Abstract
The α7 pentamer nicotinic acetylcholine receptors (nAChRs) are a target in transduction of anti-inflammatory signals from the central nervous system to the gastrointestinal (GI) tract. The aim of this study was to investigate the anti-inflammatory action of the novel α7 nAChR partial agonist encenicline and to determine the mechanism underlying its activity. Anti-inflammatory activity of encenicline was evaluated using trinitrobenzenesulfonic acid (TNBS)- and dextran sulfate sodium (DSS)-induced models of colitis. Macroscopic score, ulcer score, colon length and thickness, as well as myeloperoxidase (MPO) activity were recorded. Immunohistochemistry (IHC) was used to measure the infiltration of immune cells in the colon. Furthermore, we employed flow cytometry to determine the effect of encenicline on frequencies of FoxP3+ and interleukin (IL)-17A+ T cells in the mouse colon. Encenicline attenuated TNBS- and DSS-induced colitis in mice via α7 nAChRs, as indicated by significantly reduced macroscopic parameters and MPO activity. Treatment with encenicline significantly reduced the infiltration of macrophages, neutrophils, and B cells in the colon of TNBS-treated animals, as indicated by IHC. In the TNBS model encenicline reduced the frequency of FoxP3+ IL-17A+ T cells in the colon. In the DSS-model treatment encenicline increased the frequency of FoxP3+ T cells and reduced IL-17A+ T cells. Stimulation of α7 nAChR with partial agonist encenicline alleviates colitis via alteration of the number and/or activation status of the immune cells in the gut, emphasizing a potential role of α7 nAChRs as a target for anticolitic drugs.
Introduction
Inflammatory bowel diseases (IBD), comprising ulcerative colitis (UC), and Crohn’s disease (CD), is a group of chronic and relapsing gastrointestinal (GI) disorders manifested mainly by imbalanced immunologic response leading to an inflammatory state in the gut, disrupted motility, and abdominal pain (Sałaga et al., 2013; Sobczak et al., 2014). Although several pharmacological strategies have been employed to treat IBD, none of them has entirely succeeded. Recent studies point toward the role of α7 homopentamer nicotinic acetylocholine receptors (nAChRs) in the cholinergic modulation of immune cell activity (de Jonge and Ulloa, 2007; Snoek et al., 2010). The α7 nAChRs are located in both central and the peripheral nervous systems (de Jonge and Ulloa, 2007; Snoek et al., 2010). In the periphery, α7 nAChRs participate in cholinergic transmission and are involved in control of the heart rate, hormone secretion, GI motility, and immunomodulation (de Jonge and Ulloa, 2007).
The main neurotransmitter of the vagal nerve is acetylcholine, which is thought to control the functions of immune cells via nAChRs. Borovikova et al. (2000a) reported that acetylcholine significantly attenuated the release of proinflammatory, such as tumor necrosis factor-α (TNF-α), but not the anti-inflammatory cytokines, such as interleukin-10 (IL-10), in human macrophages in vitro. From a pharmacological point of view, nAChR agonists are more efficient than acetylcholine in inhibition of inflammatory signaling and production of proinflammatory cytokines. For example, it has been demonstrated that activation of nAChRs by nicotine reduces, in a dose-dependent way, the release of TNF-α and IL-6 from isolated mouse peritoneal macrophages stimulated with lipopolysaccharide (de Jonge et al., 2005). This observation is further supported by studies in animal models of GI tract disorders indicating that activation of vagal α7 nAChRs may effectively attenuate colitis by reduction in cytokine secretion from immune cells (de Jonge and Ulloa, 2007; Ji et al., 2014), as well as by clinical reports indicating that smoking and administration of nicotine (i.e., via patches) may provide beneficial effect on colonic inflammation in UC patients (Pullan et al., 1994).
To explore the therapeutic potential of α7 nAChRs in the treatment of IBD, we tested the anti-inflammatory properties and mechanisms of action of a novel partial α7 nAChR agonist, encenicline, which exhibits high efficacy at α7 nAChRs with a Ki value of 4.33 nM versus [125I]α-bungarotoxin in rat brain homogenate (Prickaerts et al., 2012) and is currently in the phase 3 clinical trial (ClinicalTrials.gov Identifier: NCT01969136). To assess the anti-inflammatory activity of encenicline, we used trinitrobenezenesulfonic acid (TNBS)- and dextran sulfate sodium (DSS)-induced models of colitis. To investigate the mechanism of action of encenicline, we used the α7 nAChRs antagonist methyllycaconitine (MLA). Moreover, we applied the ganglionic blocker hexamethonium (HEX) to investigate whether the blockade of peripherally-located nAChRs may abolish the potential ant-inflammatory action of encenicline. Using quantitative immunohistochemistry (IHC) and flow cytometry, we examined whether treatment with encenicline affects the number of immunocytes (macrophages, neutrophils, T cells, and B cells) and changes the ratio of pro- and anti-inflammatory T-cell subtypes in the colonic tissue.
Materials and Methods
Animals
Male BALB/c mice obtained from Animal Facility of the University of Lodz, Poland, weighing 22–26 g, were used for all experiments. Mice were housed at a constant temperature (22–24°C) and maintained in sawdust-lined plastic cages under a 12-hour light/dark cycle with free access to laboratory chow and tap water ad libitum. The study was carried out in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC) and with the institutional recommendations. The experimental protocol was approved by the Local Ethical Committee for Animal Experiments in Lodz (No. 589/2011).
Induction of Colitis and Assessment of Colonic Damage
TNBS Model.
Colitis was induced by intracolonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS), as described before (Fichna et al., 2012). Briefly, mice were lightly anesthetized with 1% isoflurane (Baxter Healthcare Corp., IL), and TNBS (4 mg in 0.1 ml of 30% ethanol in saline) was instilled into the colon through a catheter inserted into the anus (3 cm proximally). Control animals received vehicle alone (0.1 ml of 30% ethanol in saline). Preliminary experiments demonstrated that the dose of TNBS used in this study induced reproducible colitis.
DSS Model.
Colitis was induced by the addition of DSS (4% w/v; molecular weigh 40,000, Lot No. 5237K; MP Biomedicals, Aurora, OH,) to drinking water from day 0 to day 5. On days 6 and 7 animals received tap water (without DSS). Control animals received tap water throughout entire experiment. Body weight was monitored daily.
Pharmacological Treatments
We used two different therapeutic regimens in the TNBS model. In the acute TNBS model therapeutic effect of encenicline was evaluated as follows: Colitis was induced on day 0 and encenicline (3 mg/kg, i.p. twice daily) was administered from day 0 to day 2 with the first treatment 30 minutes before the induction of colitis. Animals were sacrificed on day 3 and the evaluation of colonic tissue damage was performed (Fig. 1A). The α7 nAChRs antagonist MLA (3 mg/kg i.p.) was administered 60 minutes before encenicline (n = 8).
A scheme illustrating experimental protocols and treatment regimens used in TNBS (A and B) and DSS (C and D) models.
The ganglionic blocker HEX was administered at the dose of 10 mg/kg i.p., 30 minutes prior to encenicline. In the semichronic TNBS model a curative treatment mode was applied: Inflammation was induced on day 0 and animals received encenicline treatment (3 mg/kg, i.p. twice daily) between days 3 and 6 (Fig. 1B). On day 7 mice were sacrificed and the evaluation of colonic damage was performed.
In DSS-model animals were treated with encenicline (3 mg/kg, i.p. twice daily) either from day 0 to day 6 (Fig. 1C) with the first treatment 30 minutes before the addition of DSS to the drinking water, or from day 3 to day 6 (Fig. 1D). On day 7 mice were sacrificed and the evaluation of colonic damage was performed. In all experiments control animals received vehicle (intraperitoneally) alone.
For the in vitro assay, a stock solution of encenicline in dimethyl sulfoxide (10−2 M) was prepared and diluted accordingly.
Evaluation of Colonic Damage
TNBS Model.
Animals were sacrificed by cervical dislocation. The colon was removed, opened longitudinally, rinsed with phosphate buffered saline (PBS), and immediately examined. Macroscopic colonic damage was assessed by an established semiquantitative scoring system by adding individual scores for ulcer, colonic shortening, wall thickness, and presence of hemorrhage, fecal blood, and diarrhea, as described before (Fichna et al., 2012). For scoring ulcer and colonic shortening the following scale was used: ulcer, 0.5 points for each 0.5 cm; shortening of the colon, 1 point for >15%, 2 points for >25% (with the basis being a mean length of the colon in untreated mice of 8.01 ± 0.15 cm; n = 6). The wall thickness was measured in millimeters, a thickness of n mm corresponding to n scoring points. The presence of hemorrhage, fecal blood, or diarrhea increased the score by 1 point for each additional feature. All colon samples for further experiments were collected during macroscopic evaluation process and were stored at –80°C until further processing.
DSS Model.
Mice were sacrificed by cervical dislocation 7 days after addition of DSS to the drinking water. The colon was rapidly isolated and weighed with fecal content. Colon was then opened along the mesenteric border and fecal material was removed. A total macroscopic damage score was calculated for each animal, including stool consistence (where 0 means normal well-formed fecal pellets and 3 means diarrhea), colon epithelial damage considered as number of ulcers (0–3), colon length and weight scores, considered as a percentage loss of either parameter in proportion to the control group (0, ≤5% weight/length loss; 1, 5–14% weight/length loss; 2, 15–24% weight/length loss; 3, 25–35% weight/length loss; and 4, >35% weight/length loss), where score = 0 means no inflammation (Fichna et al., 2012). The presence (score = 1) or absence (score = 0) of fecal blood was also recorded.
Determination of Tissue Myeloperoxidase Activity
The method described by Sałaga et al. (2014) was used to quantify the myeloperoxidase (MPO) activity, which is an indicator of granulocyte infiltration (Pulli et al., 2013). Briefly, 1-cm segments of colon were weighed and homogenized in hexadecyltrimethylammonium bromide buffer (0.5% in 50 mM potassium phosphate buffer, pH 6.0; 1:20 w/v), afterward, the homogenate was centrifuged (15 minutes, 13,200g, 4°C). On a 96-well plate, 200 μl of 50 mM potassium phosphate buffer (pH 6.0), containing 0.167 mg/ml of O-dianisidine hydrochloride and 0.05 μl of 1% hydrogen peroxide was added to 7 μl of supernatant. Absorbance was measured at 450 nm (iMARK Microplate Reader; Biorad, Hempstead, United Kingdom). All measurements were performed in triplicate. MPO was expressed in milliunits per gram of wet tissue, 1 unit being the quantity of enzyme able to convert 1 μmol of hydrogen peroxide to water in 1 minute at room temperature. Units of MPO activity per 1 minute were calculated from a standard curve using purified peroxidase enzyme.
Immunostaining of Immune Cells in Mouse Colon Tissue
Tissue Processing.
After the macroscopic scoring, segments of distal colon were stapled flat, mucosal side up, onto cardboard and fixed in 10% neutral-buffered formalin and stored in 4°C. Following fixation the tissue was washed extensively in 1× phosphate-buffered saline, pH 7.4, and cryoprotected via subsequent overnight incubations in PBS containing 10% and 30% sucrose, respectively. The colon samples were frozen cryosectioned on the same day. Tissue sections (12 μm) were cut on a Microm HM 500 OM cryostat (Thermo Scientific, Schwerte, Germany) and collected on Superfrost Plus slides (Menzel-Gläser, Braunschweig, Germany). Slides were air dried and stored at –20°C until further processing.
Immunohistochemistry
For IHC staining the method described by Blomster et al. (2011) was used. Briefly, slides were washed for 3 × 5 min in PBS followed by quenching of endogenous peroxidase activity with PBS containing 0.6% (v/v) H2O2 and 10% (v/v) methanol for 30 minutes at room temperature. After another round of washing, slides were immersed in blocking solution/antibody diluent containing 2% (w/v) bovine serum albumin (Sigma-Aldrich, St. Louis, MO), 5% (v/v) goat serum (Dako, Carpinteria, CA), and 0.2% (v/v) Triton X-100 in PBS for 1 hour to reduce nonspecific antibody binding. The sections were incubated overnight in a humidified chamber at 4°C with primary antibodies to visualize either macrophages (rabbit anti-Iba1, 1:1000; Wako Pure Chemical Industries, Richmond, VA), neutrophils and macrophages (rat anti-Ly6B.2; 1:100; AbD Serotec/Bio-Rad, Puchheim, Germany), T cells (rat anti-CD3; 1:100; AbD Serotec), or B cells (rat anti-CD19; 1:50; AbD Serotec ).
The following day, slides were washed extensively for 3 × 10 minutes in PBS and further incubated with the appropriate secondary antibody, i.e., biotinylated goat anti-rabbit or donkey anti-rat antibody (1:1000; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1.5 hours at room temperature. After another round of washing to remove unbound antibody, slides were incubated with an avidin–biotin complex reagent (1:200; Vector Laboratories, Burlingame, CA) for 1 hour at room temperature per manufacturer’s instructions. The staining was developed after a final round of washing by incubating the slides with 0.1% (w/v) 3,3-diaminobenzidine and 0.03% (v/v) H2O2 in Tris-HCl, pH 7.6. The reaction was stopped after approximately 4–5 minutes via immersion of the slides in distilled H2O, after which they were dehydrated through a graded series of ethanol, cleared in xylene, and coverslipped in Pertex mounting medium (HistoLab, Göteborg, Sweden). Evaluation of the staining was performed in the computerized Axo Imager Carl Zeiss light microscope.
Flow Cytometry
Frozen tissue samples were placed in 2 ml of Hanks’ balanced salt solution (HBSS) buffer with collagenase type II (200 IU/ml; Life Technologies, Carlsbad, CA) and incubated 4 hours at 37°C. Tissues were next homogenized using tissue raptor (Ultra-Turrax T 25, IKA, Staufen, Germany) at 20,000 rpm/min for 30 seconds, and 0.5 ml of trypsin solution (0.05% in HBSS) was added. Samples were incubated for 20 minutes at +37°C and filtered through 70-μm nylon mesh (BD, Franklin Lakes, NJ) to separate the dispersed cells and tissue fragments from the larger pieces. Cells were washed twice in PBS (without Ca2+/Mg2+) and centrifuged at 500g, 5 minutes, at room temperature. Cell pellet was resuspended in 1 ml of PBS and cells were counted using Turk’s staining solution. Cells were stained using the mouse Th17/Treg phenotyping kit (BD Pharmingen/BD Biosciences, San Jose, CA) following the manufacturer’s protocol. Briefly, 1 mln cells/ml was fixed for 30 minutes at 4°C at dark, permeabilized for 30 minutes at +37°C at dark, and stained with Th17/Treg phenotyping cocktail. The following antibodies were used: CD4 PerCP-Cy5.5 Clone: RM4-5, IL-17A PE Clone: TC11-18H10.1, Foxp3 Alexa Fluor 647 Clone: MF23. After each step cells were washed twice and centrifuged at 300g, 7 minutes, at room temperature. Finally, cells were resuspended in 200 μl of PBS and analyzed on a flow cytometer (LSR II Fortessa; BD Biosciences). Lymphocytes were gated using forward- and side-scatter plot, and CD4+ cells were identified as SSClowCD4+ cells. The change in frequency of FoxP3-, IL-17A-, and FoxP3 IL-17A-positive cells was analyzed.
Determination of α7 nAChRs mRNA Level in Colonic Tissue
RNA was isolated according to manufacturer’s protocol using PureLink RNA Mini Kit (Fisher Scientific, Schwerte, Germany). Briefly, tissue samples were homogenized in lysis buffer (600 μl), complemented with 1% 2-mercaptoethanol (AppliChem Inc., St. Louis, MO). Subsequently, the homogenates were centrifuged to remove the debris. Next, supernatants were placed onto ion-exchange columns and finally purified total RNA was eluted using diethyl pyrocarbonate-treated water (50 μl). To assess the purity and quantity of isolated RNA, dedicated spectrophotometer (BioPhotometer; Eppendorf, Hamburg, Germany) was used. Total isolated RNA (1 μg) was transcribed onto cDNA with First Strand cDNA synthesis kit (Fermentas, Burlington, Canada). Subsequently quantitative analysis was performed using fluorescently labeled TaqMan probe Mm01312230_m1 for mouse α7 nAChR and Mm01545399_m1 for mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT), which was used as the endogenous control (Life Technologies) on Mastercycler S realplex 4 apparatus (Eppendorf, Hamburg, Germany) and TaqMan Gene Expression Master Mix (Life Technologies) in accordance with manufacturer’s protocol. All experiments were performed in triplicate.
The Ct (threshold cycle) values for examined genes were normalized to Ct values obtained for the housekeeping gene HPRT. Relative amount of mRNA copies was calculated using following equation: 2–ΔCt × 1000.
Drugs
All drugs and reagents, unless otherwise stated, were purchased from Sigma-Aldrich (Poznan, Poland). Encenicline ((R)-7-chloro-N-quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide 13) was provided by NeuroSearch A/S (Ballerup, Denmark). DSS (MW 40,000) was purchased from MP Biomedicals (Solon, OH). [1α,4(S),6β,14α,16β]-20-Ethyl-1,6,14,16-tetramethoxy-4-[[[2-(3-methyl-2,5-dioxo-1-pyrrolidinyl)benzoyl]oxy]methyl]aconitane-7,8-diol citrate (MLA) was purchased from Tocris Bioscience (Ellisville, MO). In the in vivo tests, drugs were dissolved in 5% dimethyl sulfoxide in saline, which was used as vehicle in control experiments. The vehicles had no effect on the observed parameters in the concentrations used in this research.
Statistics
Statistical analysis was performed using Prism 5.0 (GraphPad Software Inc., La Jolla, CA). The data are expressed as means ± S.E.M. and calculated per square millimeter of tissue (for IHC experiments). Macrophages, neutrophils, B cells, and T cells were quantified from 10 randomly assigned high-powered fields (object magnification × 40) of view per animal (n = 4 mice per group), and P values were assessed by the use of the Mann–Whitney U test. Student’s t test or one-way analysis of variance (ANOVA) followed by Newman-Keuls post-hoc test were used for all other analyses. P values < 0.05 were considered statistically significant.
Results
Encenicline Protects against TNBS-Induced Colitis in Mice by Stimulation of α7 nAChRs.
To evaluate the anti-inflammatory activity of encenicline in the mouse GI tract, we used a well-established mouse model of acute colitis induced by TNBS. The intracolonic injection of TNBS resulted in reproducible increases in macroscopic damage scores and elevated MPO activity (Fig. 2). Encenicline (3 mg/kg, i.p. twice daily) significantly improved colitis as shown by lowered macroscopic score, ulcer score, bowel thickness, and MPO activity (Fig. 2, A–D), and significantly increased colon length (Fig. 2E). The anti-inflammatory activity of encenicline was significantly inhibited after preadministration of MLA (3 mg/kg, i.p., twice daily) as shown by changes in ulcer score, bowel thickness, MPO activity and colon length (Fig. 2, B–E).
The intraperitoneal administration of encenicline (3 mg/kg, twice daily) over 3 days attenuated TNBS-induced colitis in mice. Figure shows data for macroscopic score (A), ulcer score (B), bowel thickness (C), MPO activity (D), and colon length (E) after administration of encenicline alone or in the presence of MLA and for MLA alone. *P < 0.05, **P < 0.01 ***P < 0.001, compared with control mice. #P < 0.05, ##P < 0.01, ###P < 0.001, versus TNBS-treated mice. &P < 0.05, &&P < 0.01, &&&P < 0.001, compared with encenicline-treated mice. Data represent mean ± S.E.M. of 6–8 mice per group.
To investigate any effect of encenicline on established colitis, the compound was injected twice daily from day 3 following administration of TNBS. The selection of the day from which the treatment started followed literature data (Sans et al., 2001; Monteleone et al., 2012) and our previous observations indicating that clinical symptoms of colitis (e.g., changes in body weight), which represent severity of the disease, reach maximum at day 3 after TNBS injury. Treatment with encenicline resulted in a significant attenuation of intestinal inflammation as shown by significant decrease in total macroscopic score, ulcer score, bowel thickness, and MPO activity (Fig. 3, A–D). There was no difference in colon length between vehicle- and encenicline-treated animals (Fig. 3E).
The intraperitoneal administration of encenicline (3 mg/kg, twice daily) alleviates established TNBS-induced colitis in mice. Figure shows data for macroscopic score (A), ulcer score (B), bowel thickness (C), MPO activity (D), and colon length (E) after administration of encenicline. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control mice. #P < 0.05, ##P < 0.01, ###P < 0.001, versus TNBS-treated mice. Data represent mean ± S.E.M. of 6–8 mice per group.
The Ganglionic Blocker Hexamethonium Blocks the Effect of Encenicline on Colon Length and Thickness but Not on Other Macroscopic and Biochemical Parameters.
To investigate whether central or peripheral nicotinic receptors are involved in the anti-inflammatory activity of encenicline, we used a ganglionic blocker HEX (10 mg/kg i.p., 30 minutes prior to encenicline). We observed that treatment with HEX did not block the effect of encenicline on macroscopic score, ulcer score, and MPO activity (Fig. 4, A, B, D). However, HEX inhibited the beneficial effect of encenicline on colon thickness and length (Fig. 4, C and E).
The intraperitoneal administration of ganglionic blocker HEX (10 mg/kg, twice daily) does not block the anti-inflammatory effect of encenicline (3 mg/kg, twice daily) in model of TNBS-induced colitis. Figure shows data for macroscopic score (A), ulcer score (B), bowel thickness (C), MPO activity (D), and colon length (E) after administration of encenicline alone or in the presence of HEX. *P < 0.05, **P < 0.01 ***P < 0.001, as compared with control mice. #P < 0.05, ###P < 0.001, versus TNBS-treated mice. &P < 0.05, &&P < 0.01 compared with encenicline treated mice. Data represent mean ± S.E.M. of 6–8 mice per group.
Encenicline Prevents Development of DSS-Induced Colitis in Mice but Has No Healing Effect on Established DSS-Induced Colonic Inflammation.
Additionally, we characterized the anti-inflammatory activity of encenicline in a mouse model of colitis induced by DSS. We used two different dosing regimens to distinguish between potential prophylactic and healing effects of encenicline. We observed that encenicline administered at the dose of 3 mg/kg i.p., twice daily for 6 constitutive days during the DSS-treatment significantly attenuated colitis, as shown by significantly reduced macroscopic score and MPO activity and increased colon weight and length (Fig. 5, A–D). On the other hand, administration of encenicline (3 mg/kg, i.p., twice daily) from day 3 to day 6 of the experiment did not improve any of the measured parameters, indicating that the effect cannot be achieved when inflammation is established (Fig. 6 A–D).
Encenicline (3 mg/kg, i.p.) injected twice daily over 7 days attenuated DSS-induced colitis in mice. Figure shows data for macroscopic score (A), colon weight (B), MPO activity (C), colon length (D), and body weight changes (E). *P < 0.05, **P < 0.01, ***P < 0.001, as compared with control mice. #P < 0.05, ##P < 0.01 versus DSS-treated mice. Data represent mean ± S.E.M. of 6–8 mice per group.
Encenicline (3 mg/kg, i.p.) injected twice daily from day 3 to day 6 does not exhibit healing effects on established DSS-induced colitis in mice. Figure shows data for macroscopic score (A), colon weight (B), MPO activity (C), colon length (D), and body weight changes (E). *P < 0.05, **P < 0.01, ***P < 0.001, compared with control mice. Data represent mean ± S.E.M. of 6–8 mice per group.
Stimulation of α7 nAChRs Reduces the Number of Immune Cells in the Inflamed Colonic Tissue.
To investigate the infiltration of specific immune cells into the colon, we used semi-quantitative IHC staining of mouse colon tissue. Injection of TNBS resulted in a significant elevation of the number of macrophages, neutrophils, T cells, and B cells both in mucosal and submucosal layers of the colonic tissue (Fig. 7). Administration of encenicline resulted in a significant reduction in number of macrophages in both colonic mucosa and submucosa (Fig. 7A). This effect was completely reversed by preadministration of α7 nAChRs antagonist MLA (Fig. 7A). Moreover, treatment with encenicline reduced the number of neutrophils in the mucosa and submucosa (Fig. 7B). In the mucosal layer this effect was significantly reversed by MLA, but in submucosal layer the difference did not reach statistical significance (Fig. 7B). The number of T cells was not affected by administration of encenicline (Fig. 7C). Quantification of B cells showed that administration of encenicline significantly reduced their number only in the submucosal layer of the colon and this effect was not affected by pretreatment with MLA (Fig. 7D). Representative photographs of neutrophil presence in the TNBS-treated mucosa (Fig. 7E, i) and submucosa (Fig. 7E, ii). Treatment with encenicline reduced the number of neutrophils markedly (Fig. 7E, iii and iv).
Encenicline (3 mg/kg, i.p.) injected twice daily over 3 days reduces the infiltration of macrophages, neutrophils, and B-cells but not T cells in the colonic tissue. Figure shows data for quantification of macrophages (A), neutrophils (B), T cells (C), and B-cells (D) after administration of encenicline alone or in the presence of MLA. (E) Representative micrographs of mucosal (i) and submucosal (ii) layers of TNBS-treated mouse distal colon stained with neutrophil marker, as well as mucosal (iii) and submucosal (iv) layers of TNBS + encenicline-treated mouse distal colon stained with neutrophil marker. Scale bar, 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001, as compared with control mice. #P < 0.05, ##P < 0.01, versus TNBS-treated mice. &P < 0.05, &&P < 0.01, compared with encenicline-treated mice. Data represent mean ± S.E.M. of 4 mice per group.
Administration of MLA alone did not affect the number of any type of quantified immune cells with the exception of neutrophils in the submucosal layer when the number of cells was increased following MLA treatment alone (data not shown).
Stimulation of α7 nAChRs Alters the Frequency of FoxP3+ and IL-17A+ T Cells in the Colon.
Since we observed that administration of encenicline does not affect the total number of T cells in the inflamed colon, we used flow cytometry to investigate whether it alters the frequencies of different T-cell subtypes, such as FoxP3+ Treg cells and proinflammatory IL-17A T cells among all CD4+ T cells in the colonic tissue. We observed that the frequencies of all subtypes of T cells, namely FoxP3+ (Fig. 8A), IL-17A+ (Fig. 8B), and FoxP3+ IL-17A+ (Fig. 8C) T cells were elevated in the TNBS-treated groups. Treatment with encenicline significantly decreased the number of FoxP3+ IL-17A+ T cells (Fig. 8C) in the colonic tissue, but it did not affect the frequencies of FoxP3+ T cells and IL-17A+ T cells.
Effect of the intraperitoneal administration of encenicline (3 mg/kg, twice daily) on the frequency of FoxP3 and IL-17A expressing CD4+ T cells in the colon of TNBS-treated mice. Figure shows data for the frequency of (A) FoxP3+ T cells, (B) IL-17A+ T cells, and (C) FoxP3+ IL-17A+ T cells. The frequencies were assessed by flow cytometry. Cells were gated on CD4 populations. (D) Representative flow cytometric analysis is. *P < 0.05, compared with control mice. #P < 0.05, versus TNBS-treated mice. Data represent mean ± S.E.M. of 5–8 mice per group.
The frequency of FoxP3+ (Fig. 9A) T cells was significantly elevated and the frequency of IL-17A+ (Fig. 9B) T cells was reduced in the groups treated with encenicline compared with DSS-treated group. There was also a significant elevation of FoxP3+ IL-17A+ (Fig. 9C) T cells in DSS-treated animals, which was only partially reduced by treatment with encenicline.
Effect of the intraperitoneal administration of encenicline (3 mg/kg, twice daily) on the frequency of FoxP-3 and IL-17A producing CD4+ T cells in the colon of DSS-treated mice. Figure shows data for the frequency of (A) FoxP3+ T cells, (B) IL-17A+ T cells, and (C) FoxP3+ IL-17A+ T cells. The frequencies were assessed by flow cytometry. Cells were gated on CD4 populations. (D) Representative flow cytometric analysis. *P < 0.05, compared with control mice. Data represent mean ± S.E.M. of 5–8 mice per group.
α7 nAChRs mRNA Level Is Increased in TNBS-Induced Colitis.
To examine the changes in α7 nAChRs expression in the course of intestinal inflammation we determined the α7 nAChRs mRNA level in specimens obtained from control, TNBS-treated, and encenicline-treated animals. A significant upregulation of α7 nAChRs mRNA level was detected in TNBS-treated animals. Administration of encenicline partially reversed this effect but the difference did not reach statistical significance. (Fig. 10).
Determination of α7 nAChRs mRNA levels in mouse colon specimens. The level of α7 nAChRs mRNA is significantly increased in TNBS-treated mice. There is an observable difference between TNBS- and encenicline-treated animals, but it did not reach statistical significance. Data represent mean ± S.E.M. of 6 mice per group.
Discussion
The existence of the so called “cholinergic anti-inflammatory pathway” was proposed more than a decade ago (for review please see Martelli et al., 2014). Early experiments showed that the vagal nerve is responsible for the transmission of anti-inflammatory stimuli from the central nervous system to the periphery (Borovikova et al., 2000a,b). Subsequently, it has been shown that secondary lymphoid organs, such as spleen, are the primary target for vagal anti-inflammatory signals and α7 nAChRs have been implicated in their transduction (Wang et al., 2003). There are several lines of evidences showing that nicotinic receptors, and α7 nAChRs in particular, may serve as a pharmacological target for anti-IBD drugs (de Jonge and Ulloa, 2007). However, the exact mechanism of this phenomenon is not fully understood. Current hypotheses concern the interplay between centrally located receptors (both nicotinic and muscarinic) and α7 nAChRs located on peripheral immune cells, such as macrophages, neutrophils, T cells, B cells and dendritic cells.
From this study the evidence is clear that the stimulation of α7 nAChRs with the novel, selective partial agonist encenicline alleviates symptoms of experimental colitis in mice and reduces the number of immune cells infiltrating the colon. The selectivity of encenicline for the target has been validated by inhibition of some of the effects with the selective α7 nAChRs antagonist MLA. Moreover, by employing the ganglionic blocker HEX we demonstrated that central, rather than peripheral, sites are responsible for encenicline activity, because HEX did not block overall macroscopic effects of the α7 nAChR agonist on intestinal inflammation and did not block the effect on biochemical parameters. It is noteworthy, even though some of the macroscopic parameters (e.g., colon length and thickness) were worsened by HEX (compared with encenicline-treated group), the inflammatory state at the cellular level was alleviated as indicated by low MPO activity. Our observation remains in line with recent findings reported by Munyaka et al. (2014) who showed that central cholinergic activation improves DNBS-induced colitis and reduces secretion of colonic and splenic cytokines.
Previous studies showed that immune cells, such as macrophages, neutrophils, T cells, and B cells, express α7 nAChR mRNA, and thus their function may be modulated by cholinergic signaling (Yoshikawa et al., 2006; Kawashima et al., 2007; Razani-Boroujerdi et al., 2007; Koval et al., 2009; Su et al., 2010; Munyaka et al., 2014). Moreover, it has been demonstrated that α7 nAChRs are involved in the modulation of immune cell activity in the spleen (Munyaka et al., 2014). In our study, TNBS infusion caused a massive infiltration of immune cells, namely, macrophages, neutrophils, T cells, and B cells, in the mucosal and submucosal layers of the colon, and stimulation of α7 nAChRs with encenicline reversed this process. Moreover encenicline inhibited the viability of spleen-derived monocytes stimulated with lipopolysaccharide, which may explain, at least partially, the reduced number of macrophages infiltrating the colon of encenicline-treated animals (data not shown). The detailed mechanism of this process is not resolved but may involve the Jak2-STAT3 signaling and/or reduced nuclear factor (NF)-κB activation as demonstrated for isolated peritoneal macrophages (de Jonge et al., 2005; de Jonge and Ulloa, 2007).
Additionally, we observed reduced neutrophil infiltration indicated by decreased MPO activity in the colonic tissue of animals treated with encenicline. This result was confirmed by IHC staining showing a significantly lower number of neutrophils both in the mucosal and submucosal layers of the colon. These two findings correspond to the effects observed at the macroscopic level, since neutrophils are mainly responsible for the damage of the colonic tissue in the course of colitis (Lampinen et al., 2008; Knutson et al., 2013). Again pretreatment with MLA emphasized that α7 nAChRs are specifically responsible for these effects, which is in line with findings reported by Gahring et al. (2010) who showed an increased number of neutrophils infiltrating the site of inflammation in α7 nAChRs KO animals. It has also been shown that nicotine affects various neutrophil functions, e.g., superoxide anion production, chemokine secretion, chemotaxis, and integrin expression (de Jonge and Ulloa, 2007). Disruption of these processes may contribute to the effects observed in this study.
Here we show that TNBS-induced colitis is associated with strong infiltration of B cells into the mucosa and submucosa of the colon. Changes in the number of B cells were more significant in the submucosal than mucosal layer, which is probably caused by the smaller number of residual B cells in the submucosa of control animals. Under physiologic conditions the mucosa is constantly exposed to external surroundings abundant in foreign antigens (e.g., bacterial); the reservoir of B cells in this layer is necessary for production of antibodies needed to protect against pathogens. TNBS insult results not only in the accumulation of antigen-like molecules in the colon but also disrupts its mucosal barrier; thus, the recruitment and/or proliferation of B cells is observed in the deeper, submucosal layer of the colonic tissue (Lee et al., 2007). Treatment with encenicline significantly reduced the number of B cells present in the colon. This effect may be attributed to the fact that α7 nAChRs negatively regulate B-cell proliferation (Koval et al., 2009).
Of note, the effect of encenicline on B cells was not blocked by MLA. This phenomenon could be explained by a possible nonselective action of encenicline on type 3 serotonin receptors (5-HT3Rs); however, it is very improbable since its other effects were successfully blocked by MLA, and the fact that 5-HT3Rs are known to affect gastrointestinal motility rather than immunologic responses in the gut (Mawe and Hoffman, 2013). Hence, we suggest that the effect of encenicline on B cells may be indirect. It has been reported that B-cell responses are controlled by neural signaling in the spleen (Mina-Osorio et al., 2012) and that increased parasympathetic transmission decreases secretion of proinflammatory cytokines that cause the recruitment of B cells (Ghia et al., 2007; Ji et al., 2014; Munyaka et al., 2014). The reduction of B-cell infiltration may thus be a result of increased parasympathetic signaling in secondary lymphoid organs, such as spleen or gut-associated lymphoid tissue (GALT); however, the exact cellular target mediating this effect remains unknown. Moreover, it is possible that the effect of encenicline on B cells is caused by a lesser local tissue damage observed after the treatment, especially since infiltration of B cells to the site of inflammation is thought to be secondary to the inflammatory status of the tissue.
Interestingly, treatment with encenicline did not change the total number of T cells in the colonic tissue. T cells are crucially involved in the pathophysiology of intestinal inflammation and mediate various processes that lead to colonic tissue damage and maintenance of the inflammatory state, such as secretion of proinflammatory cytokines. Furthermore, inhibition of T-cell migration to the gut is one of the strategies used in the treatment of IBD in humans (Raine 2014). On the other hand, T cells have been implicated in mediation of vagal cholinergic anti-inflammatory reflex. It has been shown that in nude mice lacking T cells the stimulation of vagal nerve does not cause anti-inflammatory effect and that adoptive transfer of T cells into these animals partially restores the anti-inflammatory activity of vagal transmission (Martelli et al., 2014). Furthermore, Rosas-Ballina et al. (2011) characterized an acetylcholine-producing memory-phenotype T-cell population that is integral to the anti-inflammatory transmission and is necessary for inhibition of cytokine production by the vagus nerve.
Here, given the dual nature of T cells in the context of intestinal inflammation we examined the frequency of three different subtypes of T cells that have a potentially strong impact on the inflammation in the colon, namely FoxP3+ T, IL-17A+ T, and FoxP3+ IL-17A+ T cells. We found significant differences in the effect of encenicline on the frequency of these T-cell subtypes between TNBS and DSS models of colitis. In the TNBS model, the frequency of FoxP3+ IL-17A+ T cells was significantly reduced after treatment with encenicline, although there was no effect on other subtypes. The role of FoxP3+ IL-17A+ T cells in the inflammation is only partially understood. It has been shown that these cells are generated in the periphery at mucosal sites during inflammation and their presence has been confirmed in the lamina propria of the gut (Zhou et al., 2008; Voo et al., 2009). Moreover, their differentiation is stimulated by IL-1β, IL-2, IL-21, and IL-23, i.e., cytokines involved in the pathophysiology of intestinal inflammation (Voo et al., 2009). Although they resemble the immunosuppressive FoxP3+ phenotype (Treg cells), the expression of FoxP3+ in FoxP3+ IL-17A+ T cells is lower than that of FoxP3+ IL-17A– T cells in CD4+ population (Voo et al., 2009). Therefore, the proinflammatory function has been attributed to these cells. In line, Ueno et al. (2013) have recently found that prevalence of circulating FoxP3+ IL-17A+ T cells is increased in patients with IBD compared with healthy controls. Moreover, the conversion of Treg cells into FoxP3+ IL-17A+ T cells has been suggested as a mechanism of their generation, since the ability of Treg cells to suppress autologous T-cell proliferation was decreased in IBD patients (Ueno et al., 2013). It has also been reported that FoxP3+ IL-17A+ T cells coexpress RAR-related orphan receptor γt (RORγt), which promotes the differentiation of thymocytes into proinflammatory T-helper 17 cells (Sun et al., 2000; Dong 2008). In the DSS model, we observed an increased frequency of Treg and decreased frequency of proinflammatory IL-17A+ T cells after treatment with encenicline, which most probably contributes to the anti-inflammatory effect observed at the macroscopic level. On the other hand, it should be noted that a differential effect of encenicline on T-cell subpopulations in both models could be seen owing to the different time points of the analyses, which correlate with the kinetics of disease severity. Moreover, a less pronounced involvement of innate immune system in the acute TNBS- compared with DSS-induced colitis may be of importance. Taken together, our observations suggest that stimulation of α7 nAChRs with clinically effective partial agonist encenicline activates different anti-inflammatory mechanisms that in both models lead to the improvement of colitis. Our finding adds another layer of complexity to the knowledge of models of intestinal inflammation and differentiates these on the basis of the role of FoxP3+ IL-17A+ T cells in their pathophysiology.
Additionally, we found that α7 nAChRs mRNA is increased in the colonic tissue of TNBS-treated animals. This phenomenon is most probably caused by a massive infiltration of α7 nAChRs-expressing immune cells into the colon. Moreover, it has been shown that expression of α7 nAChRs increases in immune cells upon their activation with concanavalin A (Nizri et al., 2009), which could also account for the overall increase in α347 mRNA expression. Curiously, treatment with encenicline only partially reduced this effect. However, it has been reported that the expression of α7 nAChRs increases upon stimulation with nicotinic agonists, which may be the reason why the α7 nAChR mRNA did not go down to the control level (Van der Zanden et al., 2012).
To summarize, our study extends the knowledge of the pathogenesis of colonic inflammation and provides novel information on the mechanisms underlying the anti-inflammatory activity of the α7 nAChR partial agonist encenicline. Although there have been some doubts concerning targeting α7 nAChRs in the treatment of colitis [e.g., it has been suggested that α7 nAChRs agonists worsen the disease (Snoek et al., 2010)], we suggest that alteration of the frequencies of FoxP3+- and/or IL-17A+-expressing T cells may underlie the beneficial effects of encenicline on intestinal inflammation. Furthermore we highlight the superiority of partial over full α7 nAChR agonists in the treatment of colitis, which is supported by recent reports showing on one hand that in vitro partial α7 nAChR agonists exhibit anti-inflammatory properties (Thomsen and Mikkelsen, 2012) and on the other indicating that full agonists do not affect colitis in vivo (Snoek et al., 2010).
Moreover, we show that the duration of treatment may be another factor that affects its outcomes. Our insights in the DSS model show that longer (7-day) stimulation of α7 nAChRs is necessary to obtain significant anti-inflammatory effect in the GI tract, which may be further supported by the fact that chronic nicotine intake prevents UC in humans. In line, Van der Zanden et al. (2012) demonstrated that repeated exposure to nicotine upregulates α7 nAChRs expression in human monocytes and enhances the potency of α7 nAChRs agonists in reduction of TNF-α levels. Given the results of our study, we postulate that cholinergic brain-immune system interactions, mediated mainly by vagal nerve, regulate the abnormal immunologic responses observed in the course of IBD. Hence we encourage further development of α7 nAChR-targeting compounds with anti-inflammatory activity.
Authorship Contributions
Participated in research design: Fichna, Mikkelsen, Salaga, Blomster.
Conducted experiments: Salaga, Blomster, Piechota-Polańczyk, Zielińska, Jacenik, Cygankiewicz.
Contributed new reagents or analytic tools: Mikkelsen.
Performed data analysis: Salaga, Blomster, Piechota-Polańczyk, Zielińska, Jacenik, Cygankiewicz.
Wrote or contributed to the writing of the manuscript: Salaga, Fichna, Mikkelsen, Blomster, Krajewska.
Footnotes
- Received July 30, 2015.
- Accepted September 24, 2015.
Supported by the Iuventus Plus program of the Polish Ministry of Science and Higher Education [No. 0107/IP1/2013/72 to J.F.] and the grants from the Medical University of Lodz [502-03/1-156-02/502-14-140 to MS and #503/1-156-04/503-01 to J.F.] and National Science Centre [No. UMO-2013/11/N/NZ7/02354 to M.S., No. UMO-2013/11/B/NZ7/01301 and No. UMO-2014/13/B/NZ4/01179 to J.F.]. L.V.B. and J.D.M. are supported by grants from the Danish Research Council for Strategic Research [COGNITO] and the Novo Nordisk Foundation. Study sponsored by a Polpharma Scientific Foundation scholarship to M.S.
Abbreviations
- CD
- Crohn’s disease
- DSS
- dextran sulfate sodium
- GI
- gastrointestinal tract
- HEX
- hexamethonium
- IBD
- inflammatory bowel diseases
- IHC
- immunohistochemistry
- IL
- interleukin
- MLA
- methyllycaconitine
- MPO
- myeloperoxidase
- nAChR
- nicotinic acetylcholine receptor
- PBS
- phosphate-buffered saline
- TNBS
- 2,4,6-trinitrobenzene sulfonic acid
- TNF-α
- tumor necrosis factor-α
- UC
- ulcerative colitis
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
References
In this issue
Encenicline in Colitis
