Begoña Oliver-Martos

Killer cell immunoglobulin-like receptor genes in Spanish multiple sclerosis patients

Killer cell immunoglobulin-like receptors (KIRs) are regulators of cytolytic activity of natural killer and certain T cells through interactions with human leukocyte antigen (HLA) class I ligands. KIRs have been shown to contribute to the pathogenesis of several autoimmune diseases, but their role in multiple sclerosis (MS) is still unclear. Here we determined the influence of KIR genes and their HLA class I ligands on susceptibility to MS and on the response to interferon-beta treatment in a Spanish population. KIR and HLA genotyping were performed in 200 MS patients and 200 controls. Significantly higher frequencies were found for KIR2DL5 and KIR3DS1 genes in MS patients and the carriage of the KIR2DL1 gene was associated with a higher progression index. Moreover, the frequency of the HLA-Bw4 motif was significantly reduced in MS patients. The KIR2DL1 and HLA-C2 matches were more frequent in MS patients, whereas the KIR3DL1 and HLA-Bw4 matches were more frequent in healthy controls. Nevertheless, non significant associations were found between all the KIR genes and therapeutic response to interferon-beta. Our results confirm that the carriage of HLA-Bw4 is a protective factor in MS and suggest that KIR2DL5 and KIR3DS1 may have a predisposing role in the disease.

The CD4+ T-cell subset lacking expression of the CD28 costimulatory molecule is expanded and shows a higher activation state in multiple sclerosis

Multiple sclerosis (MS) is a chronic debilitating disease, in which T-cells are considered to play a pivotal role. CD28 is the quintessential costimulatory molecule on T-cells and its expression declines progressively with repeated stimulations, leading to the generation of CD28(-) T-cells. Our aim was to examine whether CD4(+)CD28(-) T-cells were enriched in MS patients, and characterize the phenotype of this subset in MS patients and healthy controls (HC). All these changes could provide these CD4(+)CD28(-) T-cell characteristics that might be involved in the pathogenesis of MS, turning this T-cell subset into a potential target for future therapeutic strategies.

Gene expression in IFNß signalling pathway differs between monocytes, CD4 and CD8 T cells from MS patients.

IFNß exerts its activity through the interaction with IFNAR, through activation of the JAK/STAT pathway. We analyzed the changes in IFNAR1, IFNAR2, STAT1, STAT2, Tyk2, JAK1, IRF9 and MxA gene expressions after prolonged IFNß treatment, in isolated mononuclear-cell subpopulations from MS patients, by real time PCR. The effect of IFNß on gene expression differed depending on the subpopulation assessed. The data suggest that CD8+ T cells are the most influenced by prolonged IFNß therapy as IFNAR2, Tyk2, IRF9 and Jak1 expressions were decreased, whereas MxA expression was increased in these cells.

HLA alleles as biomarkers of high-titre neutralising antibodies to interferon-β therapy in multiple sclerosis

Background Recombinant interferon β (IFNβ) is a first-line therapy for relapsing-remitting multiple sclerosis (MS), with a proven effect on the inflammatory activity. Neutralising antibodies against IFNβ (NAbs) promote a loss of IFNβ bioactivity in a titre-dependent way and their development was associated with certain human leucocyte antigen (HLA) alleles. We investigated the contribution conferred by HLA alleles on the development of NAbs in independent cohorts of Southern Europe. Methods Serum NAbs from 610 MS patients with HLA-genotype data were evaluated by cytopathic effect assay: negative tests included at least one negative result (NAb titres<20 NU/mL) after 1 year treatment; NAb-titres ≥20 NU/mL were positive tests and NAb titres ≥150 NU/mL in any test were classified as high-titre positives. Results The combined presence of DRB1*07/DQA1*02 with A*26 or B*14 was found in 20% of patients with NAbs at high titres, but only in 5.4% of NAb-negative patients (p=0.00052, OR (95% CI) 4.34 (1.85 to 10.13)). The DRB1*04:01 allele was also more frequently carried by patients with high titres of NAbs (10% vs 4.5%; p=0.046, OR (95% CI) 2.38 (0.93 to 5.92)). The alleles carried at a significantly lower frequency in patients with high persistent NAbs corresponded to the A*11 allele (3.3% vs 13.8%; p=0.023, OR (95% CI) 0.22 (0.02 to 0.87)), as well as the DRB1*03/DQA1*05/DQB1*02 haplotype (16.3% vs 26.8%; p=0.02, OR (95% CI) 0.53 (0.27 to 1.03)) and the DRB1*13/DQA1*01:03/DQB1*06:03 haplotype (2.5% vs 9.1%; p=0.045, OR (95% CI) 0.25 (0.03 to 1.02)). Conclusions 50% of the studied MS patients carried some of the five independently associated HLA allele/allele combinations described in this work. This relevant percentage of patients could benefit a therapeutic decision.

TRAIL/TRAIL Receptor System and Susceptibility to Multiple Sclerosis

The TNF-related apoptosis inducing ligand (TRAIL)/TRAIL receptor system participates in crucial steps in immune cell activation or differentiation. It is able to inhibit proliferation and activation of T cells and to induce apoptosis of neurons and oligodendrocytes, and seems to be implicated in autoimmune diseases. Thus, TRAIL and TRAIL receptor genes are potential candidates for involvement in susceptibility to multiple sclerosis (MS). To test whether single-nucleotide polymorphisms (SNPs) in the human genes encoding TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 are associated with MS susceptibility, we performed a candidate gene case-control study in the Spanish population. 59 SNPs in the TRAIL and TRAIL receptor genes were analysed in 628 MS patients and 660 controls, and validated in an additional cohort of 295 MS patients and 233 controls. Despite none of the SNPs withstood the highly conservative Bonferroni correction, three SNPs showing uncorrected p values<0.05 were successfully replicated: rs4894559 in TRAIL gene, p = 9.8×10−4, OR = 1.34; rs4872077, in TRAILR-1 gene, p = 0.005, OR = 1.72; and rs1001793 in TRAILR-2 gene, p = 0.012, OR = 0.84. The combination of the alleles G/T/A in these SNPs appears to be associated with a reduced risk of developing MS (p = 2.12×10−5, OR = 0.59). These results suggest that genes of the TRAIL/TRAIL receptor system exerts a genetic influence on MS.

Candidate gene study of TRAIL and TRAIL receptors: association with response to interferon beta therapy in multiple sclerosis patients.

TRAIL and TRAIL Receptor genes have been implicated in Multiple Sclerosis pathology as well as in the response to IFN beta therapy. The objective of our study was to evaluate the association of these genes in relation to the age at disease onset (AAO) and to the clinical response upon IFN beta treatment in Spanish MS patients. We carried out a candidate gene study of TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 genes. A total of 54 SNPs were analysed in 509 MS patients under IFN beta treatment, and an additional cohort of 226 MS patients was used to validate the results. Associations of rs1047275 in TRAILR-2 and rs7011559 in TRAILR-4 genes with AAO under an additive model did not withstand Bonferroni correction. In contrast, patients with the TRAILR-1 rs20576-CC genotype showed a better clinical response to IFN beta therapy compared with patients carrying the A-allele (recessive model: p = 8.88×10−4, pc = 0.048, OR = 0.30). This SNP resulted in a non synonymous substitution of Glutamic acid to Alanine in position 228 (E228A), a change previously associated with susceptibility to different cancer types and risk of metastases, suggesting a lack of functionality of TRAILR-1. In order to unravel how this amino acid change in TRAILR-1 would affect to death signal, we performed a molecular modelling with both alleles. Neither TRAIL binding sites in the receptor nor the expression levels of TRAILR-1 in peripheral blood mononuclear cell subsets (monocytes, CD4+ and CD8+ T cells) were modified, suggesting that this SNP may be altering the death signal by some other mechanism. These findings show a role for TRAILR-1 gene variations in the clinical outcome of IFN beta therapy that might have relevance as a biomarker to predict the response to IFN beta in MS.

Activation of the JAK-STAT Signaling Pathway after In Vitro Stimulation with IFNß in Multiple Sclerosis Patients According to the Therapeutic Response to IFNß

Interferon beta (IFNß) is a common treatment used for multiple sclerosis (MS) which acts through the activation of the JAK-STAT pathway. However, this therapy is not always effective and currently there are no reliable biomarkers to predict therapeutic response. We postulate that the heterogeneity in the response to IFNß therapy could be related to differential activation patterns of the JAK-STAT signaling pathway. Our aim was to evaluate the basal levels and the short term activation of this pathway after IFNß stimulation in untreated and IFNß treated patients, as well as according to therapeutic response. Therefore, cell surface levels of IFNAR subunits (IFNAR1 and IFNAR2) and the activated forms of STAT1 and STAT2 were assessed in peripheral blood mononuclear cells from MS patients by flow cytometry. Basal levels of each of the markers strongly correlated with the expression of the others in untreated patients, but many of these correlations lost significance in treated patients and after short term activation with IFNß. Patients who had undergone IFNß treatment showed higher basal levels of IFNAR1 and pSTAT1, but a reduced response to in vitro exposure to IFNß. Conversely, untreated patients, with lower basal levels, showed a greater ability of short term activation of this pathway. Monocytes from responder patients had lower IFNAR1 levels (p = 0.039) and higher IFNAR2 levels (p = 0.035) than non-responders just after IFNß stimulation. A cluster analysis showed that levels of IFNAR1, IFNAR2 and pSTAT1-2 in monocytes grouped 13 out of 19 responder patients with a similar expression pattern, showing an association of this pattern with the phenotype of good response to IFNß (p = 0.013). Our findings suggest that an activation pattern of the IFNß signaling pathway in monocytes could be associated with a clinical phenotype of good response to IFNß treatment and that a differential modulation of the IFNAR subunits in monocytes could be related with treatment effectiveness.

Development and validation of an ELISA for quantification of soluble IFN-β receptor: assessment in multiple sclerosis.

AIM The soluble isoform of the IFN-β receptor (sIFNAR2) can bind IFN-β and modulate its activity, although its role in autoimmune diseases remains unknown. METHODS A recombinant human sIFNAR2 protein was cloned, expressed and purified after which we developed and validated an ELISA for its quantification in human serum. Serum sIFNAR2 were assessed in multiple sclerosis (MS) patients and healthy controls. RESULTS The ELISA has a dynamic range of 3.9-250 ng/ml and a detection limit of 2.44 ng/ml. Serum sIFNAR2 were significantly lower in untreated-MS patients than in healthy controls. CONCLUSION The ELISA is suitable for quantification of sIFNAR2 in serum and should facilitate the study of sIFNAR2 in neuroimmunological diseases such as MS.

Killer-cell immunoglobulin-like receptor expression on lymphocyte subsets in multiple sclerosis patients treated with interferon-β: evaluation as biomarkers for clinical response.

BackgroundBoth the adaptative and the innate immune systems interplay in multiple sclerosis (MS) pathogeny. Killer-cell immunoglobulin-like receptors (KIRs) are key regulators of the immune response, with activating and inhibitory isoforms.ObjectiveIn this study we analysed whether the expression of KIR isoforms is implicated in MS pathogenesis and in the therapeutic response to interferon (IFN)-β.MethodsPeripheral blood samples were collected from 78 IFN-β-treated MS patients and 46 healthy controls (HC). KIR expression was evaluated by flow cytometry on natural killer (NK) and T cells.ResultsThe expression of KIRs on NK cells and T lymphocytes did not differ between MS patients and HC. IFN-β therapy decreased the expression of KIR2DL1/2DS1 and increased that of KIR2DL2/3 on NK cells. This therapy also reduced KIR2DL1/2DS1, KIR2DL2/2DL3 and KIR3DL2 expression on CD8+ T cells. The baseline evaluation of the percentage of circulating CD16+ NK cells was predictive of the clinical response to IFN-β; however, response to this therapy did not appear related to KIR expression.ConclusionsThis study shows that expression of KIR isoforms on NK and T lymphocytes correlated in different ways with IFN-β therapy, suggesting that KIR dynamics may be associated with the pathways involved in the mechanisms of action of IFN-β.

Global methylation correlates with clinical status in multiple sclerosis patients in the first year of IFNbeta treatment

The alteration of DNA methylation patterns are a key component of disease onset and/or progression. Our objective was to evaluate the differences in Long Interspersed Nuclear Element-1 (LINE-1) methylation levels, as a surrogate marker of global DNA methylation, between multiple sclerosis (MS) patients and healthy controls. In addition, we assessed the association of LINE-1 methylation with clinical disease activity in patients treated with IFNbeta (IFNβ). We found that individuals with high levels of LINE-1 methylation showed 6-fold increased risk of suffering MS. Additionally, treated MS patients who bear high LINE-1 methylation levels had an 11-fold increased risk of clinical activity. Moreover, a negative correlation between treatment duration and percentage of LINE-1 methylation, that was statistically significant exclusively in the group of patients without clinical activity, was observed. Our data suggest that in MS patients, a slight global DNA hypermethylation occurs that may be related to the pathophysiology of the disease. In addition, global DNA methylation levels could play a role as a biomarker for the differential clinical response to IFNβ.