Conceived and designed the experiments: FZ OA CID. Performed the experiments: KH JMM NH. Analyzed the data: KH JMM NH. Wrote the paper: KH JMM. Edited the manuscript: FZ OA CID.
The authors have declared that no competing interests exist.
Multiple sclerosis (MS) is an inflammatory autoimmune disease of the central nervous system. However, studies of MS and the animal model, experimental autoimmune encephalomyelitis (EAE), indicate that neuronal pathology is the principle cause of clinical disability. Thus, there is need to develop new therapeutic strategies that not only address immunomodulation but also neuroprotection. Here we show that the combination therapy of Glatiramer acetate (GA), an immunomodulatory MS therapeutic, and the neuroprotectant epigallocatechin-3-gallate (EGCG), the main phenol in green tea, have synergistic protective effects
Multiple Sclerosis (MS) is a common inflammatory autoimmune disease of the central nervous system (CNS) in which myelin-specific CD4+Th1 and Th17 cells are thought to orchestrate effector processes resulting in the destruction of the myelin sheath
Glatiramer acetate (GA, Copaxone, Copolymer 1) is an immunomodulatory agent for treatment of relapsing-remitting MS. GA is a synthetic basic random copolymer composed of tyrosine, glutamate, alanine and lysine that induces GA-specific Th2 cells which secrete anti-inflammatory cytokines in the CNS through cross-recognition with myelin autoantigens
We have recently shown that epigallocatechin-3-gallate (EGCG), the main phenol of green tea, can reduce clinical severity of EAE by both limiting brain inflammation and reducing neuronal damage
In light of the immunomodulatory and neuroprotective properties of GA and EGCG we hypothesized an additive or synergistic protective effect of GA and EGCG in inflammation as well as in neuroprotection
Combination therapy of GA and EGCG synergistically reduced neuronal cell death and promoted axonal outgrowth of primary neurons. These effects could be translated into the EAE model in which diminished clinical disease severity was associated with reduced CNS inflammation in a synergistic manner. These results strengthen the prospects of EGCG as an adjunct and well-tolerated therapy for neuroinflammatory diseases, and underscore the importance of evaluating combined anti-degenerative and anti-inflammatory treatments.
Animals were housed in the animal facility of the Neuroscience Research Center of the Charité Universitätsmedizin Berlin. Experimental procedures were approved by the regional animal study committee of Berlin (LAGeSo approval ID G0147/05). All animal work was conducted in accordance to national and international guidelines to minimize discomfort to animals.
For assessment of viability, hippocampal HT22 cells were seeded at 5,000 cells per well in 96 well plates and cultured for 24 h. On the following day, cells received fresh medium and were incubated with 10 µg/ml EGCG, 50 µg/ml GA, or both compounds for 2 h before incubation with 5 mM glutamate. In parallel, glioblastoma LN18 cells were cultured at 30,000 cells per well along with 10 µg/ml EGCG and/or 12.5 µg/ml GA, and cell death was induced by incubation with TRAIL (20 ng/ml, Alexis, San Diego, CA) and TRAIL enhancer (2 µg/ml). The crystal violet assay was performed 16 h thereafter as previously described
Entorhinal cortex slices were prepared at postnatal day 2 from mouse brains as previously described
6–8 week old female SJL/L mice (Charles River Laboratories, Sulzfeld, Germany) were immunized s.c. with 250 µg of proteolipid protein (PLP) peptide 139–151 (purity >95%; Pepceuticals, Leicester, UK) emulsified in an equal volume of PBS and complete Freund's adjuvant containing 6 mg/ml Mycobacterium tuberculosis H37Ra (Difco, Franklin Lakes, NJ). 200 ng of Bordetella pertussis toxin (List Biological Laboratories, Campell, CA) was administered i.p. at day 0 and 2. For preventive treatment, EGCG (Sigma-Aldrich, Deisenhofen, Germany) was dissolved in 0.9% NaCl. 300 µg of EGCG or vehicle (0.9% NaCl) were given by oral gavage twice daily from day-9 before immunization on. 50 µg GA (TEVA) or vehicle (Mannitol 4%) was dissolved in PBS, emulsified in incomplete Freund's adjuvant (Difco) and injected s.c. once on day −7. Therapeutic treatment with 300 µg of EGCG or vehicle twice daily, and with 150 µg GA or vehicle once daily, was started after animals reached a clinical score≥2.
Mice were monitored daily for clinical symptoms and scored as follows: 0 = no disease; 1 = complete tail paralysis; 2 = hind limb paraparesis; 3 = bilateral hind limb paraplegia; 4 = complete forelimb and hind limb paraplegia; 5 = moribund or dead animals.
Spinal cords from mice transcardially perfused with 4% paraformaldehyde were postfixed overnight, cryoprotected successively in 15% and 30% sucrose and cut into 8 cross-sectional segments. The segments were embedded together in Cryo-Embed (Ax-lab), frozen in a methylbutane/dry ice mixture and cut into 12 µm sections with a cryostat. Hematoxylin and eosin stained sections were examined by light microscopy for the presence of inflammation, using a Zeiss Observer.Z1 microscope with a Zeiss AxioCam MRm camera. Semi-quantitative assessment was done by counting the number of quadrants with inflammatory infiltrates for each of the 8 segments. Quadruplicate sections for each mouse were assessed in a blinded manner and data are presented as the percentage of total quadrants positive for inflammatory infiltrates.
EAE disease courses, histology scores, and cumulative disease activity were analyzed with the non-parametric Kruskal-Wallace ANOVA with the Dunn's post-hoc test, and the Mann-Whitney test. The cell death and axonal outgrowth assays were analyzed with 1-way ANOVA with the Bonferroni correction. Time to disease onset was analyzed with the logrank test, using the Bonferroni correction for multiple comparisons. GraphPad Prism 4 (GraphPad Software, San Diego CA) was used for the analysis. p-values<0.05 were considered significant (* p<0.05; ** p<0.01; *** p<0.001).
We asked whether the combination of EGCG and GA could have an additive or synergistic effect in preventing neuronal damage in mouse and human CNS cell lines. Therefore, we induced neuronal cell death through two mechanisms, which are known to contribute to neuronal damage in neuroinflammation, glutamate-promoted excitotoxicity and TRAIL-mediated apoptosis.
We induced excitotoxicity by incubation of murine hippocampal HT22 cells with glutamate. We preincubated the cells with different concentrations of EGCG and GA and established suboptimal doses of EGCG (10 µg/ml) or GA (50 µg/ml). Suboptimal doses of EGCG and GA showed little or no neuroprotective effect compared to control, whereas the combination of 10 µg/ml EGCG and 50 µg/ml GA significantly increased survival of HT22 cells compared to controls, as determined by the crystal violet assay (
Survival of murine HT22 neuronal cells (A) and human glioblastomal LN18 cells (B) was determined in a crystal violet assay after induction of apoptosis by incubation with Glutamate (5 mM) (A) or TRAIL (20 ng/ml) (B). EGCG (10 µg/ml) and/or GA (12.5 µg/ml) were added to the culture 2 h before apoptosis induction, *p<0.05, **p<0.001, representative of 2 experiments.
To examine whether the combination therapy is also protective in a caspase-dependent apoptotic pathway, we induced TRAIL-mediated cell death in susceptible human glioblastomal LN18 cells. Again, the combination of EGCG (10 µg/ml) and GA (12.5 µg/ml) showed a statistically significant increase in cell survival compared to control. Conversely, EGCG or GA alone did not induce a significant increase (
We next investigated whether EGCG and GA stimulate neuroregeneration using an axonal outgrowth model of entorhinal cortex explants. The explants were embedded in a three-dimensional collagen gel matrix with 5 µg/ml EGCG or 6.25 µg/ml GA alone or with the combination of 5 µg/ml EGCG and 6.25 µg/ml GA. The density of the outgrowing neurites was photo-documented at higher magnification and quantified (
Representative photomicrographs of brain slices with axonal outgrowth after addition of EGCG (5 µg/ml), GA (6.25 µg/ml) and the combination of EGCG (5 µg/ml) and GA (6.25 µg/ml) are shown. 100× magnification, scale bar: 300 µm (A). The number of axons was measured by quantifying all intersections of axons with a standardized line parallel to the brain slice border, **p<0.001 (B). The density of outgrowing axons was determined by measuring the mean intensity in a standardized area parallel to the brain slice edge, **p<0.001 (C). Levels of GDNF (D) and BDNF (E) were measured in the supernatant of outgrowth assays by ELISA, n.d. = not detectable. **p<0.001 ***p<0.0001.
The total number of axons increased significantly in the presence of EGCG and GA in combination compared to control brain slices (
Axons in slices treated with both GA and EGCG showed a significantly higher density compared to brain slices under control conditions. Again, there was no significant effect of EGCG or GA alone (
Next, we tested whether these neuroprotective and neuroregenerative effects of EGCG and GA
For preventive treatment, EGCG was given orally twice daily starting nine days before EAE induction, and GA was injected s.c. in incomplete Freund's adjuvant once seven days before immunization with PLP139–151. As expected, treatment with suboptimal doses of GA alone did not significantly decrease disease incidence and severity. However, the combination therapy significantly reduced the severity of relapsing-remitting EAE and synergistically delayed the onset of disease compared to vehicle treated animals (
For preventive treatment EGCG (300 µg) or vehicle (NaCl 0.9%) was administered orally twice daily from day −9 before immunization with PLP139–151 until day 45. GA (50 µg) or vehicle (Mannitol 4%) in incomplete Freund's adjuvant was given s.c. once on day −7 before immunization. Mean disease score of control, EGCG, GA and the combination therapy group is shown from day 0 to day 45 after immunization (A), * p<0.05 day 13,15–18,20–22,24,26–27, **p<0.01 day 14,19,23,25,28,35–39,42, n = 8 per group. Onset of disease in control, GA, EGCG and combination therapy group is presented in a Kaplan-Meier survival curve as percentage of sick mice at a certain day after immunization (B), *p<0.05. Cumulative disease activity of control, GA, EGCG and combination therapy group was calculated as the area under the curve of the clinical score plots for each individual animal (C), *p<0.05.
For the treatment of patients suffering from MS it is important to know whether the combined treatment could have a protective effect in established disease. For this therapeutic paradigm, treatment was initiated when all mice showed a clinical score≥2. EGCG (300 µg) was given twice daily, and GA (150 µg) was injected subcutaneously daily. The combination therapy enhanced recovery and prevented long-term neurological deficits, as indicated by significant reductions in the mean clinical score (
For treatment of acute disease PLP139–151-induced EAE mice were randomized into two groups after reaching a disease score of 2 and subsequently received 300 µg of oral EGCG or vehicle (0.9% NaCl) twice daily in addition to daily s.c. injections of 150 µg GA or vehicle (4% Mannitol). Mean disease score of control and combination therapy group is shown from day 0 to day 44 after immunization (A), *p<0.05 day 19, 20, 23, 24, 34, n = 6. Cumulative disease activity of control and combination therapy group was calculated (B), *p<0.05.
We then examined whether the synergistic protective effects of the preventive EGCG and GA treatment on EAE clinical scores would be reflected in the CNS inflammatory pathology. Histological examination revealed extensive inflammatory lesions throughout the spinal cord in control mice. While some reduction in the histopathology could be seen in both the EGCG and GA single treated groups, this effect was synergistic in the combination treatment group (
Representative Hematoxylin and Eosin staining of transverse spinal cord sections of vehicle, EGCG, GA and EGCG and GA treated EAE mice is shown (A). 100× magnification, scale bar 500 µm. Inflammation in standardized areas of the spinal cord of all treatment groups was assessed and is presented semi-quantitatively as percentage of spinal cord quadrants with inflammatory foci relative to all assessed quadrants (B), *p<0.05, n = 8 per group.
GA is one of the most common immunomodulatory therapies for MS. In addition to the inflammatory components of the pathology, neurodegeneration, axonal damage and deficits in neuroregeneration are important aspects of the disease and should be taken into consideration as targets for therapeutic approaches. Thus, we investigated the potential of the combination therapy of GA and the previously tested neuroprotective agent EGCG.
We showed a synergistic effect of the combination therapy of EGCG and GA on neuronal survival and axonal growth
Neuronal cell death is a major feature in the pathology of neuroinflammatory diseases such as MS. Several mechanisms have been shown to contribute to neuronal damage in neuroinflammation, such as TRAIL-mediated apoptosis and glutamate-promoted excitotoxicity. We previously reported that the death ligand TRAIL critically contributes to irreversible CNS damage during autoimmune neuroinflammation
Increase in extracellular glutamate by activated microglia during neuroinflammation can have serious consequences, as it is capable of precipitating excitotoxic cell death of neurons and oligodendrocytes by overstimulation of ionotropic glutamate receptors
Both our previous study
The mechanisms underlying GA-mediated neuroprotection involve triggering T cell reactivation and inducing T cells to switch to a protective phenotype
It has been shown that multiple endogenous repair mechanisms become activated after damage to the central nervous system in the context of traumatic injury, neurodegenerative or neuroinflammatory diseases. These include repopulation of damaged tissue by precursor cells for neurons and oligodendrocytes, and remyelination, as well as sprouting and rewiring of axon collaterals, formation of new synapses and axonal regeneration. However, these endogenous self-repair mechanisms are not sufficiently effective to reverse the damage that occurs in severe or chronic disease. In MS, where demyelination and axonal damage are major hallmarks of the disease, some efforts have been made in developing regenerative therapies that further promote remyelination, but very few studies focus on promotion of axonal growth and regeneration
In our previous study we demonstrated a clear disease-limiting effect of EGCG in EAE
An increasing body of evidence indicates that cumulative axonal loss in MS correlates with permanent clinical disability and that axonal damage begins at the earliest stages of disease
Thus, the combination therapy of GA and EGCG is a promising and safe approach for MS as the therapeutic effects of the already established agent GA might be enhanced by the neuroprotectant EGCG. The beneficial effects of EGCG and GA could also be relevant for other chronic neurodegenerative diseases, as Alzheimer's and Parkinson's disease; thus combination therapy with these compounds could have even broader clinical implications.
We thank J. Lips and B. Seeger for technical assistance, S. Duke as native speaker for help with the manuscript, and C. Pfüller for useful discussions.