The synthetic triterpenoid CDDO-methyl ester modulates microglial activities, inhibits TNF production, and provides dopaminergic neuroprotection
© Tran et al; licensee BioMed Central Ltd. 2008
Received: 06 March 2008
Accepted: 12 May 2008
Published: 12 May 2008
Recent animal and human studies implicate chronic activation of microglia in the progressive loss of CNS neurons. The inflammatory mechanisms that have neurotoxic effects and contribute to neurodegeneration need to be elucidated and specifically targeted without interfering with the neuroprotective effects of glial activities. Synthetic triterpenoid analogs of oleanolic acid, such as methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me, RTA 402) have potent anti-proliferative and differentiating effects on tumor cells, and anti-inflammatory activities on activated macrophages. We hypothesized that CDDO-Me may be able to suppress neurotoxic microglial activities while enhancing those that promote neuronal survival. Therefore, the aims of our study were to identify specific microglial activities modulated by CDDO-Me in vitro, and to determine the extent to which this modulation affords neuroprotection against inflammatory stimuli.
We tested the synthetic triterpenoid methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me, RTA 402) in various in vitro assays using the murine BV2 microglia cell line, mouse primary microglia, or mouse primary peritoneal macrophages to investigate its effects on proliferation, inflammatory gene expression, cytokine secretion, and phagocytosis. The antioxidant and neuroprotective effects of CDDO-Me were also investigated in primary neuron/glia cultures from rat basal forebrain or ventral midbrain.
We found that at low nanomolar concentrations, treatment of rat primary mesencephalon neuron/glia cultures with CDDO-Me resulted in attenuated LPS-, TNF- or fibrillar amyloid beta 1–42 (Aβ1–42) peptide-induced increases in reactive microglia and inflammatory gene expression without an overall effect on cell viability. In functional assays CDDO-Me blocked death in the dopaminergic neuron-like cell line MN9D induced by conditioned media (CM) of LPS-stimulated BV2 microglia, but did not block cell death induced by addition of TNF to MN9D cells, suggesting that dopaminergic neuroprotection by CDDO-Me involved inhibition of microglial-derived cytokine production and not direct inhibition of TNF-dependent pro-apoptotic pathways. Multiplexed immunoassays of CM from LPS-stimulated microglia confirmed that CDDO-Me-treated BV2 cells produced decreased levels of specific subsets of cytokines, in particular TNF. Lastly, CDDO-Me enhanced phagocytic activity of BV2 cells in a stimulus-specific manner but inhibited generation of reactive oxygen species (ROS) in mixed neuron/glia basal forebrain cultures and dopaminergic cells.
The neuroimmune modulatory properties of CDDO-Me indicate that this potent antioxidant and anti-inflammatory compound may have therapeutic potential to modify the course of neurodegenerative diseases characterized by chronic neuroinflammation and amyloid deposition. The extent to which synthetic triterpenoids afford therapeutic benefit in animal models of Parkinson's and Alzheimer's disease deserves further investigation.
Plant-derived triterpenoids, including oleanolic acid and ursolic acid, have been used extensively in Asian countries for their anti-inflammatory and anti-tumor properties . In an attempt to increase the potency of these natural products, over 300 synthetic derivatives of oleanolic acid were generated and tested for their ability to inhibit NO production in activated macrophages [2–4]. Some of the most potent of these, including 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO; RTA 401) and its methyl ester (CDDO-Me; RTA 402), exhibit greater than 2 × 105-fold increased potency compared to the parental compound. CDDO-Me is presently in Phase I/II clinical trials for the treatment of solid tumors. In light of their potent bioactivity, this new class of compounds has therapeutic potential in the treatment and prevention of acute and chronic inflammatory syndromes.
Although identification of the molecular targets of triterpenoids is just underway, a number of recent studies have identified key mechanisms that mediate the potent effects of triterpenoids. One of these mechanisms involves decreasing the levels of reactive oxygen species (ROS) through activation of Nrf2-dependent transcription [5, 6]. In addition, the triterpenoids directly inhibit NF-κB signaling [7, 8], a key pathway that regulates the production of a number of inflammatory mediators and their signaling cascades (e.g. TNF, IL-1β, IFNγ, TLR) . Increased levels of antioxidant enzymes produced by Nrf2 reduce the cellular levels of ROS, thereby further attenuating NF-κB signaling and the transcription of pro-inflammatory genes such as iNOS and TNF [5, 10, 11].
The role of neuroinflammation in neurodegenerative disease has been under intense investigation in recent years and there is now overwhelming evidence that inflammation-induced oxidative stress compromises neuronal survival and may contribute to the progression of neurodegenerative diseases including Parkinson's (PD) and Alzheimer's disease (AD) (reviewed in [12–18]). We reasoned that if CDDO-Me were able to suppress microglial activities that contribute to neurotoxicity while promoting those that support neuronal survival, it may be capable of exerting neuroprotective effects. Therefore, the overall purpose of these studies was to investigate the cellular basis for the anti-inflammatory properties of CDDO-Me; specifically, to identify microglial activities modulated by CDDO-Me in vitro and the extent to which this modulation protects against inflammatory stimuli.
Experimental procedures involving use of animal tissue were performed in accordance with the NIH Guidelines for Animal Care and Use and approved by the Institutional Animal Care and Use Committee (IACUC) at The University of Texas Southwestern Medical Center in Dallas. Animals were housed in a climate controlled facility staffed with certified veterinarians.
Lyophilized stocks of the synthetic triterpenoid CDDO-Me (RTA 402) were stored at -20°C until they were dissolved in DMSO.
The murine BV2 microglia cell line was generated by Dr. Bistoni and colleagues by infecting primary microglial cell cultures with the v-raf/v-myc oncogene carrying retrovirus J2. These cells retain many of the morphological, phenotypical and functional properties described for freshly isolated microglial cells . BV2 microglia were cultured in DMEM/F12 supplemented with 5% heat-inactivated fetal bovine serum (Sigma-Aldrich, St Louis MO), 1% penicillin-streptomycin, and 1% L-glutamine. The murine clonal hybrid cell line MN9D was developed by A. Heller and colleagues by somatic cell fusion of rostral mesencephalic tegmentum (RMT) from 14-day-old embryonic mice and the murine neuroblastoma cell line N18TG2 . MN9D cells were grown in DMEM (Sigma-Aldrich) supplemented with 10% FBS (Gemini, West Sacramento CA), and 1% penicillin/streptomycin. To induce terminal differentiation of MN9D cells and increase their sensitivity to apoptotic stimuli, cells were incubated with 5 mM of valproic acid in N2 (Invitrogen, Carlsbad, CA)-supplemented serum-free DMEM for 3 days. Primary microglia were harvested from postnatal day 2–4 mouse pups using previously published protocols . Briefly, brain tissue was removed, finely minced with a razor, incubated in a dissociation media containing 1 μL/mL DNAseI (Invitrogen), 1.2 U/mL dispase II (Roche), and 1 mg/mL papain (Sigma-Aldrich) in DMEM/F12 (Sigma-Aldrich) for 30–45 minutes at 37°C. After mechanical trituration, cells were centrifuged, passed through a 40 μM filter (BD Falcon, San Jose CA), and plated at 500,000 total cells/well in 6 well plates pre-coated with 0.1 mg/mL poly-D lysine (Sigma). Cells were fed every 3 days with fresh media (DMEM/F12 supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 1% penicillin-streptomycin, and 1% L-glutamine). After 14–18 days in vitro, cultures were treated with 0.0625% trypsin-EDTA (diluted in DMEM/F12) for 45 minutes at 37°C to lift astrocytes and neurons from the wells, leaving a pure culture of primary microglia. The cultures were checked for purity and found to be greater than 95% microglia as measured by cell-type specific expression of CD68 and less than 5% astrocytes as measured by GFAP immunoreactivity. Murine peritoneal macrophages were obtained by eliciting an acute peripheral inflammatory reaction with intraperitoneal injection of thioglycolate . Briefly, adult mice were injected intraperitoneally with 1 mL 3% Brewer's yeast thioglycolate. Three days later the animals were sacrificed, and 10 mL of cold sterile PBS (pH 7.4) was injected into the peritoneal cavity to wash out and recover peritoneal exudate. Cells were pelleted (1000 rpm, 5 min, 4°C), resuspended in culture media (high glucose DMEM supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA), and 1% penicillin, streptomycin, and L-glutamine) and allowed to adhere to culture plates for 6 hr. Cells were washed twice with PBS to remove non-adherent cells and growth medium was replenished.
Aggregation of amyloid beta peptide
Aβ 1–42 peptide was synthesized by Dr. Haydn Ball in the Protein Chemistry Core at UT Southwestern. Aggregation into fibrillar form was achieved by resuspending the peptide at final concentration of 100 μM into phosphate-buffered saline and incubating it at 37°C for 48 hrs. Thioflavin T fluorescence  and Congo Red binding in vitro was used to confirm fibril formation .
Microglia proliferation and viability assays
Cell viability and proliferation were assayed in the BV2 microglia cell line using the Alamar Blue reagent (Invitrogen). Cells were seeded at 2000 cells/well in a 96-well plate. Cells were serum deprived for 3–5 hours, and CDDO-Me was added 1 hour before treatment with LPS as indicated. Alamar Blue was added as per the manufacturer instructions 2 hr before absorbance was read at 570 nm and 595 nm.
Microglia activation assays
Rat embryonic ventral mesencephalon primary cultures were harvested from E14 pups, mechanically dissociated and seeded as micro-islands (25 uL of 1 × 106 cells/mL) on 4-chamber slides precoated with poly-D-lysine and laminin (BD Bioscience) in DMEM/F12 with 1% penicillin/streptomycin, glutamine, and non-essential amino acid and containing 10% fetal bovine serum (Atlanta Biologicals) and 10 ng/mL FGF-2 (R&D Systems, Minneapolis, MN). Culture media was changed after 2 days in vitro and cells were treated at day 5 in vitro with indicated compounds in DMEM containing 2.5% FBS and lacking FGF-2. Cells were fixed at 2 days post-treatment in 4% paraformaldehyde in PBS (pH 7.4) and stained with an antibody against activation marker F4/80 (1:60 dilution Serotec, Raleigh, NC) to quantify number of activated microglia. Each condition was done in triplicate; 20 random sites were visited per well; data was plotted as the average number of F4/80-positive microglia per field.
BV2 cells were plated at 500,000 cells/well in a 6-well plate in DMEM with 5% FBS and switched to serum-free media before pre-treatment with CDDO-Me or DMSO vehicle and subsequent stimulation with LPS as indicated. RNA was harvested as detailed below and levels of inflammation-related gene expression were detected on an oligonucleotide array as per manufacturer's instructions (Superarray Bioscience Corporation, Frederick, MD). Data analyses were performed using the Scatter Plot data analysis tool in the SuperArray GEArray Analysis Suite. The Scatter Plot displays the fold difference in the relative expression levels of genes between groups. Seven housekeeping genes were used for normalization. The control group was assigned to the X-Axis, and the treated group was assigned to the Y-Axis. An arbitrary boundary of 1.5-fold regulation in either direction was selected. If the fold increase was greater than boundary value, the gene names are shown in red with a plus (+) sign, and are located above the upper line. The further the sign is from the upper line, the greater the fold difference. If the fold decrease is greater than the boundary value, the genes are shown in green with a minus (-) sign, and are located below the lower line. The further the sign is from the lower line, the greater the fold difference. Black signs mean the fold change is not significant.
Real time quantitative polymerase chain reaction
Real-time quantitative PCR (QPCR) was performed as previously described . Briefly, total RNA was isolated from cultured cells or animal tissues using RNA Stat-60 (Tel-Test, Inc., Friendswood, TX), treated with DNase I (Invitrogen), and reverse transcribed using Superscript II RNase H-reverse transcriptase (Invitrogen). RNA concentration was determined by absorbance at 260 nm. Quantitative real-time PCR was performed using an ABI Prism 7900 HT Fast Detection System (Applied Biosystems Inc., Foster City, CA). Each 10 μl reaction was performed in 384-well format with 25 ng cDNA, 5 μl SYBR green PCR Master Mix, and 150 nM of each PCR primer. All reactions were performed in duplicate. Levels of mRNA expression were normalized to those of the mouse house-keeping gene cyclophilin B. Oligonucleotide primers for QPCR were obtained from Integrated DNA Technologies (Coralville, IA). The following mouse primers sequences were validated and used for gene amplification:
mTNF: forward 5'-CTG AGG TCA ATC TGC CCA AGT AC-3' and reverse 5'-CTT CAC AGA GCA ATG ACT CCA AAG-3'
mIL1β: forward 5'-CAA CCA ACA AGT GAT ATT CTC CAT G-3' and reverse 5'-GAT CCA CAC TCT CCA GCT GCA-3'
mMip1α: forward 5'-TTC ATC GTT GAC TAT TTT GAA ACC A-3' and reverse 5'-GCC GGT TTC TCT TAG TCA GGA A-3
iNOS: forward 5'-CAG GAG GAG AGA GAT CCG ATT TA-3' and reverse 5'-GCA TTA GCA TGG AAG CAA AGA-3'
BV2 cells were plated at 500,000 cells/well in a 6-well plate in DMEM containing 5% FBS and switched to serum-free media before pre-treatment with CDDO-Me or DMSO vehicle and subsequent stimulation of LPS as indicated. Conditioned Medium (CM) was collected to measure the production of seven cytokines (IFN-γ, IL-1β, IL-6, IL-10, IL-12, KC/CXCL1, and TNF) using a multiplexed immunoassay per the manufacturer instructions (Meso-Scale Discovery, Gaithersburg, MD).
Intracellular reactive oxygen species (ROS) imaging
Rat embryonic ventral mesencephalon neuron/glia cultures were prepared as published previously . At 5 days in vitro (5 DIV), they were incubated with 3 μM DCFDA (Invitrogen) in serum-free growth medium for 40 min to quantify intracellular reactive oxygen species (ROS) production by fluorescence imaging. The next day, cells were treated with the vehicle, 1 μM fibrillar amyloid beta (Aβ) 42 peptide plus or minus 10 ng/mL LPS in the presence or absence of CDDO-Me as indicated for 24 hrs. Fluorescence images were captured on an Olympus CK40 microscope with a CoolSnap CCD ES monochromatic camera with a FITC filter in place. Quantification of fluorescence intensity was performed using intensity threshold analysis of digital images on an Alpha Innotech ChemiImager 4400 (Alpha Innotech, San Leandro, CA). MN9D dopaminergic cells were plated at 50,000 cells per well in DMEM containing 10% FBS in 24 well plates. The following day cultures were differentiated in N2 supplemented serum-free DMEM containing 5 mM valproic acid. 48 hours following differentiation MN9D cells were incubated with 3 uM DCFDA in serum free DMEM for 40 min, and then returned to differentiation media. Six hours after DCFDA loading, MN9D cells were treated with 10 nM CDDO-Me (or DMSO vehicle) for 16–20 hrs before a 30 minute stimulation with either TNF or conditioned media from LPS and CDDO-Me treated BV2 microglial cultures. BV2 microglial cultures for these experiments were plated in DMEM containing 5% FBS at 800,000 cells per well in a 6-well plate. Cultures were permitted to adhere to the plastic and then were pretreated with 10 nM CDDO-Me (or DMSO vehicle) for 16–18 hrs prior to stimulation with 10 ng/mL LPS (or saline vehicle). The following day conditioned media from BV2 cultures was removed and centrifuged at 1200 × g for 4 min before addition to DCFDA-loaded, differentiated MN9D dopaminergic cultures. Fluorescence images were captured on an Olympus CK40 microscope with a CoolSnap CCD ES monochromatic camera with a FITC filter in place. Quantification of ROS accumulation was performed by counting DCFDA-positive cell bodies in four fields per condition under 20× magnification (equivalent to approximately 50% of the plated area per well) in two independent experiments. DCFDA positive cells ranged between 38 and 420 per field depending on experiment and treatment. Values for treatment conditions in each experiment were expressed as fold increase in DCFDA positive cells per field relative to DMSO vehicle, saline treated control, and averaged between independent experiments.
Cell survival/neuroprotection assays
MN9D dopaminergic cells (grown as described above), were terminally differentiated with 5 mM valproic acid in N2-supplemented serum-free DMEM 3 days prior to neuroprotection studies with CDDO-Me. MN9D cell viability was measured using the CellTiter 96 AQueous Assay reagent (Promega, Madison, WI). This reagent uses the tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) and the electron coupling reagent, phenazine methosulfate (PMS). MTS is chemically reduced into soluble formazan in metabolically active cells. MN9D cell viability was assayed by measurement of formazan absorbance at 492 nm in multi-titer 96-well plates at 492 nm during the last 2–4 hrs of a three-day incubation with soluble TNF or a two-day incubation with BV2 conditioned media in target-effector assays in which the BV2 microglia cell line was used as the effector cell and the MN9D dopaminergic ells as the target cell. Specifically, conditioned medium (CM) from LPS-treated BV2 microglia was transferred to MN9D cell cultures to induce inflammation-induced death in a dose-dependent manner.
BV2 microglia were plated at a density of 50,000 cells/well in a 96-well plate and switched to serum-free media 24 hr later for stimulation as indicated with LPS (10 ng/mL) and/or fAβ (1 μM), in the presence or absence of CDDO-Me (10 nM). After 24 hr stimulation, phagocytosis was measured by exposing the cultures to fluorescently-labeled E. coli particles (Invitrogen) for 2 hr. Cells were incubated with trypan blue and rinsed with PBS to remove non-internalized particles prior to measuring fluorescence at 480 nm excitation and 520 nm emission on a Fluoroskan multiwell plate reader.
CDDO-Me inhibits proliferation and activation of microglia
CDDO-Me suppresses transcription of inflammatory mediators in microglia and macrophages
CDDO-Me attenuates microglial-mediated neuronal oxidative stress
CDDO-Me protects the dopaminergic MN9D cell line from inflammation-induced death
CDDO-Me enhancement of microglial phagocytic activity is stimulus-specific
Chronic neurodegenerative diseases are often associated with neuroinflammatory processes that may not only occur in response to neuron loss but may also contribute to it [15, 16, 27–30]. Because certain inflammatory responses in the brain are required for clearing cellular debris, limiting tissue damage, and contributing to wound repair, it is critical that inflammatory factors and mechanisms that contribute to neurotoxicity and compromise neuronal survival be identified and selectively targeted without interfering with the neuroprotective effects of glial activities . Our previous studies established a critical role for TNF as a mediator oxidative neurotoxin- and endotoxin-induced dopaminergic neuron death in models of PD . The ability of CDDO-Me to inhibit new synthesis of TNF in microglia and to effectively reduce soluble TNF production by activated cells offers one possible mechanism by which the anti-inflammatory properties of CDDO-Me affords protection to dopaminergic cells.
In addition to its anti-inflammatory properties, CDDO-Me may be able to boost or strengthen the immune system through upregulation of IL-2 signaling. IL-2 has been shown to be critical for survival, proliferation and differentiation of T-cells into effector cells and to confer a survival advantage to CD4+ T-cells to facilitate development of a memory population [37, 38]. In chronic inflammatory syndromes characterized by persistent elevation of pro-inflammatory cytokines, localized IL-2 depletion at sites of inflammation has been shown to be the most profound effect of long term exposure to TNF ; our data suggests that therapeutic use of CDDO-Me may be able to reverse this phenomenon.
The observed antioxidant effects of CDDO-Me are not surprising given that synthetic triterpenoids have been shown to activate Nrf2, the key transcription factor that globally regulates the phase II detoxification pathway. Regardless of whether the primary antioxidant effect of CDDO-Me is mediated via direct action on neuronal populations or by suppression of glial-derived extracellular ROS production to reduce oxidative stress in neurons, the mechanisms by which synthetic triterpenoids exert antioxidant effects merit further investigation in animal models of neurodegeneration where oxidative stress is believed to be the primary mediator of neuron death. In support of this idea, it was recently reported that feeding pharmacological inducers of the phase II detoxification pathway to Drosophila parkin mutants or flies overexpressing α-synuclein suppressed the neuronal loss in both models of Parkinson's disease .
In summary, our findings indicate that in response to specific inflammatory triggers, CDDO-Me is able to differentially regulate microglial activities without compromising either microglial survival or the ability of microglia to perform basic functions (i.e. phagocytosis). Moreover, the ability of CDDO-Me to limit production and secretion of neurotoxic pro-inflammatory cytokines and to attenuate intracellular ROS accumulation strongly suggest that chronic administration of brain-permeant synthetic triterpenoids will confer neuroprotection in vivo. Several other anti-inflammatory agents have been reported to have neuroprotective properties in in vitro and in vivo models of Parkinson's disease. Specifically, the tetracycline derivative minocycline, which inhibits TNF synthesis, potently attenuates DA neuron loss resulting from nigral LPS treatment of rats . Similarly, thalidomide (a non-selective immune modulating drug that reduces TNF expression through degradation of TNF mRNA) has been demonstrated to partially attenuate dopamine depletion in an MPTP mouse model of PD . Naloxone, an opioid receptor antagonist, protected rat DA neurons against inflammatory damage through inhibition of microglia activation and superoxide generation ; and the kappa-opioid receptor agonist dynorphin A (1–17) attenuated inflammation-mediated degeneration of DA neurons in rat midbrain neuron-glia cultures . In addition, dextromethorphan (DM), an ingredient widely used in antitussive remedies, has been shown to reduce the inflammation-mediated degeneration of DA neurons through inhibition of microglial activation . Therefore, the neuroprotective properties of CDDO-Me and related compounds merit further investigation in pre-clinical animal models of PD.
Lastly, it may be of interest to determine the extent to which the CDDO-Me-induced enhancement of microglial phagocytic activity observed in our in vitro studies can be achieved in vivo with brain-permeant synthetic triterpenoids. This issue may be of particular therapeutic relevance in the treatment of AD because pro-inflammatory cytokines, including TNF, have been shown to preferentially attenuate phagocytic activity of microglia induced by fibrillar Aβ amyloid peptides (but not by IgG antibody activation of Fc Receptor) through an E prostanoid receptor-dependent mechanism . On the basis of our results, we speculate that synthetic triterpenoids will be able to promote fibrillar amyloid clearance by potentiating the phagocytic activity of microglia at plaque sites characterized by inflammation where TNF is locally elevated. If synthetic triterpenoids can promote plaque clearance, their use as an adjunct therapy to reduce amyloid burden in patients with AD may be beneficial.
This work was supported by grant 5 R01 NS049433-02 from the National Institutes of Health, National Institute of Neurological Disorders and Stroke (MGT). The authors thank J. Repa for assistance with peripheral macrophage harvest, Y. Lei for help with proliferation assays, and A. Harms in the Tansey lab for technical assistance with primary microglia cultures. The authors thank T. Wyss-Coray for BV2 cells, E.M. Johnson Jr. for MN9D cells, and W.C. Wigley and D. Ferguson at Reata Pharmaceuticals Inc. for reagents and helpful discussions.
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