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

Abstract

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-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-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.

Keywords: IFNAR; immunology; interferon; soluble receptors.

Figure 1

Immunomodulatory activity of recombinant sIFNAR2. (A) Heatmaps showing the production of secreted cytokines, chemokines and growth factors in human cells in the presence of recombinant sIFNAR2. Each metabolite was normalized by the maximum value obtained. Each column represents a subject and each row represents a metabolite. A higher intensity of the green color represents a higher production of the molecule (above). Non-supervised average linkage hierarchical clustering grouped the proteins with similar expression patterns (P) and they were numbered PI, PII, PIII or PIV in each map. The clustering was executed with Genesis software (below). (B) Secreted IL-17, IFN-ɣ, and TNF-ɑ were assessed by Luminex (pg mL⁻¹).The data have been depicted in groups (as box-plots) and individually (as lines in order to show intra-individual changes). The cells exposed to recombinant sIFNAR2 were compared to the positive control (c+) (Wilcoxon Rank test). N =19 * p< 0.01. (C) Intracellular IL-17, IFN-ɣ, and TNF-ɑ in CD45+ T cells were assessed by flow cytometry. The data show the percentage of positive cells expressing IL-17, IFN-ɣ, and TNF-ɑ. The data have been depicted in groups (as box-plots) and individually (as lines in order to show intra-individual changes). The cells exposed to recombinant sIFNAR2 were compared to the positive control (c+) (Wilcoxon Rank test). N = 20 * p< 0.01. (D) Cell viability before the fixation and permeabilization of cells for the evaluation of the production of intracellular cytokines by flow cytometry using LIVE/DEAD^® Fixable Far Red. N = 20. ns: non-significant.

Figure 2

Antiproliferative activity of recombinant sIFNAR2. The graph shows the mean percentage ± s.d. confluence of the N2a cell culture over time. The % of confluence in the presence of recombinant sIFNAR2 was compared to that of the control (Wilcoxon Rank test). *** p< 0.01. Representative images of the cell culture before (above) and after 15 h of treatment (below).

Figure 3

Antiviral activity of recombinant sIFNAR2. (A) Demonstration of the antiviral activity of recombinant sIFNAR2 by the cytopathic effect test (CPE) or bioassay. The bar chart represents the mean of the percentage of viability ± s.d. in the presence of 15 30 y 60 µg mL⁻¹ of recombinant sIFNAR2 (N = 10 for 15 and 30 µg mL⁻¹ and N = 18 for 60 µg mL⁻¹), including the viral control (VC), the cellular control (CC) and the IFN-ß standard curve (from 20 to 0.16 UI mL⁻¹). Values above the red line indicate cell protection after virus infection. The % of viability, in the presence of the different concentrations of recombinant sIFNAR2 or IFN-ß, was compared to the % of viability of the virus control (Mann Whitney test). * p < 0.01. (B) Image of a representative CPE plate performed with a A549 cell line and infected with the EMC virus (above). Non-infected cells (CC), as well as those cells that remained alive after the virus infection, were stained in blue. The assay included the following conditions: IFN-ß standard curve (from 20 to 0.16 UI mL⁻¹); (1) decreased concentrations of IFN-ß (from 20 to 0.16 UI mL⁻¹) + constant recombinant sIFNAR2 (30 µg mL⁻¹); (2, 3 and 4) eight replicates of 15, 30 and 60 µg mL⁻¹, respectively, of recombinant sIFNAR2 added the day before the infection; (5) sixteen replicates with 30 µg mL⁻¹ of recombinant sIFNAR2 added at the same time as the virus (#); CC: cellular control; and VC: viral control. Below, the bar chart represents the mean of the percentage of viability ± s.d. of each condition from the CPE plate.

Figure 4

Specificity of the antiviral activity of the recombinant sIFNAR2. (A) Buffer effect. The CPE test was performed with two dilutions of the buffer (B1 and B2) without recombinant sIFNAR2. The B1 did not allow for the growth of the monolayer and the wells were almost empty due to the lower quantity of DMEM. The B2 allowed for the growth of the monolayer, but showed no protection after virus infection. (B) Irrelevant protein. The CPE test was performed in the presence of 30 µg mL⁻¹ of albumin and no protection was observed after virus infection. For both graphs, the bar chart represents the mean of the percentage of viability ± s.d. of 8 duplicates in each condition. (C) Production/purification process. The CPE test was performed with four different batches of recombinant sIFNAR2 (1, 2, 3.1 and 3.2) at 30 µg mL⁻¹. The bar chart represents the mean of the percentage of viability ± s.d. of 8 duplicates. (D) Representative dose-response curve of recombinant sIFNAR2 (from 120 to 0.9 µg mL⁻¹) in duplicate. 120 µg mL⁻¹ did not allow for the confluence of the monolayer, and the maximum antiviral effect was achieved at 60 µg mL⁻¹. The IC50 was established at 33 µg mL⁻¹. (E) Neutralizing antibodies against IFN-ß effects (NAbs). The CPE test was performed in the presence of a high titer of NAbs (917 TRU) that blocked the IFN-ß antiviral activity, but not the recombinant sIFNAR2 (at 60 µg mL⁻¹) antiviral activity. The bar chart represents the mean of the percentage of viability ± s.d. of each condition (NAbs / IFN-ß UI mL⁻¹ or sIFNAR2 µg mL⁻¹) of 8 replicates. -NAbs: serum without neutralizing antibodies. +NAbs: serum with neutralizing antibodies. (F) Presence of IFN-ß inhibitor (B18). The CPE test was performed in the presence of B18, which abrogated the IFN-ß antiviral activity, but not the recombinant sIFNAR2 antiviral activity. The bar chart represents the mean ± s.d of the percentage of viability of each condition in 8 replicates.(G)Bioassays were performed in the presence of polymyxin B (PMX-B) in order to block the residual LPS of the recombinant sIFNAR2 preparation. Polymyxin B was used at 10 and 1 µg mL⁻¹ alone or in the presence of recombinant sIFNAR2 (at 30 µg mL⁻¹). The presence of polymyxin B did not affect the antiviral activity of recombinant sIFNAR2. The bar chart represents the mean ± s.d of the percentage of viability of each condition for three independent experiments. CC: cellular control. VC: viral control. (H)Bioassay plate showing the effect of the buffer and of an irrelevant protein (albumin), as well as how NABs and B18 inhibit the antiviral activity of IFN-ß, but not the antiviral activity of recombinant sIFNAR2. (I) Bioassay plate showing that the presence of polymyxin Bdid not affect the antiviral activity of recombinant sIFNAR2. CC: cellular control. VC: viral control.

Figure 5

The activities of human recombinant sIFNAR2 are independent of the JAK-STAT signaling pathway. (A) Percentage of pSTAT1 in unstimulated (unst.) CD4+ and CD8+ human T cells and after stimulation with IFNβ or with recombinant sIFNAR2, assessed by flow cytometry (N = 5). sIFNAR2 does not induce STAT1 phosphorylation (Wilcoxon Rank test). * p< 0.01, ns. was not significant. (B) Relative MxA gene expression in PBMC. MxA expression in unstimulated PBMCs was compared to that from PBMC stimulated with IFN-ß or sIFNAR2. (Wilcoxon Rank test). *p< 0.01, ns. not significant. sIFNAR2 did not induce MxA expression. (C) Relative MxA gene expression in the Jurkat T-cell line, including inhibitor B18. Stimulation with IFN-ß increases the MxA expression, as compared to unstimulated cells, and this induction was abrogated in the presence of B18. sIFNAR2 did not induce MxA expression. (D) Differential gene expression and canonical pathway activation analyses in the presence of recombinant sIFNAR2. Gene expression was determined by RNA-seq from HeLa cells before and after stimulation with either IFN-ß or sIFNAR2. The heatmap displays the 96 statistically differentially expressed genes (fold change > 2 and p value < 0.05) identified after the comparison to untreated cells (Left). Enriched canonical pathways after IPA analysis with the corresponding statistically differentially expressed genes determined in each case. Color indicates the pathway induction (orange) or inactivation (blue) (Right).