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Elevated mycobacterium avium subsp. paratuberculosis (MAP) antibody titer in Japanese multiple sclerosis

  • Fumie Hayashi
    Affiliations
    Department of Neurology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
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  • Noriko Isobe
    Affiliations
    Department of Neurology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
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  • Davide Cossu
    Affiliations
    Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan

    Department of Biomedical Sciences, Division of Microbiology and Virology, University of Sassari, Italy
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  • Kazumasa Yokoyama
    Affiliations
    Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan
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  • Ayako Sakoda
    Affiliations
    Department of Neurology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

    Department of Neurology, Brain and Nerve Center, Fukuoka Central Hospital, International University of Health and Welfare, Fukuoka, Japan
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  • Takuya Matsushita
    Affiliations
    Department of Neurology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
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  • Nobutaka Hattori
    Affiliations
    Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan
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  • Jun-ichi Kira
    Correspondence
    Corresponding author at: Department of Neurology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
    Affiliations
    Department of Neurology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

    Department of Neurology, Brain and Nerve Center, Fukuoka Central Hospital, International University of Health and Welfare, Fukuoka, Japan

    Translational Neuroscience Center, Graduate School of Medicine, School of Pharmacy at Fukuoka, International University of Health and Welfare, Fukuoka, Japan
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Open AccessPublished:August 25, 2021DOI:https://doi.org/10.1016/j.jneuroim.2021.577701

      Highlights

      • Immunity against mycobacterium avium subsp. paratuberculosis (MAP) was assessed.
      • IgG against MAP2694 (MAP2694-IgG) was elevated in patients with multiple sclerosis.
      • Higher MAP2694-IgG titers correlated with worse disability.
      • MAP2694-IgG was not associated with HLA-DRB1 alleles or dairy product consumption.

      Abstract

      To investigate whether antibody production against mycobacterium avium subsp. paratuberculosis (MAP) is related to clinical characteristics of multiple sclerosis (MS) and human leukocyte antigen (HLA) alleles, IgG antibody against three MAP peptides and two human peptides homologous to MAP were measured in sera from 103 MS patients and 50 healthy controls (HCs). MS patients had higher IgG levels against MAP2694295–303 (MAP2694-IgG) than HCs, while the other antibodies were comparable. Multivariate analysis demonstrated that higher MAP2694-IgG titers were associated with higher EDSS scores, but not with HLA alleles or dairy product consumption. Immune response against MAP may worsen MS disability.

      Graphical abstract

      Keywords

      Abbreviations:

      BLAST (Basic Local Alignment Search Tool), CFA (complete Freund's adjuvant), EAE (experimental autoimmune encephalomyelitis), EBV (Epstein-Barr virus), EDSS (Expanded Disability Status Scale), ELISA (enzyme-linked immunosorbent assay), HC (healthy controls), HLA (human leukocyte antigen), IgG (immunoglobulin G), IRF5 (interferon regulatory factor 5), MAP (mycobacterium avium subsp. paratuberculosis), MBP (myelin basic protein), MS (multiple sclerosis), OCBs (oligoclonal IgG bands.)

      1. Introduction

      Multiple sclerosis (MS) is a neuroinflammatory disorder that affects over 2.8 million people worldwide (
      • The Multiple Sclerosis International Federation
      Atlas of MS 3rd Edition: Mapping Multiple Sclerosis Around the World.
      ). Although the prevalence of MS in Japan is not as high as in Europe and North America, it has been increasing dramatically (
      • Osoegawa M.
      • Kira J.
      • Fukazawa T.
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      • Kikuchi S.
      • Matsui M.
      • Kohriyama T.
      • Sobue G.
      • Yamamura T.
      • Itoyama Y.
      • Saida T.
      • Sakata K.
      • Ochi H.
      • Matsuoka T.
      • Kuroiwa Y.
      • Igata A.
      • Nishitani H.
      • Chiba S.
      • Fujii Y.
      • Furukawa S.
      • Hara H.
      • Hara T.
      • Hisanaga K.
      • Ikeda S.I.
      • Izumo S.
      • Kaji R.
      • Kanda T.
      • Koh S.
      • Kusunoki S.
      • Kuwabara S.
      • Matsuo H.
      • Mizusawa H.
      • Nakamura T.
      • Nomura K.
      • Ogino M.
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      • Osame M.
      • Ota K.
      • Shimizu J.
      • Suzumura A.
      • Tabira T.
      • Tanaka K.
      • Tanaka M.
      • Yoneda M.
      • Yoshikawa H.
      • Yuki N.
      Temporal changes and geographical differences in multiple sclerosis phenotypes in Japanese: Nationwide survey results over 30 years.
      ). MS is considered a complex disease, and both genetic and environmental factors play important roles in its pathogenesis (
      • Olsson T.
      • Barcellos L.F.
      • Alfredsson L.
      Interactions between genetic, lifestyle and environmental risk factors for multiple sclerosis.
      ). Epstein-Barr virus (EBV), adolescent obesity, lack of sun exposure or low levels of vitamin D, and exposure to tobacco smoke and organic solvents have been reported as environmental risk factors for MS (
      • Olsson T.
      • Barcellos L.F.
      • Alfredsson L.
      Interactions between genetic, lifestyle and environmental risk factors for multiple sclerosis.
      ;
      • Belbasis L.
      • Bellou V.
      • Evangelou E.
      • Ioannidis J.P.A.
      • Tzoulaki I.
      Environmental risk factors and multiple sclerosis: an umbrella review of systematic reviews and meta-analyses.
      ).
      Mycobacterium avium subsp. paratuberculosis (MAP) is an intracellular bacterium causing Johne's disease, a chronic enteritis of ruminants. The clinical similarity between Johne's disease and Crohn's disease in human has long been recognized. Multiple studies, including a meta-analysis, have shown that MAP DNA and antibodies against MAP in blood were detected more frequently in patients with Crohn's disease than in controls (
      • Feller M.
      • Huwiler K.
      • Stephan R.
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      • Pfyffer G.E.
      • Jemmi T.
      • Baumgartner A.
      • Egger M.
      Mycobacterium avium subspecies paratuberculosis and Crohn’s disease: a systematic review and meta-analysis.
      ). Among autoimmune diseases, Crohn's disease shares the most genetic risk factors with MS (
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      • Quach H.
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      • Rog D.
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      • Zindler E.
      • Haines J.L.
      • Pericak-Vance M.A.
      • Ivinson A.J.
      • Stewart G.
      • Hafler D.
      • Hauser S.L.
      • Compston A.
      • McVean G.
      • De Jager P.
      • Sawcer S.J.
      • McCauley J.L.
      Analysis of immune-related loci identifies 48 new susceptibility variants for multiple sclerosis.
      ). Recently, an Italian group reported an association between MAP infection and MS in a Sardinian population: higher positivity of MAP DNA in peripheral blood of MS patients compared with that of controls and higher titers and prevalence of the antibody against MAP2694259–303, which is a predicted homolog of T-cell receptor gamma-chain/complement component 1 (
      • Cossu D.
      • Cocco E.
      • Paccagnini D.
      • Masala S.
      • Ahmed N.
      • Frau J.
      • Marrosu M.G.
      • Sechi L.A.
      Association of mycobacterium avium subsp. paratuberculosis with multiple sclerosis in sardinian patients.
      ). Although a higher frequency of antibodies against only MAP2694259–303 was also reported in Japanese MS patients compared with controls, immuno-reactivity to other MAP peptides were distinct between Italian and Japanese MS cohorts (
      • Cossu D.
      • Yokoyama K.
      • Sechi L.A.
      • Otsubo S.
      • Tomizawa Y.
      • Momotani E.
      • Hattori N.
      Humoral response against host-mimetic homologous epitopes of Mycobacterium avium subsp. Paratuberculosis in Japanese multiple sclerosis patients.
      ). Thus, it remains to be elucidated whether MAP is a common environmental pathogen related to MS across populations, or whether immuno-reactivity to MAP2694259–303 is because of cross-reactivity to other homologous antigens in Japanese patients with MS. Interestingly, Sardinians and Japanese share the major risk allele in the class II region of human leukocyte antigen (HLA), HLA-DRB1*04:05, for MS susceptibility (
      • Isobe N.
      • Oksenberg J.R.
      ;
      • Cocco E.
      • Sardu C.
      • Pieroni E.
      • Valentini M.
      • Murru R.
      • Costa G.
      • Tranquilli S.
      • Frau J.
      • Coghe G.
      • Carboni N.
      • Floris M.
      • Contu P.
      • Marrosu M.G.
      HLA-DRB1-DQB1 haplotypes confer susceptibility and resistance to multiple sclerosis in Sardinia.
      ). Therefore, it is important to know if there is any association between HLA alleles and anti-MAP antibody titers.
      In this study, we analyzed another Japanese MS cohort for any distinctive immunological reaction against MAP and assessed whether the antibody level against MAP has any relationship with clinical parameters and HLA alleles in our local Japanese cohort.

      2. Materials and methods

      2.1 Subjects and clinical assessment

      A total of 153 Japanese participants, 103 MS patients and 50 healthy controls (HCs), were enrolled. Peripheral blood was collected from all participants and serum and mononuclear cells isolated. None of the MS patients received any immune-related drugs for at least 3 months prior to sample collection. For each patient, we collected clinical and laboratory data, including sex, age at examination, age at onset, disease course, disease duration, Expanded Disability Status Scale (EDSS) scores of Kurtzke (
      • Kurtzke J.F.
      Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS).
      ), oligoclonal IgG bands (OCBs) positivity in cerebrospinal fluid, IgG index, fulfillment of the Barkhof criteria, and HLA-DRB1 allele genotypes. The study was approved by the Ethical Committee of Kyushu University. For the confirmatory studies, we added 53 HCs to the original cohort to make the patient and HC groups comparable in terms of sample size.

      2.2 Mycobacterial peptides and homologous human peptides

      Three MAP peptides (MAP2694295–303, MAP0106121–132 and MAP402718–32) and two human peptides homologous with MAP [myelin basic protein (MBP)85–98 and interferon regulatory factor-5 (IRF5)424–434], to which Italian MS patients were reported to show higher immune-reactivity than HCs (
      • Cossu D.
      • Yokoyama K.
      • Sechi L.A.
      • Otsubo S.
      • Tomizawa Y.
      • Momotani E.
      • Hattori N.
      Humoral response against host-mimetic homologous epitopes of Mycobacterium avium subsp. Paratuberculosis in Japanese multiple sclerosis patients.
      ), were used (Supplementary Table S1). MAP2694295–303 is an immunodominant epitope within the MAP2694 protein, which is a specific transmembrane protein of the mycobacterium. MAP2694295–303 has sequence homology with human T cell receptor gamma chain (
      • Cossu D.
      • Cocco E.
      • Paccagnini D.
      • Masala S.
      • Ahmed N.
      • Frau J.
      • Marrosu M.G.
      • Sechi L.A.
      Association of mycobacterium avium subsp. paratuberculosis with multiple sclerosis in sardinian patients.
      ;
      • Cossu D.
      • Masala S.
      • Frau J.
      • Mameli G.
      • Marrosu M.G.
      • Cocco E.
      • Sechi L.A.
      Antigenic epitopes of MAP2694 homologous to T-cell receptor gamma-chain are highly recognized in multiple sclerosis Sardinian patients.
      ). MAP0106121–132 has conformational homology with human MBP85–98 (
      • Mameli G.
      • Cossu D.
      • Cocco E.
      • Masala S.
      • Frau J.
      • Marrosu M.G.
      • Sechi L.A.
      Epstein-Barr virus and Mycobacterium avium subsp. paratuberculosis peptides are cross recognized by anti-myelin basic protein antibodies in multiple sclerosis patients.
      ), while MAP402718–32 is homologous to IRF5424–434 (
      • Cossu D.
      • Mameli G.
      • Galleri G.
      • Cocco E.
      • Masala S.
      • Frau J.
      • Marrosu M.G.
      • Manetti R.
      • Sechi L.A.
      Human interferon regulatory factor 5 homologous epitopes of Epstein-Barr virus and Mycobacterium avium subsp. Paratuberculosis induce a specific humoral and cellular immune response in multiple sclerosis patients.
      ).

      2.3 Enzyme-linked immunosorbent assay (ELISA)

      The levels of IgG for all five peptides were analyzed by indirect ELISA at Juntendo University. Nunc-immuno-MicroWell-96 well solid plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated overnight at 4 °C with optimal concentrations of each antigen in ELISA coating buffer (Bio-Rad, Hercules, CA, USA). The following day, plates were blocked with 250 μl Blocking One (Nacalai Tesque, Kyoto, Japan) and were incubated for 1 h at room temperature. After washing with (10 mM) phosphate-buffered saline, pH 7.0, containing 0.5% Tween 80 (PBS-T) twice, serum samples were added at 1:100 dilution in Blocking One and incubated for 2 h at room temperature. After washing three times, horseradish peroxidase-labeled goat anti-human IgG polyclonal antibody diluted (1,2000) in PBS (Southern Biotech Associates, Birmingham, AL, USA) was added to each well and incubated for 2 h at room temperature. After washing three times, 100 μl ABTS Peroxidase System (SeraCare Life Sciences, KPL, Gaithersburg, MD, USA) was added to each well and incubated for 5 min in the dark at room temperature. Plates were read at 650 nm on a Benchmark Plus Microplate Reader (Bio-Rad). The results were normalized to a positive control serum included in all experiments, the reactivity of which was set at 10,000 arbitrary units (AU)/ml. Negative controls were conducted by incubation of immobilized peptides with secondary antibody alone, and their mean values were subtracted from all samples.

      2.4 HLA typing

      High-resolution HLA allele typing for the HLA-DRB1 locus was conducted for all study participants except one MS patient because of the lack of samples.

      2.5 Intake of dairy products

      We estimated the daily intake of dairy products including milk, low-fat milk, cheese and yogurt using the Food Frequency Questionnaire, which can reveal dietary habits over the preceding year (
      • Nanri A.
      • Shimazu T.
      • Ishihara J.
      • Takachi R.
      • Mizoue T.
      • Inoue M.
      • Tsugane S.
      • Sasaki S.
      • Kobayashi M.
      • Sobue T.
      • Yamamoto S.
      • Akabane M.
      • Iitoi Y.
      • Iwase Y.
      • Takahashi T.
      • Hasegawa K.
      • Kawabata T.
      • Tsubono Y.
      • Iso H.
      • Karita S.
      • Yamaguchi M.
      • Matsumura Y.
      Reproducibility and validity of dietary patterns assessed by a food frequency questionnaire used in the 5-year follow-up survey of the Japan public health center-based prospective study.
      ).

      2.6 Search for human proteins homologous to MAP2694295–303

      Basic Local Alignment Search Tool (BLAST) is the most updated tool for homology searching (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (
      • Madden T.
      The BLAST sequence analysis tool.
      ). We used BLAST to search for human proteins homologous to MAP2694295–303.

      2.7 Statistical analysis

      Categorical variables were described with counts and percentages, while continuous variables were described as the mean with standard deviation. Sex composition was compared between MS patients and HCs groups using the chi-square test. The Mann–Whitney U test was used to compare IgG titers between binomial parameters (e.g. sex, affectation status, fulfillment of Barkhof criteria, OCB positivity, and MS-associated HLA alleles). For association analysis between MAP-related IgG and all HLA-DRB1 alleles, Bonferroni correction was applied to obtain corrected p values (pcorr) from uncorrected p values (puncorr) after multiplication by the number of HLA-DRB1 alleles. Correlations between clinical parameters and MAP2694-IgG titers were calculated using Spearman's rank correlation test. Associations of clinical and laboratory parameters as well as allele counts (doses) of the HLA-DRB1 alleles against MAP-IgG titers were assessed using a linear regression model, with disease duration and age at examination as candidate covariates. All the analyses were performed using JMP® Pro version 14.1 (SAS Institute Inc., Cary, NC, USA).

      3. Results

      3.1 Demographic features of study participants and humoral response against MAP-derived peptides and homologous human peptides

      The demographic features of the study participants are summarized in Table 1. The sex ratio was similar between MS patients and HCs but age at examination was higher in MS patients than in HCs (p = 0.0001). Of the two major MS-associated HLA-DRB1 alleles in the Japanese population, the carrier frequency of HLA-DRB1*04:05 was significantly higher in MS patients than in HCs, as expected (50.0% vs. 26.0%, p = 0.0053). The level of MAP2694295303 IgG (MAP2694-IgG) was significantly higher in MS patients than in HCs (mean ± standard deviation: 2158.7 ± 993.1 AU/ml vs. 1697.6 ± 725.4 AU/ml, p = 0.0032, Fig. 1A). However, there were no significant differences in the levels of antibodies against the other four peptides between MS patients and HCs (Fig. 1B–E). We also measured MAP2694-IgG titers in 53 additional HC samples to increase the sample size up to 103. This extended cohort confirmed the significantly higher MAP2694-IgG levels in patients with MS compared with HCs (p < 0.0001, Supplementary Fig. S1).

      3.2 Association between MAP2694-IgG titers and clinical features of MS

      Given that only MAP2694-IgG was significantly higher in MS patients compared with HCs, we next investigated any association between the MAP2694-IgG titers and clinical parameters in MS patients (Table 2). MAP2694-IgG titers did not differ by sex, OCB positivity, fulfillment of Barkhof criteria, and carrier status of HLA-DRB1*04:05 and HLA-DRB1*15:01. We further analyzed the association between MAP2694-IgG levels and all HLA-DRB1 alleles, but there were no significant differences (Supplementary Table S2). A linear regression model demonstrated that higher EDSS scores were significantly associated with higher MAP2694-IgG titers [regression coefficient (β) = 133.67, p = 0.0038, Table 2]. This EDSS association remained significant even after correction for age at examination and disease duration (p = 0.0048). Other parameters, including age at examination, disease duration, IgG index, and the allelic frequencies of the MS-associated HLA alleles, showed no significant association with MAP2694-IgG titers.
      We also conducted Spearman's rank correlation test to evaluate correlations between clinical parameters and MAP2694-IgG titers. Although no correlation was detected within the MS group between MAP2694-IgG and clinical parameters, such as age at examination (p = 0.55), disease duration (p = 0.099) and IgG index (p = 0.21), EDSS was mildly correlated with MAP2694-IgG titers in a direct way (rho = 0.224, p = 0.0231, Fig. 2). In HCs, there was no correlation between MAP2694-IgG and age at examination (p = 0.35). When we stratified MS patients by the positivity of MS-related HLA alleles to evaluate correlations between EDSS and MAP2694-IgG titers, EDSS was not correlated with MAP2694-IgG titers in the groups carrying HLA risk alleles but was weakly correlated in the groups not carrying HLA risk alleles [rho = 0.295, p = 0.0357 in HLA-DRB1*04:05 (−) MS group and rho = 0.282, p = 0.0148 in HLA-DRB1*15:01 (−) MS group, Fig. 3].
      Fig. 1
      Fig. 1Immunoglobulin G (IgG) titers against five peptides in multiple sclerosis (MS) patients and healthy controls (HCs). Serum samples from HCs (n = 50, open circle) and MS patients (n = 103, black circle) were tested for their reactivity against plate-coated (A) MAP2694295–303, (B) MAP0106c121–132, (C) MBP85–98, (D) MAP402718–32, and (E) IRF5424–434 peptides. IRF5-IgG = IgG against IRF5424–434, MAP0106c-IgG = IgG against MAP0106c121–132, MAP2694-IgG = IgG against MAP2694295–303, MAP4027-IgG = IgG against MAP402718–32, MBP-IgG = IgG against MBP85–98, NS = not significant.
      Table 1Demographics and clinical characteristics of the study cohort.
      MS patients (n = 103)HCs (n = 50)p value
      Female (%)77 (74.8)31 (62.0)0.13
      Age at examination (y)
      mean ± standard deviation.
      43.4 ± 14.933.4 ± 10.70.0001
      Age at onset (y)
      mean ± standard deviation.
      31.4 ± 11.3
      Disease typeRR: 81/SP: 9/PP: 13
      Disease duration (y)
      mean ± standard deviation.
      12.0 ± 11.6
      EDSS score
      mean ± standard deviation.
      2.7 ± 2.1
      OCBs (%)48/93 (51.6)
      IgG index
      mean ± standard deviation.
      0.77 ± 0.42
      Barkhof criteria (%)76/103 (73.8)
      Carrier frequencies of HLA-DRB1*04:05 (%)51/102 (50.0)13/50 (26.0)0.0053
      15:01 (%)28/102 (27.5)8/50 (16.0)0.16
      EDSS = Expanded Disability Status Scale, HCs = healthy controls, HLA = human leukocyte antigen, IgG = immunoglobulin G, MS = multiple sclerosis, n = number, OCBs = oligoclonal IgG bands, PP = primary progressive, RR = relapsing remitting, SP = secondary progressive, y = year.
      a mean ± standard deviation.
      Table 2MAP2694-IgG titers and their associations with clinical variables.
      MAP2694-IgG
      mean ± standard deviation.
      (AU/ml)
      p valueRegression coefficient (β)p value
      Sex
       F2083.4 ± 857.00.49
       M2381.7 ± 1312.2
      Age0.810.90
      Disease duration14.650.084
      EDSS133.670.0038
      OCBs
       positive2136.6 ± 1022.80.69
       negative2220.7 ± 998.8
      IgG index−95.740.75
      Barkhof criteria
       positive2222.1 ± 1014.30.38
       negative1980.4 ± 925.4
      HLA-DRB1*04:05
       carriers2184.1 ± 954.40.59
       non-carriers2132.4 ± 1048.6
       allele counts−14.030.94
      HLA-DRB1*15:01
       carriers2308.2 ± 1124.00.48
       non-carriers2101.49 ± 948.0
       allele counts130.790.53
      EDSS = Expanded Disability Status Scale, F = female, HLA = human leukocyte antigen, IgG = immunoglobulin G, M = male, MAP = mycobacterium avium subsp. paratuberculosis, MAP2694-IgG = IgG against MAP2694295–303, MS = multiple sclerosis, OCBs = oligoclonal IgG bands.
      a mean ± standard deviation.
      Fig. 2
      Fig. 2Correlation between Expanded Disability Status Scale (EDSS) scores and MAP2694-IgG titers in MS patients. MAP2694-IgG = IgG against MAP2694295–303.
      Fig. 3
      Fig. 3Correlation between Expanded Disability Status Scale (EDSS) scores and MAP2694-IgG titers in MS patients stratified by human leukocyte antigen (HLA) risk allele status. MAP2694-IgG = IgG against MAP2694295–303, NS = not significant.

      3.3 Association between MAP2694-IgG titers and intake of dairy products

      Because MAP is transmitted via consumed dairy products (
      • Cossu D.
      • Yokoyama K.
      • Sechi L.A.
      • Otsubo S.
      • Tomizawa Y.
      • Momotani E.
      • Hattori N.
      Humoral response against host-mimetic homologous epitopes of Mycobacterium avium subsp. Paratuberculosis in Japanese multiple sclerosis patients.
      ;
      • Otsubo S.
      • Cossu D.
      • Eda S.
      • Otsubo Y.
      • Sechi L.A.
      • Suzuki T.
      • Iwao Y.
      • Yamamoto S.
      • Kuribayashi T.
      • Momotani E.
      Seroprevalence of IgG1 and IgG4 class antibodies against Mycobacterium avium subsp. paratuberculosis in Japanese population.
      ), we checked whether MAP2694-IgG titers were associated with daily intake of dairy products. We detected no associations between MAP2694-IgG and the amounts of dairy products consumed, including milk (p = 0.78), low-fat milk (p = 0.54), cheese (p = 0.61), and yogurt (p = 0.64) (Supplementary Table S3).

      3.4 Search for human proteins homologous to MAP2694295–303

      To update possible human proteins homologous to MAP2694295–303, we performed BLAST searches. We identified that MAP2694295–303 has homology with phospholipase C, seizure protein 6, and transducin-like enhancer protein (Supplementary Table S4).

      4. Discussion

      This study demonstrated that levels of MAP2694-IgG were significantly higher in our local Japanese MS patients than in controls, although antibody levels against other MAP proteins did not differ by affectation status. This result is in concordance with the limited immuno-reactivity to MAP peptides demonstrated in a previous Japanese case-control study, showing higher prevalence of only MAP2694-IgG in Japanese MS (
      • Cossu D.
      • Yokoyama K.
      • Sechi L.A.
      • Otsubo S.
      • Tomizawa Y.
      • Momotani E.
      • Hattori N.
      Humoral response against host-mimetic homologous epitopes of Mycobacterium avium subsp. Paratuberculosis in Japanese multiple sclerosis patients.
      ). Sardinian MS patients had higher antibody levels not only against MAP2694259–303 but also against other MAP peptides compared with controls (
      • Cossu D.
      • Yokoyama K.
      • Sechi L.A.
      • Otsubo S.
      • Tomizawa Y.
      • Momotani E.
      • Hattori N.
      Humoral response against host-mimetic homologous epitopes of Mycobacterium avium subsp. Paratuberculosis in Japanese multiple sclerosis patients.
      ), which indicates real MAP infection in MS patients residing in Sardinia, where the bacteria is endemic in ruminants (
      • Cossu D.
      • Yokoyama K.
      • Hattori N.
      Bacteria–host interactions in multiple sclerosis.
      ). Actually, the prevalence of MAP in ruminants is 10%–60% in Australia and Europe (
      • Momotani E.
      Epidemiological situation and control strategies for paratuberculosis in Japan.
      ;
      • Nielsen S.S.
      • Toft N.
      A review of prevalences of paratuberculosis in farmed animals in Europe.
      ) and about 70% in the United States (
      • United States Department of Agriculture-Animal and Plant Health Inspection Service
      Johne’s Disease on U.S. Dairies, 1991–2007.
      ), whereas its prevalence in Japan is 0.2% (
      • Momotani E.
      Epidemiological situation and control strategies for paratuberculosis in Japan.
      ). MAP is one of the bacteria that causes Johne's disease in ruminants and its infection is, in part, mediated by the consumption of dairy products (
      • Otsubo S.
      • Cossu D.
      • Eda S.
      • Otsubo Y.
      • Sechi L.A.
      • Suzuki T.
      • Iwao Y.
      • Yamamoto S.
      • Kuribayashi T.
      • Momotani E.
      Seroprevalence of IgG1 and IgG4 class antibodies against Mycobacterium avium subsp. paratuberculosis in Japanese population.
      ); therefore, we studied the relationship between dairy product consumption and MAP2694-IgG, but found no association. This is consistent with a previous report that detected no association between dairy product intake and MS in Japanese patients (
      • Sakoda A.
      • Matsushita T.
      • Nakamura Y.
      • Watanabe M.
      • Shinoda K.
      • Masaki K.
      • Isobe N.
      • Yamasaki R.
      • Kira J.
      Environmental risk factors for multiple sclerosis in Japanese people.
      ). These findings collectively indicate real MAP infection in Japanese MS patients to be unlikely, although it is still possible that the genetic differences between the two populations may account for the distinct antibody production patterns to MAP on real infection.
      Alternatively, the reactivity to MAP2694259–303 may be derived from cross-reactivity to homologous proteins. Although human MBP85–98 and IRF5424–434 have some homology to MAP peptides (
      • Mameli G.
      • Cossu D.
      • Cocco E.
      • Masala S.
      • Frau J.
      • Marrosu M.G.
      • Sechi L.A.
      Epstein-Barr virus and Mycobacterium avium subsp. paratuberculosis peptides are cross recognized by anti-myelin basic protein antibodies in multiple sclerosis patients.
      ;
      • Cossu D.
      • Mameli G.
      • Galleri G.
      • Cocco E.
      • Masala S.
      • Frau J.
      • Marrosu M.G.
      • Manetti R.
      • Sechi L.A.
      Human interferon regulatory factor 5 homologous epitopes of Epstein-Barr virus and Mycobacterium avium subsp. Paratuberculosis induce a specific humoral and cellular immune response in multiple sclerosis patients.
      ), our Japanese MS patients did not show increased reactivity to these peptides as seen in Italian MS patients (
      • Cossu D.
      • Yokoyama K.
      • Sechi L.A.
      • Otsubo S.
      • Tomizawa Y.
      • Momotani E.
      • Hattori N.
      Humoral response against host-mimetic homologous epitopes of Mycobacterium avium subsp. Paratuberculosis in Japanese multiple sclerosis patients.
      ). In this regard, we found that MAP2694295–303 has high homology with phospholipase C, seizure protein 6, and transducin-like enhancer protein, in addition to that with T-cell receptor gamma-chain (
      • Cossu D.
      • Cocco E.
      • Paccagnini D.
      • Masala S.
      • Ahmed N.
      • Frau J.
      • Marrosu M.G.
      • Sechi L.A.
      Association of mycobacterium avium subsp. paratuberculosis with multiple sclerosis in sardinian patients.
      ). Phospholipase C plays a pivotal role in the arachidonic acid cascade, which is activated in MS lesions (
      • Rose J.W.
      • Hill K.E.
      • Watt H.E.
      • Carlson N.G.
      Inflammatory cell expression of cyclooxygenase-2 in the multiple sclerosis lesion.
      ). Seizure protein 6 is widely expressed throughout the brain and linked to neurodevelopmental and psychiatric disorders (
      • Pigoni M.
      • Hsia H.
      • Hartmann J.
      • Rudan Njavro J.
      • Shmueli M.D.
      • Müller S.A.
      • Güner G.
      • Tüshaus J.
      • Kuhn P.
      • Kumar R.
      • Gao P.
      • Tran M.L.
      • Ramazanov B.
      • Blank B.
      • Hipgrave Ederveen A.L.
      • Von Blume J.
      • Mulle C.
      • Gunnersen J.M.
      • Wuhrer M.
      • Rammes G.
      • Busche M.A.
      • Koeglsperger T.
      • Lichtenthaler S.F.
      Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3.
      ). Transducin-like enhancer protein performs numerous functions throughout life, interacting with several pathways and controlling gene expression. There have been no reports concerning the latter two with MS. Further studies on immunoreactivity to these homologous peptides are required to elucidate the significance of increased reactivity to MAP2694259–303 in Japanese MS.
      Interestingly, this study revealed the association between MAP2694-IgG titers and EDSS scores, which is in accord with a previous observation that EDSS scores were higher in MAP-IgG-positive patients than in antibody-negative patients, although antibody titers were not determined (
      • Yokoyama K.
      • Cossu D.
      • Hoshino Y.
      • Tomizawa Y.
      • Momotani E.
      • Hattori N.
      Anti-mycobacterial antibodies in paired cerebrospinal fluid and serum samples from Japanese patients with multiple sclerosis or neuromyelitis optica spectrum disorder.
      ). Furthermore, when MAP was used as an adjuvant instead of regular complete Freund's adjuvant (CFA) for inducing experimental autoimmune encephalomyelitis (EAE), the onset of EAE was faster and clinical scores were significantly more severe than in CFA-immunized mice (
      • Cossu D.
      • Yokoyama K.
      • Sakanishi T.
      • Momotani E.
      • Hattori N.
      Adjuvant and antigenic properties of Mycobacterium avium subsp. paratuberculosis on experimental autoimmune encephalomyelitis.
      ). Another animal study showed that oral administration of MAP activates mucosal immunity and exacerbates acute EAE in C57BL/6 J mice by modulating immune cell traffic from secondary lymphoid organs to the CNS (
      • Cossu D.
      • Yokoyama K.
      • Sakanishi T.
      • Kuwahara-Arai K.
      • Momotani E.
      • Hattori N.
      A mucosal immune response induced by oral administration of heat-killed Mycobacterium avium subsp. paratuberculosis exacerbates EAE.
      ). Accordingly, it is possible that MAP is involved in the worsening of MS pathology.
      Previous reports have shown that immune responses of MS patients to certain viruses, such as EBV, cytomegalovirus and JC virus, alter depending on the presence or absence of HLA-DRB1 risk alleles (
      • Olsson T.
      • Barcellos L.F.
      • Alfredsson L.
      Interactions between genetic, lifestyle and environmental risk factors for multiple sclerosis.
      ;
      • Hayashi F.
      • Isobe N.
      • Glanville J.
      • Matsushita T.
      • Maimaitijiang G.
      • Fukumoto S.
      • Watanabe M.
      • Masaki K.
      • Kira J.
      A new clustering method identifies multiple sclerosis-specific T-cell receptors.
      ;
      • Watanabe M.
      • Nakamura Y.
      • Isobe N.
      • Tanaka M.
      • Sakoda A.
      • Hayashi F.
      • Kawano Y.
      • Yamasaki R.
      • Matsushita T.
      • Kira J.
      Two susceptible HLA-DRB1 alleles for multiple sclerosis differentially regulate anti-JC virus antibody serostatus along with fingolimod.
      ). This indicates that HLA alleles may play roles in virus-associated immune responses that are related to MS pathogenesis. The positive association between MAP antibody titers and EDSS scores in MS patients without but not with the HLA-DRB1*04:05 allele indicates a possibility that MAP may be involved in the worsening disability of HLA-DRB1*04:05-negative MS patients. It is important to characterize exactly which HLA-DRB1 allele is associated with MAP-related worsening disability in a large-scale study. Moreover, as only HLA-DRB1 alleles were analyzed in this study, it is necessary to examine other HLA loci (e.g., HLA-A and HLA-DQB1) in the future.
      One potential limitation of our present study is that it did not include disease controls. Thus, it would be interesting to add other inflammatory diseases, such as Crohn's disease, as disease controls in future studies.
      In conclusion, MAP2694-IgG levels were significantly higher in our local Japanese MS patients than in controls, and higher MAP2694-IgG titers were significantly associated with higher EDSS scores in HLA-DRB1*04:05-negative MS patients. As no association between MAP2694-IgG and the intake of dairy products was found, additional environmental factors and the interaction between environmental and genetic factors should be explored.

      Author contributions

      F.H. and J.K. conceived the experiments. All authors contributed to the experimental design. F.H., D.C., and A.S. performed the experiments. F.H. and N.I. analyzed the results. J.K., T.M. and K.Y. provided technical advice for the analyses. F.H., N.I., D.C., K.Y. and J.K. were involved in the interpretation of the results. F.H., N.I., and J.K. drafted the manuscript. All authors reviewed the manuscript.

      Declaration of Competing Interest

      None.

      Acknowledgements

      This work was supported by a Grant-in-Aid for Scientific Research (C) (JSPS KAKENHI Grant Number 16H02657 ) from the Japan Society for the Promotion of Science . We thank Edanz Group (https://en-author-services.edanz.com/ac) for editing a draft of this manuscript.

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