Effect of Myelin Oligodendrocyte Glycoprotein (MOG35-55) on Cell Differentiation and Antibodies-Abzymes Production in Transgenic EAE-Prone Th Mice with T Cells Response during the Development of Experimental Encephalomyelitis

Research Article

J Immun Res. 2021; 7(2): 1042.

Effect of Myelin Oligodendrocyte Glycoprotein (MOG35-55) on Cell Differentiation and Antibodies-Abzymes Production in Transgenic EAE-Prone Th Mice with T Cells Response during the Development of Experimental Encephalomyelitis

Aulova KS1, Urusov AE1, Toporkova LB2, Sedykh SE1, Shevchenko YA2, Tereshchenko VP2, Sennikov SV2, Budde T3, Meuth SG4, Orlovskaya IA2 and Nevinsky GA1*

1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia

2Institute of Clinical Immunology, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia

3Westfälische Wilhelms-Universität, Institut für Physiologie I, Münster, Germany

4Klinik für Neurologie Universitätsklinikum Düsseldorf, Moorenstraße 5, Germany

*Corresponding author: Georgy A Nevinsky, Institute of Chemical Biology and Fundamental Medicine of Siberian Branch of Russian Academy of Sciences; Novosibirsk 630090, Russia

Received: July 28, 2021; Accepted: October 06, 2021; Published: October 13, 2021

Abstract

The mechanisms of multiple sclerosis development are still unknown. It was shown that the development of Experimental Autoimmune Encephalomyelitis (EAE) in EAE prone C57BL/6mice (model mimicking human multiple sclerosis) having B and T lymphocyte responses is associated with modification in the differentiation profiles of bone marrow Hematopoietic Stem Cells (HSCs) and the increase in lymphocyte proliferation. T cell responses characterize other EAE transgenic prone Th mice. Different characteristics of the autoimmune reaction in Th mice were analyzed. During the development of EAE (and inflammation processes), the differentiation profiles of Th mice bone marrow HSCs (BFU-E, CFU-E, CFU-GM, CFU-GEMM, T and B lymphocytes) were noticeably or significantly different in male and female mice before and after their immunization with myelin oligodendrocyte glycoprotein (MOG35-55).The patterns of B and T (including CD4 and CD8 cells) lymphocytes proliferation in several organs (spleen, thymus, bone marrow, blood and lymph nodes) during spontaneous (completely untreated mice) and MOG-treatment-accelerated development of EAE was also remarkably or significantly different in male and female mice. All these changes in male and female mice, despite some differences, were coupled with the increase in the concentrations of autoantibodies against DNA, myelin basic protein, and MOG, and with the increase in the relative activity of catalytic antibodies hydrolyzing these antigens. A comparison of the changes in a large number of parameters characterizing the development of EAE in Th and C57BL/6 mice was carried out. It was shown that MOG very much accelerates the development of EAE in Th mice. Despite some differences, the general patterns of the developing of spontaneous and MOG-accelerated EAE in Th male and female mice and in C57BL/6 mice are similar to a notable extent.

Keywords: Transgenic Th mice with T lymphocytes response; Experimental autoimmune encephalomyelitis development; Hematopoietic stem cells differentiation; Lymphocyte proliferation in different organs; Catalytic antibodies

Abbreviations

Abs: Antibodies; auto-Abs: Autoantibodies; AI: Autoimmune; AIDs: Autoimmune Diseases; BFU-E: Erythroid Burst-Forming Unit (early erythroid colonies); CFU-GM: Granulocyte-Macrophage Colony-Forming Unit; CFU-E: Erythroid Burst-Forming Unit (late erythroid colonies); CBA: (CBAxC57BL)F1 Mice; CFU-GEMM: Granulocyte-Erythroid-Megakaryocyte-Macrophage Colony- Forming Unit; CSF: Cerebrospinal Fluid; CNS: Central Nervous System; EPO: Erythropoietin; EAE: Experimental Autoimmune Encephalomyelitis; HSCs: Hematopoietic Stem Cells; IgG: Immunoglobulin G; IL: Interleukin; MBP: Myelin Basic Protein; MOG35-55: Myelin Oligodendrocyte Glycoprotein; MTT: Tetrazolium Dye MTT 3-(4,5-methylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; MS: Multiple Sclerosis; SDS-PAGE: Sodiumdodecyl Sulfate-Polyacrylamide Gel Electrophoresis; sc; Supercoiled; scDNA: Supercoiled DNA; SLE: Systemic Lupus Erythematosus

Introduction

Multiple Sclerosis (MS) is the pathology of the Central Nervous System (CNS) related to an increase in T lymphocytes and macrophages. The precise route of multiple sclerosis is unknown [1]. Different studies support the essential role of the destruction of myelin due to Autoimmune (AI) reactions and inflammation processes. The activated myelin-active CD4+cells could be mediators of MS. Some findings also confirm the critical role of B cells and Autoantibodies (auto-Abs) to various myelin autoantigens in MS pathogenesis [1-3] The increased amounts of autoantibodies and the stockpiling of B cells in the bone marrow Cerebrospinal Fluid (CSF), as well as the MS patient’s typical lesions, ensure key evidence for the demyelination involvement in the humoral response [4]. Studies of MS animal models indicate auto-Abs against myelin components are involved in Absdependent demyelination [3]. Autoantibodies against cell proteinoligodendrocyte progenitors could interfere with remyelination by removing or obstructing these cells [5]. Autoimmune Diseases (AIDs) were first being proposed are originated from Hematopoietic Stem Cells (HSCs) defects [6]. Later, it was identified that the spontaneous (without mice immunization with any antigens), as well as antigeninduced development of EAE in C57BL/6 [7,8] and in MRL-lpr/lpr mice of Systemic Lupus Erythematosus (SLE) [9-11], is reached due to specific immune reorganization of bone marrow hematopoietic stem cells. The bone marrow immune system defects include specific parallel changes in the profile of differentiation of bone marrow HSCs and the production of catalytic

Autoantibodies-abzymes splitting DNA, RNA, polysaccharides, proteins, and peptides. The detection of Abs with catalytic activities is the statistically most significant and earliest marker of many AIDs in humans and animals [12-17] as well as in SLE [18-20], EAE [7,8] and MS [21-28]. Enzymatic activities of abzymes are well detected at the very initial stages of the diseases (at the pre-disease onset stage) before the emerging of typical markers of different AIDs [13-17]. Titers of auto-Abs to various auto-antigens at the onset of different AIDs usually correspond to typical indices’ ranges corresponding to healthy humans and experimental mice. The appearance of multiple abzymes clear indicates the start of AIDs, while an increase in their enzymatic activities is associated with deep pathologies development. However, different AIDs development might be mediated byseveral mechanisms, eventually leading to a self-tolerance breakdown and inflammation processes. Several EAE models mimic particular aspects of human MS, including C57BL/6 mice (for a review, see [29-32]). In C57BL/6 mice, EAE passes as a spontaneous chronic- progressive disease. These mice show the specific T and B lymphocyte responses to antigens [29-32]. C57BL/6 mice were used to analyze eventual mechanisms of spontaneous DNA- and myelin oligodendrocyte glycoprotein (MOG35-55)-dependent acceleration of EAE development [7,8]. These mice’s immunizations with MOG35-55 or DNA led to a speed-up of EAE development, associated with parallel specific changes in the profiles of differentiation of bone marrow HSCs, lymphocyte proliferation, and auto- Abs production possessing Myelin Basic Protein (MBP)-, MOG- and DNA-hydrolyzing activities. Another Th model corresponding to spontaneous EAE autoimmunity of CNS exists. This model was obtained due to crossing transgenic mice with a particular myelinspecific T-Cell Receptor (TCR) and mice specific for myelin-specific immunoglobulin heavy chain knock-in mice and described in [33]. Th mice demonstrate T cell responses to various antigens and primarily MOG, resulting in the spontaneous and stimulated development of a severe EAE. It is important to understand all possible complementary parallel mechanisms of disease to explain how MS and EAE develop. Here, we have carried out for the first time the analysis of many different parameters, including abzymes, characterizing accelerated development of EAE after Th mice immunization with MOG and compared the features of the development of pathology before and after the mice immunization. In addition, the same parameters were compared for Th mice with T-response and C57BL/6 with T and B-cell responses before and after mice immunization with MOG at different stages of EAE development.

Material and Methods

Chemicals

Protein G-Sepharose and Superdex 200 HR 10/30 columns were obtained from GE Healthcare (New York, USA), bovine polymeric DNA, and other different proteins and chemicals were from Sigma- Aldrich (Munich, Germany). 18.5kDa human MBP was from the Research Center of Molecular Diagnostics and Therapy (RCMDT, Moscow, Russia) and mouse oligopeptide MOG35-55 was perched from EZBiolab (Germany). All preparations were free from any possible contaminants. Methylcellulose-based M3434 medium was from StemCell Technologies (Canada) and RBC lysis buffer from Biolegend (San Diego, CA, USA). Fetal bovine serum was from Invitrogen (Waltham, MA, USA)

Experimental animals and ethical statement

Th mice line with T-cell response was matured in Germany (Westfälische Wilhelms- Universität, Department of Neurology, Münster). The main characteristics of this line of mice, they are predisposed to the development of EAE, are described in [33]. Inbred Th mice (3 months of age at the beginning of all experiments) were grown in the Institute of Cytology and Genetics (ICG) special mouse breeding facility in standard conditions free of any pathogens. All experiments were carried out with mice pursuant to protocols of the Bioethical Committee of the Institute of Cytology and Genetics (document number 134A of 07 September 2010), satisfying the humane principles of the European Communities Council Directive (86/609/CEE) for working with animals. The Bioethical Committee of ICG supported this study. The relative overtime weight of male and female mice and degree of proteinuria (concentration of total protein in the urine, mg/ml) were analyzed as in [9-11]. For comparison, we used C57BL/6 wild-type mice described earlier [7,8,31].

Immunization of mice

Analysis of changes in various parameters, characterizing the development of EAE during 70-83 days, was carried out in the case of groups of seven female and male mice without any treatment and after their immunization with MOG. Immunization (ortreatment) of Th mice with MOG35-55 was performed using MOG35-55 and Pertussis toxin according to a previously published protocol [31] used previously in [7]. On day 1 (zero time of experiments), Th mice were treated by injection of 30μg of MOG35-55 per mouse in the back, two times in the left and right side using 20μl of Freund’s complete adjuvant containing Pertussis toxin (400ng/mouse; Mycobacterium tuberculosis). The next day 20μl of Pertussis Toxin (400ng/mouse) was additionally injected in a similar way. For different experiments, 0.7-1ml of the blood was collected after decapitation using standard approaches.

ELISA of anti-proteins and anti-DNA antibodies

Anti-DNA Abs analysis (the sera were diluted 100-fold) was performed using the ELISA test system of ORGENTEC Diagnostika (Germany) according to the manufacturer’s instructions as in [7- 9,34,35]. The relative contents of IgGs against MBP and MOG35- 55 were estimated using purified polyclonal electro phoretically homogeneous Abs as in [7-9,34,35]. After all previously described stages of the treatment of immobilized MBP and MOG and bound with them IgGs, rabbit anti-mouse Abs conjugated with horseradish peroxidase were added; all samples were incubated with H2O2 and then with tetramethylbenzidine. The optical densities of the solutions (A450; after adding H2SO4) were measured using the Uniskan II plate reader (MTX Lab Systems, USA) [7-9]. The relative A450 values of the samples were calculated from differences in A450 between experimental and control solutions containing no DNA, MBP, or MOG. More detailed data concerning the determination of anti-DNA and anti-proteins concentrations are given in Supplementary data (Part 1: “ELISA of anti-proteins and anti-DNA antibodies”).

IgG purification

Electrophoretically homogeneous IgGs from blood sera of Th mice were obtained using affinity chromatography of sera components on Protein G-Sepharose and following FPLC gel filtration of IgGs in drastic conditions (pH2.6), destroying immunocomplexes as in [7,8,21-23,34,35]. More detailed data concerning these methods are given in Supplementary data (Part 2: “IgG purification”). IgGs were protected from viral and bacterial contamination by their filtration using Millex membranes (0.1μm). Analysis of IgGs by SDS-PAGE was carried out using gradient gels (4-15%) and visualized using silver staining according to [7,8,21-23,34,35].

DNA-hydrolyzing activity assay

DNase activity of IgGs was estimated according to [9,24,25]. The mixtures (20μL) containing 20mM Tris-HCl (pH 7.5) supplemented with 20mM NaCl, 1mM Ethylenediaminetetraacetic Acid (EDTA), 5mM MgCl2, 20μg/ml Supercoiled (sc) pBluescript and 0.001-0.1 mg/ml of IgGs. The samples were incubated during 5-24 h at 37oC. The products of DNA splitting were analyzed using 0.8% agarose gels electrophoresis. Photographs of ethidium bromide-colored gels were analyzed by Gel-Pro Analyzer v9.11 (Media Cybernetics, L.P., Germany). The Relative Catalytic Activities (RAs) were calculated from the difference between the relative amount of intact Supercoiled DNA (scDNA) and its hydrolyzed-relaxed form, taking into account DNA-substrate distribution between these two bands after scDNA incubation without IgGs. All initial DNA splitting rates were analyzed from linear parts of the reaction time dependencies (30-40% of scDNA hydrolysis) and concentrations of Abs providing 30-40% of scDNA hydrolysis. A complete transition of scDNA to its relaxed form was taken for 100% of the activity. The relative activities (% of the hydrolysis) were finally recalculated to the same standard conditions; time and IgGs concentration (mole of sc DNA /min/mg of IgGs)

Protease activity assay

The mixtures (10-50μL) containing 20mM Tris-HCl buffer (pH7.5) supplemented with 0.7-1.0mg/ml of proteins (MBP or MOG), as well as 0.001-0.2 mg/mL of IgGs were incubated for 5-24 h at 37oC [7,8,21-23,34,35]. The proteins cleavage products were estimated by SDS-PAGE using 12% or 3-15% gradient gels with the following staining Coomassie R250. The gels were scanned, and hydrolyzed products were quantified by GelPro v3.1 software. IgG samples’ relative activities were evaluated from a decrease (%) in the initial MOG or MBP transited to their hydrolyzed forms. The hydrolysis of the proteins incubated without antibodies was taken into account. All initial hydrolysis rates were calculated using pseudo-first-order reaction conditions, linear regions of reactions time dependencies, and IgGs concentrations (20-40% hydrolysis of the proteins). The relative activities (% of the hydrolysis) were finally recalculated to the same standard conditions; time and IgGs concentration (mole of protein (MBP or MOG) / min/mg of IgGs).

SDS-PAGE analysis of catalytic activities

To prove that antibody activity is their own property, the analysis of DNA- and different proteins-hydrolyzing activities of Th mice IgGs after SDS-PAGE was performed as in [7,8,21-23,34,35]. These methods have been developed and published previously and are given in Supplementary data (Part 3: “SDS-PAGE analysis of catalytic activities”).

Analysis of bone marrow progenitor cells in culture

Samples of bone marrow were obtained from 14 mouse femurs, and then the ability of bone marrow cells to form different colonies was estimated as in [7,8,34,35]. The standard methylcellulosebased M3434 medium specific for mouse cells was used; four dishes per mouse (2×104 cells) were grown. The medium contained Erythropoietin (EPO), stem cell factor and interleukins IL-3 and IL-6. The relative number of CFU-GM (Granulocyte-Macrophage Colony-Forming Unit), CFU-GEMM (Granulocyte-Erythroid- Megakaryocyte- Macrophage Colony-Forming Unit), BFU-E (Erythroid Burst-Forming Unit, Early Erythroid Forming Unit) and CFU-E (Erythroid Burst-Forming Unit, Late Erythroid Forming Unit) cell colonies on the dishes was calculated after 14 days of incubation at 37oC (5% CO2) in a humidified incubator as in [7,8,34,35].

Evaluation of lymphocytes in different mouse tissue samples

The relative content of B and T lymphocytes in various organs and blood of mice was estimated by flow cytometry. Peripheral blood was obtained using mice’s standard decapitation. Sodium citrate was used as an anticoagulant. Five hundred thousand leukocytes in not more than 150μL were used for cytometry analysis. Cells were incubated with monoclonal Abs for 20min in the darkness, then the cells of blood samples were lysed using a special 10-fold volume of RBC lysis buffer for 20min, centrifuged for 10min, and washed using 500μL of PBS buffer (150mM NaCl, 17mM KH2PO4, 52mM Na2HPO4) containing 0.02% EDTA and 1% sodium azide. After samples centrifugation, 50μL of PBS buffer was added to the cell pellet, and they were analyzed by a flow cytometer. Lymphocytes were isolated from blood, bone marrow, thymus, lymph nodes, and spleen. Bone marrow was derived by rinsing the femoral cavity. Lymph nodes and thymus samples were carefully homogenized, large particles were removed and different cells were resuspended by their passing using a disposable syringe through a needle. Spleen cells were received by washing this organ with a medium-filled syringe through punctures in the spleen stroma. This approach allows the obtaining of splenocytes without impurities from the stroma of the spleen. Cells were then washed by their centrifugation with RPMI- 1640 medium (5ml) at 1500rpm for 10min. After the second centrifugation, RPMI- 1640 medium (1ml) containing 10mM HEPES, 10% fetal bovine serum, 0.5mM 2- mercaptoethanol, 2mM L-glutamine, 100μg/ ml benzylpenicillin, and 80μg/ml gentamicin were added to the cell pellets, and the cells were counted. The relative cell content in extracts of various organs was analyzed using 500 thousand cells in 100μL of PBS buffer supplemented with 10% fetal bovine serum and the conjugates of different specific monoclonal antibodies. To analyze the relative amount of different cells, specific antiCD45-BV510 (Biolegendcat # 103138), antiCD3-FITC (Biolegendcat # 100204), antiCD4-PerCP (Biolegendcat # 100432), antiCD8alpha-APC (Biolegendcat # 126614), and antiCD19-PE (Biolegendcat # 115508) antibodies were used. All staining of cells was carried out according to the manufacturer’s recommendations. Cells were incubated for 20 min with monoclonal antibodies, washed by centrifugation after adding 500μL of PBS. 50μL of PBS after centrifugation was added to the cell pellet, and the mixtures were used for analysis using the BD FacsVerse flow cytometer (BD Biosciences, SanJose, CA, USA). More than 100,000 events were collected for each sample. Gating was performed as follows: the total cell population was isolated according to cell size and granularity, and the white blood cell population was determined using the pan-white blood cell marker CD45+ and populations of CD3+ and CD3–leukocytes were obtained. In the CD3+ leukocytes population (T cells), CD4+ and CD8+T cells were determined, and in the CD3– leukocytes population, the relative content of CD19+ B cells was found. For all groups, the percentage ratio was calculated relative to the initial lymphocyte population.

Statistical analysis

The final determined values are given as the mean±S.D. of three independent experiments for each mouse (7 male and 7 female mice); the data were averaged over seven different male and female mice of each group. Some of the sets of samples did not match the Gaussian distribution. Therefore, to estimate the differences between parameters analyzed, the Mann-Whitney U test was utilized; P < 0.05 was regarded statistically significant.

Results

Choosing a model for studying EAE development

T and B lymphocytes play vitally important roles in the pathogenesis of human MS [1] and animals EAE [29-32]. B cells provide Abs important for the humoral immunity of the adaptive immune system [32]. In the bone marrow, mature B cells have membrane receptors allowing them to interact with different antigens leading to the initiation of the Abs response. Spontaneous and MOGaccelerated EAE in C57BL/6 mice are characterized by both T and B cell responses [29-32]. C57BL/6 mice spontaneous development of EAE associates with slow changes in HSCs differentiation profiles parallel with an increase in the level of lymphocyte proliferation in various organs during the initial 2-3 months [7,8,34,35]. These mice’s changes lead to the parallel production of abzymes hydrolyzing MOG, MBP and DNA [7,8,34,35]. Immunizing C57BL/6 mice with MOG accounts for a powerful acceleration of EAE development with the onset and acute phases appearing at 7 and 14-20 days after immunization, respectively [7,8,29-32,34,35]. During these phases, there was observed an increase in concentrations of auto-Abs and the appearance of abzymes hydrolyzing DNA, MBP and MOG. Such abzymes are very dangerous for mammals. Abzymes with DNase activity can penetrate through cellular and nuclear membranes, cleavage DNA of chromatin, and induced cell apoptosis [36-38]. This process results in an increase in blood concentration of DNA complexes with histones, which are the most important antigens in the production of Abs against DNA and histones [39]. Abzymes splitting MBP and MOG hydrolyze these components of nerve tissue membranes, leading to an impaired nerve impulse [13-19,21-23]. In contrast to EAE prone C57BL/6 mice having T and B cell responses [29-32], transgenic EAE prone Th mice are characterized by a T cell response [33]. Therefore, it was interesting to compare various parameters characterizing the spontaneous and MOG- accelerated development of EAE in Th and C57BL/6 mice. In the case of C57BL/6 mice, there was analyzed the development of EAE using only male mice [7, 8,34,35]. However, multiple sclerosis is less common for men than for women [40]. Only about a third of patients with MS are men, and the rest are women. In addition, the disease often occurs quite late in men and proceeds more severely. Therefore, taking this into account, in this study, we compared the development of EAE after immunization with MOG of male and female Th mice. In addition, to compare specific features of the EAE development in mice with T (Th) response and T and B (C57BL/6) responses, some previously obtained data on the analysis of C57BL/6 male mice were used [7,8,34,35].

Weight and proteinuria of EAE mice

We have first analyzed over time changes in the relative weight of Th mice before and after they immunization with MOG (Figure 1A). Interestingly, the immunization of male and female mice with MOG had minimal effect on their weight over time compared with the untreated mice. After the immunization, a decrease in weight of C57BL/6 males during 10-15 days (Supplementary S1; all supplementary Figures are given in Supplementary data) was more pronounced than in Th males (Figure 1A). Development of autoimmune pathologies in different autoimmune prone mammals, including MRL-lpr/lpr [9-11] and C57BL/6 mice [7,8,34,35], is characterized by proteinuria (3mg/ml concentration of protein in urine). Non-autoimmune CBA and BALB mice at least during 12months demonstrated the absence of proteinuria (0.1-0.12 mg/ ml) [9-11]. Autoimmune-prone healthy MRL-lpr/lpr mice before the development of SLE are characterized by low proteinuria (0.38mg/ ml) [9-11]. However, C57BL/6 male mice have demonstrated a higher level of protein concentration in urine (up to 10-12 mg/ml) [7,8,34,35]. The overtime changes in proteinuria of Th mice before and after their immunization with MOG were analyzed (Figure 1B). At time zero (3months of age) in two groups of seven male and female mice before their immunization, the average urine protein concentration was surprisingly very different: 7.6 ± 2.2 and 3.2 ± 1.0 mg/ml (P < 0.05), respectively (Figure 1B). During the spontaneous development of EAE, the concentration of proteins in the urine of Th males increased 1.6 times (to 12mg/ml), while in females, only 1.3 times (to 4.2mg/ ml); the difference in concentration is statistically significant (p=0.01). After immunization of male mice, proteinuria was increased up to day 43, similarto its growth before mice treatment and then it began to decreased (Figure 1B). The difference is statistically significant (P=0.01). In contrast to Th male mice, the immunization of C57BL/6 male mice led to a relatively flowing 1.8-fold increase in proteinuria after immunization compared with the spontaneous development of EAE (Supplementary Figure S1). A completely different situation was observed in Th female mice having at zero time lower proteinuria. In female mice from 15 to 30 days, proteinuria was sharply increased 3.2-fold (to 10.3 mg/ml) and then was decreased (Figure 1B). Thus, the immunization with MOG differently affects the proteinuria of female and male mice, which might be possibly associated with some specific features of EAE development in Th female and male mice.