Mycobacterium avium subsp. paratuberculosis Antigens Elicit a Strong IgG4 Response in Patients with Multiple Sclerosis and Exacerbate Experimental Autoimmune Encephalomyelitis

Neuroinflammation can be triggered by microbial products disrupting immune regulation. In this study, we investigated the levels of IgG1, IgG2, IgG3, and IgG4 subclasses against the heat shock protein (HSP)70533–545 peptide and lipopentapeptide (MAP_Lp5) derived from Mycobacterium avium subsp. paratuberculosis (MAP) in the blood samples of Japanese and Italian individuals with relapsing remitting multiple sclerosis (MS). Additionally, we examined the impact of this peptide on MOG-induced experimental autoimmune encephalomyelitis (EAE). A total of 130 Japanese and 130 Italian subjects were retrospectively analyzed using the indirect ELISA method. Furthermore, a group of C57BL/6J mice received immunization with the MAP_HSP70533–545 peptide two weeks prior to the active induction of MOG35–55 EAE. The results revealed a significantly robust antibody response against MAP_HSP70533–545 in serum of both Japanese and Italian MS patients compared to their respective control groups. Moreover, heightened levels of serum IgG4 antibodies specific to MAP antigens were correlated with the severity of the disease. Additionally, EAE mice that were immunized with MAP_HSP70533–545 peptide exhibited more severe disease symptoms and increased reactivity of MOG35–55-specific T-cell compared to untreated mice. These findings provide evidence suggesting a potential link between MAP and the development or exacerbation of MS, particularly in a subgroup of MS patients with elevated serum IgG4 levels.


Introduction
While the precise pathological mechanism remains unclear, the influence of pathogen exposure as an environmental trigger for multiple sclerosis (MS), a chronic disease impacting the central nervous system (CNS), has been recognized. MS is characterized by inflammatory demyelination in the early phase of relapsing-remitting MS (RR-MS), followed by progressive phases dominated by neurodegenerative processes, resulting in the continuous loss of neurons and axons [1].
Although MS is not hereditary, genetic factors play a significant role in determining susceptibility to the disease, but they alone cannot fully explain its incidence [2]. The hygiene hypothesis proposes that early childhood exposure to pathogens may provide protective immunity, while infections during adulthood could act as triggers for autoimmunity, Life 2023, 13, 1437 2 of 12 particularly in individuals with specific genetic predispositions [3]. Another possibility is the reactivation of viral or bacterial in individuals who had asymptomatic infections years earlier, potentially due to a weakened immune system or other external factors [4]. For instance, it has been suggested that the Epstein-Barr virus (EBV), which is considered the most influential risk factor for MS, could induce the expression of endogenous retroviruses from the HERV-W family, leading to the onset of MS [5]. Moreover, the variation in MS incidence prevalence across different geographical regions suggests that an abnormal immune response might be triggered by a region-specific pathogen prevalent in areas with high MS rates [1].
In the absence of a confirmed pathogen directly causing MS, the interplay between pathogens could have a significant impact on the pathogenesis of the disease. It is highly plausible that bacterial-viral coinfections could contribute to the disparity in MS risk between different regions.
Among bacterial factors, exposure to antigenic determinants of Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of paratuberculosis (commonly known as Johne's disease) in animals, has been linked to the risk of developing MS [6][7][8][9]. Multiple clinical studies conducted in various countries consistently demonstrated an association between MAP and MS, based on the detection of mycobacterial DNA, as well as the presence of humoral and antigen-specific immune responses against MAP antigens in the sera and cerebrospinal fluids of patients with RR-MS [9]. It is important to note that the bacterium has never been isolated from any MS patient, suggesting that its role in MS, particularly in regions with low paratuberculosis prevalence, may be more related to the ingestion of antigenic components through contaminated food rather than active infection [10].
The potential of MAP antigens to exacerbate the progression of experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of neuroinflammation, has been demonstrated. MAP can serve as an adjuvant, replacing Mycobacterium tuberculosis, to enhance EAE [11]. Additionally, it has been observed that oral administration of heathkilled mycobacteria activates mucosal immunity, modulates dendritic cells, and influences the trafficking of CD4 T-cells from mesenteric lymph nodes and spleen to the CNS, thereby worsening active EAE [12].
Furthermore, immune reactivity has been identified against cross-epitopes of MAP and EBV in individuals with MS, suggesting that both pathogens, through molecular mimicry, may activate a shared pathway leading to neuroinflammation in genetically susceptible individuals [13,14]. Studies have reported the presence of autoantibodies that recognize peptides from human myelin basic protein and interferon regulatory factor 5, which also cross-react with homologous peptides from EBV latent and lytic proteins, as well as MAP, in the sera and cerebrospinal fluid of MS patients [14].
In a recent study, specific antibodies against an epitope of the EBV nuclear antigen 1 (EBNA1) protein, specifically EBNA1 386-405 , which binds to the glial cell adhesion molecule (GlialCAM), exhibited increased immunoreactivity in both the serum and CSF of patients with RR-MS from the United States and German when compared to HCs [15].
In our previous study, we highlighted the high recognition of MAP_0106c 121-132 and its homologous peptide EBNA1 400-413 , which shares a 5-amino acid overlap (GRRPF) with EBNA1 386-405 , in the serum and CSF of patients with RR-MS [14]. Furthermore, these peptides were found to induce both humoral and cell-mediated responses in RR-MS patients with a history of infectious mononucleosis [16].
Through the use of the Basic Local Alignment Search Tool (BLAST), we conducted an in silico analysis and discovered regions of local similarity between EBNA1 386-405 and an epitope of MAP heath shock protein (HSP) 70, a protein that has previously been associated with MS [17].
Based on these findings, the objectives of our current study were as follows: 1.
To evaluate the humoral response against the peptide MAP_HSP70 533-545 in Japanese and Italian patients with RR-MS through in vitro experiments. To evaluate the impact of MAP_HSP70 533-545 on neuroinflammation using an active EAE model.

Peptide-Based Enzyme-Linked Immunosorbent Assays (ELISAs)
The perform the peptide-based indirect ELISA, we utilized the Imject maleimideactivated bovine serum albumin (BSA) spin kit (Thermo Fisher Scientific, Waltham, MA, USA). The kit was chosen to prevent the masking of antigenic epitopes, ensuring their accessibility for antibody binding. The procedure involved activating the BSA carrier protein with reactive sulfhydryl maleimide, purifying it, and then crosslinking it with the sulfidyl group (-SH) moiety in the cysteine-containing peptide antigen MAPHSP70 533-545 following the manufacturer's instructions.
To determine the optimal coating conditions, titration experiments were conducted. Nunc-immuno-MicroWell-96-well solid plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 50 µL/well of MAP_HSP70 533-545 peptide diluted in ELISA coating buffer (Bio-Rad, Tokyo, Japan) at a final concentration of 10 µg/mL. The plates were incubated overnight at 4 • C. Subsequently, the microplate was blocked with 200 µL/well of Blocking One (Nakalai Tesque, Kyoto, Japan) for 1 h at room temperature. Sera samples were then added to the duplicate wells, diluted 1:100 in Blocking One, and incubated for 2 h at room temperature (25 • C).
Following four washes with phosphate-buffered saline with 0.05% Tween 20 (PBS-T), the plates were incubated with 100 µL/well of horseradish peroxidase-labeled goat antihuman total IgG IgG1, IgG2, IgG3 or IgG4 antibodies (Southern Biotech Associates, Inc., Birmingham, AL, USA) for 1 h at room temperature. After incubation, the microplates were washed again, and the wells were incubated with 100 µL/well of ABTS Peroxidase System (SeraCare Life Sciences, KPL, Gaithersburg, MD, USA) for 10 min at room temperature in the dark. The optical density was measured at 650 nm using a Benchmark Plus Microplate Reader (Bio-Rad, Tokyo, Japan). Wells coated with BSA were included as a negative control, and the mean value obtained from these wells was subtracted from all other data points. The results were normalized against a positive control serum, which was included in all experiments.

Indirect ELISA for Anti-Lipopentapeptide (MAP_Lp5) Antibodies
In order to evaluate the specificity of our findings, all individuals who tested positive to MAP_HSP70 533-545 were further tested for reactivity against the specific antigen MAP_Lp5. MAP_Lp5 is a distinctive antigenic lipoprotein found in the cell wall of MAP [19].
To eliminate any potential cross-reactivity with other mycobacterial components, all sera were pre-incubated with lyophilized Mycobacterium phlei obtained from the commercially available ELISA kit, Johnelisa II kit (Kyoritsu Seiyaku Corporation, Tokyo, Japan) [20]. This step ensured that the antibody response observed was specific to the antigens of interest.

Inhibition ELISA
To determine the presence of cross-reactive antibodies between MAP_HSP70 533-545 and EBNA1 386-405 peptides in the sera of RR-MS patients, we conducted an inhibition ELISA. Serum samples were first pre-absorbed with saturating concentrations [10-15 mM] of EBNA1 386-405 , MAP_HSP70 533-545 , or scramble peptide overnight. This pre-absorption step aimed to block any cross-reactivity or binding of antibodies to these specific peptides.
After the incubation, the serum samples containing the antibody-antigen mixture were added to microplates coated with the MAP_HSP70 533-545 peptide. The plates were then subjected to an indirect ELISA, following the previously described procedure.
By pre-absorbing the serum samples with the respective peptides, any antibodies present in the samples that were specific to MAP_HSP70 533-545 and EBNA1 386-405 would already be bound to their corresponding peptide agent. As a result, the binding reaction in the wells of the ELISA microplate is reduced, and the reduction in absorbance in the wells is inversely proportional to the concentration of the analyte (cross-reactive antibodies) in the patient samples.

Animal and Mouse Immunization
Groups of 9-week-old female wild-type C57BL/6J mice, with a total of 20 mice, were obtained from Charles River Laboratories, Yokohama, Japan, Inc. The mice were housed under pathogen-free conditions with a 12-h light/dark cycle. The animal experiments were conducted in accordance with the guidelines and approval of the Institutional Animal Care and Use Committee of Juntendo University School of Medicine (No. 290238).
To induce EAE, mice were subcutaneously immunized with 200 µg of MAP_HSP70 533-545 peptide emulsified in incomplete Freund's adjuvant supplemented with 4 mg/mL of Mycobacterium tuberculosis H37Ra (CFA) two weeks prior to the induction of EAE. Placebo mice were immunized with a scrambled control peptide.
For the induction of active EAE, mice were subcutaneously immunized with 200 µg of MOG  peptide emulsified in CFA, followed by an intraperitoneal dose of 200 ng of pertussis toxin immediately after immunization and another dose 48 h later. The mice were monitored daily for clinical symptoms of EAE, and their disease severity was scored as follows: (1) for flaccid tail, (2) for impaired righting reflex and hind limb weakness, (3) for complete hind limb paralysis, (4) for complete hind limb paralysis with partial forelimb paralysis, and (5) for death.

Histological Analysis and T-Cell Proliferation Assay
Histological analysis was performed on the spinal cord sections of EAE mice euthanized during the peak (14-16 days post-immunization) of clinical symptoms. The sections were stained with hematoxylin/eosin to detect inflammatory foci. The severity of inflammation was analyzed using a semiquantitative scale, where 1 represented a small infiltrate (<10 cells/field), 2 represented a medium infiltrate (>15 cells/field), and 3 represented a large infiltrate (>100 cells/field).
T-cell proliferation was assessed by measuring the incorporation of radioactive 3 H-thymidine (1uCi/well) (PerkilnElmenr, Waltham, MA, USA) [21]. Spleen cells (4 × 10 5 cells/well) from EAE mice at the peak of clinical symptoms were cultured for 2 days with 50 µg/mL MOG  in the presence of gamma-irradiated (30.0 Gy) spleen cells (1 × 10 6 cells/mL) syngeneic to the responding T-cells. DNA synthesis was determined by measuring the radioactivity of the incorporated-3 H-thymidine with a scintillation plate counter (MicroBeta TriLux, PerkinElmer, Waltham, MA, USA) 18 h later. The proliferative response was expressed as a stimulation index (SI): counts per minute (cpm) of stimulated cells divided by counts per cpm of unstimulated cells.

Statistics
Statistical analysis was performed using Graphpad Prism 10 software (GraphPad Software, La Jolla, CA, USA). The non-parametric Mann-Whitney's u-test was used to compare ELISA results between patients and HCs. Spearman's correlation analysis was conducted to verify cross-reactivity between antibodies. Receiver operating characteristic (ROC) analysis was performed to assess the diagnostic accuracy of the ELISA and determine the cut-off for positivity with a specificity of 95%.
Clinical EAE scores were analyzed using the non-parametric Mann-Whitney u-test. Histological analysis of spinal cords was analyzed using one-way analysis of variance (ANOVA) followed by post-hoc Dunnett's multiple comparison test. T-cell proliferation was analyzed using a two-tailed Student's t-test. A p-value less than 0.05 was considered statistically significant.
To confirm the specificity of the MAP_HSP70533-545 epitope, all subject sera were preadsorbed with Mycobacterium phlei antigen and tested against MAP_Lp5 antigen. Linear regression analysis demonstrated a good correlation between the ELISA based on MAP_HSP70533-545 and the MAP_Lp5-ELISA for both Italian (r = 0.73, p < 0.005) ( Figure  1C) and Japanese (r = 0.46, p = 0.01) ( Figure 1F) subjects, indicating the specificity of MAP detection. Similarly, in the Japanese RR-MS patients, at the same cut-off level, the MAP_HSP70 533-545 peptide elicited strong antibody titers in the serum of 27 out of 65 (41%; 95% confidence interval [CI]: 23-46%), while no controls displayed positive results (p < 0.0001) ( Figure 1D). IgG1 and IgG4 antibodies were elevated in the sera of 14 (52%) and 13 sera (48%), respectively ( Figure 1E). A significant correlation between IgG4 and EDSS was also observed in Japanese RR-MS patients (r = 0.73, p < 0.0001).
To confirm the specificity of the MAP_HSP70 533-545 epitope, all subject sera were pre-adsorbed with Mycobacterium phlei antigen and tested against MAP_Lp5 antigen. Linear regression analysis demonstrated a good correlation between the ELISA based on MAP_HSP70 533-545 and the MAP_Lp5-ELISA for both Italian (r = 0.73, p < 0.005) ( Figure 1C) and Japanese (r = 0.46, p = 0.01) ( Figure 1F) subjects, indicating the specificity of MAP detection.
The indirect ELISA test showed good reproducibility, with an inter-assay coefficient of variation (CV) of 8% and an intra-assay CV of 4%.

Cross Reactivity between HSP70 and EBNA
Inhibition immunoassays were conducted to investigate the presence of cross-reactive antibodies to MAP_HSP70 533-545 and EBNA1 386-405 peptides in the sera of RR-MS patients from Italy ( Figure 2A) and Japan ( Figure 2B). The results showed that EBNA1 386-405 peptide Life 2023, 13, 1437 7 of 12 inhibited the binding signal on MAP_HSP70 533-545 coated plates by 35-47% (p < 0.0001). In comparison, the positive control (MAP_HSP70 533-545 peptide) caused a decrease in the binding signal by 64-76%, while the negative control (scramble peptide) resulted in 15-17% reduction. Furthermore, a strong correlation was observed between the levels of anti-EBNA1 366-345 antibodies and MAP_HSP70 533-545 antibodies detected by peptide-based ELISA (r = 0.82, p < 0.0034) ( Figure 2C). These findings not only support the association between EBV and MS but also suggest that MAP might be involved in the etiology or progression of the disease through a mechanism of molecular mimicry.
The indirect ELISA test showed good reproducibility, with an inter-assay coefficient of variation (CV) of 8% and an intra-assay CV of 4%.

Cross Reactivity between HSP70 and EBNA
Inhibition immunoassays were conducted to investigate the presence of crossreactive antibodies to MAP_HSP70533-545 and EBNA1386-405 peptides in the sera of RR-MS patients from Italy ( Figure 2A) and Japan ( Figure 2B). The results showed that EBNA1386-405 peptide inhibited the binding signal on MAP_HSP70533-545 coated plates by 35-47% (p < 0.0001). In comparison, the positive control (MAP_HSP70533-545 peptide) caused a decrease in the binding signal by 64-76%, while the negative control (scramble peptide) resulted in 15-17% reduction. Furthermore, a strong correlation was observed between the levels of anti-EBNA1366-345 antibodies and MAP_HSP70533-545 antibodies detected by peptide-based ELISA (r = 0.82, p < 0.0034) ( Figure 2C). These findings not only support the association between EBV and MS but also suggest that MAP might be involved in the etiology or progression of the disease through a mechanism of molecular mimicry.

MAP_HSP70533-545 Immunization Exacerbates Active EAE
To assess the encephalitogenic potential of MAP_HSP70533-545 in active EAE, groups of wild type mice (C57BL/6J) were immunized with the MAP_HSP70533-545 peptide two months prior to EAE induction. The immunized mice exhibited a slightly delayed onset of the disease (9 ± 0.4 placebo vs. 10 ± 0.5 immunized) but developed a more severe peak of the disease (3.0 ± 0.5 placebo vs. 3.5 ± 0.4 immunized) compared to non-immunized control mice ( Figure 3A). The disease incidence (9/10 placebo vs. 9/10 immunized) and clinical course were comparable to placebo controls ( Table 2).
Furthermore, histological examination of spinal cords revealed an increased infiltration of mononuclear cells in MAP_HSP70533-545-immunized mice compared to placebo-treated mice ( Figure 3C,D).

Discussion
In this study, we have provided further evidence for the involvement of mycobacteria in the pathology of MS by identifying a peptide derived from the MAP_HSP70 protein antigen that can elicit a strong immune response in patients with RR-MS. Additionally, we have demonstrated the role of this peptide in the neuroinflammation induced by the EAE model.
HSP70s are a group of highly conserved protein families that are induced under stress conditions, such as inflammation, and have been associated with neuroinflammation and neurodegeneration, particularly in RR-MS [22]. Through in silico analysis and the transcriptional profiling of MS patients compared to healthy controls, we observed a significant upregulation of several genes encoding HSP70s in various brain regions affected by MS, including the corpus callosum, hippocampus, internal capsule, and optic chiasm [22].
A separate study conducted on a cohort of 268 MS patients and 231 HCs from Sardinia demonstrated the presence of serum anti-MAP_HSP70 IgG in 23% of the patients, whereas only 6.5% of controls showed the presence of these antibodies [17]. BLASTp analysis revealed 28% amino acid identity between human HSP70 and MAP_HSP70, although no immunodominant epitope from the recombinant protein was identified.
Here, we have demonstrated the presence of cross-reactivity between MAP_HSP70 533-545 and EBNA1 386-405 , which is a peptide involved in molecular mimicry with GlialCAM [15]. GlialCAM is an adhesion molecule primarily expressed in oligodendrocytes and astrocytes [23]. Mutations in the GlialCAM protein have been associated with megalencephalic leukodystrophy, a genetic neurodegenerative disorder that affecting the white matter of the CNS, which consists of glial cells and myelinated axons [24]. The shared linear amino acid sequence or structural similarities between MAP/EBV and GlialCAM epitopes may contribute to autoimmune processes.
When exposed to bacterial or viral antigens, the immune system undergoes adaptation, including changes in antibody affinity and isotype of peripheral B cells [25]. In patients with MS, the somatic hypermutation of B cells has been observed [26], potentially leading to the production of self-reactive antibodies that strongly interact with host proteins, such as GlialCAM. This interaction may trigger an immune response against GlialCAM-expressingcells in the CNS, leading to inflammation and demyelination.
In support of this hypothesis, we have detected IgG4 antibodies in serum of RR-MS patients (~50%) directed against MAP_HSP70 533-545 , particularly in patients with higher EDSS scores. Normally, IgG4 antibodies constitute around 5% of total IgG immunoglobulins [27]. The presence of IgG4 in RR-MS patients may be attributed to chronic exposure to MAP, primarily through the fecal-oral route. In Italy, paratuberculosis is endemic in domestic livestock in Sardinia [28], and several studies have demonstrated the presence of specific antibodies and MAP DNA in patients with autoimmune disorders, including MS, Chron's diseases and type 1 diabetes [29]. As for the epidemiological situation of paratuberculosis in Japan, the disease's prevalence is relatively low compared to Western countries [30]. However, seroprevalence studies have indicated the presence of IgG1 and IgG4 antibodies against MAP antigens in the serum of healthy individuals [20], as well as IgE in individuals with allergies [31], suggesting that the Japanese population may also be exposed to the mycobacterium, likely through the consumption of contaminated dairy products [20].
Furthermore, certain HLA DRB1*0405 alleles have been associated with an increased risk of developing MS in specific countries, such as Sardinia and Japan [32,33], as well as an increased susceptibility to IgG4-related disease in the Japanese populations [34].
Although the prevalence of MS is higher in Western countries compared to East Asia [35], one hypothesis is that individuals carrying this specific haplotype and exposed to MAP antigens, including MAP_HSP70 533-545 , may be more susceptible to developing autoimmune disorders.
While there is limited literature on the effect of therapy on IgG4 in MS, a clinical study involving 29 Greek RR-MS patients demonstrated an increase in IgG4 levels after 24 months of treatment with alemtuzumab, a monoclonal antibody targeting CD52 [36]. Moreover, patients with higher IgG4 titers were more likely to develop secondary autoimmune disorders such as Crohn's disease and thyroid-related disorders [36].
We have also demonstrated the direct impact of MAP_HSP70 533-545 on the development of EAE. In C57Bl/6J mice, the exacerbation of EAE was likely attributed to autoreactive MAP_HSP70 533-545 T-cells generated in response to immunization. Previous studies on EAE have revealed that various self-antigen specific T-cells contribute to autoimmune inflammation [37]. The delayed onset but more severe disease observed in mice may be associated with the chaperoning effect of HSP70 on regulatory T (Treg) cells' function [38], potentially modulating the trafficking of Tregs between the periphery (where their accumulation may delay the onset of EAE) and the CNS (where a defective number is linked to disease severity).
Interestingly, recent research has suggested that Bacillus Calmette-Guérin vaccination may reduce the incidence of MS through cytokine-induced IL-10 secreting CD8 T-cells, indicating that certain mycobacteria may have a role in disease prevention [21].
Although we have provided evidence supporting the association between MAP and the pathogenesis of MS, further investigation is needed to determine whether the antigenic components of MAP are recognized by T-cells and/or disease-causing autoreactive antibodies or if these antigens solely contribute to epitope spreading. To assess the encephalitogenic effect of MAP_HSP70 533-545 T-cell during the effector phase of EAE, future experiments will involve inducing induce passive EAE through the adoptive transfer of these cells.
Moreover, additional research will be conducted to comprehend the mechanism of infection-mediated neuroinflammation, with a specific focus on the role of mitochondria dysfunction. Mitochondria dysfunction has been associated with the development of both MS and EAE [39,40], as well as mycobacterial infection [41]. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data underlying this article will be shared on reasonable request to the corresponding author.