Type III Interferon Induces Distinct SOCS1 Expression Pattern that Contributes to Delayed but Prolonged Activation of Jak/STAT Signaling Pathway: Implications for Treatment Non-Response in HCV Patients

Abstract

Suppressor of cytokine signaling 1 (SOCS1) has long been thought to block type I interferon signaling. However, IFN-λ, a type III IFN with limited receptor expression in hepatic cells, efficiently inhibits HCV (Hepatitis C virus) replication in vivo with potentially less side effects than IFN-α. Previous studies demonstrated that type I and type III activated Janus kinase/signal transducer and activator of transcription (Jak/STAT) signaling pathway differently, with delayed but prolonged activation by IFN-λ stimulation compared to IFNα/β. However, the molecular mechanisms underlying this observation is not well understood. Here, we found that there are distinct differences in SOCS1 expression patterns in Huh-7.5.1 cells following stimulation with IFN-α and IFN-λ. IFN-λ induced a faster but shorter expression of SOCS1. Furthermore, we confirmed that SOCS1 over-expression abrogates anti-HCV effect of both IFN-α and IFN-λ, leading to increased HCV RNA replication in both HCV replicon cells and JFH1 HCV culture system. In line with this, SOCS1 over-expression inhibited STAT1 phosphorylation, attenuated IFN-stimulated response elements (ISRE) reporter activity, and blocked IFN-stimulated genes (ISGs) expression. Finally, we measured SOCS1 mRNA expression levels in peripheral blood mononuclear cells (PBMCs) with or without IFN-α treatment from 48 chronic hepatitis C patients and we found the baseline SOCS1 expression levels are higher in treatment non-responders than in responders before IFN-α treatment. Taken together, SOCS1 acts as a suppressor for both type I and type III IFNs and is negatively associated with sustained virological response (SVR) to IFN-based therapy in patients with HCV. More importantly, faster but shorter induction of SOCS1 by IFN-λ may contribute to delayed but prolonged activation of IFN signaling and ISG expression kinetics by type III IFN.

Fig 1. Different induction kinetics of ISGs (SOCS1, ISG15, MxA) by type I and type III IFNs.

(A) Induction by 100 IU/mL IFN-α or 50 ng/mL IFN-λ for 0, 1, 4, 8, 16, 24 and 48 hours in Huh-7.5.1. (B) Induction kinetics of ISG15 after stimulation with 100 IU/mL IFN-α or 50 ng/mL IFN-λ in the absence and presence of SOCS1 over-expression. (C) Induction kinetics of MxA after stimulation with 100 IU/mL IFN-α or 50 ng/mL IFN-λ in the absence and presence of SOCS1 over-expression. Total RNA was harvested and reverse transcribed. The levels of mRNA expression were determined by quantitative real time PCR normalized to GAPDH. Data are presented as means ± SEM, n = 3. Error bars indicate standard error of mean (SEM).

Fig 2. Over-expression of SOCS1 blocked IFN-α and IFN-λ signaling pathway.

Over-expression of SOCS1 repressed IFN-induced ISRE-luciferase activity (A), decreased IFN-α and IFN-λ induced ISG15 and MxA (B) mRNA and protein levels (C) and STAT1 phosphorylation (C) in Con1b replicon cell. (D) Western blot data analyzed by Quantity One are expressed as the means of ratios of targeted genes (pSTAT1, ISG15 and MxA) /β-actin. Con1b cells were cotransfected with pCR3.1 (mock) or pCR3.1/SOCS1, pISRE-luc and pRL-TK for 24 hours and then 100 IU/mL IFN-α and 50 ng/mL IFN-λ was added to the cells for 24 hours respectively. The firefly and Renilla luciferase activity was measured. Total RNA was harvested and reverse transcribed. Cells were transfected with pCR3.1 (mock) or pCR3.1/SOCS1 for 24 hours and then treated with 100 IU/mL IFN-α and 50 ng/mL IFN-λ respectively for 24 hours and the cells were collected. Cell lysates were harvested and the levels of mRNA expression of ISG15 and MxA were determined by quantitative real time PCR normalized to GAPDH. In addition, the cell were analyzed by immunoblotting with the indicated antibodies as described in Materials and Methods. The samples for Tyrosine phosphorylation of STAT1 (pSTAT1) were harvested after incubating with IFNs for 15 min. Shown is one representative Western blot out of three performed experiments. + with;—without. Data are presented as means ± SEM, n = 3. Error bars indicate standard error of mean (SEM). “*” means p values less than 0.05; “**” p values less than 0.01; “***” means p values less than 0.001.

Fig 3. Over-expression of SOCS1 blunted both type I and type III anti-viral activity in HCV replicon cell and HCVcc system.

Over-expression of SOCS1 inhibited IFN-α and IFN-λ antiviral effects in Con1b replicon (A) and JFH-1 HCVcc system (B). Cells were transfected with pCR3.1 (mock) or pCR3.1/SOCS1 for 24 hours and then treated with 100 IU/mL IFN-α and 50 ng/mL IFN-λ respectively for 24 hours and the cells were collected. Total RNA was harvested and reverse transcribed. The levels of mRNA expression of Con1b and JFH1 were determined by quantitative real time PCR normalized to GAPDH. Data are presented as means ± SEM, n = 3. Error bars indicate standard error of mean (SEM). “*” means p values less than 0.05; “**” p values less than 0.01.

Fig 4. Differential mRNA expression levels of SOCS1 in PBMCs from non-responders (NRs) compared to those from patients with sustained virological response (Rs).

PBMCs were cultured in vitro with/without IFN-α (500 IU/mL) treatment for 8 hours and the cells were collected. Total RNA was harvested and reverse transcribed. The levels of mRNA expression of SOCS1 were determined by quantitative real time PCR normalized to GAPDH. Data are presented as means ± SEM, n = 3. Error bars indicate standard error of mean (SEM). “*” means p values less than 0.05. “NS.” means no statistical significance.