Gut microorganisms act together to exacerbate inflammation in spinal cords

Abstract

Accumulating evidence indicates that gut microorganisms have a pathogenic role in autoimmune diseases, including in multiple sclerosis1. Studies of experimental autoimmune encephalomyelitis (an animal model of multiple sclerosis)2,3, as well as human studies4,5,6, have implicated gut microorganisms in the development or severity of multiple sclerosis. However, it remains unclear how gut microorganisms act on the inflammation of extra-intestinal tissues such as the spinal cord. Here we show that two distinct signals from gut microorganisms coordinately activate autoreactive T cells in the small intestine that respond specifically to myelin oligodendrocyte glycoprotein (MOG). After induction of experimental autoimmune encephalomyelitis in mice, MOG-specific CD4+ T cells are observed in the small intestine. Experiments using germ-free mice that were monocolonized with microorganisms from the small intestine demonstrated that a newly isolated strain in the family Erysipelotrichaceae acts similarly to an adjuvant to enhance the responses of T helper 17 cells. Shotgun sequencing of the contents of the small intestine revealed a strain of Lactobacillus reuteri that possesses peptides that potentially mimic MOG. Mice that were co-colonized with these two strains showed experimental autoimmune encephalomyelitis symptoms that were more severe than those of germ-free or monocolonized mice. These data suggest that the synergistic effects that result from the presence of these microorganisms should be considered in the pathogenicity of multiple sclerosis, and that further study of these microorganisms may lead to preventive strategies for this disease.

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Fig. 1: Suppression of EAE by manipulation of the gut microbiota.
Fig. 2: Increased severity of EAE caused by colonization with OTU0002.
Fig. 3: Coordinate effects of L. reuteri and OTU0002 on EAE development.

Data availability

The 16S rRNA sequence data, shotgun sequence data, and genome sequence data of L. reuteri and OTU0002 are available from the DDBJ Sequence Read Archive under DDBJ BioProject identifiers PRJDB6615, PRJDB6620, and PRJDB6618 and PRJDB6619, respectively. Source data are provided with this paper.

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Acknowledgements

We thank T. Kanaya, T. Kato and T. Takeuchi for their technical support. E.M. is supported by the RIKEN Special Postdoctoral Researcher Program. This work was supported in part by the RIKEN Interdisciplinary Research Program ‘Integrated Symbiology’, the RIKEN Pioneering Project ‘Biology of Symbiosis’, Grants-in-Aid for Young Scientists (B) (26850090 to E.M.) and Scientific Research (A) (19H01030 to H.O.), AMED-CREST (19gm0710009h0006 to H.O.) and the Food Science Institute Foundation (to H.O.).

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Authors

Contributions

E.M. and H.O. conceived the study. E.M. designed and performed the experiments and analyses, and cowrote the manuscript. S.-W.K. and T.D.T. contributed to analysis of the bacterial genome. M.K. and N.T.-A. contributed to 16S rRNA and bacterial genome sequencing. S.O. prepared gnotobiotic mice and helped with the experiments. W.S. and M.H. performed shotgun sequencing. H.M. contributed to generating the gene-deficient bacterial mutant. H.O. directed the research and cowrote the manuscript.

Corresponding author

Correspondence to Hiroshi Ohno.

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The authors declare no competing interests.

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Peer review information Nature thanks Martin Blaser, Vijay Kuchroo and Harmut Wekerle for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Suppression of MOG-specific immune responses by oral treatment with antibiotics.

a, Schematic of treatment of EAE mice with antibiotics (ABX). PTX, pertussis toxin. b, EAE clinical scores for mice given normal (control, con) drinking water, or drinking water containing an antibiotic cocktail (ampicillin, vancomycin, neomycin and metronidazole (AVNM))) (n = 5). c, EAE clinical scores for mice given ampicillin in their drinking water (amp-oral) or by daily intraperitoneal (i.p.) injections (amp-i.p.) (n = 5). Asterisks indicate a significant difference between the orally treated group and the intraperitoneally treated group. d, Mice were killed on day 10, and splenocytes were restimulated with or without MOG35–55. After culturing for three days, the concentrations of IFNγ and IL-17 were measured by ELISA (d, n = 3). e, Small-intestinal CD4+ or CD8+ T cells from naive mice (naive), EAE mice (con) and ampicillin-treated EAE mice (amp) were co-cultured with splenic CD11c+ cells from naive mice in the presence or absence of MOG35–55 for three days. The cytokine concentrations in the supernatants were measured by ELISA (n = 4). ND, not detectable. f, Percentage and absolute numbers of TH1 and TH17 cells in the small-intestinal lamina propria (n = 5 mice). gk, Representative FACS plots (gated on CD3+CD4+ cells) and summary data of FOXP3+CD4+ regulatory T cells in the small-intestinal lamina propria (g; n = 4 mice) and other tissues (hj, n = 4 mice; k, n = 5 mice). Data are mean ± s.d. Detailed statistics of the EAE clinical scores are summarized in Supplementary Tables 7, 8. ***P < 0.001, **P < 0.01, *P < 0.05. Two-way ANOVA with Bonferroni’s (b) or Tukey’s (c) test, or one-way ANOVA with Tukey’s test (dk). Exact P values are in Source Data. Source data

Extended Data Fig. 2 Bacterial loads and composition of the small-intestine microbiota, and phylogeny of OTU0002.

a, Bacterial loads in the contents of the small intestine, as determined by qPCR of 16S rRNA (n = 5 mice). Data are mean ± s.d. ***P < 0.001, **P < 0.01, *P < 0.05. One-way ANOVA with Tukey’s test. Exact P values are in Source Data. b, Phylum-level relative abundance of small-intestine microbiota. c, Principal coordinate analysis plot of unweighted UniFrac distances. Each ellipse shows an 80% confidence interval (n = 5 mice). PC1, PC2, principle coordinates 1 and 2. d, Phylogenetic tree based on 16S rRNA gene sequences of OTU0002 and deposited isolates. e, Phylogenetic tree of OTU0002 and related uncultured bacteria. The bacterial reads from human faeces (human faeces 1 and 2) were retrieved from ref. 38 and ref. 6, respectively. f, Representative SEM images of the caecum and colon from OTU0002-monocolonized mice (n = 4). Source data

Extended Data Fig. 3 Modulation of TH17-cell function by SAA.

a, Splenocytes from germ-free mice inoculated with L. reuteri or OTU0002 were cultured in the presence or absence of MOG35–55 for three days. The cytokine concentrations in the supernatants were measured by ELISA (n = 5 mice). b, Percentage and absolute numbers of TH17 cells in the spleen (n = 5 mice). c, f, mRNA expression of the indicated genes in tissues of the small intestine (n = 5 mice). d, e, Splenocytes from specific-pathogen-free EAE mice were restimulated with MOG35–55 in the presence or absence of SAA. d, After culturing for two days, CD4+ cells were enriched by magnetic beads and the mRNA expression of the indicated genes was quantified by qPCR (n = 4 mice). e, The cytokine concentrations in the supernatants were measured by ELISA (n = 4 mice). g, Representative FACS plots (gated on CD3+ and CD4+ cells), percentage and absolute numbers of TH17 cells in the small-intestinal lamina propria of naive mice (germ-free, n = 6 mice; L. reuteri and OTU0002, n = 5 mice). Data are mean ± s.d. ***P < 0.001, **P < 0.01, *P < 0.05. Two-way ANOVA with Bonferroni’s test (a), one-way ANOVA with Tukey’s test (bg) or Kruskal–Wallis with Dunn’s test (b, number of IL-17A+CD4+ T cells; c, Saa1, Saa2 and Il12b; d, Rorc and Il17a; f, Csf2; g, number of IL-17A+CD4+ T cells). Exact P values are in Source Data. Source data

Extended Data Fig. 4 Microbiota-dependent proliferation of MOG-specific 2D2 TCR T cells in the small intestine.

a, b, Representative FACS plots (gated on CD3+ cells) and percentage of Ki67+CD4+ T cells and absolute number of CD4+ T cells in the small-intestinal lamina propria (a, n = 6 mice) and spleen (b, n = 4 mice) of wild-type and 2D2 mice. c, Percentage of Ki67+CD4+ T cells and absolute numbers of CD4+ T cells in the small-intestinal lamina propria of non-treated or AVNM-treated (+AVNM) 2D2 mice (n = 4 mice). d, Percentage of Ki67+ in 2D2 TCR (Vα3.2+Vβ11+) cells (n = 4 mice). Data are mean ± s.d. P values were calculated using two-tailed unpaired t-test. Source data

Extended Data Fig. 5 Induction of EAE by mimicry peptide.

a, Candidate mimicry proteins (matching TCR-binding residues of MOG40–48, shown in bold) were observed in the contents of the small intestines of control mice. Left, presence or absence of each candidate. The candidates observed in all five mice were used as references for the analysis shown in Fig. 3a. b, Splenic CD11c+ cells pretreated with indicated antibodies (Ab) were co-cultured with CD4+ T cells from mesenteric lymph nodes of 2D2 TCR mice in the presence of indicated peptides for four days. Representative FACS plots (gated on CD3+ cells) and summary data are shown (n = 4). c, Mesenteric lymph nodes from wild-type or 2D2 mice were stimulated with indicated peptides for four days. Representative FACS plots (gated on CD3+CD4+cells) and summary data are shown (n = 6). d, CD4+ T cells from the spinal cord of EAE wild-type mice were co-cultured with splenic CD11c+ cells from naive wild-type mice in the presence of indicated peptides for three days. The concentrations of IFNγ and IL-17 were measured by ELISA (n = 6). e, f, Wild-type mice were immunized with MOG38–50, UvrA or vehicle (PBS). EAE clinical scores are shown in e (n = 6). mRNA expression of the indicated genes in the spinal cord is shown in f (MOG, n = 5; PBS and UvrA, n = 6). g, Left, representative images of spinal cord sections stained with luxol fast blue. Right, demyelinated area in the white matter was calculated. Scale bars, 500 μm (g) (PBS, n = 3; MOG and UvrA, n = 4). h, RNA and DNA were extracted from the small intestine, caecum (Cec) and colonic contents (Col) of naive mice, and qPCR was performed using uvrA-specific primers. The RNA/DNA ratios of the uvrA gene are shown (n = 5). Data are mean ± s.d. ***P < 0.001, **P < 0.01, *P < 0.05. Two-way ANOVA with Bonferroni’s test (b), Kruskal–Wallis with Dunn’s test (c, d IFNγ, f, h) or one-way ANOVA with Tukey’s test (d IL-17, g). Exact P values are in Source Data. Source data

Extended Data Fig. 6 Effects of co-colonization with L. reuteri and OTU0002 on TH17 cells.

a, Left, representative images of spinal cord sections from EAE-induced germ-free mice or germ-free mice colonized with the indicated strains, stained with luxol fast blue (left). Scale bars, 500 μm. Right, the demyelinated area in the white matter was calculated. The combined results of two independent experiments are shown (germ-free and OTU0002, n = 8; L. reuteri + OTU0002, n = 6). b, Absolute numbers of CD4+ T cells in the spinal cord (germ-free and OTU0002, n = 8; L. reuteri + OTU0002, n = 6). c, The splenocytes and small-intestinal lamina propria cells were cultured in the presence or absence of MOG35–55 for three days. The cytokine concentrations in the supernatants were measured by ELISA (n = 5 mice). d, Percentage and absolute numbers of FOXP3+CD4+ regulatory T cells in the small-intestinal lamina propria (n = 5 mice). e, mRNA expression of the indicated genes in the small-intestine tissue (n = 5 mice). f, Percentage and absolute numbers of TH17 cells in the spleen (n = 5 mice). Data are mean ± s.d. ***P < 0.001, **P < 0.01, *P < 0.05. Kruskal–Wallis with Dunn’s test (a, e), one-way ANOVA with Tukey’s test (b, d, f) or two-way ANOVA with Bonferroni’s test (c). Exact P values are in Source Data. Source data

Extended Data Fig. 7 Coordinated effects of SFB and L. reuteri on EAE development.

a, The abundance of SFB and L. reuteri in the contents of the small intestine. qPCR with specific primers for SFB and Lactobacillus was performed (n = 5 mice). P value (two-tailed unpaired Mann–Whitney test) for the difference in SFB abundance is shown. b, c, EAE clinical scores (b) and incidence (c) of germ-free mice monocolonized with SFB and co-colonized with SFB and L. reuteri (n = 5). Asterisks indicate a significant difference between the monocolonized group and the co-colonized group. Detailed statistics of the EAE clinical scores are summarized in Supplementary Table 9. d, mRNA expression of the indicated genes in the small-intestine tissue (n = 5 mice). e, Representative FACS plots (gated on CD3+ and CD4+ cells), percentage and absolute numbers of TH17 cells in the small-intestinal lamina propria (n = 5 mice). e, Representative FACS plots (gated on CD3+ cells) and summary data of Ki67+CD4+ T cells in the small-intestinal lamina propria are shown (n = 5 mice). Data are mean ± s.d. ***P < 0.001, **P < 0.01, *P < 0.05. Two-way ANOVA with Tukey’s (b) or one-way ANOVA with Tukey’s test (df). EAE incidence was analysed by two-tailed log–rank (Mantel–Cox) test (c). Exact P values are in Source Data. Source data

Extended Data Fig. 8 Generation of uvrA-deficient L. reuteri.

a, Schematic of the genomic structure of the uvrA gene of L. reuteri (L. reuteri wild type), the targeting vector and the resultant mutant (L. reuteri ΔuvrA) generated by homologous recombination containing the erythromycin-resistance gene (ermAM). The NdeI and SpeI restriction sites are indicated by NI and SI, respectively. b, PCR analysis of genomic DNA from L. reuteri and L. reuteri ΔuvrA using uvrA- and ermAM-specific primers. c, Southern blot analysis of NdeI- and SpeI-digested genomic DNA from L. reuteri and L. reuteri ΔuvrA. Probes A and B, depicted in a, were used to detect genomic fragments present in both strains and only in L. reuteri ΔuvrA, respectively. The experiments were independently repeated two times with similar results (b, c). For gel and blot source data, see Supplementary Fig. 1.

Extended Data Fig. 9 Effect of uvrA-deficient L. reuteri on MOG-specific 2D2 TCR T cells.

a, Germ-free wild-type or 2D2 mice were monocolonized with L. reuteri wild-type strain or a uvrA-deficient strain (ΔuvrA). The percentage and absolute numbers of Ki67+CD4+ T cells are shown (wild-type mice with ΔuvrA, n = 3; wild-type mice and wild-type strain, and 2D2 mice and ΔuvrA strain, n = 4; 2D2 mice and wild-type strain, n = 5). b, Representative FACS plots (gated on CD3+CD4+ cells) and summary data of Ki67+ 2D2 TCR (Vα3.2+Vβ11+) cells in the small-intestinal lamina propria are shown (n = 5 mice). c, d, Germ-free wild-type mice were co-colonized with OTU0002 and a wild-type or uvrA-deficient L. reuteri strain, and EAE was induced. The abundance of L. reuteri and OTU0002 in the contents of the small intestine was quantified by qPCR with specific primers for Lactobacillus and Allobaculum (d) (n = 4 mice). Representative FACS plots (gated on CD3+ and CD4+), percentage and absolute numbers of TH17 cells in the small-intestinal lamina propria are shown (e) (n = 4). Data are mean ± s.d. ***P < 0.001, **P < 0.01, *P < 0.05. One-way ANOVA with Tukey’s test (a) or two-tailed unpaired t-test (b). Exact P values are in Source Data. Source data

Extended Data Fig. 10 Model of the role of microorganisms of the small intestine in EAE.

Two key microorganisms coordinately activate MOG-specific T cells in the small intestine. Mimicry peptides (UvrA) expressed in L. reuteri (OTU0001) trigger TCR signals, and OTU0002-induced pro-inflammatory factors (SAA and IL-23) increase the pathogenicity of MOG-specific TH17 cells. The activated cells may migrate into the spinal cord and induce demyelination.

Supplementary information

Supplementary Figure 1:

Supplementary Figures Raw gel and blot data from Extended Data Fig. 8b and 8c. Red boxes indicate the cropped area used in Extended Data Fig. 8b and 8c.

Reporting Summary

Supplementary Table 1:

Supplementary Table Predicted virulence factors in OTU0002 genome.

Supplementary Table 2:

Supplementary Table Sequences of primers and probes.

Supplementary Table 3:

Supplementary Table Detailed statistics of the EAE clinical scores shown in Fig. 1a. Data represent the mean ± s.d. (a). P values were calculated with one-way (a) or two-way ANOVA with Tukey’s test (b).

Supplementary Table 4:

Supplementary Table Detailed statistics of the EAE clinical scores shown in Fig. 2d. Data represent the mean ± s.d. (a). P values were calculated with one-way (a) or two-way ANOVA with Tukey’s test (b).

Supplementary Table 5:

Supplementary Table Detailed statistics of the EAE clinical scores shown in Fig. 3d. Data represent the mean ± s.d. (a). P values were calculated with one-way (a) or two-way ANOVA with Tukey’s test (b).

Supplementary Table 6:

Supplementary Table Detailed statistics of the EAE clinical scores shown in Fig. 3h. Data represent the mean ± s.d. (a). P values were calculated with two-tailed unpaired t-test (a) or two-way ANOVA with Bonferroni’s test (b).

Supplementary Table 7:

Supplementary Table Detailed statistics of the EAE clinical scores shown in Extended Data Fig. 1b. Data represent the mean ± s.d. (a). P values were calculated with two-tailed unpaired t-test (a) or two-way ANOVA with Bonferroni’s test (b).

Supplementary Table 8:

Supplementary Table Detailed statistics of the EAE clinical scores shown in Extended Data Fig. 1c. Data represent the mean ± s.d. (a). P values were calculated with one-way (a) or two-way ANOVA with Tukey’s test (b).

Supplementary Table 9:

Supplementary Table Detailed statistics of the EAE clinical scores shown in Extended Data Fig. 7b. Data represent the mean ± s.d. (a). P values were calculated with one-way (a) or two-way ANOVA with Tukey’s test (b).

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Miyauchi, E., Kim, S., Suda, W. et al. Gut microorganisms act together to exacerbate inflammation in spinal cords. Nature 585, 102–106 (2020). https://doi.org/10.1038/s41586-020-2634-9

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