Article pubs.acs.org/JAFC
Cucurbitacin E Potently Modulates the Activity of Encephalitogenic Cells Bojan Jevtić,† Neda Djedović,† Suzana Stanisavljević,† Jovana Despotović,‡ Djordje Miljković,*,† and Gordana Timotijević§ †
Department of Immunology, Institute for Biological Research “Siniša Stanković”, University of Belgrade, Belgrade, Serbia Laboratory for Molecular Biology, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia § Laboratory for Plant Molecular Biology, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia ‡
ABSTRACT: Cucurbitacin E (CucE) is a highly oxidized steroid consisting of a tetracyclic triterpene. It is a member of a Cucurbitacin family of biomolecules that are predominantly found in Cucurbitaceae plants. CucE has already been identified as a potent anti-inflammatory compound. Here, its effects on CD4+ T helper (Th) cells and macrophages, as the major encephalitogenic cells in the autoimmunity of the central nervous system, were investigated. Production of major pathogenic Th cell cytokines: interferon-gamma and interleukin-17 were inhibited under the influence of CucE. The effects of CucE on CD4+ T cells were mediated through the modulation of aryl hydrocarbon receptor, STAT3, NFκB, p38 MAPK, and miR-146 signaling. Further, production of nitric oxide and reactive oxygen species, as well as phagocytic ability, were inhibited in macrophages treated with CucE. These results imply that CucE possesses powerful antiencephalitogenic activity. KEYWORDS: cucurbitacin E, T cell, macrophage, autoimmunity, experimental autoimmune encephalomyelitis
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tion.10 They produce various pro-inflammatory cytokines, including IL-6 and TNF, as well as NO, and thus contribute to the destruction of neural tissue.10 The primary aim of this study was to investigate if CucE has a potency to counteract encephalitogenic T cells and macrophages. Indeed, CucE was shown to inhibit the production of IFN-γ and IL-17 in T cells obtained from EAE rats. The effect was mediated through the inhibition of an aryl hydrocarbon receptor and STAT3 and NFκB signaling. Moreover, CucE reduced the generation of NO and reactive oxygen species in rat macrophages, as well as their phagocytic ability.
INTRODUCTION Cucurbitacins are structurally diverse triterpenes that are identified in Cucurbitaceae but also in several other plant families.1 They have immense pharmacological potential, as they exert anti-inflammatory, antitumor, antiatherosclerotic, and antidiabetogenic effects.1 Cucurbitacin E (CucE), also known as α-elaterin and α-elaterine (empirical formula C32H44O8), seems to be a potent anti-inflammatory compound. It was shown to be efficient in reducing nitric oxide (NO), tumor necrosis factor (TNF), and interleukin (IL)-1βproduction and COX enzymes in RAW264.7 macrophages.2,3 Further, CucE was demonstrated to inhibit IL-2, TNF, and interferon (IFN)-gamma in Jurkat T.4 Finally, it was shown to be efficient in reducing TNF-induced IL-1β, IL-6, and IL-8 in synoviocyte MH7A cells.5 The effects of CucE in macrophages, T cells, and synoviocytes were mediated through the inhibition of NFκB signaling.3−5 Experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model of multiple sclerosis. Multiple sclerosis is a chronic inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS). EAE has been useful for understanding the pathogenesis of multiple sclerosis, as well as for the design of drugs for the disease.6 Autoimmune response against the CNS is initiated in lymph nodes where CD4+ T cells (T helper, Th cells) specific for CNS antigens are activated and differentiated toward IFN-γ-producing Th1 and IL-17-generating Th17 phenotype.7,8 Consequently, these Th cells invade the CNS where local antigen-presenting cells are reactivating them.9 After the reactivation, these encephalitogenic Th cells initiate and propagate inflammation within the CNS.8 Macrophages are activated to perform effector functions during the inflamma© 2016 American Chemical Society
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MATERIALS AND METHODS
Cells and Cell Cultures. Cells were isolated from Dark Agouti rats. These rats were bred and kept under conventional conditions in the animal facility of the Institute for Biological Research “Siniša Stanković”. The experimental procedures were approved by the local Ethics Committee (IBISS, No. 2-10/15). Cells of draining (popliteal) lymph nodes were obtained from rats immunized with guinea pig 50 μg/rat of MBP (obtained from Professor Alexander Flügel, University of Göttingen, Germany), emulsified with an equal volume of complete Freund’s adjuvant (CFA, Difco, Detroit, MI). CFA was supplemented with 5 mg/mL of M. tuberculosis (Difco). The animals were injected subcutaneously into hocks with 100 μL of MBP + CFA. The isolation took place 7 days post-immunization. Draining lymph node cells were cultured in RPMI-1640 culture medium (PAA Laboratories, Pasching, Austria) that was supplemented with 2% rat serum. The cells were seeded at 5 × 106/mL/well in 24-well plates (Sarstedt, Nümbrecht,
Received: Revised: Accepted: Published: 4900
February 29, 2016 May 2, 2016 May 25, 2016 May 25, 2016 DOI: 10.1021/acs.jafc.6b00951 J. Agric. Food Chem. 2016, 64, 4900−4907
Article
Journal of Agricultural and Food Chemistry Germany) and treated with MBP (10 μg/mL) and cucurbitacin E (CucE, purity ≥95%, Sigma-Aldrich, St. Louis, MO). For purification of CD4+ T cells from the lymph node cells, biotin conjugated antibody specific for CD4 (eBioscience, San Diego, CA) and IMagSAv Particles Plus (BD Biosciences, San Diego, CA) were used. CD4+ T cells were seeded in 24-well plates (1 × 106/ml/well) and cultivated with or without CucE. Spinal cord immune cells were isolated from rats immunized with rat spinal cord homogenate emulsified in CFA. For the isolation, extensive perfusion of rats with cold PBS preceded spinal cord isolation. Spinal cords were homogenized, and homogenates were centrifuged using 30/70% gradient of Percoll (Sigma-Aldrich). Following centrifugation, spinal cord immune cells were obtained from the Percoll border and washed in RPMI medium. Spinal cord infiltrating cells were seeded at 2.5 × 106/mL/well in 24-well plates and cultivated with or without CucE. For isolation of resident peritoneal cells, peritoneal lavage with 3 mL of ice-cold PBS was performed. For the cultivation of peritoneal cells, RPMI-1640 culture medium supplemented with 5% heat-inactivated fetal calf serum (FCS, PAA Laboratories) was used. The cells were seeded into 24-well plates (2 × 106/well), cultivated at 37 °C in a humidified atmosphere containing 5% CO2, and were washed two times with PBS 2 h later to eliminate nonadherent cells. The purity of adherent cells, i.e., macrophages, was assessed by flow cytofluorimetry analysis on a CyFlow Space flow cytometer (Partec, Munster, Germany). Typically, the proportion of CD3+ and CD45R+ cells in the purified populations was less than 5%. Macrophages were treated with 10 ng/mL lipopolysaccharide (LPS, Sigma-Aldrich) and CucE. Dendritic cells were obtained from progenitor bone marrow cells that were flushed from the femur. These cells were cultured in RPMI 1640 supplemented with 10% FCS (5 × 106/2 mL/well in 6-well plate) in the presence of 20 ng/mL of granulocyte-macrophage colonystimulating factor (GM-CSF, Peprotech, Rocky Hill, NJ). Bone marrow derived dendritic cells were obtained after 6 days of cultivation as nonadherent and weakly adherent cells that were transferred into 24-well plates (2 × 106/mL/well) and treated with 100 ng/mL LPS for 24 h in the absence or presence of CucE. Cell Viability Assays. Viability of draining lymph node cells and spinal cord immune cells was assessed by MTT assay. These cells were collected in tubes and centrifuged in order to remove supernatants. Cell pellets were dissolved in 0.5 μg/mL MTT (Sigma-Aldrich) solution and incubated for 30 min at 37 °C. Cells were centrifuged, and the formed formazan crystals were dissolved in DMSO. Crystalviolet (CV) test was used for the determination of macrophage viability. Macrophages were fixed with methanol and stained with 1% CV solution. Afterward, the plates were thoroughly washed, and CV was dissolved in acetic acid (33%). In both assays, the number of viable cells was determined as the absorbance of dissolved dyes. Absorbance was measured at 540 nm with a correction at 690 nm on an automated microplate reader (LKB 5060-006, LKB, Vienna, Austria). Detection of NO Release and ROS Generation. Griess reaction was used for the determination of nitrite accumulation in cell culture supernatants, as a measure of NO release. Triplicates of the supernatants were mixed with an equal volume of Griess reagent. The reagent was made as a 1:1 mixture of 0.1% naphthylethylenediaminedihydrochloride and 1% sulphanilamide in 5% H3PO4. Subsequently, the absorbance at 540 nm was determined. A standard curve was made according to the known concentrations of NaNO2. ROS generation was determined by dihydrorhodamine 123 (DHR, SigmaAldrich) staining. The cells were pretreated with CucE for 24 h, then incubated in the presence of 1 μM DHR for 30 min, and stimulated with LPS for an additional 90 min. The fluorescence was acquired via flow cytometry, and the results are presented as the percent of positive cells and as mean fluorescence intensity (mfi) of the population. Cytokine Level Measurement. ELISA was used for the measurement of cytokine concentration in cell culture supernatants. Specifically, sandwich ELISA was performed in MaxiSorp plates (Nunc, Rochild, Denmark) with paired antibodies. Samples were analyzed in duplicates for rat IL-1, rat IL-6, rat IFN-γ (R&DSystems, Minneapolis, MN), rat TNF (BD Biosciences), and murine/rat IL-17
(eBioscience). The lower limit of detection was 30 pg/mL, while the upper limit of detection was 10 ng/mL for all of the ELISA tests performed. Samples that showed values over the upper limit of detection were adequately diluted for the measurement. For the calculation of the results, standard curves were made with appropriate concentrations of the recombinant cytokines. Immunoblot. Cells were lysed with a solution of 62.5 mMTrisHCl, 2% w/v SDS, 10% glycerol, 50 mM dithiothreitol, 0.01% w/v bromophenol blue, 1 mM phenylmethanesulfonyl fluoride, 1 μg/mL aprotinine, and 2 mM EDTA (pH 6.8). Twenty micrograms of proteins was loaded into 12% SDS−polyacrylamide gel and electrophoresis was performed. Semidry transfer of the samples to polyvinylidenedifluoride membranes was performed at 5 mA/cm2 (Fastblot B43, Biorad, Muenchen, Germany). 5% w/v bovine serum albumin (BSA) (fraction V; Sigma-Aldrich) in PBS with 0.1% Tween20 was used as a blocking solution. The membranes were probed with specific antibodies for phosphorylated-IκB and total IκB, phosphorylated STAT-3 and total STAT-3 (all from Cell Signaling Technology, Boston, MA), phosphorylated-p38, and total p38 (both from Santa Cruz Biotechnology, Dallas, TX). Then, incubation with the secondary antibody (ECL donkey antirabbit HRP-linked, GE Healthcare, Buckinghamshire, England, UK) was done. Subsequently, hemiluminescence (Immobilon Western, Millipore) was performed. Photographs were made by X-ray films (Kodak, Rochester, NY), and densitometry was done with Scion Image Alpha 4.0.3.2 (Scion Corporation, Frederick, MD). Cytofluorimetry for the Detection of Cell-Surface Markers, Apoptosis, and Phagocytosis. Cells were stained with PEconjugated anti-CD4, FITC-conjugated anti-CD11b (BD Biosciences), PE-conjugated anti-CD86 (AbDSerotec, Oxford, UK), and PEconjugated anti-MHC class II antibodies (eBioscience). In order to set gates for cell marker positivity, adequate isotype control antibodies were used. Characteristically, the proportion of cells stained with the isotype control antibody was less than 1%. Detection of apoptosis was performed via AnnexinV-FITC staining (Biotium, Hayward, CA). In parallel, cells were stained with PE-conjugated anti-CD4. AnnexinVFITC positive cells were considered apoptotic. For the detection of phagocytosis, macrophages were plated in 24-well plates at 1 × 105/ well and incubated at 37 °C for 1 h. Latex beads (1 μm, yellow-green, Sigma-Aldrich) were preopsonized in PBS supplemented with 50% FCS. The preopsonized beads were added to macrophages (10 beads per cell), and cells were incubated at 37 °C for an hour in the presence or absence of CucE. Macrophages were analyzed with cytofluorimetry (PartecCyFlow Space cytometer). Results are presented as percentage of cells or mean fluorescent intensity (mfi) of the cell population. Reverse Transcription−Real Time Polymerase Chain Reaction. mi-Total RNA Isolation Kit (Metabion, Martinsried, Germany) was used for the isolation of total RNA. Reverse transcriptase was performed with miScript II RT kit (Qiagen). In a reverse transcription reaction, miRNAs and other noncoding RNAs (ncRNAs) were polyadenylated by poly(A) polymerase. For the conversion of these RNAs, as well as of naturally polyadenilated mRNAs into cDNA, oligodT priming was used in reverse transcription. For the amplification of cDNAs, Maxima SYBR Green/ROX qPCR Master Mix (Fermentas) was used. The amplifications were performed in an ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). The PCR primers (Metabion) were as follows: β-actin forward primer, 5′-GCT TCT TTG CAG CTC CTTCGT-3′; U6 forward primer, 5′TGCTTCGGCAGCACATATAC-3′; miR146 forward primer, 5′GGC GAT GAG AAC TGA ATT CCA-3′; miR-155 forward primer, 5′-GGA GGT TAA TGC TAA TTG TGA TAG-3′; and Cyp1A forward primer, 5′-GGG GAG GTT ACT GGT TCT GG-3′. The miScript Universal Primer (Qiagen) was used in all reactions as the reverse primer. For the analysis of the results, 7500 System Software was used. Relative RNA expression is determined as 2−dCt, where dCt is the Ct value of a gene of interest − the Ct value of the endogenous control (β-actin or U6). Zebrafish Husbandry and Embryo Treatments. Zebrafish (Daniorerio) Tübingen wild-type strain was used in all experiments and maintained and bred according to standard protocols.11 After breeding, 4901
DOI: 10.1021/acs.jafc.6b00951 J. Agric. Food Chem. 2016, 64, 4900−4907
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Figure 1. Effects of CucE on encephalitogenic T cells. Draining lymph node cells were stimulated with MBP and treated with various concentrations of CucE for 24 h (A−C). Subsequently, cell viability was determined by MTT assay (A), while cytokine levels were measured in the culture supernatants (B,C). The percentage of apoptotic cells in the absence (0) or presence of CucE (50 ng/mL) was determined by cytofluorimetry (D). Representative plots obtained from CucE-treated and CucE-untreated (Ctrl) cultures are presented (E). Spinal cord immune cells were cultivated in the absence (0) or presence of CucE (50 ng/mL) for 24 h (F−H). Subsequently, cell viability was determined by MTT assay (F), while cytokine levels were measured in the culture supernatants (G,H). Data are presented as the mean ± SD from repeated measurements. *p < 0.05 is statistically significantly different from 0. fertilized eggs were collected and selected under a binocular stereomicroscope (PXS-VI,Optica, Zhejiang, China). Influence of CucE on the development of zebrafish embryos was investigated during the first 72h postfertilization (hpf). The CucE treatments of embryos were done at 6 hpf in 24-well plates, 12 embryos per well in a volume of 750 μL. All the treatments were done at least in triplicate. The embryos were observed at 24, 48, and 72 hpf under a light microscope (CKX41, Olympus, Hamburg, Germany) at 40× and 100× magnification. Mortality, hatching rates, and different malformations (pericardial edema, tail circulation, nondetachment of
the tail, lack of heartbeat, lack of somite formation, yolk deformation/ edema, eye pigmentation, scoliosis, growth retardation, body pigmentation, and malformations of the head, eye, otolyths, chorda, tail, and heart) were recorded. The observed effect was considered significant if present in more than 50% of the embryos per treatment in comparison to the control group (untreated embryos). Statistical Analysis. Data are presented as the mean ± SD of values got in repetitive experiments. For statistical analysis, a Student’s t test (two-tailed) was used. Differences that reached a p value less than 0.05 were considered statistically significant. 4902
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RESULTS
CucE Inhibits the Encephalitogenic Potential of T Cells. Draining lymph node cells isolated from rats immunized with MBP were treated with MBP in vitro in order to restimulate MBP-specific, i.e., encephalitogenic T cells. Viability of draining lymph node cells cultivated for 24 h was reduced under the influence of CucE in concentrations of 100 ng/mL and higher (Figure 1A). CucE induced apoptosis in lymph node cells and purified CD4+ T cells already at a concentration of 50 ng/mL (Figure 1D,E). Production of typical encephalitogenic CD4+ T cell cytokines IFN-γ and IL-17 was inhibited dose-dependently in draining lymph node cells (Figure 1B,C). This was independent of the CucE effect on cell viability, as it was observed at a concentration of 25 ng/mL and lower. Moreover, 50 ng/mL of CucE did not affect viability, but it did efficiently inhibit IFN-γ but not IL-17 generation in spinal cord cells isolated at EAE peak (Figure 1F−H). Thus, these results show that CucE inhibits encephalitogenic T cells producing IFN-γ and IL-17. CucE Affects Intracellular Signaling in CD4+ T Cells. CD4+ T cells were refined from draining lymph nodes of rats immunized with MBP. They were treated with CucE in concentration that was efficient in reducing IFN-γ and IL-17 in CD4+ T cells (50 ng/mL) for short intervals (30 and 60 min). This was performed to explore the influence of CucE on intracellular signaling. Levels of phosphorylated and total STAT3, IκB, and p38 were determined by immunoblot. Phosphorylation (activation) of STAT3 was decreased under the influence of CucE at both time points (Figure 2A,D). Phosphorylation (deactivation) of NFκB-inhibitory molecule IκB was reduced by CucE after 60 min of cultivation (Figure 2B,D). On the contrary, p38 phosphorylation (activation) was stimulated by CucE at the same time point (Figure 2C,D). Levels of miR-146, mi-R155, and cytochrome P450 (cyp)1A were determined by RT-PCR. While miR-146 expression was inhibited, expression of miR-155 and cyp1A were increased under the influence of CucE (Figure 2E−G). These results demonstrate that CucE affects various signaling pathways important for the activity of encephalitogenic CD4+ T cells. CucE Inhibits Effector Functions of Macrophages. Macrophages were activated with LPS to generate effector molecules. Viability of macrophages was inhibited in concentrations higher than 100 ng/mL (Figure 3A). Production of nitric oxide but not of TNF, IL-6, and IL-1β was inhibited by CucE in a dose-dependent manner (Figure 3B−E). CucE applied at a concentration of 50 ng/mL did not induce apoptosis in macrophages (Figure 3F,K), nor did it affect the expression of MHC class II molecules, CD86 and CD11b (Figure 3G,K), but it inhibited the phagocytosis of macrophages (Figure 3H,K), as well as their ability to generate reactive oxygen species (Figure 3I,K). Further, expression of MHC class II molecules and CD86 was not reduced in dendritic cells under the influence of CucE (Figure 3J,L). The obtained results demonstrate that CucE potently inhibits important effector functions of activated macrophages without interfering with their viability. CucE Does Not Affect Zebrafish Development. Zebrafish embryonic morphologies were visually inspected. No developmental malformations were observed when embryos were treated with CucE concentrations 25, 50, and 100 ng/mL during the first 72 hpf (data not shown). Also, there was no significant difference in hatching rate and mortality with any of
Figure 2. Effects of CucE on intracellular signaling in CD4+ T cells. CD4+ T cells were cultivated in the absence (0) and presence of 50 ng/mL of CucE. After 30 or 60 min of cultivation, the cells were lysed, and immunoblot (A−D) or RT-PCR (E−G) was performed. Ratio of densitometric values is presented for immunoblots, while relative RNA expression is presented for RT-PCR results. Data are presented as the mean ± SD of values obtained in repeated experiments (A−C, E, and F). A representative experiment is also shown (D). *p < 0.05 is statistically significantly different from 0.
the CucE concentrations tested (25−1000 ng/mL). Embryos treated with 500 ng/mL of CucE developed yolk sac edema at 48 hpf, while bent spine and hemagglutination were observed at 72 hpf (Figure 4B,C). Widening of yolk extension was observed in the embryos treated with 1000 ng/mL of CucE at 24 hpf (Figure 4D). Also, this concentration induced yolk sac edema and signs of hemagglutination at 48 hpf (Figure 4E), and severe hemagglutination together with bent spine at 72 hpf (Figure 4F). The two highest concentrations of CucE tested on zebrafish embryos (500 and 1000 ng/mL) induced yolk sac edema in 52.8% ± 4.8% and in 64.5% ± 15.0% of examined embryos, respectively. Also, shortening of embryo body length was observed with these concentrations of CucE (Figure 4C,F,I). 4903
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Figure 3. Effects of CucE on macrophages. Macrophages were stimulated with LPS and treated with various concentrations of CucE for 24 h. Subsequently, cell viability was determined by MTT assay (A), while nitrite and cytokine levels were measured in the culture supernatants (B−E). Percentage of apoptotic cells (F), expression levels of MHC class II molecules, CD86 and CD11b (G), phagocytosis (H), and ROS levels (I) in the absence (0) or presence of CucE (50 ng/mL) were determined by cytofluorimetry. Dendritic cells were stimulated with LPS and cultivated in the absence (0) or presence of CucE (50 ng/mL) for 24 h (J). Data are presented as the mean ± SD from repeated measurements. Representative plots obtained from CucE-treated and CucE-untreated (Ctrl) macrophage (K) and dendritic cell (L) cultures are also presented. *p < 0.05 is statistically significantly different from 0.
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dibutyrate and ionomycin.4 In our study, effects of CucE on CNS antigen-specific response in primary cells were investigated. It was determined that CucE inhibited the production of IFN-γ and IL-17 in in vitro restimulated MBP-specific T cells from draining lymph nodes. Also, CucE down-regulated IFN-γ generation in T cells restimulated in vivo in the spinal cord of EAE rats. Th1 and Th17 cells are the main encephalitogenic populations in EAE and multiple sclerosis,7,12 while IFN-γ and IL-17 are their signature cytokines, respectively. Besides Th1 cells, CD8+ T cells also generate IFN-γ. Importantly, CD8+ T cells considerably contribute to the pathogenesis of multiple sclerosis.12 IFN-γ and IL-17 typically stimulate macrophages
DISCUSSION Here, it is presented that CucE potently reduces encephalitogenic activity of CD4+ T cells and effector functions of macrophages. Importantly, CucE down-regulates the production of major pro-inflammatory cytokines produced by Th1 and Th17 cells, i.e., IFN-γ and IL-17. It also inhibits the generation of NO and ROS in macrophages and prevents their phagocytic activity. Effects of CucE on T cells have not been thoroughly explored, so far. There was a study which showed that CucE significantly inhibited the production of IL-2, TNF, and IFN-γ in Jurkat cell line polyclonally stimulated with phorbol 12,134904
DOI: 10.1021/acs.jafc.6b00951 J. Agric. Food Chem. 2016, 64, 4900−4907
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Figure 4. Effects of CucE on zebrafish development. Zebrafish embryos were exposed to 500 ng/mL CucE (A−C), 1000 ng/mL CucE (D−F), or they were the untreated-control (G−I). Representative images of zebrafish embryos are shown. Magnifications are 40× (A, C, D, F, G, and I) or 100× (B, E, and H). Embryos are shown in lateral view with anterior to the left. Abbreviations: AYE, abnormal yolk extension; BS, bent spine; HE, hemagglutination; SBL, shortening of the body length; YSE, yolk sac edema.
is mediated by the p38 MAP kinase that reduces transforming growth factor β-activated protein kinase 1 (TAK1).27 From the observed results on the effects of CucE on intracellular signaling, it is reasonable to assume that the inhibitory effects of CucE on IFN-γ and IL-17 generation in CD4+ T cells are mediated through the down-regulation of STAT3 and NFκB signaling. Indeed, STAT3 signaling is essential for IL-17 generation and Th17 differentiation.28 Further, mice that are selectively deficient for STAT3 in CD4+ T cells are resistant to EAE induction.29 Also, NFκB signaling is crucial for Th1 differentiation and IFN-γ production,30−32 as well as for Th17 differentiation and activity.33 Consequently, it has been demonstrated that encephalitogenicity of CD4+ T cells is dependent on intact NFκB signaling.34 Further, the observed stimulating effect of CucE on cyp1A expression implies that CucE potentiates aryl hydrocarbon receptor signaling in CD4+ T cells. This might be of particular importance in understanding the antiencephalitogenic effects of CucE, as aryl hydrocarbon receptor signaling has been shown to be essential for the reduction of Th17 cells and stimulation of regulatory T cells.35 However, as cyp1A expression can be modulated by other signals, besides those coming through the aryl hydrocarbon receptor, further analyses are needed before concluding that CucE acts on this signaling. Also, miR-146 and miR-155 have been associated with the pathogenesis of multiple sclerosis and EAE.36−38 It was shown that miR-155 promoted Th17 cell activation and restimulation of encephalitogenic T cells36,38 in EAE, while miR-146 expression was increased in multiple sclerosis patients’ peripheral blood mononuclear cells.37 Although CucE did not inhibit miR-155 expression, it did down-regulate miR-146 in CD4+ T cells. Thus, our results imply that CucE inhibits the activation of encephalitogenic Th1 and Th17 through the reduction of STAT3, NFκB, aryl hydrocarbon receptor, and miR-146 signaling.
and neutrophils, respectively, and in this way, they execute CNS tissue destruction. Additionally, they can act directly on CNS resident cells.13 It was observed that they induced olygodendrocyte cell death and blood−brain barrier dysfunction.14,15 Therefore, the ability of CucE to restrict IFN-γ and IL-17 production and activity of MBP-reactivated Th1 and Th17 clearly indicate that this agent has antiencephalitogenic potency. Our results imply that CucE inhibits STAT3 and NFκB signaling but potentiates p38 MAPK signaling in encephalitogenic T cells. There have already been reports on the effects of CucE on the examined signaling pathways. CucE was shown to inhibit STAT3 signaling in pancreatic cancer cells,16 endothelial cells,17 bladder cancer cells,18 breast cancer cells,19 and in microglia.20 It inhibited effects on NFκB signaling in macrophage cell line RAW 264.7,3 Jurkat T cell line,4 microglia,20 and in synoviocytes.5 Importantly, increase in p38 signaling by CucE was also observed previously in Jurkat T cells.4 Also, other cucurbitacins were shown stimulatory for p38 signaling. Namely, cucurbitacin B increased the level of phosphorylated p38 in glioma cell lines,21 cucurbitacin D in the K562 cell line,22 and cucurbitacin I in B leukemic cell lines.23 Still, it was reported that CucE inhibited p38 activation in endothelial cells. In that study, the cells were pretreated with CucE for 4 h before the stimulation with vascular endothelial growth factor (VEGF). Thus, the cells used, the stimulation applied, and the way of CucE administration differ markedly between the studies, and these might explain the observed variation in the effects of CucE on p38 activation. Although p38 MAPK is generally considered as NFκB promoting, there are reports demonstrating that it down-regulates NFκB signaling. Namely, it was shown that various compounds including sodium salicylate, sorbitol, hydrogen peroxide, arsenite, and vitamin C inhibit NFκB activation through p38 MAPK signaling.24−26 It seems that the effect on the NFκB signal transduction pathway 4905
DOI: 10.1021/acs.jafc.6b00951 J. Agric. Food Chem. 2016, 64, 4900−4907
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Serbia. Tel: +381 11 20 78 390. E-mail: georgije_zw@yahoo. com.
CucE potently inhibited NO and ROS production in macrophages. The inhibitory effects of CucE on NO generation in RAW 264.7 cells stimulated with IFN-γ and LPS have already been reported.2 There was also a study showing that inducible nitric oxide synthase (iNOS) activity was reduced by CucE in LPS-stimulated microglial cells.20 Inhibitory effects of CucE on pro-inflammatory cytokines TNF, IL-6, and IL-1β were also demonstrated in microglia and RAW264.7 cells.3,20 However, in our study, there was no effect of CucE on TNF generation in macrophages, while the effects on IL-1β and IL-6 were inhibitory with lower doses and stimulatory with a dose of 100 ng/mL. This interesting finding and its relevance for the treatment of EAE should be investigated in detail in upcoming studies. The difference in the observed effects of CucE on the cytokines might be explained with the variance in cells used in the studies. NO is a free radical molecule with important roles in multiple sclerosis and EAE pathogenesis.39 This molecule exerts some positive effects in the inflamed CNS. For instance, it was shown to induce the apoptosis of autoreactive T cells invading the CNS.40,41 However, its predominant role in multiple sclerosis and EAE is deleterious.42,43 If NO interacts with O2−, peroxynitrite is formed. Peroxynitrite is an extremely vicious molecule as it is able to induce lipid peroxidation and to directly damage oligodendrocytes in this way.43 Importantly, it was demonstrated that both microglia and activated macrophages generate reactive nitrogen and oxygen species.44 Therefore, it was not a surprise that these cells were present in close proximity to injured axons in multiple sclerosis.44 Thus, the ability of CucE to inhibit both NO and ROS in macrophages is of great importance for its potential antiencephalitigenic effects. Also, we found that CucE inhibits the phagocytic activity of macrophages but does not influence the expression of MHC class II molecules and CD86 in macrophages or dendritic cells. Despite the inefficiency of CucE in reducing the expression of the major antigen-presenting molecules on macrophages, down-regulated phagocytosis in macrophages might reduce their ability to present CNS-derived antigens to T cells. Thus, this effect might prevent the efficiency of macrophages to activate T cells. Having in mind the importance of Th1 and Th17 cells and macrophages for the pathogenesis of EAE and multiple sclerosis, it seems plausible to assume that CucE will have beneficial effects in the CNS autoimmunity. This assumption has to be tested in studies on EAE and other animal models of multiple sclerosis. Importantly, the screening of CucE effects on zebrafish embryos showed the absence of developmental malformation in concentrations of CucE that were efficiently immunoregulatory in T cells and macrophages. Mild disorders of embryo development were observed only with 10-fold higher concentrations of the analyzed compound. Accordingly, the effects of CucE on T cell and macrophage viability were limited in vitro. Taken together, these results are encouraging regarding the application of CucE in vivo. Thus, studies on antiencephalitogenic effects of CucE, as well as of natural sources of CucE, such as leaf or root extracts of plants rich in this compound are warranted.
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Funding
This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (173035, 173005, 173013, and 173008). Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Kaushik, U.; Aeri, V.; Mir, S. R. Cucurbitacins - An insight into medicinal leads from nature. Pharmacogn. Rev. 2015, 9, 12−8. (2) Abdelwahab, S. I.; Hassan, L. E.; Sirat, H. M.; Yagi, S. M.; Koko, W. S.; Mohan, S.; Taha, M. M.; Ahmad, S.; Chuen, C. S.; Narrima, P.; Rais, M. M.; Hadi, A. H. Anti-inflammatory activities of cucurbitacin E isolated from Citrullus lanatus var. citroides: role of reactive nitrogen species and cyclooxygenase enzyme inhibition. Fitoterapia 2011, 82, 1190−7. (3) Qiao, J.; Xu, L. H.; He, J.; Ouyang, D. Y.; He, X. H. Cucurbitacin E exhibits anti-inflammatory effect in RAW 264.7 cells via suppression of NF-kappaB nuclear translocation. Inflammation Res. 2013, 62, 461− 9. (4) Wang, L.; Li, C.; Lin, Q.; Zhang, X.; Pan, H.; Xu, L.; Shi, Z.; Ouyang, D.; He, X. Cucurbitacin E suppresses cytokine expression in human Jurkat T cells through down-regulating the NF-kappaB signaling. Acta Biochim. Biophys. Sin. 2015, 47, 459−65. (5) Jia, Q.; Cheng, W.; Yue, Y.; Hu, Y.; Zhang, J.; Pan, X.; Xu, Z.; Zhang, P. Cucurbitacin E inhibits TNF-alpha-induced inflammatory cytokine production in human synoviocyte MH7A cells via suppression of PI3K/Akt/NF-kappaB pathways. Int. Immunopharmacol. 2015, 29, 884−90. (6) Ben-Nun, A.; Kaushansky, N.; Kawakami, N.; Krishnamoorthy, G.; Berer, K.; Liblau, R.; Hohlfeld, R.; Wekerle, H. From classic to spontaneous and humanized models of multiple sclerosis: impact on understanding pathogenesis and drug development. J. Autoimmun. 2014, 54, 33−50. (7) Petermann, F.; Korn, T. Cytokines and effector T cell subsets causing autoimmune CNS disease. FEBS Lett. 2011, 585, 3747−57. (8) Rostami, A.; Ciric, B. Role of Th17 cells in the pathogenesis of CNS inflammatory demyelination. J. Neurol. Sci. 2013, 333, 76−87. (9) Bartholomaus, I.; Kawakami, N.; Odoardi, F.; Schlager, C.; Miljkovic, D.; Ellwart, J. W.; Klinkert, W. E.; Flugel-Koch, C.; Issekutz, T. B.; Wekerle, H.; Flugel, A. Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions. Nature 2009, 462, 94−8. (10) Bogie, J. F.; Stinissen, P.; Hendriks, J. J. Macrophage subsets and microglia in multiple sclerosis. Acta Neuropathol. 2014, 128, 191−213. (11) Westerfield, M. In The Zebrafish Book; University of Oregon Press: Eugene, OR, 1995. (12) Dendrou, C. A.; Fugger, L.; Friese, M. A. Immunopathology of multiple sclerosis. Nat. Rev. Immunol. 2015, 15, 545−58. (13) Kurschus, F. C. T cell mediated pathogenesis in EAE: Molecular mechanisms. Biomed. J. 2015, 38, 183−93. (14) Vartanian, T.; Li, Y.; Zhao, M.; Stefansson, K. Interferongamma-induced oligodendrocyte cell death: implications for the pathogenesis of multiple sclerosis. Mol. Med. 1995, 1, 732−43. (15) Kebir, H.; Kreymborg, K.; Ifergan, I.; Dodelet-Devillers, A.; Cayrol, R.; Bernard, M.; Giuliani, F.; Arbour, N.; Becher, B.; Prat, A. Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation. Nat. Med. 2007, 13, 1173− 5. (16) Sun, C.; Zhang, M.; Shan, X.; Zhou, X.; Yang, J.; Wang, Y.; LiLing, J.; Deng, Y. Inhibitory effect of cucurbitacin E on pancreatic cancer cells growth via STAT3 signaling. J. Cancer Res. Clin. Oncol. 2010, 136, 603−10. (17) Dong, Y.; Lu, B.; Zhang, X.; Zhang, J.; Lai, L.; Li, D.; Wu, Y.; Song, Y.; Luo, J.; Pang, X.; Yi, Z.; Liu, M.; Cucurbitacin, E. a tetracyclic triterpenes compound from Chinese medicine, inhibits tumor
AUTHOR INFORMATION
Corresponding Author
*Institute for Biological Research “Siniša Stanković”, Department of Immunology, Despota Stefana 142, 11000 Belgrade, 4906
DOI: 10.1021/acs.jafc.6b00951 J. Agric. Food Chem. 2016, 64, 4900−4907
Article
Journal of Agricultural and Food Chemistry angiogenesis through VEGFR2-mediated Jak2-STAT3 signaling pathway. Carcinogenesis 2010, 31, 2097−104. (18) Huang, W. W.; Yang, J. S.; Lin, M. W.; Chen, P. Y.; Chiou, S. M.; Chueh, F. S.; Lan, Y. H.; Pai, S. J.; Tsuzuki, M.; Ho, W. J.; Chung, J. G.; Cucurbitacin, E. Induces G(2)/M Phase Arrest through STAT3/ p53/p21 Signaling and Provokes Apoptosis via Fas/CD95 and Mitochondria-Dependent Pathways in Human Bladder Cancer T24 Cells. Evid. Based Complement Alternat. Med. 2012, 2012, 952762. (19) Lan, T.; Wang, L.; Xu, Q.; Liu, W.; Jin, H.; Mao, W.; Wang, X. Growth inhibitory effect of Cucurbitacin E on breast cancer cells. Int. J. Clin. Exp. Pathol. 2013, 6, 1799−805. (20) Park, S. Y.; Kim, Y. H.; Park, G. Cucurbitacins attenuate microglial activation and protect from neuroinflammatory injury through Nrf2/ARE activation and STAT/NF-kappaB inhibition. Neurosci. Lett. 2015, 609, 129−36. (21) Yin, D.; Wakimoto, N.; Xing, H.; Lu, D.; Huynh, T.; Wang, X.; Black, K. L.; Koeffler, H. P. Cucurbitacin B markedly inhibits growth and rapidly affects the cytoskeleton in glioblastoma multiforme. Int. J. Cancer 2008, 123, 1364−75. (22) Liu, K.; Xing, H.; Zhang, S.; Liu, S.; Fung, M. Cucurbitacin D induces fetal hemoglobin synthesis in K562 cells and human hematopoietic progenitors through activation of p38 pathway and stabilization of the gamma-globin mRNA. Blood Cells, Mol., Dis. 2010, 45, 269−75. (23) Ishdorj, G.; Johnston, J. B.; Gibson, S. B. Cucurbitacin-I (JSI124) activates the JNK/c-Jun signaling pathway independent of apoptosis and cell cycle arrest in B leukemic cells. BMC Cancer 2011, 11, 268. (24) Schwenger, P.; Alpert, D.; Skolnik, E. Y.; Vilcek, J. Activation of p38 mitogen-activated protein kinase by sodium salicylate leads to inhibition of tumor necrosis factor-induced IkappaB alpha phosphorylation and degradation. Mol. Cell. Biol. 1998, 18, 78−84. (25) Alpert, D.; Schwenger, P.; Han, J.; Vilcek, J. Cell stress and MKK6b-mediated p38 MAP kinase activation inhibit tumor necrosis factor-induced IkappaB phosphorylation and NF-kappaB activation. J. Biol. Chem. 1999, 274, 22176−83. (26) Bowie, A. G.; O’Neill, L. A. Vitamin C inhibits NF-kappa B activation by TNF via the activation of p38 mitogen-activated protein kinase. J. Immunol. 2000, 165, 7180−8. (27) Cheung, P. C.; Campbell, D. G.; Nebreda, A. R.; Cohen, P. Feedback control of the protein kinase TAK1 by SAPK2a/p38alpha. EMBO J. 2003, 22, 5793−805. (28) Yang, X. O.; Panopoulos, A. D.; Nurieva, R.; Chang, S. H.; Wang, D.; Watowich, S. S.; Dong, C. STAT3 regulates cytokinemediated generation of inflammatory helper T cells. J. Biol. Chem. 2007, 282, 9358−63. (29) Liu, X.; Lee, Y. S.; Yu, C. R.; Egwuagu, C. E. Loss of STAT3 in CD4+ T cells prevents development of experimental autoimmune diseases. J. Immunol. 2008, 180, 6070−6. (30) Aronica, M. A.; Mora, A. L.; Mitchell, D. B.; Finn, P. W.; Johnson, J. E.; Sheller, J. R.; Boothby, M. R. Preferential role for NFkappa B/Rel signaling in the type 1 but not type 2 T cell-dependent immune response in vivo. J. Immunol. 1999, 163, 5116−24. (31) Hilliard, B.; Samoilova, E. B.; Liu, T. S.; Rostami, A.; Chen, Y. Experimental autoimmune encephalomyelitis in NF-kappa B-deficient mice:roles of NF-kappa B in the activation and differentiation of autoreactive T cells. J. Immunol. 1999, 163, 2937−43. (32) Hilliard, B. A.; Mason, N.; Xu, L.; Sun, J.; Lamhamedi-Cherradi, S. E.; Liou, H. C.; Hunter, C.; Chen, Y. H. Critical roles of c-Rel in autoimmune inflammation and helper T cell differentiation. J. Clin. Invest. 2002, 110, 843−50. (33) Ruan, Q.; Chen, Y. H. Nuclear factor-kappaB in immunity and inflammation: the Treg and Th17 connection. Adv. Exp. Med. Biol. 2012, 946, 207−21. (34) Dasgupta, S.; Jana, M.; Zhou, Y.; Fung, Y. K.; Ghosh, S.; Pahan, K. Antineuroinflammatory effect of NF-kappaB essential modifierbinding domain peptides in the adoptive transfer model of experimental allergic encephalomyelitis. J. Immunol. 2004, 173, 1344−54.
(35) Baricza, E.; Tamasi, V.; Marton, N.; Buzas, E. I.; Nagy, G. The emerging role of aryl hydrocarbon receptor in the activation and differentiation of Th17 cells. Cell. Mol. Life Sci. 2016, 73, 95−117. (36) O’Connell, R. M.; Kahn, D.; Gibson, W. S.; Round, J. L.; Scholz, R. L.; Chaudhuri, A. A.; Kahn, M. E.; Rao, D. S.; Baltimore, D. MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development. Immunity 2010, 33, 607−19. (37) Fenoglio, C.; Cantoni, C.; De Riz, M.; Ridolfi, E.; Cortini, F.; Serpente, M.; Villa, C.; Comi, C.; Monaco, F.; Mellesi, L.; Valzelli, S.; Bresolin, N.; Galimberti, D.; Scarpini, E. Expression and genetic analysis of miRNAs involved in CD4+ cell activation in patients with multiple sclerosis. Neurosci. Lett. 2011, 504, 9−12. (38) Jevtic, B.; Timotijevic, G.; Stanisavljevic, S.; Momcilovic, M.; Mostarica Stojkovic, M.; Miljkovic, D. Micro RNA-155 participates in re-activation of encephalitogenic T cells. Biomed. Pharmacother. 2015, 74, 206−10. (39) Smith, K. J.; Lassmann, H. The role of nitric oxide in multiple sclerosis. Lancet Neurol. 2002, 1, 232−41. (40) Zettl, U. K.; Mix, E.; Zielasek, J.; Stangel, M.; Hartung, H. P.; Gold, R. Apoptosis of myelin-reactive T cells induced by reactive oxygen and nitrogen intermediates in vitro. Cell. Immunol. 1997, 178, 1−8. (41) Okuda, Y.; Sakoda, S.; Fujimura, H.; Yanagihara, T. Nitric oxide via an inducible isoform of nitric oxide synthase is a possible factor to eliminate inflammatory cells from the central nervous system of mice with experimental allergic encephalomyelitis. J. Neuroimmunol. 1997, 73, 107−16. (42) Mitrovic, B.; Ignarro, L. J.; Vinters, H. V.; Akers, M. A.; Schmid, I.; Uittenbogaart, C.; Merrill, J. E. Nitric oxide induces necrotic but not apoptotic cell death in oligodendrocytes. Neuroscience 1995, 65, 531− 9. (43) Cross, A. H.; Manning, P. T.; Stern, M. K.; Misko, T. P. Evidence for the production of peroxynitrite in inflammatory CNS demyelination. J. Neuroimmunol. 1997, 80, 121−30. (44) Lassmann, H. Axonal and neuronal pathology in multiple sclerosis: what have we learnt from animal models. Exp. Neurol. 2010, 225, 2−8.
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DOI: 10.1021/acs.jafc.6b00951 J. Agric. Food Chem. 2016, 64, 4900−4907
