Purinergic P2X7 receptor antagonist ameliorates intestinal inflammation in postoperative ileus.

Postoperative ileus (POI) is a postsurgical gastrointestinal motility dysfunction caused by mechanical stress to the intestine during abdominal surgery. POI leads to nausea and vomiting reduced patient quality of life, as well as high medical costs and extended hospitalization. Intestinal inflammation caused by macrophages and neutrophils is thought to be important in the mechanism of POI. Surgery-associated tissue injury and inflammation induce the release of adenosine triphosphate (ATP) from injured cells. Released ATP binds the purinergic P2X7 receptor (P2X7R) expressed on inflammatory cells, inducing the secretion of inflammatory mediators. P2X7R antagonists are thought to be important mediators of the first step in the inflammation process, and studies in chemically induced colitis models confirmed that P2X7R antagonists exhibit anti-inflammatory effects. Therefore, we hypothesized that P2X7R plays an important role in POI. POI models were generated from C57BL/6J mice. Mice were treated with P2X7R antagonist A438079 (34 mg/kg) 30 min before and 2 hr after intestinal manipulation (IM). Inflammatory cell infiltration and gastrointestinal transit were measured. A438079 ameliorated macrophage and neutrophil infiltration in the POI model. Impaired intestinal transit improved following A438079 treatment. P2X7R was expressed on both infiltrating and resident macrophages in the inflamed ileal muscle layer. The P2X7R antagonist A438079 exhibits anti-inflammatory effects via P2X7R expressed on macrophages and therefore could be a target in the treatment of POI.

Postoperative ileus (POI) is a transient intestinal dysmotility disorder that occurs after abdominal surgery. POI causes abdominal distension, nausea, and vomiting and may be a risk factor for complications such as pulmonary embolism and thromboembolism [44, 46]. Although considerable effort has been extended to develop approaches to prevent or treat POI, 10~30% of patients develop POI after laparotomy [47]. In addition, management of POI is very costly, with total US medical expenditures for treating POI estimated at over $14.6 billion annually. POI therefore imparts a significant burden on the medical system [2, 45].

The development of POI is thought to involve three stages: a neurologic stage, an inflammatory stage, and elimination of ileus and vagal nerve over-activation [47]. The inflammatory stage is known to be immune-mediated [44], with POI developing as follows [1, 19, 41, 44, 45, 48]. First, cytotoxic factors such as adenosine triphosphate (ATP) are released from injured cells as a result of mechanical stress associated with laparotomy. These factors then activate immune cells, which in turn release inflammatory cytokines and chemokines. However, the cytotoxic factors involved in POI have not been fully identified. In the second step, monocytes and macrophages infiltrate into the intestinal muscularis and induce inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, leading to the release of PGE2 and NO, which decrease the contraction of intestinal smooth muscles and impair intestinal motility.

Purinergic receptors, which bind ATP and purine nucleotides, are classified into three groups: P1, P2X, and P2Y. The P1 and P2Y groups include G-protein–coupled receptors, whereas P2X receptors are ion channels [3]. Seven P2X receptor subtypes have been identified [4, 26]. Receptor P2X7 (P2X7R) is expressed primarily on inflammatory cells such as neutrophils, macrophages, dendritic cells, and mast cells [4, 8, 21]. P2X7 receptors in the wall of intestines exist not only mice, but also humans [7, 17]. When ATP binds and activates P2X7R, potassium efflux, and sodium and calcium influx occur [4]. ATP released from infected or stressed cells is thought to function as an endogenous signaling molecule in the regulation of inflammation and immune responses [30]. Extracellular ATP activates the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome via activation of P2X7R and induces secretion of the inflammatory cytokines interleukin 1β (IL-1β) and IL-18. ATP also functions as a secondary signal in the production of reactive oxygen species and the activation of nuclear factor–kappa B (NF-κB) [6, 32]. These observations suggest that P2X7R plays an important role in the early stages of inflammation.

The anti-inflammatory effects of P2X7R inhibitors have been confirmed using a model of colitis induced by chemical substances such as dextran sulfate sodium or trinitrobenzene sulfonic acid [18, 30]. As macrophages are known to play a central role in the pathogenesis of POI and P2X7R inhibitors have demonstrated anti-inflammatory effects in other pathologies of colitis, we hypothesized that P2X7R also plays an important role in the pathology of POI. In this study, therefore, we examined the effect of a P2X7R inhibitor on macrophages using a model of POI.

MATERIALS AND METHODS

Animals

All animal care and experimental procedures complied with the Guide for Animal Use and Care published by The University of Tokyo and were approved by the Institutional Review Board of The University of Tokyo (approval code P16-187). C57BL/6J male mice (6–9 weeks of age) were housed under controlled conditions (12-hr light/dark cycle). We have confirmed that there is no gender difference in this model of mice under the preliminary experiments (data not shown).

POI model

A POI model was developed by surgical manipulation of the distal part of the ileum of C57BL/6J mice that were fasted for 16–24 hr. All mice were anesthetized with sodium pentobarbital (50 mg/kg i.p., Somnopentyl; Kyoritsu Seiyaku Corp., Tokyo, Japan), and the animal model of POI was generated via intestinal manipulation (IM) as previously reported [2]. Briefly, the distal ileum (10 cm from the ileocecal region) was exposed and scratched three times with a sterile moist cotton applicator. In the present study, laparotomy with intestinal manipulation was defined as the POI model. This POI model is an acute model in which inflammation peaks 24 hr and heals 48–60 hr after treatment. Mice were randomly assigned to the following groups: Control, no treatment with fasting; IM, intestinal manipulation; IM + A438079 (Tocris Bioscience, Tokyo, Japan), subcutaneous injection of P2X7R antagonist (34 mg/kg) 30 min before and 2 hr after IM. A438079 was dissolved in sterile physiological saline. The concentration of A438079 administered was determined according to previous reports [18, 30], in which A438079 showed fast pharmacokinetic metabolism.

Whole-mount immunohistochemistry

Physiological salt solution was used for ileal muscle layer immunohistochemistry, as follows (all concentrations mM): NaCl 136.9, NaHCO3 23.8, glucose 5.5, KCl 5.4, CaCl2 1.5, MgCl2 1.0, and EDTA 0.01. Mice were sacrificed by exsanguination, and manipulated parts of the ileum were isolated 24 hr after IM. The ileum was opened along the mesenteric attachment, and the mucosal and submucosal layers were removed using incisive scissors and tweezers. The ileal smooth muscle layer was cut into 0.7 × 0.7 cm pieces and fixed in acetone for 5–10 min. The preparations were then washed three times with Tris-buffered saline (TBS), blocked with 2% BSA/TBS for 30 min, and incubated overnight at 4°C with primary antibodies. Preparations were then incubated with secondary antibodies after washing three times. Finally, the preparations were washed three times with TBS and examined under a confocal microscope (EZ-C1, Nikon, Tokyo, Japan). CD68-positive cells in the myenteric plexus layer of four randomly selected areas in each preparation were counted, and the average value was calculated. The same experiment was performed at least four times to calculate the mean ± SEM. The primary and secondary antibodies used in the study are listed in Table 1.

Table 1.

Antibodies used in this study

Type	Target	Host	Clone	Label	Dilution	Supplier (catalogue No.)
Primary antibody	PGP9.5	Rabbit polyclonal			1:200	UltraClone Limited, Isle of Wight, UK (RA95101)
	CD68	Rat monoclonal	FA-11		1:500	AbD Serotec, Oxford, UK (MCA1957)
	P2X7	Rabbit polyclonal			1:250	Alomone Labs, Jerusalem, Israel
Secondary antibody	Rat IgG (H+L)	Donkey		Alexa Fluor 488	1:500	Life Technologies, Gaithersburg, MD, USA (A21208)
	Rat IgG (H+L)	Goat		Alexa Fluor 594	1:500	Life Technologies (A11007)
	Rabbit IgG (H+L)	Donkey		Alexa Fluor 488	1:500	Life Technologies (SA5-10038)
	Rabbit IgG (H+L)	Donkey		Alexa Fluor 594	1:500	Life Technologies (A21207)

Myeloperoxidase staining

Whole-mount preparations were fixed with 5 or 10% neutral buffered formalin for 30 min, then washed three times with TBS for 1.5 hr. The preparations were incubated in 10 ml of TBS containing 10 mg of Hanker-Yates reagent (Polysciences, Warrington, PA, USA) and 10 μl of 30–35.5% hydrogen peroxidase (Mitsubishi Gas Chemical Co., Tokyo, Japan) for 5 min, then washed in TBS for at least 10 min. Myeloperoxidase (MPO)-positive neutrophils were counted under a microscope (Nikon ACT-1C for DXM1200) in four randomly selected areas of each preparation.

Measurement of intestinal transit

Twenty-three hours after IM, 100 μl of fluorescein isothiocyanate (FITC)-dextran solution (5 mg/ml, Molecular weight: 70,000, Sigma, Tokyo, Japan) was administered to the mice orally. One hour after oral administration, mice were euthanized, and the intestine from stomach to the end of the colon was dissected. The intestine was separated into 15 sections, as follows: Sto, stomach; S1–S10, small intestine; C1–C3, colon. The sections were cut in physiological salt solution (PSS) and then shaken vigorously for 10 sec, centrifuged at 1,500 g for 5 min at 4°C, and then each supernatant was transferred to a new tube. The supernatants were then additionally centrifuged at 11,000 g for 5 min at 4°C, and 200 μl of each resulting supernatant was transferred to wells of a 96-well plate. The fluorescence intensity of FITC was measured using an EMC-427 plate reader (JASCO Corp., Tokyo, Japan). The ratio of the fluorescence intensity of each well to the total fluorescence intensity was then calculated. In addition, the geometric center (GC) of the FITC-dextran distribution was calculated according to the following equation:

GC=sum (each fluorescence intensity ratio [%] × section number)/100.

Statistical analyses

Results are expressed as mean ± SEM. Data were statistically evaluated using one-way analysis of variance and Tukey’s test. Values of P<0.05 were considered statistically significant.

RESULTS

P2X7 blockade inhibits IM-induced macrophage and neutrophil infiltration

CD68-positive macrophages were observed by immunohistochemistry (Fig. 1A). Some resident macrophages were detected in the myenteric plexus region of the ileum in control mice. Many macrophages had infiltrated into the muscle layer by 24 hr after IM. Inhibition of P2X7R strongly inhibited macrophage infiltration into the myenteric plexus.

Fig. 1.

P2X7 receptor (P2X7R) blockade inhibited leukocyte infiltration in a postoperative ileus model in control mice. Effect of purinergic P2X7R blockade on leukocyte infiltration induced by intestinal manipulation (IM) in a postoperative ileus model in control mice. Myenteric nerve plexus inhibition via P2X7R blockade was performed by administration of A438079 (34 mg/kg, s.c.), as described in the Materials and Methods. A: Immunohistochemical or histochemical staining of CD68-positive macrophages or myeloperoxidase (MPO)-stained neutrophils in the intestine of control mice. Representative images from four independent experiments are shown. Black bar indicates 50 μm and white bar indicates 100 μm. Red and green signals indicate PGP 9.5–positive myenteric neurons and CD68-positive macrophages, respectively. B and C: Number of infiltrating macrophages (B) and neutrophils (C) in images shown in (A). * or ***, significantly different from control at P<0.05 or P<0.001; # or ##, significantly different from IM at P<0.05 or P<0.01, respectively (n=4 each). Each column shows the mean ± SEM.

We also investigated the infiltration of MPO-positive neutrophils (Fig. 1A). Almost no neutrophils were observed in the myenteric plexus region of control mice. Many neutrophils infiltrated after IM, but inhibition of P2X7R attenuated this infiltration (Fig. 1A and 1C). Administration of A438079 alone did not affect the movement of macrophages or neutrophils in the myenteric plexus region in the ileum (n=2, data not shown).

P2X7R inhibition ameliorates IM-induced intestinal dysmotility

We assessed the gastrointestinal transit capacity 24 hr after IM. In the control group, 90% of the FITC was located in sections S6–S10. However, in the IM-treated group, 72% of the FITC was located in sections Sto–S5, suggesting that IM decreased the intestinal transit capacity. In comparison, 69% of the FITC was located in sections S6–S10 in the A438079-treated group. Taken together, these data suggest that A438079 ameliorates IM-induced impaired intestinal transit capacity (Fig. 2A). In addition, we analyzed these graphs geometrically to calculate the GC of the FITC distribution. A high GC value is indicative of a high intestinal transit capacity. The IM-treated group showed a decrease in transit, but significant improvement in transit was observed in the A438079-treated group (Fig. 2B).

Fig. 2.

P2X7 receptor (P2X7R) inhibition ameliorates diminished gastrointestinal transit in a mouse model of postoperative ileus (POI). A: Data shown are mean ± SEM of the ratio (%) of fluorescein isothiocyanate (FITC) content. B: Geometric center calculated from A. ***P<0.001; significantly different from control. #P<0.05; significantly different from POI. Data shown are mean ± SEM from four independent experiments.

P2X7R is expressed on macrophages in the small intestine muscle layer

As P2X7R antagonist A438079 exhibited anti-inflammatory effects in the POI model, we examined the expression of P2X7R in the intestinal muscle layer. P2X7R was expressed on resident macrophages in the intestine of normal mice (Fig. 3). At 24 hr after IM, P2X7R was also expressed on infiltrating macrophages in the intestine of IM-treated mice (Fig. 3).

Fig. 3.

Expression of P2X7 receptor (P2X7R) on cells of the ileal muscle layer of control and IM-treated mice. Double-immunostaining of CD68 (red) and purinergic P2X7R (green) in the ileal muscle layer of control and intestinal manipulation (IM)-treated mice. Scale bar, 100 μm. Typical results are shown for three independent experiments.

DISCUSSION

POI is a transient postoperative gastrointestinal dysfunction that primarily occurs after laparotomy [45]. POI may cause increased mucosal permeability involving diarrhea and constipation and we found those symptoms in this study. In current regimens for POI prevention and treatment, lidocaine, COX-2 inhibitors, mosapride, and daikenchuto have been shown to be effective [34, 42, 44]. Lidocaine acts on the adrenal glands to reduce catecholamine release and directly stimulates the smooth muscle to enhance gastrointestinal motility. Mosapride and daikenchuto are distributed in the cholinergic nerves of the gastrointestinal wall. Mosapride exerts preventive and therapeutic effects on POI by directly or indirectly activating the 5-HT4 receptor to enhance gastrointestinal motility. By comparison, the preventive and therapeutic effects of COX-2 inhibitors on POI involve local suppression of inflammation in the gastrointestinal tract, but these agents carry the risk of NSAIDs-induced gastric ulcers and small intestinal ulcers. Therefore, promoting gastrointestinal motility is considered more effective for preventing and treating POI because it prevents gastrointestinal adhesion due to stagnation of gastrointestinal motility. It is important to inhibit only excessive inflammation because the intestinal inflammatory response is necessary for healing of the surgical site.

In this study, the P2X7R selective inhibitor A438079 significantly improved the gastrointestinal transport capacity that was suppressed by IM in POI model mice (Fig. 2). That is, our results suggest that administration of P2X7R inhibitors is effective for the prevention and treatment of POI. The decreased gastrointestinal motility of POI is caused by macrophage-induced inflammation in the muscularis of the gastrointestinal tract. These cells produce PGE2 and NO, which affect the smooth muscle cells of the gastrointestinal tract [1, 22, 31]. It has also been reported that suppressing the infiltration of inflammatory cells such as macrophages and neutrophils restores hypokinesia of the gastrointestinal tract [23, 48]. Therefore, we performed immunohistochemical staining to determine whether the improvement in gastrointestinal transport capability associated with the P2X7R inhibitor A438079 was due to the suppression of inflammatory cell infiltration in the POI model mice. These analyses indicated that the P2X7R inhibitor significantly suppressed the infiltration of both neutrophils and macrophages into the inflamed areas of the gastrointestinal tract induced by IM (Fig. 1). These results confirmed that inhibiting P2X7R using A438079 has an anti-inflammatory effect in the POI model, and this in turn reverses the slow-down in intestinal transit. We injected A438079 subcutaneously before and after surgery. The reasons for systemic administration instead of topical administration are follows: first, we want to administer it prophylactically before surgery. Second, the pathogenesis of POI is caused by local inflammation of gastrointestinal tract, but the inflammation is known to propagate throughout the gastrointestinal tract including the uninjured part through the immune pathway [11]. Therefore, we expected a suppressive effect by systemic administration.

The extracellular ATP activates P2X7R on immune cells, such as monocytes and macrophages, to release IL-1β [8, 35, 38]. However, macrophages of P2X7R or NLRP3 inflammasome knock-out mice do not release IL-1β in response to ATP [28, 29, 40], even though P2X7R activation by extracellular ATP is one of the strongest signal transduction systems for activation of NLRP3 inflammasomes and stimulation of IL-1β release [29, 33]. Other recent studies have shown that release of K+ to the extracellular milieu is involved in the activation of NLRP3 inflammasomes [14, 33, 49]. Therefore, the anti-inflammatory effects of P2X7R antagonists could be induced by suppression of NLRP3 inflammasomes, followed by downregulation of IL-1β release. It was reported that P2X7R activation induces nuclear factor-κB (NF-κB) phosphorylation and neuronal inflammation [20]. In inflammatory bowel disease, macrophages NF-κB is activated [37]; however, inhibition of NF-κB transcription decreases the release of Tumor Necrosis Factor α (TNF-α) and IL-1β [39]. In addition, NF-κB is activated by increased intracellular Ca2+ concentrations [24, 27]. As a result, iNOS is induced and NO is released, which is related to the inflammatory response [1, 19, 41]. These reports and the results of this study suggest that P2X7R induces an influx of Ca2+ [14, 25] and that P2X7R antagonists ameliorate the release of inflammatory cytokines and NO from macrophages via iNOS.

We then investigated the point at which the anti-inflammatory effect of P2X7R inhibitors is exerted in POI. A previous report revealed that macrophages play an important role in the pathogenesis of POI [48], but the mechanism of macrophage activation in POI remains unknown. Therefore, in order to clarify the role of P2X7R in the pathology of POI and the relationship to macrophages, immunohistochemical staining was performed on both P2X7R and macrophages. P2X7R was expressed on both the relatively large, dendritic-shaped resident macrophages in the gastrointestinal muscularis and the small, circular infiltrating macrophages induced by IM (Fig. 3). This suggests that macrophages are the target cells of the P2X7R-mediated anti-inflammatory activity in POI. Because POI is caused by non-infectious cytotoxic inflammation, it likely involves surgically damaged cells in the gastrointestinal and abdominal walls, such as serosal lining cells and smooth muscle cells. It is therefore likely that ATP released from the damaged cells activates P2X7R to induce local inflammation of the gastrointestinal tract, which contributes to the development of POI. As discussed above, it is thought that the anti-inflammatory effect of P2X7R inhibitors in POI involves a series of anti-inflammatory processes initiated by the inhibition of P2X7R expressed on both resident and infiltrating macrophages in the gastrointestinal muscularis. These conclusions are supported by reports demonstrating that (i) P2X7R stimulation increases IL-1β secretion by macrophages [12, 15, 36], (ii) IL-1β induces iNOS [5, 10, 13], and (iii) NO production from iNOS is responsible for the pathogenesis of POI [2, 22, 31, 43, 44, 47].

Based on the results described above, the mechanism of the anti-inflammatory effect of P2X7R inhibitors in POI appears to involve suppression of the secretion of the inflammatory cytokine IL-1β by macrophages. However, such suppression of P2X7R-mediated secretion of IL-1β has also been reported in neutrophils [25]. Neutrophil infiltration also plays an important role in the pathogenesis of POI [1, 23, 44]. Therefore, although immunohistochemical staining of P2X7R and neutrophils was not performed in this study, it is possible that P2X7R is also expressed by neutrophils and that these cells play a role in the pathogenesis of POI. P2X7R is also highly expressed on nerve cells [9], and P2X7R-mediated neural signals have also been implicated in other intestinal diseases [16]. These data suggest that P2X7R expressed on nerves may also be involved in the anti-inflammatory effect and gastrointestinal motility–promoting activity of P2X7R inhibitors in POI.

In order to investigate the above possibilities, it will be necessary to evaluate the expression of P2X7R on cells other than macrophages and examine the point of action in the prevention and treatment of POI. There is no denying the possibility that cells other than macrophages and nerve plexus cells could be involved in the pathogenesis of POI. However, immunohistochemistry experiments revealed almost complete co-staining of P2X7R-expressing cells and CD68-positive gastrointestinal wall macrophages, and no P2X7R was detected on cells other than macrophages, including cells of the intramural plexus. These results thus suggest that P2X7R expressed on intestinal macrophages plays a critical role in POI.

In summary, the present study demonstrated that A438079 significantly suppresses inflammation of the ileal muscularis and improves gastrointestinal motility by inhibiting P2X7R in the pathophysiology of POI. These data suggest that P2X7R inhibitors would be effective for preventing POI. Our data also showed that P2X7R-mediated signaling plays an important role in the pathogenesis of POI via gastrointestinal resident and infiltrating macrophages which in turn may be induce anti-inflammatory action. As a result, P2X7R inhibitors significantly increase gastrointestinal transport capacity. P2X7R should be considered effective drug discovery targets for the treatment and prevention of gastrointestinal insufficiency.

CONFLICT OF INTEREST

No conflicts of interest, financial or otherwise, are declared by the authors.