The Lactic Acid Bacterium Pediococcus acidilactici Suppresses Autoimmune Encephalomyelitis by Inducing IL-10-Producing Regulatory T Cells

BACKGROUND: Certain intestinal microflora are thought to regulate the systemic immune response. Lactic acid bacteria are one of the most studied bacteria in terms of their beneficial effects on health and autoimmune diseases; one of which is Multiple sclerosis (MS) which affects the central nervous system. We investigated whether the lactic acid bacterium Pediococcus acidilactici, which comprises human commensal bacteria, has beneficial effects on experimental autoimmune encephalomyelitis (EAE), an animal model of MS. METHODOLOGY/PRINCIPAL FINDINGS: P. acidilactici R037 was orally administered to EAE mice to investigate the effects of R037. R037 treatment suppressed clinical EAE severity as prophylaxis and therapy. The antigen-specific production of inflammatory cytokines was inhibited in R037-treated mice. A significant increase in the number of CD4(+) Interleukin (IL)-10-producing cells was observed in the mesenteric lymph nodes (MLNs) and spleens isolated from R037-treated naive mice, while no increase was observed in the number of these cells in the lamina propria. Because only a slight increase in the CD4(+)Foxp3(+) cells was observed in MLNs, R037 may primarily induce Foxp3(-) IL10-producing T regulatory type 1 (Tr1) cells in MLNs, which contribute to the beneficial effect of R037 on EAE. CONCLUSIONS/SIGNIFICANCE: An orally administered single strain of P. acidilactici R037 ameliorates EAE by inducing IL10-producing Tr1 cells. Our findings indicate the therapeutic potential of the oral administration of R037 for treating multiple sclerosis

Administration of R037 inhibits the secretion of inflammatory cytokines.

<p>Lymphocytes were isolated by draining lymph nodes (dLN) and spleens of C57BL/6 mice on day 11 after immunization and restimulating with MOG_35–55 for 72 h. IL-17, IFN-γ and IL-10 in the culture supernatants were assayed by ELISA. Decreased IL-17 production by both dLN cells and splenocytes and decreased IFN-γ by splenocytes were observed in the R037-treated group. Data are shown as mean + SEM from a representative of three independent experiments for 6 mice in the R037-treated and 8 in the control groups. **p≤0.01; *p≤0.05.</p

Administration of R037 induces CD4⁺ IL-10-producing T cells in mesenteric lymph nodes (MLNs) and spleen.

<p>(A) Lamina propria cells, MLNs and splenocytes were isolated from the unimmunized mice that were fed R037 for 2 weeks and after isolation, cells were stimulated with anti-CD3/anti-CD28 antibodies for 72 h in vitro. IL-17, interferon-γ (IFN-γ) and IL-10 in the supernatants were assayed by ELISA. For lamina propria, each bar indicates mean + SEM of triplicate samples from 2 mice in each group. For MLNs and splenocytes, each bar indicates mean + SEM of 8 mice in each group. Data are representative of more than three independent experiments for 8 mice each. * p≤0.05 (B) Lamina propria cells, MLNs and splenocytes were isolated from the mice that were fed R037 for 2 weeks. Intracellular staining of IL-10 and Foxp3 (B) in CD4⁺ T cells was analyzed by flow cytometry. Data are representative of 2 independent experiments.</p

<i>Pediococcus acidilactici</i> ameliorates EAE.

<p>(A) R037 (4 mg/day; n = 18) or PBS (n = 17; control) was administered daily by oral gavages to C57BL/6 mice from 14 days before immunization with MOG_35–55 until the end of the study. (B) R037 (0.8 mg/ml; n = 16) or PBS (n = 17) in a water bottle was administered to SJL/J mice from 14 days before immunization with PLP_131–151 until the end of the study. Data represent mean score + SEM from a representative of two independent experiments. *p≤0.05; **p≤0.01 (t-test).</p

Infiltration of MNCs into the spinal cord is reduced in R037-treated mice.

<p>Spinal cord sections obtained from control (A) or R037-treated (B) C57BL/6 mice on day 22 after immunization were analyzed by hematoxyline and eosin (H&E) staining. Scale bar = 250 µm. (C) Semiquantitative evaluation of the pathological scores was performed as described in the Methods section. Each bar indicates the mean pathological score + SEM of 8 mice from each group.</p

R037 relieves EAE severity in a therapeutic manner.

<p>R037 (0.8 mg/ml; n = 16) or PBS (n = 17; control) were administered in a water bottle to C57BL/6 mice from 11 days after immunization with MOG_35–55. The area under the curve (AUC) under the bar was significantly lower in R037-treated mice. Data represent mean score + SEM of two independent experiments. *p≤0.05.</p

Febuxostat ameliorates secondary progressive experimental autoimmune encephalomyelitis by restoring mitochondrial energy production in a GOT2-dependent manner

<div><p>Oxidative stress and mitochondrial dysfunction are important determinants of neurodegeneration in secondary progressive multiple sclerosis (SPMS). We previously showed that febuxostat, a xanthine oxidase inhibitor, ameliorated both relapsing-remitting and secondary progressive experimental autoimmune encephalomyelitis (EAE) by preventing neurodegeneration in mice. In this study, we investigated how febuxostat protects neuron in secondary progressive EAE. A DNA microarray analysis revealed that febuxostat treatment increased the CNS expression of several mitochondria-related genes in EAE mice, most notably including <i>GOT2</i>, which encodes glutamate oxaloacetate transaminase 2 (GOT2). GOT2 is a mitochondrial enzyme that oxidizes glutamate to produce α-ketoglutarate for the Krebs cycle, eventually leading to the production of adenosine triphosphate (ATP). Whereas GOT2 expression was decreased in the spinal cord during the chronic progressive phase of EAE, febuxostat-treated EAE mice showed increased GOT2 expression. Moreover, febuxostat treatment of Neuro2a cells <i>in vitro</i> ameliorated ATP exhaustion induced by rotenone application. The ability of febuxostat to preserve ATP production in the presence of rotenone was significantly reduced by GOT2 siRNA. GOT2-mediated ATP synthesis may be a pivotal mechanism underlying the protective effect of febuxostat against neurodegeneration in EAE. Accordingly, febuxostat may also have clinical utility as a disease-modifying drug in SPMS.</p></div

Febuxostat pretreatment increases ATP levels in Neuro2a cells incubated with rotenone.

<p>(A) Neuro2a cells were incubated with different concentrations of rotenone (Rot; 1 μM, 100 nM, 10 nM) for 6 h; then, cells were lysed and intracellular ATP was measured with a bioluminescence assay. (B) Neuro2a cells were treated with febuxostat (Fx; 100 μM, 10 μM) for 12 h and intracellular ATP was measured. (C) Neuro2a cells were pre-treated with febuxostat (10 μM) or vehicle (DMSO) for 12 h followed by incubation with rotenone (10 nM) for 6 h and intracellular ATP was measured. All experiments were performed in triplicate wells for each condition and repeated twice. The bars indicate amounts of ATP relative to the negative control. Error bars indicate standard error. * indicates P ≤ 0.05. One-way analysis of Student T-tests was used to compare two groups and a one-way analysis of variance (ANOVA) was used to compare more than two groups.</p

Febuxostat increases ATP production in mitochondrial dysfunction via GOT2.

<p>Neuro2a cells were transfected with control siRNA or GOT2 siRNA. RT-PCR analysis (A) and western blot analysis (B) confirmed the silencing of GOT2 gene and protein expression, respectively. (C) ATP production was assessed in Neuro2a cells that were transfected either with control or GOT2-specific siRNA and treated with DMSO or 10 μM of Fx for 12 h. (D) Neuro2a cells that were transfected with either control or GOT2-specific siRNA were stimulated with 10 nM of rotenone (Rt, 6 h) after pre-treatment with DMSO or 10 μM of Fx for 12 h. Data are mean ± SEM of triplicate cultures. Error bars indicate standard error. All data are representative of 3 independent experiments. * indicates P ≤ 0.05. One-way analysis of Student T-tests was used to compare two groups and a one-way analysis of variance (ANOVA) was used to compare more than two groups.</p

Febuxostat increases GOT2 expression in animal model of SPMS.

<p>(A) Representative alterations in gene expression detected by DNA microarray analysis in brains and spinal cords from mice with secondary progressive experimental autoimmune encephalomyelitis (SP-EAE) that were treated with febuxostat (Fx) or vehicle. Pink represents upregulation, while blue represents those downregulation of genes. (B) Schematic of an alternative pathway for ATP synthesis associated with GOT2 and the Krebs cycle. (C) RT-PCR analysis of GOT2 gene expression in spinal cords isolated from naïve, SP-EAE mice and SP-EAE mice treated with Fx (naïve; n = 3, EAE; n = 3, EAE+Fx; n = 3). (D) Western blot analysis of GOT2 protein expression in spinal cords isolated from naïve mice and SP-EAE mice treated with vehicle or Fx. β-actin was used as an internal control. (E) The relative expression level of GOT2 to β-actin for each depicted group (naïve, n = 3; SP-EAE, n = 3; SP-EAE+Fx, n = 3). (F) Immunohistochemical analysis of GOT2 expression in the lumbar cords isolated from naïve mice, SP-EAE mice or SP-EAE mice treated with Fx. Lines indicate borders between white matter and gray matter. Hematoxylin was used as a counterstain. The magnified image illustrates the dot-like expression of GOT2 in neuronal somata. (G) Immunofluorescent staining analysis of GOT2 and MAP2 or TOM20 in lumbar cords isolated from naïve mice indicates GOT2 is expressed in neuronal mitochondria.</p