Soluble Receptor Isoform of IFN-Beta (sIFNAR2) in Multiple Sclerosis Patients and Their Association With the Clinical Response to IFN-Beta Treatment

Alternative splicing; Soluble receptors; IFNAR; Interferon beta; Multiple sclerosisEmpalmament alternatiu; Receptors solubles; IFNAR; Interferó beta; Esclerosi múltipleSplicing alternativo; Receptores solubles; IFNAR; Interferón beta; Esclerosis múltiplePurpose: Interferon beta receptor 2 subunit (IFNAR2) can be produced as a transmembrane protein, but also as a soluble form (sIFNAR2) generated by alternative splicing or proteolytic cleavage, which has both agonist and antagonist activities for IFN-β. However, its role regarding the clinical response to IFN-β for relapsing-remitting multiple sclerosis (RRMS) is unknown. We aim to evaluate the in vitro short-term effects and after 6 and 12 months of IFN-β therapy on sIFNAR2 production and their association with the clinical response in MS patients. Methods: Ninety-four RRMS patients were included and evaluated at baseline, 6 and 12 months from treatment onset. A subset of 41 patients were classified as responders and non-responders to IFN-β therapy. sIFNAR2 serum levels were measured by ELISA. mRNA expression for IFNAR1, IFNAR2 splice variants, MxA and proteases were assessed by RT-PCR. The short-term effect was evaluated in PBMC from RRMS patients after IFN-β stimulation in vitro. Results: Protein and mRNA levels of sIFNAR2 increased after IFN-β treatment. According to the clinical response, only non-responders increased sIFNAR2 significantly at both protein and mRNA levels. sIFNAR2 gene expression correlated with the transmembrane isoform expression and was 2.3-fold higher. While MxA gene expression increased significantly after treatment, IFNAR1 and IFNAR2 only slightly increased. After short-term IFN-β in vitro induction of PBMC, 6/7 patients increased the sIFNAR2 expression. Conclusions: IFN-β administration induces the production of sIFNAR2 in RRMS and higher levels might be associated to the reduction of therapeutic response. Thus, levels of sIFNAR2 could be monitored to optimize an effective response to IFN-β therapy.This research was funded by grants from the Instituto de Salud Carlos III and co-funded by European Regional Development Fund (ERDF), Technological Development Project in health DTS/1800045 to BO-M. BO-M holds a contract from Red Andaluza de Investigacion Clínica y Traslacional en Neurología (Neuro-reca) (RIC-0111-2019). PA-G is supported by Promoción de Empleo Joven e Implantación de la Garantía Juvenil 2018 (PEJ2018-002719-A). JR-B is supported by grants from Red Temática de Investigación Cooperativa, Red Española de Esclerosis Multiple REEM (RD16/0015/0010). LL holds a Nicolás Monardes research contract (RC-002-2019) from the Andalusian Ministry of Health and Family. IB-M holds a pFIS contract (FI19/00139) from the Spanish Science and Innovation Ministry

Antiviral, Immunomodulatory and Antiproliferative Activities of Recombinant Soluble IFNAR2 without IFN-beta Mediation

Soluble receptors of cytokines are able to modify cytokine activities and therefore the immune system, and some have intrinsic biological activities without mediation from their cytokines. The soluble interferon beta (IFN-ss) receptor is generated through alternative splicing of IFNAR2 and has both agonist and antagonist properties for IFN-ss, but its role is unknown. We previously demonstrated that a recombinant human soluble IFN-ss receptor showed intrinsic therapeutic efficacy in a mouse model of multiple sclerosis. Here we evaluate the potential biological activities of recombinant sIFNAR2 without the mediation of IFN-ss in human cells. Recombinant sIFNAR2 down-regulated the production of IL-17 and IFN-? and reduced the cell proliferation rate. Moreover, it showed a strong antiviral activity, fully protecting the cell monolayer after being infected by the virus. Specific inhibitors completely abrogated the antiviral activity of IFN-ss, but not that of the recombinant sIFNAR2, and there was no activation of the JAK-STAT signaling pathway. Consequently, r-sIFNAR2 exerts immunomodulatory, antiproliferative and antiviral activities without IFN-ss mediation, and could be a promising treatment against viral infections and immune-mediated diseases

Glycerol valorization by etherification to polyglycerols by using metal oxides derived from MgFe hydrotalcites

This work investigates the use of MgFe mixed oxides, derived from layered double hydroxides (LDH) with Mg/Fe molar ratio ranging from 1 to 4, as base catalysts for the etherification of glycerol. LDH precursors and catalysts were characterized by XRD, XPS, CO2-TPD, NH3-TPD, N2 adsorption and DTA–TG analysis. The MgFe mixed oxides exhibit excellent textural properties, with specific surface areas close to 200 m2 g−1 and average pore diameters in the mesoporous range. This family of catalysts has shown to be active in the formation of polyglycerols from glycerol without solvent, at 220 °C, in a batch reactor. The highest conversion (41%) is found for the MgFeO4 catalyst prepared with a Mg/Fe molar ratio of 4, whereas full selectivity to diglycerols is only reached for the MgFeO1 catalyst. Only diglycerols (DGs) and triglycerols (TGs) have been detected after 24 h of reaction.The authors are grateful to financial support from the Spanish Ministry of Science and Innovation (ENE2009-12743-C04-03 project), Junta de Andalucía (P09-FQM-5070) and FEDER funds. RMT would like to thank this Ministry of Science and Innovation for the financial support under the Program Ramón y Cajal (RYC-2008-03387).Peer Reviewe

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

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

<div><p>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 <i>in vitro</i> 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.</p></div

Cross-reactivity of antibodies against interferon beta in multiple sclerosis patients and interference of the JAK-STAT signaling pathway

Abstract Interferon beta (IFNβ) therapy has immunogenic properties and induces the development of neutralizing antibodies (NAbs). From the extensive literature focused in the development of NAbs in multiple sclerosis (MS) patients, their ability to cross-react has been deficiently evaluated, despite having important consequences in the clinical practice. Here, the relation between the cross-reactivity and the NAbs titers has been evaluated in MS patients, by inhibition of the antiviral activity of IFNβ by bioassay and through the interference with the activation of the IFNß pathway (JAK-STAT), by phosphoflow. Thus, patients with intermediate-high titers of NAbs, determined by bioassay, had a 79-fold increased risk of cross-reactivity compared to patients with low titers. The cross-reactivity is also demonstrated because NAbs positive sera were able to decrease significantly the activation of pSTAT1 achieved by other different IFNβ molecules in the cells patients. Besides, a linear relationship between the STAT1 phosphorylation and NAbs titers was found. The study demonstrates that cross-reactivity increases with the titer of antibodies, which has important implications in clinical practice when switching the treatment. The direct relationship between the NAbs titer and the activation of STAT1 suggest that its determination could be an indirect method to identify the presence of NAbs

Significant correlations between Mean Fluorescence Intensities of IFNAR1, IFNAR2, pSTAT1 and pSTAT2 in unstimulated cells (MFI_us).

<p>Comparison between responders and sub-optimal responders to IFNß therapy.</p

Differences in the expression of IFNAR1, IFNAR2, pSTAT1 and pSTAT2 in unstimulated cells between untreated and treated patients.

<p>MFI_us of IFNAR1, IFNAR2, pSTAT1 and pSTAT2 in CD4+, CD8+ and CD14+ subsets from untreated and treated patients. The Mann-Whitney U test was used for the comparison between two groups.</p

Correlations between levels of expression of the different markers in the three stimulated cell subsets (CD4+ T cells, CD8+ T cells, CD14+ monocytes) in untreated and IFNß-treated MS patients.

<p>The conditions of IFNß stimulation were 1000UI/mL, 30 min. The correlation between the log₂ [MFI_s/ MFI_us] of the different markers was assessed using the Pearson correlation coefficient.</p> <p>MFI_s: mean fluorescence intensity in IFNß-stimulated cells</p> <p>MFI_us: mean fluorescence intensity in unstimulated cells</p