inhibit adenylate cyclase [49]. Our data shows an increase of positive P2Y_12_R in infected lymphocytes when compared to control (Fig. 3B). P2Y receptor activation increases migration, which may also promote clearance of damaged and apoptotic cells [49]. This is supported by a study showing that P2Y_12_ is linked to toll-like receptor 2-mediated random migration of microglia [50], what could be happening in this study.
RSV 50 μM up-express P2Y_12_R, these results can be related to antioxidant and anti-inflammatory effects of RSV in order to induce apoptosis of infected cells. P2Y11R density was also overexpressed in infected lymphocytes (Fig. 3E). Of note, P2Y_11_ activation is associated with increased intracellular cAMP concentration [51]. However, RSV treatment isolate or combinate to SMX-TMP diminished P2Y_11_R density in infected cells demonstrate that RSV has selective inhibitory effects on ADP-stimulated lymphocyte activation possibly via P2Y12R inhibiting cell growth and differentiation.
Subsequently, we considered a potential involvement of adenosine receptors as potential targets in the anti-inflammatory effect of RSV. A1 and A_2A_ receptors were analyzed by flow cytometry and western blotting techniques (Fig. 3C, F) respectively. T. gondii-infection induce down expression of A1R^+^ cells and overexpression of A_2A_R. The antagonist effects may induce its effects by blocking T cell receptor signaling after an increase in cyclic AMP following A_2A_R stimulation (Stagg and Smyth, 2010). RSV treatments isolated (10 and 50 μM) or combinate to SMX-TMP positively stimulate A1R in infected cells (Fig. 3C). The opposite can be observed for A_2A_R in infected lymphocytes (Fig. 3F). To our knowledge, our data provide the dual effect of RSV acting as immunomodulator molecule as antagonist of A1R and attenuation the inflammation thought A_2A_R. Moreover, a recently study reported that diet supplemented with RSV modulates the adenosine-mediated signaling pathways in Alzheimer Disease model, suggesting a protective role by re-sensitizing A1 and desensitizing A_2A_ mediated signaling in RSV-treated mice [52].
A limitation of the present study is the absence of pharmacological modulation using specific agonists and antagonists of P2X7 and P1 receptors, which would allow a more direct evaluation of the mechanisms involved; this comparison represents an important direction for future investigations.
Also, this study evaluate the protein density of IL-1β and IL-10 in PBMCs stimulated with the virulent T. gondii RH strain. Our results revels that at the initiation of inflammation (24hs after T. gondii-infection) eATP levels are release, probably produced by infected and damage cells. This will result in decreased ATP levels and increased adenosine levels. Increased adenosine will inhibit the inflammation and at the same time, the low levels of ATP also have an inhibiting effect on the inflammatory response. Our hypothesis can be confirmed by IL-1β, a proinflammatory and IL-10, anti-inflammatory, densities (Fig. 4C, D). In addition, ATP is known to boost the activation of T cells by amplifying the T cell receptor (TCR) induced activation and by increasing IL-2 production [53].
Resveratrol-treated cells downregulate IL-1β protein (Fig. 4C) and stimulate IL-10 secretion in infected lymphocytes (Fig. 4D) due to anti-inflammatory properties of RSV; this process occurs through Sirt1 activation as already mentioned.
Like in inflammatory responses, effects of ATP on specific immune cells appear to be concentration dependent: while low ATP concentrations activate T cells, high concentrations and prolonged stimulation of the P2X_7_ receptor induced pore formation and apoptosis (Tsukimoto et al., 2006, Trabanelli et al., 2012). The apoptosis could be mediated by ROS production and nitrosamine metabolism, which is an important antimicrobial response for the elimination of pathogens (Finkel, 2011; [54]). Interestingly, however, ROS is not induced in T. gondii-infected human lymphocytes (Fig. 4A). Nox levels were augmented in infected cells (Fig. 4B). However, RSV could protect lymphocytes from oxidative/nitrative damage induced by T. gondii and activated several defensives signaling pathways that could mediated by purinergic signaling as showed in this study.
Conclusion
Therefore, our data demonstrate that RSV upregulate purinergic signaling avoiding high amount of extracellular ATP, which leads to IL-1β secretion, ROS and nitrites generation, which consequently induced apoptosis and reduce lymphocyte viability during* T. gondii*-infection. In addition, RSV, due to anti-oxidant and anti-inflammatory biological effects combinate to SMX-TMP is suggestive of a safe therapeutically as a target for toxoplasmosis.
Supplementary Information
Below is the link to the electronic supplementary material.
Material Suplementar 1.