Interferon signaling and treatment outcome in chronic hepatitis C

Abstract

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The current standard therapy for chronic hepatitis C (CHC) consists of a combination of pegylated IFN alpha (pegIFNalpha) and ribavirin. It achieves a sustained viral clearance in only 50-60% of patients. To learn more about molecular mechanisms underlying treatment failure, we investigated IFN-induced signaling in paired liver biopsies collected from CHC patients before and after administration of pegIFNalpha. In patients with a rapid virological response to treatment, pegIFNalpha induced a strong up-regulation of IFN-stimulated genes (ISGs). As shown previously, nonresponders had high expression levels of ISGs before therapy. Analysis of posttreatment biopsies of these patients revealed that pegIFNalpha did not induce expression of ISGs above the pretreatment levels. In accordance with ISG expression data, phosphorylation, DNA binding, and nuclear localization of STAT1 indicated that the IFN signaling pathway in nonresponsive patients is preactivated and refractory to further stimulation. Some features characteristic of nonresponders were more accentuated in patients infected with HCV genotypes 1 and 4 compared with genotypes 2 and 3, providing a possible explanation for the poor response of the former group to therapy. Taken together with previous findings, our data support the concept that activation of the endogenous IFN system in CHC not only is ineffective in clearing the infection but also may impede the response to therapy, most likely by inducing a refractory state of the IFN signaling pathway.

Fig. 1.

PegIFNα2b induced regulation of gene expression in liver and PBMCs. (A) Venn diagram of genes significantly (paired t test, P < 0.05) up- or down-regulated >2-fold in response to pegIFN-α2b in >50% of the 10 RR and 6 non-RR biopsy samples. (B) Venn diagram of genes significantly (paired t test, P < 0.05) up- or down-regulated >2-fold in response to pegIFN-α2b in biopsy and PBMC samples of >50% of RR patients. (C) RR up- or down-regulate significantly more genes in the liver in response to pegIFN-α2b than non-RR patients. Shown are the mean (+SEM) number of genes changed >2-fold at significance levels P < 0.01 (lanes 1, 2, 5, and 6) and P < 0.05 (lanes 3, 4, 7, and 8) in >50% of patients within each response group in liver biopsies and PBMCs.

Fig. 2.

PegIFNα2b induced gene regulation in HCV-infected patients shows major differences between livers of RR and non-RR patients. (A) Four ISGs (Viperin, Mda5/helicard, OAS1, USP18) were chosen from the list of genes significantly regulated >2-fold between B-1 and B-2 in RR patients. In the liver of non-RR patients, expression of these genes is already high before treatment (lanes 25–30) and does not further increase after pegIFNα (lanes 31–36). In RR patients, pretreatment expression (lanes 5–14) is similar to controls (lanes 1–4), and pegIFNα induces a strong up-regulation (lanes 15–24). The y axes display absolute expression values. (B) An example of a gene (CCL8) up-regulated in liver in response to pegIFN-α2b in both RR and non-RR patients. The x axis represents individual biopsy samples. Lanes 5–14 (B-1) and 15–24 (B-2) correspond to RR patients number 1–10 in Table 1 (in the same order), and lanes 25–30 (B-1) and 31–36 (B-2) correspond to non-RR patients numbers 11–16.

Fig. 3.

RT-qPCR analysis of selected ISGs. (A) The expression level of selected ISGs in pretreatment biopsies is lower in RR than in non-RR patients. (B) Expression levels of USP18 and IFI27 in pretreatment biopsies are significantly higher in patients infected with genotype 1 (GT1) compared with GT 3. (C) Expression levels of USP18 and IFI27 are higher in non-RR patients also after stratification of the patients in a GT1/4 (“difficult-to-treat”) and a GT2/3 group. In A–C, the y axis shows expression relative to that of GAPDH. The P values were obtained with the Mann–Whitney test. N = number of patients in each group.

Fig. 4.

Analysis of Jak-STAT signaling in liver biopsies. (A) STAT1 phosphorylation in extracts of liver biopsies collected before (B-1) and after (B-2) pegIFN-α2b injection. Extracts were analyzed by Western blot analysis by using antibodies specific for PY (701)-STAT1. Signals were quantified by using Odyssey Imaging Software to calculate the integrated intensity (kilo counts × mm²). The values represent the fold increase of phosphorylation in B-2 samples. RR patient numbers are shown in blue, non-RR patients in red. Blots were stripped and reprobed for total STAT1 used as a loading control for each pair of samples. (B) Representative examples of B-1 and B-2 of RR and non-RR patients. No nuclear staining is evident in pretreatment biopsies of RR patients (Pat. 4). The light-blue color of the nuclei originates from the counterstaining with hematoxylin. Four hours after pegIFNα, most hepatocytes show strong nuclear staining. In non-RR patients (Pat. 12), weak nuclear staining is already present in pretreatment biopsies, and pegIFNα induces little change in hepatocytes. The visible increased nuclear staining is confined to Kupffer cells.