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ACTIVATION OF THE LIVER X RECEPTOR PROTECTS AGAINST HEPATIC INJURY IN ENDOTOXEMIA BY SUPPRESSING KUPFFER CELL ACTIVATION

Wang, Yun Yong*; Dahle, Maria K.*; Ågren, Joanna*; Myhre, Anders E.*; Reinholt, Finn P.; Foster, Simon J.§; Collins, Jon L.; Thiemermann, Christoph; Aasen, Ansgar O.*†; Wang, Jacob E.*†

doi: 10.1097/01.shk.0000191377.78144.d9
Basic Science Aspects
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ABSTRACT Recent reports have demonstrated that liver X receptors (LXRs) of the nuclear receptor family have anti-inflammatory effects on macrophages. Here we examine whether activation of LXR by the synthetic agonist GW3965 can ameliorate the liver injury/dysfunction caused by endotoxins in the rat. Male Wistar rats received GW3965 (0.3 mg/kg) or vehicle (50% dimethyl sulfoxide) 30 min before coadministration of lipopolysaccharide (LPS, 5 mg/kg i.v.) and peptidoglycan (1 mg/kg i.v.). Treatment with GW3965 attenuated the increase in the plasma levels of alanine aminotransferase and bilirubin (markers of liver injury/dysfunction) as well as the focal hepatocyte necrosis (histology) caused by coadministration of LPS and peptidoglycan. This protective effect of GW3965 treatment was associated with reduced infiltration of mast cells in the liver (histopathology) and reduced gene expression of the chemokines eotaxins 1 and 2, whereas MIP-2 mRNA levels were not affected. Plasma levels of tumor necrosis factor α and prostaglandin E2 were significantly attenuated by GW3965, whereas plasma interleukins 6 and 10 were not altered. High expression of LXRα mRNA was observed in Kupffer cell cultures, suggesting that Kupffer cells are targets of GW3965. Subsequent in vitro studies in Kupffer cells demonstrated that exposure to GW3965 attenuated the LPS-induced release of tumor necrosis factor α and prostaglandin E2 in a dose-dependent manner. In conclusion, this study demonstrates that activation of LXR by GW3965 protects against liver injury and dysfunction in a rat model of endotoxemia, in part by exerting an anti-inflammatory effect on Kupffer cells.

*University of Oslo, Faculty Division Rikshospitalet, Institute for Surgical Research, Oslo, Norway; Rikshospitalet University Hospital, Institute for Surgical Research, Oslo, Norway; Rikshospitalet University Hospital, Institute/Department of Pathology, Oslo, Norway; §University of Sheffield, Sheffield, UK; GlaxoSmithKline Research and Development, Research Triangle Park, NC; and The William Harvey Research Institute, London, UK

Received 28 Jul 2005; first review completed 31 Aug 2005; accepted in final form 7 Oct 2005

Address reprint requests to Jacob E. Wang, Rikshospitalet University Hospital, Institute for Surgical Research, Sognsvannsveien 20, 0027 Oslo, Norway. E-mail: j.e.wang@medisin.uio.no.

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INTRODUCTION

Sepsis denotes the inflammatory host response to infection. Whereas inflammation is normally life-preserving and pivotal for activation of host defense to invading pathogens, an exaggerated and dysregulated inflammation contributes to development of organ injury and, ultimately, mortality in patients with severe sepsis. The host response is activated when bacterial components (endotoxins) such as lipopolysaccharide (LPS) and peptidoglycan (PepG) are recognized by receptors on host immune cells, which triggers the inflammatory response (rev. in Ref. 1). Injection of LPS into animals causes a sepsis-like syndrome, comprising a number of features, which are important clinical manifestations of severe sepsis such as hypotension, systemic inflammation and ultimately, organ injury/dysfunction. PepG, a cell wall fragment primarily found in the wall of gram-positive bacteria, also causes inflammation and organ injury, alone or in combination with LPS or lipoteichoic acid (2-4). Furthermore, activation of Kupffer cells by LPS contributes to development of liver injury mediated by cytokines such as tumor necrosis factor α (TNF-α) and nitric oxide (5-7). Exuberant levels of additional inflammatory mediators are also produced during sepsis (8-11). Numerous clinical trials with drugs to block reactions within the inflammatory cascade in patients with severe sepsis have been performed over the last decade. This approach has, however, not made substantial impact on mortality, which is still unacceptable high.

Liver X receptors (LXRs) belong to the nuclear hormone receptor family and are DNA-binding transcriptional regulators. For many years, LXRs have been known to act as cellular sensors of cholesterol-regulating genes involved in lipid metabolism (12-14). The physiologic ligands of LXR are oxysterols, which are oxidized cholesterol derivatives. There are two forms of LXR: LXRα and LXRβ. Whereas LXRβ is ubiquitously expressed, LXRα is predominantly expressed in macrophages (15) and regulates a number of genes controlling reverse cholesterol transport (16). Surprisingly, it was recently demonstrated that activation of LXR has anti-inflammatory effects in macrophages (17). A synthetic agonist of LXR (GW3965) was shown to inhibit transcription of inflammatory genes such as cyclooxygenase (COX) 2, inducible nitric oxide synthase, interleukin (IL) 6, and matrix metalloproteinase 9, (17, 18) all of which are implicated in the pathogenesis of organ injury in endotoxemia. Thus, we postulated that activation of LXR may ameliorate the inflammation and organ injury caused by coadministration of LPS and PepG in the rat.

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MATERIALS AND METHODS

Materials

PepG was isolated from Staphylococcus aureus, using a method described for Bacillus subtilis (19). The isolated PepG was also enzymatically digested, resulting in the expected reverse phase-high-pressure liquid chromatography muropeptide profile with no spurious products. Before administration, the PepG aggregates were dispersed by sonication (3,000 Hz, 3 × 10 s) on ice to increase its availability to interact with cells. LPS (serotype B026) was purchased from Sigma (St. Louis, Mo).

GW3965 (3-{3-[(2-chloro-3-trifluoromethyl-benzyl)-(2,2-diphenyl-ethyl)-amino] -propoxy}-phenyl)-acetic acid) is a synthetic selective agonist of LXR (20, 21).

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Surgical procedure

This study was carried out on male Wistar rats (230-280 g) receiving a standard diet and water ad libitum. The investigation was evaluated and approved by the local ethical committee and was performed according to European legislation for animal experimentation. Animals were anesthetized with thiopentone sodium (Intraval Sodium, 120 mg/kg, i.p.), and the general surgical procedures was performed as previously described (3). After surgery, heart rate and blood pressure were allowed to stabilize for 10 to 15 min, followed by slow injection of (i) saline with 50% DMSO (sham, n = 9), (ii) saline with 50% DMSO 30 min before LPS (5 mg/kg) + PepG (1 mg/kg, n = 8), and (iii) GW3965 (0.3 mg/kg) in 50% DMSO 30 min before LPS/PepG (n = 9) into the jugular vein over a 10-min period. After 1, 3, and 6 h, blood was sampled from the carotid artery, and plasma was stored.

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Measurement of indices of organ injury/dysfunction

Six hours after coadministration of LPS and PepG, blood was collected from a catheter placed in the carotid artery. Plasma samples were analyzed for alanine aminotransferase (ALT), and bilirubin was analyzed by activity assays and Roche automated clinical chemistry analyzer (Roche Diagnostics, Indianapolis, Ind). In brief, the ALT assay is based on conversion of α-ketoglutarate and L-alanine to L-glutamate and pyruvate by bioactive ALT in the sample. The increase in pyruvate is determined in an indicator reaction catalyzed by lactate dehydrogenase where NADH is oxidized to NAD+. The rate of the photometrically determined NADH decrease is directly proportional to the rate of formation of pyruvate and thus the ALT activity. The bilirubin assay is based on conversion of bilirubin into azobilirubin under acidic conditions, which is photometrically determined.

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Histopathological assessment

Livers were fixed by immersion in 4% phosphate-buffered formalin. Samples obtained by systematic random sampling were embedded in paraffin wax, sectioned at 5 μm, and stained with hematoxylin and eosin according to a routine protocol. Coded sections were evaluated by an experienced pathologist focusing on the following parameters: focal hepatocyte injury, lobular inflammation (infiltration of neutrophils), and presence of mast cells in portal area. The parameters were semiquantified in a three-graded scale.

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Analysis of cytokines and prostaglandin E2

TNF-α (Biosource, Camarillo, Calif), IL-6 (R&D, Abingdon, UK), and IL-10 (Biosource) were analyzed by ELISA kits specific for rat cytokines. Prostaglandin E2 (PGE2, R&D, UK) was analyzed by enzyme immunoassays which is independent of species.

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Real-time reverse transcriptase-polymerase chain reaction

For Kupffer cell RNA preparation, cultures in 6-well plates (3 × 106 cells per plate) were scraped in RNeasy lysis buffer (Qiagen, Hilding, Germany). Tissue samples were immediately frozen in liquid nitrogen and grinded in a precooled mortar, and 30 mg of frozen material was homogenized by ultra Turrax (20,000/min) in RNeasy lysis buffer (Qiagen). Further isolation was performed using RNeasy mini Kit (Qiagen), according to the manufacturer's protocol. Fifty nanograms of total RNA from each sample was reverse transcribed using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, Calif), and specific mRNA levels were assessed using the ABI Prism 7900HT (Applied Biosystems) with sequence specific polymerase chain reaction (PCR) primers (Table 1). Real-time PCR was performed with 2× qPCR Master Mix for SYBR Green (Eurogentec, Liege, Belgium), 300 nmol/L sense and antisense primers, cDNA, and water up to 25 μL. The specificities of all SYBR Green assays were confirmed by melting point analysis. The concentration of 18S mRNA was used for normalization of target gene expression (18S Predeveloped Assay Reagents, Applied Biosystems). All samples were run in triplicate; standard curves were run on the same plate, and the standard curve method was used to calculate the relative gene expression.

Table 1

Table 1

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Primary cultures of Kupffer cells

Kupffer cells were isolated from rat liver as recently described (22). In brief, livers were perfused ex vivo with collagenase P (0.24 mg/mL, Roche, Mannheim, Germany) to digest the liver. The subsequent cell suspension was subjected to gradient centrifugation in Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden) to separate the nonparenchymal cells (liver endothelial cells and Kupffer cells). Nonparenchymal cells were plated on 96-well plates (0.2 million cells per well) and washed after 2 h to remove liver endothelial cells. Adherent Kupffer cells were then cultured in RPMI1640 (BioWhittaker Europe, Verviers, Belgium) in the presence of 10% fetal calf serum (GIBCO, Grand Island, NY), L-glutamine (2 mmol/L, GIBCO) and antibiotics (penicillin and streptomycin, GIBCO). Immunohistochemical assessment revealed that more than 90% of the cells stained with the macrophage marker ED2, indicative of Kupffer cells. Following 2 days in culture, Kupffer cells were exposed to GW3965 (0.01, 0.l, 1, or 10 μmol/L) 60 min before addition of LPS (1 μg/mL). All unstimulated and control samples were added an equal amount of drug solvent (DMSO, 1‰ vol/vol). Media samples were harvested for cytokine analyses after 6 h or for RNA analyses after 2 h.

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Statistics

Statistical analyses were performed by analysis of variance or t test. P ≤ 0.05 was considered significant.

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RESULTS

Effects of GW3965 on mean arterial pressure

Baseline values of mean arterial pressure ranged between 130 and 150 mmHg and were not significantly different between groups (Table 2). Coadministration of LPS and PepG to the jugular vein caused a transient reduction in mean arterial pressure lasting 30 min. Injection of GW3965 before coadministration of LPS and PepG did not influence the mean arterial pressure in the animals.

Table 2

Table 2

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Effects of GW3965 on hepatocellular injury and dysfunction

Coadministration of LPS and PepG to the rats caused significant increase in plasma levels of ALT (P ≤ 0.01) and bilirubin (P ≤ 0.01) (Fig. 1). Pretreatment with GW3965 before injection of LPS/PepG reduced the plasma levels of ALT (P ≤ 0.05) (Fig. 1A), whereas the reduction in bilirubin did not reach statistical significance (Fig. 1B).

Fig. 1

Fig. 1

Histopathological examination confirmed that coadministration of LPS and PepG caused significant focal hepatocyte injury (Fig. 1C), which resembles the liver injury caused by infections. Treatment with GW3965 caused significant reduction in hepatocellular injury, as quantified by a three-graded histopathological assessment (Fig. 1C).

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Effect of GW3965 on leukocyte recruitment

To determine the effect of LXR activation on recruitment of leukocytes to the liver, sections were evaluated by a pathologist with respect to infiltration of leukocytes. Coadministration of LPS and PepG caused a significant increase in lobular inflammation as seen by leukocyte accumulation in sections of the liver, which was not significantly inhibited by GW3965 treatment (Fig. 2A). Moreover, mast cells were observed in the periportal area of LPS/PepG-treated animals (control). Treatment with GW3965 reduced the mast cell count to basal level (Fig. 2B).

Fig. 2

Fig. 2

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Effects of GW3965 on the increase in the plasma levels of cytokines and PGE2

To test the effect of GW3965 on systemic inflammation, we measured the levels of cytokines and PGE2 in plasma samples (Fig. 3). Sham animals had low plasma levels of all cytokines tested. Coadministration of LPS and PepG caused a substantial increase in the plasma levels of TNF-α (Fig. 3A), PGE2 (Fig. 3B), IL-10 (Fig. 3C), and IL-6 (Fig. 3D), compared with sham animals. Administration of GW3965 was associated with a substantial reduction in plasma levels of TNF-α at 1 h (P ≤ 0.05) and PGE2 at 6 h (P ≤ 0.05), compared with vehicle controls. In contrast, plasma levels of IL-6 or IL-10 were not significantly altered by pretreatment of GW3965.

Fig. 3

Fig. 3

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Effects of GW3965 on chemokine gene regulation in liver

To understand how GW3965 has attenuated recruitment of mast cells to the liver, we next examined the gene activation of chemokines. MIP-2 mRNA was clearly enhanced in liver by administration of LPS/PepG but was not significantly altered by GW3965 (Fig. 4A). LPS/PepG also caused an increase in the expression of eotaxin 1 mRNA (Fig. 4B). This increase was significantly reduced in livers from rats treated with GW3965 before LPS/PepG. Expression of eotaxin 2 mRNA was also reduced by GW3965 treatment (Fig. 4C).

Fig. 4

Fig. 4

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Expression and regulation of LXRα

We hypothesized that the Kupffer cells were potential targets of GW3965 in the liver. Indeed, expression of LXRα mRNA was observed in primary cultures of rat Kupffer cells, which was substantially higher than the levels of LXRα mRNA detected in whole liver, lung, and kidney (Fig. 5A). After exposure to LPS, LXRα mRNA levels in the Kupffer cells were significantly decreased (Fig. 5B). Also, treatment of the Kupffer cells with GW3965 (1 μmol/L) caused significant induction of ABCA1 mRNA (Fig. 5C), a positive control gene for LXR signaling involved in reverse cholesterol transport.

Fig. 5

Fig. 5

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Effect of LXR activation on cytokine release in primary cultures of rat Kupffer cells

Pretreatment of Kupffer cells with GW3965 before addition of LPS attenuated the release of TNF-α (Fig. 6A) and PGE2 (Fig. 6B), whereas the levels of IL-10 (Fig. 6C) and IL-6 (Fig. 6D) were not significantly altered.

Fig. 6

Fig. 6

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DISCUSSION

When activated by physiologic ligands such as oxysterol (cholesterol derivatives), LXRs control a number of genes involved in cholesterol and glucose metabolism. Recently, convincing evidence was presented that LXRs also control genes involved in regulation of inflammation (17). On this background, we wanted to test the hypothesis that activation of LXR may protect against organ injury/dysfunction in endotoxemia. The synthetic agonist of LXR (GW3965) used in this study was originally developed for the treatment of atherosclerosis (23). The key findings reported in this manuscript are the following: in rats injected with LPS/PepG, the systemic administration of (low doses of) the specific LXR agonist GW3965 attenuated (i) the hepatic injury (increase in transaminases and histopathology), (ii) the rise in plasma TNF-α and PGE2, and (iii) the recruitment of mast cells to the liver. In addition, GW3965 also inhibited the LPS-induced release of TNF-α and PGE2 in rat Kupffer cell cultures.

ALT and bilirubin are biochemical markers of liver injury/dysfunction, which are routinely used in diagnosis of liver disease and liver failure. Almost all of the organism's ALT is located in the cytoplasm of hepatocytes, and upon injury to these cells, ALT leaks out and can be detected in serum. Our findings that ALT was reduced by treatment with GW3965, therefore, convincingly demonstrate that activation of LXR has protected the hepatocytes from injury. Bilirubin is a metabolite of hemoglobin, which is normally excreted with bile into the intestine. In liver failure or in situations of excessive hemolysis, the hepatocytes' capacity to conjugate and excrete bilirubin is reduced, leading to increase in serum bilirubin. Hence, the increase in serum bilirubin in the model of LPS-induced shock used here indicates a reduction in excretory function of the liver, which was partly restored by treatment with GW3965.

TNF-α is an early mediator in endotoxemia, with multipotent functions (24). Our finding that the increase in the plasma levels of TNF-α caused by coadministration of LPS and PepG was attenuated by GW3965 is in line with a report showing that activation of LXR inhibits the production of TNF-α in a rodent model of contact dermatitis (25). This finding is also consistent with the reported ability of GW3965 to interfere with NF-κB signaling (17). In murine macrophages, activation of LXR by GW3965 has been shown to attenuate the expression of proinflammatory genes (17). Notably, GW3965 reduced the gene activation of COX-2, inducible nitric oxide synthase, matrix metalloproteinase 9, and IL-6 induced by LPS in peritoneal murine macrophages (17). Inhibition of the expression of COX-2 protein by GW3965 may well explain the attenuation of the increase in the plasma levels of PGE2 afforded by this LXR agonist in our model of LPS/PepG shock. There is good evidence that PGE2 is a mediator of liver injury in endotoxemia (26). Thus, the reduced PGE2 levels in rats treated with GW3965 may in part explain the reduced hepatocellular injury in these animals. However, it should be noted that whereas PGE2 has proinflammatory functions in the early phase of inflammation, it serves anti-inflammatory properties on macrophages by induction of cAMP (27).

One of the major functions of the previously discussed proinflammatory cytokines is the induction of chemokines, which are chemotactic proteins directing leukocytes to a site of inflammation. This raised the question whether the LXR agonist GW3965 might suppress chemokine expression. The mRNA expression of the chemokine MIP-2 in the liver, which is important in recruitment of neutrophils to the liver (28), was not significantly suppressed by GW3965 treatment. This is partly consistent with the nonsignificant reduction of neutrophil infiltration in the liver, observed by histopathology. In contrast, we demonstrate that both eotaxins 1 and 2 mRNA in the liver were suppressed by GW3965. Eotaxins are ligands for the chemokine receptor CCR3, which is predominately expressed on eosinophils and mast cells (29-31). Consistent with the reduced expression of eotaxins, reduced number of mast cells was observed in the livers of animals treated with LXR agonist. There is good evidence to suggest that eosinophils contribute to liver injury in endotoxemia (32, 33), whereas a similar role of mast cells remains to be demonstrated. Thus, the significance of the suppressed recruitment of mast cells in animals treated with GW3965 remains elusive. Also, whether the suppressive effect of GW3965 on eotaxin gene activation is a direct effect or occurs secondary to the reduction in TNF-α or PGE2 is still not clear.

LXRα is reported to be predominantly expressed in macrophages (15). Hence, it is reasonable to hypothesize that the beneficial effects of GW3965 on liver injury/dysfunction may be secondary to interactions with the macrophages of the liver, the Kupffer cells. We confirm the expression of LXRα mRNA in Kupffer cells (34) and report that LXR-mediated gene regulation operates in these cells (induction of ABCA1 mRNA).

We also report for the first time that activation of LXR causes downregulation of TNF-α and PGE2 in these cells, in accordance with the in vivo effects of GW3965. It should be mentioned that the dose-response curve on TNF-α was bell-shaped,where low doses of GW3965 inhibited TNF-α, whereas a higher dose (1 μmol/L) was unable to do so. The effect of LXR on gene regulation is complex and is influenced by activation of coactivators and corepressors. Alternatively, whereas GW3965 is a specific agonist at low doses, it is possible that it has off-target effects at higher doses which are unrelated to LXR. Similar to our observations in vivo, GW3965 also did not affect the increase in the levels of IL-6 and IL-10 measured in the supernatants of these isolated Kupffer cells. These data demonstrate for the first time that GW3965 has direct anti-inflammatory effects on Kupffer cells.

Together, this points to a mechanism whereby GW3965 activates LXR in Kupffer cells, leading to suppression of the potent inflammatory mediators PGE2 and TNF-α, as well as suppressed expression of eotaxins. This leads to reduced recruitment of certain leukocytes such as mast cells to the liver. Our study demonstrates a novel and potentially important modality in the therapy of organ injury caused by coadministration of wall fragments of gram-negative (LPS) and gram-positive bacteria (peptidoglycan) and a novel area of interest for LXR research.

In conclusion, the present study demonstrates that activation of LXR by GW3965 reduces the liver injury and inflammation in a rat model of LPS/PepG-induced shock, in part by exerting an anti-inflammatory effect on Kupffer cells.

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ACKNOWLEDGMENTS

The authors thank Elin Sletbakk, Grethe Dyrhaug, Barbara Løken, and the Department of Medical Biochemistry, Rikshospitalet University Hospital, for skilful technical assistance. We are also grateful to the Norwegian Research Council and Stiftelsen Sophies Minde for providing financial support. This work was, in part, supported by the William Harvey Research Foundation.

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Keywords:

Liver X receptor; GW3965; liver injury; MODS; TNF-α; sepsis; rat; LPS

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