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Piroxicam accelerates development of colitis in T-cell receptor α chain-deficient mice
European Journal of Pharmacology 615 (2009) 241–245
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Immunopharmacology and Inflammation
Piroxicam accelerates development of colitis in T-cell receptor α chain-deficient mice Atsushi Nishiyori, Yasunori Nagakura, Katsuomi Ichikawa ⁎ Discovery Biology Research, Nagoya Laboratories, Pfizer Global Research and Development, Pfizer Inc., Taketoyo, Aichi, Japan
a r t i c l e
i n f o
Article history: Received 15 April 2009 Accepted 7 May 2009 Available online 14 May 2009 Keywords: Inflammatory bowel disease Colitis T-cell receptor α deficient mouse Nonsteroidal anti-inflammatory drug Piroxicam Corticosteroid
a b s t r a c t T-cell receptor α chain (TCR ·α)-deficient mice spontaneously develop colitis that resembles human ulcerative colitis; however, the incidence varies among individuals and takes place lately in the life. We have demonstrated that piroxicam induces colitis in non-colitic TCR · α-deficient mice, but not wild-type mice, within 14 days. The histological features and cytokine profiles were similar to those seen in spontaneous colitis in TCR · α-deficient mice. Dexamethasone prevented piroxicam-induced colitis concurrent with the suppression of interleukin (IL)-1β, IL-17, tumor necrosis factor α and interferon γ. This modified model of colitis could be useful for the evaluation of potential therapeutics for ulcerative colitis. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Inflammatory bowel disease, including ulcerative colitis and Crohn's disease, is a recurrent inflammatory disorder of the gastrointestinal tract (Hanauer, 2006; Crohn et al., 1984). Although the precise etiology of inflammatory bowel disease remains obscure, recent studies have clarified that environment, commensal enteric bacteria, and several human genes influence the incidence of disease through hyperactivation of immune cells within the gastrointestinal tract (Lakatos et al., 2006). Medical therapy of inflammatory bowel disease includes corticosteroids, aminosalicylates, and anti-tumor necrosis factor (TNF) α antibodies (Domenech, 2006; Regueiro et al., 2006). For further research and eventual development of inflammatory bowel disease drugs to be successful, a disease-relevant animal model is essential. Of the previously described inflammatory bowel disease animal models, the majority of the chemical-induced and spontaneous colitis models are Crohn's disease-like, while very few are ulcerative colitis-like (Kawada et al., 2007; Mizoguchi and Mizoguchi, 2008). T cell receptor α chain (TCRα)-deficient mice spontaneously develop chronic colitis that has similar features to human ulcerative colitis, including the pathohistology and cytokine expression profile within the colon (Mombaerts et al., 1993; Iijima et al., 1999; Bhan et al., 2000; Chinene et al., 2006). Thus, the TCRα-deficient mouse model of colitis may be useful as an ulcerative colitis-like model;
⁎ Corresponding author. Present address: Clinical Pharmacology, Clinical Research, Pfizer Global R&D Tokyo Laboratories, Shinjuku Bunka Quint Building, 3-22-7 Yoyogi, Shibuya-ku Tokyo 151-8589, Japan. Tel.: +81 3 5309 7071; fax: +81 3 5309 9841. E-mail address: katsuomi.ichikawa@pfizer.com (K. Ichikawa). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.05.002
however, the spontaneous development of colitis takes more than six months. In addition, the severity of disease is variable (Mizoguchi et al., 1997), limiting the ability to evaluate the efficacy of potential candidates for inflammatory bowel disease therapy. Clinical reports have suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) induce flares of colitis in humans (Bjarnason et al., 1993; Kurahara et al., 2001). NSAIDs induce apoptosis in colonic epithelial cells, allowing luminal bacteria to penetrate the colonic mucosa (Hale et al., 2005). NSAIDs are known to induce the rapid development of colitis in interleukin (IL)-10-deficient mice, a Crohn's disease-like colitis model (Berg et al., 2002). It remains unknown, however, if NSAIDs induce a similar rapid development of colitis in an ulcerative colitis model. Therefore, this study sought to determine if piroxicam accelerates the onset of colitis in TCRα-deficient mice and to characterize piroxicam-induced colitis. 2. Materials and methods 2.1. Animals and housing Male TCR·α-deficient mice on the C57BL/6J background at four-tofive weeks-old were purchased from Jackson Laboratories (Maine, USA). Male five-week-old C57BL/6J mice were purchased from CHARLES RIVER LABORATORIES JAPAN, INC. (Yokohama, Japan). Animals were housed under 12 h light/12 h dark cycles and allowed access to water and normal solid CE-7 food (CLEA Japan, Inc., Tokyo, Japan) ad libitum. Animals acclimated for at least two weeks before experimentation. All animal experiments were approved by the Animal Ethics Committee of the Nagoya Laboratories of Pfizer Japan in agreement with the internal guidelines for animal experiments and in adherence to Pfizer policy.
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2.2. Piroxicam treatment and efficacy of dexamethasone
2.3. Histological analysis of colitis
Deficient and wild-type mice were divided into three and two groups at eight-to-nine weeks-old, respectively. The one group was fed normal solid CE-7 food while the other groups received CE-7 food containing piroxicam (200 ppm) for 14 consecutive days. The remaining group of deficient mice was also treated with dexamethasone. Animals were treated orally with 3 mg/kg dexamethasone (Sigma, St. Luis, USA), suspended in 0.5% methylcellulose solution (Shin-Etsu Chemical Co., Ltd, Tokyo, Japan), or vehicle alone once daily for 14 consecutive days. After treatment the mice were anesthetized with Isoflurane (ABBOTT JAPAN, Japan) and euthanized. Colons were immediately removed, rinsed with sterile saline (Otsuka Pharmaceutical, Japan), and cut longitudinally on a sheet of paper to remove excess water. After being weighed, each colon was longitudinally halved; one piece was fixed in 4% paraformaldehyde (Wako, Japan) for histological analysis, while the other was quickly transferred into liquid nitrogen and stored at − 80 °C until homogenization. CE-7 and piroxicam-containing food were purchased from CLEA Japan, Inc.
Fixed colons were serially dehydrated with increasing concentrations of ethanol (70–100%), immersed in xylane twice, and embedded in paraffin. Sections were prepared at a 3-μm thickness using a microtome. After deparaffinization in xylane and serial hydration with decreasing concentrations of ethanol (100–70%), sections were stained with Hematoxylin and Eosin (Merck, Germany). After rinsing and dehydration, sections were enclosed with Entellan (Merck). Sections were visualized by light microscopy (Olympus, Japan); the digital images were acquired with a CCD camera (Olympus) attached to the microscope. 2.4. Quantification of cytokines in colons Halved colons were homogenized in phosphate-buffered saline (PBS, Invitrogen, USA) containing a protease inhibitor cocktail (Sigma, St. Luis, USA) according to the manufacturer's instructions. Tissues were homogenized with a polytron-homogenizer for 30 s on ice. After centrifugation of homogenates at 12,000 ×g for 15 min, supernatants were collected and stored at −80 °C until measurement. ELISA was performed according to the manufacturer's instructions (IL-12 p70:
Fig. 1. Ratios of colonic weight to length (A) and colonic histological changes (B) in TCRα-deficient and wild-type mice fed normal food (control) or food containing piroxicam for 14 days. Dexamethasone (DEX, 3 mg/kg) or vehicle alone was administered orally to TCRα-deficient mice once daily starting on the first day of piroxicam treatment for 14 days. WT; wild type, KO; TCRα-deficient mice. A) The ratio of colonic weight to length was increased in the piroxicam-treated TCRα-deficient mice in comparison to control TCRαdeficient and piroxicam-treated wild-type mice. Dexamethasone suppressed the increase in this ratio. Columns and error bars represent the means ± S.E.M. of four to seven animals, respectively. Statistical significance was examined by unpaired t-test. ns, not significant, ##P b 0.01, ⁎⁎P b 0.01. B) Representative histological changes (Hematoxyline and Eosin staining) of the middle colons of TCRα-deficient and wild-type mice. Only the colons of piroxicam-treated TCRα-deficient mice manifested overt colitis, with ulcers, the infiltration of leukocytes, mucosal hyperplasia, the loss of crypts, and frequent crypt abscesses. A typical crypt abscess is shown in the inset image. As with untreated wild-type controls, neither piroxicam-treated wild-type mice nor TCRα-deficient mice fed a normal diet exhibited features of colitis. Dexamethasone suppressed the development of histological changes in TCRα-deficient mice. Scale bars indicate 200 μm in all images except the inset, in which the scale bar equals 50 μm.
A. Nishiyori et al. / European Journal of Pharmacology 615 (2009) 241–245
BioSource, California, USA; IL-23, eBioscience, San Diego, USA; IL-1β, IL-4, IL-5, IL-10, IL-17, TNFα, and IFNγ, R&D systems, Minneapolis, USA). Absorbance was measured using a spectrometer (Molecular devices, California, USA); concentrations were calculated using SoftMax Pro version 4.8 software (Molecular devices). 2.5. Statistical analysis Our statistical analyses utilized an unpaired-t test and an unpairedt test with Welch's correction using GraphPad Prism version 4.02 software for Windows (GraphPad software Inc., USA). The analyses varied by individual case.
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3. Results 3.1. Effect of piroxicam treatment on the ratio of colonic weight to length in TCRα-deficient mice We treated TCRα-deficient mice with piroxicam in their food to determine if the onset of colitis is accelerated. After 14 days of piroxicam treatment, TCRα-deficient mice exhibited a significant increase in the ratio of colonic weight to length in comparison to the deficient mice fed normal food (n = 7 piroxicam-group, n = 4 control group, P b 0.01 by unpaired t-test, Fig. 1A). In contrast, there were no significant differences in the ratio of colonic weight to length in wild-
Fig. 2. Cytokine expression profiles of the colons of TCRα-deficient and wild-type mice fed for 14 days with normal food or food containing piroxicam. Dexamethasone (DEX, 3 mg/ kg) or vehicle alone was administered orally to TCRα-deficient mice once daily for 14 days starting on the first day of piroxicam treatment. Piroxicam treatment induced the robust expression of IL-1β, IL-17, TNFα, and IFNγ in TCRα KO mice while the levels of IL-12 p70 and IL-23 were significantly decreased. In contrast, we did not observe any differences in the levels of these cytokines in wild-type mice with or without piroxicam, except for IL-12 p70. The levels of IL-4 and IL-10 were detectable, but unchanged, in all treatment groups (data not shown). IL-5 could not be detected in any of the treatment groups (data not shown). Dexamethasone markedly inhibited the increases seen in IL-1β, IL-17, TNFα, and IFNγ in TCRα KO mice. The columns and bars represent the means ± S.E.M. of four to seven animals, respectively. ##P b 0.01 versus untreated TCRα KO, ⁎P b 0.05 and ⁎⁎P b 0.01 versus the vehicle-treated group by unpaired t-test with Welch's correction. ud; undetected, WT; wild-type, KO; TCRα-deficient mice.
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type mice with or without piroxicam (n = 4, P N 0.05 by unpaired t-test, Fig. 1A). 3.2. Histology of colons of TCRα deficient mice following piroxicam-treatment Following piroxicam treatment, TCRα-deficient mice developed mucosal ulcers, the infiltration of leukocytes into mucosa and submucosa, mucosal hyperplasia, loss of goblet cells, and discontinuous crypt abscesses from the proximal to distal colon; wild-type mice did not manifest any such histological changes (Fig. 1B). Similar histological changes characteristic of colitis were also observed in the colitis that developed spontaneously in TCRα-deficient mice, although the spontaneous colitis affected the proximal to distal colon continuously (data not shown). In contrast, untreated TCRα-deficient and control wild-type mice did not exhibit any inflammation of the colon (Fig. 1B). 3.3. Cytokine profile in colons of TCRα-deficient mice following piroxicam treatment We examined the expression of colonic cytokines, including IL-1β, IL-4, IL-5, IL-10, IL-12 p70, IL-17, IL-23, TNFα, and IFNγ. In the colons of TCRα-deficient mice fed normal food, we observed expression of IL-12 p70 and IL-23, faint expression of IL-1β, IL-4, IL-10, and IL-17, and minimal expression of IL-5 and TNFα (Fig. 2). In the colons of piroxicam-treated TCRα-deficient mice, we detected the robust upregulation of IL-1β, IL-17, TNFα, and IFNγ expression. IL-12 p70 and IL-23 were significantly reduced after piroxicam treatment in TCRα KO mice (Fig. 2). The expression of IL-4, IL-5 and IL-10 remained unchanged (data not shown). In contrast, there were no significant differences in the colonic expression of these cytokines in wild-type mice with or without piroxicam, except for IL-12 p70 (Fig. 2). 3.4. Pharmacological efficacy of dexamethasone in piroxicamaccelerated colitis model of TCRα-deficient mice We addressed the clinical relevance of piroxicam-accelerated colitis as a model of human inflammatory bowel disease by examining the efficacy of dexamethasone, a glucocorticoid, to reverse colonic inflammation. Treatment with dexamethasone (3 mg/kg) for 14 days significantly suppressed the increase in colonic weight per length observed in the vehicle-treated group (n = 6, P b 0.01 by an unpaired t-test, Fig. 1A). The histological inflammatory changes seen in the colon were markedly suppressed by dexamethasone treatment, with only a faint loss of goblet cells, rare infiltration of leukocytes into the mucosa and submucosa, the absence of crypt abscesses, and no mucosal hyperplasia (Fig. 1B). In addition, dexamethasone definitively suppressed the induction of IL-1β, IL-17, TNFα, and IFNγ in the colons of animals treated with vehicle alone, and then normalized the reduction of IL-12 p70 and IL-23 in the colons (Fig. 2). 4. Discussion As the colonic epithelial barrier plays a critical role in defending against the invasion of luminal bacteria into the mucosa, destruction of this barrier allows luminal bacteria to gain access to the colonic lamina propria, resulting in the activation of the mucosal immune response and causing colonic inflammation. NSAIDs, such as piroxicam, inhibit the synthesis of prostaglandins, soluble mediators that function in the maintenance of the epithelial barrier. Piroxicam also directly induces the apoptosis of epithelial cells (Hale et al., 2005). Thus, treatment with NSAIDs weakens mucosal barrier function. IL-10-deficient mice, a Crohn's disease-like model, take several months to develop spontaneous colitis; treatment for two weeks with NSAIDs, such as piroxicam and sulindac, are reported to accelerate the development of colitis to a
clinical condition similar to spontaneous colitis (Berg et al., 2002). Hale et al. (2005) reported that piroxicam treatment facilitates the invasion of luminal bacteria into the colonic mucosa in IL-10-deficient mice, triggering the induction of colitis. It remains unknown, however, if a similar rapid development of colitis can be induced in an ulcerative colitis model. In this study, we demonstrated that treatment with piroxicam for two weeks can induce uniform colitis in TCR ·α-deficient mice. The histological features seen included ulcers, the infiltration of leukocytes into the mucosa and submucosa, mucosal hyperplasia, loss of goblet cells, and crypt abscesses, all ulcerative colitis-like features similar to those seen in the spontaneous colitis that develops in TCR·αdeficient mice. As reported in spontaneous colitis (Mombaerts et al., 1993; Iijima et al., 1999; Bhan et al., 2000; Chinene et al., 2006), we observed increased protein levels of IL-1β, TNFα, and IFNγ in the inflamed colons of piroxicam-treated TCRα-deficient mice, while no increases in IL-4, IL-5, IL-12 p70, or IL-23 production were seen. The observation that IL-12 p70 and IL-23 expression levels were not increased during colitis is consistent with human ulcerative colitis, but not Crohn's disease (Fuss et al., 2006). We also observed a marked increase in IL-17 expression in the inflamed colon of TCR·α-deficient mice treated with piroxicam, consistent with the increased levels seen in spontaneous colitis (Hegazi et al., 2006) and human ulcerative colitis (Fujino et al., 2003). This cytokine has been implicated as a pivotal player in the pathogenesis and/or maintenance of inflammatory bowel disease (reviewed in Zhang et al., 2007). Recent studies have demonstrated that IL-17-secreting Th17 cells play a critical role in experimental autoimmune encephalomyelitis (Ivanov et al., 2006) and in animal models of arthritis (Sato et al., 2006). Th17 cells differentiate under the control of IL-23 (McKenzie et al., 2006), transforming growth factor-β and IL-6 (Bettelli et al., 2006; Veldhoen et al., 2006). Though expression levels of neither IL-12 p70 nor IL-23 increased but rather decreased when other cytokines including IL-17 and IL-1 elevated, not IL-23 itself but rather the expression level of IL-23 receptors may be pivotal for differentiation of Th17 cells because this study demonstrated that IL-23 is originally detected in the colon even in normal mice. This idea may be supported by a report that the identification in inflammatory bowel disease patients of associations in IL-23 receptors and regions that include other genes in the IL-23/ Th17 pathway has highlighted the importance of proper IL-23/Th17 pathway regulation in intestinal immune homeostasis (Abraham and Cho, 2009). It is of interest to address the role of IL-23 and IL-12 p70 in pathogenesis and maintenance of this TCRα-deficient mouse colitis model and human ulcerative colitis, because the present study demonstrated that dexamethasone prevented incidence of colitis and in parallel maintained levels of IL-23 and IL-12 p70. In this study, dexamethasone was able to prevent the development of colitis by suppressing the increases in Th1 and Th17 cytokines in piroxicam-treated TCR·α-deficient mice, although many models of colitis are not responsive to glucocorticoids in contrast to human disease. In summary, we have demonstrated that piroxicam-induced colitis in TCRα-deficient mice has many common characteristics with spontaneous colitis, including similar histological features and cytokine profiles. In addition, the development of colitis is significantly inhibited by dexamethasone. Consequently, piroxicam-induced colitis in TCRαdeficient mice provides a useful ulcerative colitis-like model in which to develop potential candidates for the treatment of inflammatory bowel disease. Acknowledgments The authors thank Drs. Nigel Bunnett (University of California, San Francisco), John Furness (University of Melbourne), and Yvette Taché (University of California, Los Angeles) for their invaluable comments on this study. We acknowledge Dr. Makoto Nagaoka (Pfizer inc.) for the excellent technical support and helpful comments.
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Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Immunopharmacology and Inflammation
Piroxicam accelerates development of colitis in T-cell receptor α chain-deficient mice Atsushi Nishiyori, Yasunori Nagakura, Katsuomi Ichikawa ⁎ Discovery Biology Research, Nagoya Laboratories, Pfizer Global Research and Development, Pfizer Inc., Taketoyo, Aichi, Japan
a r t i c l e
i n f o
Article history: Received 15 April 2009 Accepted 7 May 2009 Available online 14 May 2009 Keywords: Inflammatory bowel disease Colitis T-cell receptor α deficient mouse Nonsteroidal anti-inflammatory drug Piroxicam Corticosteroid
a b s t r a c t T-cell receptor α chain (TCR ·α)-deficient mice spontaneously develop colitis that resembles human ulcerative colitis; however, the incidence varies among individuals and takes place lately in the life. We have demonstrated that piroxicam induces colitis in non-colitic TCR · α-deficient mice, but not wild-type mice, within 14 days. The histological features and cytokine profiles were similar to those seen in spontaneous colitis in TCR · α-deficient mice. Dexamethasone prevented piroxicam-induced colitis concurrent with the suppression of interleukin (IL)-1β, IL-17, tumor necrosis factor α and interferon γ. This modified model of colitis could be useful for the evaluation of potential therapeutics for ulcerative colitis. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Inflammatory bowel disease, including ulcerative colitis and Crohn's disease, is a recurrent inflammatory disorder of the gastrointestinal tract (Hanauer, 2006; Crohn et al., 1984). Although the precise etiology of inflammatory bowel disease remains obscure, recent studies have clarified that environment, commensal enteric bacteria, and several human genes influence the incidence of disease through hyperactivation of immune cells within the gastrointestinal tract (Lakatos et al., 2006). Medical therapy of inflammatory bowel disease includes corticosteroids, aminosalicylates, and anti-tumor necrosis factor (TNF) α antibodies (Domenech, 2006; Regueiro et al., 2006). For further research and eventual development of inflammatory bowel disease drugs to be successful, a disease-relevant animal model is essential. Of the previously described inflammatory bowel disease animal models, the majority of the chemical-induced and spontaneous colitis models are Crohn's disease-like, while very few are ulcerative colitis-like (Kawada et al., 2007; Mizoguchi and Mizoguchi, 2008). T cell receptor α chain (TCRα)-deficient mice spontaneously develop chronic colitis that has similar features to human ulcerative colitis, including the pathohistology and cytokine expression profile within the colon (Mombaerts et al., 1993; Iijima et al., 1999; Bhan et al., 2000; Chinene et al., 2006). Thus, the TCRα-deficient mouse model of colitis may be useful as an ulcerative colitis-like model;
⁎ Corresponding author. Present address: Clinical Pharmacology, Clinical Research, Pfizer Global R&D Tokyo Laboratories, Shinjuku Bunka Quint Building, 3-22-7 Yoyogi, Shibuya-ku Tokyo 151-8589, Japan. Tel.: +81 3 5309 7071; fax: +81 3 5309 9841. E-mail address: katsuomi.ichikawa@pfizer.com (K. Ichikawa). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.05.002
however, the spontaneous development of colitis takes more than six months. In addition, the severity of disease is variable (Mizoguchi et al., 1997), limiting the ability to evaluate the efficacy of potential candidates for inflammatory bowel disease therapy. Clinical reports have suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) induce flares of colitis in humans (Bjarnason et al., 1993; Kurahara et al., 2001). NSAIDs induce apoptosis in colonic epithelial cells, allowing luminal bacteria to penetrate the colonic mucosa (Hale et al., 2005). NSAIDs are known to induce the rapid development of colitis in interleukin (IL)-10-deficient mice, a Crohn's disease-like colitis model (Berg et al., 2002). It remains unknown, however, if NSAIDs induce a similar rapid development of colitis in an ulcerative colitis model. Therefore, this study sought to determine if piroxicam accelerates the onset of colitis in TCRα-deficient mice and to characterize piroxicam-induced colitis. 2. Materials and methods 2.1. Animals and housing Male TCR·α-deficient mice on the C57BL/6J background at four-tofive weeks-old were purchased from Jackson Laboratories (Maine, USA). Male five-week-old C57BL/6J mice were purchased from CHARLES RIVER LABORATORIES JAPAN, INC. (Yokohama, Japan). Animals were housed under 12 h light/12 h dark cycles and allowed access to water and normal solid CE-7 food (CLEA Japan, Inc., Tokyo, Japan) ad libitum. Animals acclimated for at least two weeks before experimentation. All animal experiments were approved by the Animal Ethics Committee of the Nagoya Laboratories of Pfizer Japan in agreement with the internal guidelines for animal experiments and in adherence to Pfizer policy.
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A. Nishiyori et al. / European Journal of Pharmacology 615 (2009) 241–245
2.2. Piroxicam treatment and efficacy of dexamethasone
2.3. Histological analysis of colitis
Deficient and wild-type mice were divided into three and two groups at eight-to-nine weeks-old, respectively. The one group was fed normal solid CE-7 food while the other groups received CE-7 food containing piroxicam (200 ppm) for 14 consecutive days. The remaining group of deficient mice was also treated with dexamethasone. Animals were treated orally with 3 mg/kg dexamethasone (Sigma, St. Luis, USA), suspended in 0.5% methylcellulose solution (Shin-Etsu Chemical Co., Ltd, Tokyo, Japan), or vehicle alone once daily for 14 consecutive days. After treatment the mice were anesthetized with Isoflurane (ABBOTT JAPAN, Japan) and euthanized. Colons were immediately removed, rinsed with sterile saline (Otsuka Pharmaceutical, Japan), and cut longitudinally on a sheet of paper to remove excess water. After being weighed, each colon was longitudinally halved; one piece was fixed in 4% paraformaldehyde (Wako, Japan) for histological analysis, while the other was quickly transferred into liquid nitrogen and stored at − 80 °C until homogenization. CE-7 and piroxicam-containing food were purchased from CLEA Japan, Inc.
Fixed colons were serially dehydrated with increasing concentrations of ethanol (70–100%), immersed in xylane twice, and embedded in paraffin. Sections were prepared at a 3-μm thickness using a microtome. After deparaffinization in xylane and serial hydration with decreasing concentrations of ethanol (100–70%), sections were stained with Hematoxylin and Eosin (Merck, Germany). After rinsing and dehydration, sections were enclosed with Entellan (Merck). Sections were visualized by light microscopy (Olympus, Japan); the digital images were acquired with a CCD camera (Olympus) attached to the microscope. 2.4. Quantification of cytokines in colons Halved colons were homogenized in phosphate-buffered saline (PBS, Invitrogen, USA) containing a protease inhibitor cocktail (Sigma, St. Luis, USA) according to the manufacturer's instructions. Tissues were homogenized with a polytron-homogenizer for 30 s on ice. After centrifugation of homogenates at 12,000 ×g for 15 min, supernatants were collected and stored at −80 °C until measurement. ELISA was performed according to the manufacturer's instructions (IL-12 p70:
Fig. 1. Ratios of colonic weight to length (A) and colonic histological changes (B) in TCRα-deficient and wild-type mice fed normal food (control) or food containing piroxicam for 14 days. Dexamethasone (DEX, 3 mg/kg) or vehicle alone was administered orally to TCRα-deficient mice once daily starting on the first day of piroxicam treatment for 14 days. WT; wild type, KO; TCRα-deficient mice. A) The ratio of colonic weight to length was increased in the piroxicam-treated TCRα-deficient mice in comparison to control TCRαdeficient and piroxicam-treated wild-type mice. Dexamethasone suppressed the increase in this ratio. Columns and error bars represent the means ± S.E.M. of four to seven animals, respectively. Statistical significance was examined by unpaired t-test. ns, not significant, ##P b 0.01, ⁎⁎P b 0.01. B) Representative histological changes (Hematoxyline and Eosin staining) of the middle colons of TCRα-deficient and wild-type mice. Only the colons of piroxicam-treated TCRα-deficient mice manifested overt colitis, with ulcers, the infiltration of leukocytes, mucosal hyperplasia, the loss of crypts, and frequent crypt abscesses. A typical crypt abscess is shown in the inset image. As with untreated wild-type controls, neither piroxicam-treated wild-type mice nor TCRα-deficient mice fed a normal diet exhibited features of colitis. Dexamethasone suppressed the development of histological changes in TCRα-deficient mice. Scale bars indicate 200 μm in all images except the inset, in which the scale bar equals 50 μm.
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BioSource, California, USA; IL-23, eBioscience, San Diego, USA; IL-1β, IL-4, IL-5, IL-10, IL-17, TNFα, and IFNγ, R&D systems, Minneapolis, USA). Absorbance was measured using a spectrometer (Molecular devices, California, USA); concentrations were calculated using SoftMax Pro version 4.8 software (Molecular devices). 2.5. Statistical analysis Our statistical analyses utilized an unpaired-t test and an unpairedt test with Welch's correction using GraphPad Prism version 4.02 software for Windows (GraphPad software Inc., USA). The analyses varied by individual case.
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3. Results 3.1. Effect of piroxicam treatment on the ratio of colonic weight to length in TCRα-deficient mice We treated TCRα-deficient mice with piroxicam in their food to determine if the onset of colitis is accelerated. After 14 days of piroxicam treatment, TCRα-deficient mice exhibited a significant increase in the ratio of colonic weight to length in comparison to the deficient mice fed normal food (n = 7 piroxicam-group, n = 4 control group, P b 0.01 by unpaired t-test, Fig. 1A). In contrast, there were no significant differences in the ratio of colonic weight to length in wild-
Fig. 2. Cytokine expression profiles of the colons of TCRα-deficient and wild-type mice fed for 14 days with normal food or food containing piroxicam. Dexamethasone (DEX, 3 mg/ kg) or vehicle alone was administered orally to TCRα-deficient mice once daily for 14 days starting on the first day of piroxicam treatment. Piroxicam treatment induced the robust expression of IL-1β, IL-17, TNFα, and IFNγ in TCRα KO mice while the levels of IL-12 p70 and IL-23 were significantly decreased. In contrast, we did not observe any differences in the levels of these cytokines in wild-type mice with or without piroxicam, except for IL-12 p70. The levels of IL-4 and IL-10 were detectable, but unchanged, in all treatment groups (data not shown). IL-5 could not be detected in any of the treatment groups (data not shown). Dexamethasone markedly inhibited the increases seen in IL-1β, IL-17, TNFα, and IFNγ in TCRα KO mice. The columns and bars represent the means ± S.E.M. of four to seven animals, respectively. ##P b 0.01 versus untreated TCRα KO, ⁎P b 0.05 and ⁎⁎P b 0.01 versus the vehicle-treated group by unpaired t-test with Welch's correction. ud; undetected, WT; wild-type, KO; TCRα-deficient mice.
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type mice with or without piroxicam (n = 4, P N 0.05 by unpaired t-test, Fig. 1A). 3.2. Histology of colons of TCRα deficient mice following piroxicam-treatment Following piroxicam treatment, TCRα-deficient mice developed mucosal ulcers, the infiltration of leukocytes into mucosa and submucosa, mucosal hyperplasia, loss of goblet cells, and discontinuous crypt abscesses from the proximal to distal colon; wild-type mice did not manifest any such histological changes (Fig. 1B). Similar histological changes characteristic of colitis were also observed in the colitis that developed spontaneously in TCRα-deficient mice, although the spontaneous colitis affected the proximal to distal colon continuously (data not shown). In contrast, untreated TCRα-deficient and control wild-type mice did not exhibit any inflammation of the colon (Fig. 1B). 3.3. Cytokine profile in colons of TCRα-deficient mice following piroxicam treatment We examined the expression of colonic cytokines, including IL-1β, IL-4, IL-5, IL-10, IL-12 p70, IL-17, IL-23, TNFα, and IFNγ. In the colons of TCRα-deficient mice fed normal food, we observed expression of IL-12 p70 and IL-23, faint expression of IL-1β, IL-4, IL-10, and IL-17, and minimal expression of IL-5 and TNFα (Fig. 2). In the colons of piroxicam-treated TCRα-deficient mice, we detected the robust upregulation of IL-1β, IL-17, TNFα, and IFNγ expression. IL-12 p70 and IL-23 were significantly reduced after piroxicam treatment in TCRα KO mice (Fig. 2). The expression of IL-4, IL-5 and IL-10 remained unchanged (data not shown). In contrast, there were no significant differences in the colonic expression of these cytokines in wild-type mice with or without piroxicam, except for IL-12 p70 (Fig. 2). 3.4. Pharmacological efficacy of dexamethasone in piroxicamaccelerated colitis model of TCRα-deficient mice We addressed the clinical relevance of piroxicam-accelerated colitis as a model of human inflammatory bowel disease by examining the efficacy of dexamethasone, a glucocorticoid, to reverse colonic inflammation. Treatment with dexamethasone (3 mg/kg) for 14 days significantly suppressed the increase in colonic weight per length observed in the vehicle-treated group (n = 6, P b 0.01 by an unpaired t-test, Fig. 1A). The histological inflammatory changes seen in the colon were markedly suppressed by dexamethasone treatment, with only a faint loss of goblet cells, rare infiltration of leukocytes into the mucosa and submucosa, the absence of crypt abscesses, and no mucosal hyperplasia (Fig. 1B). In addition, dexamethasone definitively suppressed the induction of IL-1β, IL-17, TNFα, and IFNγ in the colons of animals treated with vehicle alone, and then normalized the reduction of IL-12 p70 and IL-23 in the colons (Fig. 2). 4. Discussion As the colonic epithelial barrier plays a critical role in defending against the invasion of luminal bacteria into the mucosa, destruction of this barrier allows luminal bacteria to gain access to the colonic lamina propria, resulting in the activation of the mucosal immune response and causing colonic inflammation. NSAIDs, such as piroxicam, inhibit the synthesis of prostaglandins, soluble mediators that function in the maintenance of the epithelial barrier. Piroxicam also directly induces the apoptosis of epithelial cells (Hale et al., 2005). Thus, treatment with NSAIDs weakens mucosal barrier function. IL-10-deficient mice, a Crohn's disease-like model, take several months to develop spontaneous colitis; treatment for two weeks with NSAIDs, such as piroxicam and sulindac, are reported to accelerate the development of colitis to a
clinical condition similar to spontaneous colitis (Berg et al., 2002). Hale et al. (2005) reported that piroxicam treatment facilitates the invasion of luminal bacteria into the colonic mucosa in IL-10-deficient mice, triggering the induction of colitis. It remains unknown, however, if a similar rapid development of colitis can be induced in an ulcerative colitis model. In this study, we demonstrated that treatment with piroxicam for two weeks can induce uniform colitis in TCR ·α-deficient mice. The histological features seen included ulcers, the infiltration of leukocytes into the mucosa and submucosa, mucosal hyperplasia, loss of goblet cells, and crypt abscesses, all ulcerative colitis-like features similar to those seen in the spontaneous colitis that develops in TCR·αdeficient mice. As reported in spontaneous colitis (Mombaerts et al., 1993; Iijima et al., 1999; Bhan et al., 2000; Chinene et al., 2006), we observed increased protein levels of IL-1β, TNFα, and IFNγ in the inflamed colons of piroxicam-treated TCRα-deficient mice, while no increases in IL-4, IL-5, IL-12 p70, or IL-23 production were seen. The observation that IL-12 p70 and IL-23 expression levels were not increased during colitis is consistent with human ulcerative colitis, but not Crohn's disease (Fuss et al., 2006). We also observed a marked increase in IL-17 expression in the inflamed colon of TCR·α-deficient mice treated with piroxicam, consistent with the increased levels seen in spontaneous colitis (Hegazi et al., 2006) and human ulcerative colitis (Fujino et al., 2003). This cytokine has been implicated as a pivotal player in the pathogenesis and/or maintenance of inflammatory bowel disease (reviewed in Zhang et al., 2007). Recent studies have demonstrated that IL-17-secreting Th17 cells play a critical role in experimental autoimmune encephalomyelitis (Ivanov et al., 2006) and in animal models of arthritis (Sato et al., 2006). Th17 cells differentiate under the control of IL-23 (McKenzie et al., 2006), transforming growth factor-β and IL-6 (Bettelli et al., 2006; Veldhoen et al., 2006). Though expression levels of neither IL-12 p70 nor IL-23 increased but rather decreased when other cytokines including IL-17 and IL-1 elevated, not IL-23 itself but rather the expression level of IL-23 receptors may be pivotal for differentiation of Th17 cells because this study demonstrated that IL-23 is originally detected in the colon even in normal mice. This idea may be supported by a report that the identification in inflammatory bowel disease patients of associations in IL-23 receptors and regions that include other genes in the IL-23/ Th17 pathway has highlighted the importance of proper IL-23/Th17 pathway regulation in intestinal immune homeostasis (Abraham and Cho, 2009). It is of interest to address the role of IL-23 and IL-12 p70 in pathogenesis and maintenance of this TCRα-deficient mouse colitis model and human ulcerative colitis, because the present study demonstrated that dexamethasone prevented incidence of colitis and in parallel maintained levels of IL-23 and IL-12 p70. In this study, dexamethasone was able to prevent the development of colitis by suppressing the increases in Th1 and Th17 cytokines in piroxicam-treated TCR·α-deficient mice, although many models of colitis are not responsive to glucocorticoids in contrast to human disease. In summary, we have demonstrated that piroxicam-induced colitis in TCRα-deficient mice has many common characteristics with spontaneous colitis, including similar histological features and cytokine profiles. In addition, the development of colitis is significantly inhibited by dexamethasone. Consequently, piroxicam-induced colitis in TCRαdeficient mice provides a useful ulcerative colitis-like model in which to develop potential candidates for the treatment of inflammatory bowel disease. Acknowledgments The authors thank Drs. Nigel Bunnett (University of California, San Francisco), John Furness (University of Melbourne), and Yvette Taché (University of California, Los Angeles) for their invaluable comments on this study. We acknowledge Dr. Makoto Nagaoka (Pfizer inc.) for the excellent technical support and helpful comments.
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