Activation of the aryl hydrocarbon receptor pathway may ... - Nature

Immunology and Cell Biology (2010) 88, 685–689 & 2010 Australasian Society for Immunology Inc. All rights reserved 0818-9641/10 $32.00 www.nature.com/icb

SHORT COMMUNICATION

Activation of the aryl hydrocarbon receptor pathway may ameliorate dextran sodium sulfate-induced colitis in mice Takeyuki Takamura1, Daisuke Harama1, Shuji Matsuoka2, Naomi Shimokawa1, Yuki Nakamura1, Ko Okumura3, Hideoki Ogawa3, Masanori Kitamura4 and Atsuhito Nakao1,3 The aryl hydrocarbon receptor (AhR) recognizes numerous small xenobiotic and natural molecules, such as dioxin and natural chemicals, and is involved in the metabolism of these compounds. AhR also has a regulatory role in inflammatory responses. This study investigated whether the activation of the AhR pathway affects dextran sodium sulfate (DSS)-induced colitis, an ulcerative colitis-like model, in mice. DSS-induced colitis was ameliorated by pretreatment with a potent AhR activator, 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), in mice. In addition, the mice pretreated with TCDD showed increased prostaglandin E2 (PGE2) production in the colon, and inhibition of PGE2 production by indomethacin abrogated the inhibitory effects of TCDD on DSS-induced colitis. Collectively, the activation of the AhR pathway by TCDD may ameliorate DSS-induced colitis, at least in part, through PGE2 production. Immunology and Cell Biology (2010) 88, 685–689; doi:10.1038/icb.2010.35; published online 16 March 2010 Keywords: aryl hydrocarbon receptor; colitis; mouse; prostaglandin

The aryl hydrocarbon receptor (AhR) recognizes numerous small xenobiotic and natural molecules, such as dioxin and natural chemicals, and is involved in the metabolism of these compounds.1 On ligand binding, AhR in the cytosol translocates to the nucleus, dimerizes with the AhR nuclear translocator, interacts with the xenobiotic responsive element in the promoter regions of target genes and initiates the transcription of the target genes, such as those of the xenobiotic-metabolizing enzymes, for example, cytochrome P450 family 1A1 (CYP1A1).1 Aryl hydrocarbon receptor was first discovered as a mediator of dioxin toxicity, but, at present, the AhR pathway is thought to have diverse roles in many cellular functions including the immune system.2,3 The AhR pathway has been recently shown to be an important regulator of inflammation and immunity.4,5 For instance, Ahr has an essential role in the negative regulation of the lipopolysaccharide (LPS) signaling pathway through interaction with Stat1.6 However, it remains unclear whether the AhR pathway has a role in inflammatory bowel diseases. This study investigated whether activation of the AhR pathway affects inflammatory bowel disease-related experimental colitis. For this purpose, we examined the effects of a potent AhR activator, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the development of dextran sodium sulfate (DSS)-induced colitis in mice.7 This model is

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characterized by epithelial disruption, resulting in luminal bacterial translocation and subsequent infiltration of neutrophils and other acute immune cells.7 These features recapitulate the events that lead to acute mucosal injury of human ulcerative colitis, and the model exhibits many signs which are very similar to those seen in the disease (for example, body weight loss, shortening of the large intestine, mucosal ulceration and increased proinflammatory cytokine production). Although DSS-induced colitis clearly differs from human ulcerative colitis, including the initiating events as well as the clinical course of the disease, it can be considered as an appropriate animal model to investigate the acute phase of the disease pathogenesis.8 RESULTS AND DISCUSSION The single oral administration of TCDD (5 lg kg1) induces the persistent activation of the AhR pathway in the mouse colon without any apparent toxicity The TCDD is a persistent AhR activator with a half-life of B2 weeks in mice.9 We have previously shown using AhR signaling reporter mice that a single oral administration of TCDD (5 mg kg1) induces dramatic, persistent activation of the AhR pathway in mice at least for 3 weeks.9 A strong systemic activation of the AhR pathway is observed at 7 days after the TCDD administration in mice.10 On the basis of that study, mice were pretreated orally with 5 mg kg1 TCDD

of Immunology, University of Yamanashi Faculty of Medicine, Yamanashi, Japan; 2Department of Pathology, Juntendo University School of Medicine, Tokyo, Japan; Research Center, Juntendo University School of Medicine, Tokyo, Japan and 4Department of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan Correspondence: Professor A Nakao, Department of Immunology, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan. E-mail: [email protected] Received 17 January 2010; revised 15 February 2010; accepted 16 February 2010; published online 16 March 2010 3Atopy

AhR activation may inhibit experimental colitis T Takamura et al 686

As expected, the mRNA expression of CYP1A1, a major target gene of the AhR/xenobiotic responsive element pathway,1 increased in the colon of the mice pretreated with TCDD on days 1, 7 and

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or vehicle (day 0) and, 7 days later, colitis was induced by feeding 3% DSS dissolved in drinking water for 7 days (days 7–14); the mice were killed 3 days later for evaluations (day 17) (Figure 1a).

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Figure 1 Activation of the AhR pathway by TCDD may ameliorate DSS-induced colitis in mice. (a) Protocol to determine the effects of TCDD or vehicle on DSS-induced colitis: mice are pretreated with TCDD (orally) on day 0 and, 7 days later, colitis was induced by feeding 3% DSS dissolved in drinking water for 7 days (days 7–14). (b) Real-time PCR for CYP1A1 expression in the colon tissue obtained from the mice pretreated with TCDD on days 1, 7 and 17. (c) Body weight changes in mice with DSS-induced colitis with or without TCDD pretreatment. (d) Colon length in mice with DSS-induced colitis with or without TCDD pretreatment on day 17. (e) Inflammation scores in mice with DSS-induced colitis with or without TCDD pretreatment on day 17. (f) Real-time PCR for heme oxygenase 1 (HO-1), myeloperoxidase (MPO) and tumor necrosis factor-a (TNF-a) expression in the colon tissue obtained from the mice with DSS-induced colitis with or without TCDD pretreatment on day 17. (g) TNF-a levels measured by enzyme-linked immunosorbent assay in the colon tissue obtained from mice with DSS-induced colitis with or without TCDD pretreatment on day 17. Values represent the mean±s.d. (n¼4 per group in b–g). *Po0.05 compared with the corresponding controls. Representative results from three independent experiments are shown (b–g). Immunology and Cell Biology


17 (Figure 1b), thus suggesting the AhR pathway to be activated persistently in the mouse colon during the current experimental protocol. In addition, the mice pretreated orally with 5 mg kg1 TCDD did not show any lethality at least 30 days after the TCDD treatment (data not shown). Furthermore, pretreatment of mice with 5 mg kg1 TCDD did not affect the white blood cell counts, blood chemicals (alanine transaminase and lactate dehydrogenase) and CD4/CD8 T-cell and B-cell populations in the thymus and spleen on day 7 (Supplementary Figure 1a–d). These results suggested that the single oral administration of TCDD (5 mg kg1) to mice induced the persistent activation of the AhR pathway in the colon without any apparent systemic and immunological toxicity.

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Pretreatment of mice with TCDD inhibits DSS-induced colitis We then examined the effects of TCDD (5 mg kg1) on the development of DSS-induced colitis in mice on day 17. The mice pretreated with 5 mg kg1 TCDD showed less body weight loss, colon shrinkage and reduced inflammation in comparison with the control dimethyl sulfoxide-pretreated mice (Figures 1c–e). The expression levels of proinflammatory molecular markers, heme oxygenase-1 (one of the major acute phase proteins elevated in DSS-induced colitis),11 myeloperoxidase (a peroxidase enzyme most abundantly present in neutrophils) and tumor necrosis factor-a (TNF-a) mRNAs in the colon were consistently less in TCDD-pretreated mice than in dimethyl sulfoxide-pretreated mice (Figure 1f). Reduced levels of TNF-a protein in the colon were also observed in the mice pretreated

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TCDD-induced prostaglandin E2 production may contribute to the amelioration of DSS-induced colitis by TCDD Prostaglandin E2 (PGE2) is an important protective factor against injury of the gastrointestinal tract,12 and the blockade of PGE2 production by a nonselective cyclooxygenase 1 and cyclooxygenase 2 inhibitor, indomethacin, exacerbates DSS-induced colitis in mice when it is administered during DSS treatment.13,14 In addition, the activation of AhR induces PGE2 production in certain types of cells, both in vitro and in vivo.15,16 Therefore, the effects of TCDD on PGE2 expression in the mouse colon were examined to investigate the possible mechanisms underlying the inhibitory effects of TCDD on DSS-induced colitis. Mice pretreated with TCDD (5 mg kg1) showed an increase in PGE2 levels in the colon 1 week after the single oral administration (day 7) (Figure 2a). To investigate the roles of the TCDD-induced PGE2 production in the mouse colon, mice were pretreated with TCDD (day 0), together with indomethacin (days 0–3) and, 7 days later, colitis was induced by feeding 3% DSS for 7 days as described earlier (days 7–14). We confirmed that mice pretreated with indomethacin alone (days 0–3) did not show any exacerbation of DSS-induced colitis (Figure 2b). Importantly, simultaneous treatment of indomethacin with TCDD abrogated the inhibitory effects of TCDD on DSS-induced colitis, indicated by inflammation scores,

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with TCDD (Figure 1g). These results suggested that the pretreatment of mice with TCDD ameliorated DSS-induced colitis in mice.

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Figure 2 TCDD-induced PGE2 production may contribute to the amelioration of DSS-induced colitis by TCDD. (a) PGE2 levels measured by enzyme-linked immunosorbent assay in the colon tissue obtained from the mice 1 week after TCDD treatment (protocol day 7). (b) Inflammation scores in mice with DSS-induced colitis with or without TCDD pretreatment (day 0), together with indomethacin (days 0–3) on day 17. (c) Real-time PCR for heme oxygenase 1 (HO-1), myeloperoxidase (MPO), and tumor necrosis factor-a (TNF-a) expression in the colon tissue obtained from the mice with DSS-induced colitis with or without TCDD pretreatment (day 0) simultaneously with indomethacin (days 0–3) on day 17. Values represent the mean±s.d. (n¼4 per group in a–c). *Po0.05 compared with the corresponding controls. Representative results from two independent experiments are shown (a–c). Immunology and Cell Biology


and heme oxygenase-1, myeloperoxidase and TNF-a mRNA expression in the mouse colon (Figures 2b and c). These results suggested that the earlier blockade of TCDD-induced PGE2 production by indomethacin abrogated the suppressive effects of TCDD pretreatment on DSS-induced colitis. Thus, TCDD-induced PGE2 production may contribute to the amelioration of DSS-induced colitis by TCDD. The TCDD is one of the most extensively studied classes of environmental chemicals examined for immunotoxicity in experimental animals and humans.17 For instance, TCDD can cause thymic atrophy, myelotoxicity, inhibition of lymphocyte functions and increased susceptibility to challenge with infectious agents in rodents.17 In this study, we found that the single oral administration of TCDD (5 mg kg1) to mice induced the persistent activation of the AhR pathway in the colon, but the TCDD dose did not induce any apparent systemic and immunological toxicity, as judged by white blood cell counts, thymic and splenic profiles of lymphocyte components, serum levels of liver enzymes and mortality (Supplementary Figure 1). Thus, the inhibition of DSS-induced colitis by TCDD seems not to be associated with immunotoxicity of the xenobiotics. The precise mechanisms by which TCDD inhibits DSS-induced colitis remain to be determined. It is thought that oral administration of DSS induces disruption of intestinal epithelial cells, resulting in luminal bacterial translocation and subsequent infiltration of neutrophils and other acute immune cells, thereby developing colitis in rodents.8 In addition, it is reported that DSS-induced colitis can occur in lymphocyte-deficient mice, suggesting that innate, but not acquired, immune responses have an important role in the development of DSS-induced colitis, in particular, in the acute model.18 Thus, the inhibition of DSS-induced colitis by TCDD may be associated with the protective effects of TCDD on epithelial barrier functions or inhibitory effects of TCDD on innate immune responses. The current findings (Figure 2) suggest that increased intestinal PGE2 production by TCDD and subsequent augmentation of epithelial barrier functions may contribute, at least in part, to the inhibitory effects on DSS-induced colitis in mice. It has been shown that TCDD treatment can affect innate immune responses in rodents.17 For instance, at high doses of TCDD that can mediate its acute toxicity in mice (350–500 mg kg1), TCDD can increase serum TNF-a levels and endotoxin enhances this effect.19,20 In contrast, Kimura et al.6 most recently showed that the activation of endogenous AhR pathway inhibited LPS-induced inflammatory responses in macrophages. Considering the findings by Kimura et al., it is possible that the inhibition of DSS-induced colitis by TCDD may be due to the suppression of LPS-activated macrophage responses. However, we found that TCDD treatment did not affect LPS-induced TNF-a expression in mouse peritoneal macrophages in vitro (Supplementary Figure 2). Therefore, at present, we would think that increased intestinal PGE2 production by TCDD is likely to be more important to the inhibitory effects on DSS-induced colitis in mice than the inhibition of macrophage or other innate immune cell functions by TCDD. The possibility that inhibition of acquired immune responses by TCDD contributes to the suppression of DSS-induced colitis cannot be excluded either. However, as stated above, acute DSS-induced colitis can occur in lymphocyte-deficient mice.18 In addition, Lawrence and Kerkvliet16 previously showed that exposure of TCDD (15 mg kg1) to C57BL/6 mice increased PGE2 production in the peritoneal cavity during the immune responses to allogenic P815 tumor cells, whereas the blockade of PGE2 by indomethacin did not reverse the suppressive effects of TCDD on cytotoxic T lymphocyte responses to P815 tumor cells and antibody production, suggesting Immunology and Cell Biology

that TCDD-induced PGE2 production did not affect acquired immune responses in vivo. Thus, given the efficient effects of indomethacin in our experimental system, we think that suppression of DSS-induced colitis by TCDD is unlikely to be due to the inhibition of acquired immune responses. Several environmental factors have been suggested to activate the AhR pathway, including cigarette smoking.1,21 Interestingly, smoking has been suggested to have beneficial effects on ulcerative colitis, although the mechanisms remain unclear.22,23 Given the current findings, the activation of AhR pathway might explain the preventive effects of cigarette smoke on ulcerative colitis. Actually, we found that repeated exposure of mice to cigarette smoke induced activation of AhR in the mouse colon as well as in the lung, as judged by the induction of CYP1A1 mRNA (Supplementary Figure 3). It will thus be interesting to investigate whether environmental factors influencing ulcerative colitis can affect the intestinal AhR signaling pathway. In summary, this study showed that the earlier activation of the AhR pathway by TCDD inhibited DSS-induced colitis in mice, possibly through PGE2 production. These results suggest that activation of the AhR pathway may have an inhibitory role in DSS-induced colitis in mice, although this concept should be tested in more detail using AhR-deficient mice in future studies. METHODS Mice Female 4- to 6-week-old C57BL/6 mice were purchased from Japan SLC (Tokyo, Japan) and these mice were bred under specific pathogen-free conditions. All animal experiments were approved by the institutional review board of the University of Yamanashi.

Induction of colitis Mice were pretreated orally with either dimethyl sulfoxide or 5 mg kg1 per mouse TCDD (Wako Pure Chemical Co, Osaka, Japan; day 0) (n¼4 per group for each experiment). After 7 days, colitis was induced by feeding 3% DSS dissolved in distilled drinking water (molecular weight 5000; Wako Pure Chemical Co) for 7 days, followed by feeding distilled water for 3 days. For some experiments, indomethacin (Sigma Aldrich Inc, St Louis, MO, USA) was also added to the drinking water at a dose of 4 mg kg1 per day and administered to the mice during protocol days 0–2. This dose of indomethacin was reported to inhibit PGE2 production in mice in vivo.13,24 The degree of inflammation was histologically scored as previously described.25 Briefly, scores of inflammation were 0, no increased inflammatory infiltrates; 1, focal mild inflammation; 2, diffuse mild inflammation; 3, cryptic abscess formation; and 4, diffuse dense inflammation.

Quantitative real-time PCR Quantitative PCR analysis using cDNA from the mouse tissue specimens was performed using the AB7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions, using primers and probes for mouse CYP1A1, heme oxygenase-1, myeloperoxidase, TNF-a and glyceraldehyde-3-phosphate dehydrogenase (Applied Biosystems) as previously described.26 The ratio of each gene to that of glyceraldehyde-3-phosphate dehydrogenase was calculated, and the relative expression levels are shown.

Enzyme-linked immunosorbent assay The amounts of TNF-a or PGE2 in the mouse colon tissue were determined using the mouse TNF-a enzyme-linked immunosorbent assay kits (R&D Inc, Minneapolis, MN, USA) or PGE2 competitive enzyme-linked immunosorbent assay kit (Thermo Scientific Inc, Waltham, MA, USA), respectively, according to the manufacturers’ instructions.

Statistical analysis Values represent the mean±s.d. The statistical analysis was performed using un-paired Student’s t-test. A value of Po0.05 was considered to be significant.


CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank Ms Yuko Ohnuma and Mutsuko Hara for valuable general assistance. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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