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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-&szlig, ) receptor is generated through alternative splicing of IFNAR2 and has both agonist and antagonist properties for IFN-&szlig, but its role is unknown. We previously demonstrated that a recombinant human soluble IFN-&szlig, 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-&szlig, in human cells. Recombinant sIFNAR2 down-regulated the production of IL-17 and IFN-ɣ 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-&szlig, 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-&szlig, mediation, and could be a promising treatment against viral infections and immune-mediated diseases.

Background: The soluble isoform of the interferon-β (IFN-β) receptor (sIFNAR2) could modulate the activity of both endogenous and systemically administered IFN-β. Previously, we described lower serum sIFNAR2 levels in untreated multiple sclerosis (MS) than in healthy controls (HCs). Objective: To assess sIFNAR2 levels in a new cohort of MS patients and HCs, as well as in patients with clinically isolated syndrome (CIS) and with other inflammatory neurological disorders (OIND) and to assess its ability as a diagnostic biomarker. Methods: The cross-sectional study included 148 MS (84 treatment naive and 64 treated), 87 CIS, 42 OIND, and 96 HCs. Longitudinal study included 94 MS pretreatment and after 1 year of therapy with IFN-β, glatiramer acetate (GA), or natalizumab. sIFNAR2 serum levels were measured by a quantitative ELISA developed and validated in our laboratory. Results: Naive MS and CIS patients showed significantly lower sIFNAR2 levels than HCs and OIND patients. The sensitivity and specificity to discriminate between MS and OIND, for a sIFNAR2 cutoff value of 122.02 ng/mL, were 70.1%, and 79.4%, respectively. sIFNAR2 increased significantly in IFN-β-treated patients during the first year of therapy in contrast to GA- and natalizumab-treated patients who showed non-significant changes. Conclusion: The results suggest that sIFNAR2 could be a potential diagnostic biomarker for MS.

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.

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. Yes

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.