Dynamic expression profiling of type I and type III interferon-stimulated hepatocytes reveals a stable hierarchy of gene expression

Abstract

Despite activating similar signaling cascades, the type I and type III interferons (IFNs) differ in their ability to antagonize virus replication. However, it is not clear whether these cytokines induce unique antiviral states, particularly in the liver, where the clinically important hepatitis B and C viruses cause persistent infection. Here, clustering and promoter analyses of microarray-based gene expression profiling were combined with mechanistic studies of signaling pathways to dynamically characterize the transcriptional responses induced by these cytokines in Huh7 hepatoma cells and primary human hepatocytes. Type I and III IFNs differed greatly in their level of interferon-stimulated gene (ISG) induction with a clearly detectable hierarchy (IFN-β > IFN-α > IFN-λ3 > IFN-λ1 > IFN-λ2). Notably, although the hierarchy identified varying numbers of differentially expressed genes when quantified using common statistical thresholds, further analysis of gene expression over multiple timepoints indicated that the individual IFNs do not in fact regulate unique sets of genes. The kinetic profiles of IFN-induced gene expression were also qualitatively similar with the important exception of IFN-α. While stimulation with either IFN-β or IFN-λs resulted in a similar long-lasting ISG induction, IFN-α signaling peaked early after stimulation then declined due to a negative feedback mechanism. The quantitative expression hierarchy and unique kinetics of IFN-α reveal potential specific roles for individual IFNs in the immune response, and elucidate the mechanism behind previously observed differences in IFN antiviral activity. While current clinical trials are focused on IFN-λ1 as a potential antiviral therapy, the finding that IFN-λ3 invariably possesses the highest activity among type III IFNs suggests that this cytokine may have superior clinical activity.

Figure 1. IFN treatment generates a stable hierarchy of gene expression

Huh7 cells were treated with either IFN-α, IFN-β (500 U/ml), or IFN-λ1, IFN-λ2, IFN-λ3 (10 ng/ml), and gene expression was measured at 0, 0.5, 1, 2, 4, 6, 12, and 24 hours post stimulation. a) A total of 434 probesets (rows) were found to be significantly differentially-expressed for at least one time point by at least one IFN (columns). Hierarchical clustering identified six separate groups (numbered boxes). b) The number of genes significantly induced or suppressed by individual IFNs at each time point. c) Comparison of log2 expression values for genes induced after 12 hours of stimulation with IFN-λ1, IFN-λ2 or IFN-λ3. Equivalent expression is indicated by the 1-to-1 line (dotted line), while the solid line shows the line of best fit with slope set to 1.

Figure 2. IFN treatment induces six distinct temporal expression patterns

Hierarchical clustering was carried out on the set of differentially expressed probesets using a distance matrix based on the pairwise correlation. A set of 6 groups was identified using manual analysis of the group structure (same as shown in Figure 1A). Each line indicates the average log₂ fold change for genes in each group.

Figure 3. Temporal clusters are associated with common transcription factors

a) The number of genes in each group is represented as a sum of genes previously described as confirmed ISGs (dark grey), and other genes or potential ISGs (light grey). b) Over-representation of genes containing the indicated transcription factor binding sites within each group was calculated using a hypergeometric test. The log₁₀ (p-value) is shown for significantly enriched sites.

Figure 4. RT-qPCR validation confirms unique pattern of activity for IFN-α-induced ISGs

a) Huh7 cells were treated with IFN-α, IFN-β (500 U/ml), IFN-λ1, IFN-λ2, or IFN-λ3 (10 ng/ml), and gene expression of six ISGs from groups 4, 5, and 6 were quantified using RT-qPCR (bottom), and compared to fold-changes from the microarray data (top). Huh7 cells were treated with 10, 100, 500, or 2500 U/ml of IFN-α or IFN-β (b), or with 1 or 10 ng/ml of IFN-λ1, IFN-λ2, or IFN-λ3 (c) at fixed time points, and mRNA expression of MX1, IRF9, IFIT3 and CXCL10 were quantified by RT-qPCR.

Figure 5. IFN stimulation of primary human hepatocytes confirms hierarchy of IFN activity over time

Primary human hepatocytes were seeded into collagen-coated 12-well plates before treatment with IFN-α, IFN-β (500 U/ml), IFN-λ1, IFN-λ2, or IFN-λ3 (10 ng/ml) for 0, 1, 4, 6, 12, or 24 hours. The expression of six ISGs from groups 4, 5, and 6 were quantified using RT-qPCR.

Figure 6. Type I IFNs induced ISG expression in a serine phosphorylation independent manner

a) The levels of pSTAT1 S727 were measured at various time points after exposure of Huh7 cells to IFN-α (grey) and IFN-β (black). The mean (points) ± standard error (bars) was calculated from four independent experiments. b) Cells were treated with kinase inhibitors before stimulation with IFN-α and IFN-β for 6 hours. Cell lysates were used in Western Blotting and probed for serine phosphorylation. c) The relative amounts of pSTAT1 S727 in b) were quantified and normalized to an untreated control. d) Expression of selected ISGs was measured at various time points using RT-qPCR with (black) or without (grey) the presence of rottlerin following treatment with the type I IFNs.