DOI: 10.7272/q6n58jh2
title: Gut Microbiota from Multiple Sclerosis patients triggers spontaneous autoimmune encephalomyelitis in mice --shotgun data--

description: There is emerging evidence that the commensal microbiota has a role in the
 pathogenesis of multiple sclerosis (MS), a putative autoimmune disease of
 the central nervous system. Here, we compared the gut microbial
 composition of 34 monozygotic twin pairs discordant for MS. While there
 were no major differences in the overall microbial profiles, we found a
 significant increase in some taxa such as Akkermansia in untreated MS
 twins. Furthermore, most notably, when transplanted to a transgenic mouse
 model of spontaneous brain autoimmunity, MS twin-derived microbiota
 induced a significantly higher incidence of autoimmunity than the healthy
 twin-derived microbiota. The microbial profiles of the colonized mice
 showed a high intra-individual and remarkable temporal stability with
 several differences, including Sutterella, an organism shown to induce a
 protective immunoregulatory profile in vitro. Immune cells from mouse
 recipients of MS-twin samples produced less IL-10 compared to immune cells
 from mice colonized with healthy twin samples. IL-10 may have a regulatory
 role in spontaneous CNS autoimmunity, as neutralization of the cytokine in
 mice colonized with healthy twin fecal samples increased disease
 incidence. These findings provide first evidence that MS-derived
 microbiota contain factors that precipitate an MS-like autoimmune disease
 in a transgenic mouse model. They hence encourage the detailed search for
 protective and pathogenic microbial components in human MS.
description: Study design. MZ twins were recruited by
 launching a national televised appeal as well as internet notification in
 Germany with support from the German Multiple Sclerosis Society (DMSG).
 Inclusion criteria for study participation were met for MZ twins with an
 MS diagnosis according to the revised McDonald criteria or clinically
 isolated syndrome (CIS) in one twin only. Exclusion criteria were
 antibiotic, glucocorticosteroidal or immunosuppressive treatment,
 gastrointestinal infection or diet irregularities in the 3 months prior to
 study entry. In total, 34 pairs (see Table 1) visited the outpatient
 department at the Institute of Clinical Neuroimmunology in Munich for a
 detailed interview on past and present medical, family and social history,
 a neurological examination as well as a nutrition questionnaire. To
 confirm the MS diagnosis, medical records including MRI scans were
 obtained and reviewed. Fecal samples were either directly collected in
 hospital or taken at home, stored at -20°C and transferred to the hospital
 in cooling bags. Finally, all samples were stored at -80°C. Of all
 participants buccal swabs for zygosity testing were taken. The study was
 approved by the local Ethics Committee of the Ludwig-Maximilians
 University Munich and all participants gave written informed consent.
 Metagenomic analysis of human and mouse
 samplesWe performed metagenomic sequencing of the gut
 microbiome in 16 pairs of identical twins, each composed of a sibling
 affected by MS and one unaffected sibling. In addition, stool samples from
 25 germ-free mice that were colonized with four twin pair samples also
 underwent metagenomic sequencing. Each human and mouse fecal sample
 produced at least 30 million paired-end DNA reads of length 100 base
 pairs. Sequence quality, evaluated using FastQC, was high in the majority
 of sequences across all samples. We used the HMP Unified Metabolic
 Analysis Network (HUMAnN2) tool to calculate the relative abundance of
 specific microbes, gene families, and metabolic pathways. This software
 pipeline uses MetaPhlAn2 to obtain a list of abundant organisms by
 aligning sequences to genes unique to known bacterial species. DNA
 sequences are subsequently aligned to genomes of the identified organisms
 using the Bowtie 2 aligner and an annotated pangenome database,
 ChocoPhlAn. Unmapped DNA reads undergo translated alignment to the
 bacterial proteome using the software Diamond and a large protein
 database, UniRef50. The product of sequence alignment is a quantitative
 relative abundance of specific protein families. The HUMAnN2 software
 subsequently uses this information to determine the number complete copies
 of specific metabolic pathways using MetaCyc, a database mapping metabolic
 reactions to pathways. After obtaining quantitative measurements of gut
 bacterial abundance, gene families, and metabolic pathways, we performed
 an association testing to identify pathogenic and protective factors in
 multiple sclerosis. We calculated the pairwise sum of absolute differences
 of microbial relative abundance between two individuals to determine if
 gut bacterial flora is more similar between twins with discordant
 phenotype compared to pairs unrelated individuals. We used logistic
 regression (adjusting for twin pair and number of genome equivalents
 sequenced per sample) to examine associations between each gut bacterial
 variable with MS phenotype. We corrected for multiple comparisons, and
 adjusted p-values using a false discovery rate of 5%. We applied the same
 rigorous statistical approach to mouse samples, and also adjusted for the
 twin pair from which mice were colonized. Colonization
 of germ-free RR mice with human MS-twin-derived fecal samples.
 For the human to mouse fecal transfer experiments we
 selected a subgroup of 5 discordant twin pairs, mainly based on pragmatic
 criteria such as relatively young age (20-40 yrs), female sex and either
 no treatment or only treatment with interferon-beta (Table S1). One gram
 of human fecal material was suspended in 15 ml pre-reduced PBS (PBS
 supplemented with 0.1% L-Cysteine hydrochloride monohydrate) and vortexed
 at room temperature for 5 min. Large insoluble particles were allowed to
 settle by gravity for 5 min. Supernatant was transferred to an anaerobic
 crimped tube (Sigma-Aldrich). Pre-reduced glycerol (containing 0.1%
 L-Cysteine hydrochloride monohydrate) was added to a final concentration
 of 20% and tubes were frozen at -80°C. Tubes were sprayed thoroughly with
 Virkon (V.P. Produkte) before being transferred to the gnotobiotic
 isolators. Germ-free RR mice were gavaged with approximately 300 µl of
 fecal bacterial suspension. In addition, mice colonized with healthy twin
 fecal material were injected with 250 µg anti-IL-10 (JES5-2A5; BioXcell)
 or isotype control antibodies weekly once. All animal procedures were in
 accordance with the guidelines of the Committee on Animals of the Max
 Planck Institute of Neurobiology and the Max Planck Institute of
 Immunobiology and Epigenetics with a license from the Regierung von
 Oberbayern as well as the Regierungspräsidium Freiburg.
description: Two zipped files have been uploaded (one of human and one for
 mice). Each zipped file contains 10 individual files.
 Bacterial relative abundance at the genus (1), species
 (2), and all taxonomic classifications (3):1.
 humann2_metaphlan_bugs_list.genus.tsv2.
 humann2_metaphlan_bugs_list.species.tsv3.
 humann2_metaphlan_bugs_list.tsvBacterial gene family
 relative abundance across all bacteria (4) and for each bacteria (5):
 4.
 humann2_genefamilies_community.tsv5.
 humann2_genefamilies.tsvBacterial metabolic pathway
 abundance across all bacteria (6) and for each bacteria (7), and bacterial
 metabolic pathway coverage across all bacteria (8) and for each bacteria
 (9): 6.
 humann2_pathabundance_community.tsv7.
 humann2_pathabundance.tsv8.
 humann2_pathcoverage_community.tsv9.
 humann2_pathcoverage.tsvSample IDs, phenotype, twin
 pair, and genome equivalents calculated using MicrobeCensus (https://github.com/snayfach/MicrobeCensus):10. metagenomic_ids_genome_equivalents.tsvNote: A description of HUMAnN2 output files is here:https://bitbucket.org/biobakery/humann2/wiki/Home#markdow...