Research ArticleArticle

E6130, a Novel CX3C Chemokine Receptor 1 (CX3CR1) Modulator, Attenuates Mucosal Inflammation and Reduces CX3CR1+ Leukocyte Trafficking in Mice with Colitis

Hisashi Wakita, Tatsuya Yanagawa, Yoshikazu Kuboi and Toshio Imai
Molecular Pharmacology November 2017, 92 (5) 502-509; DOI: https://doi.org/10.1124/mol.117.108381

Abstract

The chemokine fractalkine (CX3C chemokine ligand 1; CX3CL1) and its receptor CX3CR1 are involved in the pathogenesis of several diseases, including inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis, rheumatoid arthritis, hepatitis, myositis, multiple sclerosis, renal ischemia, and atherosclerosis. There are no orally available agents that modulate the fractalkine/CX3CR1 axis. [(3S,4R)-1-[2-Chloro-6-(trifluoromethyl)benzyl]-3-{[1-(cyclohex-1-en-1-ylmethyl)piperidin-4-yl]carbamoyl}-4-methylpyrrolidin-3-yl]acetic acid (2S)-hydroxy(phenyl)acetate (E6130) is an orally available highly selective modulator of CX3CR1 that may be effective for treatment of inflammatory bowel disease. We found that E6130 inhibited the fractalkine-induced chemotaxis of human peripheral blood natural killer cells (IC50 4.9 nM), most likely via E6130-induced down-regulation of CX3CR1 on the cell surface. E6130 had agonistic activity via CX3CR1 with respect to guanosine 5′-3-O-(thio)triphosphate binding in CX3CR1-expressing Chinese hamster ovary K1 (CHO-K1) membrane and had no antagonistic activity. Orally administered E6130 ameliorated several inflammatory bowel disease–related parameters in a murine CD4+CD45RBhigh T-cell-transfer colitis model and a murine oxazolone-induced colitis model. In the CD4+CD45RBhigh T-cell transfer model, E6130 inhibited the migration of CX3CR1+ immune cells and decreased the number of these cells in the gut mucosal membrane. These results suggest that E6130 is a promising therapeutic agent for treatment of inflammatory bowel disease.

Introduction

Inflammatory bowel disease is a group of idiopathic, chronic intestinal inflammatory diseases that primarily includes two distinct conditions: Crohn’s disease and ulcerative colitis, which are both the result of inappropriate immune responses to antigens produced by commensal microorganisms. Although the signs of Crohn’s disease and ulcerative colitis manifest primarily in the gastrointestinal tract, these diseases affect the entire human body (Baumgart and Sandborn, 2007). Therapeutic treatments that inhibit cytokines are currently in widespread clinical use; however, the serious adverse events and lack of long-term efficacy of these treatments remain serious concerns.

Infiltration of leukocytes from the peripheral circulation into tissues such as the gut mucosa is an important aspect of immune surveillance. To enter the mucosal tissue from the blood, leukocytes must cross the endothelial barrier. They do this by interacting with cytokine or other proinflammatory stimuli-activated endothelial cells via either leukocyte cell-surface chemokine receptors (and their respective endothelial and mucosal ligands) or via integrins and immunoglobulin superfamily cellular adhesion molecules (Charo and Ransohoff, 2006; Thomas and Baumgart, 2012).

Chemokines and their receptors are also important means of orchestrating tissue-specific and cell type–selective leukocyte trafficking (Nishimura et al., 2009). For example, fractalkine (also known as CX3C motif ligand 1; CX3CL1) is a chemokine prominently expressed by epithelial and endothelial cells that functions as both an adhesion molecule and a chemotactic factor (Bazan et al., 1997; Nishimura et al., 2002). Fractalkine binds to its sole receptor, CX3C chemokine receptor 1 (CX3CR1), which is expressed on the surfaces of monocytes, macrophages, dendritic cells, microglia, natural killer (NK) cells, and cytotoxic effector T-cells that contribute to the development of chronic inflammation (Imai et al., 1997; Yoshie et al., 2001). In patients with inflammatory bowel disease, the expression of fractalkine by intestinal epithelial cells and microvascular cells and of CX3CR1 by peripheral blood T-cells increases with the level of disease activity. Thus, the fractalkine/CX3CR1 axis appears to be directly involved in the pathogenesis of inflammatory bowel disease (Brand et al., 2006; Kobayashi et al., 2007; Sans et al., 2007).

Here, we characterized [(3S,4R)-1-[2-Chloro-6-(trifluoromethyl)benzyl]-3-{[1-(cyclohex-1-en-1-ylmethyl)piperidin-4-yl]carbamoyl}-4-methylpyrrolidin-3-yl]acetic acid (2S)-hydroxy(phenyl)acetate (E6130), a previously identified novel modulator of CX3CR1. We found that E6130 inhibited fractalkine-induced chemotaxis and induced down regulation of CX3CR1 on the cell surface in NK cells from human peripheral blood. E6130 also prevented the trafficking of CX3CR1-expressing leukocytes into colonic tissue in a murine CD4+CD45RBhigh T cell–transfer colitis model, and it ameliorated other inflammatory bowel disease–related parameters in both the murine CD4+CD45RBhigh T-cell-transfer colitis model and a murine oxazolone-induced colitis model. Together, these results suggest that E6130 is a promising agent for treating the inflammatory bowel disease.

Materials and Methods

Animals.

BALB/c mice (female, 7–10 weeks old) were obtained from Charles River Laboratories Japan (Tokyo, Japan). Severe combined immunodeficiency (SCID) mice (female, 6 weeks old) were obtained from CLEA Japan (Tokyo, Japan). The mice were group-housed under controlled conditions (temperature, 22 ± 3°C; humidity, 55% ± 5%; 12-hour light/dark cycle) with ad libitum access to water and standard pelleted food. All animal experiments were approved by the Committee for the Welfare of Laboratory Animals, Eisai Co. Ltd.

Test Compound and Reagents.

E6130 was synthesized by Eisai Co., Ltd. at its plant located in Kashima, Japan (Yoshida et al., 2013). The chemical structure of E6130 is shown in Fig. 1. Oxazolone was purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human fractalkine was purchased from R&D Systems (Minneapolis, MN).

Fig. 1.
Fig. 1.

Chemical structure of E6130.

CX3CR1 Down-Regulation Assay.

Assay medium (RPMI 1640 medium containing 10% fetal bovine serum, 80 μl/well) and E6130 (10 μl/well) were added to each well of a 96-well culture plate. Written informed consent was obtained from each subject before blood draw. Human peripheral blood was drawn from the forearm vein of three healthy male volunteers, and an aliquot (10 μl) was added to each well.

After incubation for 30 minutes at 37°C under an atmosphere of 5% CO2, the human peripheral blood cells were washed twice with flow cytometry (FCM) buffer: phosphate-buffered saline (PBS) containing 1% fetal bovine serum, 1 mM EDTA, and 0.1% bovine serum albumin. The cells were incubated with FcR Blocking Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 minutes on ice to block Fc receptors and then stained for CD56 (NK cell surface marker) and CX3CR1 by incubation with allophycocyanin-conjugated anti-CD56 monoclonal antibody (Miltenyi Biotec) and phycoerythrin-conjugated anti-CX3CR1 monoclonal antibody (BioLegend, San Diego, CA), respectively, for 30 minutes on ice. The cells were then washed once and resuspended in FCM buffer.

After incubation for 10 minutes on ice with 7-amino actinomycin D (Sigma-Aldrich) to discriminate dead cells, the fluorescence intensity of the stained cells was determined by using a FACSCanto cell analyzer (Becton Dickinson, Franklin Lakes, NJ). Loss of CX3CR1 staining on the cell surface was assumed to represent the induction of down-regulation of CX3CR1 on the cell surface. The mean fluorescence intensity (MFI) of the CX3CR1+ cell population among the total NK cell population was also determined by using a FACSCanto cell analyzer. The percentage MFI of the control was determined by using the following formula:Embedded Imagewhere “test well” refers to wells containing E6130 and staining antibodies, “control well” refers to wells containing staining antibodies only, and “background well” refers to wells containing neither the staining antibodies nor E6130.

Isolation of NK Cells from Human Peripheral Blood.

An aliquot (25 ml) of human peripheral blood was added to a plastic centrifugation tube containing 100 U of heparin sodium (Ajinomoto, Tokyo, Japan). An aliquot (8 ml) of physiologic saline containing 6% dextran (Nacalai Tesque, Kyoto, Japan) was added to the tube, and the mixture was allowed to stand at room temperature for 30 minutes to allow sedimentation of the erythrocytes. The supernatant was transferred to another plastic centrifugation tube, mixed with an equivalent volume of PBS, and then centrifuged at 1800 rpm for 7 minutes at room temperature. The resultant hemocyte fraction was suspended in 4 ml of PBS, and the suspension was superposed on 4 ml of Ficoll-Paque Plus (GE Healthcare Life Sciences, Tokyo, Japan).

After centrifugation of the suspension at 2200 rpm for 30 minutes at room temperature, the cells in the intermediate layer (peripheral blood mononuclear cells) were collected, suspended in PBS, and centrifuged at 1800 rpm for 7 minutes. The supernatant was removed, and the precipitate was suspended in PBS containing 5 mM EDTA and 0.5% bovine serum albumin. NK cells were purified from the peripheral blood mononuclear cell population by means of negative selection using an NK cell isolation kit (Miltenyi Biotec) and a MACS LS column (Miltenyi Biotec). Cells passing through the column were considered NK cells and were collected, washed, and suspended in chemotaxis buffer: RPMI 1640 medium containing 10% fetal bovine serum, 50 μM 2-mercaptoethanol, and 10 mM HEPES.

Chemotaxis Assay.

Human fractalkine solution (dissolved in chemotaxis buffer, 60 μl/well) and chemotaxis buffer (480 μl/well) were added to the lower wells of a 24-well chemotaxis assay chamber (Boyden chamber, pore size: 5.0 μm) for a final concentration of 0.2 nM. E6130 solution (60 μl/well) was also added to the lower wells. A mixture of NK cell suspension and E6130 was added to the upper wells, and the chambers were incubated for 2 hours at 37°C under an atmosphere of 5% CO2. After incubation, the contents of the upper wells were discarded, and the assay chambers were centrifuged at 1800 rpm for 5 minutes. The supernatant was removed, and a CellTiter-Glo reagent (Promega Japan, Tokyo, Japan) was added to the precipitate.

The chemiluminescence intensity was measured by using a Wallac 1420 ARVOsx multilabel counter (PerkinElmer, Yokohama, Japan). The number of cells in each well was calculated by using a standard curve fitting the regression line for cell number. The percentage of cells exhibiting chemotaxis was determined by using the following formula:Embedded Imagewhere “test well” refers to wells containing fractalkine and E6130, “blank well” refers to wells containing neither fractalkine nor E6130, and “control well” refers to wells containing fractalkine only.

Guanosine 5′-O-(3-[35S]Thio)Triphosphate Binding Assay.

The scintillation proximity assay guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) experiments were conducted using CHO-K1 membranes stably expressing human chemokine receptors incubated in assay buffer (20 mM HEPES [pH 7.4], 100 mM NaCl, 10 μg/ml saponin, and 1 mM MgCl2).

In the agonistic activity assay, E6130 or reference ligand, assay buffer, the CHO-K1 membrane–GDP mix, [35S]GTPγS, and wheat germ agglutinin-polyvinyl toluene (WGA-PVT) beads (Perkin Elmer, Waltham, MA) mix were successively added to the wells of an OptiPlate (Perkin Elmer).

In the antagonistic activity assay, E6130 or reference ligand and CHO-K1 membrane–GDP mix were successively added to the wells of an OptiPlate, and the plates were incubated for 15 minutes at room temperature. A reference agonist at its historical EC80 was then added, followed by the addition of [35S]GTPγS and wheat germ agglutinin-polyvinyl toluene (WGA-PVT) beads.

In both assays, the plates were covered with a top seal, shaken on an orbital shaker for 2 minutes, and then incubated for 1 hour at room temperature. The plates were then centrifuged for 10 minutes at 2000 rpm and incubated at room temperature for 1 hour. Each well was counted for 1 minute by using a PerkinElmer TopCount reader.

PathHunter β-Arrestin Recruitment Assay.

For the β-arrestin recruitment assay, we used PathHunter cell lines (DiscoveRx, Fremont, CA) (Zhao et al., 2008; Bassoni et al., 2012). These cell lines were expanded from freezer stocks in T25 flasks according to standard procedures and were maintained in selective growth medium before the assay. Once it was established that the cells were healthy and growing normally, the cells were passaged from the flasks by using cell dissociation reagent and seeded into white-walled, clear-bottomed, 384-well microplates. The cells were seeded at a density of 5000/well in a total volume of 20 μl and were allowed to adhere and recover overnight before the addition of E6130.

In the agonistic activity assay, intermediate dilutions of E6130 stock solutions were generated such that 5 μl of E6130 could be added to each well to produce a final concentration of dimethylsulfoxide of 1% of the total volume. The cells were incubated in the presence of E6130 at 37°C for 90 minutes.

In the antagonistic activity assay, agonist dose curves were constructed and used to determine the EC80 values. The cells were preincubated with antagonist; this was followed by the agonist challenge, as follows: 5 μl of E6130 was added to the cells, and the plates were incubated at 37°C for 30 minutes. Then, 5 μl of agonist at its EC80 was added to the cells, and the plates were incubated at 37°C for 90 minutes.

The assay signal was generated through the addition of 12.5 or 15.0 μl (50% v/v) of PathHunter Detection reagent cocktail for the agonistic and antagonistic activity assays, respectively, followed by incubation for 1 hour at room temperature. The microplates were then read with a ParkinElmer EnVision multilabel plate reader.

Dose curves in the presence of E6130 were plotted by using GraphPad Prism or Activity Base software (GraphPad Software, San Diego, CA). In the agonistic activity assay, the percentage activity was calculated by using relative light units (RLU) and the following formula:

Embedded Image

In the antagonistic activity assay, the percentage inhibition was calculated by using the following formula:

Embedded Image

CD4+CD45RBhigh T-Cell Preparation and T-Cell-Transfer Colitis Model.

CD4+CD45RBhigh T cells were isolated from BALB/c splenocytes. BALB/c mice were euthanized by means of cervical dislocation, and their spleens were removed. The spleens were passed through a cell strainer (BD Biosciences, San Jose, CA), and a single-cell suspension of splenocytes was prepared. After hemolysis, the CD4+ T-cell population was purified from the splenocytes by means of negative selection by using a mouse CD4+ T-cell Isolation Kit II (Miltenyi Biotec) and a MACS LS column.

For the preparation of CD4+CD45RBhigh T cells, the isolated total CD4+ T cells were labeled with phycoerythrin-conjugated mouse anti-CD4 monoclonal antibody (eBioscience, San Diego, CA) and fluorescein isothiocyanate-conjugated mouse anti-CD45RB monoclonal antibody (eBioscience), and two-color cell sorting was performed by using a FACSAria cell sorter (Becton Dickinson). The sorted CD4+CD45RBhigh T cells were suspended in PBS and intravenously transferred into SCID mice. After 2 weeks, E6130 or vehicle (0.5% methyl cellulose) was orally administered once daily to the SCID mice from day 14 to day 27. Stool consistency and the body weight of all mice were recorded on days 14, 16, 18, 20, 22, 24, 26, and 28. On day 28, the mice were euthanized, their colons were removed, and the weight and length of each colon was recorded. Stool consistency was scored as follows: 0 = normal, 1 = soft stool (well-formed pellets), 2 = soft stool (very soft but formed pellets), 3 = loose stool (pasty stool), and 4 = diarrhea (liquid stool that sticks to the anus). Relative body weight was calculated by using the following formula:

Embedded Image

Next, the colons were washed with PBS and then incubated for 30 minutes at 37°C in PBS containing 1 mM dithiothreitol, 1 mM EDTA, and 1% fetal calf serum to remove epithelial cells. The remaining tissue was washed with PBS, cut into 1–2-mm sections, and incubated for 2.5 hours at 37°C in RPMI 1640 containing 0.5 mg/ml collagenase A, 1 μg/ml DNase, and 5% fetal calf serum.

After incubation, the suspension was passed through a cell strainer, and the resultant cell suspension was centrifuged at 700g for 7 minutes at room temperature. The resultant enterocyte precipitate was suspended in 4 ml of PBS, and the suspension was superposed on Percoll (GE Healthcare Life Sciences).

After centrifugation at 700g for 20 minutes at room temperature, the cells in the intermediate layer were collected, suspended in PBS, and centrifuged at 700g for 7 minutes at 4°C. The supernatant was removed, and the precipitate was suspended in FACS buffer (PBS containing 1 mM EDTA, 0.1% bovine serum albumin, and 1% fetal calf serum). The CX3CR1+ cells were labeled with phycoerythrin-conjugated goat anti-CX3CR1 polyclonal antibody (R&D Systems), and the proportion of CX3CR1+ cells was determined with a FACSCanto cell analyzer (Becton Dickinson).

Pharmacokinetics and Histologic Analysis in T-Cell-Transfer Colitis Model.

For the pharmacokinetic study, the sorted CD4+CD45RBhigh T cells, as described earlier, were suspended in PBS and intravenously transferred into SCID mice. After 2 weeks, E6130 or vehicle was orally administered once daily to the SCID mice from days 15 to 30. Blood samples were withdrawn at 30 minutes, and at 2 and 6 hours after administration of E6130 on day 29. After centrifugation of the mouse blood, the concentrations were quantitatively determined by a mass spectrometer equipped with a high-performance liquid chromatography system. Plasma protein binding was evaluated in plasma at 2 hours by the equilibrium method.

For histologic analysis, the colon was collected from mice on day 30, fixed in 10% neutral buffered formalin, embedded in paraffin, and processed for microscopic examination of H&E-stained sections.

Oxazolone-Induced Colitis Model.

BALB/c mice were sensitized with oxazolone on day 0 by painting 3% oxazolone ethanol solution onto the skin of the abdomen. Five days after sensitization, oxazolone solution was intrarectally injected into the mice under isoflurane anesthesia to induce colitis. In the vehicle group, ethanol/water (1:1) vehicle solution was intrarectally injected into the mice instead of oxazolone solution.

E6130 was orally administered 1 hour before the intrarectal injection of oxazolone. The daily administration of E6130 continued from days 5 to 7. Two hours after the administration of E6130 on day 7, the mice were euthanized by cervical dislocation, their colons were removed, and the length (millimeters) of the colons was recorded. Body weight ratio was calculated by using the following formula:

Embedded Image

Statistical Analysis.

Data are presented as mean ± S.E.M. For the stool score and relative body weight of a murine CD4+ CD45RBhigh T-cell transfer colitis model, the differences between the CD4+CD45RBhigh T-cell-transferred group and total-CD4+ T-cells- or E6130-treated group were analyzed by repeated measures analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD) test.

In other parameters, the differences between the CD4+CD45RBhigh T-cell-transferred group and total-CD4+ T-cells- or E6130-treated group were analyzed by one-way ANOVA followed by Fisher’s LSD test. We used Bonferroni correction for a final multiple comparison with correction for inflation of type 1 error, and the significance level was set to 0.001 (0.05/48).

In the murine oxazolone-induced colitis model, the differences between the oxazolone-injected group and the vehicle (ethanol/water)-injected or E6130-treated group were analyzed by one-way ANOVA followed by Fisher’s LSD test. We used Bonferroni correction for a final multiple comparison with correction for inflation of type 1 error, and the significance level was set to 0.00625 (0.05/8).

All statistical analyses were performed with GraphPad Prism software.

Results

Effect of E6130 on Fractalkine-Induced Chemotaxis by NK Cells Isolated from Human Peripheral Blood.

Previously, E6130 (structure in Fig. 1) was identified from a G-protein coupled receptor (GPCR)-focused library by using a fluorometric microvolume assay technology scanner designed to perform high-throughput screening assays using multiwell plates (Yoshida et al., 2013). The library was screened by using an in vitro chemotaxis assay and an in vivo murine inflammatory bowel disease model (unpublished results).

We used a cell-based assay to assess the effects of E6130 on fractalkine-induced chemotaxis. Human NK cells were used because most NK cells in human peripheral blood express CX3CR1 on their surfaces and NK cells can be easily isolated in large numbers. The NK cells were preincubated with E6130, and chemotaxis was induced by incubation with fractalkine for 2 hours by using a multiwell chemotaxis chamber. Marked migration of NK cells was observed against 0.2 nM fractalkine (number of cells in blank well: 887 ± 324; number in fractalkine-containing well: 8615 ± 2936). However, E6130 inhibited the chemotaxis of NK cells, with an IC50 value of 4.9 nM (Fig. 2A).

Fig. 2.
Fig. 2.

Effects of E6130 on fractalkine-induced chemotaxis and down-regulation of CX3CR1 on the cell surface in human peripheral blood natural killer cells. (A) Effects of E6130 on fractalkine-induced chemotaxis in human peripheral blood natural killer cells. Data are presented as mean ± S.E.M. from three independent experiments performed in duplicate for the blank and control groups, and performed in a single well for the test compound group. (B) Effects of E6130 on the induction of down-regulation of CX3CR1 on the cell surface in human peripheral blood natural killer cells. Data are presented as mean ± S.E.M. (n = 3).

Effect of E6130 on CX3CR1 Down-Regulation on the Cell Surface of Human Peripheral Blood NK Cells.

E6130 exhibited no antagonistic activity on [35S]GTPγS binding via CX3CR1 (Table 1). Therefore, we hypothesized that E6130 prevents fractalkine from binding to the receptor by inducing down-regulation of CX3CR1 on the cell surface, leading to inhibition of the fractalkine-induced chemotaxis of human leukocytes. To test this hypothesis, we incubated human peripheral blood with E6130 for 30 minutes at 37°C and then stained with monoclonal antibodies to CD56 and CX3CR1. The fluorescence intensity of CX3CR1 on CD56+ NK cells was evaluated by means of flow cytometry. E6130 induced the down-regulation of CX3CR1 on the cell surface of CD56+ NK cells with an EC50 value of 5.2 nM (Fig. 2B).

View this table:
TABLE 1

Agonistic and antagonistic activities of E6130 (10 μM) toward chemokine receptors

Effect of E6130 on GTPγS Binding via CX3CR1.

When activated, CX3CR1 changes conformation to expose a G-protein complex binding site. Once this G-protein complex is bound, the Gα subunit releases GDP and binds GTP. Agonist-activated GPCR induces receptor phosphorylation mediated by GPCR kinases, leading to the recruitment of β-arrestin. Upon receptor phosphorylation and β-arrestin binding, most GPCRs internalize into clathrin-coated vesicles. Therefore, next we examined the effects of E6130 on [35S]GTPγS binding and β-arrestin recruitment by CX3CR1 in CX3CR1-expressing CHO-K1 membrane. E6130 had agonistic activity with respect to [35S]GTPγS binding (EC50 = 133 nM) and β-arrestin recruitment (EC50 = 2.4 μM) through CX3CR1 (Fig. 3). No antagonistic activity of E6130 was observed in the presence of fractalkine (IC50 value: >10 μM) (data not shown).

Fig. 3.
Fig. 3.

Agonistic activity of E6130 against CX3CR1 with respect to GTPγS binding and β-arrestin recruitment. Agonistic activity of E6130 against CX3CR1 in (A) an [35S]GTPγS binding assay and (B) an β-arrestin recruitment assay. The agonistic activity of E6130 is expressed as a percentage of the activity of the reference agonist (fractalkine) at its EC100 concentration.

The agonistic and antagonistic activities of E6130 with respect to [35S]GTPγS binding and β-arrestin recruitment were also examined by using CHO-K1 membranes expressing human chemokine receptors. E6130 had agonistic activity through chemokine orphan receptor 1 for β-arrestin recruitment at 10 μM. However, no significant agonistic or antagonistic activity (EC50 and IC50 values: >10 μM) of E6130 was observed with respect to [35S]GTPγS binding or β-arrestin recruitment through any other chemokine receptor (Table 1).

Effects of E6130 in a Murine CD4+CD45RBhigh T-Cell-Transfer Colitis Model.

The in vivo efficacy of E6130 after oral administration was assessed by using a murine CD4+CD45RBhigh T-cell-transfer colitis model. E6130 (10 or 30 mg/kg) was orally administered to the SCID mice once a day. Oral availability of E6130 was confirmed with pharmacokinetic study in the mice (Table 2). The total CD4+ T-cell population was used as the negative control. After stool consistency and body weight were recorded, the colons were excised, and their weight and length were determined because the colon length-to-weight ratio correlates well with histopathologic scores (Ostanin et al., 2009). The number of CX3CR1+ lymphocytes in the lamina propria was determined by means of flow cytometry.

View this table:
TABLE 2

Plasma concentrations of E6130 after oral administration in mice

Data are shown as the mean values ± S.E.M. (n = 3–6). Plasma protein binding of E6130 in mice: 93.3%.

Oral administration of E6130 significantly (P < 0.001) improved stool consistency from day 18 at doses of 10 and 30 mg/kg compared with CD4+CD45RBhigh T-cell-transferred group, and significantly (P < 0.001) ameliorated body weight loss from day 22 at doses of 10 and 30 mg/kg (Fig. 4, A and B). Furthermore, oral administration of E6130 significantly (P < 0.001) ameliorated the increased colon weight-to-length ratio as well as the increase in the number of CX3CR1+ lymphocytes in the lamina propria at doses of 10 and 30 mg/kg (Fig. 4, C and D). These effects of E6130 were histopathologically confirmed by a marked reduction in mucosal thickness due to much less inflammatory cell infiltration and crypt hyperplasia in treated animals (Fig. 4, E and F).

Fig. 4.
Fig. 4.

Effect of E6130 in a murine CD4+CD45RBhigh T-cell-transfer colitis model. (A) Stool score was assessed on the indicated days in CD4+CD45RBhigh T-cell-transfer mice given vehicle (■), 10 mg/kg E6130 (●), 30 mg/kg E6130 (▲), or total CD4+ T cells (⋄). (B) CD4+CD45RBhigh T-cell-transfer mice were administered vehicle (■), 10 mg/kg E6130 (●), or 30 mg/kg E6130 (▲). Mice that received total CD4+ T cells were treated with vehicle (⋄). Body weight is expressed as the percentage of the body weight at day 14. *P < 0.001 versus vehicle in CD4+CD45RBhigh T-cell-transferred group (repeated measures ANOVA followed by Fisher’s LSD test in each time slice). (C) On day 28 after T-cell transfer, the mice were euthanized, and the colons were excised. Leukocytes were extracted from the colon tissue by using collagenase. CX3CR1+ cells were labeled with phycoerythrin-conjugated anti-CX3CR1 antibody, and the size of the CX3CR1+ cell population was determined by using a FACSCanto cell analyzer. (D) The colon weight-to-length ratio of each mouse was assessed on day 28 after T-cell transfer. The results are presented as mean ± S.E.M. (n = 8/group). *P < 0.001 versus vehicle in CD4+CD45RBhigh T-cell-transferred group (one-way ANOVA followed by Fisher’s LSD test). Histopathology of colons from vehicle (E) or E6130 at 30 mg/kg (F) treated CD4+CD45RBhigh T-cell-transfer colitis model. The colons exhibit massive thickening of intestinal mucosa with inflammatory cell infiltration and reactive hyperplasia of mucosal epithelial cells, with cell debris in the glandular lumen of the vehicle-treated animal and almost normal structure of colon in the E6130-treated animal. Scale bar: 100 μm, H&E stain.

Effects of E6130 in a Murine Oxazolone-Induced Colitis Model.

The in vivo efficacy of E6130 after oral administration was assessed in a murine oxazolone-induced colitis model. Body weight loss and shrinkage of the colon are well recognized as features of the hapten-induced colitis model (Kojima et al., 2004; Ishiguro et al., 2010). At a dose of 30 mg/kg, E6130 significantly (P < 0.00625) prevented colon-length shortening and significantly (P < 0.00625) suppressed body weight loss on day 7 (Fig. 5).

Fig. 5.
Fig. 5.

Effect of E6130 in a murine oxazolone-induced colitis model. (A) Mice were euthanized, colons were excised, and colon length was measured. (B) Mice that intrarectally received oxazolone solution were administered vehicle (0.5% methyl cellulose solution) (■), 3 mg/kg E6130 (▼), 10 mg/kg E6130 (●), or 30 mg/kg E6130 (▲). Mice that received same vehicle (ethanol/water) were treated with vehicle (0.5% methyl cellulose solution) (⋄). Body weight is expressed as the percentage of body weight at day 5. Data are presented as mean ± S.E.M. (n = 9/group). *P < 0.00625 versus vehicle in the oxazolone-injected group (one-way ANOVA followed by Fisher’s LSD test).

Discussion

Inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis are characterized by a chronic clinical course of relapse and remission. In both Crohn’s disease and ulcerative colitis, leukocyte infiltration into the intestine is a fundamental event in the development and progression of the disease. Indeed, CX3CR1+CD4+ T cells are increased in the peripheral blood and inflamed tissues of patients with inflammatory bowel disease. Fractalkine is expressed by epithelial cells in the colonic tissue, and the expression is greater in the inflamed tissues of patients with Crohn’s disease or ulcerative colitis (Kobayashi et al., 2007), suggesting that the fractalkine/CX3CR1 axis plays an important role in the pathogenesis of inflammatory bowel disease. Therefore, inhibition of the trafficking of CX3CR1+ leukocytes represents a potential means of treating inflammatory bowel disease.

We have found that E6130 had inhibitory activity against fractalkine-induced chemotaxis in NK cells isolated from human peripheral blood. The down-regulation of CX3CR1 on the cell surface, as induced by E6130, mainly contributes to the inhibition of fractalkine-induced chemotaxis, because the down-regulation of CX3CR1-inducing activity of E6130 (EC50 5.2 nM) was more potent than its agonistic activity with respect to GTPγS binding (EC50 133 nM) and β-arrestin recruitment (EC50 2.4 μM). E6130 exhibited no antagonistic activity on [35S]GTPγS binding via CX3CR1. We cannot completely rule out the possibility that E6130 acts through a mechanism other than CX3CR1, because E6130 was shown to affect ACKR3/CMKOR1 (atypical chemokine receptor 3/chemokine orphan receptor 1) in β-arrestin recruitment at a high concentration of E6130 (10 μM) (Table 1). The in vitro data suggest that E6130 induced down-regulation of CX3CR1 on the cell surface, thereby inhibiting the interaction between fractalkine and CX3CR1, leading to inhibition of fractalkine-induced chemotaxis.

Numerous efforts have been made to clarify the in vivo role of the fractalkine/CX3CR1 axis in intestinal inflammation, but it remains unclear whether the fractalkine/CX3CR1 axis contributes to the progression of colitis. Kostadinova et al. (2010) demonstrated that knockout of CX3CR1 in mice suppressed the development of dextran sulfate sodium–induced colitis and inhibited the trafficking of inducible nitric oxide synthase–expressing macrophages into the mucosa. Likewise, Niess and Adler (2010) demonstrated in a CD4+ T-cell adoptive-transfer colitis model that CX3CR1gfp/gfp/RAG2−/− mice had fewer colitis signs than RAG2−/− mice, fewer dendritic cells in the mesenteric lymph nodes, and reduced serum interferon gamma and interleukin-17 concentrations. In contrast, Medina-Contreras et al. (2011) demonstrated that deletion of CX3CR1 aggravates the signs of colitis in mice, and Kayama et al. (2012) demonstrated the importance of intestinal CX3CR1high myeloid cells for the prevention of intestinal inflammation. These inconsistent results may be due to a lack of expression of CX3CR1 in a specific stage during fetal development or an excessive compensation by other chemokine pathway, or a possibility that both too much and too little receptor might predispose to pathologic conditions.

Therefore, to fully understand the mechanistic role of the fractalkine/CX3CR1 axis in intestinal inflammation, the fractalkine/CX3CR1 axis needs to be interrupted at the exact time at which inflammation begins. In our murine CD4+CD45RBhigh T-cell-transfer colitis model, we used E6130 to interrupt the fractalkine/CX3CR1 axis during intestinal inflammation and found that E6130 suppressed the signs of colitis by inhibiting the infiltration of CX3CR1+ leukocytes into inflamed colon tissue.

Massive infiltration of leukocytes—including lymphocytes, macrophages, and neutrophils—into inflamed colonic mucosa is a hallmark of human inflammatory bowel disease (Nishimura et al., 2009; Thomas and Baumgart, 2012). In this regard, chemokine/receptor axes play central roles in the trafficking of leukocytes from the blood into inflamed intestinal mucosa (Nishimura et al., 2009). Previous studies have also shown the importance of the chemokine/receptor axes in the onset of T-cell-dependent, hapten-induced experimental colitis (Khan et al., 2006; Bento et al., 2008). Khan et al. (2006) reported the critical role of monocyte chemoattractant protein-1 (MCP-1) in a dinitrobenzenesulfonic acid–induced colitis model. MCP-1 is a ligand for the CC chemokine receptors CCR2, CCR4, and CCR11, and these receptors are expressed by monocytes, macrophages, and certain subsets of T lymphocytes. Khan et al. (2006) revealed that knockout of the gene encoding MCP-1 in mice attenuates the onset and severity of the signs of colitis by reducing the infiltration of F4/80+ macrophages and CD3+ lymphocytes into inflamed mucosa.

Most chemokines, including MCP-1, can bind to several receptors, and most receptors bind many chemokines, but fractalkine binds only to CX3CR1. Because the expression of MCP-1 mRNA is significantly decreased in CX3CR1-deficient mice compared with wild-type mice, it is possible that E6130 suppresses the expression of MCP-1 in inflamed colonic tissues (Morimura et al., 2016). This suggests that the fractalkine/CX3CR1 axis exists upstream of other chemokine/receptor axes that together form a specialized system that regulates chemokine-induced leukocyte infiltration into the colonic mucosa.

In conclusion, we have characterized E6130, a novel CX3CR1 modulator. We found that E6130 induced down-regulation of CX3CR1 on the cell surface, leading to suppression of fractalkine-induced chemotaxis of CX3CR1+ human NK cells. Oral administration of E6130 ameliorated inflammatory bowel disease–related parameters in both a murine CD4+CD45RBhigh T-cell-transfer colitis model and a murine oxazolone-induced colitis model. In the murine CD4+CD45RBhigh T-cell-transfer colitis model, E6130 inhibited the trafficking of CX3CR1+ immune cells into the mucosa and decreased the number of these cells in the gut mucosal membrane. Thus, E6130 is a potentially useful therapeutic agent for treatment of inflammatory bowel diseases such as ulcerative colitis and Crohn’s disease.

Acknowledgments

The authors thank I. Yoshida, T. Okabe, Y. Matsumoto, N. Watanabe, Y. Ohashi, Y. Onizawa, and H. Harada (Eisai Co. Ltd.) for synthesizing and providing us with samples of E6130.

Authorship Contributions

Participated in research design: Wakita, Yanagawa, Kuboi.

Conducted experiments: Wakita, Yanagawa, Kuboi.

Performed data analysis: Wakita, Yanagawa, Kuboi.

Wrote or contributed to the writing of the manuscript: Wakita, Imai.

Footnotes

Abbreviations

ANOVA
analysis of variance
CCR
CC chemokine receptor
CHO-K1
Chinese hamster ovary K1
CX3CR1
CX3C chemokine receptor 1; E6130, [(3S,4R)-1-[2-Chloro-6-(trifluoromethyl)benzyl]-3-{[1-(cyclohex-1-en-1-ylmethyl)piperidin-4-yl]carbamoyl}-4-methylpyrrolidin-3-yl]acetic acid (2S)-hydroxy(phenyl)acetate
GPCR
G-protein coupled receptor
LSD
least significant difference
MCP-1
monocyte chemoattractant protein-1
MFI
mean fluorescence intensity
NK cells
natural killer cells
PBS
phosphate-buffered saline
RLU
relative light units
SCID
severe combined immunodeficiency
[35S]GTPγS
guanosine 5′-O-(3-[35S]thio)triphosphate

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Characterization of a Modulator of Chemokine Receptor CX3CR1

Hisashi Wakita, Tatsuya Yanagawa, Yoshikazu Kuboi and Toshio Imai
Molecular Pharmacology November 1, 2017, 92 (5) 502-509; DOI: https://doi.org/10.1124/mol.117.108381
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