Type I Interferons Protect from Toll-like Receptor 9-Associated Liver Injury and Regulate IL-1 Receptor Antagonist in Mice

Animals and experimental protocol

The B6.129F2 and C57Bl/6 wild-type (WT) mice were purchased from Jackson Laboratory. IRF7-deficient (IRF7-/-) mice on B6.129F2 background were provided by Tadagatsu Tanaguichi (Tokyo) and type I interferon α/β receptor 1-deficient (IFNAR1−/−) mice on the C57Bl/6 background were the kind gift of Jonathan Sprent (Scripps Research Institute, La Jolla, CA). All animals (3–6/experimental group) were 6–8 weeks old and received proper care in agreement with animal protocols approved by the Institutional Animal Use and Care Committee of the University of Massachusetts Medical School.

We employed a previously described model of TLR9-associated liver injury induced by administration of TLR9 and TLR2 ligands.^{15, 16} After acclimatization, WT, IRF7−/− and IFNAR1−/− mice were injected intraperitoneally (i.p.) with saline or the combination of 2.5 mg/kg unmethylated DNA rich in cytidine-phosphate-guanosine (CpG, ODN1826 murine TLR9 ligand; InvivoGen, San Diego, CA), and 5 mg/kg lipoteichoic acid (LTA, from Staphylococcus aureus; Sigma, Saint Louis, MO). Three days after the above priming stimulus, the mice were injected i.p. with either saline or 0.5 mg/kg lipopolysaccharide (LPS, from Escherichia coli 0111:B4, Sigma, St. Louis, MO) and sacrificed as indicated. Some C57Bl/6 WT mice received a single i.p. injection of 100,000 IU human pegylated interferon alpha-2b (pegIFNα2, Pegintron, Schering, Kenilworth, NJ) two hours prior to LPS. Others were pretreated with recombinant human interleukin-1 receptor antagonist (IL-1ra) 25 mg/kg i.p. every six hours (Anakinra, Amgen, Thousand Oaks, CA) for 24 hours before CpG+LTA, and the treatment with IL-1ra was ongoing until sacrifice. Serum was separated by centrifugation. Livers were snap frozen, stored in RNAlater (Qiagen GmbH, Hilden, Germany) or fixed in 10% neutral-buffered formalin. ALT was quantified by biochemical assay (D-Tek Analytical Laboratories Inc, San Diego, CA).

Histopathology analysis

Sections of formalin-fixed, paraffin-embedded livers were stained with hematoxylin and eosin (H&E), and assessed for inflammatory infiltrate; area of inflammatory infiltrates was calculated with Microsuite (Olympus Soft Imaging Solution GmbH, Munster, Germany) image analysis software in 20 high power fields.

Isolation of hepatocytes and liver mononuclear cells

Animals received anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg); the livers were perfused with saline solution followed by in vivo digestion, as we previously described.¹⁹ The hepatocytes and liver mononuclear cells (LMNCs) were purified by centrifugation at slow speed (500g) and in Percoll gradient, respectively.

Phenotype analysis by flow cytometry

Cells were washed in PBS and incubated with anti-CD68 (FITC), anti-CD11c (FITC) or anti-PDCA1 (Alexa Fluor 647) antibodies for 30 minutes on ice. After incubation, cells were washed with PBS, fixed in paraformaldehyde and analyzed by flow cytometry. All antibodies were from eBioscience (eBioscience, Inc., San Diego, CA).

In vitro cell culture

Primary hepatocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 1% insulin, transferrin, and selenium supplement on collagen-coated plates (Becton Dickinson Labware, Bedford, MA). Primary LMNCs were cultured in DMEM with 10% FBS. Hepatocytes were treated with recombinant murine IFN-α2b (500 IU/mL, eBioscience, San Diego, CA), murine IL-1ra (100 pg/mL, R&D Systems, Minneapolis, MN), murine IL-1β (100 IU/mL, Peprotech Inc., Rocky Hill, NJ) or murine TNF-α (0-100 ng/mL, Peprotech Inc., Rocky Hill, NJ). LMNCs were treated with mouse IFN-α2b or LPS (100 ng/mL, Sigma, St. Louis, MO).

Hepatocyte cytotoxicity assay

Lactate dehydrogenase (LDH) release from the hepatocyte into the culture supernatants was measured using the LDH-cytotoxicity assay kit (Abcam, Cambridge, MA), and normalized to total LDH (determined after treatment of cells with detergent-based lysis solution).

Cytokine and chemokine measurement

Serum concentrations of the secreted forms of interleukin-1β (IL-1β) and IL-1 receptor antagonist (IL-1ra) were analyzed with ELISA (R&D Systems, Minneapolis, MN). Monocyte chemotactic protein 1 (MCP-1) in the liver lysate was measured with ELISA from R&D, and TNF-α in cell culture supernatants was analysed using ELISA from BD Biosciences (BD Biosciences, San Jose, CA).

RNA analysis

RNA was extracted from liver tissue using the RNeasy kit (Qiagen Sciences, Maryland, USA) and on-column DNase digestion was performed using the DNase Set (Qiagen GmbH, Hilden, Germany); cDNA was synthesized with Reverse Transcription System (Promega Corp., Madison, WI). Real-time quantitative polymerase chain reaction (qPCR) was performed using the iCycler iQ Cycler (Bio-Rad Laboratories, Inc., Hercules, CA) and specific primers (Supplementary Table 1).

Statistical Analysis

The data are presented as mean ± SEM. Comparison of the means was performed using Student’s T-test or Kruskall-Wallis test, when appropriate. P values less than 0.05 were considered significant.

Type I IFNs are induced in TLR9-associated liver injury

TLR9 signaling induces Type I IFNs, which may play a role in tissue injury and regeneration. ^{20, 21} We have previously demonstrated that MyD88, a common adaptor to TLR9, TLR2 and TLR4 is critical in sensitization to liver injury.¹⁶ Here we employed a model of TLR9-associated liver injury owed to sensitization by the synergistic effect of the TLR9 ligand CpG and the TLR2 ligand LTA, followed by a second hit with LPS,^{15, 16} and asked if the integrity of the Type I IFN pathway was involved in liver damage. We found that combined administration of TLR9 and TLR2 ligands induced and sensitized the liver to injury by the TLR4 ligand LPS, as indicated by increased serum ALT levels in WT mice (Fig. 1A). More importantly, TLR9+TLR2 ligands induced expression of IFNα (Fig. 1B) and IFNβ (Fig. 1C) mRNA in the liver and expression of both IFNα and IFNβ was further upregulated upon TLR4 stimulation in WT mice. We also found that expression of the Type I IFN-inducible gene, interferon regulated gene ISG15, was induced by TLR9+TLR2 ligand treatment and the expression was further increased upon subsequent LPS stimulation (Fig. 1D). Collectively, these data suggested that Type I IFNs and IFN-triggered signaling pathways were upregulated in TLR9-associated liver injury.

Deficiency in Type I IFN induction exacerbates liver injury

Our data indicated activation of Type I IFN signaling in TLR9-associated liver injury. While both TLR9 and TLR2 ligands activate production of proinflammatory cytokines, TLR9 stimulation also triggers Type I IFNs.¹⁷ Type I IFN production induced by TLR9 is largely dependent on activation of intracellular pathways involving interferon regulatory factor-7 (IRF7).²² To define the importance of Type I IFN in TLR9-associated liver injury, we tested IRF7-deficient mice.

In sharp contrast to their wild-type littermates, IRF7-deficient mice showed exacerbation of TLR9-associated liver injury, as indicated by serum ALT elevations (Fig. 2A). Histopathology analysis (Fig. 2B) of IRF7-deficient livers revealed an about 5-fold increase in the number of inflammatory infiltrates (10.33 ± 3.71 in IRF7-KO vs. 1.75 ± 0.48 in WT, P = 0.026) and an about 2.5-fold increase in total area of inflammatory infiltrates (0.67 ± 0.05 vs. 0.28 ± 0.05 mm², P = 0.017), compared to wild-type controls.

The increased TLR9-associated liver injury in IRF7-deficient mice was accompanied by deficient induction of IFNα (Fig. 2C) and IFNβ (Fig. 2D), compared to controls. LPS stimulation could partially overcome the deficit in IFNα and IFNβ expression in TLR9+TLR2 primed mice, presumably by the direct effect of TLR4 signaling on Type I IFN induction.²³ However, there was decreased induction of the IFN-inducible genes, ISG15 (Fig. 2E) and IP-10 (Fig. 2F), in IRF7-deficient mice and further expression of these molecules upon stimulation with LPS was limited. These data supported our hypothesis that Type I IFN induction plays a role in the pathogenesis of TLR9-induced liver damage and suggested a protective role for Type I IFNs in liver injury.

Deficiency in Type I IFN signaling exacerbates liver injury

The effects of Type I IFNs are mediated by a cognate receptor composed of two chains, IFNAR1 and IFNAR2, both of which are essential to initiate production of the interferon-stimulated genes (ISGs).²⁴ To differentiate between the direct and indirect protective effects of Type-I IFNs in TLR9-associated liver injury, we tested mice deficient in type I interferon receptor expression (IFNAR1-deficient mice).

Similar to findings in IRF7-deficient mice, mice deficient in IFNAR1 expression showed significantly increased serum ALT (Fig. 3A), widespread liver inflammation, about 5-fold increase in the number of inflammatory foci (4.80 ± 0.49 in IFNAR1-KO vs. 1.0 ± 0.58 in WT, P = 0.018) and about 7-fold increase in total area of inflammatory infiltrate (0.40 ± 0.11 vs. 0.06 ± 0.04 mm², P = 0.036) after TLR9+TLR2 stimulation compared to wild-type controls (Fig. 3B).

Type I IFNs per se are IFN-sensitive and are strictly regulated by a self-initiated amplification loop involving IRF7 and both IFN receptor chains.²⁴ Consistent with this mechanism, IFNAR1-deficient mice showed substantially lower induction of liver IFNα (Fig. 3C), IFNβ (Fig. 3D) and minimal expression of ISG15 (Fig. 3E) and IP-10 (Fig. 3F), compared to controls. This difference was not overcome by LPS stimulation. Collectively, these data suggested that both Type I IFN induction and IFN-induced signaling may play a protective role in the liver by limiting inflammatory infiltrate and liver injury.

Deficiency of Type I IFN signaling results in decreased dendritic cell recruitment to the liver in TLR9-associated injury

The exaggerated liver inflammatory infiltrate in IRF7- and IFNAR1-deficient mice suggested a role for Type I IFNs in recruitment of inflammatory cells in TLR9-associated liver injury. We thus aimed to define the cell composition of these inflammatory infiltrates. Flow cytometric analysis of liver mononuclear cells (LMNCs) showed equal enrichment of CD68⁺ monocytes/macrophages in the livers of both WT and IFNAR1-deficient mice after TLR9+TLR2 priming (Fig. 4A). Similarly, liver expression of chemokines MCP-1, MCP-2, MIP-1α and MIP-1β (Suppl. Fig. 1A-E) and chemokine receptors CCR1, CCR2 and CCR5 (Suppl. Fig. 1F-H), which are involved in macrophage recruitment, was upregulated in TLR9-associated liver injury to a comparable extent in both WT and IFNAR1-deficient mice.

In contrast to WT mice, LMNCs isolated from mice deficient in IFNAR1 showed a significantly lower proportion of CD11c⁺ (myeloid) and PDCA1⁺ (plasmacytoid) dendritic cells (DC) (Fig. 4B). This finding was associated with a significantly decreased liver expression of the chemokine ligand 21 (CCL-21) (Fig. 4C), a key molecule involved in recruitment of DC, ²⁵ and decreased expression of the CCL-21 receptor CCR7 (Fig. 4D). These data support our hypothesis that in TLR9-associated liver injury, Type I IFNs are required for dendritic cell recruitment to the liver.

Deficient Type I interferon signaling results in an imbalance in IL-1β / IL-1 receptor antagonist induction in TLR9-associated liver injury

The protective effect of Type I IFN induction and signaling against TLR9-associated liver injury implied the involvement of Type I IFN-dependent anti-inflammatory factors. We thus analyzed the expression of another IFN-dependent gene, IL-1 receptor antagonist (IL-1ra), which is a natural endogenous antagonist of the proinflammatory interleukin-1β (IL-1β) at the receptor level.²⁶ Pro-IL-1β gene in the liver (Fig. 5A) and IL-1β protein (Fig. 5B) in the serum were significantly induced by TLR9+TLR2 ligands and further upregulated by LPS to a comparable extent in WT, IRF7- and IFNAR-deficient mice (Fig. 5A,B). Induction of IL-1ra mRNA in the liver and of the secreted IL-1ra protein in serum were decreased both in IRF7- and IFNAR1-deficient mice treated with CpG+LTA, compared to controls (Fig. 5C,D). More important, sensitization via TLR9+TLR2 ligands resulted in less IL-1ra protein production upon stimulation with LPS in both IRF7- and IFNAR1-deficient mice compared to their wild-type controls (Fig. 5D). This finding suggested that aggravated TLR9-associated liver injury in Type I IFN deficient animals is associated with a significant imbalance in IL-1β/IL-1ra signaling.

IL-1ra protects hepatocytes from IL-1β-dependent sensitization to cell death induced by TNF-α

Our data suggested a protective effect of IL-1ra in TLR9-associated liver injury. To gain a mechanistic insight, we first investigated the source of IL-1ra in the liver. We observed that WT primary hepatocytes and LMNCs stimulated with IFN-α in vitro produced significantly more IL-1ra, compared to non-stimulated cells (Fig. 6A). Next, we asked whether deficiency of Type I IFNs results in differential production of inflammatory cytokines by LMNCs. In vivo priming of mice with TLR9+TLR2 ligands sensitized LMNCs to ex vivo stimulation with LPS and resulted in significantly greater induction of inflammatory cytokines TNF-α (Fig. 6B) and IL-1β (Fig. 6C), compared with LPS-stimulated LMNCs of non-sensitized mice. In contrast to WT mice, LMNCs isolated from IRF7- and IFNAR-deficient mice showed significantly increased induction of TNF-α upon ex vivo stimulation with LPS (Fig. 6B); induction of IL-1β did not significantly differ between genotypes (Fig. 6C).

Consequently, we asked whether IL-1ra could play a protective role in hepatocyte death induced by synergistic activities of TNF-α and IL-1β. Treatment of primary WT hepatocytes with TNF-α did not induce hepatocyte death (Fig. 6D), consistent with previous reports that healthy WT hepatocytes are resistant to stimulation with TNF-α alone.²⁷ However, pretreatment with IL-1β sensitized hepatocytes to TNF-α induced death (Fig. 6D). More important, the sensitizing effect of IL-1β was significantly reduced by IL-1ra (Fig. 6D). Stimulation with IL-1β + TNF-α lead to a significantly greater extent of hepatocyte death in IRF7- and IFNAR1-deficient hepatocytes, compared to WT; significant protection from hepatocyte death was observed after co-treatment with IL-1ra in all genotypes (Fig. 6E).

Taken together, these data suggested that Type I IFNs induce IL-1ra production both in hepatocytes and LMNCs, and that IL-1ra protects hepatocytes from IL-1β-dependent sensitization to TNF-α-induced cell death.

Type I interferon induces endogenous IL-1ra in vivo and ameliorates TLR9-associated liver injury

To evaluate the protective role of Type I IFN in TLR9-associated liver injury, we first administered pegylated IFNα2a (pegIFNα2) to WT mice and observed a significant induction of serum IL-1ra (Suppl. Fig. 2). Next, we showed that pegIFNα2 administered to TLR9+TLR2-primed mice two hours prior to LPS significantly prolonged survival (Fig. 7A) and ameliorated TLR9-associated liver injury (Fig. 7B), compared to control mice. Furthermore, pegIFNα2 prevented aggravation of LPS-induced liver injury after TLR9+TLR2-priming (Fig. 7B). Importantly, serum IL-1ra positively correlated with the length of survival (Suppl. Fig. 3A), and negatively correlated with the extent of liver injury (Suppl. Fig. 3B). Accordingly, histopathology analysis revealed reduction in liver inflammatory infiltrate and the extent of necrosis in mice pretreated with pegIFNα2 compared to controls (Fig. 7C). Taken together, these data suggest that type I IFNs induce IL-1ra which has hepatoprotective and anti-inflammatory effects in TLR9-induced liver injury.

IL-1ra ameliorates TLR9-associated liver injury

To demonstrate the direct protective effect of IL-1ra, we initiated treatment with recombinant IL-1ra in WT mice 24 hours prior to administration of TLR9+TLR2 ligands and continued with IL-1ra throughout the experiment. Mice pretreated with IL-1ra showed significantly prolonged survival after injection of LPS (Fig. 7D), which was associated with lower ALT levels (Fig. 7E) and reduction in liver inflammatory infiltrate and extent of necrosis compared to controls (Fig. 7F). Collectively, these data suggested that Type I IFNs are needed to elicit a strong anti-inflammatory response by induction of IL-1ra, and that Type I IFN-regulated IL-1β/IL-1ra system is key to the protective role of Type I IFNs in liver injury.