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. 2011 Feb;140(2):697-708.e4.
doi: 10.1053/j.gastro.2010.08.020. Epub 2010 Aug 19.

Type I interferons protect from Toll-like receptor 9-associated liver injury and regulate IL-1 receptor antagonist in mice

Jan Petrasek  1 , Angela Dolganiuc, Timea Csak, Evelyn A Kurt-Jones, Gyongyi Szabo
Affiliations

Type I interferons protect from Toll-like receptor 9-associated liver injury and regulate IL-1 receptor antagonist in mice

Jan Petrasek et al. Gastroenterology. 2011 Feb.

Abstract

Background & aims: Liver inflammation and injury are mediated by the innate immune response, which is regulated by Toll-like receptors (TLR). Activation of TLR9 induces type I interferons (IFNs) via the interferon regulatory factor (IRF)-7. We investigated the roles of type I IFNs in TLR9-associated liver injury.

Methods: Wild-type (WT), IRF7-deficient, and IFN-α/β receptor 1 (IFNAR1)-deficient mice were stimulated with TLR9 or TLR2 ligands. Findings from mice were verified in cultured hepatocytes and liver mononuclear cells (LMNCs) as well as in vivo experiments using recombinant type I IFN and interleukin-1 receptor antagonist (IL-1ra).

Results: Type I IFNs were up-regulated during TLR9-associated liver injury in WT mice. IRF7- and IFNAR1-deficient mice, which have disruptions in type I IFN production or signaling, respectively, had increased liver damage and inflammation, decreased recruitment of dendritic cells, and increased production of tumor necrosis factor α by LMNCs. These findings indicate that type I IFNs have anti-inflammatory activities in liver. IL-1ra, which is produced by LMNCs and hepatocytes, is an IFN-regulated antagonist of the proinflammatory cytokine IL-1β; IRF7- and IFNAR1-deficient mice had decreased levels of IL-1ra compared with WT mice. IL-1ra protected cultured hepatocytes from IL-1β-mediated sensitization to cytotoxicity from tumor necrosis factor α. In vivo exposure to type I IFN, which induced IL-1ra, or administration of IL-1ra reduced TLR9-associated liver injury; the protective effect of type I IFNs therefore appears to be mediated by IFN-dependent induction of IL-1ra.

Conclusions: Type I IFNs have anti-inflammatory effects mediated by endogenous IL-1ra, which regulates the extent of TLR9-induced liver damage. Type I IFN signaling is therefore required for protection from immune-mediated liver injury.

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Figures

Fig. 1
Fig. 1. Type I IFNs are induced in TLR9-associated liver injury
Wild-type mice were injected i.p. with 2.5 mg/kg CpG DNA and 5 mg/kg LTA. Three days later mice were injected with saline or 0.5 mg/kg LPS i.p. and sacrificed after 2 hours. Serum ALT levels (A) were measured and messenger RNA levels of (B) liver interferon α-4 (IFNA4), (C) interferon β (IFNB) and (D) interferon-stimulated gene 15 (ISG15) were analyzed by real-time PCR and normalized to 18s. Values are shown as mean ± SEM fold increase over saline-primed group (3–6 mice per group). Numbers in graphs denote p values. *) p < 0.05 vs. saline-stimulated control mice; #) p < 0.05 vs. LPS-stimulated control mice
Fig. 2
Fig. 2. Deficiency of Type I IFN induction exacerbates liver injury
B6.129F2 wild-type mice and IRF7-deficient mice were treated with CpG DNA + LTA ± LPS as in figure 1. Serum ALT levels were measured (A). Assessment of liver inflammatory infiltrate (B) was performed in histology samples stained with H&E. Arrows point at inflammatory infiltrates, magnification 200x. mRNA levels of liver (C) interferon α-4 (IFNA4), (D) interferon β (IFNB), (E) interferon-stimulated gene 15 (ISG15) and (F) IP-10 were analyzed by real-time PCR. Values are shown as mean ± SEM fold increase over saline-primed control group (3–6 mice per group). *) p < 0.05 vs. saline-primed wild-type mice; #) p < 0.05 vs. saline-primed IRF7-/- mice
Fig. 3
Fig. 3. Deficiency of Type I IFN signaling exacerbates liver injury
C57Bl6 wild-type mice and IFNAR1-deficient mice were treated with CpG DNA + LTA ± LPS as in figure 1. Serum ALT levels were measured (A), and H&E-stained samples assessed for liver inflammatory infiltrate (B) was performed in histology samples stained with H+E. Arrows point at inflammatory infiltrates, magnification 200x. mRNA levels of liver (C) interferon α-4 (IFNA4), (D) interferon β (IFNB), (E) interferon-stimulated gene 15 (ISG15) and (F) IP-10 were analyzed by real-time PCR. Values are shown as mean ± SEM (3–6 mice per group). *) p < 0.05 vs. saline-primed wild-type mice; #) p < 0.05 vs. saline-primed IFNAR1-/- mice
Fig. 4
Fig. 4. Deficiency of Type I IFN signaling results in decreased dendritic cell recruitment to the liver in TLR9-associated liver injury
(A,B) Wild-type and IFNAR1-deficient mice were injected i.p. with CpG DNA + LTA ± LPS as indicated. Liver mononuclear cells were isolated, stained with anti-CD68 (A), anti-CD11c and anti-PDCA1 (B) monoclonal antibodies and analyzed using flow cytometry (N= 5 mice per group). mRNA levels of (C) liver chemokine ligand 21 (CCL-21) and (D) chemokine receptor 7 (CCR7) were analyzed by real-time PCR. Values are shown as mean ± SEM. *) p < 0.05 vs. saline-primed wild-type mice; #) p < 0.05 vs. saline-primed IFNAR1-deficient mice
Fig. 5
Fig. 5. Deficient Type I interferon signaling results in an imbalance in IL-1β / IL1 receptor antagonist induction in TLR9-associated liver injury
Mice were treated with CpG DNA + LTA ± LPS as in figure 1. mRNA levels of liver pro-interleukin-1β (IL-1β) and interleukin 1-receptor antagonist (IL-1ra) were analyzed by real-time PCR (A,C). Serum IL-1ra levels were measured by ELISA (B,D). Values are shown as mean ± SEM fold increase over saline-primed group (3-6 mice per group). *) p < 0.05 vs. saline-primed wild-type B6.129F2 or C57Bl6 mice; #) p < 0.05 vs. saline-primed IRF7-/- or IFNAR1-/- mice
Fig. 6
Fig. 6. IL-1ra protects hepatocytes from IL-1β dependent sensitization to cell death induced by TNF-α
(A) Hepatocytes and liver mononuclear cells (LMNCs), isolated from WT mice, were stimulated with murine IFN-α2b, and IL-1ra in supernatants was measured with ELISA (N=5 mice per group). (B,C) WT, IRF7- and IFNAR1-deficient mice were treated with i.p. with CpG DNA + LTA. Three days later, LMNCs were isolated, ex vivo stimulated with LPS, and TNF-α and IL-1β in supernatant were measured after 6 hours. Representative values from total N= 4 mice per group are shown. *,#,§,†) p < 0.05 vs WT cells from the respective treatment group. (D) Primary WT hepatocytes were ex vivo pretreated with murine IL-1ra and IL-1β. After four hours, murine TNF-α was added. LDH release into cell culture supernatant was measured at 24 hours and normalized to total LDH. Representative values from total N= 4 mice are shown. *) p < 0.05 vs. cells not treated with IL-1β; #) p < 0.05 vs. cells not treated with IL-1ra. (E) Primary hepatocytes from WT, IRF7- and IFNAR1-deficient mice were isolated and treated ex vivo as indicated for 4 hours, followed by murine TNF-α. LDH release into cell culture supernatant was measured at 24 hours and normalized to total LDH. Representative values from total N= 4 mice per group are shown. *,#) p < 0.05 vs. WT hepatocytes of the respective treatment group.
Fig. 7
Fig. 7. Type I interferon induces endogenous IL-1ra and ameliorates TLR9-associated liver injury
(A-C) Wild-type mice were injected with CpG DNA + LTA i.p., followed 3 days later by saline or 100,000 IU pegIFNα2, and followed by LPS i.p. for the last 2 hours. Survival (A) and serum ALT were analysed at indicated time points (B). Livers were stained with H&E, magnification 200x (C). Values are shown as mean ± SEM (11 mice per group). *) p < 0.05 vs. control mice, #) p < 0.05 vs. control mice treated with pegIFNα2. (D-F) Wild-type mice were treated with saline or with recombinant IL-1ra 25 mg/kg i.p. every six hours. Twenty-four hours after IL-1ra initiation, mice were injected with CpG DNA + LTA, followed by LPS 3 days later. Survival (D) and serum ALT (E) were analysed at indicated time points. Livers were stained with H&E, magnification 200x (F). Values are shown as mean ± SEM (15 mice per group). *) p < 0.05 vs. control mice, #) p < 0.05 vs. control mice treated with IL-1ra
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