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. Author manuscript; available in PMC: 2022 Jun 1.
Published in final edited form as: J Med Virol. 2020 Oct 5;93(6):3813–3823. doi: 10.1002/jmv.26188

Epstein Barr virus-immortalizedＢlymphocytes exacerbate experimental autoimmune encephalomyelitis in xenograft mice

Pascal Polepole 1, Alison Bartenslager 2, Yutong Liu 4, Thomas M Petro 5, Samodha Fernando 2, Luwen Zhang 1,3,*
PMCID: PMC7738365  NIHMSID: NIHMS1617934  PMID: 32543727

Abstract

Multiple sclerosis (MS) is the most common autoimmune disorder affecting the central nervous system. Epstein-Barr virus (EBV) is a causative agent for infectious mononucleosis (IM) that is associated with MS pathogenesis. However, the exact mechanism by which EBV, specifically in IM, increases the risk for MS remains unknown. EBV immortalizes primary B lymphocytes in vitro and causes excessive B lymphocyte proliferation in IM in vivo. In asymptomatic carriers, EBV-infected B lymphocytes still proliferate to certain degrees, the process of which is tightly controlled by the host immune systems. Experimental autoimmune encephalomyelitis (EAE) mimics key features of MS in humans and is a well-established rodent model for human MS. We have found that xenografts of EBV-immortalized B lymphocytes, which partially resemble the hyperproliferation of EBV-infected cells in IM, exacerbate autoimmune responses in myelin oligodendrocyte glycoprotein-induced EAE in C57BL/6 mice. After remission, an additional challenge with EBV-immortalized cells induces a relapse in EAE. Moreover, xenografts with EBV-immortalized cells tighten the integrity of blood brain barrier (BBB) in thalamus and hypothalamus areas of the mouse brains. Genomic sequences of prokaryotic 16S rRNA presented in feces reveal that EBV-immortalized cells significantly change the diversities of microbial populations. Our data collectively suggest that EBV-mediated proliferation of B lymphocytes may be a risk factor for the exacerbation of MS, which are associated with gut microbiome changes and BBB modulations. Furthermore, multiple xenografts of EBV-immortalized cells into in C57BL/6 mice could sever as a useful model for human relapsing-remitting MS with predictable severity and timing.

Keywords: Epstein Barr virus, multiple sclerosis, experimental autoimmune encephalomyelitis, gut microbiome, magnetic resonance imaging

1. Introduction

Multiple sclerosis (MS) is the most common autoimmune disorder affecting the central nervous system. Pathogenically, the insulating myelinations of nerve cells in the brain and spinal cord are damaged. Damaged myelin disrupts neuronal communication, resulting in a range of signs and symptoms, including physical, mental, and sometimes psychiatric problems 1. Although the clinical presentation and course of the disease are highly variable, several MS types are recognized, including relapsing-remitting MS (RRMS) and secondary progressive MS (SPMS) 2,3. Despite decades of research, the causes of MS, are not yet well understood.

Epstein–Barr virus (EBV) is a member of human herpesvirus family that is associated with several human cancers 4,5. EBV mainly targets B lymphocytes and epithelial cells. EBV has two major infection stages: lytic and latent infections. The lytic replication cycle results in the production of infectious virions. In latency, only a portion of EBV’s genes are expressed. In vitro, EBV infects and immortalizes B lymphocytes efficiently and generate continuously growing lymphoblastoid cell lines (LCL) and establish a type III latency 4,6. In most cases EBV infection is asymptomatic but some individuals develop self-limiting infectious mononucleosis (IM), whose symptoms are initiated by overproliferation of EBV-infected B lymphocytes, majority of which are type III latency 4,6–9.

Several lines of evidence strongly support the notion that EBV infection is a risk factor for MS pathogenesis: MS patients have universal EBV seropositivity, higher EBV antibody levels in the blood and higher concentrations of virions in the saliva 10–12. Moreover, MS patients tend to have reduced EBV-specific CD8+ T-cell immunity, but increased EBV-positive B lymphocytes and plasma cells in the brains. Furthermore, spontaneous EBV-induced immortalization of peripheral blood B lymphocytes from MS patients are increased 13–15.

While the association between MS and EBV, particular in IM, is fairly strong, the exact mechanism by which EBV increases the risk of MS remains unknown. Unfortunately, EBV does not infect rodent cells and there is no murine EBV equivalent discovered yet. Therefore, there is a need for generating a rodent MS model incorporating EBV.

Experimental autoimmune encephalomyelitis (EAE) is a well-characterized animal model to study the etiology and pathogenesis of MS 16,17. By mimicking hyperproliferation of B-lymphocytes in IM in vivo, we have discovered that xenografts of EBV-immortalized B lymphocytes exacerbate autoimmune responses in a rodent EAE model. Also, the phenotype of EAE is associated with significant gut microbiota changes and tightening of blood brain barrier (BBB) in certain areas of the mouse brains. The results suggest that overproliferation of EBV-infected B lymphocytes may be a factor for MS development. Moreover, a novel model for RRMS with predictable severity may be generated.

2. Methods

2.1. Active Induction of EAE and Cell lines

C57BL/6J and APP-null (app−/−; B6.129S7-Apptm1Dbo/J) mice were purchased from Jackson Laboratories. All mice were bred and housed at the American Association for Accreditation of Laboratory Animal Care-accredited facility under specific-pathogen-free conditions. C57BL/6 mice are a common strain for modelling EAE, while the amyloid precursor protein (APP)-null strain was derived from C57BL/6 but is more sensitive for EAE induction 18. All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) and Institutional Biosafety Committee (IBC) of the University of Nebraska–Lincoln and followed federal guideline. All research staffs were trained in animal care or handling. Cells were washed with PBS and desired numbers in PBS were injected into mice in an intraperitoneal (i.p.) manner. Myelin oligodendrocyte glycoprotein peptide (MOG35–55) were synthesized from Novopep Inc in large quantities. An emulsion containing 100 μg of MOG peptide in complete Freund’s adjuvant (CFA; Mycobacterium tuberculosis H37Ra, 4 mg/mL) was injected subcutaneously on either hind flank as two injections. In addition, 200 ng of pertussis toxin was injected intraperitoneally (ip) on the same day as MOG-CFA, and another dose was administered after 48 h following standard protocol 19. Clinical EAE symptoms in mice were evaluated daily with: 0.5=partially limp tail,1=paralyzed tail, 2=paralyzed tail and hind limb paresis, 2.5=paralyzed tail and one hind limb, 3=both hind limbs paralyzed, 3.5=both hind limbs paralyzed, fore limbs partially paralyzed, 4=complete hind and fore limb paralysis, 5=moribund 20. The severity of EAE was scored on daily basis by two researchers, and occasionally a third researcher was used for calibrations. All animal welfare considerations were taken, including efforts to minimize suffering and distress. When a weight loss of 10–15% within a few days or EAE scores 4 or above, those mice were euthanized immediately. The whole experiments took 2–3 months to finish.

EBV-immortalized B lymphocytes from a SPMS patient (MS-LCL) were obtained from Multiple Sclerosis Genetic Susceptibility Project at University of California, San Francisco (UCSF). The immortalized cells were amplified and passaged in tissue culture system constantly and exhibited EBV type III latency based on EBV gene expression and morphology (data not sown). MS-Human EBV-positive Sav I and Sav III cell lines were gifts from Dr. Jeff Sample. The two lines are isogenic with different EBV-latencies (Sav I: type I latency; and SavIII, type III latency). The cells were grown and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (GIBCO Cat#: 11875135) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals, cat#: S11150) and 1X penicillin-streptomycin (PS) (Thermo Fisher Scientific Cat#: 15140163) in a humidified chamber with 5% CO2 at 37°C.

2.2. Western Blot Analysis with Enhanced Chemiluminescence (ECL)

Cells were collected and dissolved in 2x SDS loading buffer. Separation of proteins on SDS-PAGE was carried out following the standard protocol. After the proteins were transferred to a nitrocellulose (BIO-RAD, Cat# 1620112), or immobilon membrane (Millipore, IPVH00010), the membranes were placed with Ponceau S stain solution (0.5% Ponceau S in 1% acetic acid) for 30 seconds to 1 minute. Several changes of water for 30 seconds to 1 minute each were used for destaining the membranes. Stop the destaining as soon as the background got slight pink. The intensity of the bands on the membrane could be used for the rough quantifications of proteins on PVDF or nitrocellulose membranes 21. The membrane was then blocked with 5% nonfat dry milk in TBST (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 0.05% Tween-20) at room temperature for 30 minutes. It was washed briefly with TBST, and incubated with individual mouse serum in 1% milk in TBST for 1 hour at room temperature, or overnight at 4 °C. The mouse plasmas were obtained from participating mice after euthanization. Briefly, whole blood was collected and put into EDTA-treated tubes. Cells were removed by centrifugation for 15 minutes at 1,500x g. The resulting supernatant was designated plasma. After washing the membrane with TBST three times (10 minutes each), it was incubated with the secondary antibody at room temperature for 1 h. The goat anti-mouse IgG-HRP antibody was purchased from Santa Cruz Biotechnology (sc-2005). The membrane was then washed three times with TBST, treated with ECL detection reagents, and exposed to BlueBlot™HS film from Life Science Products (XR-0810-100).

2.3. Microbial DNA Extraction

To collect the fecal samples, mice were placed in an empty sterile cage for 5–10 min and freshly dropped fecal pellets were collected aseptically into sterile tubes. To obtain the cecum contents, mice were euthanized and sacrificed. Ceca were immediately collected and stored at −80°C until use. DNA was extracted from fecal samples using the Omega Mag-Bind® Soil DNA 96 Kit (Omega Bio-tek Inc., Norcross, GA) according to the manufacturer’s protocol. Isolated DNA was stored at −20°C until used for bacterial community analysis. The bacterial community was evaluated using the V4 region of the 16S rDNA. Briefly, the V4 region of the 16S rDNA gene was amplified using barcoded universal primers and was multiplexed and sequenced using the MiSeq Illumina platform using 250bp paired end sequencing using the V2 500 cycle reagent kits. Library preparation PCR contained, 0.75 Units of Terra PCR Direct Polymerase Mix, 1X Terra PCR Direct Buffer, 0.4 uM indexed primers, and 10 – 25 ng of DNA. Cycling conditions included, initial denaturation of 98 °C for 2 min, followed by 25 cycles of 98°C for 10 s, 55°C for 30 s, and 68°C for 30 s; and a final extension of 68 °C for 4 min. The resulting PCR amplicons were normalized using the Charm Biotech Normalization Kit (Charm Biotech, San Diego, CA) following the manufacturer’s protocol. Normalized libraries were pooled and were further purified using NucleoSpin® Gel and PCR Clean-up (Macherey-Nagal Inc, Bethlehem, PA). The resulting libraries were subjected to 250bp pair end sequencing using the Illumina MiSeq System (Illumina, San Diego, CA) using the V2 500 cycle sequencing kit as described by the manufacturer.

2.4. Data Processing pipeline

The DADA2 v1.10.1 pipeline was used to identify bacterial community changes as described previously 22. In short, low-quality reads with a Q score of ≥25 were removed and the reads were trimmed to a consistent length of 200 bp. Error rates were estimated to distinguish rare variants from sequencing or base calling errors and contigs were assembled using the forward and reverse reads to generate contigs for the V4 region. Furthermore, quality filtering was performed to remove sequences with ambiguous bases, incorrect contig length, or assembly. Taxonomy was assigned using SILVA reference alignment database v132 and a phylogenetic tree was generated in MOTHUR (v.1.42.1). The resulting normalized (DeSeq2) ASV table was used for Principal Coordinate Analysis (PCoA) using a weighted unifrac matrix within R by using the package ggplot2 (Wickham 2009). The weighted Unifrac distance matrix was used to perform multivariate analysis of variance (PERMANOVA) within the R package vegan.

2.5. Magnetic Resonance Imaging (MRI)

MRI was performed on a 7 Tesla small animal scanner (Bruker PharmaScan 70/16, Billerica, MA) operated using ParaVison 6.01. A Bruker made mouse head quadrature RF coil was used for signal transmitting and receiving. Mice were anesthetized by inhalation of 1–2% isoflurane in oxygen during scanning, and respiration and body temperature were monitored (Model 1025 Monitoring & Gating System, Small Animal Instruments, Inc., Stony Brook, NY). T2-weighted coronal brain images were first obtained using Rapid Acquisition with Relaxation Enhancement (RARE) as an anatomical reference (TR/TE = 4200/48 ms, FOV = 20 × 20 mm2, matrix size = 256 × 192, 21 slices, slice thickness = 0.5 mm, and averaging = 2). T1-weighted and T1 mapping scans were performed before and after intraperitoneal (i.p.) administration of gadolinium (MultiHance, Bracco Imaging) at 0.2 mM/kg. Modified driven equilibrium Fourier transform (MDEFT) was used for T1-weighted imaging with TR/TE/TI = 2600/2.2/950 ms, flip angle = 15°, FOV = 20 × 20 mm2, matrix size = 256 × 256, 15 slices and slice thickness = 0.5 mm. T1 mapping data acquired using variable TR RARE (RAREVTR) with 6 TRs from 9 to 0.4 s. Other parameters included TE = 8 ms, FOV = 20 × 20 mm2, matrix size = 96 × 128, 15 slices with slice thickness = 0.5 mm. T1 maps were calculated using an in-house developed fitting program written in IDL. T1 values on somatosensory cortex, hippocampus, striatum, thalamus and hypothalamus using region-of-interest (ROI) analysis.

2.6. Statistical analysis

Mann-Whitney U one-tailed test analyses were done with the use of Mann-Whitney U Test Calculator (https://www.socscistatistics.com/tests/mannwhitney/default2.aspx). General statistical analyses were performed by functions implemented in Microsoft Excel Program. Two group comparisons were done with Student’s two-tailed unpaired t-test. The p values of ≤ 0.05 were considered statistically significant and highly significant if p ≤ 0.01 in all analyses.

3. Results

3.1. EBV-immortalized cells exacerbate EAE responses in mice

During IM, EBV-infected B lymphocytes proliferate excessively and elicit strong immune responses, as they are viewed as foreign by the human immune system 9,23,24. One of immune responses is the induction of heterophile antibodies. The well-established monospot test for IM diagnosis is based on the detections of heterophile antibodies 25. There are several types of EBV-infected cell types and latency III latency cells are one of the predominant ones during IM 8,26. Healthy EBV-carriers may occasionally have expansions of EBV-infected B lymphocytes and those cells are quickly removed by the EBV-specific immune cells 6,26–29. The occasional expansions of EBV-infected cells are believed to be type III latency cells as EBV-specific CTLs are predominantly against type III latency antigens in EBV carriers30. If immune system had been suppressed, EBV-infected cells (type III latency) may quickly expand as in the case of post-transplant lymphoproliferative disorder 31,32. We reasoned that xenografts of EBV-immortalized B lymphocytes (type III latency) into mice could partially mimic the overproliferation of EBV-infected cells in vivo during mononucleosis and in a healthy EBV-carrier.

EBV-immortalized B lymphocytes from a SPMS patient (MS-LCL) were xenografted into C57BL/6 mice with intraperitoneal injections (i.p.). The age of mice was chosen at 4 weeks, which might be similar to human adolescent, a highly susceptible period for IM 33. First, we found that C57BL/6 could tolerate 107 MS-LCL cells without any apparent effects for a certain period of times. The 107 MS-LCL cells were injected into mice every 10–14 days until euthanization (i.p., 5–7 times). No EAE symptoms appeared in mice given MS-LCL cells alone (data not shown). Therefore, EBV-immortalized cells per se did not induce EAE in C57BL/6 mice under our experimental conditions.

To test whether EBV is a priming factor for MS 34, we xenografted MS-LCL into C57BL/6 mice twice, and then the mice were immunized with MOG peptide using a standard EAE protocol (Figure 1A) 19. The dosages for MOG peptide were screened first and the amounts for minimum EAE inductions were chosen (data not shown). As shown in Figure 1B, the MS-LCL inoculation enhanced EAE clinical scores. After EAE remission, EBV-infected cells or PBS were administrated into the mice again without MOG peptide. In this case MS-LCL alone stimulated an EAE relapse (Figure 1B; data after Day 29). Therefore, the xenografts of EBV-infected cells could enhance the EAE development in a statistically significant manner.

Figure 1: Exacerbation of EAE in mice xenografted with EBV-immortalized cells.

Figure 1:

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A, Mice are injected twice with EBV-immortalized cells (i.p.), then MOG immunization, and followed by another injection of EBV-immortalized cells when EAE score is low. Drawing is not on scale. B, the 4-week old C57BL/6 mice were first injected (i.p) with 107 EBV-immortalized SPMS-patient’s B lymphocytes (MS-LCL) (dashed lines; two litters, 3 males, 4 females) or PBS (solid lines; two litters, 3 males, 3 females) twice at Day −31 and Day −22 respectively. EAE induction was following standard protocol with MOG peptide. Mean clinical scores and standard error of the mean (SEM) are shown. After the apparent remission of EAE (Day 29, indicated by an arrow), third injection (i.p) of MS-LCL (dashed line) or PBS (solid line) respectively was carried out. Mouse feces were collected at D16, D28, D36, and D50. At the D51, mice were euthanized and ceca were collected for microbiome analyses. At the end of the study, survival rates for all groups were 100%. Total 6 out 7 mice with MS-LCL injections, while 2 out 6 PBS-injected, developed EAE. The detailed information about mice and treatments are described in the Supplemental Table 1. Mann-Whitney U one-tailed analysis. *, p≤ 0.05; **, p ≤ 0.01. C, APP-null mice (7-week old) were immunized with MS-LCL (dashed lines with blue dots; three litters: 3 males, 4 females) or PBS (solid lines with orange dots; three litters: 2 males, 3 females). All cells and reagents used were the same, and treatments were done simultaneously as in Panel B. At Day 29, third injection of MS-LCL or PBS respectively was carried out. At the end of the study, total 6 out 7 mice with MS-LCL injections, while 5 out 5 mice with PBS-injected, developed EAE. A higher rate of mortality was seen in APP-null mice with MS-LCL (3 out 7) compared with PBS (death 0 out 5, however one euthanized after reaching EAE score 4). No statistical significant differences were noticed between the two groups.

To verify the enhancement was not a dosage issue, we used a different mouse line that is known to be more sensitive to EAE. APP is a protein involved in the pathogenesis of brain diseases 35. APP-null mice are more sensitive for EAE inductions 18. APP-null mice were concurrently used for the same experiments. No statistical significant enhancements were observed in APP-null mice administered MS-LCL with the same protocol (Figure 1C). The phenotypes of APP-null mice were as previously reported: earlier EAE onset, higher clinical scores (compare PBS-treatments between APP-null and C57BL/6), and without remission (Figure 1C) 18. The data in APP-null mice confirmed that the dosages for EAE inductions were properly selected in the C57BL/6 mice.

Because we have used MS patient-derived EBV-immortalized B lymphocytes, it is therefore possible that the MS-specific factor(s) derived from these B cells is involved. This possibility is eliminated as EBV-immortalized cells from healthy non-MS individuals also have similar effects (data not shown). All those data suggest that mimicking IM in mice may potentiate EAE development.

3.2. Challenge with EBV-immortalized cells after EAE remission induces a relapse.

It is reasonable to assume that the occasional overproliferation of EBV-positive cells is present in remission stages of MS patients. As EBV-immortalized cells might be involved in exacerbating EAE (Figure 1B), we addressed whether there was an enhancement in the relapse without prior treatment of EBV-immortalized cells. C57BL/6 mice were primed with MS-LCL or PBS prior to initiation of EAE protocol. After EAE remission, both groups were challenged with the same MS-LCL cells. As seen before, EBV-immortalized MS-LCL caused EAE relapse without further injection of MOG peptide. It was apparent that the additional challenge induced a relapse (secondary EAE) predominantly in EAE mice primed with MS-LCL (Figure 2A). Because of the sample size, we did additional experiments to address whether prior exposure to MS-LCL was necessary for a relapse. As shown in Figure 2B, the prior MS-LCL treatment was required for the relapse without further MOG treatment. Furthermore, we observed that clinical peak scores of the relapses basically aligned with the previous clinical scores of the mice induced by MOG peptides.

Figure 2: EBV-immortalized cells initiate second EAE response after remission.

Figure 2:

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A, C57BL/6 mice were first immunized with MS-LCL as in Figure 1A (dashed lines; one litter: 2 males, 2 females) or PBS (solid lines; one litter: 1 male, 2 females) twice. MOG-CFA and others were injected as described in the Methods. The severity of EAE was scored on daily basis. After the apparent remission of EAE, all mice were injected (i.p.) with the same MS-LCL at Day 27, indicated by an arrow. At the end of the study, survival rates were 100%. Total 4 out 4 mice with MS-LCL injections, while 2 out 3 PBS-injected, developed EAE. Mean EAE clinical scores and standard error of the mean (SEM) were shown. Student t tests were performed by the use of Microsoft Excel. *, p ≤ 0.05; **, p ≤ 0.01. B, C57BL/6 mice were immunized with MS-LCL (two litters: 4 males, 4 females) or PBS (two litters: 3 males, 3 females) and followed by MOG-CFA injection as in Panel A. At Day 8 and 9, mice were examined by a MRI machine. All mice were injected (i.p.) with MS-LCL at remission stage. At the end of the study, survival rates were 100%. Total 7 out 8 mice with MS-LCL injections (average maximum score: 1.92), while 5 out 6 PBS-injected (average maximum score: 1.12), developed EAE. The first peak clinical scores were used as a base for calculation of the secondary induction efficiency (percentage). Mean secondary induction percentages and standard error of the mean (SEM) were shown. Mann-Whitney U one-tailed analysis. *, p≤ 0.05; C, the plasmas from MS-LCL injected and PBS control mice (after three injections) were used for Western Blot analysis as primary antibodies with identical conditions. SavIII and MS-LCL are not the same lines but they have the same type III EBV latency. Each lane contained approximately the same amounts of proteins as determined by ponceau S staining (data not shown), and the protein markers were used to facilitate the cutting the membrane into pieces. Each piece was used for incubation with one plasma at the same dilutions. They were pieced together for final exposure. Molecular weight makers (in kDa) are shown.

To test if the mice had immune responses to EBV-immortalized cells, we collected plasmas from several MS-LCL xenografted or PBS-treated mice, and used them for Western Blot analysis with identical conditions. The plasmas reacted with MS-LCL lysates pretty strongly (data not shown), which indicate that MS-LCL induced immune responses against itself. Next whether MS-LCL xenografted mice had a specific antibody to EBV latent proteins was examined. Sav I and SavIII are isogenic lines with different EBV-latencies: SavI has only one EBV protein expression (type I latency), while SavIII has eight or more viral protein expressed (type III latency)4,36,37. SavIII and MS-LCL are not the same lines but they have the same EBV latency type. As shown in Figure 2C, a MS-LCL xenografted mouse had strong antibody responses to EBV-infected human B lymphocytes. Unfortunately, no convincing evidence that specific antibodies to EBV latent antigens generated as the EBV-negative ones also react with the plasmas. Moreover, different plasmas provided different patterns of reactions (data not shown). However, the data in Figure 2C clearly showed that xenografted mice had immune response to human B lymphocytes.

3.3. EBV-immortalized cells elicit changes in gut microbiome associated with EAE progression

Recent evidence suggests alterations in the gut microbiome of MS patients may be a contributor towards the promotion of inflammatory cytokines and overall inflammation 38. Moreover, changes in microbiota composition may result in imbalances in metabolites produced and nutrients available leading to diseases. As such, modulation of the gut microbiome can be viewed as a potential novel therapeutic avenue 38–40.

To evaluate the possible associations between the gut microbiota and EAE progression, we performed longitudinal evaluation of the gut microbiota composition as described in Figure 1B and Supplemental Table 1. Basically the feces were collected on the Day 16, 28, 36, and 50. Ceca were collected on the Day 51 after euthanization. The V4 region of the 16S rDNA gene was amplified using barcoded universal primers and was multiplexed as previously described41. Resulting amplicons were sequenced using a MiSeq Illumina platform with 250-bp paired end sequencing41. The eubacterial community of the fecal microbiota was analyzed using Amplicon Sequence Variant (ASV) using the Dada2 pipeline 42. Alpha diversity metrics between the two treatment groups displayed significant differences in bacterial diversity across time periods (p= 0.026 – 0.001). Additionally, beta-diversity analysis using weighted-UniFrac 43 demonstrated significant differences in microbial community compositions between PBS and MS-LCL treated mice as a whole (p<0.009) and in a longitudinal manner (p<0.001) (Fig 3A). The changes in microbial community were associated with time of sampling, including higher abundance of phylum Verrucomicrobia during the initial sampling period followed by a decrease in abundance of this taxa over time. Additionally, the phyla Firmicutes and Bacteriodetes fluctuated over time (Fig 3B). Further analysis of bacterial taxa following the first MS-LCL injection demonstrated an increase of Ruminocococceae, and Muribaculaceae families in comparison to PBS treated mice. However, different trends were observed with the third MS-LCL treatment (D29): an increase of families of Lachinospiraceae and Ruminococaceae, and a decrease in Akkermansiaceae family (Fig 3C). Of note that Akkermansiaceae has apparent anti-inflammatory functions 44–46. Four female mice (F16-F19, Supplemental Table 1) from the same litter were maintained in the same cage. They had received the same MS-LCL treatment but presented with different clinical scores that could be classified into two groups (Supplemental Figure 1). The differential analysis of bacterial taxa between the two phenotypes in this cage displayed differences in taxa abundance of Lachinospireceae (Fig 3D). In summary, bacterial community composition significantly changed with MS-LCL xenografts.

Figure 3. Gut microbiota community changes in response to Xenografts of EBV-immortalized cells.

Figure 3.

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Fecal samples were collected as described in Supplemental Table 1 and the microbial community was analyzed using 16S rDNA sequencing of the V4 region using the MiSeq Illumina platform. The eubacterial community of the fecal microbiota was analyzed using Amplicon Sequence Variant (ASV) using the Dada2 pipeline. A, Beta-diversity plot showing microbial community differences by treatment and time. Colors represent individual mouse identities, shape represents time and size of shape represents treatment. The individual identities and treatments are listed in the Supplemental Table 1. B, Heatmap showing the relative abundances of the most abundant phyla in mice sampled five times. Phylum assignments are shown with different colors. The F13-F25 represents the identities of individual mice. The number followed represents the dates of the collections. 1=D16, 2=D28, 3=D36, 4=D50, and 5=D51 (Figure 1B). Treatments are also indicated at the bottom. C, Heatmap of differential ASVs identified in each treatment group at the time when differences of EAE scores were observed. Collection dates are shown on the top, and the treatments are shown at the bottom. Mouse numbers are not shown but can be identified from the position: from left to right: F13->F19 in MS-LCL treatments, and F20->F25 in PBS controls as in Panel B. D, Differential ASVs are identified in the cage where the four mice from the same litter, same sex (F), and same treatment (MS-LCL). The identities of the mice are as shown. The F16 and F18 mice had higher EAE clinical scores (Supplemental Figure 1). The collection dates are shown on the top.

3.4. Xenografts of EBV-immortalized cells enhance the integrity of blood brain barriers (BBB) in mice

The BBB is a highly selective permeability barrier that separates circulating blood from the brain’s extracellular fluid in the CNS. Because of tight junctions, BBB may prevent the entry of potential neurotoxins. It is generally accepted that some cells/factors must cross the BBB and enter the CNS for MS pathogenesis 47.

Because EBV-infected cells were capable of enhancing EAE scores (Figures 1 and 2), whether those cells could affect BBB permeability before the onset of EAE was examined. Mice were xenografted with MS-LCL or PBS, followed by EAE induction. Before the onset of EAE (Days 8 and 9), the BBB integrities of the mice were assessed with gadolinium (Gd)-enhanced magnetic resonance imaging (MRI). The reason to choose MRI over other methods is that MRI can monitor the BBB integrity in a non-invasive manner. Several areas in the mouse brains were examined systemically, including striatum (STR), hippocampus (HP), thalamus (TH), hypothalamus (HY) and cerebral cortex (CTX). T1-weighted images were taken prior to and after Gd injection and compared. The post-contrast signal intensity in the PBS-control mice might be higher than the MS-LCL-injected mice, suggesting that higher Gd deposition in the PBS control mice (Figure 4A). Furthermore, T1 values on various areas were measured using region-of-interest (ROI) analyses. The T1 shortening in a region suggests the Gd entry of brain parenchyma and a strong indicator of comprised BBB integrity 48. Gd-induced T1-shortening was found in all animals by comparing pre- and post-Gd T1 values, but was only statistically significant on TH and STR in PBS control mice (Figure 4B). The results suggested that the MOG/CFA injection per se could enhance BBB disruptions in TH and STR areas. The comparisons of PBS- and MS-LCL-treated groups showed that post-Gd T1 values in PBS-treated mice are significantly lower than in MS-LCL-treated mice on HY and TH (Figure 4B). The results suggested EBV-immortalized cells induced tighter BBB integrities in the HY and TH regions under the current experimental conditions. This result underscores that extent and regional specificity of BBB damage may differ among individual EAE models with different methods for detections.

Figure 4: EBV-immortalized cells may tighten the BBB in certain areas of mouse brains.

Figure 4:

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A, Mice were treated as described in details in Figure 2B. At Day 8 and 9 after MOG-CFA inductions, mice were examined by MRI. No mice had shown any EAE signs at the time of examinations. Typical pre- and post-contrast T1-weighted images of PBS- and LCL-treated mice are shown. Top row: a PBS-treated mouse, bottom row: a MS-LCL-treated mouse. Striatum (STR), hippocampus (HP), thalamus (TH), hypothalamus (HY) and cerebral cortex (CTX) arears are indicated. Scale bar: 2 mm. B, MRI obtained T1 values on HY, TH, STR, SM, CTX, and HP measured using ROI analyses. The T1 shortening on all regions suggests gadolinium entry of brain parenchyma. The T1 shortening effect is significant on TH and STR in PBS-treated mice. T1 values on HY and TH in PBS-treated mice are significantly lower than in LCL-treated mice. Mann-Whitney U one-tailed analysis. *, p≤ 0.05;

4. Discussion

4.1. Potential novel rodent model for human RRMS

Approximately 85–90% of individuals with MS are initially diagnosed as RRMS. A relapsing-remitting EAE (RR-EAE) model, representing a course of exacerbations and remissions as occurs in human MS, will be extremely useful for both basic research and translational studies. Pathophysiological RR-EAE can be induced by passive transfer of the proteolipid protein (PLP) reactive cell lines that involve the immunization of donor mice with PLP peptides. Additionally, MOG, myelin basic protein (MBP) peptides and MBP reactive T cell clones are also used to induce RR-EAE 16,49–51.

By mimicking behavior of EBV-infected B-lymphocytes in IM in vivo, we have shown that xenografts of EBV-immortalized B lymphocytes could induce a second EAE response (relapse) (Figures 1, 2). The relapse can be reproducibly generated from remitting mice. The unique feature of the system is that severity of the relapse can be predicted based on previous clinical scores (Figure 2B). In addition, onset time of the relapses can also be predicted. This model might be an additional beneficial one for RR-EAE and furthermore RRMS in humans. However, more biochemical and pathogenic studies are warranted to verify the current one as a human RRMS model.

4.2. EBV-mediated B cell proliferation in MS development

There are several possible explanations for the EAE exacerbation by EBV-infected B cells (Figures 1,2). One of them is that the EBV-induced proliferation of B lymphocytes or elevated presence of B lymphocytes, is a possible factor in MS development. In a healthy individual, hyperproliferation of B lymphocytes may be a stress factor for the immune system, which is likely to initiate a counteracting response. The process to reduce the excessive B lymphocytes may be a trigger for exacerbation of MS. The linkage between EBV and MS may be related to the fact that EBV induces overproliferation of B cells during primary IM and small but frequent B cell proliferation during asymptomatic phase. Of note, MS is associated with deficiencies in EBV-specific cytotoxic T lymphocyte (CTL) and uncontrolled lytic replication 13,52,53. CTL deficiency would allow the expansion of EBV-infected cells as in immunodeficient situations, and higher lytic replication would lead to more EBV virions that promote B cells proliferation after primary infection 4. Therefore, both associations may result in a hyperproliferation of EBV-infected B cells in vivo. It is noteworthy that clinically reductions in B lymphocytes is viewed as a good treatment for MS 54. It is therefore tempting to speculate that EBV-mediated B cell proliferation in vivo is a factor in the development or exacerbation of MS.

Our hypothesis about EBV’s role in MS is different from current hypothesized ones 13,52, in that it puts the overproliferation of B lymphocytes, rather than EBV per se, as a potential risk factor for MS development. Majority of literature support the notion that MS is associated with IM. Primary EBV infection may account for 90% of the IM cases, and a growing number of pathogens, such as cytomegalovirus, have been reported to cause mononucleosis-like illnesses 24,55,56. The literature related IM and MS might need re-evaluated with careful etiology of IM examinations. The bacterial infection might play an important role in precipitating relapse in MS 57,58. Of note bacterial infections may cause B cell proliferation 59. Therefore, our hypothesis about EBV, B lymphocytes, and MS may extend to other infectious agents or beyond.

It is surprising that the challenge of EBV-infected cells actually tightens BBB before the onset of EAE (Figure 4). Because preventing BBB untightening is associated with reduced EAE in at least SJL mice 60, our study suggests that extent and regional specificity of BBB damage differ between individual EAE models and/or at different stages.

Xenografts of EBV-immortalized cells were clearly associated with the modulation of the gut microbiome components (Figure 3A). It is possible that gut microbiome changes may result in changes in microbial metabolic end products in the gut that are associated with EAE61–64. From this limited study, Akkermansiaceae is apparently decreased after xenografts, and the supplementation of the bacteria, which is apparently safe and effective for other disease 65, might be good for inhibition of EAE/MS.

In summary, our data collectively suggest that overproliferation of EBV-infected B lymphocytes may be a risk factor for the exacerbation of MS, whereby changes are associated with the gut microbiome and BBB. Furthermore, the data herein suggest that xenografts of EBV-immortalized cells might be a useful adjunct for RR-EAE model with a predictable severity and timing in C57BL/6 mice.

Supplementary Material

Supplemental figure 1
2

Highlights.

  • Mimic IM with EBV-immortalized cells enhances EAE in C57BL/6 mice

  • Changes in BBB and microbiome are associated with the enhancement

  • Multiple exposure of EBV-immortalized cells could sever as a model for human relapsing-remitting MS with predictable severity and timing

Acknowledgement

We thank Multiple Sclerosis Genetic Susceptibility Project at UCSF for providing EBV-immortalized MS patient’s B lymphocytes, Dr. Jeff Sample for Sav I and III lines, and Dr. Jay Reddy for reviewing the manuscript. This work was made possible by grants from the Laymen Award, Revision Award from UNL, National Institute of General Medical Sciences, 1U54GM115458, which funds the Great Plains IDeA-CTR Network (LZ), Nebraska Research Initiative (YL), and USDA National Institute of Food and Agriculture (2018-67015-27496, 2018-68003-27545, and 1000579) (SCF). PP was a Fogarty fellow supported in part by the NIH Fogarty International Center grant D43TW010354.

Footnotes

Conflict of Interest Statement: The authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest.

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Supplementary Materials

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2
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