1521-0103/352/3/455–461$25.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

http://dx.doi.org/10.1124/jpet.114.217844 J Pharmacol Exp Ther 352:455–461, March 2015

Fexofenadine Regulates Nuclear Factor-kB Signaling and Endoplasmic Reticulum Stress in Intestinal Epithelial Cells and Ameliorates Acute and Chronic Colitis in Mice Seong-Joon Koh, Ji Won Kim, Byeong Gwan Kim, Kook Lae Lee, Jaeyoung Chun, and Joo Sung Kim Department of Internal Medicine, Seoul National University Boramae Hospital (S.-J.K., J.W.K., B.G.K., K.L.L.) and Liver Research Institute (J.C., J.S.K.), Seoul National University College of Medicine, Seoul, Korea Received June 26, 2014; accepted December 18, 2014

Introduction Inflammatory bowel disease (IBD) is defined as chronic and relapsing gut inflammation resulting from ongoing activation of the immune response in the mucosal immune system that is driven by normal luminal flora (Podolsky, 2002). The pathogenesis of IBD involves interplay among various factors, including genetic susceptibility, the external environment, intestinal microflora, and inappropriate immune responses (Baumgart and Carding, 2007). There have been great advances in IBD treatment, such as the use of anti–tumor necrosis factor (TNF)-a agents. However, although anti–TNF-a agents are effective in the treatment of IBD, only one third or less of cases achieve remission, and the therapeutic response of most of these patients diminishes during lifelong disease course, which suggests that a nonrelapsing cure for IBD remains elusive (Billioud et al., 2011). Therefore, new therapeutic agents need to be developed.

This work was supported by Seoul National University Hospital Research Fund [Grant 04-2013-0730]. dx.doi.org/10.1124/jpet.114.217844.

DNA-binding activity. C/EBP homologous protein mRNA expression was enhanced in the presence of TNF-a, and it was dampened by pretreatment of fexofenadine. In addition, the induction of ER stress markers caspase-12 and p-eukaryotic initiation factor 2 (eIF2)-a was significantly suppressed by the pretreatment of fexofenadine. Administration of fexofenadine significantly reduced the severity of DSS-induced murine colitis, as assessed by the disease activity index, colon length, and histology. In addition, the DSS-induced phospho-IkB kinase activation was significantly decreased in fexofenadinepretreated mice. Finally, fexofenadine significantly reduced the severity of colitis and the immunoreactivity of caspase-12 and p-eIF2-a in IL-102/2 mice as compared with controls. These results suggest that fexofenadine is a potential therapeutic agent for the treatment of inflammatory bowel disease.

Intestinal epithelial cells (IECs) lie at the interface between intestinal microbes and lamina propria hematopoietic cells, suggesting that they play a key role in intestinal mucosal homeostasis (Blumberg et al., 2008). The intestinal epithelium consists of a mucosal barrier, which isolates the host from the hostile luminal pathogens presented by intestinal microbes (Kagnoff and Eckmann, 1997). In addition, IECs produce a variety of chemokines, adhesion molecules, and inflammatory mediators when exposed to bacterial products and surface molecules such as lipopolysaccharides (Jung et al., 1995; Jobin et al., 1997). The molecules synthesized during IEC stimulation result from a highly integrated cascade complex that includes signal transduction to the nucleus through the activation of a number of protein kinases and phosphatases. Of the members of this complex cascade, nuclear factor-kB (NF-kB) plays a key role in the regulation of genes associated with cytokine production, epithelial permeability, and cellular apoptosis (Jobin and Sartor, 2000). In addition, therapeutic agents for IBD are known to have an inhibitory effect on NF-kB signaling in IECs (Auphan et al., 1995; Kaiser et al., 1999). This evidence suggests that the

ABBREVIATIONS: CHOP, C/EBP homologous protein; DSS, dextran sulfate sodium; eIF2, eukaryotic initiation factor 2; ER, endoplasmic reticulum; IBD, inflammatory bowel disease; IEC, intestinal epithelial cell; IKK, IkB kinase; IL, interleukin; NF-kB, nuclear factor-kB; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SPF, specific pathogen-free; TNBS, 2,4,6-trinitrobenzene sulfonic acid; TNF, tumor necrosis factor; UPR, unfolded protein response. 455

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ABSTRACT The aim of this study was to evaluate the effect of fexofenadine on intestinal inflammation. HCT116 and COLO205 cells were pretreated with fexofenadine and then stimulated with tumor necrosis factor (TNF)-a. Interleukin (IL)-8 expression was determined by real-time reverse-transcription polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay. DNA-binding activity of nuclear factor-kB was assessed by electrophoretic mobility shift assay. The molecular markers of endoplasmic reticulum (ER) stress were evaluated by Western blot analysis and PCR. In the acute colitis model, mice were given 4% dextran sulfate sodium (DSS) for 5 days with or without fexofenadine. IL-102/2 mice were used to evaluate the effect of fexofenadine on chronic colitis. Fexofenadine significantly inhibited the upregulated expression of IL-8 in HCT116 and COLO205 cells stimulated with TNF-a. Fexofenadine suppressed nuclear factor-kB

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Materials and Methods Cell Culture and Treatments. The human epithelial cells COLO205 (American Type Culture Collection, Rockville, MD) and HCT116 (KCBL; 10247) were used between passages 15 and 30. Cells were grown as described previously (Koh et al., 2011). Fexofenadine (Sigma-Aldrich, St. Louis, MO) and histamine (Sigma-Aldrich) were dissolved in phosphate-buffered saline (PBS). Cells were pretreated with various concentrations of fexofenadine or with PBS and were stimulated with TNF-a. Reverse-Transcription Polymerase Chain Reaction, RealTime Reverse-Transcription Polymerase Chain Reaction, and Enzyme-Linked Immunosorbent Assay. Real-time polymerase chain reaction (PCR) was performed as described previously (Koh et al., 2011). Briefly, 1 mg total cellular RNA was isolated from COLO205 and HCT116 cells using TRIzol (GIBCO, Gaithersburg, MD) and was reverse-transcribed and amplified using the LightCycler 480 DNA SYBR Green I Master system (Roche Applied

Science, Penzberg, Germany) and the LightCycler 480 II system (Roche Diagnostics, Rotkreuz, Switzerland) with specific primers for human interleukin (IL)-8 and b-actin. Amplifications were performed in triplicate, and data were normalized to b-actin levels. Reversetranscription PCR amplification of CHOP gene was performed in a thermal cycler. PCR products were separated on 2% Nusieve agarose gel (FMC Bioproducts, Rockland, ME) and stained with ethidium bromide. The amount of IL-8 secretion in HCT116 cells was measured with enzyme-linked immunosorbent assay kit according to the supplier’s instruction (Invitrogen, Carlsbad, CA). Electrophoretic Mobility Shift Assay. HCT116 cells were harvested, and nuclear extracts were separated as described previously (Koh et al., 2011). After determining protein concentration in the extracts using Bradford assay (Bio-Rad, Hercules, CA), electrophoretic mobility shift assays were done with a commercial kit (Promega, Madison, WI), as described previously (Koh et al., 2011). Immunoblot Assay. HCT116 cells were washed with ice-cold PBS and lysed in 0.5 ml lysis buffer (150 mM NaCl, 20 mM Tris at pH 7.5, 0.1 Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/ml aprotinin), as described previously (Koh et al., 2011). The concentrations of protein in the lysates were determined by using the Bradford assay (Bio-Rad). Fifty micrograms of protein per lane was size-fractionated on a 12% polyacrylamide minigel and transferred to a nitrocellulose membrane (0.45 mm pore size). Anti-IkBa, phospho-IkBa, caspase-12, phospho-eukaryotic initiation factor 2 (eIF2)-a, and anti–b-actin (all from Cell Signaling Technology, Danvers, MA) were used as primary antibodies. Peroxidase-conjugated anti-mouse IgG was used as the secondary antibody. Target proteins were detected with the Luminescent Image Analyzer, LAS 4000 (FujiFilm, Tokyo, Japan). Dextran Sulfate Sodium–Induced Acute Murine Colitis. Specific pathogen-free (SPF) mice (C57BL/6NCrljBgi female mice, 6–7 weeks old) were purchased from Orient (Seongnam, Korea) and housed according to the guidelines approved by the Institutional Animal Care and Use Committee of Seoul National University Hospital. Mice had ad libitum access to water and standard rodent food until they reached the desired age (7–8 weeks) and body weight (20–22 g). Mice were maintained on a 12-hour light/dark cycle under SPF conditions. Dextran sulfate sodium (DSS; MP Biochemical, Irvine, CA; mol. wt.: 35,000–50,000) was used to induce acute colitis, as described previously (Cheon et al., 2006; Koh et al., 2011). Five mice in each group were randomly assigned after they were weighed. Mice assigned to the negative control group received filtered water alone. DSS (4%) was dissolved in drinking water and administered for 5 days. Fexofenadine (i.e., 2 and 10 mg/kg per day) was dissolved in PBS (100 ml) and administered once daily by oral gavage by a researcher, beginning 2 days before DSS administration. Vehicle-treated mice were administered with 100 ml PBS once daily by oral gavage. To assess the unexpected effect of fexofenadine in the murine colitis model, fexofenadine (2 mg/kg per day) was administered without DSS exposure in one group of mice. The disease activity index, body weight, stool consistency, and the presence of bloody stool or blood around the anus were assessed daily by another researcher blinded to the study groups. Mice were euthanized on day 8. Macroscopic and Histologic Evaluation. Postmortem, the entire colon was removed from the cecum to the anus. The gross appearance and colon length were evaluated; the removed tissues were fixed in 10% buffered formalin embedded in paraffin and stained with H&E. A quantitative histologic evaluation was performed by a pathologist who was blind to the group assignment, as described previously (Koh et al., 2011). Briefly, the three parameters, including the severity of intestinal inflammation, the extent of mucosal injury, and crypt damage, were evaluated. Immunohistochemical Staining. Immunohistochemical analysis was performed as previously described (Koh et al., 2011). Briefly, slides were immersed in Tris/EDTA buffer (pH 9.0), heated in a decloaking chamber at 125°C for 3 minutes, and then cooled for

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modulation of NF-kB activity in IECs could be a potential target for IBD treatment. The unfolded protein response (UPR) is an intracellular mechanism to cope with endoplasmic reticulum (ER) stress. ER stress results from any situation that causes the accumulation of misfolded or unfolded proteins within the ER. When misfolded or unfolded proteins accumulate within the ER, UPR signaling is initiated, which leads to the activation of three ER transmembrane proteins, including inositol-requiring enzyme, pancreatic ER kinase, and activating transcription factor-6 (Kaser and Blumberg, 2010). Thereby, the expansion of its protein-folding function leads to attenuation of the accumulation of misfolded or unfolded proteins. However, chronic ER stress leads to the activation of proapoptotic UPR pathway, including the C/EBP homologous protein (CHOP) and c-Jun-N-terminal kinase pathways (Tabas and Ron, 2011). Therefore, persistent ER stress due to chronic environmental stress or ineffective UPR signaling can lead to compromised cellular homeostasis, resulting in apoptosis (Shkoda et al., 2007). Recently, ER stress in IECs has been implicated in the development of IBD. Patients with IBD exhibited increased ER stress in ileal and colonic specimens based on mucosal biopsy (Hu et al., 2007). In addition, a genome-wide association study identified genetic abnormalities in several genes, such as XBP1, AGR2, and ORMDL3, which encode proteins related to ER stress (Zheng et al., 2006; McGovern et al., 2010). Although the precise mechanism remains obscure, IECs may require an appropriate UPRmediated signal cascade to resolve the ER stress in response to a chronic environmental antigen or cytokine exposure. Fexofenadine is an antihistamine agent that has been prescribed for patients with allergic rhinitis, urticaria, and hay fever (Greaves and Tan, 2007). A previous study demonstrated that H1 receptor antagonists, including diphenhydramine and desloratadine, inhibited NF-kB signaling via both H1 receptor–dependent and –independent mechanisms in lung epithelial cells (Roumestan et al., 2008). In addition, fexofenadine inhibited histamine-stimulated NF-kB activity in COS-7 cells (Wu et al., 2004). Although these studies suggest a role for fexofenadine in anti-inflammatory effects, little information is available regarding fexofenadinemediated attenuation of intestinal inflammation and its mechanism. Therefore, the aim of this study was to evaluate the effect of fexofenadine on intestinal inflammation.

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Mann-Whitney U test. P values ,0.05 were considered statistically significant.

Results

10–20 minutes. After addition of 3% hydrogen peroxide, sections were incubated for 10 minutes. After washing with Tris-buffered saline with Tween 20 (pH 7.6), the slides were stained with rabbit polyclonal anti–phospho-IkB kinase (IKK)-a/b antibody (Cell Signaling Technology), anti–caspase-12, and anti–phospho-eIF2-a in an autoimmunostainer (Autostainer 2D; Laboratory Vision, Fremont, CA) for 1 hour at room temperature, according to the manufacturer’s instructions. The stained slides were washed with Tris-buffered saline with Tween 20 three times and incubated with secondary antibody for 30 minutes. After the slides were reacted with streptavidin for 20 minutes, the reaction was visualized by 3,39-diaminobenzidine tetrahydrochloride staining for 5 minutes, and the slides were then counterstained with Meyer’s hematoxylin. For phospho-IKK-a/b immunohistochemistry, each slide was evaluated for immunoreactive intensity on a 0 to 41 scale, as previously described (Koh et al., 2011). Chronic Colitis in IL-10 Knockout Mice. Seven- to eight-weekold SPF male C57BL/6 IL-10 knockout (IL-102/2) mice were obtained from the Center for Animal Resource and Development (Seoul, Korea). Five mice in each group were randomly assigned after they were weighed. Because IL-102/2 mice on a C57BL/6 strain background develop colitis lately and the onset and severity of colitis are variable, we used piroxicam (Sigma-Aldrich)-induced colitis model (Berg et al., 2002). Piroxicam-containing chow was fed to IL-102/2 mice for 10 days at a dose of 200 ppm. IL-102/2 mice were administered daily either vehicle or fexofenadine (5 mg/kg per day) by oral gavage for the next 2 weeks. Postmortem, histopathological analysis was carried out based on a scoring system, as previously described (Berg et al., 2002; Koh et al., 2014). Statistical Analysis. Differences between groups were analyzed based on analysis of variance with Bonferroni correction or the

Fig. 2. Effect of fexofenadine on NF-kB DNA-binding activity in TNF-a– stimulated HCT116 cells. HCT116 cells were pretreated with the indicated concentration of fexofenadine for 18 hours and then stimulated with TNF-a (10 ng/ml) for 30 minutes. NF-kB DNA-binding activity in the nuclear extracts was assessed by electrophoretic mobility shift assay. The data are representative of more than three independent experiments.

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Fig. 1. Effects of fexofenadine on IL-8 expression and protein secretion in HCT116 and COLO 205 cells stimulated with TNF-a. (A) HCT116 cells were pretreated with fexofenadine for 24 hours and then stimulated with TNF-a (50 ng/ml) for 4 hours. IL-8 mRNA expression was measured by real-time reverse-transcription PCR. Levels are normalized to b-actin. Data are expressed as fold change in mRNA transcript levels relative to the unstimulated control (mean 6 S.E.M., n = 5). (B) COLO205 cells were pretreated with the indicated concentration of fexofenadine for 24 hours and then stimulated with TNF-a (50 ng/ml) for 4 hours. IL-8 mRNA expression was measured by real-time reverse-transcription PCR. Levels are normalized to b-actin. Data are expressed as fold change in mRNA transcript levels relative to the unstimulated control (mean 6 S.E.M., n = 5). (C) HCT116 cells were pretreated with fexofenadine for 20 hours and then stimulated with TNF-a (20 ng/ml) for 24 hours. Secretion of IL-8 was measured by enzyme-linked immunosorbent assay. Differences between groups were analyzed based on analysis of variance with Bonferroni correction. *P , 0.05 compared with TNF-a alone.

Fexofenadine Inhibits TNF-a–Induced IL-8 Expression and Secretion in HCT116 and COLO205 Cells. Stimulation of HCT116 cells with TNF-a for 4 hours resulted in an approximately 12-fold increase in IL-8 gene expression compared with unstimulated control cells. However, pretreatment with fexofenadine significantly suppressed the TNF-a– induced expression of IL-8 in a dose-dependent manner (Fig. 1A). This result was confirmed in COLO205 cells (Fig. 1B). In addition, pretreatment with fexofenadine significantly decreased the TNF-a–induced IL-8 protein secretion in HCT116 cells (Fig. 1C). Fexofenadine Suppresses NF-kB DNA-Binding Activity in TNF-a–Stimulated HCT116 Cells. Because the transcription factor NF-kB is a key component of the TNF-a– induced IL-8 gene expression signaling pathway in IECs, we next performed an electrophoretic mobility shift assay to assess whether fexofenadine inhibits TNF-a–mediated NF-kB activity (Yuk et al., 2011). Stimulation of HCT116 cells with TNF-a resulted in strong activation of NF-kB DNA-binding activity. Pretreatment with fexofenadine reduced the NF-kB DNA-binding activity induced by TNF-a stimulation (Fig. 2). Fexofenadine Alleviates TNF-a–Induced ER Stress in IECs. Based on the protective effect of the ER chaperone response in IECs, we performed an in vitro study to evaluate whether fexofenadine could alleviate ER stress in IECs. We found that TNF-a (50 ng/ml) induced ER stress in IECs, as monitored by the expression of ER stress markers, including CHOP, caspase-12, and p-eIF2-a. We confirmed this result using COLO205 and Caco-2 cell lines (data not shown). Pretreatment of fexofenadine significantly reduced CHOP gene expression in HCT116 cells (Fig. 3A). In addition, the induction of the ER stress markers caspase-12 and p-eIF2-a in IECs was significantly reduced by the pretreatment of fexofenadine (Fig. 3B). Histamine Did Not Affect IL-8 Expression and Activate NF-kB Signaling in IECs. Because histamine has been reported to activate NF-kB signaling (Bakker et al., 2001), we evaluated the effect of histamine on NF-kB

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Discussion signaling and IL-8 expression in IECs stimulated with or without TNF-a. As shown in Fig. 4, histamine did not significantly affect the increased expression of IL-8. In addition, histamine did not induce IkBa phosphorylation/ degradation. Fexofenadine Attenuates DSS-Induced Acute Murine Colitis. Based on the in vitro study, we believe that fexofenadine inhibits NF-kB signaling and ER stress, regardless of histamine signaling. Therefore, we conducted an in vivo test to confirm the anti-inflammatory effect of the fexofenadine. Acute colitis was induced by DSS administration for 5 days. Oral administration of fexofenadine (2 and 10 mg/kg per day) significantly attenuated the severity of colitis as evaluated according to body weight reduction, disease activity index, and reduction in colon length (Table 1). Histopathological analysis was performed in a blinded manner using a histologic scoring system. Administration of DSS in the PBS-treated group induced remarkable histologic changes, such as inflammatory cell infiltration, crypt damage, and glandular loss. However, treatment with fexofenadine significantly reduced the histologic damage. Histologic grading also showed that fexofenadine significantly reduced the overall colitis score, compared with the scores of the PBS-treated controls (Fig. 5A). To evaluate the unexpected effect of fexofenadine in the murine colon, we simultaneously administered fexofenadine (2 mg/kg per day) without DSS exposure in a group of mice. Administration of fexofenadine did not induce clinical and histologic changes (data not shown). Fexofenadine Reduces IkB Kinase Activity in Mouse Colonic Epithelium. To confirm the effect of fexofenadine on NF-kB signaling in vivo, we investigated the effect of fexofenadine on IKK activity in the DSS colitis model. As shown in Fig. 5B, administration of DSS remarkably induced IKK activity in the colonic epithelium. However, treatment with fexofenadine significantly attenuated IKK activity in colonic mucosa.

Recently, an interesting case of remission induction was reported in a patient with ulcerative colitis who was treated with a combination of fexofenadine, disodium cromoglycate, and a hypoallergenic amino acid–based formula (Raithel et al., 2007). In addition, a synthetic prodrug of fexofenadine with D-glucosamine attenuated colonic inflammation in 2,4, 6-trinitrobenzene sulfonic acid (TNBS)–induced colitis in a rat model (Dhaneshwar and Gautam, 2012). Although these studies suggest that fexofenadine has an anti-inflammatory effect, its basic mechanism on intestinal inflammation remains obscure. Therefore, we aimed to elucidate the effect of fexofenadine on NF-kB signaling in IECs and performed in vivo experiments using DSS-induced acute colitis and chronic

Fig. 4. Effects of histamine on IL-8 mRNA expression and IkBa phosphorylation/degradation in IECs. (A) HCT116 cells were pretreated with the indicated concentration of histamine for 24 hours and then stimulated with TNF-a (50 ng/ml) for 30 minutes. IL-8 mRNA expression was measured by reverse-transcription PCR. (B) HCT116 cells were pretreated with the indicated concentration of histamine for 24 hours and then stimulated with TNF-a (50 ng/ml) for 30 minutes. Immunoblot analysis for phospho-IkBa, IkBa, and actin was performed. The data are representative of more than three independent experiments.

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Fig. 3. Effect of fexofenadine on ER stress markers in IECs. (A) HCT116 cells were pretreated with the indicated concentration of fexofenadine and then stimulated with TNF-a (50 ng/ml) for 4 hours. CHOP mRNA expression was measured by reverse-transcription PCR. (B) HCT116 cells were pretreated with the indicated concentration of fexofenadine for 24 hours and then stimulated with TNF-a (50 ng/ml) for 60 minutes. Immunoblot analysis for caspase-12 and phospho-eIF2-a, and b-actin was performed. The data are representative of more than three independent experiments.

Fexofenadine Ameliorates the Severity of Chronic Colitis in IL-102/2 Mice. Administration of piroxicam for 10 days resulted in body weight reduction and induced severe colitis. In the PBS-treated group, extracted colons showed severe edemas and ulcerations. However, treatment with fexofenadine attenuated the severity of bowel changes. The histologic analyses showed remarkable changes in PBStreated mice, such as ulceration, infiltration of various inflammatory cells, and tranmural inflammation. In contrast, mice treated with fexofenadine showed reduced mucosal damage and infiltration of inflammatory cells, resulting in significant attenuation of the histopathology score (Fig. 6). Fexofenadine Reduces the Immunoreactivity of Caspase-12 and p-eIF2-a in Mouse Colonic Epithelium. Because a previous study demonstrated that IECs of inflamed IL-102/2 mice reveal activated ER stress (Shkoda et al., 2007), we performed immunohistochemistry using caspase-12 and p-eIF2-a antibody. PBS-treated mice exhibited remarkably increased immunoreactivity of caspase-12 and p-eIF2-a in the colonic epithelium. However, treatment with fexofenadine reduced the immunoreactivity of ER stress markers in colonic mucosa (Fig. 7).

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TABLE 1 Clinical indices of DSS-induced acute murine colitis treated with or without fexofenadine Percent indicates body weight change at day 7 compared with day 0. Data are mean 6 S.D. of five mice. Differences between groups were analyzed based on analysis of variance with Bonferroni correction.

Control DSS + PBS DSS + FEX (2 q.d.) DSS + FEX (10 q.d.) FEX (2 q.d.)

Body Weight Change

Colon Length

% of day 0

cm

101.9 87.2 95.4 95.2 104.5

6 6 6 6 6

0.9 4.3* 3.0** 2.5** 0.6

9.3 6.4 7.8 7.6 8.6

6 6 6 6 6

0.4 0.9* 0.4** 0.6** 0.5

Disease Activity Index

— 2.8 6 0.5* 1.8 6 0.3** 1.8 6 0.5** —

FEX (2 q.d.), fexofenadine 2 mg/kg per day; FEX (10 q.d.), fexofenadine 10 mg/kg per day. *P , 0.05 compared with control group; **P , 0.05 compared with DSS + PBS group.

inhibitory mechanism of fexofenadine on NF-kB signaling in IECs. A previous study reported that fexofenadine reduced the severity of TNBS-induced colitis in rats (Dhaneshwar and Gautam, 2012). TNBS-induced colitis model has been widely used by a number of groups over the past 10 years (Wirtz et al., 2007). It has advantages for identifying proinflammatory cytokines and inflammatory mediators as well as epithelial repair (Han et al., 2009; Pereira-Fantini et al.,

Fig. 5. Histologic evaluations of the colons in the DSS-induced acute murine colitis and immunohistochemical staining for in vitro IKK. (A) Mice were divided into four groups, as follows: control, DSS and vehicle, or DSS and fexofenadine (2 or 10 mg/kg per day), as described in Materials and Methods. The total histologic score was derived from the severity and extent of total inflammation and crypt damage (mean 6 S.D.). The colons of DSS-treated mice showed complete destruction of the epithelial architecture with loss of crypts and epithelial integrity, and intense inflammatory cellular infiltration. Treatment with fexofenadine attenuated the severity of DSS-induced colitis. Results are representative of at least three separate examined sites (magnification, 100). (B) Immunoreactivity index for phospho-IKK-a/b (mean 6 S.D.) and representative colon samples treated with or without fexofenadine of colonic epithelium in DSS-induced murine colitis. Tissue specimens were stained immunohistochemically with anti–phospho-IKK-a/b. DSS exposure resulted in a significant increase in the score for phospho-IKK-a/b staining compared with control mice. However, administration of fexofenadine (2 mg/kg per day) significantly reduced the degree of phospho-IKK-a/b staining in colonic samples (mean 6 S.D.). Differences between groups were analyzed based on Mann-Whitney U test. *P , 0.05 compared with vehicle. Fexofenadine 2 mg, fexofenadine 2 mg/kg per day; Fexofenadine 10 mg, fexofenadine 10 mg/kg per day.

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colitis in a genetically susceptible IL-102/2 mice model. In the present study, fexofenadine inhibited NF-kB signaling and alleviated ER stress markers in IEC cell lines, resulting in a significant reduction in IL-8 expression. In addition, fexofenadine attenuated acute colitis in the DSS-induced mice model, as a result of the inhibition of IKK activity in IECs. These results suggest that fexofenadine reduced the severity of colitis by blocking NF-kB signaling in IECs. To our knowledge, this is the first study to elucidate the direct

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2010). However, TNBS-induced colitis is limited as a model because it is a chemically-induced and self-limiting colitis (Koboziev et al., 2011). In addition, the epithelial insult induced by TNBS rectal administration may be affected by the concentration or duration applied. Moreover, determining the therapeutic efficacy of fexofenadine on intestinal inflammation is critical for clinical applications in patients with IBD. Therefore, we performed in vivo studies using two different animal models. Fexofenadine attenuated acute colitis in the DSS-induced mice model. Subsequently, we used IL-102/2 mice to evaluate the reproducibility in the reduction of intestinal inflammation and to assess the treatment effect of fexofenadine on chronic colitis in genetically engineered mice. Our results showed that fexofenadine significantly reduced the severity of chronic colitis in IL-102/2 mice. Based on the

Fig. 7. Immunohistochemical staining for caspase-12 and p-eIF2-a in colonic tissue of IL-102/2 mice. Tissue specimens were stained immunohistochemically with caspase-12 and p-eIF2-a. PBS-treated mice exhibited remarkably increased immunoreactivity of caspase-12 and p-eIF2-a in the colonic epithelium. However, treatment with fexofenadine reduced the immunoreactivity of ER stress markers in colonic mucosa. Fexofenadine, fexofenadine 5 mg/kg per day.

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Fig. 6. Histologic evaluation of the proximal and distal colons in IL-102/2 mice treated with fexofenadine. (A) Histologic score of the proximal colons with colitis. Mice were treated with vehicle or fexofenadine (5 mg/kg per day), as described in Materials and Methods. Morphologic changes, including ulcerations, loss of crypt, edematous change of submucosa, and massive infiltration of inflammatory cells, were found in the vehicletreated mice. However, fexofenadine remarkably attenuated the severity of colitis. (B) Histologic score and representative photos of the distal colons with colitis. Results are representative of at least three separate examined sites. Differences between groups were analyzed based on Mann-Whitney U test. *P , 0.05 compared with vehicle. Fexofenadine 5 mg, fexofenadine 5 mg/kg per day.

results of these in vitro and in vivo experiments, we believe that fexofenadine is a candidate for clinical trials in the treatment of IBD. Previous studies have indicated that the suppression of NF-kB signaling in IECs plays a central role in the control of intestinal inflammation (Cheon et al., 2008; Koh et al., 2011). More importantly, the mechanism of conventional drugs used for treating IBD, such as corticosteroid and 5-aminosalicylic acid, involves the inhibition of NF-kB signaling in IECs (Auphan et al., 1995; McGovern et al., 2010). Recently, ER stress in IECs was proposed as a cause or consequence of IBD (Kaser and Blumberg, 2010). Interestingly, a previous report demonstrated that an increase in misfolded or unfolded proteins resulted in the activation of NF-kB signaling (Pahl and Baeuerle, 1995). Although the precise mechanism regarding NF-kB activation induced by ER stress remains unclear, ER stress can directly activate the NF-kB pathway through pancreatic ER kinase-eIF2-a–mediated attenuation of the translocation of IkB (Deng et al., 2004). In the present study, NF-kB signaling was inhibited by the pretreatment of fexofenadine. In addition, fexofenadine suppressed ER stress markers, including p-eIF2-a both in vitro and in vivo. These results indicate that fexofenadine attenuated intestinal inflammation not only by directly blocking NF-kB signaling, but also by suppressing ER stress. In addition, a previous study reported that chemical chaperones reduced protein misfolding and attenuated both DSS-induced colitis and IL-10 2 /2 –associated colitis in mice (Cao et al., 2013). Therefore, fexofenadine-mediated attenuation of intestinal inflammation may be associated with the UPR in murine colitis. Histamine has been implicated in the regulation of intestinal inflammation (Raithel et al., 2007). Histamine is known to modulate the maturation and migration of inflammatory cells, such as eosinophils, neutrophils, lymphocytes, and dendritic cells (Bachert, 2002). In addition, histamine has been shown to activate, via the H1 receptor, intracellular transcription factors, such as NF-kB, resulting in increased release of proinflammatory cytokines, chemokines, and adhesion molecules in various cells, including epithelial cells (Bakker et al., 2001). However,

Effects of Fexofenadine in Intestinal Inflammation

Acknowledgments

The authors thank In-Soon Chae and Gah Young Lee for excellent technical assistance. Authorship Contributions

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Address correspondence to: Dr. Joo Sung Kim, Department of Internal Medicine, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul, Korea 110-744. E-mail: [email protected]

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in the present study, histamine did not significantly affect the increased expression of IL-8. In addition, histamine did not induce IkBa phosphorylation/degradation. Based on these results, we believe that fexofenadine attenuated intestinal inflammation by direct inhibition of NF-kB signaling in IECs, regardless of histamine signaling. We think that our study has a couple of limitations. In the present study, we could not provide dose-dependent efficacy of fexofenadine in both two animal models. However, we think that dose dependency was not found in our study because of the following reasons. Fexofenadine reduced intestinal inflammation by NF-kB signaling, regardless of histamine signaling. In addition, the administration of 4% DSS induces acute and very severe intestinal inflammation, which may result in a masking of the dose-dependent efficacy. In addition, different TNF-a concentration and incubation times were chosen for the experiments. However, we think that these differences may occur because molecules synthesized during IEC stimulation, such as TNF-a, result from a highly integrated complex cascade. In conclusion, we found that fexofenadine inhibits the TNFa–induced NF-kB pathway and ER stress in IECs, and attenuates acute murine colitis and chronic colitis in mice, which suggests that fexofenadine is a potential therapeutic agent for IBD.

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