Volume 140, Issue 2, Pages 697-708.e4 (February 2011) Type I Interferons Protect From Toll-Like Receptor 9–Associated Liver Injury and Regulate IL-1 Receptor Antagonist in Mice  Jan Petrasek, Angela Dolganiuc, Timea Csak, Evelyn A. Kurt–Jones, Gyongyi Szabo  Gastroenterology  Volume 140, Issue 2, Pages 697-708.e4 (February 2011) DOI: 10.1053/j.gastro.2010.08.020 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Type I IFNs are induced in TLR9-associated liver injury. WT mice were injected IP 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 IP and sacrificed after 2 hours. Serum ALT levels (A) were measured and mRNA levels of (B) IFNA4, (C) IFNB, and (D) 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 < .05 vs saline-stimulated control mice; #P < .05 vs LPS-stimulated control mice Gastroenterology 2011 140, 697-708.e4DOI: (10.1053/j.gastro.2010.08.020) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Deficiency of type I IFN induction exacerbates liver injury. B6.129F2 WT mice and IRF7-deficient mice were treated with CpG DNA plus LTA and/or LPS as in Figure 1. (A) Serum ALT levels were measured. (B) Assessment of liver inflammatory infiltrate was performed in histology samples stained with H&E. Arrows indicate inflammatory infiltrates; original magnification 200×. mRNA levels of liver (C) IFNA4, (D) IFNB, (E) 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 < .05 vs saline-primed WT mice; #P < .05 vs saline-primed IRF7−/− mice. Gastroenterology 2011 140, 697-708.e4DOI: (10.1053/j.gastro.2010.08.020) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Deficiency of type I IFN signaling exacerbates liver injury. C57Bl/6 WT mice and IFNAR1-deficient mice were treated with CpG DNA plus LTA and/or LPS as in Figure 1. (A) Serum ALT levels were measured. (B) Assessment of liver inflammatory infiltrate was performed in histology samples stained with H&E. Arrows indicate inflammatory infiltrates; original magnification 200×. mRNA levels of liver (C) IFNA4, (D) IFNB, (E) ISG15, and (F) IP-10 were analyzed by real-time PCR. Values are shown as mean ± SEM (3–6 mice per group). *P < .05 vs saline-primed WT mice; #P < .05 vs saline-primed IFNAR1−/− mice. Gastroenterology 2011 140, 697-708.e4DOI: (10.1053/j.gastro.2010.08.020) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Deficiency of type I IFN signaling results in decreased dendritic cell recruitment to the liver in TLR9-associated liver injury. (A and B) WT and IFNAR1-deficient mice were injected IP with CpG DNA plus LTA and/or LPS as indicated. Liver mononuclear cells were isolated, stained with (A) anti-CD68 and (B) anti-CD11c and anti-PDCA1 monoclonal antibodies, and analyzed using flow cytometry (n = 5 mice per group). mRNA levels of (C) liver CCL-21 and (D) chemokine receptor 7 (CCR7) were analyzed by real-time PCR. Values are shown as mean ± SEM. *P < .05 vs saline-primed WT mice; #P < .05 vs saline-primed IFNAR1-deficient mice. Gastroenterology 2011 140, 697-708.e4DOI: (10.1053/j.gastro.2010.08.020) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Deficient type I IFN signaling results in an imbalance in IL-1β/IL-1ra induction in TLR9-associated liver injury. Mice were treated with CpG DNA and LTA and/or LPS as in Figure 1. (A and C) mRNA levels of liver pro-IL-1β and IL-1ra were analyzed by real-time PCR. (B and D) Serum IL-1ra levels were measured by ELISA. Values are shown as mean ± SEM fold increase over saline-primed group (3–6 mice per group). *P < .05 vs saline-primed WT B6.129F2 or C57Bl/6 mice; #P < .05 vs saline-primed IRF7−/− or IFNAR1−/− mice. Gastroenterology 2011 140, 697-708.e4DOI: (10.1053/j.gastro.2010.08.020) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 IL-1ra protects hepatocytes from IL-1β–dependent sensitization to cell death induced by TNF-α. (A) Hepatocytes and 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 and C) WT, IRF7-deficient, and IFNAR1-deficient mice were treated IP with CpG DNA plus LTA. Three days later, LMNCs were isolated and ex vivo stimulated with LPS, and TNF-α and IL-1β in supernatant were measured after 6 hours. Representative values from a total of n = 4 mice per group are shown. *,#,§,†P < .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 4 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 a total of n = 4 mice are shown. *P < .05 vs cells not treated with IL-1β; #P < .05 vs cells not treated with IL-1ra. (E) Primary hepatocytes from WT, IRF7-deficient, 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 a total of n= 4 mice per group are shown. *,#P < .05 vs WT hepatocytes of the respective treatment group. Gastroenterology 2011 140, 697-708.e4DOI: (10.1053/j.gastro.2010.08.020) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 Type I IFN induces endogenous IL-1ra and ameliorates TLR9-associated liver injury. (A–C) WT mice were injected with CpG DNA plus LTA IP, followed 3 days later by saline or 100,000 IU pegIFNα2, and followed by LPS IP for the last 2 hours. (A) Survival and (B) serum ALT level were analyzed at indicated time points. (C) Livers were stained with H&E; original magnification 200×. Values are shown as mean ± SEM (11 mice per group). *P < .05 vs control mice; #P < .05 vs control mice treated with pegIFNα2. (D–F) WT mice were treated with saline or with recombinant IL-1ra 25 mg/kg IP every 6 hours. Twenty-four hours after initiation of IL-1ra, mice were injected with CpG DNA plus LTA, followed by LPS 3 days later. (D) Survival and (E) serum ALT level were analyzed at indicated time points. Livers were stained with H&E; original magnification 200× (F). Values are shown as mean ± SEM (15 mice per group). *P < .05 vs control mice; #P < .05 vs control mice treated with IL-1ra. Gastroenterology 2011 140, 697-708.e4DOI: (10.1053/j.gastro.2010.08.020) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Induction of chemokines and chemokine receptors involved in monocyte/macrophage recruitment in TLR9-associated liver injury. C57Bl/6 WT mice and IFNAR1-deficient mice were treated with CpG DNA plus LTA and/or LPS as in Figure 1. mRNA levels of liver (A) macrophage chemotactic protein 1 (MCP-1), (C) macrophage chemotactic protein 2 (MCP-2), (D) macrophage inflammatory protein 1a (MIP-1a), (E) macrophage inflammatory protein 1ß (MIP-1ß), (F) chemokine receptor 1 (CCR1), (G) chemokine receptor 2 (CCR2), and (H) chemokine receptor 5 (CCR5) were analyzed by real-time PCR. (B) Liver MCP-1 protein was measured using ELISA. Significantly decreased liver expression of MCP1, MCP2, MIP-1a, and MIP-1ß was observed in IFNAR1-mice stimulated with LPS compared with WT mice. Values are shown as mean ± SEM (3–6 mice per group). *P < .05 vs saline-primed WT mice; #P < .05 vs saline-primed IFNAR1-deficient mice Gastroenterology 2011 140, 697-708.e4DOI: (10.1053/j.gastro.2010.08.020) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 Induction of endogenous IL-1ra by type I IFNs in vivo. WT mice were injected with 100,000 IU pegIFNa2 IP, and IL-1ra in serum was measured at indicated time points (n = 4 mice). Numbers denote P values compared with baseline. Gastroenterology 2011 140, 697-708.e4DOI: (10.1053/j.gastro.2010.08.020) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 Serum IL-1ra positively correlates with survival and negatively correlates with liver damage in TLR9-associated liver injury. WT mice were injected with CpG DNA plus LTA IP. Three days later, mice received saline or pegIFNa2, followed by LPS IP for the last 2 hours. Serum IL-1ra was correlated with (A) the length of survival and (B) serum ALT level. n = 6–7 mice per group. Gastroenterology 2011 140, 697-708.e4DOI: (10.1053/j.gastro.2010.08.020) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 4 Proposed mechanism of the protective role of type I IFNs in TLR9-associated liver injury. TLR9, TLR2, and TLR4 ligands stimulate liver macrophages and dendritic cells to produce inflammatory cytokines. TLR9 ligands stimulate dendritic cells to produce type I IFNs in an IRF7-dependent manner. Type I IFNs bind to type I IFN receptors (IFNAR1) and induce IL-1ra in hepatocytes and in liver mononuclear cells. IL-1ra binds to IL-1 receptor (IL-1R) and inhibits IL-1 dependent sensitization to TNF-α–induced hepatocyte death. Blue and black arrows depict interaction pathways; red arrows depict pathways of potential therapeutic targets. Gastroenterology 2011 140, 697-708.e4DOI: (10.1053/j.gastro.2010.08.020) Copyright © 2011 AGA Institute Terms and Conditions