Intestinal specimens were obtained from 15 patients with IBD. Normal tissue specimens were obtained from patients receiving colonoscopy for colorectal cancer screening. The tissues were frozen in liquid nitrogen and stored at −80°C for reverse transcription-quantitative PCR (RT-qPCR) analyses, or were embedded in optimum cutting temperature (OCT) compound for immunofluorescence staining. All studies involving samples obtained from human participants were approved by the Ethics Committee of the Humanitas Clinical and Research Center of Shanghai East Hospital, Shanghai, China. Written informed consent was obtained from each patient or their family before initiating the study protocol.

A total of 30 C57BL/6 male mice (8 to 10 weeks old, weighing 20 g) were purchased from Slaccas Laboratory Animal Co., Ltd. (Shanghai, China) and all experiments were performed under specific pathogen-free conditions at the Animal Resources facilities of Shanghai East Hospital. All animal experiments were performed following approval by the Animal Care and Use Committee of Shanghai East Hospital.

The mice were randomly divided into 3 groups as follows: the control group, DSS treatment group and the DSS plus ketanserin treatment group (n=10 in each group). The mice were administered 4% (wt/vol) DSS (MP Biomedicals, Solon, OH, USA) dissolved in their drinking water for 7 days to induce acute experimental colitis. Ketanserin (10 mg/kg; Janssen Pharmaceuticals, Beijing, China) or phosphate-buffered saline (PBS; vehicle control; administered to the mice in the DSS treatment group) were administered intra-peritoneally once daily for 3 days when the administration of DSS began. The weight of the mice was recorded daily using an electronic weighing system (Practum 612-1CN; Sartorius AG, Goettingen, Germany). On day 7, the mice were sacrificed by an intraperitoneal injection of 4% sodium pentobarbital and the colons were obtained for the measurement of colon length. For histological analysis, the colons were excised and fixed in 4% paraformaldehyde. Subsequentoy, 1 cm of the distal colon of each mouse was paraffin-embedded. The paraffin-embedded sections were cut (4-μM-thick) using a microtome and stained with hematoxylin and eosin. For gene and protein expression analyses, the colons were immediately frozen in liquid N₂ and stored at −80°C.

Total RNA was extracted from the colon tissues or lamina propria macrophages (LP-macrophages) and bone marrow derived-macrophages (BMDMs) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using the PrimeScript™ RT reagent kit (Takara, Shiga, Japan). Messenger RNA (mRNA) transcripts were analyzed by quantitative PCR using SYBR^® Premix Ex Taq™ (Takara) with an Applied Biosystems StepOne/StepOnePlus Real-Time PCR System. Gene expression was normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and the relative expression levels were quantified using the 2^−ΔΔCT method. The primer pairs used are listed in Table I.

Colon proteins were extracted using RIPA buffer supplemented with protease inhibitors (Shanghai Shenggong Co., Ltd., Shanghai, China). A total of 40 μg protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the gels were then electrotransferred onto nitrocellulose filter membranes (NC; Whatman, Alameda, CA, USA). The membranes were incubated with antibodies against total NF-κB (p65; Cat. no. 8424; 1:1,000), phosphorylated (p-)NF-κB (p65; Cat. no. 3033; 1:1,000), or GAPDH (Cat. no. 8884; 1:1,000) (all from Cell Signaling Technology, Beverly, MA, USA) overnight at 4°C. The membranes were then incubated with an IRDye 800CW-conjugated secondary antibody (Rockland, Hamburg, Germany) for 1 h at room temperature. Images were acquired using an Odyssey infrared imaging system (LI-COR Biosciences, Inc., Lincoln, NE, USA).

Briefly, the colons were longitudinally cut and washed in PBS containing 1% fetal bovine serum and 1 mM DTT for 10 min at room temperature 3 times. Subsequently, the colon tissues were digested in complete RPMI-1640 medium (Invitrogen) supplemented with 0.5 mg/ml collagenase type VIII (Sigma-Aldrich, St. Louis, MO, USA), 20 mg/ml DNase I (Roche Diagnostics, Mannheim, Germany) for 60 min at 37°C in a shaking water bath. The cell suspension was passed through a 100–70 μm filter and then resuspended in 1.077 g/cm³ iso-osmotic metrizamide medium (Accurate Chemical & Scientific Corp., Westbury, NY, USA). Following centrifugation at 1,500 × g for 15 min at room temperature, the low-density fraction was collected. For FACS analysis of the colon samples, the cells were stained with antibodies to CD45 (17-0451), CD11c (12-0114), MHC-II (56-5321), F4/80 (15-4801), CD11b (11-0112), Ly-6G (Gr-1; 53-5931) (all from eBioscience, San Diego, CA, USA) and CD206 (141711) (from BioLegend, San Diego, CA, USA). The influx of neutrophils, dendritic cells (DCs) and macrophages was determined by the frequency of macrophages, neutrophils and DCs in CD45⁺ cells in the LP of colons.

To isolate the LP-macrophages from the lamina propria of the mouse colons, the colons were washed with PBS and cut into small sections. The latter were then incubated with 5 mM EDTA and 3% FCS in Ca²⁺- and Mg²⁺-free Hanks balanced salt solution for 30 min at 37°C with stirring. The small sections were collected and were then digested with RPMI-1640 containing 5% FCS, 1 mg/ml collagenase type IV and 0.1 mg/ml DNase for 1 h at 37°C. The liberated cells were collected through a stainless steel sieve. Following centrifugation (500 × g, 4°C) and washing with PBS 3 times, the intestinal macrophages were enriched by positive selection with anti-CD11b Dynabeads following the manufacturer's instructions (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The cells obtained were tested for the expression of macrophage markers by flow cytometry (CD45⁺CD11b⁺CD11c⁻F4/80⁺); cells with a purity >85% were used in the experiments.

LP-macrophages (5×10⁴) were cultured in complete RPMI-1640 medium and treated with 100 ng/ml LPS for 6 h. Supernatants were collected for the measurement of cytokine levels and the attached cells were lysed for RNA extraction. Briefly, the levels of the cytokines, IL-1β, IL-6, IL-10 and TNF-α, were evaluated in the supernatant from the macrophage cultures by enzyme-linked immunosorbent assay (ELISA), following the manufacturer's instructions (R&D Systems, Inc., Minneapolis, MN, USA).

The migration of the LP-macrophages induced by C-X-C motif chemokine 12 [CXCL12, also known as stromal cell-derived factor 1 (SDF-1)] was assessed using a 48-well microchemotaxis chamber (Neuro Probe, Gaithersburg, MD, USA). CXCL12 (50 nM; 250-20B, PeproTech, Rocky Hill, NJ, USA) was placed in wells (30 μl) of the lower compartment, and LP-macrophages (50 μl of a 5×10^*4/ml suspension) were seeded in the wells of the upper compartment. The chamber was incubated in a humidified environment at 37°C with 5% CO₂ for 4 h. The membrane was removed, fixed in methanol, and stained with crystal violet solution for 30 min followed by destaining with water. The cells that had migrated across the membrane were counted using a microscope (Leica TCS SP8 CARS confocal microscope, Leica Microsystems GmbH, Wetzlar, Germany). The chemotaxis index was calculated as the ratio of the number of cells that had migrated towards the chemoattractant divided by the number of cells that had migrated towards the medium.

The LP-macrophages (5×10^*4) were infected with E. coli at an MOI of 1:10 for 1 h in complete RPMI-1640 medium without antibiotics, and then incubated in complete medium containing 40 μg/ml gentamycin for 120 min. The cells were then lysed and seeded on LB agar plates. Following overnight incubation at 37°C, bacterial colonies (CFUs) were counted as a measure of intracellular bacteria.

For the human tissues, 4-μm-thick frozen sections of intestinal specimens were fixed in cold acetone for 10 min at −20°C and blocked with 5% BSA for 1 h at room temperature, then incubated with a primary rabbit anti-human 5-HT_2AR antibody (ab66049; rabbit polyclonal to 5-HT_2AR; reacts to mouse, rat, human; Abcam, Cambridge, UK; 1:500) overnight at 4°C. Mouse anti-human CD68 antibody (MCA5709; mouse anti-human CD68, monoclonal antibody; AbD Serotec, Kidlington, UK; 1:500) (overnight at 4°C) was subsequently used to detect the macrophages. For the murine colon tissues, 5-HT_2AR expression was detected by overnight incubation at 4°C with rabbit anti-mouse 5-HT_2AR antibody (ab66049; rabbit polyclonal to 5-HT_2AR; reacts to mouse, rat, human; Abcam; 1:300). Subsquently, rat anti-mouse CD68 antibody (MCA1957GA; rat anti-mouse CD68, monoclonal antibody; AbD Serotec; 1:500) was used overnight at 4°C. For both analyses, Alexa-Fluor 488- and Alexa-Fluor 555-conjugated antibodies [goat anti-rabbit IgG (ab150077, goat anti-rat IgG (ab150158) and goat anti-mouse IgG (ab150118); all from Abcam, Cambridge, UK; 1:1,000; 1 h at room temperature] were used as secondary antibodies, followed by incubation with 1 μg/ml DAPI (20 min, room temperature). The sections were finally visualized under a confocal microscope (Olympus, Tokyo, Japan). Images were captured using FluoView software.

siRNA targeting the 5-HT_2AR (Genepharm Biotech, Shanghai, China) was synthesized (GACAACUGUCGUGAUUAUUTT) and control siRNA (UUCUCCGAACGUGUCACGUTT) was also used. BMDMs were isolated from bone marrow cells obtained from the mice. Three C57BL/6 male mice (8 to 10 weeks old, weighing 20 g) were sacrificed as described for the mice in the other experiments, and bone marrow cells were isolated from femurs and cultured with RPMI-1640 supplemented with 10% FBS, 1% penicillin and streptomycin (Invitrogen) and 10% L929 conditioned medium. The culture fluid was exchanged for fresh culture medium every 4 days. Under these conditions, adherent macrophage monolayers were obtained within 8–10 days. The BMDMs were then transfected with the siRNAs as previously described (17). Following transfection, the BMDMs (1×10⁶/ml) were pre-incubated with ketanserin (10 μM) for 10 min and then stimulated with LPS (100 ng/ml) and 5-HT (100 nmol/l) for 12 h. RNA was then extracted for PCR.

The statistical significance of the differences between the treatment and control groups was determined using a Student's t-test. Data were analyzed with one-way analysis of variance (ANOVA), followed by the Student's t-test for experiments involving only 2 groups, and Dunnett's t-test for experiments involving >2 groups. All data are expressed as the means ± standard deviation (SD). Statistical significance was set at P<0.05.

We first examined the expression of 5-HT_2AR in patients with IBD and in mice with DSS-induced experimental colitis. The 5-HT_2AR mRNA level was increased in the patients with IBD (Fig. 1A) compared to the normal tissue specimens, and 5-HT_2AR protein expression was also elevated in the patients with IBD, as detected by immunofluorescence staining (Fig. 1B). 5-HT_2AR is known to be expressed on the surface of lymphocytes, natural killer (NK) cells and monocytes/macrophages/DCs (18,19). We observed a specific co-localization of 5-HT_2AR with the CD68 macro-with the CD68 macrophage marker (Fig. 1B), reflecting that 5-HT_2AR expression is enhanced in macrophages. The 5-HT_2AR expression level was also upregulated in the mice with DSS-induced experimental colitis (Fig. 1C and D) and was co-localized with CD68 postivity (Fig. 1E). These data suggest that the synthesis of 5-HT_2AR is induced in the inflamed colon and that it is mostly expressed in macrophages.

To investigate whether ketanserin reduces susceptibility to colitis, we induced colitis in mice using DSS. The mice with DSS-induced colitis exhibited a continuous decrease in body weight from day 4 to day 7 and shortened colon lengths. By contrast, the administration of ketanserin during the induction of colitis significantly prevented the decrease in body weight (Fig. 2A) and colon shortening (Fig. 2B). A histological examination of the colons of the mice with DSS-induced colitis revealed severe inflammation with ulcerative lesions, loss of crypts and the infiltration of inflammatory cells, whereas treatment with ketanserin alleviated these histological changes and damage to the colon, characterized by a decrease in the loss of architecture, fewer ulcerative lesions, and a decrease in inflammatory cell infiltration into the inflamed mucosa (Fig. 2C and D). Our data therefore suggest that ketanserin exerts a potent therapeutic effect, ameliorating DSS-induced colitis.

DSS is directly toxic to the colonic epithelium and triggers inflammation in the gut with high levels of inflammatory mediators, such as iNOS, IL-1β and IL-6 (20,21). We thus evaluated the effects of ketanserin on the production of inflammatory mediators in the colonic mucosa by RT-qPCR. Compared with the vehicle-treated mice with colitis (DSS group), the ketanserin-treated mice exhibited significantly lower mRNA levels of keratinocyte-derived chemokine (KC; a major chemoattractant for neutrophils), iNOS, TNF-α, IL-1β and IL-6 (Fig. 2E). Moreover, the transcript levels of IL-10 were found to be higher in the ketanserin-treated mice. The levels of CXCL12, the major chemoattractant for DCs, remained unaltered.

Innate immune cells are the major sources of inflammatory mediators in DSS-induced colitis (22,23). We thus investigated whether the effects of ketanserin alter the infiltration of innate immune cells, thus influencing the inflammatory response in the colon. In the mice with DSS-induced colitis, the percentage of neutrophils, macrophages and DCs in the CD45⁺ cells was significantly increased in the colonic lamina propria on day 7 compared with the control mice (Fig. 3A). Consistently, treatment with ketanserin resulted in a decrease in the influx of neutrophils and macrophages into the inflamed colons of the mice (Fig. 3B). Minor differences in the relative percentage of DCs in the CD4⁺ T cell population were observed. We also found that the colonic macrophages in the ketanserin-treated group expressed higher levels of CD206, a marker of M2 macrophages, compared to the vehicle-treated mice with colitis (DSS group; Fig. 3A and C).

As the ketanserin-treated mice exhibited a reduced influx of macrophages into the colonic lamina propria, we then determined whether ketanserin suppresses the chemotaxis of macrophages. As CXCL12 is a critical regulator of macrophage migration (24,25), the migration of LP-macrophages through an 8-μm filter towards CXCL12 (50 nM) placed in the lower chamber was examined. The chemotaxis index indicated that ketanserin significantly reduced the migration of macrophages towards CXCL12 (Fig. 4A and B).

It has been shown that 5-HT modulates the activity of the phagocytosis of bacteria by macrophages through 5-HT receptors (26). Thus, to determine whether ketanserin affects the phagocytic ability of macrophages, LP-macrophages were infected with E. coli and viable intracellular bacteria CFUs were counted in order to evaluate bacterial phagocytosis. We found that the ability of the LP-macrophages to phagocytize E. coli in the ketanserin-treated mice with colitis did not differ from that in the vehicle-treated mice with colitis (DSS group; Fig. 4C).

We then evaluated the release of cytokines in the supernatants of LP-macrophages obtained from the inflamed colon. In line with our hypothesis, the secretion of TNF-α, IL-1β and IL-6 was significantly decreased in the ketanserin-treated mice (Fig. 5A–C). In addition, the ketanserin-treated mice produced higher amounts of IL-10 (Fig. 5D).

Since the LP-macrophages from the ketanserin-treated mice with colitis exhibited a high level of CD206, a marker of M2 macrophages (as shown by FACS analysis), we therefore measured the mRNA levels of iNOS, CD32 and IL-12 p40, as M1 polarization markers, and the levels of CD206 and IL-10, as M2 polarization markers in the LP-macrophages in all 3 groups. Of note, the LP-macrophages from the ketanserin-treated mice exhibited a notably decreased expression of iNOS, CD32 and IL-12 p40 (Fig. 5E), but elevated levels of CD206 and IL-10 (Fig. 5F). Taken together, these data indicate that ketanserin modulates LP-macrophage function by reducing the production of pro-inflammatory cytokines and promoting M2 as opposed to M1 polarization.

In order to determine whether 5-HT_2AR plays a role in the effects of ketanserin, we used 5-HT_2AR siRNA to downregulate the expression of 5-HT_2AR in BMDMs. The LPS/5HT-induced expression of the pro-inflammatory cytokines, IL-1β (Fig. 6A), TNF-α (Fig. 6B), IL-6 (Fig. 6C) and iNOS (Fig. 6D), was inhibited by treatment with ketanserin in the BMDMs. The knockdown of 5-HT_2AR by siRNA partly abolished the inhibitory effects of ketanserin on the expression of these pro-inflammatory cytokines in the BMDMs stimulated with LPS plus 5-HT (Fig. 6). In a previous study, it was suggested that the anti-inflammatory effects of ketanserin are partly dependent on the arterial baroreflex (13). In the present study, we found that ketanserin decreased the levels of inflammatory cytokines in BMDMs, suggesting that the anti-inflammatory effects of ketanserin are not entirely dependent on the baroreflex. Moreover, these effects were dimished by transfection with siRNA targeting 5-HT_2AR, thus demonstrating that the inhibitory effects of ketanserin are partly-dependent on 5-HT_2AR.

NF-κB is a critical transcription factor for the inflammatory response (27). It functions as a pro-inflammatory factor and participates in the pathophysiology of IBD (28). Thus, in order to elucidate the mechanisms responsible for the anti-inflammatory effects of ketanserin, we examined its effects on the activation of the NF-κB pathway in BMDMS. Changes in the levels of p-NF-κB p65 in the BMDMs treated with or without ketanserin were evaluated by western blot analysis. As demonstrated in Fig. 6E and F, ketanserin markedly decreased the protein level of p-p65 compared with the BMDMs not treated with ketanserin. These data indicate that 5-HT_2AR/NF-κB may play a role in the anti-inflammatory effects of ketanserin in macrophages.