Effects of Coq10 on the Ratio of Th1/Th2 in Experimental Autoimmune Encephalomyelitis Model

Effects of CoQ10 on the ratio of Th1/Th2 in Experimental Autoimmune Encephalomyelitis model of Multiple Sclerosis in C57BL/6

Maryam Soleimani 1, Seyed Behnameddin Jameie 1&2*,Mahmood Brati 3, Mahdi Mehdizadeh 1, Mahdieh kerdary 1

1 Departmentof Anatomy, Faculty of Medicine, Iran University of Medical Sciences, Tehran, I.R.Iran

2 Department of Medical Basic Sciences, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, I.R.Iran

3 Department of Pharmaceutical Biotechnology, Shahid beheshti University of Medical Sciences, Tehran, I.R.Iran

*Corresponding author; Tel: +982186705801; Fax: +982188622593; E-mail:

Abstract

Background:Multiple Sclerosis (MS) is known as progressive inflammatory disease that lead to disability. Inflammatory factors such as certain type of interleukins, inflammatory cells and cell mediated mechanisms are involve in its etiology. Experimental Autoimmune Encephalomyelitis (EAE) is common animal model of MS. Because of the importance role of inflammatory factors and oxidative stress, recently antioxidant received more attention. CoQ10 acts as an antioxidant. To our knowledge there is lack of enough research on COQ10 ,MS and Th1/Th2 ratio, so the present research designed.

Methods: Thirty C57BL/6 female adult mice were used . The animals randomly divided into trial and control groups. To induce MS, routine procedure of EAE was used and scoring was done based clinical signs. By detecting score one, CoQ10 administration started (10mg/kg/three weeks). By using ELISA and RT- PCR the brain level of TNF-α, IL-10, IL-4 and IL-12 studied.

Results:Significant decrease of clinical symptoms in trial groups comparing control animals was seen(Pv< 0.05). We also found that the level of the TNF-α significantly decreased following CoQ10 administration versus IL-10. By comparing and counting, the ratio of Th1/Th2 interleukins in treated animals was significantly less than non-treated (p=0.01).

Conclusion:It could be concluded that CoQ10 is capable to suppress the inflammatory agents that enhances MS. Whether this property is clinically valuable in human is not clear and need more research.

Key words:EAE, Multiple Sclerosis, CoQ10, Th1/Th2

Introduction

Multiple Sclerosis (MS) is the most important central nervous system progressive immune-mediated disease. Epidemiological studies as cited anywhere showed that the incidence of MS dramatically has been raised up among different societies especially in developing country. Reasons for such an alteration are still unknown. From physiopathological point of view it is shown that MS mainly characterized by infiltration of perivascular CD4+Tcell and mononuclear cell that leads to demyelination of axonal tracks in central nervous system[1]. Regarding the role of T cells in MS, it is considered as an autoimmune disease in which the myelin proteins including myelin basic protein (MBP), myelin proteolipid protein (PLP) and/or myelin-oligodendrocyte glycoprotein (MOG) are attacked and destroyed by T cells or other immune responses[2]. The course of the disease is mainly depending on the stage and process of demyelination. Immunhistochemical studies have confirmed the presence of the pro-inflammatory cytokines including TNF-a and IL-12 in chronic MS plaques, but not in the CNS of controls or in the peripheral blood mononuclear cells (PBMCs) of MS patients.[3] The cytokines produced by activated CD4+ T helper cells not only determine the onset or progression of disease but also use to categorize the T helper cells. “Th1” and “Th2” cells were “important regulators of the class of immune response[4,5]. Th1cells secrete pro-inflammatory cytokines such as interferon-g (IFN-g), tumor necrosis factor-a (TNF-a) and the Th2release cytokines such as interleukin-4 (IL-4), IL-5 and IL-13 whichrespectively activate macrophages to clear intracellular pathogens and aid in antibody class-switching and removal of extracellular infectious agents. The role of other interleukins such as IL-12 in MS pathogenesis also emphasized[6].

Regarding growing evidences of the importance of various type of IL in MS, recently the ratio or balance of Th1/Th2 received more attention, although the hypothesis dated back to the late 80s.

Among he certain kind of interleukins, IL-10, IL-4, IL-12 and TNF-a are seem to be more important in onset, severity and progression of MS[6]. IL-10 which produced by monocytes, macrophages, B cells, and Th2 not only inhibits the production of other cytokines, such as IL-1 and TNF-a, but also ceases the proliferation of T cells[7]. It is reported that IL-10 mRNA was continuously expressed throughout the course of EAE in SJL mice immunized with PLP (Begolka et al., 1998)[8]. IL-4 that release as the same as IL-10 acts in near the same way inhibits the activation of Th1 cells. Some studies showed that IL-4 has been implicated as a suppressor cytokine in EAE.IL-12 that is critical for the differentiation of Th1 cells was found to be elevated immediately prior to the onset of disease in a monophasic EAE rat model (Issazadeh et al., 1995a, 1996)[9] as well as in a relapsing murine model (Bright et al., 1998; Issazadeh et al., 1998)[10]. TNF-a production is associated with a Th1 response and classically induces activation of a variety of cell types and expression of adhesion molecules, chemokines, and cytokines.The expression of TNF-a in the CNS parallels the disease course in EAE (Issazadeh et al., 1995a, 1996, 1998; Begolka & Miller, 1998)[11]. Kuroda et al in 1991 reported that injections of TNF-a lead to significant prolongation of clinical EAE and more severe cellular infiltration in the spinal cord. However, surprisingly, TNF-a-deficient mice develop a more severe form of EAE (Liu et al., 1998)[12], characterized by significantly more inflammation and demyelination. Interleukins are considered to be one of the important items in onset and progression of inflammation[13]. From this point of viewinflammatory processes with infiltrating leukocytes play a crucial role in the pathology of the MS lesion mediated by the production of inflammatory mediators which also involve reactive oxygen species (ROS).Excessive release of free radicals may play an important role in MS pathogenesis and promote trans-endothelial leukocyte migration that lead to oligodendrocyte damage and as axonal degeneration [14,15]. Free radicals such as nitric oxide (NO), ROS and/or Reactive Nitrogen Species (RNS), which mainly produced by macrophages, microglia and astrocytes respectively considered to cause damage of neurons, axons, myelin and oligodendrocyte[16]. This process also may involve mitochondrial damage. Based on these findings it seems logic that other non-inflammatory mechanisms such as mitochondrial dysfunction may contribute to neurodegeneration in MS (Kalman et al., 1996, 1999, 2002; Kalman and Leist, 2003;Vyshkina et al., 2005; Kalman, 2006)[17]in their study reported the functional involvement of mitochondrion in the development of MS lesions. Mitochondriaare involved in the development of lesions and define the fate of effectors immune cells in the central nervous system (CNS)[18]. Regarding the importance of effective treatment of MS in order to suppress or decrease the rate of progression certain therapies suggested. As it is supposed that the inflammation through dysfunction of mitochondrionand production of ROS is one of the important causes in MS pathology recent therapy strategy focused on antioxidant therapies. Different antioxidants such as vitamin D, E and CoQ10 are introduced during the last decades. Ubiquinone CoQ10 is a vital cofactor in complexes I to III of the mitochondrial electron transport chain, which acts as an electron acceptor and is a key component of the mitochondrial respiratory chain for adenosine triphosphate synthesis[19,20].In addition to its unique role in mitochondria, CoQ10 is a potent antioxidant and scavenging free radicals and inhibiting lipid per oxidation[21]. CoQ10 has bioenergetics and anti-inflammatory effects, and protects against apoptosis of neurons and it protects cells from oxidative stress in vivo[22]. Some research showed that CoQ10 modulate cytokine production by human peripheral blood mononuclear cells (PBMC)[23].

Whether the ratio of Th1/Th2 that finally determines the outcome of EAE model of MS is affected by CoQ10 administration is not clear yet, in order to answer this question the present research designed.

Materials and methods

Biological models. Thirty 10-12 weeks adult female C57Bl/6 mice (18-20 gr; Pasteur Institute of Iran) were used. The animals randomly divided into four groups (n=6) including EAE, EAE + CoQ10, EAE+ sesame oil and control. All the procedures used in this study approved by The Committee of Ethics in Animal Research of Iran University of Medical Sciences.

Induction of EAE. To induce EAE, the routine procedures introduced by others were used as follows: The animals were immunized with 1:1 ratio of MOG 35-55 (Alexis, Switzerland) dissolved in Complete Freund’s adjuvant (CFA) containing 0.4 mg of mycobacterium tuberculosis (Sigma-Aldrich, USA). For this purpose 300μg of MOG dissolved in 100μl PBS and mixed with equal volume of CFA. On day 0, each animal received 200μl, two single shot, of MOG-CFA emulsion subcutaneously into two sites of the upper flanks. The supplement immune adjuvant, pertussis toxin (PTX), (Sigma-Aldrich, USA) at the dose of 200μl containing 400ng of PTX, was injected i.p immediately after MOG-CFA emulsion, the same injection repeated 48 hourslater.

Clinical EAE score. To approve the onset and stage of progression of the diseasethe standard scoring system sited elsewhere was used. Based on this method of scaling: clinical signs of no symptoms, distal weak or spastic tail, completely limp tail, limp tail and hind-limb weakness,unilateral partial hind-limb paralysis, bilateral partial hind-limb paralysis, complete bilateral hind-limb paralysis, completehind-limb and unilateral partial forelimb paralysis, moribund and dead equaled to 0, 0.5, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4 and 5 respectively [20]

CoQ10 administration. Thereafter the animal show score 1, intraperitoneal injection of CoQ10 ( Sigma- Aldrich)at the dose of 10 mg/kg in 0.5 ml sesame oil was started and continued for 3 weeks. For the control animals only sesame oil was used in same method.

Tissue preparation. Perfusion and fixation by aldehyde solutions was done transcardially via left ventricle. Brains were removed and post-fixed in same solution overnight. After that the brains exposed to tissue processing and in turn paraffin embedding. By using rotatory microtome coronal sections of 4µ prepared. To study the myelination in certain part of the brain including corpus callosum, Luxol Fast Blue staining with Nissl contra staining was used. The total surface of demyelinated regions was calculated by Infinity Software.

Study of the level of Cytokine.The animals were sacrificed by lethal dose of ketamine and xylazine. Brain tissue removed and rapidly transferred to liquid nitrogen. To homogenized the tissue solution including (1x Lysis Buffer: 1x TBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.004% sodium azide.) combine with 10 μl PMSF solution, 10 μl sodium orthovanadate solution and 10–20 μl protease inhibitor cocktail solution per ml of 1x RIPA Lysis buffer mixed to prepare complete RIPA ( Santa Cruze). Use 3 ml complete RIPA per gram of tissueBrain homogenate was spun at 10,000× g for 10 min at 4 °C and supernatants were collected and stored −70 °C. The concentration of IL-12, TNF-α,IL-4and IL-10 in the supernatants of brain extraction, at 1:10 dilution in 1% BSA in phosphate buffered saline (PBS), was assayed in an ELISA set-up using commercially available antibodies and the concentrations according to the procedures supplied by the manufacturer (eBiosciences ,Austra).

RNA extraction and RT-PCR. Animals were sacrificed by decapitation within a few seconds after being picked up from their home cage. Brain removed using aseptic techniques, placed in sterile tubes, freeze on dry ice. Total RNA extraction was performed using RNX-plus (Cinnagen, Iran) according to the protocol. The RNA samples were re-suspended in 30 µl of nuclease-free water. The concentration and quantification of total RNA was measured with spectrophotometer, with the OD260/OD280 ratio of all RNA samples 1.9–2.0 and OD260/OD230 ratio up to 2. The first strand cDNA was synthesized with the First Strand cDNA Synthesis Kit (Bioneer kit, K-2101, Korea). For each reaction, 1 µg (1 µl) RNA was used for reverse transcription, in a mixture of 20 pmoles (1 µl) random primer, and 18µl DEPC-D.W with a final volume of 20µl. The mixture was incubated at 15 ◦C for 1 minute, 50 ◦C for 60 minutes, and heated at 95 ◦C for 5 minutes to terminate the reaction. The cDNA was subsequently stored at −20 ◦C. qPCR was performed in a volume 1 µl of Primer and 1 µl from Template also add 3 µl DEPC.D.W with 5 µl Mastermix (AccuPower® 2X GreenStarTM qPCR Master Mix, Bioneer kit, Korea) . All PCR reactions were in the following condition: initial 95◦C for 15 minutes followed by 40 cycles at 95 ◦C for 15 s and 60 ◦C for 30 s. The PCR primers for each gene were shown in Table 2. Each sample was tested in duplicated. The values were normalized against the housekeeping genes GAPDH (glyceraldehyde-3 phosphatedehydrogenase) (Table 1).

Statistical analysis. By using SPSS 21 and statistical exams, data analyzed and presented as mean ±SEM. The result of the real time PCR was analyzed by a twosided Student’s t-test. All mean differences were considered significant if p <0.05.Analyses were conducted using SPSS for Windows® v.21 (SPSS Inc., Chicago, USA).

Results

Scoring. Loss of weight which consider as one of the important markers for confirmation of model, significantly occurred in EAE induced animals comparing to control and sham vehicle. The maximum mean score for the EAE + CQ10 animals was significantly lower than the animals of EAE (Pv< 0.05, 1.6±0.54, 2.5±0.9 respectively).

Histological study. EAE caused significant demyelination in certain area of the brain like corpus callosum. Comparing EAE group with the animals of EAE+ CoQ10 (42.56 ± 37.12 %, 31.19±39.14% respectively) showed significant less demyelination in later group (Fig.1). For the animals received only sesame oil the result showed significant less demyelination compering with EAE and EAE + CoQ10 (Fig.2).

Cytokine analysis. Level of brain cytokines including IL-12, TNF-α (TH1) and IL-10, IL4 (TH2) measured on post-immunization day 21. Level of TNF-α and IL-12 was higher but not significant in untreated group compare with the control animals (Fig. 3). Our finding showed that CoQ10 administration significantly decreased level of TNF-α comparing untreated animal (p<0.01) (Fig. 3), this finding is the same for control animals (p<0.05) (Fig.). Conversely, elevated level of IL-10 found in treated animals comparing with not treated (p<0.001) and control ones (p<0.05) (Fig. 3). Regarding level of IL-12 and IL-4 no significant difference observed between untreated and control groups. Put these findings together showed that the ratio of Th1/Th2 in EAE animals was higher than sesame and CoQ10 treated animals (Fig. 4).

Cytokine gene expression. Pro-inflammatory cytokines expression analyzed with REST software (Fig. 5). IL-12 mRNA and TNF-α mRNA expression were increased remarkably in EAE model. Comparingcontrol group, no significant increase of IL-4 anti-inflammatory cytokines mRNA expression in EAE was found. Strong increasing expression of IL-10 mRNA did not occurred in EAE animals. However, a noticeable decline but not significant of IL-10 mRNA expression was seen in sesame and CoQ10 treatment group. Compared to control group, IL-4 mRNA expression in sesame oil group was obviously higher. Regarding IL-10 mRNA expression, it obviously expressed lower in EAE mice. Significant lower expression of TNF-α mRNA was also observed in the CoQ10 and sesame oil.

Discussion

MS is still considered as an autoimmune disease, for better treatment in order to decrease or suppress the rate of progression of the disease number of immunomodulatory and immunosuppressive agents have been used. Involvement of interleukins in MS pathophysiology has reported by other researchers. Clinical findings from human cases also supported Th1 involvement in MS Kaplan et al showed elevated level of Th1 cytokine such as IL-12 in MS patient. Same other reports also confirm serum elevation of IFN-gamma (Th1) and IL-4 (Th-2) in the patients with progressive MS[7][4]. Issazadeh et al reported the low expression of IL-4 in EAE model of MS.IL-10 is produced by monocytes, macrophages, B cells, and Th2 cells. It inhibits the production of several cytokines, including IL-1 and TNF-a and the proliferation of T cells in vitro[9].Cannella et al showed that the administration of anti-IL-10 monoclonal antibody in murine EAE model worsen the rate of disease[24]. Begolka et al in 1998 showed IL-10 mRNA was continuously expressed throughout the course of EAE in SJL mice immunized with PLP[25]. It is also shown that IL-10-deficient C57BL/6 mice are more susceptible and developed a more severe form of EAE compared with IL-4-deficient mice (Bettelli et al., 1998)[26].Racke et al in 1994 reported that intraperitoneal administration of IL-4 reduces clinical severity of EAE. IL-4 mRNA was undetectable until disease reduction in SJL mice immunized with PLP (Begolka et al., 1998)[8]. However, little expression of IL-4 in the CNS has been found in other models of EAE (Issazadeh et al., 1995a, 1995b, 1996)[9]. Some studies showed that IL-4 has been implicated as a suppressor cytokine in EAE. Controversy about IL-4 still remains unsolved and our data showed unchanged level of IL-4 in EAE, although it increased after treatment by CoQ10. Regarding TNF-α it is shown that mice transgenic for expression of TNF-a leads to more oligodendrocyte apoptosis and demyelination (Akassoglou et al., 1998)[27].These results approved inflammatory role for TNF-α and suggest that absence of TNF might enhance EAE. It also seems that expression of TNF-α parallel the disease course in EAE [28]. In humans, elevated serum of TNF-α and PBMC secreting TNF-α have been reported in MSpatients[29]. As most evidences focused on inflammatory roles of cytokines in etiology of MS, therapy went to certain type of anti-inflammatory agents or drugs. (Kalman et al., 2007) showed functional involvement of mitochondrion in the development of MS[17]. It is shown that any mitochondrial dysfunction results in a number ofcellular consequences including: (1) decreased ATP production; (2) increased reliance on alternative anaerobic energy sources; and (3) increased production of reactive oxygen species (ROS) [30]. Although there are no abundant evidences for benefits of antioxidant therapy for MS, more attention paysto antioxidants use for that. During the last decade many researchers focused on CoQ10 with treatment indications for migraine, chronic tinnitus, hypertension, heart failure,atherosclerosis, age dependent disorders and cancer[31]. There is considerable amount of data suggesting that CoQ10 has beneficial effects on certain pathological or normal conditions. Because of its role in proton/electron translocation in mitochondria and lysosomes, it has the potential to protect mitochondria from free radical damage and is thought to be capable of preventing apoptosis[2].Regarding the use of CoQ10 on expression of IL-4, IL-10, IL-12 and TNF-α in EAE our findings differ from or in agreement with others. For example Fuller et al reported that CoQ10 suppressed the production of inflammatory mediators like IL-6 (Bessler et al 2010)[23], their finding was similar to what we found for TNF-αwhich is known as an inflammatory factor. Following CoQ10 administration IL-4 (Th2) remained unchanged but the expression of IL-10 (Th2) increased significantly. IL-10 which synthesized locally in brain and acts to suppress or limit clinical signs of stroke, multiple sclerosis, Alzheimer's disease, meningitis, and the behavioral changes that occur during bacterial infections. Similar to what reported by (Zhouet al in 2001) the expression of IL-10 elevated during the course of most important diseases in CNS we also found same result[32].Base on literature, IL-10 (Th2) is capable of inhibiting synthesis of pro-inflammatory cytokines like IFN-γ, IL-2, IL-3, and TNFα made by cells such as macrophages and regulatory T-cells and thus promotes survival of neurons and all glial cells in the brain by blocking the effects of pro-apoptotic cytokines and by promoting expression of cell survival signals [33]. IL-10 also limits inflammation in the brain via three major pathways including reducing synthesis of proinflammatory cytokines, suppressing cytokine receptor expression, and inhibiting receptor activation (Zhou et al 2000)[32]. Ozenci et al Regarding the use of IFN-β as the drug of choice for MS it is approved that the balance or rebalance between TNF-α andIL-10 is the major role of drug[29]. In fact the normalization of TNF-α from elevation and IL-10 from decreased level accepted as one reason for the effect of the IFN-β. From this point of view our findings following CoQ10 therapy clearly showed same phenomenon as using IFN-β. How CoQ10 could act in near same way to INF-β is questionable. It is shown that the beneficial clinical effects of CoQ10 on human health happen via certain different mechanisms. Among these mechanisms, immunomodulation properties of CoQ10 received more attention especially for some immune based diseases such as MS. As previously mentioned , Fuller et al reported that CoQ10 could suppress the increased production of certain inflammatory mediators such as IL-6 [34]. In addition to the mentioned mechanism, it is shown that CoQ10 insert synergetic effects when combined with certain drugs in patients with cancer. Administration of Tamoxifen with CoQ10 in breast cancer reduced the time of treatment[35]. Restoration of number of cultured damaged oligodendrocyte following administration of CoQ10 reported by Cammer et al[36]. About the mechanisms it is generally accepted that CoQ10 not only produce sub-cellular energy but also acts as antioxidant that prevents lipid peroxidation and scavenges superoxide anions [37]. Reduction of exercise induced muscular injury following oxidative stress reported by Kon et al [38]. Antioxidant property of CoQ10 is well-known and it is shown that CoQ10 can diffuse within biological membrane and it can leak out the inner membrane of mitochondria (Cammer 2002)[36]. NAC which is known as an antioxidant can block the effects of TNF-α in Hela cells. It acts via interaction with kinases involved in cellular signaling pathway (Cammer 2002)[36]. Same result for inhibitory effects of CoQ10 on TNF-α reported by Bessler et al in 2010. It is possible that CoQ10 acts in the same way to block the effects of pro-inflammatory cytokines. Other mechanisms such as increasing the capability of phagocytic cells to reach the targets, elevation of the level of the circulatory anti-inflammatory antibodies, preventing apoptosis are supposed to be important in fundamental role of CoQ10 function in cross talk with immune system elements[39]. Based on our findings it is also possible that CoQ10 may act via involvement in regulating circulatory level of pro or anti-inflammatory cytokines. The mechanisms needs more study to known.