The peripheral endocannabinoid system and its association with biomarkers of inflammation in untreated patients with multiple sclerosis.

BACKGROUND AND PURPOSE: The endocannabinoid system (ECS) has been found altered in patients with multiple sclerosis (MS). However, whether the ECS alteration is present in the early stage of MS remains unknown. First, we aimed to compare the ECS profile between newly diagnosed MS patients and healthy controls (HCs). Next, we explored the association of the ECS, biomarkers of inflammation, and clinical parameters in newly diagnosed MS patients. METHODS: Whole blood gene expression of ECS components and levels of endocannabinoids in plasma were measured by real-time quantitative polymerase chain reaction and ultra-high-pressure liquid chromatography-mass spectrometry, respectively, in 66 untreated MS patients and 46 HCs. RESULTS: No differences were found in the gene expression or plasma levels of the selected ECS components between newly diagnosed MS patients and HCs. Interferon-γ, encoded by the gene IFNG, correlated positively (ρ = 0.60) with the expression of G protein-coupled receptor 55 (GPR55), and interleukin1ß (IL1B) correlated negatively (ρ = -0.50) with cannabinoid receptor 2 (CNR2) in HCs. CONCLUSIONS: We found no alteration in the peripheral ECS between untreated patients with MS and HC. Furthermore, our results indicate that the ECS has a minor overall involvement in the early stage of MS on inflammatory markers and clinical parameters when compared with HCs.

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.

GPR15+ T cells are Th17 like, increased in smokers and associated with multiple sclerosis.

Smoking is a risk factor for the development and progression of multiple sclerosis (MS); however, the pathogenic effects of smoking are poorly understood. We studied the smoking-associated chemokine receptor-like molecule GPR15 in relation to relapsing-remitting MS (RRMS). Using microarray analyses and qPCR we found elevated GPR15 in blood cells from smokers, and increased GPR15 expression in RRMS. By flow cytometry we detected increased frequencies of GPR15 expressing T and B cells in smokers, but no difference between patients with RRMS and healthy controls. However, after cell culture with the autoantigens myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein, frequencies of MBP-reactive and non-proliferating GPR15+CD4+ T cells were increased in patients with RRMS compared with healthy controls. GPR15+CD4+ T cells produced IL-17 and were enriched in the cerebrospinal fluid (CSF). Furthermore, in the CSF of patients with RRMS, GPR15+ T cells were associated with CCR6+CXCR3+/CCR6-CXCR3+ phenotypes and correlated positively with concentrations of the newly identified GPR15-ligand (GPR15L), myelin degradation and disability. In conclusion, we have identified a proinflammatory cell type linking smoking with pathogenic immune cell functions in RRMS.

DNA methylation as a mediator of HLA-DRB1*15:01 and a protective variant in multiple sclerosis.

The human leukocyte antigen (HLA) haplotype DRB1*15:01 is the major risk factor for multiple sclerosis (MS). Here, we find that DRB1*15:01 is hypomethylated and predominantly expressed in monocytes among carriers of DRB1*15:01. A differentially methylated region (DMR) encompassing HLA-DRB1 exon 2 is particularly affected and displays methylation-sensitive regulatory properties in vitro. Causal inference and Mendelian randomization provide evidence that HLA variants mediate risk for MS via changes in the HLA-DRB1 DMR that modify HLA-DRB1 expression. Meta-analysis of 14,259 cases and 171,347 controls confirms that these variants confer risk from DRB1*15:01 and also identifies a protective variant (rs9267649, p < 3.32 × 10-8, odds ratio = 0.86) after conditioning for all MS-associated variants in the region. rs9267649 is associated with increased DNA methylation at the HLA-DRB1 DMR and reduced expression of HLA-DRB1, suggesting a modulation of the DRB1*15:01 effect. Our integrative approach provides insights into the molecular mechanisms of MS susceptibility and suggests putative therapeutic strategies targeting a methylation-mediated regulation of the major risk gene.

Smoking affects the interferon beta treatment response in multiple sclerosis.

OBJECTIVE: To investigate whether smoking in patients with relapsing-remitting multiple sclerosis (RRMS) treated with interferon beta (IFN-ß) is associated with the relapse rate and whether there is an interaction between smoking and human leukocyte antigen (HLA)-DRB1*15:01, HLA-A*02:01, and the N-acetyltransferase-1 (NAT1) variant rs7388368A. METHODS: DNA from 834 IFN-ß-treated patients with RRMS from the Danish Multiple Sclerosis Biobank was extracted for genotyping. Information about relapses from 2 years before the start of treatment to either the end of treatment or the last follow-up visit was obtained from the Danish Multiple Sclerosis Treatment Register. Smoking information came from a comprehensive questionnaire. RESULTS: We found that the relapse rate in patients with RRMS during IFN-ß treatment was higher in smokers compared to nonsmokers, with an incidence rate ratio (IRR) of 1.20 (95% confidence interval [CI] 1.021-1.416, p = 0.027) and with an IRR increase of 27% per pack of cigarettes per day (IRR 1.27, 95% CI 1.056-1.537, p = 0.012). We found no association or interaction with HLA and the NAT1 variant. CONCLUSION: In this observational cohort study, we found that smoking is associated with increased relapse activity in patients with RRMS treated with IFN-ß, but we found no association or interaction with HLA or the NAT1 variant.

Multiple sclerosis risk loci and disease severity in 7,125 individuals from 10 studies.

OBJECTIVE: We investigated the association between 52 risk variants identified through genome-wide association studies and disease severity in multiple sclerosis (MS). METHODS: Ten unique MS case data sets were analyzed. The Multiple Sclerosis Severity Score (MSSS) was calculated using the Expanded Disability Status Scale at study entry and disease duration. MSSS was considered as a continuous variable and as 2 dichotomous variables (median and extreme ends; MSSS of ≤5 vs >5 and MSSS of <2.5 vs ≥7.5, respectively). Single nucleotide polymorphisms (SNPs) were examined individually and as both combined weighted genetic risk score (wGRS) and unweighted genetic risk score (GRS) for association with disease severity. Random-effects meta-analyses were conducted and adjusted for cohort, sex, age at onset, and HLA-DRB1*15:01. RESULTS: A total of 7,125 MS cases were analyzed. The wGRS and GRS were not strongly associated with disease severity after accounting for cohort, sex, age at onset, and HLA-DRB1*15:01. After restricting analyses to cases with disease duration ≥10 years, associations were null (p value ≥0.05). No SNP was associated with disease severity after adjusting for multiple testing. CONCLUSIONS: The largest meta-analysis of established MS genetic risk variants and disease severity, to date, was performed. Results suggest that the investigated MS genetic risk variants are not associated with MSSS, even after controlling for potential confounders. Further research in large cohorts is needed to identify genetic determinants of disease severity using sensitive clinical and MRI measures, which are critical to understanding disease mechanisms and guiding development of effective treatments.

Oligoclonal band phenotypes in MS differ in their HLA class II association, while specific KIR ligands at HLA class I show association to MS in general.

Multiple sclerosis (MS) patients have been reported to have different HLA class II allele profiles depending on oligoclonal bands (OCBs) in the cerebrospinal fluid, but HLA class I alleles and killer cell immunoglobulin-like receptor (KIR) ligands have not been studied. We investigated the association of HLA alleles and KIR ligands according to OCB status in MS patients (n=3876). Specific KIR ligands were associated with patients when compared to controls (n=3148), supporting a role for NK cells in MS pathogenesis. HLA class I alleles and KIR ligands did not differ between OCB phenotypes, but HLA class II associations were convincingly replicated.

JC polyomavirus infection is strongly controlled by human leucocyte antigen class II variants.

JC polyomavirus (JCV) carriers with a compromised immune system, such as in HIV, or subjects on immune-modulating therapies, such as anti VLA-4 therapy may develop progressive multifocal leukoencephalopathy (PML) which is a lytic infection of oligodendrocytes in the brain. Serum antibodies to JCV mark infection occur only in 50-60% of infected individuals, and high JCV-antibody titers seem to increase the risk of developing PML. We here investigated the role of human leukocyte antigen (HLA), instrumental in immune defense in JCV antibody response. Anti-JCV antibody status, as a surrogate for JCV infection, were compared to HLA class I and II alleles in 1621 Scandinavian persons with MS and 1064 population-based Swedish controls and associations were replicated in 718 German persons with MS. HLA-alleles were determined by SNP imputation, sequence specific (SSP) kits and a reverse PCR sequence-specific oligonucleotide (PCR-SSO) method. An initial GWAS screen displayed a strong HLA class II region signal. The HLA-DRB1*15 haplotype was strongly negatively associated to JCV sero-status in Scandinavian MS cases (OR = 0.42, p = 7×10(-15)) and controls (OR = 0.53, p = 2×10(-5)). In contrast, the DQB1*06:03 haplotype was positively associated with JCV sero-status, in Scandinavian MS cases (OR = 1.63, p = 0.006), and controls (OR = 2.69, p = 1×10(-5)). The German dataset confirmed these findings (OR = 0.54, p = 1×10(-4) and OR = 1.58, p = 0.03 respectively for these haplotypes). HLA class II restricted immune responses, and hence CD4+ T cell immunity is pivotal for JCV infection control. Alleles within the HLA-DR1*15 haplotype are associated with a protective effect on JCV infection. Alleles within the DQB1*06:03 haplotype show an opposite association. These associations between JC virus antibody response and human leucocyte antigens supports the notion that CD4+ T cells are crucial in the immune defence to JCV and lays the ground for risk stratification for PML and development of therapy and prevention.

Association between DPP6 polymorphism and the risk of progressive multiple sclerosis in Northern and Southern Europeans.

BACKGROUND: In this study, we investigated the role of the dipeptidyl-peptidase-6 (DPP6) gene in the etiopathogenesis of progressive forms of multiple sclerosis (PrMS). This gene emerged as a candidate gene in a genome-wide association study (GWAS) performed in an Italian sample of PrMS and controls in which two SNPs located in the gene (rs6956703 and rs11767658) showed evidence of association (nominal p-value<10(-4)) (Martinelli-Boneschi et al.) [18]. Moreover, the gene is highly expressed in the central nervous system, and it has been found to be associated with sporadic cases of amyotrophic lateral sclerosis which shares some feature with PrMS. METHODS: We genotyped 19 SNPs selected using a direct and tagging approach in 244 Italian PrMS and 225 controls, and we measured the expression levels of the gene in 13 PrMS cases and 25 controls. RESULTS: Five out of 19 SNPs were found to be associated with the disease (adjusted p<0.05), and they have been tested in an independent sample of 179 primary progressive MS and 198 controls from Northern Europe. None of the SNPs was replicated, but combined analysis confirmed the presence of association for rs2046748 (p=2.5×10(-3),OR=1.82, 95%CI=1.24-2.69). CONCLUSIONS: These results, inflated by the limited sample size determined by the rarity of this condition, suggest a possible role of this gene in the susceptibility to PrMS, at least in Southern Europeans. Moreover, DPP6 was over-expressed in PrMS patients compared to controls.

Genetic association of multiple sclerosis with the marker rs391745 near the endogenous retroviral locus HERV-Fc1: analysis of disease subtypes.

We have previously described the occurrence of multiple sclerosis (MS) to be associated with human endogenous retroviruses, specifically the X-linked viral locus HERV-Fc1. The aim of this study was to investigate a possible association of the HERV-Fc1 locus with subtypes of MS. MS patients are generally subdivided into three categories: Remitting/Relapsing and Secondary Progressive, which together constitute Bout Onset MS, and Primary Progressive. In this study of 1181 MS patients and 1886 controls we found that Bout Onset MS was associated with the C-allele of the marker rs391745 near the HERV-Fc1 locus (p = 0.003), while primary progressive disease was not. The ability to see genetic differences between subtypes of MS near this gene speaks for the involvement of the virus HERV-Fc1 locus in modifying the disease course of MS.

The etiology of multiple sclerosis: genetic evidence for the involvement of the human endogenous retrovirus HERV-Fc1.

We have investigated the role of human endogenous retroviruses in multiple sclerosis by analyzing the DNA of patients and controls in 4 cohorts for associations between multiple sclerosis and polymorphisms near viral restriction genes or near endogenous retroviral loci with one or more intact or almost-intact genes. We found that SNPs in the gene TRIM5 were inversely correlated with disease. Conversely, SNPs around one retroviral locus, HERV-Fc1, showed a highly significant association with disease. The latter association was limited to a narrow region that contains no other known genes. We conclude that HERV-Fc1 and TRIM5 play a role in the etiology of multiple sclerosis. If these results are confirmed, they point to new modes of treatment for multiple sclerosis.

Polymorphisms of innate pattern recognition receptors, response to interferon-beta and development of neutralizing antibodies in multiple sclerosis patients.

BACKGROUND: Interferon-beta therapy of patients with relapsing-remitting multiple sclerosis involves repeated 'immunizations' with exogenous protein solutions. Innate pattern recognition receptors play an important role in immune responses towards foreign substances and may thus be related to treatment outcome. OBJECTIVE: To determine the genotypes at 42 single nucleotide polymorphism loci in selected pattern recognition receptors for 567 prospectively followed relapsing-remitting multiple sclerosis patients treated with recombinant interferon-beta, and test for relationships to several outcome parameters, including formation of interferon-beta neutralizing antibodies. RESULTS: The results suggest an association between the rs5743810 polymorphism (Ser249Pro) of TLR6 and development of neutralizing antibodies after 24 months of therapy in males (p = 0.00002), but not in females (p = 0.2). This association survived crude Bonferroni correction (p (corrected) = 0.02). Additional associations were observed in carriers of the TLR2-rs5743708 and NOD2-rs3135499 SNPs (time to relapse), the TLR7-rs179008 and NOD1-rs2075820 SNPs (time to disease progression) and the TLR4-rs7873784, TLR9-rs5743836, and NOD2-rs2066842 SNPs (frequency of neutralizing antibodies development). All of these, however, failed to survive correction for multiple testing. There were no significant differences between interferon-beta responders and non-responders for any of the investigated single nucleotide polymorphisms. CONCLUSIONS: The rs5743810 polymorphism of TLR6 may be involved in development of anti-interferon-beta antibodies in males, although further studies are required to firmly establish this.

A role for VAV1 in experimental autoimmune encephalomyelitis and multiple sclerosis.

Multiple sclerosis, the most common cause of progressive neurological disability in young adults, is a chronic inflammatory disease. There is solid evidence for a genetic influence in multiple sclerosis, and deciphering the causative genes could reveal key pathways influencing the disease. A genome region on rat chromosome 9 regulates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Using interval-specific congenic rat lines and association of single-nucleotide polymorphisms with inflammatory phenotypes, we localized the gene of influence to Vav1, which codes for a signal-transducing protein in leukocytes. Analysis of seven human cohorts (12,735 individuals) demonstrated an association of rs2546133-rs2617822 haplotypes in the first VAV1 intron with multiple sclerosis (CA: odds ratio, 1.18; CG: odds ratio, 0.86; TG: odds ratio, 0.90). The risk CA haplotype also predisposed for higher VAV1 messenger RNA expression. VAV1 expression was increased in individuals with multiple sclerosis and correlated with tumor necrosis factor and interferon-gamma expression in peripheral blood and cerebrospinal fluid cells. We conclude that VAV1 plays a central role in controlling central nervous system immune-mediated disease and proinflammatory cytokine production critical for disease pathogenesis.

The SH2D2A gene and susceptibility to multiple sclerosis.

We previously reported an association between the SH2D2A gene encoding TSAd and multiple sclerosis (MS). Here a total of 2128 Nordic MS patients and 2004 controls were genotyped for the SH2D2A promoter GA repeat polymorphism and rs926103 encoding a serine to asparagine substitution at amino acid position 52 in TSAd. The GA(16)-rs926103()A haplotype was associated with MS in Norwegians (OR 1.4, P=0.04). A similar trend was observed among Danes. In the independent Norwegian, Danish and Swedish sample sets the GA(16) allele showed a combined OR of 1.13, P=0.05. Thus, the present study shows that the SH2D2A gene may contribute to susceptibility to MS.