A novel probiotic mixture exerts a therapeutic effect on experimental autoimmune encephalomyelitis mediated by IL-10 producing regulatory T cells

Abstract

Background: Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). One potential therapeutic strategy for MS is to induce regulatory cells that mediate immunological tolerance. Probiotics, including lactobacilli, are known to induce immunomodulatory activity with promising effects in inflammatory diseases. We tested the potential of various strains of lactobacilli for suppression of experimental autoimmune encephalomyelitis (EAE), an animal model of MS.

Methodology/principal findings: The preventive effects of five daily-administered strains of lactobacilli were investigated in mice developing EAE. After a primary screening, three Lactobacillus strains, L. paracasei DSM 13434, L. plantarum DSM 15312 and DSM 15313 that reduced inflammation in CNS and autoreactive T cell responses were chosen. L. paracasei and L. plantarum DSM 15312 induced CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) in mesenteric lymph nodes (MLNs) and enhanced production of serum TGF-beta1, while L. plantarum DSM 15313 increased serum IL-27 levels. Further screening of the chosen strains showed that each monostrain probiotic failed to be therapeutic in diseased mice, while a mixture of the three lactobacilli strains suppressed the progression and reversed the clinical and histological signs of EAE. The suppressive activity correlated with attenuation of pro-inflammatory Th1 and Th17 cytokines followed by IL-10 induction in MLNs, spleen and blood. Additional adoptive transfer studies demonstrated that IL-10 producing CD4(+)CD25(+) Tregs are involved in the suppressive effect induced by the lactobacilli mixture.

Conclusions/significance: Our data provide evidence showing that the therapeutic effect of the chosen mixture of probiotic lactobacilli was associated with induction of transferable tolerogenic Tregs in MLNs, but also in the periphery and the CNS, mediated through an IL-10-dependent mechanism. Our findings indicate a therapeutic potential of oral administration of a combination of probiotics and provide a more complete understanding of the host-commensal interactions that contribute to beneficial effects in autoimmune diseases.

Figure 1. Distinct probiotic strains prevents EAE and suppresses MOG-reactive T cells.

C57BL/6 mice received either L. paracasei DSM 13434, L. plantarum DSM 15312, L. plantarum DSM 15313 L. paracasei PCC 101, L. delbrueckii DSM 20081 (10⁹ cfu, daily) or vehicle as control, starting 12 days prior to immunization for EAE. (A) The mean clinical score for each group of mice (n = 8) is shown. Data are representative of three separate experiments. (B) Spleen cell cultures from the probiotic-treated or control mice were restimulated in vitro with MOG_35–55 peptide. MOG specific T cell proliferation was conducted with [³H]thymidine incorporation assay and each bar represents the mean stimulation index (± SEM) from triplicate measurements (n = 3). (C) Immunohistochemical staining for CD4⁺ T cells in sections from spinal cord isolated from L. paracasei DSM 13434-, L. plantarum DSM 15312-treated and control EAE animals at day 25 of immunization. In the histogram, each bar represents the mean percentage of stained area (± SEM). * represents a p-value≤0,05.

Figure 2. L. paracasei reduces secretion of pro-inflammatory and promotes secretion of anti-inflammatory cytokines in EAE mice.

(A–E) Spleen cell cultures from probiotic-treated or control mice were restimulated in vitro with MOG_35–55 peptide, supernatants were collected and the levels of pro-inflammatory Th1 cytokines, TNF-α and IFN-γ, and Th2 cytokines, IL-4, IL-10 and TGF-β1, were determined using ELISA kits. Data are representative of two experiments. Each bar represents mean ± SEM of three samples per group. * represents a p-value ≤0,05 and ** a p-value ≤0,01.

Figure 3. Administration of three potential probiotic strains suppresses inflammatory autoimmune disorders in mice with established EAE.

C57BL/6 mice were immunized and scored for clinical signs of EAE. The animals were orally treated, starting 14 days after the onset of EAE, with L. paracasei DSM 13434 (n = 18), a mixture of lactobacilli (Lacto-mix) containing L. paracasei DSM 13434, L. plantarum DSM 15312 and DSM 15313 (n = 18) or saline (vehicle) as control (n = 18). (A) The mean clinical score for each group of mice is shown. Data represent pooled values from two independent experiments. The presence of infiltrating CD4⁺ T cells into the spinal cord parenchyma and perivascular cuffs was assessed by immunohistochemical staining in sections from (B) control EAE and (C) Lacto-mix-treated animals after 20 days of therapeutic treatment. (D) In the histogram, each bar represents the mean percentage of stained area (± SEM) from 5 mice per treatment group. Levels of pro-inflammatory cytokines (E) TNF-α, (F) IFN-γ, (G) IL-17, and (H) anti-inflammatory IL-10 were measured in supernatants of MOG_35–55 peptide restimulated spleen cell cultures from the Lacto-mix-treated (n = 3) or control mice (n = 3), after 20 days of therapeutic treatment. Data are representative of two experiments. Each bar represents mean ± SEM of three samples per group. * represents a p-value ≤0,05 and ** a p-value ≤0,01.

Figure 4. Probiotic treatment suppresses IL-17 expression and favors Treg emergence in an IL-10-dependent manner.

C57BL/6 wild type (WT) or IL-10-KO mice were immunized and observed for clinical signs of EAE. Fourteen days after the onset of EAE, animals were orally treated with a mixture of lactobacilli (Lacto-mix) or saline as control. (A) The mean clinical score for each group of mice is shown. Data represent pooled values from two independent experiments. Histograms show the expression of (B) IL-17 and (C) IL-10 by CD4⁺ T cells freshly isolated from the brain of Lacto-mix-treated WT (thick line), IL-10 KO (dashed line), or saline-treated control mice (thin line), after 20 days of therapeutic treatment. (D) MLNs and spleens isolated from these animals were analysed by flow cytometry for expression of CD4, CD25 and Foxp3. Presented FACS plots show the percentage of CD4⁺Foxp3⁺ cells of the total CD4⁺ T cells (numbers in quadrants) in control EAE (top panel), Lacto-mix-treated WT (middle panel), or IL-10 KO (bottom panel) mice. The presence of infiltrating Foxp3⁺ cells into the cerebellum parenchyma and perivascular cuffs was assessed by immunohistochemical staining of sections from (E) control EAE, (F) Lacto-mix-treated WT, (G) Lacto-mix-treated IL-10 KO. (H) In the histogram, each bar represents the mean percentage of Foxp3 stained area (± SEM) from 3 mice per treatment group. Data are representative of two experiments. * represents a p-value ≤0,05.

Figure 5. A synergistic combination of probiotics induces Treg cell expansion and systemic IL-10 production.

Healthy C57BL/6 mice were daily fed with L. paracasei DSM 13434, L. plantarum DSM 15312, L. plantarum DSM 15313, a mixture of all three (Lacto-mix) or saline (control) during 14 days (n = 5). MLNs and spleens isolated from these animals were analysed for expression of CD3, CD4, CD25 and Foxp3 by flow cytometry. (A) The proportion of CD4⁺CD25⁺Foxp3⁺ stained cells was examined on the total gated CD3⁺ lymphocytes from MLNs (top) and spleen cells (bottom). Blood serum from these animals was analysed for different cytokines using ELISA kits. Shown are data from (B) IL-10, (C) TGF-β1 and (D) IL-27 analysis. Data are mean ± SEM and representative of two experiments. * represents a p-value ≤0,05 and ** a p-value ≤0,01.

Figure 6. Probiotic-induced Treg cells suppress EAE in an IL-10-dependent fashion.

Naïve C57BL/6 wild type (WT) or IL-10-KO mice were treated with either the probiotic Lacto-mix or saline (control) during 14 days. MLN cells were isolated from these animals and administered intravenously into recipient mice which were immunized for EAE 24 h later. (A) EAE development in recipient mice after adoptive cell transfer of total MLN cells (isolated from mice treated with either saline or Lacto-mix), MLNs (isolated from WT mice treated with Lacto-mix) depleted for CD4⁺CD25⁺ cells, isolated CD4⁺CD25⁺ cells (from MLNs of Lacto-mix treated WT mice), isolated CD4⁺CD25⁺ cells (from MLNs of Lacto-mix treated IL-10 KO mice) (n = 3−5). (B) Serum levels of IL-10 were measured by ELISA in plasma obtained from recipient animals at 28 days after cell transfer. Data are representative of two experiments. Each bar represents mean ± SEM of three samples per group. * represents a p-value ≤0,05 and ** a p-value ≤0,01.