The FASEB Journal • Research Communication
Interleukin-18 is a crucial determinant of vulnerability of the mouse rectum to psychosocial stress Kensei Nishida, Mai Kamizato, Tomoko Kawai, Kiyoshi Masuda, Keiko Takeo, Shigetada Teshima-Kondo, Toshihito Tanahashi, and Kazuhito Rokutan1 Department of Stress Science, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan Psychosocial factors are important determinants of disease manifestations, treatment efficacy, and prognosis of functional and inflammatory bowel disorders. Isolation of C57BL/6J mice from their 4 brothers growing in the same cage reduced goblet cells and MUC2 expression with a peak on day 8 in the rectum, but not in the colon. Gene expression analysis using a whole mouse genome microarray showed that the stress induced a 10-fold larger change in the gene expression in the rectum (722 genes) than in the colon (72 genes). The Ingenuity Pathway Analysis (IPA) application organized the rectum-specific 711 genes into stress response-related pathways. Nuclear factor-!B-related cytokine networks constructed with IPA showed selective up-regulation of interleukin (IL)-18 mRNA expression, which was also confirmed by real-time polymerase chain reaction. The stress produced active forms of caspase 1, IL-18, and a negative regulator for goblet cells, Notch 1, only in the rectum. IL-18-knockout mouse rectum had significantly increased goblet cells and MUC2 mucin, compared with wild-type mouse rectum. The absence of IL-18 completely blocked the stress-induced changes in gene expression and the goblet cell responses in the rectum. Thus, IL-18 may be a crucial determinant for the vulnerability of the rectum to psychosocial stress.— Nishida, K., Kamizato, M., Kawai, T., Masuda, K., Takeo, K., Teshima-Kondo, S., Tanahashi, T., Rokutan, K. Interleukin-18 is a crucial determinant of vulnerability of the mouse rectum to psychosocial stress. FASEB J. 23, 1797–1805 (2009)
ABSTRACT
Key Words: intestinal mucosal barrier ! goblet cells ! MUC2 ! inflammatory cytokines ! Notch 1 Psychosocial stressors activate complex neuroendocrine immune pathways and modify disease activities of inflammatory bowel disease (IBD) (1– 4) and irritable bowel syndrome (IBS) (5). Exposure to various stressors is known to impair the integrity and mucosal barrier of the gastrointestinal tract, at least in part, by decreased production and release of mucins (6, 7). However, the molecular mechanism linking psychosocial stress and impairment of mucosal barrier functions remains to be elucidated. 0892-6638/09/0023-1797 © FASEB
Mucins play a central role in maintaining homeostasis of the intestine through interfacing with food, water, and luminal microorganisms. There are two groups of mucins: membrane-associated and secretory gel-forming mucins (8). MUC2 is the major core polypeptide of membrane-associated mucins in the intestine. Several recent studies have highlighted the important role of MUC2 in defense against inflammation and carcinogenesis in the colon. MUC2 deficiency leads to spontaneous inflammation of the mouse colon and exacerbation of experimental colitis (9). MUC2-knockout mice spontaneously develop colon cancer by 1 yr of age (10). Mice with two noncomplementing missense mutations of the MUC2 gene also spontaneously develop severe colitis in the distal colon in association with a diminished mucus barrier and ER stress due to mucin misfolding (11). IBD is characterized by depleted goblet cells and a reduced mucus layer. Patients with ulcerative colitis (UC) are known to have decreased expression of MUC2 in the colon (12). There is also growing evidence suggesting involvement of cytokines in homeostasis of the colon and in pathogenesis of IBD and IBS (5, 13). A pleiotropic interleukin (IL)-18 is thought to be one of the crucial mediators (14 –18), since it activates various signal pathways, including those engaged in cell proliferation/survival (19, 20). Moreover, our recent report has shown that acute psychological stress stimulates IL-18 secretion from the adrenal gland, which is indispensable to subsequent production of cytokines, including IL-6 (21). These findings led us to speculate that IL-18 may play a pivotal role in the regulation of mucosal barrier functions under psychosocially stressful conditions. Using mice exposed to isolation stress, we show here that goblet cells and MUC2 mucin production in the rectum is extremely sensitive to psychosocial stress. Thus, IL-18 may be one of the crucial determinants for the vulnerability of the rectum. 1
Correspondence: Department of Stress Science, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan. E-mail:
[email protected] doi: 10.1096/fj.08-125005 1797
MATERIALS AND METHODS Animals and isolation stress All mice were treated in accordance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all procedures were approved by the Animal Care Committee of the University of Tokushima. C57BL/6J and IL-18-knockout (IL-18!/!) mice (C57BL/6 background) were purchased from Nippon CLEA (Shizuoka, Japan) and Jackson Laboratories (Bar Harbor, ME, USA), respectively. Mice were maintained under an artificial 12-h dark-light cycle (lights on at 8:30 AM) at a constant temperature of 23 " 2°C and 65% humidity under specific pathogen-free conditions. Offspring born in our animal center were used in this study. Five male littermates were separated from their mother at 4 wk of age and housed in a cage (225#338#140 mm). At 10 wk of age, 2 mice were sacrificed to use as controls, and each of the remaining 3 mice was housed in an independent cage (225#338#140 mm) for up to 16 days. To avoid nonspecific effects of handling, adequate amounts of laboratory chow (Oriental Yeast, Tokyo, Japan) were given on the starting day of experiments and on day 8, and an automatic water supply system was used. Food consumption and body weight were measured on days 8 and 16. These mice were sacrificed after anesthesia with diethyl ether at 10:00 AM on the indicated days. Histological examinations
Gene expression analysis Mucosal tissues from the colon (middle portion, between 2.5 and 3.5 cm from the ileocecum) and the rectum (7 mm total length from the anus) prepared from wild-type and IL-18!/! mice were immediately incubated with the RNAlater stabilization reagent (Ambion, Austin, TX, USA) for 24 h and stored at !80°C until analysis. Total RNA was isolated using the RNeasy Mini kit (Qiagen, Hiden, Germany). The quality of the purified RNA was assessed by Agilent 2100 Bioanalyzer using an RNA 6000 Nano Labchip kit (Agilent Technologies, Palo Alto, CA, USA). An equal amount of RNA purified from 5 mice of each group was pooled. Synthesis, amplification, and labeling of complementary RNA (cRNA) with Cy3 dye were done according to the manufacturer’s protocols. Prepared cRNA was applied to the whole mouse genome oligo DNA microarray (4#44k; Agilent). Hybridization was performed at 62°C for 12 h. After washing, fluorescence intensity at each spot was assayed using a scanner (G2565BA Microarray Scanner; Agilent). Signal intensities of Cy3 were quantiVol. 23
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Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was prepared from colorectal tissues using the ISOGEN reagent (Nippon Gene, Tokyo, Japan). Contaminating DNA was removed with deoxyribonuclease (Ambion). Complementary DNA was synthesized using 2 $g of RNA and the PrimeScript RTase Synthesis kit (Takara, Tokyo, Japan). qRT-PCR was performed by the ABI 7500 instrument using specific primer and probe sets for the SYBR Green or TaqMan Probe Detection assay (Applied Biosystems, Foster City, CA, USA) (Supplemental Table S1). Ribosomal 18S RNA was used as an endogenous quantity control. Western blot analysis
Colon and rectum tissues were fixed with phosphate-buffered formalin, embedded in paraffin, and cut into 4-$m serial sections. These sections were stained with hematoxylin and eosin (H&E) or Alcian blue. For immunohistochemistry, endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide in 80% methanol for 30 min at 25°C. After blocking nonspecific binding sites with 4% purified milk casein, the sections were incubated overnight at 4°C with a 1:50 dilution of an antibody against mouse MUC2 (sc-13312; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a 1:100 dilution of an antibody against mouse trefoil factor 3 (TFF3) (sc-18273; Santa Cruz Biotechnology) in phosphatebuffered saline containing 1% purified milk casein and 0.01% (vol/vol) Triton X-100. Bound antibodies were detected using diaminobenzidine and an avidin-biotin horseradish peroxidase kit (Vector Laboratories, Burlingame, CA, USA), and then the sections were counterstained with hematoxylin.
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fied and analyzed by subtracting backgrounds using Feature Extraction 9.5 software (Agilent). We selected 18,247 genes having fluorescence intensities higher than a cutoff value of 100 among all samples. These data were normalized by GeneSpring 7.3 software (Agilent). The Ingenuity Pathway Analysis (IPA) application (Ingenuity Systems, Mountain View, CA, USA) was used to organize differentially expressed genes into networks of interacting genes and to identify modules of functionally related genes that correspond to pathways (22). A detailed description is given in the online repository (http://www.ingenuity.com).
Colorectal mucosa was homogenized in ice-cold lysis buffer, pH 7.4, consisting of 50 mM Tris-HCl, 150 mM NaCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and a protease inhibitor mixture (Roche, Indianapolis, IN, USA). Supernatants (25 $g protein/lane) obtained by centrifugation of the homogenates at 15,000 g for 15 min at 4°C were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a 12% (for IL-18) or a 5–10% gradient polyacrylamide gel (for MUC2) and transferred onto a polyvinylidene difluoride membrane. A 1:1000 diluted antibody against mouse IL-18 (MBL, Nagoya, Japan) (21), MUC2 (Santa Cruz Biotechnology), caspase 1 (Santa Cruz Biotechnology), or cleaved Notch 1 (Val1744; Cell Signaling Technology, Danvers, MA, USA) was used to measure the amounts of respective proteins. For O-glycosidase treatment, colonic mucosa from a wild-type mouse was homogenized in ice-cold sodium phosphate buffer, pH 6.0, containing 0.5% Nonidet P-40 and a protease inhibitor mixture (Roche). Supernatants (2 mg protein/ml), obtained by centrifugation of the homogenates at 15,000 g for 15 min at 4°C, were treated with 1 mU/ml O-glycosidase (Roche) for 12 h at 37°C. After centrifugation at 15,000 g for 5 min at 4°C, MUC2 mucin fragments were assessed by Western blot analysis with the anti-MUC2 antibody. After bound antibodies were detected with an enhanced chemiluminescence Western blot analysis detection kit (Amersham Pharmacia, Piscataway, NJ, USA), the polyvinylidene difluoride membrane was reblotted using an anti-%-actin antibody (Abcam, Cambridge, MA, USA), for a loading control (23). Measurement of plasma IL-18 levels Plasma IL-18 levels were measured using the ELISA kit (MBL, Nagoya, Japan), according to the manufacturer’s protocol.
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RESULTS Effects of stress on goblet cells and MUC2 expression The lack of physical contact with socially reared counterparts by postweaning isolation generates a range of behavioral and physiological reactions in rodents, which are known to affect the emotional reactivity for several weeks or longer periods depending on the study protocols (24 –26). In our conditions, isolation reduced food intake by &20% (data not shown) and caused body weight loss (&1.2%) during the 16 days (Supplemental Fig. S1A) without significant increases in plasma adrenocorticotropic hormone (ACTH) (Supplemental Fig. S1B) and corticosterone (Supplemental Fig. S1C) levels. The isolated mice did not show any signs of sickness. Even under this mild stress, MUC2 mRNA levels started to decline on day 4 and remained consistently reduced on days 8 and 16 in the rectum (Supplemental Fig. S1D). We also examined a time course of rectal and colonic MUC2 mRNA expression over a period of 10 to 16 wk of age and confirmed that there was no age-dependent change in the expression (data not shown). Although MUC2 mRNA levels in the rectum were decreased to around 50% of control levels on day 8 (Fig. 1A), mRNA level of another goblet cell product, TFF3, was maintained (Fig. 1B), and mRNA expression of an absorptive cell marker of the colon (CA1, carbonic anhydrase 1) (27) was rather significantly increased (Fig. 1C) in the rectum. In contrast, expression
of all these transcripts was up-regulated 2- to 4-fold in the colon (Fig. 1A–C). Isolation stress did not cause any manifested infiltration of inflammatory cells in lamina propria (Fig. 1D, E), and myeloperoxidase mRNA was not detected in the colon and the rectum (data not shown). However, H&E and Alcian blue stains showed that isolation stress decreased goblet cells in the rectum, particularly in the upper half of rectal glands (Fig. 1E). Consequently, isolation selectively reduced the percentage of Alcian blue-positive cells in total glandular epithelial cells of the rectum (Fig. 1F). Immunoblotting showed that the anti-MUC2 antibody recognized 200- and 250-kDa proteins (Fig. 2A). O-Glycosidase treatment decomposed these proteins and concomitantly increased MUC2-containing, deglycosylated, and autocatalytically cleaved products with lower molecular masses (Fig. 2A, a and b) (28). On the basis of these findings, we assessed MUC2 mucin levels by measuring the amounts of the 200- and 250-kDa proteins. Consistent with qRT-PCR and histological findings, isolation stress increased MUC2 mucin contents in the colon, whereas it decreased the contents in the rectum (Fig. 2B, C). These results suggest that the mouse rectum may be highly vulnerable to psychosocial stress. Gene expression signatures in the rectum before and after isolation Reflecting the anatomical and functional differences, 1840 genes were differentially expressed between the
Figure 1. Effects of isolation stress on goblet cells and expression of MUC2, TFF3, and carbonic anhydrase type 1 (CA1) mRNAs in the colon and the rectum. A–C) Before (control; open column) and 8 days after isolation (solid column), total RNA was extracted from the colon (middle portion, between 2.5 and 3.5 cm from the ileocecum) or the rectum. MUC2 (A), TFF3 (B), and CA1 (C) mRNA levels were measured by qRT-PCR and normalized to the amount of ribosomal 18S RNA. Values are means " sd, n ' 6. *P ( 0.05; ANOVA and Bonferroni test. D, E) Before and after stress, colonic (D) and rectal (E) mucosas were subjected to H&E (a, b) or Alcian blue staining (c, d). Scale bars ' 100 $m. F) Percentage of Alcian blue-positive cells in total glandular epithelial cells per crypt was calculated by counting at least 20 crypts. Values are means " sd, n ' 20. *P ( 0.05; ANOVA and Bonferroni test. PSYCHOSOCIAL STRESS RESPONSE OF THE RECTUM
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were not significantly modified. It was of interest to note that IL-18 mRNA was selectively up-regulated in the rectum (Fig. 4A). An equal amount of RNA purified from 5 mice of each group was mixed and used for microarray analysis. Considering individual variations, we selected differentially expressed genes whose mRNA levels differed by ,2-fold. However, the microarray data did not exclude the possibility that expression of the other cytokine genes might be significantly changed by (2-fold. qRTPCR showed that the mouse rectum constitutively expressed a higher amount of IL-18 mRNA than the colon and validated that isolation stress further increased its mRNA levels &2-fold in the rectum, but not in the colon (Fig. 4B). In contrast, qRT-PCR also confirmed that isolation did not significantly change mRNA levels of TNF-*, IFN+, IL-1%, and IL-13 in the colon and the rectum (Fig. 4C–F). IL-6 and IL-4 mRNA were not detected in our experimental conditions. Presence of an active form of IL-18 in the rectum after isolation Figure 2. Western blot analysis of MUC2 mucin. A) Soluble proteins of colonic mucosa were prepared from wild-type mice and subjected to immunoblot analysis using an antiMUC2 antibody. The antibody recognized 200- and 250-kDa proteins (arrowheads). Treatment with O-glycosidase decomposed these immunoreactive bands and concomitantly increased the low-molecular-mass fragments (a, b). B) Amounts of 200- and 250-kDa proteins in colonic and the rectal mucosas before and 8 days after isolation were measured using %-actin as loading control. Data are representative results from 8 mice/group. C) Data were quantitated by densitometry and expressed as fold changes, compared with those of unstressed control samples. Values are means " sd; n ' 8. *P ( 0.05; ANOVA and Bonferroni test. MW, molecular mass.
colon and the rectum of unstressed wild-type mice. Analysis of the 1840 genes with IPA indicated that the rectum was likely to have altered activities of 6 biofunctional pathways: cancer (P'3.25E-16), inflammatory disease (P'7.83E-10), gastrointestinal disease (P'1.73E-06), organismal injury and abnormalities (P'1.08E-05), infectious disease (P'1.64E-03), and immunological disease (P'2.59E-03) (Supplemental Fig. S2). More important, isolation stress changed 10 times more transcripts in the rectum (722 genes), compared with the colon (72 genes) (Fig. 3A). Expression of only 11 genes was commonly modified in both the rectum and the colon. IPA showed that the 711 rectum-specific stress-responsive genes significantly modified stress-associated canonical pathways (Fig. 3B). In contrast, the 61 colon-specific stress-responsive genes did not form any of these canonical pathways. IPA also revealed that the 711 rectum-specific stressresponsive genes formed a proinflammatory cytokine network; mRNA levels of many genes linked to nuclear factor-)B (NF-)B) were modified (Fig. 4A). Among the key cytokines included, mRNA levels of tumor necrosis factor * (TNF-*), interferon-+ (IFN+), IL-1%, and IL-6 1800
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IL-18 is produced as a 24-kDa precursor that is processed to an 18-kDa bioactive form by caspase 1. As
Figure 3. Venn diagram and functional analysis for a data set of differentially expressed genes after isolation stress. RNA was prepared from the colon (middle) and rectum of mice before and 8 days after isolation stress and subjected to microarray analysis. A) Isolation stress altered expression of 72 and 722 genes by ,2-fold in the colon and rectum, respectively. Eleven genes overlapped. B) Canonical pathway analysis with the IPA application shows that the 711 rectumspecific stress-responsive genes significantly modified the listed pathways by Fisher’s exact test. Threshold, P ' 0.05.
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Figure 4. Isolation stress-induced changes in proinflammatory cytokine networks. A) Using the 711 genes identified as rectum-specific stress-responsive genes, proinflammatory cytokine networks were formed with the IPA application. Genes up-regulated and down-regulated by 2-fold are red and green, respectively. B–F) Levels of IL-18 (B), tumor necrosis factor * (TNF-*) (C), interferon-+ (IFN+) (D), IL-1% (E), and IL-13 (F) mRNAs were measured by qRT-PCR using ribosomal 18S RNA as an endogenous quantity control. Values are means " sd; n ' 6. *P ( 0.05; ANOVA and Bonferroni test.
shown in Fig. 5A, B, the rectum constitutively possessed a higher amount of the immature 24-kDa IL-18 than the colon. Isolation stress significantly increased this 24-kDa IL-18, and at the same time, the 18-kDa active form could be detected only in the rectum (Fig. 5A, B). In association with the appearance of the active IL-18, caspase 1 was also cleaved into active forms only in the rectum (Fig. 5C). We also confirmed that isolation did not change plasma IL-18 levels during the initial 8 days (Fig. 5D).
Absence of stress responses in the IL-18"/" mouse rectum The potential role of IL-18 in the stress response of the rectum was examined using IL-18!/! mice. The IL18!/! mouse rectum constitutively increased Alcian blue-positive, MUC2-expressing, or TFF3-expressing cells (Fig. 6A) and the percentage of goblet cells (Fig. 6E), in association with 2-fold increases in MUC2 mucin (Fig. 6B) and MUC2 mRNA (Fig. 6C) levels. The entire
Figure 5. Effects of isolation stress on IL-18 levels. A) Levels of IL-18 proteins in colonic and rectal mucosas of unstressed or stressed wild-type mice were measured by immunoblot analysis with an anti-IL-18 antibody, using %-actin as a loading control. A mature recombinant mouse IL-18 (rIL-18) and soluble proteins from macrophages (m-) activated by lipopolysaccharide were used as positive controls. Soluble colonic proteins from IL-18!/! mouse were used as a negative control. B) Data were quantitated by densitometry and expressed as fold changes and then compared with those of unstressed respective tissues. Values are means " sd; n ' 8. *P ( 0.05; ANOVA and Bonferroni test. C) Levels of caspase 1 were measured by immunoblotting with an anti-caspase 1 antibody. Soluble proteins were prepared from the rectal mucosa of wild-type mice exposed to restraint stress and used as a positive control (lane 5). Same sample was used to check specificity of the antibody after preabsorption test using a 50-M excess of the synthetic polypeptide antigen (Ag) (lane 6). Arrows indicate procaspase 1 and cleaved caspase 1 proteins with molecular masses of 20 and 10 kDa. D) Blood was collected in EDTA-containing tubes by heart puncture under anesthesia with diethyl ether before (0) and on the indicated days after isolation. Plasma IL-18 levels were measured by ELISA. Values are means " sd; n ' 6. Cont., control; Iso., isolation; MW, molecular mass. PSYCHOSOCIAL STRESS RESPONSE OF THE RECTUM
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Figure 6. Goblet cells and expression of MUC2 and TFF3 in IL-18!/! mice. A) Colon (middle) and rectum of wild-type or IL-18!/! mice were subjected to Alcian blue stain or immunohistochemistry with an antibody against MUC2 or TFF3. Images are representative sections from 6 animals. Scale bars ' 100 $m. B) Levels of MUC2 mucin in the middle colon and rectum of wild-type (open column) and IL-18!/! (gray column) mice were measured as in Fig. 2, and amount of MUC2 mucin was quantitated by densitometry. C, D) MUC2 (C) and TFF3 (D) mRNA levels in the colon (proximal, middle, and distal portions) and rectum of unstressed wild-type (open column), unstressed IL-18!/! (gray column), or stressed IL-18!/! mice (solid column) were measured by qRT-PCR as in Fig. 1. E) Percentage of Alcian blue-positive cells in total glandular epithelial cells per crypt in the middle colon and the rectum was calculated. Values are means " sd; n ' 6. *P ( 0.05; ANOVA and Bonferroni test. MW, molecular mass.
colon of IL-18!/! mice also increased MUC2 mucin (Fig. 6B), as well as MUC2 (Fig. 6C) and TFF3 mRNA (Fig. 6D) levels without changing the goblet cell population (Fig. 6E). More interestingly, isolation stress did not modify the MUC2 mRNA expression in the rectum or in the colon of IL-18!/! mice (Fig. 6C) and did not cause depletion of goblet cells in the rectum (Fig. 6E). Deficiency of IL-18 constitutively changed expression of 230 and 88 genes in the rectum and colon, respectively (Supplemental Fig. S3). qRT-PCR (Fig. 7) as well as microarray analysis demonstrated that the IL-18!/! mouse rectum up-regulated genes encoding goblet cell differentiation factors, including atonal homologue 1 (MATH1) (29), Krueppel-like factor 4 (KLF4) (30), E74-like factor 3 (ELF3) (31), and caudal type homeobox 2 (CDX2) (32) (Fig. 7A–D) and down-regulated a gene encoding its suppressor (NOTCH1) (Fig. 7E) (33–35). It should be noted that the absence of IL-18 almost completely blocked the stress-induced change in gene expression in the rectum: isolation modified expression of only 52 genes in the IL-18!/! mouse rectum (Supplemental Fig. S3A). Among the differentiation factors, only NOTCH1 mRNA expression was significantly up-regulated in the 1802
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rectum in response to the stress (Fig. 7E). This gene was included in the NF-)B network (Fig. 4A). Western blot analysis using the antibody against cleaved Notch 1 showed that the Notch intracellular domain (NICD) was detected only in the wild-type mouse rectum after the stress (Fig. 7F, left), which was absent in IL-18!/! mice (Fig. 7F, right). DISCUSSION Psychosocial factors are important determinants of disease manifestations, treatment efficacy, and prognosis of IBD and IBS (1–5). The isolation stress used in this study may be one of the appropriate models for studying the colorectal response to psychosocially stressful events. This model was mild, but was enough to disclose the differences between the colon and the rectum in sensitivity and vulnerability to the psychosocial stress. In response to isolation stress, colonic mucosa increased mucosal defense factors, MUC2 and TFF3, whereas rectal mucosa mitigated goblet cell-dependent barrier functions. We also examined whether different types of psychological stress pro-
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Figure 7. Expression of goblet cell growth/ differentiation regulators in the colorectum of wild-type and IL-18!/! mice before or after isolation. Total RNA was prepared from colonic and rectal mucosas of wild-type and IL-18!/! mice before and 8 days after isolation. A–E) Levels of atonal homologue 1 (MATH1) (A), Krueppel-like factor 4 (KLF4) (B), E74-like factor 3 (ELF3) (C), caudal type homeobox 2 (CDX2) (D), and NOTCH1 (E) mRNAs were measured by qRT-PCR using specific primer sets for the SYBR Green Detection assay (see Supplemental Table S1) using ribosomal 18S RNA as an endogenous quantity control. Values are means " sd, n ' 6. *P ( 0.05; ANOVA and Bonferroni test. F) Appearance of the Notch intracellular domain (NICD) in the rectum and colon of wild-type (left) or IL-18!/! mice (right) before and 8 days after isolation were measured by Western blot analysis using an antibody against cleaved Notch 1 (Val1744). Cont., control; Iso., isolation.
duced similar colorectal responses. For this purpose, C57BL/6J mice were exposed to immobilization stress at 10 wk of age (see Supplemental Fig. S4) (21). The immobilization stress rapidly increased plasma ACTH, corticosterone, and IL-18 levels. Even under the relatively more severe conditions, the colon significantly increased MUC2, TFF3, and CA1 mRNA levels, while the rectum selectively reduced goblet cells and MUC2 mucin in association with selective up-regulation of IL-18 mRNA expression (Supplemental Fig. S4). Thus, the immobilization stress model also suggests a crucial role of IL-18 in the vulnerability of goblet cell-dependent mucosal barrier of the rectum. Reflecting the different embryological origins (midgut and hindgut), the colon (middle portion) and the rectum have anatomical differences, including circulatory, neuronal, and lymphatic systems. The rectum is exposed to higher concentrations of luminal microflora or irritant residues than the colon. It is known that the rectum and the colon have different mucosal immune system and metabolism. The stress-induced barrier dysfunction of the rectum might be important from a clinical point of view. About 30% of total colorectal cancers develop in the rectum (36). In the case of UC, inflammation occasionally originates from the rectum and subsequently spreads over the colon. Further, MUC2 mucin was reduced in the colonic mucosa of UC patients (12, 37). These clinical observations also suggest that the rectum may be highly sensitive to various environmental stressors. PSYCHOSOCIAL STRESS RESPONSE OF THE RECTUM
From microarray data, expression of 1840 genes in total was significantly changed in the rectum, compared with that in the colon. The biofunctional analysis of the differentially expressed genes suggest that the rectum may have different activities of distinct pathways related to cancer, inflammatory disease, gastrointestinal disease, organismal injury and abnormality, immunological disease, and infectious disease. In fact, isolation stress altered expression of 10 times more genes in the rectum, compared with that in the colon. It was possible that the change in cell composition (loss of goblet cells) caused the gene expression changes. However, IPA revealed that the listed canonical pathways significantly modified by the rectum-specific, stress-responsive genes (711 genes) were associated with stress responses. Thus, isolation stress was likely to profoundly affect expression of stress-responsive genes in association with the depletion of goblet cells. Psychological stress produces the complex and overlapped neural circuitry in the enteric and central nervous system; therefore, several mechanisms, including the corticotropin-releasing hormone receptors (CRHRs) and their ligands (38), mast cell-derived factors (39), reactive nitrogen and oxygen species, and possibly others, are involved in the stress-initiated manifestations of the rectum. Because we could not detect any significant changes in gene expression related to CRHRs and mast cell factors in our model, we focused on proinflammatory cytokine networks connected to NF-)B activation and found that IL-18 mRNA expression was selectively up-regulated in the rectum. The mouse rectum consti1803
tutively expressed higher amounts of IL-18 mRNA and its protein than the colon. Only the rectum responded to the stress, increased IL-18 mRNA and immature IL-18 levels, and produced the bioactive forms of caspase 1 and IL-18. IL-18!/! mice increased MUC2 and TFF3 mRNA levels &2-fold in the entire colon and the rectum, compared with wild-type mice. Although IL-18!/! mice did not change the percentage of goblet cells in colonic glands, the goblet cell population in rectal glands was increased to levels equivalent to that in colonic glands. These results suggest a potential role of IL-18 as a negative regulator of goblet cells. This idea was also supported by the findings that the absence of IL-18 up-regulated genes encoding positive regulators for growth/differentiation of the goblet cell lineage such as MATH1 (29), KLF4 (30), and ELF3 (31), while it down-regulated expression of a negative regulator gene, NOTCH1 (33–35). Among them, Notch 1 may be one of the crucial factors involved in the stress-induced decline of goblet cells, since cleaved, bioactive Notch 1 was specifically produced in the rectum of the stressed wild-type mice. Notch 1 has been suggested to regulate the proliferation, cell fate specification, and differentiation of intestinal cells (33–35). The stress-induced loss of goblet cells in association with the increased expression of an absorptive cell marker gene (CA1) in the rectum may be explained, at least in part, by the activation of Notch signals. Finally, we demonstrated that the absence of IL-18 almost completely blocked the goblet cell response and the gene expression changes in the rectum. It was possible that the 230 differentially expressed genes in the IL-18!/! mouse rectum might modify the subsequent response to isolation. Of the 230 genes, 54 genes also participated in the stress response of the wild-type mouse rectum (Supplemental Fig. S3A). However, the IL-18!/! mouse rectum changed expression of all these genes in the same direction as observed in the wild-type mouse rectum after isolation stress. Thus, the 230 genes were not likely to contribute to the disappearance of stress response in the IL-18!/! mouse rectum. There is still limitation to interpret the results of IL-18!/! mice. The wild-type and IL-18!/! mice came from different sources. Since colonic flora will be influenced by that of the dam, differences in the microflora between the IL-18!/! and C57BL/6J mice may influence the results. IL-18 is a modulator of immune functions and has proinflammatory, proapoptotic, and proatherogenic activities (20, 40, 41). Several lines of evidence suggest an important role of IL-18 in inflammation and damage of the colon (14 –16), including Crohn’s disease and UC (17, 18). Recently, it has been suggested that IL-18 may participate in the regulation of the hypothalamic-pituitary-adrenal axis (41). We showed that acute psychological stress (immobilization stress) stimulated IL-18 secretion from the adrenal cortex in an ACTHdependent manner, which was indispensable to subsequent production of IL-6 (21). In the present experi1804
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ments, isolation stress did not increase plasma ACTH levels enough to increase plasma IL-18 levels. IL-18 is released as a mature form. Immunoblot analysis showed that mature IL-18 was not detected in the adrenal gland exposed to isolation stress (data not shown), whereas immobilization stress clearly induced mature IL-18 in the adrenal gland (21). These results indicated that locally produced IL-18 was likely to play a crucial role in the stress response of the rectum. Notch 1 signals might be involved in the regulation of the IL-18-mediated loss of goblet cells. At present, however, there is no evidence showing the direct interaction between IL-18 and Notch 1 signals. In preliminary experiments, we observed that treatment of a colon cancer cell line (HT29) with IL-18 did not change expression of MATH1, KLF4, ELF3, CDX2, and NOTCH1 mRNAs (data not shown). We also confirmed that IL-18 treatment did not down-regulate MUC2 mRNA expression in goblet-like cells (HT29-MTX) (42) (data not shown). These preliminary experiments suggest that IL-18 dependently produced, undefined mediators may impair the goblet cell-dependent mucosal barrier functions of the rectum. The present study clearly shows the sensitivity of the rectum to psychosocial stress and suggests an important role of locally produced IL-18 in the vulnerability of the rectum. Although the precise mechanism for the action is still unknown, IL-18 may be one of the potential therapeutic targets for stress-related disorders, such as IBS or IBD. Transcript profiling: GEO repository for expression microarray data; accession number GSE11910 (http://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?token'jpaftigiiywasvs&acc'GSE11910).
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