Demyelination and axonal damage are critical processes in the pathogenesis of multiple sclerosis (MS). Oxidative stress and pro-inflammatory cytokines elicited by inflammation mediates tissue damage. To monitor the demyelination and axonal injury associated with microglia activation we employed a model using cerebellar organotypic cultures stimulated with lipopolysaccharide (LPS). Microglia activated by LPS released pro-inflammatory cytokines (IL-1β, IL-6 and TNFα), and increased the expression of inducible nitric oxide synthase (iNOS) and production of reactive oxygen species (ROS). This activation was associated with demyelination and axonal damage in cerebellar cultures. Axonal damage, as revealed by the presence of non-phosphorylated neurofilaments, mitochondrial accumulation in axonal spheroids, and axonal transection, was associated with stronger iNOS expression and concomitant increases in ROS. Moreover, we analyzed the contribution of pro-inflammatory cytokines and oxidative stress in demyelination and axonal degeneration using the iNOS inhibitor ethyl pyruvate, a free-scavenger and xanthine oxidase inhibitor allopurinol, as well as via blockage of pro-inflammatory cytokines using a Fc-TNFR1 construct. We found that blocking microglia activation with ethyl pyruvate or allopurinol significantly decreased axonal damage, and to a lesser extent, demyelination. Blocking TNFα significantly decreased demyelination but did not prevented axonal damage. Moreover, the most common therapy for MS, interferon-beta, was used as an example of an immunomodulator compound that can be tested in this model. In vitro, interferon-beta treatment decreased oxidative stress (iNOS and ROS levels) and the release of pro-inflammatory cytokines after LPS stimulation, reducing axonal damage. The model of neuroinflammation using cerebellar culture stimulated with endotoxin mimicked myelin and axonal damage mediated by the combination of oxidative stress and pro-inflammatory cytokines. This model may both facilitate understanding of the events involved in neuroinflammation and aid in the development of neuroprotective therapies for the treatment of MS and other neurodegenerative diseases.

Primary progressive multiple sclerosis is a poorly understood disease entity with no specific prognostic biomarkers and scarce therapeutic options. We aimed to identify disease activity biomarkers in multiple sclerosis by performing an RNA sequencing approach in peripheral blood mononuclear cells from a discovery cohort of 44 untreated patients with multiple sclerosis belonging to different clinical forms and activity phases of the disease, and 12 healthy control subjects. A validation cohort of 58 patients with multiple sclerosis and 26 healthy control subjects was included in the study to replicate the RNA sequencing findings. The RNA sequencing revealed an interleukin 1 beta (IL1B) signature in patients with primary progressive multiple sclerosis. Subsequent immunophenotyping pointed to blood monocytes as responsible for the IL1B signature observed in this group of patients. Functional experiments at baseline measuring apoptosis-associated speck-like protein containing a CARD (ASC) speck formation showed that the NOD-leucine rich repeat and pyrin containing protein 3 (NLRP3) inflammasome was overactive in monocytes from patients with primary progressive multiple sclerosis, and canonical NLRP3 inflammasome activation with a combination of ATP plus lipopolysaccharide was associated with increased IL1B production in this group of patients. Primary progressive multiple sclerosis patients with high IL1B gene expression levels in peripheral blood mononuclear cells progressed significantly faster compared to patients with low IL1B levels based on the time to reach an EDSS of 6.0 and the Multiple Sclerosis Severity Score. In agreement with peripheral blood findings, both NLRP3 and IL1B expression in brain tissue from patients with primary progressive multiple sclerosis was mainly restricted to cells of myeloid lineage. Treatment of mice with a specific NLRP3 inflammasome inhibitor attenuated established experimental autoimmune encephalomyelitis disease severity and improved CNS histopathology. NLRP3 inflammasome-specific inhibition was also effective in reducing axonal damage in a model of lipopolysaccharide-neuroinflammation using organotypic cerebellar cultures. Altogether, these results point to a role of IL1B and the NLRP3 inflammasome as prognostic biomarker and potential therapeutic target, respectively, in patients with primary progressive multiple sclerosis.

Genome-wide association studies (GWAS) have revealed that different diseases share susceptibility variants. Twelve single-nucleotide polymorphisms (SNPs) previously associated with different immune-mediated diseases in GWAS were genotyped in a Caucasian Spanish population of 2864 multiple sclerosis (MS) patients and 2930 controls. Three SNPs were found to be associated with MS: rs1678542 in KIF5A (P=0.001, odds ratio (OR)=1.13, 95% confidence interval (CI)=1.05–1.23); rs3184504 in SH2B3 (P=0.00001, OR=1.19, 95% CI=1.10–1.27) and rs763361 in CD226 (P=0.00007, OR=1.16, 95%CI=1.08–1.25). These variants have previously been associated with rheumatoid arthritis and type 1 diabetes. The SH2B3 polymorphism has additionally been associated with systemic lupus erythematosus. Our results, in addition to validating some of these loci as risk factors for MS, are consistent with shared genetic mechanisms underlying different immune-mediated diseases. These data may help to shape the contribution of each pathway to different disorders.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• 6‐Mercaptopurine (6‐MP) and azathioprine (AZA) are both inactive prodrugs that require intracellular activation into the active 6‐thioguanine nucleotides (6‐TGNs).• This metabolic process undergoes three different competitive pathways that are catalysed by three different enzymes; xanthine oxidase (XO), thiopurine methyltransferase (TPMT) and inosine triphosphatase (ITPA), all of which exhibit genetic polymorphisms.• Although the impact of genetic variation in theTPMTgene on treatment outcome and toxicity has been demonstrated, the role of other polymorphisms remains less well known.WHAT THIS STUDY ADDS• New information on the allelic variation of these three enzymes (XO, TPMT and ITPA) and their influence on 6‐MP/AZA metabolism and toxicity.• Confirmation of the association ofTPMTpolymorphism with haematological toxicity.• Identified potential genetic characteristics that may contribute to higher risk of adverse events (such asITPA IVS2+21A→Cmutation).AIMS: To examine the allelic variation of three enzymes involved in 6‐mercaptopurine/azathioprine (6‐MP/AZA) metabolism and evaluate the influence of these polymorphisms on toxicity, haematological parameters and metabolite levels in patients with acute lymphoblastic leukaemia (ALL) or inflammatory bowel disease (IBD).METHODS: Clinical data and blood samples were collected from 19 ALL paediatric patients and 35 IBD patients who were receiving 6‐MP/AZA therapy. All patients were screened for seven genetic polymorphisms in three enzymes involved in mercaptopurine metabolism [xanthine oxidase,inosine triphosphatase(C94→AandIVS2+21A→C) andthiopurine methyltransferase]. Erythrocyte and plasma metabolite concentrations were also determined. The associations between the various genotypes and myelotoxicity, haematological parameters and metabolite concentrations were determined.RESULTS: Thiopurine methyltransferasevariant alleles were associated with a preferential metabolism away from 6‐methylmercaptopurine nucleotides (P = 0.008 in ALL patients,P = 0.038 in IBD patients) favouring 6‐thioguanine nucleotides (6‐TGNs) (P = 0.021 in ALL patients). Interestingly, carriers ofinosine triphosphatase IVS2+21A→Cvariants among ALL and IBD patients had significantly higher concentrations of the active cytotoxic metabolites, 6‐TGNs (P = 0.008 in ALL patients,P = 0.047 in IBD patients). The study confirmed the association ofthiopurine methyltransferaseheterozygosity with leucopenia and neutropenia in ALL patients and reported a significant association betweeninosine triphosphatase IVS2+21A→Cvariants with thrombocytopenia (P = 0.012).CONCLUSIONS: Pharmacogenetic polymorphisms in the 6‐MP pathway may help identify patients at risk for associated toxicities and may serve as a guide for dose individualization.