IFN-λ4 Attenuates Antiviral Responses by Enhancing Negative Regulation of IFN Signaling

Abstract

Type III IFNs are important mediators of antiviral immunity. IFN-λ4 is a unique type III IFN because it is produced only in individuals who carry a dG allele of a genetic variant rs368234815-dG/TT. Counterintuitively, those individuals who can produce IFN-λ4, an antiviral cytokine, are also less likely to clear hepatitis C virus infection. In this study, we searched for unique functional properties of IFN-λ4 that might explain its negative effect on hepatitis C virus clearance. We used fresh primary human hepatocytes (PHHs) treated with recombinant type III IFNs or infected with Sendai virus to model acute viral infection and subsequently validated our findings in HepG2 cell line models. Endogenous IFN-λ4 protein was detectable only in Sendai virus-infected PHHs from individuals with the dG allele, where it was poorly secreted but highly functional, even at concentrations < 50 pg/ml. IFN-λ4 acted faster than other type III IFNs in inducing antiviral genes, as well as negative regulators of the IFN response, such as USP18 and SOCS1 Transient treatment of PHHs with IFN-λ4, but not IFN-λ3, caused a strong and sustained induction of SOCS1 and refractoriness to further stimulation with IFN-λ3. Our results suggest unique functional properties of IFN-λ4 that can be important in viral clearance and other clinical conditions.

Fig. 1. IFN-λ4 is produced in PHH in response to SeV infection

(A) Expression of IFNL4 and other type-III IFNs in PHH, 6 or 24 hrs post-SeV infection. Expression is presented in relation to IFNL4 genotypes - with dG allele (dG/dG, n=2 and dG/TT, n=5) vs. TT/TT, n=4; results are plotted as ΔΔCt values (log2 scale) after normalization to endogenous controls (GAPDH and ACTB) and to uninfected samples (0 hrs). Shown individual values and group means (black bars). (B) Western blotting of lysates of PHH (n=4) infected with SeV for 6 or 24 hrs, showing a band corresponding in size to glycosylated IFN-λ4. Recombinant IFN-λ4 (0.1 and 1 ng), produced in E.coli and therefore non-glycosylated, is used for comparison. Deglycosylation treatment with PNGaseF resulted in a band of ~19 kDa. M - protein size marker. Donor IDs correspond to Supplementary Table. (C) Expression of IFNL4 and SeV RNA in samples shown in (B). Gene expression is presented as ΔCt values (log2 scale) after normalization to endogenous controls (GAPDH and ACTB).

Fig. 2. IFN-λ4 activity requires secretion but at very low levels

(A) Representative flow cytometry plots for expression of IFN-λ3-GFP and IFN-λ4-GFP induced with doxycycline (Dox) for 24 hrs in corresponding stable HepG2 cell lines; expression of IFN-λ4-GFP was readily detectable, while IFN-λ3-GFP was detectable only after treatment with GolgiStop for 4 hrs to block protein secretion. (B) Kinetics of ISRE-Luc activation in stable HepG2 cells expressing IFN-λ4-GFP or IFN-λ3-GFP co-cultured in a 1:1 ratio with HepG2-ISRE-Luc cells. (C) Concentration of GFP measured by ELISA (n=2) in culture media of the experiment described in (B); shown one of two independent experiments. (D) Western blotting for GFP in culture media of stable inducible HepG2 cells expressing IFN-λ4-GFP (not detectable) or IFN-λ3-GFP (detectable, marked by arrow) from experiment described in (B). M - protein size marker. (E) The effect of secreted IFN-λ4 is tested in stable HepG2 cells expressing IFN-λ4-GFP co-cultured in a 1:1 ratio with HepG2-ISRE-Luc cells. The effect of both secreted and intracellular IFN-λ4 is tested in stable HepG2 cells expressing IFN-λ4-GFP and ISRE-Luc reporter. Cells were induced with doxycycline (Dox) for 24 hrs to express IFN-λ4-GFP in the presence of 20 μg/ml of rabbit antibodies - blocking anti-IFN-λ4 or IgG control, shown one of two independent experiments.

Fig. 3. IFN-λ4 is associated with earlier antiviral response SeV infection

(A) Expression of select ISGs and IFNs in PHH, 6 or 24 hrs post-SeV infection, presented in relation to IFNL4 genotypes, with dG allele (dG/dG, n=2 and dG/TT), n=5 vs. TT/TT, n=4. (B) Expression of SeV in corresponding samples from (A). For (A) gene expression is presented as ΔΔCt values after normalization to endogenous controls (GAPDH and ACTB) and uninfected samples (0 hrs), while for (B) expression is presented as ΔCt values after normalizing to endogenous controls only. Both ΔCt and ΔΔCt values are presented on log2 scale, shown individual values and group means (black bars). *P<0.05. LLOD, lower limit of detection.

Fig. 4. Activity of IFN-λ4 is rapid but transient compared to other type-III IFNs

Detection of the custom recombinant IFN-λ4 (350 ng) with (A) Coomassie staining and (B) Western blotting using the monoclonal mouse and rabbit α-IFN-λ4 antibodies. All methods detected a single band at ~19 KDa, which corresponds to the estimated size of IFN-λ4. M indicates protein size marker. (C) Western blotting for pY701-STAT1 and STAT1 in HepG2 cells treated for 30 min with different concentrations of IFN-λ4. (D) Western blotting as in (C) but in the presence of 10 μg/ml of the blocking α-IL-10R2 mouse monoclonal (mAb) or goat polyclonal (pAb) antibodies that are expected to block IFN-λ4 signaling and decrease pY701-STAT1 expression. (E). ISRE-Luc activity in HepG2-ISRE-Luc cells treated with 11 concentrations of all four type-III IFNs in a 24-hour time course. Vertical dashed lines indicate the peak ISRE-Luc induction observed at 10 hrs. (F) The ratio of average activity at 4 hrs and (G) at 24 hrs to the peak at 10 hrs, shown for top 6 concentrations of each type-III IFN. (H). Similar stability of IFN-λ3 and IFN-λ4 demonstrated by preincubation of recombinant IFNs at 37 °C for indicated time points followed by treatment HepG2-ISRE-Luc cells for 8 hrs. ***P<0.001.

Fig. 5. IFN-λ4 shows faster activity than IFN-λ3 in a SeV infection model

(A) SeV RNA quantified by qRT-PCR and presented as Ct values (log 2 scale) in HepG2 cells, 3, 6, 12 and 24 hrs post-SeV infection (B) Change in SeV RNA in HepG2 cells infected with SeV after pretreatment with different concentrations of IFN-λ3 or IFN-λ4 for 10 hrs. SeV RNA values are normalized to expression of endogenous controls (GAPDH and ACTB) and presented as % of SeV RNA levels in untreated cells. Shown one of two independent experiments, each with three biological replicates. (C) ISRE-Luc induction in HepG2-ISRE-Luc cells treated with IFN-λ3 or IFN-λ4 in a 24-hrs time course. (D) Schematic representation of SeV infection experiment that includes pretreatment with IFN-λ3 or IFN-λ4. (E-F) SeV RNA in PHH (E) or stable HepG2 cells (F) pretreated or induced to express IFN-λ4-GFP or IFN-λ3-GFP, and then infected with SeV. Results are presented as in (B). *P<0.05, **P<0.01, ***P<0.001.

Fig. 6. Transcriptional profiling identifies ISGs differentially induced by IFN-λ3 and IFN-λ4

(A) Heatmap for expression of select ISGs measured by an antiviral qRT-PCR array in HepG2 cells treated with IFN-λ4 or IFN-λ3 for indicated time points. Red and green colors indicate higher expression of ISG induced by IFN-λ4 treatment, or IFN-λ3 treatment, respectively. (B) Expression of select ISGs in PHH obtained from three liver donors and treated with IFN-λ3 or IFN-λ4. Donor IDs correspond to Supplementary Table. (C). Expression of select ISGs in stable HepG2 cells induced to express IFN-λ3-GFP or IFN-λ4-GFP. Expression is presented as ΔΔCt values (log 2 scale) that are normalized to endogenous controls (GAPDH and ACTB) and uninfected samples (0 hrs). Plots show individual biological replicates; *P<0.05, **P<0.01, ***P<0.001.

Fig. 7. IFN-λ4 is a potent inducer of negative regulators of IFN response

(A). Western blot analysis for pY701-STAT1, STAT1, pY690-STAT2 and STAT2 in HepG2 cells and PHH treated with IFN-λ3 and IFN-λ4, shown for one of three independent experiments. (B). Expression of select transcripts in HepG2 cells and PHH treated with IFN-λ3 or IFN-λ4, and stable HepG2 cells induced to produce IFN-λ3-GFP or IFN-λ4-GFP. Gene expression is presented as ΔΔCt values (log 2 scale) that are normalized to endogenous controls (GAPDH and ACTB) and untreated samples. (C) Western blot analysis of USP18 and SOCS1 after treatment of PHH with IFN-λ3 or IFN-λ4 in a time course. Positive (+) control represents lysate of HEK293 cells transfected with SOCS1 plasmid, negative (–) control represents untransfected HEK293 cells. (D) Expression of SOCS1 in PHH treated with different concentrations of all four type-III IFNs for 8 hrs. Gene expression analyzed as described in (B). (E) Expression of select transcripts in PHH obtained from one liver donor and treated with IFN-λ3 or IFN-λ4 in different combinations. X and Y represent PHH pretreated with IFN-λ3 or IFN-λ4, respectively, before restimulating with IFN-λ3. Gene expression is analyzed as described in (B). Results represent one of three independent experiments, each in biological duplicates. (F) Western blot analysis of SOCS1 in PHH transfected with either scrambled siRNA (Scr siRNA) or SOCS1 siRNA for 48 hrs and then stimulated with IFN-λ4 for 24 hrs; 50 nM or proteasome inhibitor MG132 was added to PHH 2 hrs prior to harvesting. (G) Expression of SOCS1 and select ISGs in PHH transfected with scrambled or SOCS1 siRNA. 48 hrs after transfection, cells were treated with IFN-λ3 (20 ng/ml) or IFN-λ4 (50 ng/ml) for 24 hrs. After 24 hrs, all the samples were restimulated with IFN-λ3 (20 ng/ml) for 8 hrs, and analyzed for gene expression. Expression is presented as ΔΔCt values (log 2 scale) that are normalized to endogenous control (GAPDH) and uninfected samples (0 hrs). siRNA knock-down resulted in a decrease of SOCS1 expression by a ΔΔCt value of 1.2, which corresponds to a 2.3-fold decrease. Plots show individual biological replicates from one of two independent experiments; *P<0.05, **P<0.01, ***P<0.001.

Fig. 8. IFN-λ4 is associated with higher expression of USP18 and SOCS1 during viral infection

(A) Expression of SOCS1 and USP18 in PHH, 6 or 24 hrs post-SeV infection, presented in relation to IFNL4 genotypes, with dG allele (dG/dG, n=2 and dG/TT, n=5) vs. TT/TT, n=4, and plotted as ΔΔCt values (log2 scale) that are normalized to endogenous controls (GAPDH and ACTB) and to uninfected samples (0 hrs). *P<0.05. (B) Pre- and post- treatment liver biopsies from 17 HCV patients were analyzed for expression of MX1, ISG15, OAS2 and USP18. The results are presented as ΔCt values (log 2 scale) normalized by expression of endogenous control (GAPDH) and presented according to IFNL4 genotype groups. P-values for pre- and post-treatment expression levels are based on paired T-tests within genotype groups, and unpaired T-tests between genotype groups. Shown individual values and group means (black bars).