Type I Interferon Supports Inducible Nitric Oxide Synthase in Murine Hepatoma Cells and Hepatocytes and during Experimental Acetaminophen-Induced Liver Damage

Cytokine regulation of high-output nitric oxide (NO) derived from inducible NO synthase (iNOS) is critically involved in inflammation biology and host defense. Herein, we set out to characterize the role of type I interferon (IFN) as potential regulator of hepatic iNOS in vitro and in vivo. In this regard, we identified in murine Hepa1-6 hepatoma cells a potent synergism between pro-inflammatory interleukin-β/tumor necrosis factor-α and immunoregulatory IFNβ as detected by analysis of iNOS expression and nitrite release. Upregulation of iNOS by IFNβ coincided with enhanced binding of signal transducer and activator of transcription-1 to a regulatory region at the murine iNOS promoter known to support target gene expression in response to this signaling pathway. Synergistic iNOS induction under the influence of IFNβ was confirmed in alternate murine Hepa56.1D hepatoma cells and primary hepatocytes. To assess iNOS regulation by type I IFN in vivo, murine acetaminophen (APAP)-induced sterile liver inflammation was investigated. In this model of acute liver injury, excessive necroinflammation drives iNOS expression in diverse liver cell types, among others hepatocytes. Herein, we demonstrate impaired iNOS expression in type I IFN receptor-deficient mice which associated with diminished APAP-induced liver damage. Data presented indicate a vital role of type I IFN within the inflamed liver for fine-tuning pathological processes such as overt iNOS expression.

Especially under conditions of glutathione depletion, hepatotoxicity by xenobiotics is frequently mediated at least partly by the highly reactive NO metabolite peroxynitrite (5).
Regulation of iNOS expression and activity occurs foremost on the level of gene transcription with pathways activating nuclear factor (NF)-κB and signal transducer and activator of transcription (STAT)-1 being of particular importance. Depending on the biochemical and cellular context STAT1 is able to support iNOS expression as STAT1 homodimer or as part of a protein complex together with STAT2 and IRF9 known as interferon (IFN)-stimulated gene factor (ISGF)-3 (1,2,(6)(7)(8)(9). Particularly in non-leukocytic cells such as renal mesangial cells and hepatocytes, iNOS mRNA is amplified by NO-driven feedforward mechanisms (10,11). Whereas a crucial role for IFNγ concerning iNOS induction is established in diverse cell types including hepatocytes (12), information on the role of immunoregulatory type I IFN (including IFNα/β) as cofactor for induction of hepatocyte iNOS is currently lacking. Of note, IFNα/β is reported to efficiently drive monocyte/macrophage-derived iNOS in humans and mice (9,(13)(14)(15). Moreover, in combination with interleukin (IL)-22, IFNα upregulates expression of iNOS in human DLD1 colon carcinoma cells (16). Herein, we set out to further characterize in vitro and in vivo the role of type I IFN for murine hepatic iNOS regulation by using the cellular model of IFNβ-stimulated hepatoma cells (Hepa1-6, Hepa56.1D) and hepatocytes and by investigating murine acetaminophen (paracetamol, APAP)-induced sterile liver inflammation in the context type I IFN receptor-deficient mice.
Thereafter, the liver was perfused with HBSS with Ca 2+ and Mg 2+ [supplemented with 15 mM HEPES, 5 mM CaCl2, 0.13 mg/ml collagenase IV (Sigma-Aldrich, Darmstadt, Germany), 100 U/ml penicillin, and 100 µg/ml streptomycin] for additional 10 min. The liver was carefully removed from the abdominal cavity, placed in a Petri dish on ice in DMEM (supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin) and opened with a forceps. Liver cells were resuspended and put over a 100 µm cell strainer (Becton Dickinson, Heidelberg, Germany). After two rounds of centrifugation (5 min at 50 g and 4°C) and resuspension, cell viability was determined by trypan blue dye exclusion, and cells were seeded in DMEM (supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin) on collagen G-coated plates (Biochrom, Berlin, Germany). Adherent hepatocytes were washed after 4 h with PBS and fresh Williams Medium E [supplemented with 10% FCS, 2 mM l-Alanyl-l-Glutamin (Biochrom), 2 ng/ml insulin, 100 ng/ml dexamethasone, 100 U/ml penicillin, and 100 µg/ml streptomycin] was added. Stimulation with recombinant cytokines for analysis of iNOS expression was performed 16 h thereafter. Dexamethasone was not removed from standard supplemented Williams Medium E because this glucocorticoid is known to inhibit hepatocyte apoptosis (18,19) thereby supporting viability. This protocol was adhered to despite the prospect that dexamethasone at this concentration is capable of reducing NF-κB thus partly affecting hepatocyte iNOS (20).

chromatin immunoprecipitation (chiP)
Chromatin immunoprecipitation was performed as previously described (21). For immunoprecipitation, an IgG control or a specific STAT1 antibody was used (rabbit polyclonal antibody; Santa Cruz Biotechnology, Heidelberg, Germany Murine APAP (500 mg/kg)-induced liver injury was performed as described (23). Briefly, fasted male mice obtained i.p. injection of either warm 0.9% NaCl (B. Braun, Melsungen, Germany) or 500 mg/kg (dissolved in warm 0.9% NaCl) APAP. Mice that obtained NaCl are depicted as control mice (ctrl) throughout the manuscript. Mice had free access to food and water. After 6 h (only wt-mice) or 24 h (wt-and IFNAR −/− mice), mice underwent isoflurane (Abbott, Wiesbaden, Germany) anesthesia and were sacrificed thereafter. Blood was taken from the retroorbital venous plexus. Serum alanine aminotransferase (ALT) activity was quantified according to manufacturer's instructions (Reflotron, Roche Diagnostics, Mannheim, Germany). Serum was stored at −80°C. For RNA and protein analysis, liver tissue was snap frozen in liquid nitrogen and stored at −80°C. For histological analysis, liver tissue was perfused with PBS via the portal vein followed by overnight incubation in 4.5% buffered formalin. Thereafter, tissue was embedded in paraffin for histologic analysis. Paraffinembedded liver sections (4 µm) were stained with hematoxylin (Applichem, Darmstadt, Germany). The degree of histopathological liver injury was quantified by Keyence BZ-II Analyzer software (Neu-Isenburg, Germany). Specifically, computer-aided analysis of tissue necrosis was performed by using similarly located liver sections obtained from 9 wt-and 9 IFNAR-deficient individual mice (n = 9 per genotype) treated with APAP (500 mg/ kg, 24 h). One complete liver section per individual mice underwent analysis. The software quantifies the degree of liver necrosis by identifying necrotic areas based on differences in hematoxylin staining. Results are presented as text-only and expressed as (%-reduction) of liver necrosis observed in IFNAR-deficient mice compared with wt-mice.

immunohistochemical Detection of inOs
Paraffin-embedded liver sections (4 µm) were used for detection of iNOS. Briefly, sections were deparaffinized and unmasked by heat treatment (Target Retrieval Solution; Dako, Glostrup, Denmark). Sections were stained using either a self-made inhouse (24,25) or a commercially available (Enzo Life Sciences, Lörrach, Germany) rabbit polyclonal antimurine iNOS antibody overnight at 4°C. Notably, both iNOS detecting antibodies generated analogous iNOS staining in livers of APAP-treated mice. For detection, goat antirabbit ABC staining system (Santa Cruz Biotechnology) and the 3,3′-diaminobenzidine Substrate Kit for Peroxidase (Sigma-Aldrich) were used. Sections were counterstained with hematoxylin.

immunoblot analysis
Tissue homogenates were generated as previously described (23). Briefly, cells or liver homogenates were generated using lysis buffer [150 mM NaCl, 1 mM CaCl2, 25 mM Tris-Cl (pH 7.4), 1% Triton X-100], supplemented with protease inhibitor cocktail (Roche Diagnostics) and DTT, Na3VO4, PMSF (each 1 mM), and NaF (20 mM). Thereafter, SDS-PAGE and immunoblotting were performed. To detect iNOS or pSTAT1 together with GAPDH on the same blot, the blot was cut. Antibodies: iNOS, rabbit polyclonal antibody (Enzo Life Sciences); GAPDH, rabbit polyclonal antibody (Trevigen, Gaithersburg, USA); pSTAT1 (Tyr-701), rabbit polyclonal antibody (Cell Signaling, Frankfurt, Germany). Quantifications of immunoblots were performed by Quantity-One analysis software (Bio-Rad, Munich, Germany). As a "positive control" for iNOS expressing murine C57BL/6 tissue, cutaneous wound lysates obtained 3 days after skin wounding were used. At that time point iNOS protein expression in the wounded skin peaks (26). Wound lysates of iNOS-deficient mice from the same time point were analyzed as "negative control. " Those cutaneous wound lysates were kindly provided by Dr. Itamar Goren and Prof. Stefan Frank (University Hospital, Goethe University Frankfurt, pharmazentrum frankfurt). All animal experiments were approved by the Regierungspräsidium Darmstadt (Germany).

resUlTs iFnβ amplifies hepatocyte inOs expression in cell culture
Whereas type I IFN is an established inducer of iNOS in monocytes/macrophages (9,(13)(14)(15), information on effects of IFNα/β on hepatocyte iNOS is scarce. Notably, one earlier report demonstrated that iNOS in human hepatoma Huh7 cells is not induced by IFNα as sole stimulus. Interactions with other cytokines were not investigated in that earlier report (27). To further investigate this matter, murine Hepa1-6 hepatoma cells were exposed to IFNβ in the presence or absence of NF-κB-activating cytokines IL-1β and TNFα (28). The combination IL-1β plus TNFα was employed since pilot experiments in Hepa1-6 cells (data not shown) demonstrated that this combination synergizes for induction of the prototypic hepatocyte-derived NF-κB-dependent chemokine MIP2 (29). Herein, we demonstrate that IFNβ in cooperation with aforementioned pro-inflammatory cytokines potentiates iNOS expression ( Figure 1A) and activity as detected by nitrite release (Figure 1B). Potentiation of nitrite release by coincubation with IFNβ was stable over a 48 h time period (Figure 1C).
Notably, in accord with the aforementioned report (27), IFNβ  failed to induce iNOS as sole stimulus (Figures 1A-C). Detailed analysis furthermore revealed that triplet stimulation by IL-1β/ TNFα/IFNβ is superior to that by either IL-1β/IFNβ or TNFα/ IFNβ ( Figure 1D) and that potentiation of iNOS is likewise detectable in the context of IFNβ preincubation ( Figure 1E). Amplification of hepatocyte iNOS by IFNβ was not confined to Hepa1-6 cells. Synergism between IL-1β/TNFα and IFNβ for iNOS expression was actually even more pronounced in alternate murine Hepa56.1D hepatoma cells ( Figure 1F). Moreover, IFNβ likewise potentiated iNOS in murine primary C57BL/6 hepatocytes which was readily detectable on mRNA (Figure 2A) and nitrite level (Figure 2B).
In order to characterize molecular mechanisms of murine hepatic iNOS gene induction by IFNβ, we chose to focus herein on Hepa1-6 hepatoma cells. Activation of the transcription factor STAT1 is key to immunoregulation by IFNα/β (30,31). STAT1 activation, assessed by analysis of Tyr-701 phosphorylation (pSTAT1), was readily detectable in Hepa1-6 cells under the influence of IFNβ (Figure 3A). Within the murine iNOS promoter, a specific region (−912 to −1,029 bp relative to the TSS) was found to mediate STAT1-induced iNOS transcriptional activation which may be achieved by STAT1-homodimers (in response to IFNγ or type I IFN) or by the type I IFN/ISGF3 axis. In this region (Figure 3B, upper panel), adjacent STAT1-binding elements are located. Namely, a dual GAS/IFN-stimulated response element (ISRE) sequence (−951 to −935 bp)-binding STAT1 homodimers or ISGF3-and an additional ISRE site (−924 to −912 bp)-likewise potentially binding ISGF3 (8,9,31). STAT1 binding to this region was investigated herein to further characterize mechanisms mediating IFNβ potentiation of iNOS in Hepa1-6 hepatoma cells. In the presence of IL-1β/TNFα, ChIP analysis revealed STAT1 binding to this site in response to IFNβ which suggests enhanced STAT1-dependent transcriptional activity at the iNOS promoter. Unexpectedly, IFNβ as single stimulus failed to initiate STAT1 binding in Hepa1-6 cells. This was in contrast to IFNγ and may propose pivotal action of ISGF3 signaling in the context of stimulation by IFNβ. Data indicate that, in Hepa1-6 cells, signaling by IL-1β/TNFα enforces IFNβ-induced STAT1 binding to the iNOS promoter thereby enabling synergistic gene induction. As expected, IL-1β/ TNFα-stimulation, without IFNβ, did not mediate STAT1 binding to this promoter region (Figure 3B, lower panel). In order to further deepen the connection between IL-1β/TNFα and IFNβrelated STAT1 binding to the hepatocyte iNOS promoter, NF-κB activation was inhibited by exposing cells to the IKK inhibitor IKK-VII (32). As detected by ChIP analysis, STAT1 binding to the aforementioned region of the iNOS promoter (−912 to −1,029 bp relative to the TSS) was significantly impaired under the influence of IKK-VII ( Figure 3C). Data indicate that NF-κB activation by IL-1β/TNFα, likely mediated by an active proximal NF-κB site at −85 to −76 bp relative to the TSS (8), supports STAT1 binding to the iNOS promoter in murine Hepa1-6 hepatoma cells.

Type i iFn supports expression of inOs during aPaP-induced liver injury
To investigate regulation of hepatic iNOS by type I IFN in vivo, the model of moderate APAP-induced acute liver inflammation was used. Liver injury and thus associated necroinflammation in this model is at its peak at around 24 h after APAP administration. Notably, 48 h after onset of intoxication serum ALT levels, indicative of liver necrosis, drop to approximately or below 20% of those detectable at 24 h with liver morphology displaying regeneration and recovery from injury (33)(34)(35)(36)(37)(38). In light of these characteristics, we chose to focus herein on the 24 h time point after APAP administration. Notably, this APAP toxicity is associated with a cytokine response that includes upregulation of IL-1β and TNFα (23,39). Previous reports demonstrated that, during murine APAP intoxication, iNOS protein is well detectable (33,40) in hepatocytes at regions with centrilobular injury (41,42). Evaluation of iNOS knockout mice indicated that iNOS-derived NO may promote injury during early intoxication (detected by serum ALT) (43,44). In contrast, hepatotoxicity was found to be independent from iNOS analyzed histochemically after 24 h. The role of iNOS in APAP-induced liver injury appears complex since NO also inhibits generation of poisonous N-acetyl-p-benzoquinone imine from APAP and reduces superoxide anion-dependent lipid peroxidation (43).
In the present study, we confirm increased hepatic iNOS protein in APAP-treated mice (Figure 4A; with densitometric quantification, right panel). Immunohistochemistry likewise corroborated iNOS protein expression by hepatocytes during APAP intoxication which was absent in ctrl-mice ( Figure 4B).
To determine the relevance of type I IFN for iNOS expression in the context of APAP-induced liver injury, experiments were performed by using IFNAR −/− mice. Those mice are unable to respond to type I IFN (22,31). In fact, induction of hepatic iNOS protein was impaired in IFNAR-deficient mice (Figure 5A; with densitometric quantification, right panel). In a next step, expression of hepatic IFNα/β was determined in order to further assess type I IFN action during APAP intoxication. Notably, we did not observe upregulation of hepatic IFNα/β mRNA as detected 6 h (data not shown) or 24 h after administration of APAP. However, basal constitutive type I IFN was readily detectable in all liver specimens investigated. Figure 5B demonstrates constitutive hepatic IFNα expression as detected by standard PCR using primers targeting the entire panel of murine α-subtypes. Those results were confirmed by realtime PCR for detection of IFNα2 ( Figure 5C, left panel), IFNα4 (Figure 5C, middle panel), and IFNα5 ( Figure 5C, right panel). Likewise, hepatic IFNβ was expressed constitutively (Figure 5D). Besides iNOS, IFNAR-deficient mice exposed to APAP likewise displayed decreased hepatic mRNA of STAT1-dependent CXCL9 (Figure 5E). We and others have previously reported that STAT1 gene expression is regulated by IFN/STAT1-driven positive feedback regulation (16,31,(45)(46)(47). Accordingly, decreased hepatic STAT1 mRNA was likewise detected in IFNAR-deficient mice ( Figure 5F) which likely contributes to downregulation of STAT1-inducible genes such as iNOS.
Finally, the role of IFNAR concerning the severity of APAP-induced liver injury was assessed. This issue is in fact controversially discussed. Whereas a previous study reported no effects of IFNAR deficiency on intoxication (35), another study observed pathological action of type I IFN. Specifically, administration of IFNAR-neutralizing antibodies diminishes murine APAP-induced liver damage. Moreover, intoxication is enhanced in genetically engineered mice displaying impaired IFNAR degradation but ameliorated when degradation is enforced by pharmacological means (48). In support of this latter view, herein, reduced APAP toxicity connected to IFNAR deficiency which was observed by analysis of serum ALT ( Figure 6A) and histological software-aided evaluation 24 h after administration of 500 mg/kg APAP (29.6 ± 5.3% reduction of liver necrosis in IFNAR-deficient versus wt mice; n = 9, P < 0.01 by unpaired Student's t-test). Figure 6B displays histochemistry of representative APAP-induced hepatic injury in wt and IFNAR −/− mice, respectively.

DiscUssiOn
Type I IFN is a key cytokine component of innate immunity supposed to affect course of disease particularly in viral but also during bacterial infections and, due to a substantial immunoregulatory potential, likewise in sterile inflammation (30,(49)(50)(51)(52).
Herein, type I IFN is characterized in vitro and in vivo as significant determinant of hepatic iNOS expression having the potential to determine disease outcome during liver inflammation. On a cellular level, we demonstrate that IFNβ potently synergizes with the prototypic inflammatory cytokines IL-1β/TNFα for induction of iNOS in primary murine hepatocytes and murine Hepa56.1D as well as Hepa1-6 hepatoma cells. As determined in this latter cellular model, IFNβ directs STAT1 binding to a critical regulatory site within the murine iNOS promoter (−912 to −1,029 bp relative to the TSS) (8,9), a process that, in Hepa1-6 hepatoma cells, demanded simultaneous pro-inflammatory signaling by IL-1β/TNFα. Notably, a recent report identified in murine bone marrow-derived macrophages (BMDM) an additional distal STAT1 binding region 30 kB upstream of the iNOS TSS which may be able to regulate gene expression (53). However, using IL-1β/TNFα/IFNβ-stimulated Hepa1-6 cells we confirm evident binding of STAT1 to the aforementioned proximal promoter region. Combined with previous studies (8,9), present data thus emphasize the relevance of this proximal STAT1-binding region for iNOS induction. Interestingly, IFNγ but not IFNβ as single stimulus mediated STAT1 binding to this site-suggesting a role for ISGF3. In contrast to the present observations using Hepa1-6 cells, IFNβ as single stimulus induces STAT1 binding to this proximal binding region in BMDM (9) which indicates cell type-specific mechanisms at work. The unexpected requirement of IL-1β/TNFα signaling for STAT1 binding to the iNOS promoter observed herein is a further cell-type specific facet of the well-described STAT1/NF-κB synergism that drives inflammatory/antimicrobial gene expression (53). Regulation of CXCL9 in murine NIH3T3 fibroblasts may serve as a leading case in that context. There, efficient STAT1 binding to the CXCL9 promoter demands TNFα costimulation and downstream STAT1/NF-κB (e) Hepatic CXCL9 mRNA was determined by realtime PCR and normalized to GAPDH (mean ± SEM; wt, n = 8 individual mice; IFNAR −/− , n = 9; *P < 0.05). (F) Hepatic STAT1 mRNA was determined by realtime PCR and normalized to GAPDH (mean ± SEM; n = 9 individual mice per genotype; *P < 0.05). (a,e,F) Statistical analysis, raw data were analyzed by Student's t-test.
bridging by CREB binding protein-though STAT1 homodimers are involved in this case (54). The notion of cooperative transcription factor binding, specifically NF-κB enforcing STAT1 binding to the iNOS promoter in IL-1β/TNFα/IFNβ-stimulated Hepa1-6 cells, was confirmed herein by pharmacological inhibition of NF-κB. Upregulation of iNOS in vivo during murine APAP-induced liver injury and sterile inflammation in fact largely depended on type I IFN signaling which associated with pronounced liver injury. Whereas hepatic type I IFN, as assessed by analysis of IFNα/β expression, was not upregulated during APAP intoxication, basal type I IFN was well detectable in murine liver tissue, an observation that agrees with previous reports on constitutive murine hepatic IFNα (55) and IFNβ (56), respectively. Notably, the liver is regarded a major target for constitutively produced type I IFN in healthy mice (57) and low-level "physiological" expression of type I IFN also applies to human liver tissue and hepatocytes (58,59). By generally promoting signal transduction mechanisms related to cellular activation, constitutive low-level expression of type I IFN is supposed to prime diverse tissues for immunological alertness (45) which may in particular apply to the liver as crucial host/environment-interface serving "firewall" functions (60).
It must, however, be emphasized that regulatory properties of type I IFN during hepatic inflammation are multilayered and context dependent. This is exemplified by the general ability of type I and II IFN to potently upregulate anti-inflammatory IL-1 receptor antagonist (IL-1Ra) (61) which, by inhibiting IL-1 biological activity, enables protection in murine models of nucleic acid (virus)-induced liver damage (22,62). Interestingly, the role of IL-1 in APAP-induced liver injury is actually discussed controversially with disease aggravating action (63,64), no significant role (65), or even protective functions (66) being ascribed to this cytokine. Of note, administration of IL-1Ra did not affect disease in the current protocol of APAP-induced liver injury (Bachmann and Mühl, unpublished data), an observation supporting aforementioned previous report (65). Data thus indicate that putative upregulation of potentially protective IL-1Ra by surplus type I IFN falls short in the current pathophysiological context.
Data presented not only relate to sterile inflammation as seen in APAP intoxication but likewise connect to infectious diseases such as viral hepatitis. Interestingly, hepatocytes express iNOS protein during chronic hepatitis C virus (HCV) infection (67) which, according to data presented herein, should be supported by induction of endogenous type I IFN in response to the virus (68). Moreover, HCV patients responding most efficiently to IFNα therapy likewise display increased serum nitrite/nitrate levels (69), an established surrogate marker of iNOS activation during infectious diseases (70).
Taken together, current knowledge and data presented herein suggest that IFNβ supports hepatocyte iNOS by dual complementary action. That is, IFN signaling directly triggers STAT1 biological activity, a process further enhanced by feed-forward upregulation of STAT1 gene expression. Data also suggest a pathogenic role for constitutive type I IFN during the course of APAP intoxication which is regarded a prototypic model for drug-induced injury and sterile inflammation at the liver compartment.

eThics sTaTeMenT
All animal experiments using C57Bl/6 mice (male, 9-10-week-old, wt, and IFNAR −/− mice) were carried out in accordance with the recommendations of the Animal Protection Agency of the Federal State of Hessen (Regierungspräsidium Darmstadt, Germany). The protocol was approved by the Regierungspräsidium Darmstadt (Germany).

aUThOr cOnTriBUTiOns
HM analyzed the data, designed the study, wrote the paper, and performed manuscript editing. MB performed all experiments, analyzed the data, and contributed to manuscript writing and editing. ZW analyzed the data, provided mice, and technical support. TP provided crucial technical support. JP analyzed the data, provided reagents (antibodies), and contributed to manuscript editing.

acKnOWleDgMenTs
The work of the author is supported by a grant from the DFG (MU 1284/6-1 to HM). Authors gratefully acknowledge expert technical assistance of Dorothea Kreuz. Authors are indebted to Dr. Itamar Goren and Prof. Stefan Frank (pharmazentrum Frankfurt, Germany) for providing positive/negative control lysates for iNOS expressing murine tissue.