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.

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 samples

We 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.

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.tsv

2. humann2_metaphlan_bugs_list.species.tsv

3. humann2_metaphlan_bugs_list.tsv

Bacterial gene family relative abundance across all bacteria (4) and for each bacteria (5):

4. humann2_genefamilies_community.tsv

5. humann2_genefamilies.tsv

Bacterial 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.tsv

7. humann2_pathabundance.tsv

8. humann2_pathcoverage_community.tsv

9. humann2_pathcoverage.tsv

Sample IDs, phenotype, twin pair, and genome equivalents calculated using MicrobeCensus (https://github.com/snayfach/MicrobeCensus):

10. metagenomic_ids_genome_equivalents.tsv

Note: A description of HUMAnN2 output files is here:

https://bitbucket.org/biobakery/humann2/wiki/Home#markdow...