|Posted on December 4, 2020 at 6:00 PM|
Natural triterpenes modulate immune-inflammatory markers of experimental autoimmune encephalomyelitis: therapeutic implications for multiple sclerosis
R Martín,* M Hernández, C Córdova, and ML Nieto
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BACKGROUND AND PURPOSE
Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory demyelinating diseases that develop as a result of deregulated immune responses causing glial activation and destruction of CNS tissues. Oleanolic acid and erythrodiol are natural triterpenes that display strong anti-inflammatory and immunomodulatory activities. Oleanolic acid beneficially influences the course of established EAE. We now extend our previous observations to erythrodiol and address the efficacy of both compounds to protect against EAE, given under different regimens.
The utility of both triterpenes in disease prevention was evaluated at a clinical and molecular level: in vivo through their prophylactic administration to myelin oligodendrocyte protein-immunized C57BL/6 mice, and in vitro through their addition to stimulated-BV2 microglial cells.
These triterpenes protected against EAE by restricting infiltration of inflammatory cells into the CNS and by preventing blood–brain barrier disruption. Triterpene-pretreated EAE-mice exhibited less leptin secretion, and switched cytokine production towards a Th2/regulatory profile, with lower levels of Th1 and Th17 cytokines and higher expression of Th2 cytokines in both serum and spinal cord. Triterpenes also affected the humoral response causing auto-antibody production inhibition. In vitro, triterpenes inhibited ERK and rS6 phosphorylation and reduced the proliferative response, phagocytic properties and synthesis of proinflammatory mediators induced by the addition of inflammatory stimuli to microglia.
CONCLUSIONS AND IMPLICATIONS
Both triterpenes restricted the development of the characteristic features of EAE. We envision these natural products as novel helpful tools for intervention in autoimmune and neurodegenerative diseases including MS.
Keywords: encephalomyelitis, neuroimmunology, inflammation, microglia, pharmacology, triterpenes
Multiple sclerosis (MS) is an autoimmune demyelinating disease directed against myelin proteins of the brain and spinal cord, and is considered as one of the major neurological diseases in young adults (Noseworthy et al., 2000). The precise cause of MS is unknown, but one theory is that it might be triggered by exposure to a viral infection or environmental influences. The disease takes dissimilar courses in different people and can go into four main pathological subtypes, even leading to death in the very progressive form (Lassmann et al., 2001).
Experimental autoimmune encephalomyelitis (EAE) induced in susceptible strains of animals provides the best available model for understanding events in MS and to test new drugs that could lead to novel therapies (Steinman, 1999). MS/EAE pathogenesis is driven mostly by a Th1-mediated autoimmune response. The development of the disease includes breakdown of the blood–brain barrier (BBB), infiltration of the CNS – brain and spinal cord – by myelin-reactive T cells and macrophages, activation of resident CNS cells (microglia and astrocytes), demyelination and axonal loss (Merrill and Benveniste, 1996; Benveniste, 1997; Engelhardt, 2006).
Microglial cells are active participants throughout the MS disease process. ‘Activated’ microglia produces inflammatory cytokines, free radicals and attracts immune cells into the CNS. A diffuse activation of microglia throughout the brain serves as a source of inflammation inside the CNS in chronic MS/EAE, while at latter stages of the disease a chronically activated microglia is associated with impaired neural function (Rasmussen et al., 2007).
Other components of the immune system that play crucial roles in MS/EAE pathogenesis include dendritic and B cells, antibodies, as well as inflammation-related enzymes, cytokines and chemokines. Thus, COX-2 and inducible nitric oxide synthase (iNOS) enzymes and pro-inflammatory cytokines such as IFN-γ, TNF-α or IL-17 are considered to be pathogenic, while the Th2 cell-related cytokines IL-4 and IL-10 have been shown to down-regulate the immune response in acute EAE (Hafler, 2004; Imitola et al., 2005; Sospedra and Martin, 2005). Much progress has been made over the past decade in elucidating the causes and molecular basis of MS, but in spite of the extensive research performed to develop new pharmacotherapeutic approaches to slow down the disease progression, there are still no optimal therapies available, due to both unwanted side effects of the drugs and the clinical and immunopathological heterogeneity of this disease (Hemmer et al., 2006).
Oleanolic acid and erythrodiol are two natural triterpenes of the oleanane group present in many vegetables, including the leaves and fruits of Olea europea (the olive tree). They have been recognized to have hepatoprotective, anti-inflammatory and antihyperlipidemic properties. Indeed, oleanolic acid has been promoted in China as an oral drug for human liver disorders. Data correlated well with the traditional use of O. europea in African and European Mediterranean countries, where this plant has been utilized widely in folk medicine as a diuretic, hypotensive, hypoglycaemic, emollient, febrifuge and tonic, for urinary and bladder infections, for headaches, as well as a therapy for inflammatory pain (Dold and Cocks, 1999). Recently, a number of synthetic oleanane triterpenoid derivatives have been synthesized based on oleanolic acid with more potent activities, some of which are currently being developed for the treatment of chronic kidney diseases (Pergola et al., 2011) or as an attractive new therapeutic option for cancer patients by enhancing the effect of immunotherapy (Nagaraj et al., 2010). In the last years, a variety of novel pharmacological properties of triterpenoids have been reported: (i) beneficial effects on cardiovascular system due to antioxidant and vasorelaxant activities (Rodriguez-Rodriguez et al., 2006); (ii) interaction with cytochrome P450s; (iii) anti-proliferative activities on tumoural cells by activating apoptotic programmes (Martín et al., 2007; 2009); (iv) effects on intracellular redox balance and protective effects against lipid peroxidation; as well as (v) immunomodulatory effects (Marquez-Martin et al., 2006). Besides, we have shown that oleanolic acid has a therapeutic effect on an experimental model of MS (Martín et al., 2010), demonstrating that i.p. administration of oleanolic acid, in mice with established EAE, is capable of reducing important biomarkers related to EAE disease. However, the potential of these biologically active molecules on maintenance of health has not been addressed in depth, although disease prevention is a major goal on public health, particularly because of the shifting of the concept from ‘disease care’ to ‘health care’. Therefore, it has been of interest in the present study to assess the influence of early administration of oleanolic acid and erythrodiol, an intermediate from which oleanolic acid is formed and on which no previous data exist, on health promotion in our EAE model. Our findings confirmed that both erythrodiol and oleanolic acid markedly slowed the clinical manifestations of the disease and we were able to correlate the magnitude of improvement for EAE with the decrease of the immuno-inflammatory responses.
Disease induction and treatment
All animal care and experimental protocols were reviewed and approved by the Animal Ethics Committee of the University of Valladolid and complied with the European Communities directive 86/609/ECC and Spanish legislation (BOE 252/34367-91, 2005) regulating animal research. C57BL/J6 mice (from Charles River Laboratories, Barcelona, Spain) were housed in the animal care facility at the Medical School of the University of Valladolid and provided food and water ad libitum.
EAE was induced in 8 to 10-week-old female C57BL/J6 mice by subcutaneous immunization with 100 µg of myelin oligodendrocyte glycoprotein (MOG)35–55 peptide (MEVGWYRSPFSRVVHLYRNGK; from Dr F. Barahona, CBM, Madrid) emulsified in complete Freund's adjuvant containing 0.4 mg Mycobacterium tuberculosis (H37Ra; Difco, Detroit, MI, USA) on day 0. Additionally, mice received 300 ng of Pertussis toxin i.p. on days 0 and 2. Clinical signs of EAE were assessed daily in a double-blind manner on a scale of 0 to 5, with 0.5 points for intermediate clinical findings: grade 0, no abnormality; grade 0.5, partial loss/reduced tail tone, assessed by inability to curl the distal end of the tail; grade 1, tail atony; grade 1,5, slightly/moderately clumsy gait, impaired righting ability or combination; grade 2, hind limb weakness; grade 2,5, partial hind limb paralysis; grade 3, complete hind limb paralysis; grade 3,5, complete hind limb paralysis and fore limb weakness; grade 4, tetraplegic; grade 5, moribund state or death. Scores from two investigators, both unaware of the treatments, were averaged. Data were plotted as daily mean clinical score for all animals in a particular treatment group. Scores of asymptomatic mice (score = 0) were included in the calculation of the daily mean clinical score for each group. Mice scoring at level 4 for 2 days were automatically given a disease severity grade of 5 and killed.
Triterpene treatment procedure
MOG-Immunized mice were treated daily with 50 mg kg–1 day–1 of oleanolic acid or erythrodiol by i.p. injection beginning at different times.
Groups OA0 and ERY0: triterpene treatment started at the immunization day.
Groups OA-7 and ERY-7: triterpene treatment started on day -7, before EAE induction.
Groups OA12 and ERY12: triterpene treatment started on day 12 after EAE induction.
Control groups (without EAE induction):
Group control, C: treated daily with 0.2% w/v DMSO.
Groups OA and ERY: healthy mice treated with the triterpenes for the same time as the corresponding EAE mice.
Animals were studied at two different times:
30 days after immunization, when EAE mice showed hind limb paralysis, or
at the day when severe symptoms (score 5) in each animal group were apparent. This was at day 40 in untreated EAE mice and at day 110 for triterpene-treated EAE mice, after immunization.
Control mice (without EAE induction) were also injected daily with oleanolic acid or erythrodiol for an equivalent period of time.
Oleanolic acid and erythrodiol (Extrasynthese, Genay Cedex, France) were first dissolved in 2% w/v DMSO and then diluted with PBS for each experiment (the final concentration of DMSO was 0.2%, w/v).
Spinal cord tissue was obtained from five representative animals of the different experimental groups on day 30 after immunization. Tissues were fixed and embedded in paraffin, cut on a microtome (5 µm thicknesses), stained with eosin-haematoxylin. Histological examination was performed with a Nikon Eclipse 90i (Nikon Instruments, Inc., Amstelveen, the Netherlands) connected to a DXM1200C digital camera (Nikon Instruments Inc). Sections from 4–10 segments per mouse were examined by one investigator, without knowledge of the treatments.
Intravital microscopy in mouse brain
Intravital microscopy of the mouse cerebromicrovasculature was performed as previously described (Martín et al., 2010). Briefly, mice were anaesthetized at day 30 post-immunization by i.p. injection of a mixture of 100 mg·kg−1 ketamine and 10 mg·kg−1 xylazine, and the tail vein was cannulated for administration of fluorescent dyes. A craniotomy was performed using a high-speed drill (Dremel, Madrid, Spain) and the dura matter was removed to expose the underlying pial vasculature. The mouse was maintained at 37°C throughout the experiment and the exposed brain was continuously superfused with artificial CSF buffer at 37°C.
Leukocytes were fluorescently labelled by i.v. administration of rhodamine 6G (5 mg·kg−1 body weight) and visualized by a Zeiss Axioplan 2 imaging microscope (Hertfordshire, UK) connected to an AxioCam MR digital camera using the AxioVision AC imaging software and an Acroplan 20x/0.50W Ph2 lens. Eight different post-capillary venules of diameter between 30 and 70 µm were chosen for observation. Rolling leukocytes were defined as white cells moving at a velocity less than that of erythrocytes. Leukocytes remaining stationary for 30 s or longer were considered adherent to the venular endothelium. Leukocyte adhesion was expressed as cells/mm2 of venular surface area, as shown previously (Martín et al., 2010).
Evaluation of cytokines and MOG-specific antibodies by elisa
Anti-MOG-specific IgM and IgG isotypes were detected in serum samples collected from animals on day 30 after immunization, using elisa. In brief, 96-well polystyrene microtitre plates were coated with 0.5 mg per well of MOG35–55 peptide diluted in PBS overnight in a humidified chamber followed by PBS washing and blocking for 1 h with 5% BSA in PBS. Wells were incubated in duplicate with serum samples diluted 1:60 in PBS for 2 h at room temperature. After washing, HRP-labelled rat anti-mouse IgM, anti-mouse IgG, anti-mouse IgG1 and anti-mouse IgG2a (1:2000) from Serotec (Sigma-Aldrich, St Louis, MO, USA) were subsequently added for 90 min. After another washing, adding the substrate, and arresting the reaction with 0.1N HCl, absorbance was read at 450 nm. Data are expressed as mean optical density at 450 nm.
Leptin levels in serum samples and spinal cord tissue were determined by elisa (RayBiotech, Norcross, GA, USA). For cytokine quantification (IL-4, IL-6, IL-10, IL-17, TNF-α, and IFN-γ), cell culture medium, serum and spinal cord tissue were analysed by elisa according to the manufacturer's protocols (eBioscience, San Diego, CA, USA). Spinal cords were removed on day 30 after immunization or at the severe stage of the disease (score 5), weighed and then frozen at −80°C. SC tissue was homogenized by using a tissue homogenizer (Cole-Parmer Instrument, Vernon Hills, IL, USA) in an ice bath in 0.5 mL ice-cold PBS supplemented with 0.4 M NaCl, 0.05% Tween 20, 0.5% BSA and a protease inhibitor cocktail: 20 µg·mL−1 of leupeptin, 20 KI units of aprotinin, 0.1 mM phenylmethylsulphonyl fluoride (Sigma-Aldrich), and centrifuged at 3000× g for 10 min at 4°C. Supernatant were stored at −80°C until cytokine assays were performed. Total protein was assayed using the Bradford method. A 50 to 100 µL sample of each supernatant was used for tests.
Data were processed and expressed as pg of cytokine per mg of spinal cord wet weight, or pg of cytokine per mL for serum samples.
BBB permeability measurement
To evaluate BBB disruption, we measured the extravasation of Evans blue (EB) dye as a marker of albumin extravasation. At 30–31 days following EAE induction, mice were injected i.p. with 1 mL of 4% w/v EB. After 4 h, mice were killed, perfused, and brain and spinal cords were removed. Dye was extracted for 2–3 days in formamide (4 mL·g−1 of wet tissue) at room temperature. Extracted dye concentration was determined by measuring the absorbance at 650 nm. CNS tissue was dried 24 h at 60°C and weighed. Calculations were based on external standard readings and extravasated dye was expressed as mg of EB per mg dried weight of tissue.
Murine BV-2 cells, an immortalized murine microglia cell line, exhibit phenotypic and functional properties comparable with those of primary microglia and hippocampal neurons (Bocchini et al., 1992). BV-2 cells (a gift from Prof J. Bethea, Miller School of Medicine, Miami, FL, USA) were cultured in Dulbecco's modified Eagle's medium high sucrose, supplemented with 10% fetal bovine serum (FBS), 100 U·mL−1 penicillin and 100 µg·mL−1 streptomycin, and kept at 37°C in 5% CO2. Cells were seeded in 96-well plates (5 × 104 cells per well) or 60 mm culture dishes (3 × 106 cells per well.).
Cell proliferation was quantified by using the Promega kit (Madison, WI, USA), Cell Titer 96® Aqueous One Solution Cell Proliferation Assay, according to the manufacturer's recommendations. Briefly, cells were seeded in 96-well plates and serum starved for 24 h. Then, cells were treated in triplicate with IFN-γ, leptin or LPS, in the presence or absence of the triterpenes. After 24 h of incubation, formazan product formation was assayed by recording the absorbance at 490 nm in a 96-well plate reader (OD value). Formazan is measured as an assessment of the number of metabolically active cells and expressed in percentages relative to FBS-stimulated cells. Cell viability was assessed by Trypan blue exclusion.
Western blot analysis
Cells were washed with PBS and harvested in Laemmli SDS sample buffer. Protein extracts were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% BSA-TBST at room temperature and then incubated for 18 h at 4°C with the indicated antibodies including ERK 1/2 (Zymed Laboratories, South San Francisco, CA, USA), rabbit p-ERK1/2, p-rS6 (Cell Signaling Technology, Danvers, MA, USA), COX-2 (sc-1745, Santa Cruz Biotech, Santa Cruz, CA, USA), actin (sc-8432, Santa Cruz Biotech) and iNOS (BD Biosciences, Lexington, KY, USA). After washing with TBST buffer, a 1:2.000 (v/v) dilution of horseradish peroxidase-labelled IgG was added at room temperature for 1 h. The blots were developed using enhanced chemiluminescence.
Cells were stimulated in serum-free media with or without 100 UI·mL−1 of IFN-γ, 1 µg·mL−1 of LPS or 0.5 µM of leptin for 24 h, in the presence or absence of different doses of oleanolic acid or erythrodiol and then exposed to 0.1 mg·mL−1 of FITC-labelled dextran (MW 40 000) for 2 h. Non-internalized particles were removed by vigorous washing with cold PBS (pH 7.4) prior to measuring fluorescence at 480 nm excitation and 520 nm emission on either a Flow Cytometer (Gallios™; Beckman Coulter, Fullerton, CA, USA) or a Fluoroskan multiwell plate reader (TECAN Genios Pro; Tecan Group Ltd, Zurich, Switzerland). Cultures without fluospheres were used (blank wells) as background. Each culture condition was done in triplicate, and three independent experiments were performed. To confirm that the fluospheres were accumulated intracellularly, a Leica TCS SP5X confocal microscope was used with the Leica LAS AF acquisition software (Wetzlar, Germany) and a ×60 oil objective.
Statistical analysis was performed with the GraphPad Prism Version 4 software (San Diego, CA, USA) by anova. Analyses were performed using repeated measures anova (or two-way anova) for comparison of clinical parameters, and one-way anova for comparison of parameters such as cytokines, extravasation, leukocytes and MOG antibodies. A post hoc analysis was made by the Bonferroni's multiple comparison test. P < 0.05 was considered statistically significant.
Effects of preventive treatment with oleanolic acid or erythrodiol on clinical EAE
Female C57BL/6 mice exhibit active EAE after immunization with the MOG35–55 peptide. In this experimental model we compared the effects of two pentacyclic triterpenes, oleanolic acid and erythrodiol given at a dose (50 mg·kg−1) previously proven to be both safe and therapeutically relevant in rodents (Jeong, 1999; Senthil et al., 2007; Martín et al., 2010) in two regimens: 7 days before immunization (day -7; OA-7, ERY-7) or at the day of induction (day 0; OA0, ERY0). The clinical analysis of the different groups of animals is shown in Figure 1. The placebo-treated animals developed neurological symptoms of active EAE after 12 to 31 days, consisting of tail limpness and a mild-to-moderate paraparesis, as well as progressive weight loss. Interestingly, when oleanolic acid or erythrodiol were administered from the day of induction, clinical disease was markedly less severe and mice had a later onset of the clinical signs compared with untreated animals with EAE (Figure 1A). First neurological symptoms (score 1) were observed at day 11 with mean day of onset 13.5 ± 2 in untreated EAE mice, while OA0 or ERY0 animals showed no clinical signs at that time and a similar score (tail atony) was first reached on day 27 (mean values 33 ± 2 and 34 ± 2 days respectively). When the triterpenes were given as a pre-treatment, starting 1 week before EAE induction, clinical disease remained mostly suppressed for the duration of the experiment (until day 30 post-induction)........... https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3419913/
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