C57/BL6J male mice were obtained from Janvier Labs (Le Genest Saint-Isle, France) and housed under specific pathogen-free conditions with ad libitum access to food and water. All mice were housed under specific pathogen-free conditions at the INSERM animal facility (Zootechnie US-006), which is accredited by the French Ministry of Agriculture (accreditation number A-31 55508) to perform experiments on live mice. All experimental protocols were approved by the local ethics committee and are in compliance with the French and European regulations on care and protection of the Laboratory Animals (EC Directive 2010/63).

The mAbs used for flow cytometry were as follows: RM4-5 (anti-mouse CD4), IM7 (anti-mouse CD44), H57-597 (anti-mouse TCRαβ), and FJK-165 (anti-mouse Foxp3). The fluorescent conjugated antibodies were purchased from e-Biosciences, BD Biosciences, and Biolegend. Antibodies used for ELISA were as follows: AN18 (anti-IFN-γ), purified anti-mouse IL-17A (BD Biosciences), purified anti-mouse GM-CSF (BD Biosciences), XMG1.2 (anti-IFN-γ biotin), biotin anti-mouse IL-17A (BD Biosciences), and biotin anti-mouse GM-CSF (BD Biosciences).

The strains used in this study are the probiotic E. coli Nissle 1917 (ECN) and the archetypal K12 E. coli strain MG1655. Both strains were engineered to exhibit a mutation in the rpsL gene, which is known to confer resistance to streptomycin (27). Before oral administrations, ECN and MG1655 strains were grown for 6 h in LB broth supplemented with streptomycin (50 µg/ml) at 37°C with shaking. This culture was diluted 1:100 in LB broth without antibiotics and cultured overnight at 37°C with shaking. Bacterial pellets from this overnight culture were resuspended in sterile PBS to the concentration of 10⁹ cfu/ml. Bacteria were amplified from −80°C frozen stocks to ensure genetic stability.

The MOG_35–55 (MEVGWYRSPFSRVVHLYRNGK) peptide was purchased from Polypeptide Laboratories (San Diego, CA, USA). 8- to 12-week-old mice were immunized subcutaneously with 50 µg of MOG_35–55 peptide emulsified in complete Freund’s adjuvant (BD Difco, Franklin Lakes, NJ, USA) containing 500 µg of Mycobacterium tuberculosis (Strain H37, Difco). Mice were injected intravenously with 200 ng of pertussis toxin (List Biological Laboratories, Campbell, CA, USA) at days 0 and 2 post-immunization. Mice were daily treated with ECN, MG1655, or PBS starting 7 days before immunization (Figure S1 in Supplementary Material). Bacteria were administered every day by gavage at the dose of 10⁸ cfu/animal, prepared in 100 µl of PBS. Bacteria were freshly prepared every day as already described. Clinical scores were recorded daily as follow: 0, no signs of disease; 1, loss of tone in the tail; 2, hind limb paresis; 3, hind limb paralysis; 4, tetraplegia; and 5, moribund (28).

Feces homogenates were prepared in 600 µl of isotonic saline solution using Precellys tissue homogenizer (Bertin Technologies). Ten-fold serial dilutions of homogenates were plated on MacConkey agar plates (Biovalley) supplemented or not with streptomycin. Plates were incubated at 37°C and 5% CO₂ and cfu numbers were enumerated after 24 h. Colonies found growing on MacConkey’s agar plates without antibiotic were considered to be enterobacteria-like bacteria belonging to the family Enterobacteriaceae.

In vivo intestinal permeability assessment was performed using FITC–dextran. Briefly, food and water were withdrawn for 3 h, and mice were gavaged with FITC–dextran (625 mg/kg body weight of FITC–dextran, MW: 4 kDa, FD4, Sigma-Aldrich). Serum was collected after retro-orbital puncture and fluorescence intensity was measured at 485/525 nm using an automatic Infinite M200 microplate reader (Tecan). FD4 concentrations were determined from standard curves generated by serial dilution of FD4. FD4 recovery upon time was calculated by linear regression of sample fluorescence, taking account of mice body weight (29).

Ex vivo intestinal permeability was performed in Ussing chamber. Biopsies from ileum or colon were placed in a chamber exposing 0.196 cm² of tissue surface to 1.5 ml of circulating oxygenated Ringer solution at 37°C (30). Ileum and colon paracellular permeability were assessed by measuring the mucosal-to-serosal flux of 4 kDa FITC–dextran (Sigma-Aldrich) for 1 h (29). Results are expressed as flux of FITC–dextran per square centimeters of mucosa per hour.

Total mRNA was extracted from ileum and colon mucosa scrapping by Trizol-Chloroform precipitation. mRNA was subsequently purified using Rneasy Plus kit (Qiagen) following the manufacturer’s instructions. For reverse transcription PCR, the iScript cDNA synthesis kit (Bio-Rad) was used. Real-time PCR was carried out using iQSYBR Green Supermix (Bio-Rad) for Reg3γ, Reg3β, ZO-1, Claudin-8, and IL-6. Primers are presented in Supplementary Table S1. Mouse glyceraldehyde 3-phosphate dehydrogenase served as endogenous control. PCR was done in duplicate and threshold cycle (Ct) values of the target genes were normalized to the endogenous control. Differential expression was calculated using the 2^−ΔΔCt methods and expressed as fold induction as compared to the expression in non-immunized animals (31).

Mice were anesthetized with ketamine and perfused with cold PBS. Brain and spinal cord were collected separately, homogenized and digested with collagenase D (2.5 mg/ml, Roche Diagnostics), Dnase I (10 µg/ml), and TLCK (1 µg/ml, Roche, Basel, Switzerland) for 30 min at 37°C. Cells were then washed, suspended in 37% Percoll, and layered on 70% Percoll. After a 20-min centrifugation at 2,000 rpm, the mononuclear cells were collected from the interface washed and resuspended in culture medium. For tetramer staining, cells from brain, spinal cord, and LNs were incubated for 2 h at room temperature with I-Ab MOG_38–49 Tetramer-PE (NIH tetramer core facility) and then stained for surface markers before flow cytometry analysis. For functional analysis, 3 × 10⁶ total lymph node cells were stimulated with different concentrations of MOG_35–55 for 72 h. Cytokines were then quantified in the supernatants.

Enzyme immunoassays were used to measure cytokines in culture supernatants. 96-well plates were coated for 2 h at 37°C with anti-IFN-γ, anti-IL-17, or anti-GM-CSF in carbonate buffer 0.05 M pH 9.6. Culture supernatants or standards were incubated 2 h at 37°C. The plates were then incubated for 2 h with a secondary biotinylated antibody specific for each cytokine, followed by 20 min incubation with streptavidin–phosphatase alkaline at 37°C. Finally, plates were revealed by phosphatase alkaline substrate, and absorbance was measured at 450/540 nm. IL-10 and TNF were tested using CBA technology.

Statistical evaluation of differences between the experimental groups was determined by using two-way analysis of variance followed by a Bonferroni posttest for clinical monitoring and intestinal permeability assay; or with Mann–Whitney U test for the other assays. All tests were performed with GraphPad Prism 5.04 (GraphPad Software Inc., San Diego, CA, USA). All data are presented as mean ± SEM. A p value ≤0.05 was considered significant.

To study the impact of ECN treatment on the development of CNS inflammation, we compared the susceptibility of PBS- and ECN-treated mice to EAE according to the protocol shown in Figure S1 in Supplementary Material. When immunized by MOG_35–55 peptide, PBS-treated animals developed a classical disease characterized by a progressive ascendant paralysis. Notably, when daily treated with ECN, mice developed a less severe disease, as illustrated by reduced clinical score (Figure 1A) and mortality (Figure 1B). The day of onset was similar between both groups (Figure 1C), while the incidence (Figure 1D) and the severity of disease progression were drastically reduced (Figures 1E,F) upon ECN treatment. Interestingly, mice treated with E. coli strain MG1655 developed similar disease progression, incidence and severity as PBS-treated animals (Figures 2A–F), suggesting that the protective effect observed with ECN is due to its intrinsic properties. The absence of probiotic effect with MG1655 is unlikely related to defective gut colonization, since E. coli intestinal loads were equivalent regardless of the type of strain (Figures 1G and 2G).

Peripheral activation and subsequent migration of autoreactive CD4⁺ T cells into the CNS are critical steps in the pathogenesis of EAE and MS (32, 33). 14 days after EAE induction, leukocytes were isolated from the spinal cord or peripheral lymphoid organs and were analyzed. Interestingly, the total numbers of CD4⁺ and MOG-specific CD4⁺ T cells were significantly decreased in the spinal cord (Figure 3A) of ECN-treated animals. In contrast, the numbers of these cells increased in inguinal (draining) (Figure 3B), mesenteric (Figure 3C), and cervical (Figure 3D) lymph nodes in ECN-treated animals. We further checked whether oral treatment with ECN could modulate the effector function of MOG-specific CD4⁺ T cells. Draining lymph node T cells collected on day 14 (Figure 4) or day 30 (Figure S2 in Supplementary Material) after immunization were stimulated with MOG_35–55 for 72 h and analyzed for the production of various cytokines. Treatment with ECN significantly reduced the production of IFN-γ, GM-CSF, IL-17, and TNF-α (Figures 4A–D; Figure S2 in Supplementary Material). This effect was not observed when T cells originated from MG1655-treated mice (Figure S2 in Supplementary Material). Intriguingly, treatment with ECN significantly increased the production of IL-10 by MOG-specific CD4⁺ T cells (Figure 4E), which is a hallmark of Tregs (34, 35). Moreover, draining lymph nodes from ECN-treated mice exhibited a significantly higher number of CD4⁺Foxp3⁺ cells (Figure 4F). Taken together, these data demonstrate that ECN protection of the host from CNS inflammation is associated to dampened inflammatory cytokine production and reduced migration of MOG-specific T cells from the periphery to the CNS.

It has been recently reported that intestinal permeability is increased during the course of EAE (36). In agreement with these studies, mice exhibited a robust and time-dependent enhanced intestinal permeability when immunized with MOG_35–55, as assessed in vivo by the luminal to blood passage of fluorescent marker [FITC–dextran 4 kDa (FD4)]. This was observed before the clinical onset, i.e., 7 days after immunization and at the peak of the disease, 14 days after immunization (Figure 5A). Interestingly, we observed a significant correlation between colon and ileum permeability and EAE-clinical score 14 days after immunization (Figures 5B,C), suggesting that intestinal barrier dysfunction is an important parameter accounting for the severity of autoimmune inflammation.

The effectiveness of ECN as a probiotic organism has been correlated to its ability to strengthen the intestinal barrier function (37). To investigate the impact of ECN treatment on intestinal barrier function during EAE progression, we measured the intestinal permeability of mice treated daily with ECN, 7 and 14 days after MOG_35–55 immunization. The oral treatment with ECN significantly reduced the FD4 passage from the intestinal lumen to the blood compartment, demonstrating that ECN is able to preserve the intestinal barrier function during EAE (Figure 5D). In contrast, mice treated with MG1655 developed similar intestinal barrier function impairment as PBS-treated animals (Figure 5D). To gain further insight into EAE-induced intestinal barrier dysfunction, colon and ileum biopsies were mounted in Ussing chambers and analyzed for the paracellular flux of FD4. Seven and fourteen days after immunization, PBS-treated mice exhibited an increase of both colon and ileum permeability (Figures 5E,F). Such an increase in colon and ileum intestinal permeability was not modulated by MG1655 treatment, while they were significantly reduced by oral treatment with ECN (Figures 5E,F). To investigate the underlying mechanisms, we first analyzed several components of the intestinal barrier function. Fourteen days after MOG_35–55 immunization, we observed a downregulation of mRNA coding for the antimicrobial peptides Reg3γ (Figure 6A) and Reg3β (Figure 6B), as well as for the tight-junction proteins Claudin-8 (Figure 6C) and ZO-1 (Figure 6D), whereas expression of the prototypal pro-inflammatory cytokine IL-6 was increased (Figure 6E). These alterations of the intestinal barrier function were reduced upon ECN treatment as compared to PBS or MG1655-treated animals (Figure 6; Figure S3 in Supplementary Material).