Influence of Canonical and Non-Canonical IFNLR1 Isoform Expression on Interferon Lambda Signaling

Abstract

Interferon lambdas (IFNLs) are innate immune cytokines that induce antiviral cellular responses by signaling through a heterodimer composed of IL10RB and the interferon lambda receptor 1 (IFNLR1). Multiple IFNLR1 transcriptional variants are expressed in vivo and are predicted to encode distinct protein isoforms whose function is not fully established. IFNLR1 isoform 1 has the highest relative transcriptional expression and encodes the full-length functional form that supports canonical IFNL signaling. IFNLR1 isoforms 2 and 3 have lower relative expression and are predicted to encode signaling-defective proteins. To gain insight into IFNLR1 function and regulation, we explored how altering relative expression of IFNLR1 isoforms influenced the cellular response to IFNLs. To achieve this, we generated and functionally characterized stable HEK293T clones expressing doxycycline-inducible FLAG-tagged IFNLR1 isoforms. Minimal FLAG-IFNLR1 isoform 1 overexpression markedly increased IFNL3-dependent expression of antiviral and pro-inflammatory genes, a phenotype that could not be further augmented by expressing higher levels of FLAG-IFNLR1 isoform 1. Expression of low levels of FLAG-IFNLR1 isoform 2 led to partial induction of antiviral genes, but not pro-inflammatory genes, after IFNL3 treatment, a phenotype that was largely abrogated at higher FLAG-IFNLR1 isoform 2 expression levels. Expression of FLAG-IFNLR1 isoform 3 partially augmented antiviral gene expression after IFNL3 treatment. In addition, FLAG-IFNLR1 isoform 1 significantly reduced cellular sensitivity to the type-I IFN IFNA2 when overexpressed. These results identify a unique influence of canonical and non-canonical IFNLR1 isoforms on mediating the cellular response to interferons and provide insight into possible pathway regulation in vivo.

Keywords: antiviral; inflammation; innate immunity; interferon lambda receptor 1; interferons.

Figure 1

FLAG-IFNLR1 isoforms and co-receptor IL10RB. Schematic depicting the structure of each FLAG-tagged IFNLR1 isoform and IL10RB. Box1 and Box2 depict the Jak1 binding domain that is fully present in FLAG-IFNLR1 isoform 1, truncated in FLAG-IFNLR1 isoform 2, and absent in FLAG-IFNLR1 isoform 3. Image created with BioRender.com using default settings to represent a transmembrane protein.

Figure 2

Doxycycline-inducible expression of FLAG-IFNLR1 isoforms in HEK293T cells. (A,B) Dox-inducible expression of IFNLR1 isoforms in stable HEK293T clone cell lysates (A) and culture supernatant (B) was detected by western blot using anti-FLAG antibody. Predicted non-glycosylated and glycosylated proteins are denoted by red and blue arrows, respectively. (C) Percent of FLAG-positive cells after 24 h induction ±dox (100 ng/mL) followed by permeabilization (perm) or no permeabilization (non-perm), staining with anti-FLAG antibody, and analysis by flow cytometry (representative data, three independent experiments).

Figure 3

FLAG-Iso1 overexpression augments the cellular response to IFNL3. (A) Quantitation of percent pSTAT1+ in HEK293T-EV and FLAG-Iso1 cells after 0, 1, and 4 h of IFNL3 treatment (100 ng/mL) (representative data, two independent experiments). (B) HEK293T-EV and FLAG-Iso1 cells were co-transfected with plasmids encoding Firefly Luciferase under control of an ISRE promoter and Renilla Luciferase under control of a CMV promoter. Cells were treated ±dox (100 ng/mL) for 24 h prior to mock or IFNL3 (100 ng/mL) treatment for 24 h and harvested for dual luciferase assay (representative data, three independent experiments). (C) HEK293T-EV and FLAG-Iso1 cells were treated ±dox (100 ng/mL) for 24 h prior to IFNL3 treatment for 24 h (0, 1, 10, or 100 ng/mL), then harvested for qRT-PCR analysis of VIPERIN and GAPDH (representative data, four independent experiments). Statistical significance represented by lone asterisks reflect comparisons between identically treated EV and FLAG-Iso1 cells. Statistical significance represented by bars and asterisks reflect comparisons between -dox and +dox conditions within each cell line. Error bars represent standard error of the mean. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. NS = not significant.

Figure 4

FLAG-Iso2 overexpression differentially influences the cellular response to IFNL3 based on receptor abundance. (A) pSTAT1 (two independent experiments), (B) ISRE activity (five independent experiments), and (C) VIPERIN expression (three independent experiments) were analyzed as described in Figure 3. Statistical significance represented by lone asterisks reflect comparisons between identically treated EV and FLAG-Iso2 cells. Statistical significance represented by bars and asterisks reflect comparisons between −dox and +dox conditions within each cell line. Error bars represent standard error of the mean. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. NS = not significant.

Figure 5

The cellular response to IFNL3 is inversely proportional to FLAG-Iso2 abundance. Two independent FLAG-Iso2 stable lines were transfected with CMV-Renilla and ISRE-Firefly plasmids and dox-treated (dose range between 0–100 ng/mL) for 24 h. Cells were then treated with IFNL3 (100 ng/mL) for 24 h prior to dual luciferase assay. (A) FLAG-Iso2 clonal line characterized in Figure 4 and (B) an additional independent FLAG-Iso2 clonal line (representative data, two independent experiments). Statistical analysis compares IFNL3 relative to mock-treated cells at a given dox concentration. Error bars represent standard error of the mean. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. NS = not significant.

Figure 6

FLAG-Iso3 expression partially augments IFNL signaling. (A) pSTAT1 (two independent experiments), (B) ISRE activity (three independent experiments), and (C) VIPERIN expression (three independent experiments) were analyzed as in Figure 3. Statistical significance represented by lone asterisks reflect comparisons between identically treated EV and FLAG-Iso3 cells. Statistical significance represented by bars and asterisks reflect comparisons between −dox and +dox conditions within each cell line. Error bars represent standard error of the mean. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. NS = not significant.

Figure 7

FLAG-Iso1 overexpression uniquely supports expression of the pro-inflammatory gene CXCL10. HEK293T stable lines were induced ±dox (100 ng/mL) for 24 h, then mock or IFNL3 (100 ng/mL) treated for an additional 24 h, then evaluated by qRT-PCR for CXCL10 expression relative to GAPDH (representative data, two independent experiments). Error bars represent standard error of the mean. ** = p < 0.01, NS = not significant.

Figure 8

Relative expression of FLAG-IFNLR1 isoforms differentially influences IFNL3-dependent gene expression. HEK293T WT and stable lines were induced ±dox (100 ng/mL) for 24 h prior to treatment ±IFNL3 (100 ng/mL) for an additional 24 h. RNA was collected and gene counts were quantitated using NanoString analysis (nCounter Human Immunology v2 Panel). Data are shown as log transformed normalized counts of differentially expressed genes (>2-fold change in any condition).

Figure 9

Overexpression of FLAG-Iso1 partially reduces the cellular response to the type-I IFN ligand IFNA2. (A) Quantitation of percent pSTAT1+ in HEK293T-EV and FLAG-IFNLR1 isoform cells after 0, 1, and 4 h of IFNA2 treatment (100 ng/mL) (representative data, two independent experiments). (B) HEK293T stable lines were ±dox (100 ng/mL) treated for 24 h, then mock or IFNA2 (100 ng/mL) treated for 24 h, then harvested for qRT-PCR analysis of VIPERIN and GAPDH (representative data, two independent experiments). Statistical significance represented by bars and asterisks reflect comparisons between the indicated conditions. Error bars represent standard error of the mean. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. NS = not significant.