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. 2016 May 27;90(12):5549-5560.
doi: 10.1128/JVI.02557-15. Print 2016 Jun 15.

Lipopolysaccharide and Tumor Necrosis Factor Alpha Inhibit Interferon Signaling in Hepatocytes by Increasing Ubiquitin-Like Protease 18 (USP18) Expression

Sonya A MacParland  1 , Xue-Zhong Ma  1 , Limin Chen  1   2 , Ramzi Khattar  1 , Vera Cherepanov  3 , Markus Selzner  1 , Jordan J Feld  3   4 , Nazia Selzner  1 , Ian D McGilvray  5
Affiliations

Lipopolysaccharide and Tumor Necrosis Factor Alpha Inhibit Interferon Signaling in Hepatocytes by Increasing Ubiquitin-Like Protease 18 (USP18) Expression

Sonya A MacParland et al. J Virol. .

Abstract

Inflammation may be maladaptive to the control of viral infection when it impairs interferon (IFN) responses, enhancing viral replication and spread. Dysregulated immunity as a result of inappropriate innate inflammatory responses is a hallmark of chronic viral infections such as, hepatitis B virus and hepatitis C virus (HCV). Previous studies from our laboratory have shown that expression of an IFN-stimulated gene (ISG), ubiquitin-like protease (USP)18 is upregulated in chronic HCV infection, leading to impaired hepatocyte responses to IFN-α. We examined the ability of inflammatory stimuli, including tumor necrosis factor alpha (TNF-α), lipopolysaccharide (LPS), interleukin-6 (IL-6) and IL-10 to upregulate hepatocyte USP18 expression and blunt the IFN-α response. Human hepatoma cells and primary murine hepatocytes were treated with TNF-α/LPS/IL-6/IL-10 and USP18, phosphorylated (p)-STAT1 and myxovirus (influenza virus) resistance 1 (Mx1) expression was determined. Treatment of Huh7.5 cells and primary murine hepatocytes with LPS and TNF-α, but not IL-6 or IL-10, led to upregulated USP18 expression and induced an IFN-α refractory state, which was reversed by USP18 knockdown. Liver inflammation was induced in vivo using a murine model of hepatic ischemia/reperfusion injury. Hepatic ischemia/reperfusion injury led to an induction of USP18 expression in liver tissue and promotion of lymphocytic choriomeningitis replication. These data demonstrate that certain inflammatory stimuli (TNF-α and LPS) but not others (IL-6 and IL-10) _target USP18 expression and thus inhibit IFN signaling. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with USP18 representing a potential _target for intervention in various inflammatory states.

Importance: Inflammation may prevent the control of viral infection when it impairs the innate immune response, enhancing viral replication and spread. Blunted immunity as a result of inappropriate innate inflammatory responses is a common characteristic of chronic viral infections. Previous studies have shown that expression of certain interferon-stimulated genes is upregulated in chronic HCV infection, leading to impaired hepatocyte responses. In this study, we show that multiple inflammatory stimuli can modulate interferon stimulated gene expression and thus inhibit hepatocyte interferon signaling via USP18 induction. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with the induction of USP18 representing a potential _target for intervention in various inflammatory states.

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Figures

FIG 1
FIG 1
USP18 expression is upregulated by TNF-α and LPS. Huh7.5 cells were treated with LPS (100 ng/ml), TNF-α (20 ng/ml), IL-6 (100 ng/ml), IL-10 (10 ng/ml), or IFN-α (100 U/ml) for the times indicated, and ISG induction was measured by several approaches. (A and B) After stimulation with LPS, TNF-α, IL-6, IL-10, and IFN-α over a 24-h time course, RNA was harvested, and real-time PCR was performed. USP18 mRNA (A) and Mx1 mRNA (B) expression was determined and normalized to the expression of actin. Each data point was pooled from three samples. Error bars represent the standard errors of the mean (SEM) for duplicate PCRs. (C) Following stimulation with LPS, TNF-α and IFN-α for 12 h, Huh7.5 cells were lysed and USP18 protein expression was determined by Western blotting. (D and F) After stimulation with LPS, TNF-α, or SHAM for 24 h, Huh7.5 cells were fixed, permeabilized, and stained with an anti-USP18 monoclonal antibody, followed by a secondary anti-mouse FITC-labeled antibody. Representative flow plots (D) and representative histograms (E) show the degree of USP18 induction in Huh7.5 cells after LPS or TNF-α treatment. (F) Summary data showing the percentage of USP18-positive Huh7.5 cells (i) and the USP18 mean fluorescence intensity (MFI) (ii) in Huh7.5 cells after LPS or TNF-α treatment. The means ± the SEM from four independent replicates are plotted. For panels A to C, an unstimulated control (Sham) was included to identify background USP18 expression. A P value of <0.05 was considered significant.
FIG 2
FIG 2
TNF-α and LPS block IFN-α signaling in Huh7.5 hepatoma cells. Human Huh7.5 hepatoma cells were cultured in the presence or absence of LPS (100 ng/ml) or TNF-α (20 ng/ml) for 24 h and either left untreated or treated with IFN-α (100 U/ml) for 6 h (IFN-α Final). The cells were harvested, and qPCR was performed for Mx1 as an index of IFN-α signaling. The data were generated from pooled triplicate experiments analyzed in duplicate. Error bars represent the SEM for duplicate PCRs.
FIG 3
FIG 3
USP18 knockdown restores ISG induction after LPS and TNF-α stimulation. To look at the role of USP18 in the IFN-α refractory state induced by inflammatory stimuli, we examined the induction of STAT-1 phosphorylation in Huh7.5 cells with or without USP18 knockdown (A) and the expression of ISG mRNA (Mx1) in primary mouse hepatocytes and the ability of USP18 knockdown to restore ISG induction after LPS and TNF-α stimulation (B). (A) Huh7.5 cells were transfected with anti-USP18 siRNA or control irrelevant siRNA. Huh7.5 cells were then pretreated with LPS (100 ng/ml), TNF-α (20 ng/ml), or IFN-α (100 U/ml) or left untreated for 24 h and then exposed to IFN-α or left untreated for an additional 2 h. As controls, Huh7.5 cells were treated with LPS, TNF-α, and IFN-α for 2 h only. The expression of USP18, pSTAT1, STAT1, and ISG15 conjugates and actin proteins was measured by Western blotting. (B) Primary murine hepatocytes from USP18+/+ mice were isolated, transfected with anti-USP18 siRNA or control irrelevant siRNA, and pretreated with IFN-α (100 IU/ml), LPS (100 ng/ml), or TNF-α (20 ng/ml) for 24 h (IFN-α Pre, LPS Pre, or TNF-α Pre, respectively). After being washed, the cells were cultured in the presence or absence of IFN-α for 6 h (IFN-α Final). Mx1 ISG mRNA expression was measured by qPCR and normalized to the expression of the HPRT housekeeping gene. The data were generated from pooled triplicate experiments analyzed in duplicate. Error bars represent the SEM for duplicate PCRs.
FIG 4
FIG 4
ISG15 deubiquitination does not play a role in USP18-mediated IFN-α refractoriness. To examine the role of ISG15 deubiquitination in the IFN-α refractory state induced by inflammatory stimuli, we used UBE1L knockdown and then measured TNF-α- and LPS-induced IFN refractoriness. (A) USP18+/+ murine hepatocytes were transfected with anti-UBE1L siRNA or control irrelevant siRNA were stimulated with IFN-α (100 IU/ml), LPS (100 ng/ml), or TNF-α (20 ng/ml) for 24 h and then left untreated or were treated with an additional dose of IFN-α for 6 h. Expression of free ISG, ISG15 conjugates, UBE1L, and actin proteins were measured by Western blotting. (B) USP18+/+ murine hepatocytes transfected with anti-UBE1L siRNA or control irrelevant siRNA were pretreated with IFN-α (100 IU/ml), LPS (100 ng/ml), or TNF-α (20 ng/ml) for 24 h (IFN-α Pre, LPS Pre, or TNF-α Pre, respectively) and then left untreated or treated with an additional dose of IFN-α for 6 h (IFN-α Final). Mx1 ISG mRNA expression was measured by qPCR and normalized to the expression of the HPRT housekeeping gene. The data were generated from pooled triplicate experiments analyzed in duplicate. Error bars represent the SEM for duplicate PCRs.
FIG 5
FIG 5
IFN-α, LPS, and TNF-α do not induce type 1/2 IFN in primary murine hepatocytes but do induce USP18. Primary murine hepatocytes were treated with IFN-α (100 IU/ml; black bars), LPS (100 ng/ml; light gray bars), TNF-α (20 ng/ml; dark gray bars), or mock treated (white bars) for the times indicated and then lysed and assessed for IFN-α, IFN-γ, and Mx1 expression by qPCR. ISG mRNA expression was normalized to expression of the HPRT and actin housekeeping genes prior to determining the fold increase versus mock-treated samples. The data were generated from pooled triplicate experiments analyzed in duplicate. Error bars represent the SEM for duplicate PCRs.
FIG 6
FIG 6
Experimental hepatic ischemia/reperfusion induces USP18 expression and enhances LCMV replication. To examine the in vivo effect of an inflammatory stimulus on USP18 expression and viral replication, a hepatic ischemia/reperfusion model was used. (A) Partial (70%) hepatic ischemia was induced for 60 min, after which the portal vascular clamp was removed. Control animals (Sham) underwent anesthesia and a laparotomy alone. Animals were euthanized 6 and 24 h after ischemia/reperfusion (I/R), and the affected liver segments were removed. USP18 mRNA expression was determined by qPCR in whole liver tissue and normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT) expression. Data are expressed as means ± the SEM with n = 3 to 4 mice/group. A P value of <0.05 was considered significant. (B) After 60 min of HIRI or sham laparotomy, mice were infected with 2 × 106 PFU of LCMV WE. Liver lobes were harvested at day 7 postinfection. Viral titers were examined on MC57 cells using a focus-forming assay. Data are expressed as means ± the SEM with n = 3 to 10 mice/group. A P value of <0.05 was considered significant.
FIG 7
FIG 7
Inhibition of NF-κB activation impairs LPS- and TNF-α-stimulated USP18 induction. To investigate the link between LPS/TNF-α stimulation and USP18 induction, inhibitors of NF-κB activation were added to primary mouse hepatocytes for 30 min prior to stimulation with 20 ng of TNF-α/ml or 100 ng of LPS/ml for 6 h (the inhibitor names, _targets, and concentrations used are given in Table 1). Inhibitor-treated cells were also left unstimulated as a control. As a control for USP18 induction, hepatocytes were stimulated with 100 U of IFN-α/ml for 6 h. USP18 (A) and IL-1β (B) mRNA expression levels were evaluated by PCR and normalized to the HPRT housekeeping gene expression prior to determining the fold increase versus mock-treated samples. The data were generated from pooled triplicate experiments analyzed in duplicate. Error bars represent the SEM for duplicate PCRs.
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