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. 2018 Mar 9;359(6380):1156-1161.
doi: 10.1126/science.aar7201.

Translocation of a Gut Pathobiont Drives Autoimmunity in Mice and Humans

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Free PMC article

Translocation of a Gut Pathobiont Drives Autoimmunity in Mice and Humans

S Manfredo Vieira et al. Science. .
Free PMC article

Abstract

Despite multiple associations between the microbiota and immune diseases, their role in autoimmunity is poorly understood. We found that translocation of a gut pathobiont, Enterococcus gallinarum, to the liver and other systemic tissues triggers autoimmune responses in a genetic background predisposing to autoimmunity. Antibiotic treatment prevented mortality in this model, suppressed growth of E. gallinarum in tissues, and eliminated pathogenic autoantibodies and T cells. Hepatocyte-E. gallinarum cocultures induced autoimmune-promoting factors. Pathobiont translocation in monocolonized and autoimmune-prone mice induced autoantibodies and caused mortality, which could be prevented by an intramuscular vaccine targeting the pathobiont. E. gallinarum-specific DNA was recovered from liver biopsies of autoimmune patients, and cocultures with human hepatocytes replicated the murine findings; hence, similar processes apparently occur in susceptible humans. These discoveries show that a gut pathobiont can translocate and promote autoimmunity in genetically predisposed hosts.

Trial registration: ClinicalTrials.gov NCT02394964.

Figures

Fig. 1
Fig. 1. Effect of antibiotics on autoimmunity and E. gallinarum translocation to the liver
(A) Vancomycin (VANC), ampicillin (AMP), metronidazole (METR), neomycin (NEO), or control water (CTRL) were provided in the drinking water of (NZW × BXSB)F1 mice starting at 6 weeks of age (n = 15 each). Mice were followed for 30 weeks or until death from autoimmunity. (B and C) Serum anti-dsDNA (B) and anti-RNA (C) IgG at 16 weeks of age. (D) Serum levels of orally administered FITC-dextran as an indicator of gut barrier leakiness (n = 8 each). (E to G) Cultures of tissues from 16-week-old mice showed a selective growth of E. gallinarum in the mesenteric veins (E), MLN (F), and liver (G) (n = 7 each). (H) An E. gallinarum–specific FISH probe detects E. gallinarum in liver (scale bars, 30 μm) of E. gallinarum–monocolonized C57BL/6 mice 3 weeks after colonization in comparison to germ-free mice. One representative section from one mouse is shown from multiple sections with E. gallinarum signals within the tissues, representative of three mice in total. Data are presented as mean ± SD in (B) to (G); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; log-rank test and Gehan-Breslow-Wilcoxon test in (A), analysis of variance (ANOVA) followed by Bonferroni multiple-comparisons test in (B) to (G).
Fig. 2
Fig. 2. RNA expression profiling and plasmacytoid dendritic cell frequencies in small intestine from germ-free mice monocolonized with E. gallinarum, E. faecalis, or B. thetaiotaomicron
Germ-free C57BL/6 mice were monocolonized with E. gallinarum (EG), E. faecalis (EF), or B. thetaiotaomicron (BT) for RNA-seq and fluorescence-activated cell sorting (FACS) analyses of the small intestine. (A) RNA-seq was performed with ileal tissue isolated from 14-week-old monocolonized mice (n = 3 each). Heat map shows transcripts differentially expressed in the ileum 8 hours after commensal delivery. (B to J) Reverse transcription quantitative PCR (RT-qPCR) analysis of ileum RNA (n = 6 each) as described in (A). (K) Plasmacytoid dendritic cell (pDC) and conventional dendritic cell (cDC) frequencies in the small intestinal lamina propria of 12-week-old germ-free mice (n = 4 each) were evaluated 3 weeks after monocolonization by FACS analysis. (L) Confocal imaging of gut tissues was performed as described in the supplementary materials. Localizations of TJ proteins are shown in green for occludin, JAM-A, claudin-3, and claudin-5. Images are representative of six different mice. DAPI, 4′,6-diamidino-2-phenylindole. Scale bars, 40 μm. Data are presented as mean ± SD in (B) to (K); *P < 0.05, **P < 0.01, ****P < 0.0001; ANOVA followed by Bonferroni multiple-comparisons test in (B) to (K).
Fig. 3
Fig. 3. Induction of hepatic AhR by E. gallinarum, AhR antagonism in (NZW × BXSB)F1 mice, and Th17 and autoantibody induction in E. gallinarum monocolonized C57BL/ 6 mice
(A to F) E. gallinarum, E. faecalis, and B. thetaiotaomicron lysates or isolated RNA were cocultured with hepatocytes from 14-week-old (NZW × BXSB) F1 mice (n = 3 each), and expression of ERV gp70, the autoantigen β2GPI, IFN-α, and AhR was measured 6 hours later. (G and H) Serum anti-RNA (G) and anti-dsDNA (H) IgG of 16-week-old (NZWxBXSB)F1 mice (n = 4 each) gavaged with vehicle or EG and treated with AhR antagonist CH223191 or mock, as described in the supplementary materials. (I to L) C57BL/6 germ-free (GF) mice were monocolonized with E. gallinarum, E. faecalis, and B. thetaiotaomicron at 12 weeks of age (n = 6 to 10 mice) and evaluated 3 weeks later for integrity of the gut barrier with FITC-dextran (I), and for translocation to the mesenteric veins (J), MLNs (K), and liver (L). (M and N) C57BL/6 germ-free mice (n = 4 to 10 mice) were monocolonized as in (I), and anti-RNA (M) and anti-dsDNA (N) IgG autoantibodies were measured 8 weeks later. (O and P) Th17 cells and Th1 cell frequencies were determined by intracellular FACS analysis of IL-17A and IFN-g in small intestinal lamina propria (O) and MLN (P) CD45+ CD4+ CD44+ T cells from germ-free and monocolonized mice (n = 5 each). Data are presented as mean ± SD in (A) to (P); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ANOVA followed by Bonferroni multiple-comparisons test.
Fig. 4
Fig. 4. Gut barrier function and E. gallinarum in liver biopsies of autoimmune patients with anti E. gallinarum serum reactivities
(A and B) Feces from patients with SLE (n = 9) were screened for increased albumin (A) and calprotectin (B) as signs of an impaired gut barrier. NHD, normal healthy donors (n = 9). (C) Multiplex PCR for eubacterial [1383 base pairs (bp)], Enterococcus genus (112 bp), and E. gallinarum (173 bp) DNA on sterilely obtained and processed liver biopsies from cadaveric liver transplant donors (CTRL) or SLE patients. Lanes 10 to 12, bacterial strains as indicated; lane 13, water. (D) 16S rDNA sequencing of controls as in (C), autoimmune hepatitis (AIH) patients, and non-AIH cirrhosis (CIR) patients. (E) Liver biopsies from CTRL, AIH, and CIR patients (n = 5 to 6 each) were tested for E. gallinarum (EG) DNA by qPCR and normalized to any eubacterial (EUB) signal. (F to J) RT-qPCR of human primary hepatocytes (n = 3 each) stimulated with E. gallinarum, E. faecalis, or B. thetaiotaomicron as in Fig. 3. (K and L) SLE (n = 15) and AIH (n = 17) sera were screened for anti–E. gallinarum RNA IgG (K) and anti-human RNA IgG (L) by enzyme-linked immunosorbent assay (ELISA). (M) SLE and AIH serum IgG levels against human, E. gallinarum, E. faecalis, or B. thetaiotaomicron RNA normalized to NHD sera. (N and O) Correlation between anti–E. gallinarum RNA IgG and autoantibodies in SLE (N) and AIH (O) patients. Data are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Student t test in (A) and (B), ANOVA followed by Bonferroni multiple-comparisons test in (E) to (M), and Pearson correlation in (N) and (O).

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