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

Partial suppression of M1 microglia by Janus kinase 2 inhibitor does not protect against neurodegeneration in animal models of amyotrophic lateral sclerosis

BACKGROUND: Accumulating evidence has shown that the inflammatory process participates in the pathogenesis of amyotrophic lateral sclerosis (ALS), suggesting a therapeutic potential of anti-inflammatory agents. Janus kinase 2 (JAK2), one of the key molecules in inflammation, transduces signals downstream of various inflammatory cytokines, and some Janus kinase inhibitors have already been clinically applied to the treatment of inflammatory diseases. However, the efficacy of JAK2 inhibitors in treatment of ALS remains to be demonstrated. In this study, we examined the role of JAK2 in ALS by administering a selective JAK2 inhibitor, R723, to an animal model of ALS (mSOD1(G93A) mice). FINDINGS: Orally administered R723 had sufficient access to spinal cord tissue of mSOD1(G93A) mice and significantly reduced the number of Ly6c positive blood monocytes, as well as the expression levels of IFN-γ and nitric oxide synthase 2, inducible (iNOS) in the spinal cord tissue. R723 treatment did not alter the expression levels of Il-1β, Il-6, TNF, and NADPH oxidase 2 (NOX2), and suppressed the expression of Retnla, which is one of the markers of neuroprotective M2 microglia. As a result, R723 did not alter disease progression or survival of mSOD1(G93A) mice. CONCLUSIONS: JAK2 inhibitor was not effective against ALS symptoms in mSOD1(G93A) mice, irrespective of suppression in several inflammatory molecules. Simultaneous suppression of anti-inflammatory microglia with a failure to inhibit critical other inflammatory molecules might explain this result. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12974-014-0179-2) contains supplementary material, which is available to authorized users

¹¹C-Acetate PET Imaging in Patients with Multiple Sclerosis

<div><p>Background</p><p>Activation of glial cells is a cardinal feature in multiple sclerosis (MS) pathology, and acetate has been reported to be selectively uptaken by astrocytes in the CNS. The aim of this study was to investigate the efficacy of PET with ¹¹C-acetate for MS diagnosis.</p><p>Materials and Methods</p><p>Six patients with relapsing-remitting MS and 6 healthy volunteers (HV) were enrolled. The ¹¹C-acetate brain uptake on PET was measured in patients with MS and HV. Volume-of-interest analysis of cerebral gray and white matter based on the segmentation technique for co-registered MRI and voxel-based statistical parametric analysis were performed. Correlation between ¹¹C-acetate uptake and the lesion number in T1- and T2- weighted MR images were also assessed.</p><p>Results</p><p>The standardized uptake value (SUV) of ¹¹C-acetate was increased in both white and gray matter in MS patients compared to HV. Voxel-based statistical analysis revealed a significantly increased SUV relative to that in the bilateral thalami (SUVt) in a broad area of white matter, particularly in the subcortical white matter of MS patients. The numbers of T2 lesions and T1 black holes were significantly correlated with SUV of ¹¹C-acetate in white and gray matter.</p><p>Conclusions</p><p>The ¹¹C-acetate uptake significantly increased in MS patients and correlated to the number of MRI lesions. These preliminary data suggest that ¹¹C-acetate PET can be a useful clinical examination for MS patients.</p></div

Beneficial effects of fingolimod in MS patients with high serum Sema4A levels.

We previously demonstrated that patients with multiple sclerosis (MS) of high serum Sema4A levels are resistant to IFN-β therapy. To further elucidate the role of serum Sema4A as a biomarker for therapeutic stratification in MS patients, it is important to clarify the efficacy of other disease-modifying drugs (DMD) in those with high serum Sema4A levels. Thus, in this study we investigated whether fingolimod has beneficial effects on MS patients with high Sema4A levels. We retrospectively analyzed annualized relapse rate (ARR) and Expanded Disability Status Scale (EDSS) change in 56 relapsing-remitting multiple sclerosis (RRMS) patients who had been treated with fingolimod, including those who switched from IFN-β therapy. The levels of Sema4A in the sera were measured by sandwich ELISA. The implications of Sema4A on the efficacy of fingolimod were investigated by administering recombinant Sema4A-Fc and fingolimod to mice with experimental autoimmune encephalomyelitis (EAE). Retrospective analysis of MS cohort (17 high Sema4A and 39 low Sema4A) demonstrated the effectiveness of fingolimod in those with high serum Sema4A levels, showing reduction of ARR (from 1.21 to 0.12) and EDSS progression (from 0.50 to 0.04). Consistent with this observation, improvement in the disease severity of EAE mice receiving recombinant Sema4A-Fc was also observed after fingolimod treatment. These data suggest that fingolimod could serve as a candidate DMD for managing the disease activity of MS patients with high Sema4A levels

Correlation between ¹¹C-acetate SUV and the number of MRI lesions in patients with MS.

<p>Correlation between ¹¹C-acetate SUV in WM or GM and the number of T1 black holes (A, C) or T2 lesions (B, D) in each hemisphere of the six MS patients. SUV: standardized uptake value.</p

¹¹C-acetate CNS biodistribution.

<p>(A)Mean standardized uptake value (SUV) of each lesion. (B) Relative SUV compared to that of the thalamus (SUVt). Data are expressed as the mean ± standard error of the mean (SEM) (n = 6). The Mann–Whitney <i>U</i> test showed a significant difference in the median between the HV and MS groups (*:p<0.0055 after Bonferroni correction). HV  =  healthy volunteers, MS  =  multiple sclerosis.</p

¹¹C-acetate uptake distribution and quantification in MS patients.

<p>(A)Spatially normalized group mean images of ¹¹C-acetate SUVt automatically segmented based on MRI. VOI analysis summarizing the mean SUVt in WM (B) and GM (C), and the WM/GM SUV ratio (D) in the HV and MS groups. The identical analysis performed using spill-in-free VOIs are also shown (E–G). The p-value was calculated using the analysis of covariance to adjust the variance of age. (H) The SPM analysis result is overlaid onto the T1-weighted brain MRI template. Colored voxels indicate T-scores representing significantly increased ¹¹C-acetate uptake (SUVt) in patients with MS compared to HV patients. The spatially normalized PET images were smoothed for the analysis using a 12-mm FWHM isotropic Gaussian kernel. The significance thresholds are corrected for multiple comparisons at the cluster level with a p-value of 0.05 (family-wise error correction). SUV: standardized uptake value.</p

Xanthine Oxidase Mediates Axonal and Myelin Loss in a Murine Model of Multiple Sclerosis

<div><p>Objectives</p><p>Oxidative stress plays an important role in the pathogenesis of multiple sclerosis (MS). Though reactive oxygen species (ROS) are produced by various mechanisms, xanthine oxidase (XO) is a major enzyme generating ROS in the context of inflammation. The objectives of this study were to investigate the involvement of XO in the pathogenesis of MS and to develop a potent new therapy for MS based on the inhibition of ROS.</p><p>Methods</p><p>XO were assessed in a model of MS: experimental autoimmune encephalomyelitis (EAE). The contribution of XO-generated ROS to the pathogenesis of EAE was assessed by treating EAE mice with a novel XO inhibitor, febuxostat. The efficacy of febuxostat was also examined in <i>in vitro</i> studies.</p><p>Results</p><p>We showed for the first time that the expression and the activity of XO were increased dramatically within the central nervous system of EAE mice as compared to naïve mice. Furthermore, prophylactic administration of febuxostat, a XO inhibitor, markedly reduced the clinical signs of EAE. Both <i>in vivo</i> and <i>in vitro</i> studies showed infiltrating macrophages and microglia as the major sources of excess XO production, and febuxostat significantly suppressed ROS generation from these cells. Inflammatory cellular infiltration and glial activation in the spinal cord of EAE mice were inhibited by the treatment with febuxostat. Importantly, therapeutic efficacy was observed not only in mice with relapsing-remitting EAE but also in mice with secondary progressive EAE by preventing axonal loss and demyelination.</p><p>Conclusion</p><p>These results highlight the implication of XO in EAE pathogenesis and suggest XO as a target for MS treatment and febuxostat as a promising therapeutic option for MS neuropathology.</p></div

Febuxostat inhibits redox pathways of macrophages and microglia <i>in vitro</i>.

<p>(A, B) J774 macrophage cells were stimulated with LPS and IFN-γ or anti-CD40+ IFN-γ with or without febuxostat; (A) The supernatant was used to analyze NO expression using Griess reaction. **p≤0.01. (B) The cells were stained with anti-iNOS antibody. (C, D) Murine microglial GMI-M6-3 cells were stimulated with LPS (1 µg/ml) under various concentrations of febuxostat. (C) Concentrations of NO in the supernatant were assessed after a 24-hour incubation period. *p≤0.05, **p≤0.01. (D) The microglia incubated with LPS (control) and with LPS +100 µM of febuxostat (Fx) were stained with anti-iNOS antibody. Representative pictures of iNOS-positive cells are shown in the left panels. The right panel shows the semi-quantitative analysis of iNOS-positive cells in each group. *p≤0.05. The error bars denote standard deviations. (E) J774 macrophages (upper panels) and GMI-M6-3 microglia (lower panels) were left unstimulated (Sham) or were stimulated with IFN-γ+LPS (Control) or IFN-γ+LPS+febuxostat (Fx), then stained with XO antibody. Scale bars: 100 µm.</p