Changes in the Expression of TGF-Beta Regulatory Pathway Genes Induced by Vitamin D in Patients with Relapsing-Remitting Multiple Sclerosis.

Vitamin D is an environmental factor related to multiple sclerosis that plays a significant role in immune regulation. TGF-ß is a superfamily of cytokines with an important dual effect on the immune system. TGF-ß inhibits the Th1 response while facilitating the preservation of regulatory T cells (FOXP3+) in an immunoregulatory capacity. However, when IL-6 is present, it stimulates the Th17 response. Our aim was to analyze the regulatory effect of vitamin D on the in vivo TGF-ß signaling pathway in patients with relapsing-remitting multiple sclerosis (RRMS). A total of 21 patients with vitamin D levels < 30 ng/mL were recruited and supplemented with oral vitamin D. All patients were receiving disease-modifying therapy, with the majority being on natalizumab. Expression of SMAD7, ERK1, ZMIZ1, BMP2, BMPRII, BMP4, and BMP5 was measured in CD4+ lymphocytes isolated from peripheral blood at baseline and one and six months after supplementation. SMAD7 was overexpressed at six months with respect to baseline and month one. ERK1 was overexpressed at six months with respect to month one of treatment. No significant differences in expression were observed for the remaining genes. No direct correlation was found with serum vitamin D levels. BMPRII expression changed differentially in non-natalizumab- versus natalizumab-treated patients. Changes were observed in the expression of ERK1, BMP2, and BMP5 based on disease activity measured using the Rio-Score, BMP2 in patients who had relapses, and BMP5 in those whose EDSS worsened. Our results suggest indirect regulation of vitamin D in TGF-ß pathway genes in patients with RRMS.

Serum Short-Chain Fatty Acids and Associations With Inflammation in Newly Diagnosed Patients With Multiple Sclerosis and Healthy Controls.

Multiple sclerosis (MS) is a chronic immune-mediated disease characterized by demyelination and neuroaxonal damage in the central nervous system. The etiology is complex and is still not fully understood. Accumulating evidence suggests that our gut microbiota and its metabolites influence the MS pathogenesis. Short-chain fatty acids (SCFAs), such as acetate, propionate and butyrate, are metabolites produced by gut microbiota through fermentation of indigestible carbohydrates. SCFAs and kynurenine metabolites have been shown to have important immunomodulatory properties, and propionate supplementation in MS patients has been associated with long-term clinical improvement. However, the underlying mechanisms of action and its importance in MS remain incompletely understood. We analyzed serum levels of SCFAs and performed targeted metabolomics in relation to biomarkers of inflammation, and clinical and MRI measures in newly diagnosed patients with relapsing-remitting MS before their first disease modifying therapy and healthy controls (HCs). We demonstrated that serum acetate levels were nominally reduced in MS patients compared with HCs. The ratios of acetate/butyrate and acetate/(propionate + butyrate) were significantly lower in MS patients in a multivariate analysis (orthogonal partial least squares discriminant analysis; OPLS-DA). The mentioned ratios and acetate levels correlated negatively with the pro-inflammatory biomarker IFNG, indicating an inverse relation between acetate and inflammation. In contrast, the proportion of butyrate was found higher in MS patients in the multivariate analysis, and both butyrate and valerate correlated positively with proinflammatory cytokines (IFNG and TNF), suggesting complex bidirectional regulatory properties of SCFAs. Branched SCFAs were inversely correlated with clinical disability, at a nominal significance level. Otherwise SCFAs did not correlate with clinical variables or MRI measures. There were signs of an alteration of the kynurenine pathway in MS, and butyrate was positively correlated with the immunomodulatory metabolite 3-hydroxyanthranilic acid. Other variables that influenced the separation between MS and HCs were NfL, ARG1 and IL1R1, D-ribose 5-phosphate, pantothenic acid and D-glucuronic acid. In conclusion, we provide novel results in this rapidly evolving field, emphasizing the complexity of the interactions between SCFAs and inflammation; therefore, further studies are required to clarify these issues before supplementation of SCFAs can be widely recommended.

A novel fumarate, isosorbide di-(methyl fumarate) (IDMF), replicates astrocyte transcriptome responses to dimethyl fumarate (DMF) but specifically down-regulates genes linked to a reactive phenotype.

Dimethyl fumarate (DMF) has emerged as a first-line treatment for the relapsing-remitting multiple sclerosis (RRMS) subtype. It is hypothesized that DMF has anti-inflammatory and antioxidant effects although mechanisms are not fully understood. This study used RNA-seq to profile gene expression responses to DMF in cultured astrocytes. Responses were compared with those of isosorbide di-(methyl fumarate) (IDMF), a newly designed fumarate that may partially replicate DMF activity with fewer adverse effects. Both compounds altered the expression of MS-associated genes, including those near MS susceptibility loci and genes dysregulated in MS patient astrocytes. The shared DMF/IDMF transcriptome response involved altered expression of antioxidant genes (e.g., HMOX1) and genes linked to extracellular matrix integrity (TIMP3, MMP9) and migration of pro-inflammatory cells into CNS (CCL2). IDMF-specific transcriptome responses included down-regulation of mitotic genes associated with a proliferative reactive astrocyte phenotype (ICAM1) and repression of genes encoding NF-kappaB subunits (NFKB2, RELA, RELB) and NF-kappaB targets (NCAPG, CXCL1, OAS3). Overall, these results identify astrocyte-centered mechanisms that may contribute to the established efficacy of DMF as an RRMS treatment. Furthermore, our findings support a rationale for further studies of IDMF as a novel fumarate, which may have unique suppressive effects on astrocyte reactivity and glial scar formation. [200 words].

Zinc-ion binding and cytokine activity regulation pathways predicts outcome in relapsing-remitting multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disease characterized by an unpredictable clinical course with intermittent relapses that lead over time to significant neurological disability. Clinical and radiological variables are limited in the ability to predict disease course. Peripheral blood genome scale analyses were used to characterize MS patients with different disease types, but not for prediction of outcome. Using complementary-DNA microarrays we studied peripheral-blood gene expression patterns in 53 relapsing-remitting MS patients. Patients were classified into good, intermediate and poor clinical outcome established after 2-year follow-up. A training set of 26 samples was used to identify clinical outcome differentiating gene-expression signature. Supervised learning and feature selection algorithms were applied to identify a predictive signature that was validated in an independent group of 27 patients. Key genes within the predictive signature were confirmed by quantitative reverse transcription-polymerase chain reaction in an additional 10 patients. The analysis identified 431 differentiating genes between patients with good and poor clinical outcome (change in neurological disability by the expanded disability status scale was -0.33 +/- 0.24 and 1.6 +/- 0.35, P = 0.0002, total number of relapses were 0 and 1.80 +/- 0.35, P = 0.00009, respectively). An optimal set of 29 genes was depicted as a clinical outcome predictive gene expression signature and classified appropriately 88.9% of patients. This predictive signature was enriched by genes related biologically to zinc-ion binding and cytokine activity regulation pathways involved in inflammation and apoptosis. Our findings provide a basis for monitoring patients by prediction of disease outcome and can be incorporated into clinical decision-making in relapsing-remitting MS.