Despite activating similar signaling cascades the type I and type III

Despite activating similar signaling cascades the type I and type III interferons (IFNs) differ in their ability to antagonize virus replication. 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 time points 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. (the gene that encodes IFN-λ3) that are strongly correlated with the GDC-0068 resolution of chronic HCV infection. With the advantages of restricted receptor expression when compared to IFN-α and stronger activity when compared to IFN-λ1 IFN-λ3 may prove itself to be a worthwhile subject to test for future HCV clinical studies. Another intriguing aspect of our findings is the difference GDC-0068 between the patterns of ISG GDC-0068 expression in IFN-α and IFN-β-treated cells. It is generally accepted that IFN-α and IFN-β are very similar both in terms of early signaling events and antiviral activity (8). However we show that with prolonged stimulation this correlation begins to break down with expression in IFN-α-treated cells decreasing rapidly after 8 hours of stimulation. This pattern is consistent for the hundreds of Rabbit Polyclonal to CAGE1. ISGs examined by microarray and broadly extends previous observations made in a limited number of genes (10). Importantly we also observed a similar pattern in human hepatocytes suggesting that in the liver IFN-β induces a stronger and more persistent response compared to IFN-α. In an effort to understand the factors that contribute to the unique pattern of IFN-α-induced ISG expression we examined the phosphorylation kinetics of STAT1 the primary transcription factor associated with type-I IFN signaling. Although we did not observe the differences in STAT1 tyrosine phosphorylation which had been previously reported (10) IFN-β gave rise to significantly GDC-0068 higher levels of STAT1 serine phosphorylation when compared to IFN-α especially at later time points. Subsequently after identifying PKC-δ as the predominant kinase involved in S727 phosphorylation we found that the inhibition of PKC-δ activity by rottlerin led to decreased S727 phosphorylation but did not negatively affect the expression GDC-0068 of the canonical ISGs represented by MX1 IRF9 IFIT3 and CXCL10. Several previous studies have asserted a positive role for S727 phosphorylation on STAT1 in type I IFN-induced gene expression (28 33 although these papers utilized an ISRE-luciferase construct to represent the typical ISG induction by type I IFNs potentially ignoring any inherent differences in the promoter regions of different ISGs. Here in our data it seems that tyrosine phosphorylation is still the dominant factor in GDC-0068 determining ISG induction. Despite this finding however it is still possible that a small subset of genes is affected by serine phosphorylation. In summary we find that treatment with the type I and type III IFNs creates a stable hierarchy of IFN-stimulated gene expression in Huh7 cells and primary hepatocytes with IFN-β being the most active followed by IFN-α IFN-λ3 IFN-λ1 and IFN-λ2. This pattern was consistent from 30 minutes up to 24 hours after stimulation. The activity of IFN-α closely resembles that of IFN-β but after 6 hours post stimulation the expression profiles of the stimulated ISGs diverge apparently due at least in part to a negative feedback mechanism. In contrast to IFN- α IFN-β or IFN-λ stimulation resulted in a similar long-lasting ISG expression increase. In conclusion our results demonstrate that IFN signaling in human hepatocytes is represented by a stable hierarchy resulting from differing cytokine activity levels correlated with STAT1 phosphorylation states. This study deepens our understanding of the dynamics.