Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Oct 3;114(40):10719-10724.
doi: 10.1073/pnas.1711233114. Epub 2017 Sep 11.

Gut microbiota from multiple sclerosis patients enables spontaneous autoimmune encephalomyelitis in mice

Kerstin Berer  1 , Lisa Ann Gerdes  2 , Egle Cekanaviciute  3 , Xiaoming Jia  3 , Liang Xiao  4 , Zhongkui Xia  4 , Chuan Liu  4 , Luisa Klotz  5 , Uta Stauffer  6 , Sergio E Baranzini  3   7 , Tania Kümpfel  2 , Reinhard Hohlfeld  2   8 , Gurumoorthy Krishnamoorthy  9   10 , Hartmut Wekerle  9   8
Affiliations

Gut microbiota from multiple sclerosis patients enables spontaneous autoimmune encephalomyelitis in mice

Kerstin Berer et al. Proc Natl Acad Sci U S A. .

Abstract

There is emerging evidence that the commensal microbiota has a role in the pathogenesis of multiple sclerosis (MS), a putative autoimmune disease of the CNS. 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 intraindividual 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 than 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 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.

Keywords: experimental autoimmune encephalomyelitis; germ-free mice; gut microbiome; multiple sclerosis; twin study.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. S1.
Fig. S1.
Immune-phenotyping of PBMCs from twin pairs did not reveal profound changes in T cell subset composition. Frozen PBMC specimens from all twin pairs (n = 34) were thawed and immediately subjected to 10-color flow cytometry for quantification of T cell subsets including CD4 T cells, Th17 cells (defined by simultaneous expression of CCR4+, CCR6+, CD161+, CD146+), Th17 memory cells, Th1 cells (defined as CCR4−, CCR6−, CD183+), Th1 memory cells, peripheral regulatory T cells (defined as FoxP3+ Helios−), and thymic regulatory T cells (defined as FoxP3+ Helios+). (A) CD4 T cells are depicted as percentage of T cells. (B–E) All other populations are depicted as percentage of CD4 T cells. Each dot represents an individual measurement, and horizontal lines and whiskers indicate mean ± SEM. *P < 0.05 (Mann–Whitney u test).
Fig. 1.
Fig. 1.
No overt differences in alpha or beta diversity were detected by comparing the fecal microbial profiles of healthy twins with those of MS twins. (A) Phylogenetic (alpha) diversity of fecal microbiota in healthy twins (HD, black; n = 34) and MS twins (MS, green; n = 34). (B) PCoA of weighted UniFrac community distances for fecal microbiota of healthy twins (black; n = 34) and MS twins (green; n = 34).
Fig. S2.
Fig. S2.
Differential abundance of Akkermansia muciniphila in fecal samples of healthy twins (HD; n = 34) compared with co-twins with untreated MS (MS untreated; n = 17) or treated MS (MS treated; n = 17). Each dot represents an individual stool sample, and horizontal lines and whiskers indicate mean ± SEM. *P < 0.05 (Mann–Whitney u test).
Fig. S3.
Fig. S3.
Comparison of the metagenomic profiles of healthy and MS twins revealed a high similarity of the gut microbiomes between discordant twins. Pairwise analysis of gut microbial composition (sum of absolute differences in genus relative abundance between two individuals) shows that gut bacterial flora are more similar within twin pairs discordant for MS (discordant twins, n = 32) than within pairs of unrelated individuals with MS (unrelated cases; n = 16), unrelated healthy individuals (unrelated controls; n = 18) or unrelated healthy and MS individuals (unrelated individuals discordant phenotypes; n = 34).
Fig. 2.
Fig. 2.
Human microbiota can be efficiently transferred to mouse recipients. (A) Design of humanized gnotobiotic mouse experiment: 6-wk-old, germ-free RR SJL/J mice were gavaged with fecal samples from the healthy twin or the MS twin from a MZ twin pair. Fecal samples for 16S rRNA sequencing analysis were harvested at 2 and 6 wk after colonization. Humanized gnotobiotic RR mice were observed for the development of clinical signs of EAE for 12 wk. (B) Phylogenetic (alpha) diversity of fecal microbiota in human twin pairs (purple; n = 8) and mouse recipients (gray; n = 47). ****P < 0.0001 (Mann–Whitney u test). (C) PCoA of weighted UniFrac community distances comparing fecal microbiota of human donors (purple; n = 8) and mouse recipients (gray; n = 47). (D) PCoA of weighted UniFrac community distances for fecal microbiota of mice transplanted with microbiota from healthy (n = 23) or MS (n = 24) twins. (E) Mean relative abundances of bacterial genera in fecal samples of mice that received microbiota of healthy donors (HD; n = 20–26) or MS patients (MS; n = 22–26) at 2 and 6 wk after transplantation.
Fig. 3.
Fig. 3.
Intestinal microbiota plays a pivotal role in CNS-specific autoimmunity. (A) Incidence of spontaneous EAE in humanized gnotobiotic RR SJL/J mice. Germ-free RR SJL/J mice were gavaged with fecal material from healthy (HD; n = 18) or MS (MS; n = 20) twins using in total five MZ twin pairs. *P < 0.05 (Gehan–Breslow–Wilcoxon test). (B) Relative abundance of Sutterella in fecal samples of mice that received the microbiota of healthy donor (HD; n = 23) or an MS patient (MS; n = 24) using in total five MZ twin pairs. Each circle represents an individual stool sample, and the horizontal line and whiskers indicate mean ± SEM. ***P < 0.001 (Mann–Whitney u test). (C) Cytokine production by splenocytes from humanized gnotobiotic SJL/J mice. Spleen cells were stimulated for 72 h with 1 µg/mL anti-CD3 antibody or 1 µg/mL anti-CD3 and 0.5 µg/mL anti-CD28 antibody. Levels of IFN-γ, IL-17, and IL-10 in the supernatants were measured by ELISA. Bars display mean ± SEM; n = 12–16 mice per group. Data were pooled from three independent experiments. *P < 0.05; **P = 0.0069 (Mann–Whitney u test). (D) IL-10 neutralization increases spontaneous EAE in humanized gnotobiotic RR SJL/J mice. Germ-free RR SJL/J mice were gavaged with fecal material from healthy twins and were treated weekly with either isotype control antibodies (n = 10) or neutralizing anti–IL-10 antibodies (n = 11). Data were pooled from two independent experiments.
Fig. S4.
Fig. S4.
Shotgun metagenomic sequencing of mouse fecal pellets uncovered several metabolic pathways associated with MS. Examination of gut microbiome metabolic pathways identified by metagenomic sequencing in 25 germ-free mice colonized by stool from four twins discordant for MS. Pathways that remained significantly associated with MS after multiple testing correction included chondroitin sulfate degradation (beta = 22,600, P = 4.5 × 10−6), pyruvate fermentation to acetone (beta = 13,400, P = 1.3 × 10−5), and superpathway of l-tyrosine biosynthesis (beta = 11,200, P = 1.5 × 10−5).
Fig. S5.
Fig. S5.
No differences were seen in immune cell populations of mice receiving control or MS patient fecal material. Bar graphs display frequencies (± SEM) of dendritic cells (CD45+CD11c+), macrophages (CD45+CD11b+), B cells (CD45+B220+), as well as regulatory T cells (CD4+FoxP3+) in spleen and small intestinal lamina propria (siLP) of germ-free SJL/J mice receiving fecal material from healthy twins (HD) or MS patients (MS). n = 10–13 mice per group. Data were pooled from three independent experiments.
Fig. S6.
Fig. S6.
No differences were seen in serum autoantibody titers in recipients of healthy twin (HD; n = 16) or MS twin (MS; n = 19) microbiota. Serum MOG-specific IgG1 or IgG2a antibodies were measured by sandwich ELISA. Values shown are the absorbances at 405 nm. Bars depict mean ± SEM.
Fig. S7.
Fig. S7.
No differences were seen in the expression of cytokines and tight junction proteins in recipients of healthy (HD) or MS twin microbiota. Expression levels of tight junction proteins (A) and cytokines (B) were measured by real-time qPCR. (Left) Expression levels in small intestine (ileum). (Right) Expression levels in colon. n = 3 mice per group. Bars depict mean ± SEM.
Fig. 4.
Fig. 4.
Gut bacteria from healthy twins trigger an antiinflammatory T cell response. (A) IL-10 production of CD4+ T cells isolated from PBMCs of selected twin pairs. T cells were stimulated for 96 h with 1 µg/mL anti-CD3 and anti-CD28 antibodies. Levels of IL-10 (seven pairs, two singletons) in the supernatants were measured by ELISA. *P < 0.05 (Wilcoxon test). (B) Cytokine profiles of CD4+ T cells isolated from PBMCs of selected twin pairs Depending on PBMC availability and quality, eight twin pairs were selected for further in vitro stimulation assays, and data are depicted for each cytokine where stimulation-dependent production above the individual detection level could be documented. T cells were stimulated for 48 h with 5 µg/mL PHA. Levels of IFN-γ (five pairs, three singletons), IL-17 (eight pairs), and IL-4 (seven pairs, one singleton) in the supernatants were measured by the Luminex Bead-based Multiplex Assay.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wekerle H. Nature plus nurture: The triggering of multiple sclerosis. Swiss Med Wkly. 2015;145:w14189. - PubMed
    1. Sawcer S, Franklin RJ, Ban M. Multiple sclerosis genetics. Lancet Neurol. 2014;13:700–709. - PubMed
    1. Simon KC, Munger KL, Ascherio A. XVI European Charcot Foundation lecture: Nutrition and environment: Can MS be prevented? J Neurol Sci. 2011;311:1–8. - PMC - PubMed
    1. Berer K, Krishnamoorthy G. Microbial view of central nervous system autoimmunity. FEBS Lett. 2014;588:4207–4213. - PubMed
    1. Wekerle H, Hohlfeld R. Gut Microbiota in Multiple Sclerosis: A Bioreactor Driving Brain Autoimmunity. Elsevier; Amsterdam: 2016. pp. 113–123.

Publication types

MeSH terms