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

Abstract

Purpose: 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.

Keywords: IFNAR; alternative splicing; interferon beta; multiple sclerosis; soluble receptors.

Figure 1

IFN-β levels and longitudinal assessment of serum sIFNAR2 levels in MS patients. (A) Serum IFN-β levels measured in MS patients basally and after IFN-β therapy onset (N=59). Significant p-value is shown with asterisk. (B) Graphs comparing serum sIFNAR2 levels at baseline and after 6 and 12 months of IFN-β therapy onset in two independent cohorts (Cohort 1 N=59, Cohort 2 N=35). Each point represents an individual, and horizontal bars indicate the median values and interquartile ranges. Significant p-values are shown with asterisk.

Figure 2

Longitudinal assessment of serum sIFNAR2 levels in responder and non-responder patients. Graphs comparing serum sIFNAR2 levels at baseline and after 6 and 12 months of IFN-β therapy onset in responder (N=22) and non-responder patients (N=19). Each point represents an individual, and horizontal bars indicate the median values and interquartile ranges. Significant p-values are shown with asterisk.

Figure 3

Longitudinal assessment of IFNAR1, IFNAR2, sIFNAR2 and MxA gene expression. RNA relative expression of IFNAR1, IFNAR2, sIFNAR2 and MxA in PBMC from MS patients before and after 6 months of IFN-β therapy onset (N=41), assessed by real time-PCR using GAPDH as raeference gene. Significant p-values are shown with asterisk.

Figure 4

IFNAR2 splice variants under IFN-β treatment. (A) Correlation analysis between the transmembrane (IFNAR2) and soluble (sIFNAR2) transcripts before (r=0.636 p< 0.001) and after 6 months of IFN-β therapy onset (r=0.749 p<0.001). (B) Comparison of the relative expression of IFNAR2 versus sIFNAR2 before and after 6 months of IFN-β therapy onset, using GAPDH as a raeference gene (N=41). Significant p-values are shown with asterisk.

Figure 5

Longitudinal assessment of IFNAR1, IFNAR2, sIFNAR2 and MxA gene expression in responder and non-responder patients. (A) Heat map representing the gene expression data of IFNAR1, IFNAR2, sIFNAR2, and MxA between responder and non-responder patients, at baseline and 6 months after the onset of IFN-β therapy. Unchanged proteins are displayed in black, up-regulated proteins are displayed in green while the down-regulated proteins are depicted in red. (B) Bar chart representing the gene expression for responders (N=22) and non-responders (N=19) using GAPDH as raeference gene. Significant p-values are shown with asterisk.

Figure 6

Longitudinal assessment of PSEN1, PSEN2, and ADAM17 gene expression. RNA relative expression of PSEN1, PSEN2, and ADAM17 in PBMC from MS patients before and after 6 months of IFN-β treatment onset (N=41), assessed by real time-PCR using GAPDH as raeference gene. Significant p-values are shown with an asterisk. Logarithms are used to normalize and scale data for representation.

Figure 7

In vitro study of IFNAR1, IFNAR2, sIFNAR2 and MxA gene expression. Representation of RNA relative expression of IFNAR1, IFNAR2, sIFNAR2 and MxA in PBMC from untreated MS patients cultured with or without IFN-β stimulus (N=7) during 8 and 24 hours. (A) sIFNAR2 expression in PBMC from each patient after 24 hours of culture in the presence or in the absence of IFN-β. (B) Boxplots comparing sIFNAR2 and MxA expression in unstimulated PBMC and IFN-β-stimulated PBMC during 8 and 24 hours of culture. Significant p values are marked with an asterisk. Logarithms are used to normalize and scale data for representation.