Oral Administration of the Probiotic Strain Escherichia coli Nissle 1917 Reduces Susceptibility to Neuroinflammation and Repairs Experimental Autoimmune Encephalomyelitis-Induced Intestinal Barrier Dysfunction

Abstract

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) with an increasing incidence in developed countries. Recent reports suggest that modulation of the gut microbiota might be one promising therapy for MS. Here, we investigated whether the probiotic Escherichia coli strain Nissle 1917 (ECN) could modulate the outcome of experimental autoimmune encephalomyelitis (EAE), a murine model of MS. We evidenced that daily oral treatment with ECN, but not with the archetypal K12 E. coli strain MG1655, reduced the severity of EAE induced by immunization with the MOG_35-55 peptide. This beneficial effect was associated with a decreased secretion of inflammatory cytokines and an increased production of the anti-inflammatory cytokine IL-10 by autoreactive CD4 T cells, both in peripheral lymph nodes and CNS. Interestingly, ECN-treated mice exhibited increased numbers of MOG-specific CD4⁺ T cells in the periphery contrasting with severely reduced numbers in the CNS, suggesting that ECN might affect T cell migration from the periphery to the CNS through a modulation of their activation and/or differentiation. In addition, we demonstrated that EAE is associated with a profound defect in the intestinal barrier function and that treatment with ECN, but not with MG1655, repaired intestinal permeability dysfunction. Collectively, our data reveal that EAE induces a disruption of the intestinal homeostasis and that ECN protects from disease and restores the intestinal barrier function.

Keywords: Escherichia coli Nissle 1917; central nervous system; encephalitogenic T-cell; experimental autoimmune encephalomyelitis; intestinal permeability; probiotic.

Figure 1

ECN-treated mice develop less severe experimental autoimmune encephalomyelitis (EAE). (A) Daily clinical score monitoring after MOG_35–55 immunization. (B) Mortality during EAE progression. (C) Onset day assessment. (D) Disease incidence at day 30 after immunization. (E) Cumulative score was calculated as the sum of all EAE-clinical scores developed during the 30 days by each mouse per group. (F) Maximal scores were determined as the mean of maximal score reached by each mouse per group. (G) Escherichia coli enumeration in feces twice a week after immunization. Results are from three independent experiments and are depicted as means ± SEM (n = 40 mice per group), *p < 0.05; ****p < 0.001 comparing PBS-group (white bars) and ECN-group (black bars).

Figure 2

MG1655-treated mice are not protected from severe experimental autoimmune encephalomyelitis (EAE). (A) Daily clinical score monitoring after MOG_35–55 immunization. (B) Mortality during EAE progression. (C) Onset day assessment. (D) Disease incidence at day 30 after immunization. (E) Cumulative score was calculated as the sum of all EAE-clinical scores developed during the 30 days by each mouse per group. (F) Maximal scores were determined as the mean of maximal score reached by each mouse per group. (G) Escherichia coli enumeration in feces twice a week after immunization. Data are from two independent experiments and are depicted as means ± SEM (n = 30 per group), **p < 0.01 comparing PBS-group (white bars) and MG1655-group (gray bars).

Figure 3

ECN-treated mice exhibit reduced migration of CD4⁺ T cell from the periphery to the central nervous system during the acute phase of experimental autoimmune encephalomyelitis. Absolute number of TCRαβ⁺CD4⁺ and TCRαβ⁺CD4⁺ MOG-specific Tetramer⁺ T cells isolated from the spinal cord (A), draining lymph nodes (B), mesenteric lymph nodes (C), and cervical lymph nodes (D) 14 days after MOG_35–55 immunization. Data are from three independent experiments and are depicted as means ± SEM (n = 20 per group for total CD4⁺ T cells and n = 5 per group for Tetramer⁺ T cells), *p < 0.05; **p < 0.01; ***p < 0.001 comparing PBS-groups (white bars) and ECN-groups (black bars).

Figure 4

MOG-specific T cells present altered cytokine production in ECN-treated mice. Draining lymph node cells collected on day 14 after MOG_35–55 immunization from PBS- or ECN-treated mice were stimulated in vitro with MOG_35–55. Supernatants were collected after 72 h, and the secretion of IFN-γ (A), GM-CSF (B), IL-17 (C), TNF (D), and IL-10 (E) was determined by ELISA or CBA. (F) Absolute number of TCRαβ⁺CD4⁺CD25⁺FoxP3⁺ regulatory T cells isolated from draining lymph nodes. Results are from three independent experiments and are depicted as means ± SEM (n = 12–14 per group for cytokines and n = 10–15 for FoxP3⁺ T cells). *p < 0.05; **p < 0.01 comparing PBS-group (white bars) and ECN-group (black bars).

Figure 5

Experimental autoimmune encephalomyelitis (EAE) severity is associated with increased intestinal permeability prevented by ECN treatment. FD4 recovery in the serum at day 0, 7, and 14 after MOG_35–55 immunization of mice daily treated with PBS (A). Data are from three independent experiments and are expressed as means ± SEM (n = 20 per group), *p < 0.05; **p < 0.01; ***p < 0.001. Correlation between EAE-clinical scores and intestinal permeability of the colon (B) and the ileum (C) at day 14 after MOG_35–55 immunization of mice daily treated with PBS. (D) FD4 recovery in the serum at day 7 and 14 after MOG_35–55 immunization of mice treated daily with PBS (white), ECN (black), or MG1655 (gray). Colon (E) and ileum (F) biopsies were mounted in Ussing chambers at day 7 and 14 after MOG_35–55 immunization, and paracellular permeability was monitored. Data are from two independent experiments and are expressed as means ± SEM (n = 8 per group), *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 6

ECN treatment protects from experimental autoimmune encephalomyelitis-mediated alteration of the intestinal barrier function. Real-time PCR analysis of Reg3γ (A), Reg3β (B), Claudin-8 (C), ZO-1 (D), and IL-6 (E) in the ileum mucosa 14 after MOG_35–55 immunization of mice daily treated with PBS, ECN or left unimmunized. Data are from two independent experiments and are expressed as means ± SEM (n = 8 per group), *p < 0.05; **p < 0.01; ***p < 0.001; comparing PBS-groups (white bars), ECN-groups (black bars), and unimmunized group (dashed bars).