Experimental Neurology 257 (2014) 50–56
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Regular Article
Neuroprotective dimethyl fumarate synergizes with immunomodulatory interferon beta to provide enhanced axon protection in autoimmune neuroinflammation Christiane Reick a,b,1, Gisa Ellrichmann a,⁎,1, Jan Thöne a, Robert H. Scannevin c, Carsten Saft a, Ralf A. Linker d,2, Ralf Gold a,b a
Department of Neurology St. Josef-Hospital, Ruhr-University Bochum, Germany International Graduate School of Neuroscience, Ruhr-University Bochum, Germany c BiogenIdec, Inc. Cambridge, 02142 MA, USA d Department of Neurology, University of Erlangen, Germany b
a r t i c l e
i n f o
Article history: Received 15 December 2013 Revised 26 March 2014 Accepted 4 April 2014 Available online 13 April 2014 Keywords: Multiple sclerosis EAE Immunomodulation Neuroprotection Interferon-beta Dimethyl fumarate Combination therapy
a b s t r a c t Introduction: Despite recent advances in development of treatments for multiple sclerosis, there is still an unmet need for more effective and also safe therapies. Based on the modes of action of interferon-beta (IFN-β) and dimethyl fumarate (DMF), we hypothesized that anti-inflammatory and neuroprotective effects may synergize in experimental autoimmune encephalomyelitis (EAE). Methods: EAE was induced in C57BL/6 mice by immunization with MOG35–55-peptide. Murine IFN-β was injected s.c. every other day at 10.000 IU, and DMF was provided at 15 mg/kg by oral gavage twice daily. Control mice received PBS injections and were treated by oral gavage with the vehicle methylcellulose. Mice were scored daily by blinded observers and histological, FACS and cytokine studies were performed to further elucidate the underlying mechanism of action. Results: Combination therapy significantly ameliorated EAE disease course in comparison to controls and monotherapy with IFN-β. Histological analyses showed a significant effect on axon preservation with almost twice as much axons present in inflamed lesions as compared to control. Remarkably, the effect on axonal preservation was more pronounced under combination therapy than with both monotherapies. Neither monotherapy nor combination therapy demonstrated modulation of cytokines and frequency of antigen presenting cells. Discussion: Combination of IFN-β and DMF resulted in greater beneficial effects with improved tissue protection as compared to the respective monotherapies. Further combination studies of these safe therapies in human disease are warranted. © 2014 Elsevier Inc. All rights reserved.
Introduction Multiple sclerosis (MS) is a chronic neurological disease of the central nervous system (CNS) of presumed autoimmune origin that is characterized by inflammation-driven demyelination, and subsequent axonal damage and progressive neurological deficits. Studies in MS and experimental autoimmune encephalomyelitis (EAE), an established animal model mimicking several pathophysiological features of MS, demonstrated that demyelinating lesions are accompanied by T cell and macrophage infiltration (Barnabe-Heider and Miller, 2003; Gold et al., 2006; Weiner, 2009). Subsequent to T cell and macrophage
⁎ Corresponding author at: Department of Neurology, Ruhr University Bochum, D-44791 Bochum, Germany. Fax: + 49 234 509 2414. E-mail address: [email protected] (G. Ellrichmann). 1 These authors share first authorship. 2 Current address.
http://dx.doi.org/10.1016/j.expneurol.2014.04.003 0014-4886/© 2014 Elsevier Inc. All rights reserved.
activation and infiltration, these cells release pro-inflammatory cytokines, interact with antigen presenting cells (APCs) and are involved in destruction of the myelin sheet, which is associated with chronic axonal damage (Herrero-Herranz et al., 2008). For as long as 20 years interferon-beta (IFN-β) has been an established immunomodulatory agent in patients with relapsing remitting MS (RRMS). In phase III studies IFN-β showed beneficial effects on relapse rate, disease progression and development of new CNS lesions as shown by magnetic resonance imaging (MRI) (Burks, 2005; Jacobs, 1996). So far, different mechanisms of action have been described for IFN-β with a focus on downregulation of macrophage and microglia activity (Mendes and Sa, 2011). Dimethyl fumarate (DMF; study name BG-12) has shown beneficial effects in both EAE and in models of neurodegenerative diseases as well as in two phase III studies in patients with RRMS (Ellrichmann et al., 2011; Ghoreschi et al., 2011; Linker et al., 2011). So far, different mechanisms of action, including neuroprotection, have been described. DMF
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was shown to mediate neuroprotection by inducing the nuclear factor E2-related factor 2 (Nrf2) pathway and thereby addressing toxic-oxidative stress (Ghoreschi et al., 2011; Johnson et al., 2008; Williamson et al., 2012). In APCs, DMF induced glutathione depletion and thus stimulated type II dendritic cells (DCs) with an impaired secretion of interleukin- (IL) 12 and IL-23. Simultaneously, DMF induced both IL-4 producing T helper (Th) 2 cells and generated type II dendritic cells (DCs) that produce IL-10 (Ghoreschi et al., 2011). Based on their different modes of action we postulated that IFN-β and DMF synergize in early and early chronic EAE. We show that the combination of IFN-β and DMF resulted in a synergistic effect at the clinical level, as reflected by decreased disease severity of EAE. This effect was confirmed histologically by reduced inflammation and in particular preservation of axonal density in the spinal cord of EAE mice. The beneficial effects of combination therapy with IFN-β and DMF may result from effects on different pathophysiological pathways in EAE. Material and methods Animal models 8–10 week old female C57BL6/J mice were obtained from the Harlan Laboratories (Borchen, Germany, nowadays Rossdorf, Germany) and kept under standardized, pathogen free conditions at the local animal facility, Ruhr-University, Bochum, Germany. Food and water were given ad libitum to all animals. Experiments were approved by the local authorities for animal experimentation (approval no. 8.8750.10.32.08.032). EAE induction and treatment An emulsion consisting of myelin oligodendrocyte glycoprotein 35– 55 (MOG35–55) in phosphate buffered saline (100 μg) and an equal volume of complete Freund's adjuvant containing 100 μg of Mycobacterium tuberculosis (Difco, BD Bioscience) was prepared and subcutaneously injected at the tail base of 8–10 week old female C57BL6/J mice. In addition to immunization with MOG35–55 emulsion, on days 0 and 2, 100 ng pertussis toxin (List, Quadratec) was administered i.p. DMFtreated animals received 15 mg/kg body weight (bw) DMF dissolved in 0.08% methylcellulose twice a day per oral gavage. Mice treated with murine IFN-β received an s.c. injection of 10.000 units every other day. IFN-β was dissolved in endotoxin free recombinant mouse serum albumin (MSA) 1%. Preventive treatment started at the day of immunization. Animals were weighed and clinically monitored daily. The blinded observer used a 10-scale score (Linker et al., 2002) for clinical signs. Mice were sacrificed on day 10 for ELISA and FACS and on day 18 for histological analysis. For ELISA and FACS spleens and lymph nodes (superficial cervical lymph nodes, deep cervical lymph nodes, lumbar lymph nodes) were removed to prepare a single cell suspension. For histological studies, mice were deeply anesthetized with ketamine and were transcardially perfused with 4% paraformaldehyde.
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Mac-3 (1:200; BD Pharmingen, Heidelberg, Germany). Bielschowsky silver impregnation was employed to assess axonal pathology and Luxol Fast Blue staining was used to assess demyelination as described previously (Linker et al., 2011). Axonal damage was additionally visualized by double staining with CNPase and the neurofilament antibody SMI31 (1:1000; Invitrogen, Karlsruhe, Germany) with anti-mouse Alexa 488and Cy3-conjugated secondary antibodies. The sections double labeled for neurofilaments (SMI31) and CNPase using immunofluorescence were analyzed on a fluorescence microscope (Olympus BX51). To calculate the inflammatory index, spinal cord cross sections were stained with haemalaun. Histological and immunohistochemical quantifications were performed by a blinded observer as described earlier (Linker et al., 2011). Splenocyte and lymphocyte culture and ELISA experiments Splenocytes were cultured in 24-well plates at a concentration of 5 × 106 cells/ml of complete GluMax medium (GIBCO®, Invitrogen, Germany), containing 10% heat inactivated fetal calf serum (FCS), 1% sodium pyruvate, 1% L-glutamine, 1% MEM-NEAA and 1% penicillin/ streptomycin. Cells were stimulated with 10 μg/ml MOG or 1.25 μg Concanavalin A (ConA) for 24, 48 and 72 h at 37 °C, 5% CO2. At the indicated time points, supernatants were collected and centrifuged to eliminate cellular debris. Cell culture supernatants were stored at − 20 °C and brought to room temperature immediately before the measurement of different cytokines, including IL-6 (BD OptEIA 555240, Pharmingen, Germany), IL-10 (R&D Systems DY417, Heidelberg, Germany), IL-17 (R&D Systems DY421, Heidelberg, Germany) and IFN-γ (R&D Systems DY485, Heidelberg, Germany) according to manufacturers' protocols. Results shown are the mean of triplicates ± SEM. Flow cytometry (FACS) experiments FACS analyses were performed using a FACS Canto II and CellQuest software (BD). Splenic single cell suspensions and lymphocyte from lymph nodes were incubated for 20 min at 4 °C and washed with FACS buffer, centrifuged and resuspended in FACS buffer. Monoclonal antibodies purchased from BD (Heidelberg, Germany) and Miltenyi Biotech (Gladbach, Germany) were used to detect CD11b (clone M1/70) CD11c (clone HL3), CD80 (clone 16-10A1), CD86 (clone GL1) and MHC-II (clone 2G9) and CD25CD4 in lymph nodes. Statistical analysis All histological analyses were performed completely blinded with respect to treatment. Data are presented as mean ± SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) or Kruskal–Wallis test (all analyses done by Graph Pad Prism 6, San Diego, CA, USA). As post-hoc tests Bonferroni's Multiple Comparison Tests were performed. A probability level (p-value) of *p b 0.05, **p b 0.01 and ***p b 0.001 was considered to be statistically significant for all tests. All error bars represent SEM.
Immunohistochemistry Results Immunohistochemistry was performed on 5 μm paraffin embedded spinal cord cross sections (lumbar, thoracal and cervical part). If necessary, antigen unmasking was performed via boiling of sections in citric acid buffer. After inhibition of nonspecific binding with 10% bovine serum albumin (BSA), sections were incubated overnight at 4 °C with the appropriate primary antibody in 1% BSA. After blocking of endogenous peroxidase with 0.3% H2O2, the peroxidase-based ABC detection system (Vectastain, Vector Laboratories, via Linaris, Wertheim, Germany) was employed with diaminobenzidine as the chromogenic substrate. Specificity of staining was confirmed by omitting the primary antibody as a negative control. T cells were labeled by rat anti-CD3 (1:200; Serotec, Düsseldorf, Germany), macrophages by rat anti-mouse
Combination of DMF and IFN-β improves the clinical disease course in EAE mice To compare monotherapy of DMF and IFN-β with a combination of both compounds, we analyzed clinical symptoms and disease severity of MOG-EAE in C57BL/6 mice until the early chronic phase of the disease. The incidence of EAE was 100% in the DMF-group (25/25) and in the IFN-β-group (24/24) versus 22/24 in the combination-group (92%) and 19/23 in the sham treated control group (83%). The difference was statistically not significant. There were no unexpected adverse events upon daily inspection of mice with no differences in mortality
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or body weight between all treatment groups (data not shown). Mice receiving combination therapy displayed a significantly less severe EAE disease course compared to the vehicle only control group (Fig. 1). Combination therapy also yielded a significantly greater beneficial effect on clinical course vs. IFN-β alone. Whilst the combination group only exhibited limp tail and unsteadiness of gait, IFN-β treated mice suffered from paraparesis and mice in the vehicle only control group displayed severe gait ataxia. These analyses were confirmed in two further independent experiments with similar group size. Combination treatment improved histological outcome in EAE To correlate clinical data with histology, we next quantified inflammatory cell infiltration in the CNS as well as demyelination and axonal damage. Evaluation of axonal densities revealed a significant preservation of axons in mice treated with combination therapy, as compared to monotherapy and vehicle only (Figs. 2E–H and Table 1). Comparing DMF/IFN-β and vehicle only, inflammatory cell infiltration was significantly reduced after combination therapy. Combination therapy reduced macrophage infiltration as compared to control and IFN-β monotherapy (Figs. 2I–Q, Table 1). Luxol Fast Blue staining showed significantly less demyelination in mice treated with DMF/ IFN-β combination therapy as compared to control animals and a trend towards preservation of myelin versus mice treated with DMF or IFN-β alone (Figs. 2R–U and Table 1). Furthermore, using confocal laser scanning microscopy after staining for neurofilament and CNPase, we could show that treatment with DMF/IFN-β led to preservation of intact axons containing phosphorylated, SMI31-positive axons in EAE lesions (Figs. 3A–D). Combination of DMF/IFN-β treatment partly stimulated cytokine production ex vivo We then investigated the concomitant immunological processes under the different therapeutic regimes. To this end, we assessed IL-6, IL-10, IL-17 and IFN-γ secretion upon ex vivo stimulation of splenocytes at day 10 (Figs. 4A–D). Subsequent to ConA stimulation DMF/IFN-β combination therapy increased IFN-γ levels significantly as compared to vehicle (Fig. 4A). IL-6, IL-10 and IL-17 were also affected after treatment with both, DMF and IFN-β, in contrast to vehicle only in the acute phase underlining the same trend (Figs. 4B–D) shown in IFN-γ levels. FACS analysis of immune cell phenotypes To determine whether IFN-β, DMF or the combination of both had any effect on overall T cell activation and differentiation, APCs from
Fig. 1. Combination of IFN-β and DMF ameliorated EAE disease course. Acute clinical course of EAE in C57BL/6 female mice after monotherapy with IFN-β (n = 24) or DMF (n = 25) or combination therapy DMF/IFN-β (n = 24) versus control (n = 23). Combination treatment yielded a less severe course of disease compared to IFN-β monotherapy and control mice. Data represent the mean of three independent experiments ± SEM.
each group were analyzed by flow cytometry from spleen and lymph node in the acute phase (CD11b+, CD11b/c+, CD11c+). MHC II expression on CD11b+ cells or CD11b/c+ cells was not different between the control group, monotherapies and DMF/IFN-β treated EAE mice (data not shown). T cell proliferation studies To investigate the effect of DMF, IFN-β or the combination of both on MOG35–55-specific T cell responses, isolated splenocytes of immunized animals were cultured in the presence of MOG35–55 or ConA. After incubation with [3H] thymidine for 16 h proliferation was measured by [3H] thymidine incorporation. In all three treatment groups, no significant inhibition of T cell proliferation was observed (data not shown). Discussion This study demonstrates that a combination of DMF and IFN-β can ameliorate EAE disease clinical course more effectively compared to sham treatment and IFN-β monotherapy. In contrast, there was no superiority of combination therapy compared to DMF monotherapy. In line to clinical data, histological analyses revealed less inflammation, demyelination and in particular preservation of axonal damage. We hypothesized that combination of IFN-β and DMF with putatively complementary mechanisms acts synergistic in EAE: this combination may tackle both anti-inflammatory and central neuroprotective effects. Basically cross-connectivity between neurodegeneration and inflammation under autoimmune inflammatory conditions has already been described (Trapp and Nave, 2008): With its anti-inflammatory, anti-oxidative and pro-metabolic potential in a variety of cell types, DMF may inhibit immune response and hereby exert a beneficial effect on inflammatory activity (Scannevin et al., 2012; Schilling et al., 2006) IFN-β reduces inflammation by positively influencing the balance of pro- and anti-inflammatory mechanisms in the periphery, and also entry of monocytic cells into the CNS via MMP activity (Floris et al., 2002; Schmidt et al., 2001). Additionally, IFN-β may increase the production of nerve growth factor with the potential capacity to improve neuronal survival and repair (Kieseier, 2011). Thus a combination of compounds has the potential to maximize beneficial effects and thus induce synergies in EAE. Focusing on the clinical course of EAE the effectiveness of combination therapy with DMF/IFN-β may be explained by histopathological effects acting on both inflammation, especially on macrophages and on degeneration, especially axonal damage. We observed higher axonal densities and milder demyelination in all treated groups but the most marked beneficial effect was seen in the DMF/IFN-β treated group. Activated macrophages and microglia can be found nearby degenerating axons, myelin sheaths and oligodendrocytes (Ferguson et al., 1997; Kornek et al., 2000; Trapp et al., 1998). Prior studies in EAE reported that axonal damage in mice was reduced after DMF therapy (Linker et al., 2011). Axonal damage has the strongest impact on the development of irreversible neurological symptoms in MS (Ferguson et al., 1997; Kornek et al., 2000; Trapp and Nave, 2008). Common pathways between neurodegeneration and inflammation under autoimmune inflammatory conditions have already been demonstrated (Trapp and Nave, 2008) and it is known that inflammation is a driving effect for degeneration. In the current study an appropriate trend for this effect was confirmed. Here, axon protection or more correctly axonal sparing may be the consequence of the simultaneous anti-inflammatory/immunomodulatory properties of the combination of DMF/IFN-β. We demonstrated that DMF and IFN-β treatment alone preserved axonal loss compared to sham treatment but a combination of both substances yielded more pronounced effects, highlighting putative synergies mainly on axonal targets. Additional effects of combination treatment were seen on myeloid cells, especially on macrophage infiltration. IFN-β is still one of the
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Fig. 2. Histological analyses. Representative spinal cord cross-sections of mice on day 18 post immunization (p.i.). Note infiltrates (arrows) after Haemalaun staining (A to D) in control mice (A), IFN-β treated mice (B) and DMF-treated mice (C). Combination therapy diminishes number and occurrence of infiltrates (D). (E to H) Bielschowsky silver staining from control (E), IFN-β-(F), DMF- (G) and DMF/IFN-β (H) treated C57BL/6 mice. In contrast to the control group (E) there is less axonal loss after treatment (F to H). CD3 staining (I–M) revealed inflammatory T cell infiltrates. Further histological analyses showed a stronger infiltration (arrows) after Mac-3 staining (N–Q) and increased demyelination (framed area) after Luxol Fast Blue staining (R to U) in control- (N and R) and IFN-β- (O and S) or DMF-group (P and T). Scale bar = 100 μm for A to Q and 500 μm for R to U.
Table 1 Histological analyses: quantification and p-values. A
Control
DMF
Inflammatory index Demyelination of white matter in % ± SEM Axonal density ± SEM CD3 positive cells/mm2 ± SEM Mac3 positive cells/mm2 ± SEM
5.3 8.1 2.3 305 596
3.7 4.2 3.3 264 532
B Control
IFN-β DMF
IFN-β DMF DMF/IFN-β DMF DMF/IFN-β DMF/IFN-β
± ± ± ± ±
1.3 1.5 0.2 24 33
± ± ± ± ±
IFN-β 0.5 1.1 0.2 33 30
2.4 3.8 3.7 286 533
± ± ± ± ±
DMF/IFN-β 0.6 0.8 0.3 16 34
1.2 1.6 4.6 228 385
± ± ± ± ±
0.5 0.6 0.2 24 36
Inflammatory Index
Demyelination
Axonal density
CD3 positive cells/mm2
Mac3 positive cells/mm2
n.s. n.s. p*** b 0.001 n.s. n.s. n.s.
n.s n.s p** b 0.01 n.s n.s n.s
p** b 0.01 p* b 0.05 p*** b 0.001 n.s. n.s. p** b 0.01
n.s n.s n.s. n.s n.s n.s
n.s n.s p** b 0.01 n.s p* b 0.05 n.s
Blinded quantification (A) of the inflammatory index after HE-staining, CD3 positive cells, Mac-3 positive macrophages/microglia, demyelination after Luxol Fast Blue staining and axonal densities after silver impregnation were performed on day 18 post immunization marking the acute phase of the disease (n = 12 per group). Data are presented as cells/mm2 for CD3, Mac-3, percent demyelination for Luxol Fast Blue and relative axonal densities for silver impregnated axons. P-values upon comparison of the different groups are calculated by Kruskal–Wallis test with Bonferroni post testing as depicted in B. n.s. = not significant.
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Fig. 3. Combination of DMF and IFN-β protects axon density. DMF/IFN-β protects axons in EAE lesions. Representative fluorescence microscopy images of lesions from spinal cord crosssections are shown (scale bar = 50 μm, CNPase positive myelin is depicted in red, SMI31 positive, naked axons in green). Dashed white lines denote lesion borders in A–D. As compared to controls (A), therapy with DMF (C) and IFN-β (D) alone leads to some and DMF/IFN-β therapy (B) leads to significant preservation of SMI31-positive, naked axons in lesions (green). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
mainstay therapies used for MS-treatment. In line with our results, several other publications have shown beneficial but limited effects of IFNβ in EAE models (Floris et al., 2002; Ruuls et al., 1996; Schmidt et al., 2001; Yu et al., 1996). While the mechanisms of IFN-β are pleiotropic, immunomodulatory properties of IFN-β are well established (Nelson et al., 1996; Tuohy et al., 2000), with a special focus on myeloid cells, most notably monocytes (Prinz et al., 2008). Similar to IFN-β, DMF also acts on myeloid cells (Linker et al., 2010; Schilling et al., 2006). In line with results of Schilling et al., a reduction in macrophage infiltration in the spinal cord was observed after DMF treatment without relevant inhibition of T cell infiltration. In addition to their antigen presenting function macrophages/microglia cells are active players in myelin destruction in EAE (Bauer et al., 1994; Ghoreschi et al., 2011). The role of macrophages/microglia as active players in myelin destruction in EAE as well as in MS (Bauer et al., 1996) seems to be tackled by combination of DMF/IFN-β during EAE. We found a reduction of macrophages/microglia in situ under combination treatment, whereas IFN-β therapy exhibit less reduced numbers of macrophages/microglia. Hereby, the less severe clinical course in EAE might additionally be explained besides the more impressive effect on axons. Macrophages and microglia are known to be important effector cells for tissue injury in inflammatory diseases of the CNS. We believe that the synergistic effect of combination therapy with DMF/IFN-β is based on its two modes of action resulting in axon protection: 1. a direct effect via the Nrf2 pathway, that after activation due to DMF/IFN-β increases levels of detoxifying enzymes and in consequence reduces neurotoxicity, and 2. an indirect effect via modulation of macrophages making a contribution to axonal damage. The causal chain is induced by axonal damage (Linker et al., 2005; Nikic et al., 2011) that in turn is essential since axonal injury and neuronal loss are correlated with the progression of disease (Ferguson et al., 1997; Trapp et al., 1998). Thus our data argue for a clinical trial with IFN-β plus DMF in RRMS patients. Both substances are characterized to be safe and well-
tolerated. They have been used for many years so that potential side effects are well-known and manageable. Additive immunosuppressive effects are not expected. Although the exact mechanisms of preservation of axons after combination therapy are not dissected in total, we propose a beneficial net effect of combination therapy also for MS patients. Therefore, clinical studies in RRMS seem to be very reasonable and promising.
Conclusion In conclusion the presented data underline the different putative immunomodulatory mechanisms of IFN-β after systemic use with strong emphasis on anti-inflammatory effects resulting in axonal sparing. Combination treatment of DMF/IFN-β in an autoimmune experimental model improves the clinical course of EAE. This may result from putative neuroprotective activities of DMF (Barnabe-Heider and Miller, 2003; Yamada et al., 2001) which add to the immunomodulatory effects of both drugs. Establishment of both, effective and safe treatment regimes in MS therapy is a major goal and different combinations of immunomodulatory agents have been tested so far (Brod et al., 2000; Calabresi et al., 2002; Gold, 2008; Jolivalt et al., 2003; Soos et al., 2002; Vollmer et al., 2004; Weilbach et al., 2004). To better understand the underlying mechanisms, further studies are warranted.
Authors' contributions CR carried out the experiments, analyzed histological and immunological results and drafted the manuscript. RAL, CS and RG provided general support and participated in the design of the study. JT supported FACS analysis. RAL and GE conceived the study, and participated in its design and coordination and surveyed and supported the draft of the manuscript. RHS is a full time employee of Biogen Idec, Inc. All authors read and approved the final manuscript.
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Fig. 4. Cytokine levels. Levels of interferon-γ (IFN-γ) are higher in ConA stimulated splenocytes of DMF/IFN-β treated mice in the acute phase (A). DMF/IFN-β treatment also increases IL-6 (B) and anti-inflammatory IL-10 (C) after ConA stimulation as compared to vehicle. Different treatment regimes do not influence expression of the mentioned cytokine after MOG35–55 stimulation. Data shown are representative of three independent experiments showing the same result.
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Competing interests RL and RG received travel support, speaker's honoraria and research grants from BiogenIdec. RG also has board activities for BiogenIdec. RHS is an employee of BiogenIdec; the company involved in marketing of BG-12 (dimethyl fumarate). The other authors declare no competing interests. Acknowledgments The skillful technical support of Bernadette Jesionek and Silvia Seubert is highly appreciated. The original enthusiasm of Prof. Altmeyer and Prof. Przuntek, Bochum, in introducing FAE to therapy of MS is gratefully acknowledged. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.expneurol.2014.04.003. References Barnabe-Heider, F., Miller, F.D., 2003. Endogenously produced neurotrophins regulate survival and differentiation of cortical progenitors via distinct signaling pathways. J. Neurosci. 23 (12), 5149–5160. Bauer, J., Sminia, T., Wouterlood, F.G., Dijkstra, C.D., 1994. Phagocytic activity of macrophages and microglial cells during the course of acute and chronic relapsing experimental autoimmune encephalomyelitis. J. Neurosci. Res. 38 (4), 365–375. Bauer, J., Ruuls, S.R., Huitinga, I., Dijkstra, C.D., 1996. The role of macrophage subpopulations in autoimmune disease of the central nervous system. Histochem. J. 28 (2), 83–97. Brod, S.A., Lindsey, J.W., Wolinsky, J.S., 2000. Combination therapy with glatiramer acetate (copolymer-1) and a type I interferon (IFN-alpha) does not improve experimental autoimmune encephalomyelitis. Ann. Neurol. 47 (1), 127–131. Burks, J., 2005. Interferon-beta1b for multiple sclerosis. Expert. Rev. Neurother. 5 (2), 153–164. Calabresi, P.A., et al., 2002. An open-label trial of combination therapy with interferon beta-1a and oral methotrexate in MS. Neurology 58 (2), 314–317. Ellrichmann, G., et al., 2011. Efficacy of fumaric acid esters in the R6/2 and YAC128 models of Huntington's disease. PLoS ONE 6 (1), e16172. Ferguson, B., Matyszak, M.K., Esiri, M.M., Perry, V.H., 1997. Axonal damage in acute multiple sclerosis lesions. Brain 120 (Pt 3), 393–399. Floris, S., et al., 2002. Interferon-beta directly influences monocyte infiltration into the central nervous system. J. Neuroimmunol. 127 (1–2), 69–79. Ghoreschi, K., et al., 2011. Fumarates improve psoriasis and multiple sclerosis by inducing type II dendritic cells. J. Exp. Med. 208 (11), 2291–2303. Gold, R., 2008. Combination therapies in multiple sclerosis. J. Neurol. 255 (Suppl. 1), 51–60. Gold, R., Linington, C., Lassmann, H., 2006. Understanding pathogenesis and therapy of multiple sclerosis via animal models: 70 years of merits and culprits in experimental autoimmune encephalomyelitis research. Brain 129 (Pt 8), 1953–1971. Herrero-Herranz, E., Pardo, L.A., Gold, R., Linker, R.A., 2008. Pattern of axonal injury in murine myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis: implications for multiple sclerosis. Neurobiol. Dis. 30 (2), 162–173. Jacobs, L., 1996. Magnetic resonance imaging in clinical therapeutic trials of multiple sclerosis. West. J. Med. 164 (6), 531–532. Johnson, J.A., et al., 2008. The Nrf2-ARE pathway: an indicator and modulator of oxidative stress in neurodegeneration. Ann. N. Y. Acad. Sci. 1147, 61–69.
Jolivalt, C.G., Howard, R.B., Chen, L.S., Mizisin, A.P., Lai, C.S., 2003. A novel nitric oxide scavenger in combination with cyclosporine A ameliorates experimental autoimmune encephalomyelitis progression in mice. J. Neuroimmunol. 138 (1–2), 56–64. Kieseier, B.C., 2011. The mechanism of action of interferon-beta in relapsing multiple sclerosis. CNS Drugs 25 (6), 491–502. Kornek, B., et al., 2000. Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions. Am. J. Pathol. 157 (1), 267–276. Linker, R.A., et al., 2002. CNTF is a major protective factor in demyelinating CNS disease: a neurotrophic cytokine as modulator in neuroinflammation. Nat. Med. 8 (6), 620–624. Linker, R.A., et al., 2005. EAE in beta-2 microglobulin-deficient mice: axonal damage is not dependent on MHC-I restricted immune responses. Neurobiol. Dis. 19 (1–2), 218–228. Linker, R.A., et al., 2010. Functional role of brain-derived neurotrophic factor in neuroprotective autoimmunity: therapeutic implications in a model of multiple sclerosis. Brain 133 (Pt 8), 2248–2263. Linker, R.A., et al., 2011. Fumaric acid esters exert neuroprotective effects in neuroinflammation via activation of the Nrf2 antioxidant pathway. Brain 134 (Pt 3), 678–692. Mendes, A., Sa, M.J., 2011. Classical immunomodulatory therapy in multiple sclerosis: how it acts, how it works. Arq. Neuropsiquiatr. 69 (3), 536–543. Nelson, P.A., et al., 1996. Effect of oral beta interferon on subsequent immune responsiveness. Ann. N. Y. Acad. Sci. 778, 145–155. Nikic, I., et al., 2011. A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis. Nat. Med. 17 (4), 495–499. Prinz, M., et al., 2008. Distinct and nonredundant in vivo functions of IFNAR on myeloid cells limit autoimmunity in the central nervous system. Immunity 28 (5), 675–686. Ruuls, S.R., et al., 1996. The length of treatment determines whether IFN-beta prevents or aggravates experimental autoimmune encephalomyelitis in Lewis rats. J. Immunol. 157 (12), 5721–5731. Scannevin, R.H., et al., 2012. Fumarates promote cytoprotection of central nervous system cells against oxidative stress via the nuclear factor (erythroid-derived 2)-like 2 pathway. J. Pharmacol. Exp. Ther. 341 (1), 274–284. Schilling, S., Goelz, S., Linker, R., Luehder, F., Gold, R., 2006. Fumaric acid esters are effective in chronic experimental autoimmune encephalomyelitis and suppress macrophage infiltration. Clin. Exp. Immunol. 145 (1), 101–107. Schmidt, J., Sturzebecher, S., Toyka, K.V., Gold, R., 2001. Interferon-beta treatment of experimental autoimmune encephalomyelitis leads to rapid nonapoptotic termination of T cell infiltration. J. Neurosci. Res. 65 (1), 59–67. Soos, J.M., et al., 2002. Cutting edge: oral type I IFN-tau promotes a Th2 bias and enhances suppression of autoimmune encephalomyelitis by oral glatiramer acetate. J. Immunol. 169 (5), 2231–2235. Trapp, B.D., Nave, K.A., 2008. Multiple sclerosis: an immune or neurodegenerative disorder? Annu. Rev. Neurosci. 31, 247–269. Trapp, B.D., et al., 1998. Axonal transection in the lesions of multiple sclerosis. N. Engl. J. Med. 338 (5), 278–285. Tuohy, V.K., et al., 2000. Modulation of the IL-10/IL-12 cytokine circuit by interferon-beta inhibits the development of epitope spreading and disease progression in murine autoimmune encephalomyelitis. J. Neuroimmunol. 111 (1–2), 55–63. Vollmer, T.L., et al., 2004. An open-label safety and drug interaction study of natalizumab (Antegren) in combination with interferon-beta (Avonex) in patients with multiple sclerosis. Mult. Scler. 10 (5), 511–520. Weilbach, F.X., Chan, A., Toyka, K.V., Gold, R., 2004. The cardioprotector dexrazoxane augments therapeutic efficacy of mitoxantrone in experimental autoimmune encephalomyelitis. Clin. Exp. Immunol. 135 (1), 49–55. Weiner, H.L., 2009. The challenge of multiple sclerosis: how do we cure a chronic heterogeneous disease? Ann. Neurol. 65 (3), 239–248. Williamson, T.P., Johnson, D.A., Johnson, J.A., 2012. Activation of the Nrf2-ARE pathway by siRNA knockdown of Keap1 reduces oxidative stress and provides partial protection from MPTP-mediated neurotoxicity. Neurotoxicology 33 (3), 272–279. Yamada, M., et al., 2001. Differences in survival-promoting effects and intracellular signaling properties of BDNF and IGF-1 in cultured cerebral cortical neurons. J. Neurochem. 78 (5), 940–951. Yu, M., Nishiyama, A., Trapp, B.D., Tuohy, V.K., 1996. Interferon-beta inhibits progression of relapsing-remitting experimental autoimmune encephalomyelitis. J. Neuroimmunol. 64 (1), 91–100.
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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
Regular Article
Neuroprotective dimethyl fumarate synergizes with immunomodulatory interferon beta to provide enhanced axon protection in autoimmune neuroinflammation Christiane Reick a,b,1, Gisa Ellrichmann a,⁎,1, Jan Thöne a, Robert H. Scannevin c, Carsten Saft a, Ralf A. Linker d,2, Ralf Gold a,b a
Department of Neurology St. Josef-Hospital, Ruhr-University Bochum, Germany International Graduate School of Neuroscience, Ruhr-University Bochum, Germany c BiogenIdec, Inc. Cambridge, 02142 MA, USA d Department of Neurology, University of Erlangen, Germany b
a r t i c l e
i n f o
Article history: Received 15 December 2013 Revised 26 March 2014 Accepted 4 April 2014 Available online 13 April 2014 Keywords: Multiple sclerosis EAE Immunomodulation Neuroprotection Interferon-beta Dimethyl fumarate Combination therapy
a b s t r a c t Introduction: Despite recent advances in development of treatments for multiple sclerosis, there is still an unmet need for more effective and also safe therapies. Based on the modes of action of interferon-beta (IFN-β) and dimethyl fumarate (DMF), we hypothesized that anti-inflammatory and neuroprotective effects may synergize in experimental autoimmune encephalomyelitis (EAE). Methods: EAE was induced in C57BL/6 mice by immunization with MOG35–55-peptide. Murine IFN-β was injected s.c. every other day at 10.000 IU, and DMF was provided at 15 mg/kg by oral gavage twice daily. Control mice received PBS injections and were treated by oral gavage with the vehicle methylcellulose. Mice were scored daily by blinded observers and histological, FACS and cytokine studies were performed to further elucidate the underlying mechanism of action. Results: Combination therapy significantly ameliorated EAE disease course in comparison to controls and monotherapy with IFN-β. Histological analyses showed a significant effect on axon preservation with almost twice as much axons present in inflamed lesions as compared to control. Remarkably, the effect on axonal preservation was more pronounced under combination therapy than with both monotherapies. Neither monotherapy nor combination therapy demonstrated modulation of cytokines and frequency of antigen presenting cells. Discussion: Combination of IFN-β and DMF resulted in greater beneficial effects with improved tissue protection as compared to the respective monotherapies. Further combination studies of these safe therapies in human disease are warranted. © 2014 Elsevier Inc. All rights reserved.
Introduction Multiple sclerosis (MS) is a chronic neurological disease of the central nervous system (CNS) of presumed autoimmune origin that is characterized by inflammation-driven demyelination, and subsequent axonal damage and progressive neurological deficits. Studies in MS and experimental autoimmune encephalomyelitis (EAE), an established animal model mimicking several pathophysiological features of MS, demonstrated that demyelinating lesions are accompanied by T cell and macrophage infiltration (Barnabe-Heider and Miller, 2003; Gold et al., 2006; Weiner, 2009). Subsequent to T cell and macrophage
⁎ Corresponding author at: Department of Neurology, Ruhr University Bochum, D-44791 Bochum, Germany. Fax: + 49 234 509 2414. E-mail address: [email protected] (G. Ellrichmann). 1 These authors share first authorship. 2 Current address.
http://dx.doi.org/10.1016/j.expneurol.2014.04.003 0014-4886/© 2014 Elsevier Inc. All rights reserved.
activation and infiltration, these cells release pro-inflammatory cytokines, interact with antigen presenting cells (APCs) and are involved in destruction of the myelin sheet, which is associated with chronic axonal damage (Herrero-Herranz et al., 2008). For as long as 20 years interferon-beta (IFN-β) has been an established immunomodulatory agent in patients with relapsing remitting MS (RRMS). In phase III studies IFN-β showed beneficial effects on relapse rate, disease progression and development of new CNS lesions as shown by magnetic resonance imaging (MRI) (Burks, 2005; Jacobs, 1996). So far, different mechanisms of action have been described for IFN-β with a focus on downregulation of macrophage and microglia activity (Mendes and Sa, 2011). Dimethyl fumarate (DMF; study name BG-12) has shown beneficial effects in both EAE and in models of neurodegenerative diseases as well as in two phase III studies in patients with RRMS (Ellrichmann et al., 2011; Ghoreschi et al., 2011; Linker et al., 2011). So far, different mechanisms of action, including neuroprotection, have been described. DMF
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was shown to mediate neuroprotection by inducing the nuclear factor E2-related factor 2 (Nrf2) pathway and thereby addressing toxic-oxidative stress (Ghoreschi et al., 2011; Johnson et al., 2008; Williamson et al., 2012). In APCs, DMF induced glutathione depletion and thus stimulated type II dendritic cells (DCs) with an impaired secretion of interleukin- (IL) 12 and IL-23. Simultaneously, DMF induced both IL-4 producing T helper (Th) 2 cells and generated type II dendritic cells (DCs) that produce IL-10 (Ghoreschi et al., 2011). Based on their different modes of action we postulated that IFN-β and DMF synergize in early and early chronic EAE. We show that the combination of IFN-β and DMF resulted in a synergistic effect at the clinical level, as reflected by decreased disease severity of EAE. This effect was confirmed histologically by reduced inflammation and in particular preservation of axonal density in the spinal cord of EAE mice. The beneficial effects of combination therapy with IFN-β and DMF may result from effects on different pathophysiological pathways in EAE. Material and methods Animal models 8–10 week old female C57BL6/J mice were obtained from the Harlan Laboratories (Borchen, Germany, nowadays Rossdorf, Germany) and kept under standardized, pathogen free conditions at the local animal facility, Ruhr-University, Bochum, Germany. Food and water were given ad libitum to all animals. Experiments were approved by the local authorities for animal experimentation (approval no. 8.8750.10.32.08.032). EAE induction and treatment An emulsion consisting of myelin oligodendrocyte glycoprotein 35– 55 (MOG35–55) in phosphate buffered saline (100 μg) and an equal volume of complete Freund's adjuvant containing 100 μg of Mycobacterium tuberculosis (Difco, BD Bioscience) was prepared and subcutaneously injected at the tail base of 8–10 week old female C57BL6/J mice. In addition to immunization with MOG35–55 emulsion, on days 0 and 2, 100 ng pertussis toxin (List, Quadratec) was administered i.p. DMFtreated animals received 15 mg/kg body weight (bw) DMF dissolved in 0.08% methylcellulose twice a day per oral gavage. Mice treated with murine IFN-β received an s.c. injection of 10.000 units every other day. IFN-β was dissolved in endotoxin free recombinant mouse serum albumin (MSA) 1%. Preventive treatment started at the day of immunization. Animals were weighed and clinically monitored daily. The blinded observer used a 10-scale score (Linker et al., 2002) for clinical signs. Mice were sacrificed on day 10 for ELISA and FACS and on day 18 for histological analysis. For ELISA and FACS spleens and lymph nodes (superficial cervical lymph nodes, deep cervical lymph nodes, lumbar lymph nodes) were removed to prepare a single cell suspension. For histological studies, mice were deeply anesthetized with ketamine and were transcardially perfused with 4% paraformaldehyde.
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Mac-3 (1:200; BD Pharmingen, Heidelberg, Germany). Bielschowsky silver impregnation was employed to assess axonal pathology and Luxol Fast Blue staining was used to assess demyelination as described previously (Linker et al., 2011). Axonal damage was additionally visualized by double staining with CNPase and the neurofilament antibody SMI31 (1:1000; Invitrogen, Karlsruhe, Germany) with anti-mouse Alexa 488and Cy3-conjugated secondary antibodies. The sections double labeled for neurofilaments (SMI31) and CNPase using immunofluorescence were analyzed on a fluorescence microscope (Olympus BX51). To calculate the inflammatory index, spinal cord cross sections were stained with haemalaun. Histological and immunohistochemical quantifications were performed by a blinded observer as described earlier (Linker et al., 2011). Splenocyte and lymphocyte culture and ELISA experiments Splenocytes were cultured in 24-well plates at a concentration of 5 × 106 cells/ml of complete GluMax medium (GIBCO®, Invitrogen, Germany), containing 10% heat inactivated fetal calf serum (FCS), 1% sodium pyruvate, 1% L-glutamine, 1% MEM-NEAA and 1% penicillin/ streptomycin. Cells were stimulated with 10 μg/ml MOG or 1.25 μg Concanavalin A (ConA) for 24, 48 and 72 h at 37 °C, 5% CO2. At the indicated time points, supernatants were collected and centrifuged to eliminate cellular debris. Cell culture supernatants were stored at − 20 °C and brought to room temperature immediately before the measurement of different cytokines, including IL-6 (BD OptEIA 555240, Pharmingen, Germany), IL-10 (R&D Systems DY417, Heidelberg, Germany), IL-17 (R&D Systems DY421, Heidelberg, Germany) and IFN-γ (R&D Systems DY485, Heidelberg, Germany) according to manufacturers' protocols. Results shown are the mean of triplicates ± SEM. Flow cytometry (FACS) experiments FACS analyses were performed using a FACS Canto II and CellQuest software (BD). Splenic single cell suspensions and lymphocyte from lymph nodes were incubated for 20 min at 4 °C and washed with FACS buffer, centrifuged and resuspended in FACS buffer. Monoclonal antibodies purchased from BD (Heidelberg, Germany) and Miltenyi Biotech (Gladbach, Germany) were used to detect CD11b (clone M1/70) CD11c (clone HL3), CD80 (clone 16-10A1), CD86 (clone GL1) and MHC-II (clone 2G9) and CD25CD4 in lymph nodes. Statistical analysis All histological analyses were performed completely blinded with respect to treatment. Data are presented as mean ± SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) or Kruskal–Wallis test (all analyses done by Graph Pad Prism 6, San Diego, CA, USA). As post-hoc tests Bonferroni's Multiple Comparison Tests were performed. A probability level (p-value) of *p b 0.05, **p b 0.01 and ***p b 0.001 was considered to be statistically significant for all tests. All error bars represent SEM.
Immunohistochemistry Results Immunohistochemistry was performed on 5 μm paraffin embedded spinal cord cross sections (lumbar, thoracal and cervical part). If necessary, antigen unmasking was performed via boiling of sections in citric acid buffer. After inhibition of nonspecific binding with 10% bovine serum albumin (BSA), sections were incubated overnight at 4 °C with the appropriate primary antibody in 1% BSA. After blocking of endogenous peroxidase with 0.3% H2O2, the peroxidase-based ABC detection system (Vectastain, Vector Laboratories, via Linaris, Wertheim, Germany) was employed with diaminobenzidine as the chromogenic substrate. Specificity of staining was confirmed by omitting the primary antibody as a negative control. T cells were labeled by rat anti-CD3 (1:200; Serotec, Düsseldorf, Germany), macrophages by rat anti-mouse
Combination of DMF and IFN-β improves the clinical disease course in EAE mice To compare monotherapy of DMF and IFN-β with a combination of both compounds, we analyzed clinical symptoms and disease severity of MOG-EAE in C57BL/6 mice until the early chronic phase of the disease. The incidence of EAE was 100% in the DMF-group (25/25) and in the IFN-β-group (24/24) versus 22/24 in the combination-group (92%) and 19/23 in the sham treated control group (83%). The difference was statistically not significant. There were no unexpected adverse events upon daily inspection of mice with no differences in mortality
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or body weight between all treatment groups (data not shown). Mice receiving combination therapy displayed a significantly less severe EAE disease course compared to the vehicle only control group (Fig. 1). Combination therapy also yielded a significantly greater beneficial effect on clinical course vs. IFN-β alone. Whilst the combination group only exhibited limp tail and unsteadiness of gait, IFN-β treated mice suffered from paraparesis and mice in the vehicle only control group displayed severe gait ataxia. These analyses were confirmed in two further independent experiments with similar group size. Combination treatment improved histological outcome in EAE To correlate clinical data with histology, we next quantified inflammatory cell infiltration in the CNS as well as demyelination and axonal damage. Evaluation of axonal densities revealed a significant preservation of axons in mice treated with combination therapy, as compared to monotherapy and vehicle only (Figs. 2E–H and Table 1). Comparing DMF/IFN-β and vehicle only, inflammatory cell infiltration was significantly reduced after combination therapy. Combination therapy reduced macrophage infiltration as compared to control and IFN-β monotherapy (Figs. 2I–Q, Table 1). Luxol Fast Blue staining showed significantly less demyelination in mice treated with DMF/ IFN-β combination therapy as compared to control animals and a trend towards preservation of myelin versus mice treated with DMF or IFN-β alone (Figs. 2R–U and Table 1). Furthermore, using confocal laser scanning microscopy after staining for neurofilament and CNPase, we could show that treatment with DMF/IFN-β led to preservation of intact axons containing phosphorylated, SMI31-positive axons in EAE lesions (Figs. 3A–D). Combination of DMF/IFN-β treatment partly stimulated cytokine production ex vivo We then investigated the concomitant immunological processes under the different therapeutic regimes. To this end, we assessed IL-6, IL-10, IL-17 and IFN-γ secretion upon ex vivo stimulation of splenocytes at day 10 (Figs. 4A–D). Subsequent to ConA stimulation DMF/IFN-β combination therapy increased IFN-γ levels significantly as compared to vehicle (Fig. 4A). IL-6, IL-10 and IL-17 were also affected after treatment with both, DMF and IFN-β, in contrast to vehicle only in the acute phase underlining the same trend (Figs. 4B–D) shown in IFN-γ levels. FACS analysis of immune cell phenotypes To determine whether IFN-β, DMF or the combination of both had any effect on overall T cell activation and differentiation, APCs from
Fig. 1. Combination of IFN-β and DMF ameliorated EAE disease course. Acute clinical course of EAE in C57BL/6 female mice after monotherapy with IFN-β (n = 24) or DMF (n = 25) or combination therapy DMF/IFN-β (n = 24) versus control (n = 23). Combination treatment yielded a less severe course of disease compared to IFN-β monotherapy and control mice. Data represent the mean of three independent experiments ± SEM.
each group were analyzed by flow cytometry from spleen and lymph node in the acute phase (CD11b+, CD11b/c+, CD11c+). MHC II expression on CD11b+ cells or CD11b/c+ cells was not different between the control group, monotherapies and DMF/IFN-β treated EAE mice (data not shown). T cell proliferation studies To investigate the effect of DMF, IFN-β or the combination of both on MOG35–55-specific T cell responses, isolated splenocytes of immunized animals were cultured in the presence of MOG35–55 or ConA. After incubation with [3H] thymidine for 16 h proliferation was measured by [3H] thymidine incorporation. In all three treatment groups, no significant inhibition of T cell proliferation was observed (data not shown). Discussion This study demonstrates that a combination of DMF and IFN-β can ameliorate EAE disease clinical course more effectively compared to sham treatment and IFN-β monotherapy. In contrast, there was no superiority of combination therapy compared to DMF monotherapy. In line to clinical data, histological analyses revealed less inflammation, demyelination and in particular preservation of axonal damage. We hypothesized that combination of IFN-β and DMF with putatively complementary mechanisms acts synergistic in EAE: this combination may tackle both anti-inflammatory and central neuroprotective effects. Basically cross-connectivity between neurodegeneration and inflammation under autoimmune inflammatory conditions has already been described (Trapp and Nave, 2008): With its anti-inflammatory, anti-oxidative and pro-metabolic potential in a variety of cell types, DMF may inhibit immune response and hereby exert a beneficial effect on inflammatory activity (Scannevin et al., 2012; Schilling et al., 2006) IFN-β reduces inflammation by positively influencing the balance of pro- and anti-inflammatory mechanisms in the periphery, and also entry of monocytic cells into the CNS via MMP activity (Floris et al., 2002; Schmidt et al., 2001). Additionally, IFN-β may increase the production of nerve growth factor with the potential capacity to improve neuronal survival and repair (Kieseier, 2011). Thus a combination of compounds has the potential to maximize beneficial effects and thus induce synergies in EAE. Focusing on the clinical course of EAE the effectiveness of combination therapy with DMF/IFN-β may be explained by histopathological effects acting on both inflammation, especially on macrophages and on degeneration, especially axonal damage. We observed higher axonal densities and milder demyelination in all treated groups but the most marked beneficial effect was seen in the DMF/IFN-β treated group. Activated macrophages and microglia can be found nearby degenerating axons, myelin sheaths and oligodendrocytes (Ferguson et al., 1997; Kornek et al., 2000; Trapp et al., 1998). Prior studies in EAE reported that axonal damage in mice was reduced after DMF therapy (Linker et al., 2011). Axonal damage has the strongest impact on the development of irreversible neurological symptoms in MS (Ferguson et al., 1997; Kornek et al., 2000; Trapp and Nave, 2008). Common pathways between neurodegeneration and inflammation under autoimmune inflammatory conditions have already been demonstrated (Trapp and Nave, 2008) and it is known that inflammation is a driving effect for degeneration. In the current study an appropriate trend for this effect was confirmed. Here, axon protection or more correctly axonal sparing may be the consequence of the simultaneous anti-inflammatory/immunomodulatory properties of the combination of DMF/IFN-β. We demonstrated that DMF and IFN-β treatment alone preserved axonal loss compared to sham treatment but a combination of both substances yielded more pronounced effects, highlighting putative synergies mainly on axonal targets. Additional effects of combination treatment were seen on myeloid cells, especially on macrophage infiltration. IFN-β is still one of the
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Fig. 2. Histological analyses. Representative spinal cord cross-sections of mice on day 18 post immunization (p.i.). Note infiltrates (arrows) after Haemalaun staining (A to D) in control mice (A), IFN-β treated mice (B) and DMF-treated mice (C). Combination therapy diminishes number and occurrence of infiltrates (D). (E to H) Bielschowsky silver staining from control (E), IFN-β-(F), DMF- (G) and DMF/IFN-β (H) treated C57BL/6 mice. In contrast to the control group (E) there is less axonal loss after treatment (F to H). CD3 staining (I–M) revealed inflammatory T cell infiltrates. Further histological analyses showed a stronger infiltration (arrows) after Mac-3 staining (N–Q) and increased demyelination (framed area) after Luxol Fast Blue staining (R to U) in control- (N and R) and IFN-β- (O and S) or DMF-group (P and T). Scale bar = 100 μm for A to Q and 500 μm for R to U.
Table 1 Histological analyses: quantification and p-values. A
Control
DMF
Inflammatory index Demyelination of white matter in % ± SEM Axonal density ± SEM CD3 positive cells/mm2 ± SEM Mac3 positive cells/mm2 ± SEM
5.3 8.1 2.3 305 596
3.7 4.2 3.3 264 532
B Control
IFN-β DMF
IFN-β DMF DMF/IFN-β DMF DMF/IFN-β DMF/IFN-β
± ± ± ± ±
1.3 1.5 0.2 24 33
± ± ± ± ±
IFN-β 0.5 1.1 0.2 33 30
2.4 3.8 3.7 286 533
± ± ± ± ±
DMF/IFN-β 0.6 0.8 0.3 16 34
1.2 1.6 4.6 228 385
± ± ± ± ±
0.5 0.6 0.2 24 36
Inflammatory Index
Demyelination
Axonal density
CD3 positive cells/mm2
Mac3 positive cells/mm2
n.s. n.s. p*** b 0.001 n.s. n.s. n.s.
n.s n.s p** b 0.01 n.s n.s n.s
p** b 0.01 p* b 0.05 p*** b 0.001 n.s. n.s. p** b 0.01
n.s n.s n.s. n.s n.s n.s
n.s n.s p** b 0.01 n.s p* b 0.05 n.s
Blinded quantification (A) of the inflammatory index after HE-staining, CD3 positive cells, Mac-3 positive macrophages/microglia, demyelination after Luxol Fast Blue staining and axonal densities after silver impregnation were performed on day 18 post immunization marking the acute phase of the disease (n = 12 per group). Data are presented as cells/mm2 for CD3, Mac-3, percent demyelination for Luxol Fast Blue and relative axonal densities for silver impregnated axons. P-values upon comparison of the different groups are calculated by Kruskal–Wallis test with Bonferroni post testing as depicted in B. n.s. = not significant.
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Fig. 3. Combination of DMF and IFN-β protects axon density. DMF/IFN-β protects axons in EAE lesions. Representative fluorescence microscopy images of lesions from spinal cord crosssections are shown (scale bar = 50 μm, CNPase positive myelin is depicted in red, SMI31 positive, naked axons in green). Dashed white lines denote lesion borders in A–D. As compared to controls (A), therapy with DMF (C) and IFN-β (D) alone leads to some and DMF/IFN-β therapy (B) leads to significant preservation of SMI31-positive, naked axons in lesions (green). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
mainstay therapies used for MS-treatment. In line with our results, several other publications have shown beneficial but limited effects of IFNβ in EAE models (Floris et al., 2002; Ruuls et al., 1996; Schmidt et al., 2001; Yu et al., 1996). While the mechanisms of IFN-β are pleiotropic, immunomodulatory properties of IFN-β are well established (Nelson et al., 1996; Tuohy et al., 2000), with a special focus on myeloid cells, most notably monocytes (Prinz et al., 2008). Similar to IFN-β, DMF also acts on myeloid cells (Linker et al., 2010; Schilling et al., 2006). In line with results of Schilling et al., a reduction in macrophage infiltration in the spinal cord was observed after DMF treatment without relevant inhibition of T cell infiltration. In addition to their antigen presenting function macrophages/microglia cells are active players in myelin destruction in EAE (Bauer et al., 1994; Ghoreschi et al., 2011). The role of macrophages/microglia as active players in myelin destruction in EAE as well as in MS (Bauer et al., 1996) seems to be tackled by combination of DMF/IFN-β during EAE. We found a reduction of macrophages/microglia in situ under combination treatment, whereas IFN-β therapy exhibit less reduced numbers of macrophages/microglia. Hereby, the less severe clinical course in EAE might additionally be explained besides the more impressive effect on axons. Macrophages and microglia are known to be important effector cells for tissue injury in inflammatory diseases of the CNS. We believe that the synergistic effect of combination therapy with DMF/IFN-β is based on its two modes of action resulting in axon protection: 1. a direct effect via the Nrf2 pathway, that after activation due to DMF/IFN-β increases levels of detoxifying enzymes and in consequence reduces neurotoxicity, and 2. an indirect effect via modulation of macrophages making a contribution to axonal damage. The causal chain is induced by axonal damage (Linker et al., 2005; Nikic et al., 2011) that in turn is essential since axonal injury and neuronal loss are correlated with the progression of disease (Ferguson et al., 1997; Trapp et al., 1998). Thus our data argue for a clinical trial with IFN-β plus DMF in RRMS patients. Both substances are characterized to be safe and well-
tolerated. They have been used for many years so that potential side effects are well-known and manageable. Additive immunosuppressive effects are not expected. Although the exact mechanisms of preservation of axons after combination therapy are not dissected in total, we propose a beneficial net effect of combination therapy also for MS patients. Therefore, clinical studies in RRMS seem to be very reasonable and promising.
Conclusion In conclusion the presented data underline the different putative immunomodulatory mechanisms of IFN-β after systemic use with strong emphasis on anti-inflammatory effects resulting in axonal sparing. Combination treatment of DMF/IFN-β in an autoimmune experimental model improves the clinical course of EAE. This may result from putative neuroprotective activities of DMF (Barnabe-Heider and Miller, 2003; Yamada et al., 2001) which add to the immunomodulatory effects of both drugs. Establishment of both, effective and safe treatment regimes in MS therapy is a major goal and different combinations of immunomodulatory agents have been tested so far (Brod et al., 2000; Calabresi et al., 2002; Gold, 2008; Jolivalt et al., 2003; Soos et al., 2002; Vollmer et al., 2004; Weilbach et al., 2004). To better understand the underlying mechanisms, further studies are warranted.
Authors' contributions CR carried out the experiments, analyzed histological and immunological results and drafted the manuscript. RAL, CS and RG provided general support and participated in the design of the study. JT supported FACS analysis. RAL and GE conceived the study, and participated in its design and coordination and surveyed and supported the draft of the manuscript. RHS is a full time employee of Biogen Idec, Inc. All authors read and approved the final manuscript.
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Fig. 4. Cytokine levels. Levels of interferon-γ (IFN-γ) are higher in ConA stimulated splenocytes of DMF/IFN-β treated mice in the acute phase (A). DMF/IFN-β treatment also increases IL-6 (B) and anti-inflammatory IL-10 (C) after ConA stimulation as compared to vehicle. Different treatment regimes do not influence expression of the mentioned cytokine after MOG35–55 stimulation. Data shown are representative of three independent experiments showing the same result.
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Competing interests RL and RG received travel support, speaker's honoraria and research grants from BiogenIdec. RG also has board activities for BiogenIdec. RHS is an employee of BiogenIdec; the company involved in marketing of BG-12 (dimethyl fumarate). The other authors declare no competing interests. Acknowledgments The skillful technical support of Bernadette Jesionek and Silvia Seubert is highly appreciated. The original enthusiasm of Prof. Altmeyer and Prof. Przuntek, Bochum, in introducing FAE to therapy of MS is gratefully acknowledged. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.expneurol.2014.04.003. References Barnabe-Heider, F., Miller, F.D., 2003. Endogenously produced neurotrophins regulate survival and differentiation of cortical progenitors via distinct signaling pathways. J. Neurosci. 23 (12), 5149–5160. Bauer, J., Sminia, T., Wouterlood, F.G., Dijkstra, C.D., 1994. Phagocytic activity of macrophages and microglial cells during the course of acute and chronic relapsing experimental autoimmune encephalomyelitis. J. Neurosci. Res. 38 (4), 365–375. Bauer, J., Ruuls, S.R., Huitinga, I., Dijkstra, C.D., 1996. The role of macrophage subpopulations in autoimmune disease of the central nervous system. Histochem. J. 28 (2), 83–97. Brod, S.A., Lindsey, J.W., Wolinsky, J.S., 2000. Combination therapy with glatiramer acetate (copolymer-1) and a type I interferon (IFN-alpha) does not improve experimental autoimmune encephalomyelitis. Ann. Neurol. 47 (1), 127–131. Burks, J., 2005. Interferon-beta1b for multiple sclerosis. Expert. Rev. Neurother. 5 (2), 153–164. Calabresi, P.A., et al., 2002. An open-label trial of combination therapy with interferon beta-1a and oral methotrexate in MS. Neurology 58 (2), 314–317. Ellrichmann, G., et al., 2011. Efficacy of fumaric acid esters in the R6/2 and YAC128 models of Huntington's disease. PLoS ONE 6 (1), e16172. Ferguson, B., Matyszak, M.K., Esiri, M.M., Perry, V.H., 1997. Axonal damage in acute multiple sclerosis lesions. Brain 120 (Pt 3), 393–399. Floris, S., et al., 2002. Interferon-beta directly influences monocyte infiltration into the central nervous system. J. Neuroimmunol. 127 (1–2), 69–79. Ghoreschi, K., et al., 2011. Fumarates improve psoriasis and multiple sclerosis by inducing type II dendritic cells. J. Exp. Med. 208 (11), 2291–2303. Gold, R., 2008. Combination therapies in multiple sclerosis. J. Neurol. 255 (Suppl. 1), 51–60. Gold, R., Linington, C., Lassmann, H., 2006. Understanding pathogenesis and therapy of multiple sclerosis via animal models: 70 years of merits and culprits in experimental autoimmune encephalomyelitis research. Brain 129 (Pt 8), 1953–1971. Herrero-Herranz, E., Pardo, L.A., Gold, R., Linker, R.A., 2008. Pattern of axonal injury in murine myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis: implications for multiple sclerosis. Neurobiol. Dis. 30 (2), 162–173. Jacobs, L., 1996. Magnetic resonance imaging in clinical therapeutic trials of multiple sclerosis. West. J. Med. 164 (6), 531–532. Johnson, J.A., et al., 2008. The Nrf2-ARE pathway: an indicator and modulator of oxidative stress in neurodegeneration. Ann. N. Y. Acad. Sci. 1147, 61–69.
Jolivalt, C.G., Howard, R.B., Chen, L.S., Mizisin, A.P., Lai, C.S., 2003. A novel nitric oxide scavenger in combination with cyclosporine A ameliorates experimental autoimmune encephalomyelitis progression in mice. J. Neuroimmunol. 138 (1–2), 56–64. Kieseier, B.C., 2011. The mechanism of action of interferon-beta in relapsing multiple sclerosis. CNS Drugs 25 (6), 491–502. Kornek, B., et al., 2000. Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions. Am. J. Pathol. 157 (1), 267–276. Linker, R.A., et al., 2002. CNTF is a major protective factor in demyelinating CNS disease: a neurotrophic cytokine as modulator in neuroinflammation. Nat. Med. 8 (6), 620–624. Linker, R.A., et al., 2005. EAE in beta-2 microglobulin-deficient mice: axonal damage is not dependent on MHC-I restricted immune responses. Neurobiol. Dis. 19 (1–2), 218–228. Linker, R.A., et al., 2010. Functional role of brain-derived neurotrophic factor in neuroprotective autoimmunity: therapeutic implications in a model of multiple sclerosis. Brain 133 (Pt 8), 2248–2263. Linker, R.A., et al., 2011. Fumaric acid esters exert neuroprotective effects in neuroinflammation via activation of the Nrf2 antioxidant pathway. Brain 134 (Pt 3), 678–692. Mendes, A., Sa, M.J., 2011. Classical immunomodulatory therapy in multiple sclerosis: how it acts, how it works. Arq. Neuropsiquiatr. 69 (3), 536–543. Nelson, P.A., et al., 1996. Effect of oral beta interferon on subsequent immune responsiveness. Ann. N. Y. Acad. Sci. 778, 145–155. Nikic, I., et al., 2011. A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis. Nat. Med. 17 (4), 495–499. Prinz, M., et al., 2008. Distinct and nonredundant in vivo functions of IFNAR on myeloid cells limit autoimmunity in the central nervous system. Immunity 28 (5), 675–686. Ruuls, S.R., et al., 1996. The length of treatment determines whether IFN-beta prevents or aggravates experimental autoimmune encephalomyelitis in Lewis rats. J. Immunol. 157 (12), 5721–5731. Scannevin, R.H., et al., 2012. Fumarates promote cytoprotection of central nervous system cells against oxidative stress via the nuclear factor (erythroid-derived 2)-like 2 pathway. J. Pharmacol. Exp. Ther. 341 (1), 274–284. Schilling, S., Goelz, S., Linker, R., Luehder, F., Gold, R., 2006. Fumaric acid esters are effective in chronic experimental autoimmune encephalomyelitis and suppress macrophage infiltration. Clin. Exp. Immunol. 145 (1), 101–107. Schmidt, J., Sturzebecher, S., Toyka, K.V., Gold, R., 2001. Interferon-beta treatment of experimental autoimmune encephalomyelitis leads to rapid nonapoptotic termination of T cell infiltration. J. Neurosci. Res. 65 (1), 59–67. Soos, J.M., et al., 2002. Cutting edge: oral type I IFN-tau promotes a Th2 bias and enhances suppression of autoimmune encephalomyelitis by oral glatiramer acetate. J. Immunol. 169 (5), 2231–2235. Trapp, B.D., Nave, K.A., 2008. Multiple sclerosis: an immune or neurodegenerative disorder? Annu. Rev. Neurosci. 31, 247–269. Trapp, B.D., et al., 1998. Axonal transection in the lesions of multiple sclerosis. N. Engl. J. Med. 338 (5), 278–285. Tuohy, V.K., et al., 2000. Modulation of the IL-10/IL-12 cytokine circuit by interferon-beta inhibits the development of epitope spreading and disease progression in murine autoimmune encephalomyelitis. J. Neuroimmunol. 111 (1–2), 55–63. Vollmer, T.L., et al., 2004. An open-label safety and drug interaction study of natalizumab (Antegren) in combination with interferon-beta (Avonex) in patients with multiple sclerosis. Mult. Scler. 10 (5), 511–520. Weilbach, F.X., Chan, A., Toyka, K.V., Gold, R., 2004. The cardioprotector dexrazoxane augments therapeutic efficacy of mitoxantrone in experimental autoimmune encephalomyelitis. Clin. Exp. Immunol. 135 (1), 49–55. Weiner, H.L., 2009. The challenge of multiple sclerosis: how do we cure a chronic heterogeneous disease? Ann. Neurol. 65 (3), 239–248. Williamson, T.P., Johnson, D.A., Johnson, J.A., 2012. Activation of the Nrf2-ARE pathway by siRNA knockdown of Keap1 reduces oxidative stress and provides partial protection from MPTP-mediated neurotoxicity. Neurotoxicology 33 (3), 272–279. Yamada, M., et al., 2001. Differences in survival-promoting effects and intracellular signaling properties of BDNF and IGF-1 in cultured cerebral cortical neurons. J. Neurochem. 78 (5), 940–951. Yu, M., Nishiyama, A., Trapp, B.D., Tuohy, V.K., 1996. Interferon-beta inhibits progression of relapsing-remitting experimental autoimmune encephalomyelitis. J. Neuroimmunol. 64 (1), 91–100.
