More than 2.5 million people live with multiple sclerosis (MS) worldwide, and women are estimated to be afflicted twice to thrice as much as men ( 1 ). In addition, MS is an autoimmune disease associated with chronic inflammatory demyelination of the central nervous system (CNS), and its clinical symptoms include ataxia, loss of coordination, sensory impairment, cognitive dysfunction, and fatigue ( 2 ). Effective treatments have not been developed due to disease complexity and heterogeneity, and almost all the therapeutics tested in MS patients are based on concepts derived from experimental autoimmune encephalomyelitis (EAE) data ( 3 ). It is established that T self-reactive cells have the potential to differentiate into effector subsets of Th1 and Th17 ( 4 ). Effector CD4+T cell subsets play an important role in Multiple Sclerosis (MS) ( 5 ). Interleukin-27 (IL-27) suppresses Th (Th1, Th2, and Th17) cells and dampens autoimmunity and tissue inflammation by promoting the generation of Type 1 regulatory T cells (Tr1) ( 6 ).
The search for alternative therapeutic strategies for the treatment of MS continues and conflicting evidence in the literature indicates that bee venom (BV) has anti-inflammatory and antinociceptive effects on inflammatory reactions ( 7 ). Although research on BV started almost a hundred years ago, its mechanism of action is still uncertain. Some researchers found that immunotherapy using BV could lead to the production of some interleukins including IL10, while others reported the decreased amount of IL-4 and IL-5 ( 8 ). This study aimed to analyze the effect of BV on the progress cessation of EAE-induced mice.
2. Materials and Methods
2.1. Animal Model
The experiments were carried out on 8-10 weeks old female C57BL/6 mice (weighing 18-20 g) purchased from Razi Vaccine and Serum Research Institute, Karaj, Iran. Mice were housed according to institutional guidelines and had access to food and water. They were randomly divided into three groups. Group 1 included six healthy mice, as the group of control. Group 2 included six EAE Induced mice using myelin oligodendrocyte glycoprotein (MOG). Group 3 included six BV-treated mice following EAE induced by MOG35-55 .
2.2. EAE Induction and Treatment
Mice were injected subcutaneously with 50 µg MOG 35-55 peptide (Sigma-Aldrich, USA) in Complete Freund’s Adjuvant (CFA) containing 1 mg of killed Mycobacterium tuberculosis emulsion (Sigma-Aldrich, USA). This was followed by two intraperitoneal injections of 200 ng of pertussis toxin (Sigma-Aldrich, USA) on day 0 and the second day ( 9 ). The BV was dissolved in normal saline and was provided by Razi Vaccine and Serum Research Institute, Iran. The pharmacological dose of BV was calculated based on its effective dose in animal studies ( 9 ). The BV (3µg/0.1ml per mouse) was injected intraperitoneally in group 3 of animals ( 10 ). The BV treatment was performed two times (with 3 days interval) in the first week of the experiment. The clinical signs and weight of each mouse were monitored daily. The motor deficit was evaluated using a ten-point standardized rating scale, in which 0=no clinical signs; 1=partially limp tail; 2=paralyzed tail; 3=hind limb paresis and uncoordinated movements; 4=one hind limb paralysis; 5=both hind limbs paralysis; 6=hind limbs paralysis, weakened forelimbs; 7=hind limbs paralysis, one forelimb paralysis; 8=hind limbs paralysis, both forelimbs paralysis; 9=moribund; 10=death.
2.3. Cytokine and Histological Analysis
The blood was collected from the mice heart at the end of the experiment on day 26th. Serum was separated by centrifuge and preserved at -80 cͦ for future analysis. The serum IL-27 level was determined using a mouse IL-27 ELISA Kit [CUSABIO, Cat, No. CSB- E08466m]. Reading of Optical Density (OD) was performed at 450 nm within 5 min and the obtained data were analyzed according to the standard curve.
Mice in all three groups were euthanized on day 26th of the experiment. They were perfused with PBS followed by 10% paraformaldehyde for tissue fixation. The fixed brain tissues were embedded in paraffin. Paraffin-embedded sections were cut at 6 µm thickness and mounted on silane-coated standard glass microscope slides. Histological evaluation was performed by staining with hematoxylin and eosin (H&E) for inflammatory cell infiltration analyses. The intensity of inflammatory cell infiltration was assessed according to the protocol ( 11 ).
2.4. Statistical Analysis
Data were expressed as mean±SEM, and comparisons between groups were made through one-way ANOVA with Tukey’s test. A p-value less than 0.05 (P<0.05) was considered statistically significant. Statistical analysis was performed using the SPSS software (Version 16).
No change in signs and symptoms of clinical disorders was observed in the negative control group (group 1) during the experiment. The first EAE signs appeared eight days following the immunization of mice (groups 2 and 3) with MOG+CFA and PTx. The maximal degree of paralysis, indicating an acute phase, occurred around day 14 when the average clinical score reached 2.5. From this time on the animals showed a decrease in the clinical score and were partially recovered from paralysis. However, the recovery was not complete and the disease stabilized around day 20 with an average score of 1.25 (Figure 1). Variation in body weight showed an expected course characterized by a significant weight drop during the acute phase. However, weight recovery was observed in the chronic disease phase (Figure 2). Mice in groups 2 and 3 showed signs of decreased activity, nutritional behavior, and weight loss from day eight post-immunization. Clinical analysis of BV-treated mice subjected to EAE revealed no significant differences between the two groups from the onset until the peak of the disease (about 14 days post-immunization), and both groups showed higher clinical scores (ranging between 2.0 and 2.5). However, the reduction of clinical score started in BV-treated mice (group 3) from day 20 and reached score 1 on day 25, while the clinical score remained at 1.5 in non-treated mice (group 2).
3.1. Brain Parenchymal Lesions
In an attempt to establish a correlation between clinical signs and morphological changes in the mice brain, histological analysis of mice brain sections was conducted on day 26 after mice scarification, and the sections were analyzed using light microscopy. Typical mark lesions characterized by an intense perivascular inflammatory infiltrate were observed in the brain of all the mice in group 2 (Figure 3b). However, a visual inspection indicated that both the groups, EAE-induced mice and EAE-induced mice treated with BV presented comparable degrees of inflammation. The brain parenchyma of the control group showed no penetration of inflammatory cells (Figure 3a). However, an inflammatory infiltration mainly composed of mononuclear leukocytes which was noticed in the parenchyma, and inflammatory cells existed around the blood vessels. Likewise, EAE-induced mice treated with BV showed only mild inflammation around the meninges (Figure 3c). Therefore, the histological analysis indicated a significantly decreased intensity of pathological changes in the EAE-induced mice treated with BV (Figure 3).
3.2. Serum Cytokine Expression (IL-27)
In the present study, serum levels of IL–27 were evaluated in all 3 groups of mice on the last day of the experiment. The normal level of IL-27 in serum was found to be 127.26±35.72 pg/mL in the negative control group of mice. The results revealed a significant decrease (P<0.05) in the serum levels of IL-27 in the positive control group (EAE group) which was found to be 73±22.72 pg/mL. However, a highly significant (P<0.001) rise in IL-27 serum level was observed following BV treatment in group 3 of animals, which was found to be 232.23±38.3 pg/mL (Figure 4).
As mentioned previously, MS is an autoimmune disease of the NCS with penetration of macrophages and lymphocytes into CNS, which leads to demyelination. Although the etiology of this disease is still unknown, it seems that T cells and B cells play a significant role in attacking myelinated parts of the nervous system. This study has been conducted on C57BL/6 mice, and it is a known fact that these mice are susceptible to EAE induction using MOG- CFA- PTX. MOG along with CFA acts as an antigenic component. The PTx we used to enhance EAE in mice ( 12 ).
The results of the present study showed a marked change in the physical activity of mice that received MOG- CFA- PTx. It is reported that PTx is an immune-adjuvant utilized to effectively promote an inflammatory response in animal models with multiple autoimmune diseases and is critically involved in the pathogenesis of EAE ( 13 ). Administration of PTx is reported to be a pro-inflammatory cascade of IL-6, TGF-β, and Th17, in the CNS which have been essential in the development of EAE ( 14 ). The first EAE signs appear eight days after the disease induction. The maximal degree of paralysis, indicating an acute phase on day 14±1 when the average clinical score reached 2.5. The positive control group of mice, as well as BV, treated EAE induced mice were sacrificed on day 26 for histological evaluation. These findings were in line with the results obtained by Peon, Ledesma-Soto ( 14 ). According to some researchers, the first signs of neurological disease generally include weight loss and loss of activity between days 10 and 17 following EAE induction. However, based on the evidence, signs are observed somewhat earlier (starting from 5 to 7 days after cell transfer) using the adoptive transfer method ( 15 ).
Based on the obtained results in this study, serum level of IL-27 was lower in the EAE group, compared to healthy mice in the group of control, suggesting that IL-27 has the potential to be an effective response modifier for immunotherapy of autoimmune disease. These results are in line with those of other studies that reported a reduction in serum IL-27, following EAE induction ( 16 ). However, the IL-27 serum level increased in BV-treated mice, compared to healthy ones and EAE group, which suggested the effectiveness of BV in interfering with the disease progress. On the other hand, the effect of BV on EAE-induced mice was investigated considering inflammatory cells and pathological changes in CNS in the present study. Based on the evidence, EAE is a disease mediated primarily by the action of Th1 and Th17 lymphocytes, which release several pro-inflammatory cytokines and chemokines with consequent mobilization and activation of peripheral leukocytes to the CNS parenchyma ( 17 ). The peak of the disease is characterized by the prevalence of effector T cells (Th1 and Th17). In fact, recent studies indicate a positive correlation between the presence of regulatory T cells and the regression of EAE symptoms, and IL-27 potently suppressed the expansion of encephalitogenic Th17 cells in culture ( 18 ). Recent studies also identified different types of inflammatory infiltrates in CNS, and Th1 cells have been shown to correlate with a predominant infiltration of monocyte into the CNS, while Th17 cells were associated with a higher proportion of neutrophil infiltration into the CBS ( 19 ). Additionally, the clinical symptoms of Th1- and Th17- mediated EAE were found to be different: Th1 cells induced classic EAE, whereas Th17 cells induced an EAE with a more sever clinical phonotype ( 20 ).
Medicinal properties of bee products have been known from ancient times, and today the BV is used extensively for the treatment of arthritis and other inflammatory, autoimmune, and destructive diseases. The BV includes some kinds of peptides, enzymes, active amines, and other components, which can be effective in the treatment of various diseases. Melittin is one of the most effective and well-known anti-inflammatory factors in the BV ( 21 ). Adolapin is another effective anti-inflammatory substance that suppresses the activity of the cyclooxygenase enzyme ( 22 ). It seems that BV plays a role in maintaining homeostasis in the human body’s immune system and nervous system, since BV therapy can regulate two immunologically opposite conditions, including allergic disorders (Th2 dominant) and autoimmune diseases (Th1 dominant) ( 23 ). Other T cell populations, such as Th17 cells and Tregs have emerged as a key player in BV-induced modulation of the immune and nervous system ( 24 ). Several recent studies reported that BV or bvPLPA2 could upregulate peripheral Tregs and/or suppress Th17 responses in various animal models ( 25 ).
In conclusion, based on the results obtained in the present study, the treatment of EAE-induced mice with BV resulted in a decrease in the disease symptoms and pathological changes and an increase in IL-27serum level. This activity of BV is due to its anti-inflammatory and immune-modulatory effects.
Based on results obtained in the present study, BV may be a suitable candidate for the treatment of inflammatory diseases, such as MS.
Study concept and design: Sh. J. and A. Z. M.
Acquisition of data: Sh. J.
Analysis and interpretation of data: A. Z. M.
Drafting of the manuscript: Sh. O.
Critical revision of the manuscript for important intellectual content: A. Z. M.
Statistical analysis: L. M.
Administrative, technical, and material support: Sh. J. and A. Z. M.
All the animal experiments were carried out in accordance with the recommendations of the declaration of Helsinki and the internationally accepted principles for the use of experimental animals.
Conflict of Interest
The authors declare that they have no conflict of interest.
The authors would like to thank Dr. Pejman Mortazavi (Associated Professor of Veterinary Pathology, Science and Research Branch, Islamic Azad University, Tehran, Iran) for the analysis and interpretation of pathological findings that improved the manuscript significantly.
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