3A). Accordingly, USP18 protein was detectable from 16 hours on at all time points by Western blot and IHC analyses (Figure (Figure3,3, B and C). Presumably for technical reasons, we could not detect SOCS1 or SOCS3 proteins at any time point, despite testing several different antibodies. Romidepsin FDA Figure 3 The negative regulator USP18 is continuously upregulated during the entire week after pegIFN-��2b injection. We conclude that pegIFN-��2b induces transient activation of the Jak/STAT signaling pathway in hepatocytes because of the rapid induction of SOCS1, SOCS3, and USP18 and that the signaling pathway remains refractory to ongoing stimulation by circulating pegIFN-��2b because of the persistent induction of USP18. pegIFN-��2b�Cinduced genes fall into four robust classes with distinct temporal expression patterns.
We assessed pegIFN-��2b�Cregulated gene expression with transcriptome analysis using Affymetrix U133 Plus 2.0 arrays. Pairwise comparison of pretreatment and on-treatment biopsies revealed a greater than 2-fold induction in two-thirds of samples of hundreds of genes, with a peak at 16 hours (Figure (Figure4A4A and Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI70408DS1). Likewise, up to 200 genes were downregulated (Supplemental Table 2). To gain insight into the temporal expression patterns of ISGs induced by pegIFN-��2b in the human liver, we analyzed the transcriptome data using a Bayesian clustering algorithm.
The algorithm produced four robust clusters of upregulated genes, which were termed early (144 genes), intermediate (31 genes), late (299 genes), and very late ISGs (20 genes) (Figure (Figure4B4B and Supplemental Table 1). For over 95% of all upregulated genes, the peak mRNA levels occurred 4 or 16 hours after injection, followed by a steady decline over the remaining 128 hours of treatment (Figure (Figure4B).4B). Because of the limited amount of tissue obtained by percutaneous liver biopsies, we could not comprehensively analyze ISG protein expression. We therefore measured the protein expression of three exemplary ISGs. USP18 protein expression peaked at the 16-hour time point and then gradually declined, but remained induced up to the 144-hour time point. We found that USP18 mRNA expression peaked already at 4 hours, but was also persistently induced up to the 144-hour time point (Figure (Figure3).
3). STAT1 mRNA was induced up to the 96-hour time point, whereas STAT1 protein expression was still increased at 144 hours (Supplemental Figure 1). We found a very good correlation between IP10 mRNA expression in the liver and IP-10 Batimastat protein concentration in the serum (Supplemental Figure 1). Taken together, we found a reasonably good correlation between mRNA and protein expression of ISGs in this limited set of exemplary ISGs. Figure 4 pegIFN-��2b�Cinduced genes fall into four robust classes with distinct temporal expression patterns.
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