GW0742

Dose-dependent effects of peroxisome proliferator-activated receptors β/δ agonist on systemic inflammation after haemorrhagic shock

A B S T R A C T
Introduction: PPARβ/δ agonists are known to modulate the systemic inflammatory response after sepsis. In this study, inflammation modulation effects of PPARβ/δ are investigated using the selective PPARβ/δ agonist (GW0742) in a model of haemorrhagic shock (HS)-induced sterile systemic inflammation.Methods: Blood pressure-controlled (35 ± 5 mmHg) HS was performed in C57/BL6 mice for 90 min. Low-dose GW0742 (0.03 mg/kg/BW) and high-dose GW0742 (0.3 mg/kg/BW) were then administered at the beginning of resuscitation. Mice were sacrificed 6 h after induction of HS. Plasma levels of IL-6, IL-1β, IL-10, TNFα, KC, MCP- 1, and GM-CSF were determined by ELISA. Myeloperoxidase (MPO) activity in pulmonary and liver tissues was analysed with standardised MPO kits.Results: In mice treated with high-dose GW0742, plasma levels of IL-6, IL-1β, and MCP-1 were significantly increased compared to the control group mice. When compared to mice treated with low-dose GW0742 plasma levels of IL-6, IL-1β, GM-CSF, KC, and MCP-1 were significantly elevated in high-dose-treated mice. Low-dose GW0742 treatment was associated with a non-significant downtrend of inflammatory factors in mice with HS. No significant changes of MPO activity in lung and liver were observed between the control group and the GW0742 treatment groups.Conclusion: This study identified dose-dependent effects of GW0742 on systemic inflammation after HS. While high-dose GW0742 substantially enhanced the systemic inflammatory response, low-dose GW0742 led to a downtrend of pro-inflammation cytokine expression. The exact mechanisms are yet unknown and need to be assessed in further studies.

1.Introduction
Peroxisome proliferator-activated receptors (PPARs) are transcrip- tion factors that are be activated by binding of its corresponding li- gands. There are three different PPAR isoforms: PPARα, PPARβ/δ, and PPARγ have been found so far [1]. Once the PPARs are activated by their specific ligands, they will form a heterodimer with the Retinoid X receptor (RXR). The PPAR–RXR complex binds to PPAR response ele- ments in target genes and then induces gene transcription [2]. Early studies indicated that PPARs can regulate genes that are involved in lipid and glucose metabolism. Many kinds of artificial PPARs agonists, for example, fenofibrate and thiazolidinedione (TZD) drugs, were de- veloped to treat metabolic diseases. The inflammation modulation property was then reported in further studies [3,4].PPARβ/δ is ubiquitously expressed in many tissues, such as lung, liver, kidney, spleen, and blood vessels [5,6]. These tissues are also involved in the systemic inflammatory process induced by haemor- rhagic shock. GW0742 is a synthetic and selective agonist with high affinity to PPARβ/δ receptors [7]. Several studies have reported that GW0742 protects against inflammation caused by septic shock and many pathogens [8–10]. There is also reliable evidence that GW0742 reduces myocardial ischaemia/reperfusion local injury in rats. Kapoor et al. [11] reported that GW0742 treatment caused a significant re- duction in myocardial infarction size in rats, with ischaemia/reperfu- sion injury at 0.03 mg/kg/BW but not at 0.003 mg/kg/BW. Moreover, studies have also identified that PPARs could modulate the expression of genes in a dose-dependent manner [12]. However, the studies about how PPARβ/δ modulates systemic inflammatory response following a haemorrhagic shock (HS) or trauma are rare. We hypothesise that the GW0742 treatment has a systemic inflammatory modulation effect on sterile inflammation as well.
Therefore, the main aim of the present study was to analyse the effects of GW0742 on systematic inflammation after HS. Secondly, we also aimed to investigate the dose-dependent effects of this agonist in a sterile inflammatory response.

2.Materials and methods
All experimental protocols were approved by the North Rhine- Westphalian State Agency for Nature, Environment and Consumer Protection (LANUV) (no. 84-02.04. 2015. A205). All mice involved in this study were cared for and used according to the National Institutes of Health (NIH) animal care guidelines.C57/BL6 male mice were bought from Charles Rivers Laboratories in Germany, being 8–10 weeks old and with 20–25 g body weight. Before starting the experiments, all mice were housed in the animal research centre at RWTH Aachen University for two weeks with a 12-h light followed by 12-h dark cycle. Animals were housed in plastic cages with adequate laboratory food and water. Mice were anaesthetised by firstly inhaling isoflurane (Abbott Laboratories, Wiesbaden, Germany) followed by intraperitoneal injection of Pentobarbital (70 mg/kg; Merial GmbH, Hallbergmoos, Germany). Before the induction of HS, mice were injected with Buprenorphine (0.5 mg/kg; Reckitt-Benckiser, Bergheim, Germany) subcutaneously.Mice enduring HS were randomly divided into three groups (n = 8). The control group (animals with HS) received 100 μL of normal saline (NS) solution with 10% dimethyl sulfoxide (DMSO), group HS+ low- dose GW0742 (0.03 mg/kg·BW) (Groups LGW), and group HS+ high- dose GW0742 (0.3 mg/kg·BW) (Groups HGW); all the GW0742 was dissolved in 100 μL NS with 10% DMSO. Prior studies have shown that a dosage of 0.03 mg/kg/BW attenuated the systemic inflammation after sepsis stimulus [8]. GW0742 was administered via an arterial catheter 90 min after the induction of HS.

The sample collection was conducted six hours after the HS induction. All mice were kept anaesthetised throughout the experiment period.The operation procedure of HS on mice has been described in pre- vious studies [13,14] (Fig. 1). Briefly, mice subjected to HS were fixed in a supine position, and the skin of the left inguinal region was shaved and sterilised with iodophor disinfectants. The left femoral artery was exposed via an incision on the upper thigh, then the femoral artery was half dissected and subsequently cannulated with a polyethylene ca- theter (Becton, Dickinson and Company, USA). The tube was rinsed with heparin solution (Ratiopharm GmbH, Germany) before the can- nulation to prevent blood clots. The mean arterial pressure (MAP) of the mice was monitored by a digital blood pressure monitor (TSE Systems, Bad Homburg, Germany). Blood was discharged from the mice slowly via the femoral arterial catheter for 15 min. The catheter was then closed when the MAP reduced to 35 mmHg. Mice were resuscitated 90 min later by infusion of 0.9% NS. Subsequently, the catheter was removed, followed by an artery ligation and skin closure. The mice then entered a recovery phase of 4.5 h.At the end of the recovery period, a thoracotomy was performed on mice, and blood samples were collected via cardiac puncture. Previous studies have shown that many pro-inflammatory cytokines (such as interleukin-6 [IL-6], and IL-8) are released in the first six hours [15]. Heparinised blood samples were centrifuged at 5000 rpm for 10 min at 4 °C. The bottom cellular components of blood were discarded and the plasma was collected and stored at −80 °C. The plasma levels of in- flammatory cytokines and factors, including IL-1β, IL-6, IL-10, kerati- nocyte-derived chemokine (KC, IL-8 in humans), monocyte chemotactic protein 1 (MCP-1), tumour necrosis factor (TNF)-α, and granulocyte macrophage colony stimulating factor (GM-CSF), were evaluated using standardised ELISA kits (R & D System Inc., USA).Lung and liver tissues were taken from the mice at the end of the recovery period and immediately frozen in liquid nitrogen. The MPO activity in the lung and liver tissues was quantified by Myeloperoxidase (MPO)-enzyme-linked immunosorbent assay kits (MPO ELISA kit, Hycultec GmbH Beutelsbach, Germany). The procedures of tissue homogenisation and ELISA experiments were performed according to the manufacturer’s instructions.SPSS Inc., USA). All values in the figures and text were expressed as mean ± SEM of observations (five to six animals per group). A normal distribution of all the data was examined by both the Shapiro-Wilk test and the Kolmogorov-Smirnov test. We compared groups using ANOVA if normal distribution could be assumed. Comparison between the two groups was done with the independent sample T-test. We tested for significance at alpha < 0.05. 3.Results We found a significantly higher plasma level of IL-6 in mice sub- jected to high-dose GW0742 (738.4 pg./ml ± 87.9) compared to control mice (297.7 pg./ml ± 134.9, p = 0.03) and low-dose GW0742-treated mice (122.5 pg./ml ± 26.1, p = 0.0001) 6 h after HS. Low-dose GW0742-treated animals showed no significant decrease of plasma IL-6 level compared to the control group (p = 0.28) (Fig. 2A). The expression of IL-1β in shocked mice treated with high-doseGW0742 (107.8 pg./ml ± 13.9) was significantly increased compared to control mice (65.4 pg./ml ± 11.7, p = 0.04) and low-dose GW0742- treated mice (58.9 pg./ml ± 14.3, p = 0.04). Low-dose GW0742 treatment resulted in a slight decrease of IL-1β compared to the control group (p = 0.73) at 6 h (Fig. 2B).Compared to shocked mice without treatment (185.7 pg./ ml ± 64.6), MCP-1 levels in high-dose GW0742-treated mice were significantly increased (1189 pg./ml ± 447.4, p = 0.04), whereas the plasma MCP-1 levels declined slightly in low-dose GW0742-treated animals (115.1 pg./ml ± 10.27, p = 0.35) (Fig. 2C). No significant differences in plasma GM-CSF and KC levels were observed between the control group and GW0742-treated groups (both in LGW group and HGW group). However, compared to low-dose GW0742-treated ani- mals, the GM-CSF and KC plasma levels in high-dose GW0742-treated animals were significantly elevated (Fig. 2D and E). Six hours after the HS, the plasma levels of TNF-α and IL-10 in shocked mice and GW0742- treated animals were obtained, and they are shown in Fig. 2F and G. We did not observe significant differences in plasma TNF-α and IL-10 levels among the control, LGW, and HGW groups (Fig. 2F and G).MPO activity in both lung (group HGW: 1726 ± 346.1 pg./mg; group LGW: 1765 ± 269.5 pg./mg) and liver (group HGW: 446.2 ± 309.8 pg./mg; group LGW: 949.3 ± 451.0 pg./mg) did not show any significant change compared to the control group (lung:1752 ± 176.1 pg./mg; liver: 276.2 ± 32.81 pg./mg) (Fig. 3). 4.Discussion Many studies have highlighted the role of PPARs in regulating in- flammatory responses. However, most of the systemic inflammation models used in previous studies were induced by pathogens. To our knowledge, there are only a few studies focusing on the role of GW0742 on sterile systemic inflammation. This is the first study to examine the role of GW0742 in systemic inflammation by using a HS mice model.This analysis revealed the following main results:(1)High dosage of GW0742 (0.3 mg/kg·BW) significantly enhanced the systemic release of pro-inflammatory cytokines, including IL-6, IL- 1β, and MCP-1.(2)Low-dose treatment of GW0742 (0.03 mg/kg·BW) induced a non- significant downtrend of plasma inflammation cytokine levels in mice with HS.(3)Both low and high dosage of GW0742 did not show any effect onthe remote organ neutrophil infiltration (mirrored by MPO activity) 6 h after the traumatic stimulus.Results with plasma inflammatory cytokines levels, in which it was decreased, though not significantly, by low-dose GW0742 and was in- creased significantly by high-dose GW0742, suggest that GW0742 may has dual effects in modulating HS induced inflammation. In prior stu- dies, the dual functions of GW0742 have also been reported. Nandhikonda et al. [16] indicated that an agonistic effect of GW0742 displayed at low concentration turned to an antagonistic effect at high concentration. In this study, low concentrations of GW0742 enhanced the interactions between many kinds of nuclear receptors, including PPARγ, vitamin D receptor, thyroid hormone receptor β and its coac- tivators, and the interactions were inhibited by high concentrations of GW0742. However, the exact mechanisms are not clear. It has been suggested that GW0742 binds onto both PPARβ/δ and its coactivators [16,17]. PPARs coactivators play a crucial role in facilitating the transcription of PPARs-regulated genes [18]. In the context of low concentrations of GW0742, PPARβ/δ coactivators sufficiently bind to their receptors. However, the presence of high concentrations of GW0742, leads to a competition between GW0742 and coactivators. In this case, the effect of PPARβ/δ activation can be inhibited. Moreover, other studies indicate that GW0742 can also inhibit the gene expression of PPARβ/δ coactivators. It has been reported that the mRNA expres- sion of PGC-1α, one of the PPARβ/δ coactivators, was significantly decreased by GW0742 [19]. The effects of PPARβ/δ can also be in- hibited due to the lack of its coactivators.Based on these findings, we speculate that the expression of PPARβ/δ coactivators in HS mice might be severely decreased by a high dose of GW0742, or there might be an excessive binding between GW0742 and PPARβ/δ coactivators at the dosage of 0.3 mg/kg·BW, but not at the dosage of 0.03 mg/kg·BW. These could be the reasons for the dual ef- fects of GW0742 exhibited in this study.Elevated circulating levels of IL-6 and IL-1β have been shown after HS or septic shock in mice and humans [20–23]. Both IL-6 and IL-1β are known as pro-inflammatory cytokines that play an important role in the process of the immune response after haemorrhage and septic shock [24–26]. IL-6 has been recognised as a useful marker of severity during trauma and septic diseases [14,22]. Recent studies have shown pro- tective and dose-dependent effects of PPARβ/δ agonists and plasma IL-6 and IL-1β levels [9,10,27,28]. Kapoor et al. [11] reported thatGW0742 (0.03 mg/kg·BW i.v. injection) considerably attenuated myo- cardial ischaemia/reperfusion injury in rats. Haskova et al. [29] showed that GW0742 dose-dependently inhibited the pulmonary neu- trophil infiltration and cytokine production in mice challenged with lipopolysaccharides (LPS). In this analysis, a 0.03 mg/kg·BW (oral ad- ministration) dose did not significantly inhibit cell infiltration, whereas the minimal effective dose was shown to be 0.3 mg/kg·BW. Moreover, in a porcine septic shock model [30], GW0742 (0.03 mg/kg·BW i.v. injection) failed to down-regulate the plasma level of IL-6 at different time points; the lacking efficacy of GW0742 in this study was attributed to the lower PPAR β/δ receptor expression in vessels with athero- sclerosis. The discrepant results between these studies may be due to differences in animal species, inflammation models, and the drug ad- ministration route. Our findings showed that GW0742 (injected via an arterial catheter) at a dose of 0.03 mg/kg·BW did not markedly inhibit and a dose of 0.3 mg/kg·BW even stimulated IL-6 release in mice 6 h after HS.The results of this study seem to contradict those reported byZingarelli et al. [31], who indicated that, in the context of poly- microbial sepsis, treatment with GW0742 (0.5 mg/kg·BW) significantly reduced the plasma levels of TNF-α and IL-6 at 6 h and IL-1β levels at 18 h. One of the reasons might be the differences in immune stimulus between our HS model and that of Zingarelli et al. [31]. In this study, the inflammatory response was activated by polymicrobial infection. Animals in our study sustained a shock period of 90 min before GW0742 treatment. Activation of the immune system by pathogen in- fection and HS results in a pro-inflammatory response, and numerous pro-inflammatory molecules are expressed and released [32]. However, in the context of HS, the cell-mediated immune function has been de- scribed to be markedly depressed [33–35]. The pro-inflammatory cy- tokines IL-6 and IL-1β are released by a variety of immune cells (pri- marily monocytes, and macrophages) and organs (e.g. liver) [36–39]. The capacity of mononuclear macrophages to release IL-1 and IL-6 is significantly depressed after HS [40–43], a mononuclear cell mi- tochondrial dysfunction is viewed as one of the reasons for im- munosuppression after HS [42]. It has been noted that GW0742 is re- lated to mitochondrial biogenesis in many kinds of cells and organs [44–46]. Moreover, the hypoxia environment could cause the death of macrophages [47]. PPARβ/δ agonists have been demonstrated to have a positive effect on suppression of macrophage cell death under hypoxia [48].In recent studies, PPARβ/δ agonist is regarded as a context-depen-dent immune function regulator. PPARβ/δ agonists possess an anti-in- flammatory function, but also have immune stimulation effects under hypoxia stress [48]. The elevated pro-inflammatory cytokine levels of IL-6 and IL-1β after GW0742 treatment in our study indicated that high- dose GW0742 enhances the immune response in mice with HS. These changes might be associated with positive effects of GW0742 on mac- rophage proliferation and mitochondrial biogenesis under hypoxia. However, further studies are necessary to prove the immunity function potentiation possibilities of GW0742.MCP-1 and/or CCL2 are key chemokines that regulate the recruit- ment of monocytes and macrophages to sites of injury [49]. Biswas et al. [50] reported that IL-6 is a strong inducer of MCP-1 expression and secretion in mononuclear macrophages. Our data showed that both IL-6 and MCP-1 plasma levels were increased after the treatment of high-dose GW0742. According to Biswas et al.’s study [50], the elevated MCP-1 levels might be induced by IL-6. However, our data cannot elucidate the time sequence relation between IL-6 and MCP-1, because the plasma levels of inflammation cytokines were measured at only one time point.MPO is an enzyme mainly released by activated neutrophils that ischaracterised by pro-inflammatory properties [51]. MPO activity has also been used as a marker of neutrophil infiltration into tissues [52]. It seems that there is a discrepancy between the elevated levels of pro- inflammatory cytokines in plasma and the unaltered MPO activities in liver and lung tissues in this study. There are several possible ways to explain these findings. First, KC has been described as the most im- portant chemoattractant for neutrophils [53–55]. Since the plasma KC levels in GW0742 treated mice did not changed in our experiment, this stimulus might be not enough for significant neutrophil infiltration. Second, endothelial cellular adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) for example, are critical for neutrophil infiltration [56]. In the context of HS, the expression of ICAM-1 on endothelial cells is significantly upregulated and massively activated [57], the ICAM-1 induced neutrophil infiltration may reach an upper limit point, which cannot be exaggerated further by the pro-inflammatory cytokines in- crease. Furthermore, in human cells, PPARs suppresses the MPO mRNA expression by binding to an Alu receptor response element (AluRRE) in the MPO promoter region [58–61]. But the AluRRE in mouse cells is missing, which means the MPO activity in mouse cannot be regulated by PPARs [58,59], which means the MPO activity in mouse organs would not change no matter treated with low dose or high dose GW0742.In the present study, the liver MPO activity was not changed byGW0742 treatment, but it is hard to say there is no immune response in the liver. According to previous studies there is inflammation taking place in liver and lung [62,63]. Our focus in this analysis was mainly on the systemic inflammatory response after HS. The 6-h time point de- monstrates the peak of the systemic inflammation. However, for organ injury and changes, long observation periods are necessary to demon- strate relevant changes within the organs. Moreover, only one para- meter (MPO) has been chosen for organ inflammation in our experi- ment. Of course, other analyses (histology, local markers and tissue samples) are also needed to prove this hypothesis.The liver is a unique immunological organ which has about 80% of all macrophages in the body [64]. The resident macrophages in the liver, which are called Kupffer cells, are predominantly activated in the acute stage after HS; plasma concentrations of many kinds of in- flammatory cytokines, such as TNF-α, IL-1-α, and IL-6, are increased within 4 h after reperfusion [65]. The plasma levels of inflammatory cytokines were predominantly influenced by Kupffer cells [63]. Based on this evidence, we speculate that Kupffer cells in the liver might be the main resources for the production of pro-inflammatory cytokines in this study.All the plasma samples were collected at only one time point. Therefore, we cannot draw conclusions about the dynamic cytokine changes over time. Moreover, it must be noted that PPARβ/δ expression levels and the immune status were not observed in mice 6 h following HS. We assume that HS may influence PPARβ/δ expression. This needs to be studied in further analyses. 5.Conclusions Our results show that high-dose GW0742 is feasible to increase the pro-inflammatory cytokine plasma levels in mice with HS, whereas low- dose treatment shows no significant trend to down-regulate the in- flammation system. These results reflected the complexity of the in- flammation regulation mechanisms. Future research should investigate the different mechanisms or molecular pathways induced by different dosages of PPARβ/δ agonists with our standardised HS GW0742 model.