Corticotropin-Releasing Hormone Deficiency Is Associated with ...

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Endocrinology 149(7):3403–3409 Copyright © 2008 by The Endocrine Society doi: 10.1210/en.2007-1703

Corticotropin-Releasing Hormone Deficiency Is Associated with Reduced Local Inflammation in a Mouse Model of Experimental Colitis Je´roˆme Gay,* Efi Kokkotou,* Michael O’Brien, Charalabos Pothoulakis, and Katia P. Karalis Division of Endocrinology (J.G., K.P.K.), Children’s Hospital, Gastrointestinal Neuropeptide Center (E.K., C.P.), Gastroenterology Division, Beth Israel Deaconess Medical Center, and Department of Pediatrics and Nutrition (E.K., C.P., K.P.K.), Harvard Medical School, Boston, Massachusetts 02215; and Department of Pathology (M.O.), Boston University Medical Center, Boston, Massachusetts 02118 CRH, the hypothalamic component of the hypothalamic-pituitary adrenal axis, attenuates inflammation through stimulation of glucocorticoid release, whereas peripherally expressed CRH acts as a proinflammatory mediator. CRH is expressed in the intestine and up-regulated in patients with ulcerative colitis. However, its pathophysiological significance in intestinal inflammatory diseases has just started to emerge. In a mouse model of acute, trinitrobenzene sulfonic acid-induced experimental colitis, we demonstrate that, despite low glucocorticoid levels, CRH-deficient mice develop substantially reduced local inflammatory responses. These

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HE HYPOTHALAMIC-pituitary adrenal (HPA) axis and the immune system interact to maintain homeostasis during the course of inflammation. Proinflammatory cytokines such as TNF␣, IL-1␤, and IL-6 activate the HPA axis via CRH-dependent and independent pathways leading to increased ACTH secretion from the pituitary and glucocorticoid release from the adrenal gland (1). Consequently, glucocorticoid restrains further induction of inflammatory mediators and thus prevents the propagation of the inflammatory response. In addition to the antiinflammatory effects of hypothalamic CRH through induction of glucocorticoids, CRH secreted peripherally by immune cells, nerve fibers, and possibly additional cell types may act locally as a proinflammatory mediator (2). Indeed, in rodents, increased expression of CRH has been found in inflamed tissues during carrageenin-induced granulomas (2), inflammatory arthritis (3), experimental autoimmune uveoretinitis (4), chronic granulomatous enterocolitis (5), and Clostridium difficile toxin A-mediated ileitis (6, 7). In humans, peripheral CRH up-regulation has been described in rheumatoid arthritis (8), ulcerative colitis (9), Hashimoto thyroiditis (10), endometriosis (11), and psoriasis (12). The study of CRH-deficient (Crh⫺/⫺) mice (13, 14) has First Published Online April 10, 2008 * J.G. and E.K. contributed equally to this work. Abbreviations: CRHR, CRH receptor; HPA, hypothalamic-pituitary adrenal; MCP, monocyte chemoattractant protein; MPO, myeloperoxidase; NPY, neuropeptide-Y; POMC, proopiomelanocortin; TNBS, trinitrobenzene sulfonic acid; ucn, urocortin. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

effects were shown by histological scoring of tissue damage and neutrophil infiltration. At the same time, CRH deficiency was found to be associated with higher serum leptin and IL-6 levels along with sustained anorexia and weight loss, although central CRH has been reported to be a strong appetite suppressor. Taken together, our results support an important proinflammatory role for CRH during mouse experimental colitis and possibly in inflammatory bowel disease in humans. Moreover, the results suggest that CRH is involved in homeostatic pathways that link inflammation and metabolism. (Endocrinology 149: 3403–3409, 2008)

further contributed to the elucidation of the peripheral proinflammatory role of CRH, as opposed to its centrally mediated antiinflammatory effects. For example, with criteria the degree of local inflammation and expression of proinflammatory mediators, Crh⫺/⫺ mice develop less severe experimental autoimmune encephalomyelitis (15), carrageenininduced granuloma (16), and turpentine-induced hind limb abscess (17). Most recent findings from our group (7) and others (18) point to a significant role for CRH in intestinal inflammation because Crh deficiency or pharmacological antagonism were found to be associated with attenuated intestinal mucosal damage and neutrophil accumulation after intraluminal administration of the potent enterotoxin C. difficile toxin A (7). In addition to these, a previous correlational study had shown in a rat model of granulomatous enterocolitis abundant CRH expression in the chronically inflamed cecum, which was localized mainly in inflammatory cells, mesenchymal cells, and the myenteric plexus (5). Moreover, increased CRH has been described in the colon of patients with ulcerative colitis (9). However, the significance of CRH-dependent pathways in the pathophysiology of inflammatory bowel disease remains largely unexplored. Crohn’s disease and ulcerative colitis, collectively known as inflammatory bowel disease, represent lifetime diseases associated with significant morbidity, mortality, and increased health care costs (19). Among the various models of experimental colitis, trinitrobenzene sulfonic acid (TNBS)-induced intestinal inflammation resembles Crohn’s disease in many aspects (20, 21). In addition, this particular model of experimental colitis has been associated with stimulation of the HPA axis, evident by increased circulating levels of corticosterone and adrenal

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enlargement (22). In the present study, we examined the direct contribution of CRH in the local and systemic inflammatory responses during the course of acute TNBS-induced experimental colitis using the Crh⫺/⫺ mouse. Materials and Methods Animals Eight- to 10-wk-old (22–27 g) Crh⫺/⫺ female mice and their wild-type littermates, maintained in a C57B6 ⫻ 129 genetic background by breeding within the population (23), were used in our experiments. Animal housing and care was done according to National Institutes of Health (NIH) guidelines, and all experimental procedures were approved by the Animal Care and Use Committee of Children’s Hospital in Boston. Mice were housed individually at least 48 h before starting the experimental procedure, under controlled conditions (temperature 22 C; 12 h light, 12 h dark cycle; lights on at 0700 h). For the experiments involving daily monitoring of body weight and food intake, mice and their administered food were weighed before fasting and before TNBS administration (see below) and every 24 h for a total of 2 d after injection. Careful inspection of cages for spillage was done daily.

Induction of TNBS-colitis Mice were fasted for 16 h before induction of colitis while supplemented with 5% dextrose in their drinking water to prevent hypoglycemia and hypothermia. Mice were anesthetized by ip injection of 2.5% avertin (0.01 ml/g body weight), and TNBS-colitis was induced in mice as previously described (24). Briefly, a 50-␮l enema of TNBS (250 mg/kg; Fluka, Buchs, Switzerland) in 35% ethanol was infused into the colonic lumen (3.5 cm from the anal verge) via a 1-ml syringe (Becton Dickinson, Franklin Lakes, NJ) fitted with a polyethylene cannula (Intramedic PE-20 tubing; Becton Dickinson). The mice were maintained in a supine Trendelenberg position until recovery from anesthesia to prevent leakage of the intracolonic instillation of TNBS. Preliminary experiments showed that a dose of 250 mg/kg TNBS produced a robust inflammatory response in this strain of mice, whereas lower doses (100 and 150 mg/kg) failed to induce significant colitis. Control mice received 35% ethanol. Two days after TNBS exposure, the animals were killed by cervical dislocation after blood collection and the distal colon was removed, weighed, and measured. Samples were then immediately fixed in 10% formalin to assess microscopic scores of inflammation, or snap-frozen in liquid nitrogen and subsequently kept at ⫺80 C for protein or RNA extraction. Hypothalamus was also dissected and cryopreserved.

Assessment of colonic inflammation The degree of intestinal inflammation was evaluated in hematoxylin and eosin-stained transverse colonic histological sections under a light microscope (24). Three sections per animal and three views per section were examined in a blinded fashion by an experienced pathologist (M.O.), and mucosal ulceration, atrophy, and submucosal edema, inflammatory cell infiltrate, and vascular dilation were scored from 0 to 3 (maximum score 15). The activity of the enzyme myeloperoxidase (MPO), a granulocyte-associated enzyme, was measured as previously described (25). Full-thickness segments of colon (3 cm long) were suspended in phosphate buffer [50 mm (pH 6.0)] and homogenized on ice using a Polytron (10 sec at maximal speed setting), followed by three cycles of freeze-thaw. Homogenates were then centrifuged at 10,000 ⫻ g for 15 min at 4 C, and pellets were resuspended in a phosphate buffer containing 0.5% hexadecyl trimethylammonium bromide (Sigma, St. Louis, MO) followed by sonication on ice. Lysates were clarified by centrifugation at 10,000 ⫻ g for 15 min at 4 C and diluted in phosphate buffer containing 0.67 mg/ml o-dianisidine dihydrochloride (Sigma) and 0.0005% of hydrogen peroxide. MPO from human neutrophils (Sigma; 0.1 U per 100 ␮l) was used as a standard. Changes in absorbance at 450 nm were recorded every 10 sec over a 2-min period using a spectrophotometer (␭ 20, UV/VIS spectrophotometer; PerkinElmer, Norwalk, CT). Protein concentration was determined by the modified method of Lowry (detergent compatible assay, Bio-Rad, Hercules, CA), and MPO activity was normalized by protein content. Results are ex-

Gay et al. • CRH-Deficiency and Inflammation

pressed as percent of MPO activity in the wild-type, TNBS-exposed mice (100%).

Gene expression analysis RNA from colon or hypothalamus was extracted using the TRI reagent (Sigma). For semiquantitative PCR analysis, 2 ␮g of total RNA were used to generate cDNA by the Moloney murine leukemia virus reverse transcriptase in the presence of random hexamer primers (Life Technologies Inc., Rockville, MD). Mouse-specific primers for IL-1␤, IL-6, and ␤-actin were obtained from a commercial source (Biosource International, Camarillo, CA). The sequences of the sense and antisense primers and the PCR conditions are described below in the following order: primer name, sequence, size of product (base pairs), cycles, annealing temperature (C). The sequences are: ␤-actin, sense, 5⬘-TCAGAAGGACTCCTATGTGG-3⬘, antisense, 5⬘-TCTCTTTGATGTCACGCACG-3⬘, 500, 25, 55; IL-1␤, sense, 5⬘-TTGACGGACCCCAAAAGATG3⬘, antisense, 5⬘-AGAAGGTGCTCATGTCCTCA-3⬘, 204, 26, 55; IL-6, sense, 5⬘-TGGAGTCACAGAAGGAGTGGCTAAG-3⬘, antisense, 5⬘TCTGACCACAGTGAGGAATGTCCAC-3⬘, 155, 30, 62. PCR products were resolved in a 1.3% agarose gel, and band intensity was analyzed using the NIH image analysis software. Real-time quantitative RT-PCR for neuropeptide Y (NPY) and proopiomelanocortin (POMC) genes was performed using the one-step RT-PCR kit and an automated ABI 7700 sequence detector system (PE Applied Biosystems, Foster City, CA). RNA input was adjusted to 100 ng per 50-␮l reaction. The following primers at a final concentration of 900 nm each and probes at 100 nm concentration were used: NPY, forward primer, 5⬘-TCAGACCTCTTAATGAAGGAAAGCA-3⬘, reverse primer, 5⬘-GAGAACAAGTTTCATTTCCCATCA-3⬘, probe, 6-FAM-CCAGAACAAGGCTTGAAGACC-CTTCCAT-TAMRA; POMC, forward primer, 5⬘-CTGCTTCAGACCTCCATAGATGTG-3⬘, reverse primer, 5⬘-CAGCGAGAGGTCGAGTTTGC-3⬘, probe, 6-FAM-CAACCTGCTG GCTTGCATCCGG-TAMRA. The POMC or NPY gene was amplified in a multiplex reaction with rodent VIC-labeled glyceraldehyde-3-phosphate dehydrogenase (Applied Biosystems) as internal control. Samples were run in duplicate, and results are expressed as normalized arbitrary mRNA units.

Plasma hormone and cytokine measurements Blood samples were collected in heparinized tubes with minimal stress before and after each experiment via retroorbital eye bleeding of conscious mice and placed on ice. Blood samples were then centrifuged (2000 rpm; 4 C; 10 min) and plasma aliquoted and stored at ⫺80 C until further use. Plasma corticosterone levels were measured using a commercial RIA kit (ICN Pharmaceuticals Inc., Orangeburg, NY). Plasma IL-6 was measured by ELISA (R&D Systems Inc., Minneapolis, MN) and leptin by RIA (Linco Research Inc., St. Charles, MO) according to the manufacturer’s instructions.

Statistical analysis In all experiments, each group consisted of three to five mice, and each individual experiment was performed three times. Data were analyzed by one-way ANOVA followed by the Tukey’s post hoc multiple comparison test using the STATVIEW statistical software (Abacus Concepts, Berkeley, CA). Results are expressed as mean ⫾ se.

Results TNBS-mediated intestinal inflammation is attenuated in the Crh⫺/⫺ mice

The degree of colonic inflammation of the Crh⫹/⫹ and Crh⫺/⫺ mice in response to TNBS treatment was evaluated by histological analysis, myeloperoxidase activity, and measurement of the thickness of the colonic wall. Administration of TNBS caused mucosal erosions, submucosal edema, and extensive neutrophil accumulation in the Crh⫹/⫹ mice, whereas minimal pathology was detected in the Crh⫺/⫺ mice (Fig. 1A). The overall histological severity of colitis estimated


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wall thickness due to infiltration and edema. In Figure 1D is shown a statistically significant increase in weight to length ratio in TNBS-treated Crh⫹/⫹ mice, whereas there was no significant difference in the weight to length ratio between vehicle and TNBS-treated Crh⫺/⫺ mice. Despite the lack of a significant effect of TNBS in the latter marker in the Crh⫺/⫺ mice, we could not confirm a significant difference at the weight to length ratio between the two genotypes after TNBS treatment. Intestinal IL-1␤ mRNA levels were severalfold up-regulated in the Crh⫹/⫹ TNBS-treated mice, with significantly lower up-regulation (50%) in the Crh⫺/⫺ ones (P ⬍ 0.05, Fig. 2A), consistent with attenuated local inflammatory responses in the latter. However, IL-6 intestinal mRNA expression was equally up-regulated in both genotypes (Fig. 2B).

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Baseline corticosterone, IL-6, and leptin plasma levels did not differ among Crh⫹/⫹ and Crh⫺/⫺ mice (Fig. 3), consistent with previous reports (16, 17, 23). At 48 h after the induction of colitis, plasma corticosterone levels were significantly elevated in the TNBS-exposed Crh⫹/⫹ mice, compared with the ones treated with vehicle (43.35 ⫾ 3.17 vs. 7.36 ⫾ 1.96 ␮g/dl; P ⬍ 0.001), whereas a smaller, although significant, rise was detected in Crh⫺/⫺ mice (20.40 ⫾ 6.79 vs. 2.23 ⫾ 0.26 ␮g/dl; P ⬍ 0.05) (Fig. 3A). Interestingly, at the same time point, vehicle

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FIG. 1. TNBS-induced intestinal inflammation is attenuated in the Crh⫺/⫺ mice. A, Mice were evaluated 48 h after intracolonic administration of TNBS. Representative hematoxylin and eosin-stained histological sections of colonic tissue from TNBS-treated Crh⫹/⫹ and Crh⫺/⫺ mice (original magnification, ⫻200). B, Histological scoring of TNBS-induced intestinal inflammation. C, MPO activity (percent of TNBS-treated Crh⫹/⫹) in colonic tissue lysates from TNBS-exposed Crh⫹/⫹ and Crh⫺/⫺ mice. D, Colitis-associated thickness of the colonic wall as evaluated by colon weight to length ratio (grams per centimeter). **, P ⬍ 0.01; ***, P ⬍ 0.001.

by well-established criteria (26) was significantly higher in the Crh⫹/⫹ mice (9.17 ⫾ 0.91), compared with the Crh⫺/⫺ mice (1.50 ⫾ 0.96, P ⬍ 0.001) (Fig. 1B). MPO activity, the levels of which reflect the degree of neutrophil infiltration, was remarkably lower in the Crh⫺/⫺ mice (P ⬍ 0.01) in agreement with the histological findings in their inflamed intestine (Fig. 1C). The third marker of intestinal inflammation we evaluated was the weight to length ratio, representing the colon

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FIG. 2. Cytokine expression in the colonic tissue of TNBS-treated Crh⫹/⫹ and Crh⫺/⫺ mice. A, Intestinal IL-1␤ mRNA up-regulation was higher in the Crh⫹/⫹ mice. B, Intestinal IL-6 mRNA expression was equally up-regulated in Crh⫹/⫹ and Crh⫺/⫺ mice. *, P ⬍ 0.05.


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FIG. 4. Prolonged anorexia in the Crh⫺/⫺ mice up to 48 h after TNBS (250 mg/kg) exposure. A, Body weight loss, expressed as percent of initial body weight, was comparable between Crh⫹/⫹ and Crh⫺/⫺ mice. B, Suppression of food intake after TNBS treatment was more severe in the Crh⫺/⫺ mice.

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FIG. 3. Up-regulation of systemic inflammatory markers in the Crh⫺/⫺ mice in response to TNBS treatment despite their reduced local inflammation. A, Plasma corticosterone. B, Plasma IL-6. C, Plasma leptin. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001.

Significant weight loss was apparent within 24 h after induction of colitis and was sustained for the following 24 h in both Crh⫹/⫹ and Crh⫺/⫺ mice (Fig. 4A). At each time point, body weight reduction reflected the food intake of the mice during the previous day. Food intake in Crh⫹/⫹ mice was dramatically reduced the first 24 h of inflammation and gradually increased thereafter, whereas severe reduction of food intake was found in TNBS-exposed Crh⫺/⫺ mice up to 48 h after induction of colitis (Fig. 4B), despite their significantly attenuated inflammatory response. To further understand the paradoxical anorectic response of the Crh⫺/⫺ mice, we next measured hypothalamic expression of NPY and POMC, two major opposing regulators of feeding behavior. We found up-regulation of the NPY (Fig. 5A) and down-regulation of POMC (Fig. 5B) only in the Crh⫹/⫹ mice at 48 h after the induction of colitis, consistent with the observed changes in their food intake (Fig. 4B). However, the Crh⫺/⫺ mice had



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FIG. 5. Crh⫺/⫺ mice have blunted hypothalamic neuropeptide responses at 48 h after TNBS treatment. A, NPY mRNA expression. B, POMC mRNA expression. The pattern in Crh⫹/⫹ mice is consistent with a recovery in their food intake. *, P ⬍ 0.05.

blunted hypothalamic neuropeptide responses, in a milieu of high circulating levels of leptin and IL-6. Discussion

In this study, we provide evidence that CRH plays a proinflammatory role in the acute TNBS-induced experimental colitis based on the significantly attenuated intestinal inflammatory responses in the Crh⫺/⫺ mice (Fig. 1). More specifically, our results revealed significant effects of CRH deficiency in several markers of colitis, including lower MPO activity. This finding indicates a diminished infiltration of the colon by neutrophils, in agreement with the histological scoring (Fig. 1B), and local expression of IL-1␤ (Fig. 2A). However, CRH deficiency did not protect mice from colitisassociated systemic effects such as up-regulation of circulating IL-6 (Fig. 3B), anorexia, and the corresponding weight loss (Fig. 4). Our results are consistent with previous reports describing attenuated responses in the Crh⫺/⫺ mice in different experimental models of inflammation (15, 17), including acute bacterial toxin-mediated enteritis (7). There are several potential mechanisms by which native CRH can contribute to peripheral inflammation, including colitis. Previous studies have demonstrated the presence of CRH receptors in various types of immune cells and on colonocytes. In mouse thymocytes (27), human peripheral blood mononuclear cells (28), human endothelial cels, and

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human and mouse monocytes (29) (Karalis, K. P., unpublished observations), CRH activates nuclear factor-␬B and stimulates the expression of various proinflammatory mediators, including TNF␣; IL-1, -6, and -8; regulated upon activation, normal T cell expressed, and secreted; and monocyte chemoattractant protein-1. In the human and mouse intestinal mucosa and human colonocytes in particular, CRH receptor (CRHR) expression has been shown to be up-regulated during various inflammatory conditions (6, 30). Most importantly, in HT-29 human colonic epithelial cells, like in immune cells, we found a CRHR-dependent activation of the nuclear factor-␬B and MAPK signaling pathways and stimulation of IL-8 and monocyte chemoattractant protein-1 expression (30). Potential peripheral sources of CRH in our model include enteric nerves, sensory neurons, and immune cells. Taken together, these observations underscore the importance of CRH-mediated neuroimmune interactions in the intestine. The observed CRH effects could be direct or via interaction with additional neuropeptides, i.e. substance P (7, 31). CRH-independent activation of the HPA axis and glucocorticoid release evident in the present (Fig. 3A) and previous studies might be mediated by inflammatory cytokines such as IL-6 (16, 17, 32). Indeed, a greater than normal IL-6 induction was found in the plasma of TNBS-treated Crh⫺/⫺ mice (Fig. 3B). This compensatory IL-6 response in the absence of CRH is not unique to this colitis model and has been observed in inflammation induced in mice by diverse stimuli, such as turpentine, lipopolysaccharide, or a viral infection (17, 32–34). Moreover, Bethin et al. (32) demonstrated that, compared with CRH deficiency, the combined CRH/ IL-6 deficiency resulted in further attenuation of glucocorticoid release in response to an immunological challenge. A series of in vivo and in vitro studies have also shown that IL-6 can stimulate the HPA axis at different levels (34, 35). This suggests the likelihood of a similar role in stimulating corticosterone release in the described studies (Fig. 3A). Leptin is an additional hormone shown to be implicated in intestinal inflammation in both rodents and humans (36 – 39). As has been previously shown, Crh⫺/⫺ mice have lower basal leptin levels, a defect corrected by administration of CRH (23). Interestingly, in this study we found significantly up-regulated plasma leptin levels 48 h after TNBS exposure only in the Crh⫺/⫺ mice and not in the similarly treated Crh⫹/⫹ mice (Fig. 3C). This observation might reflect increased sensitivity of the Crh⫺/⫺ mice to positive regulators of leptin expression induced during TNBS-colitis or a direct negative effect of CRH on leptin expression (40 – 43). Our results are also consistent with the recent observation that prolonged fasting elevated leptin levels in the Crh⫺/⫺ mice, rather than falling as in normal mice (23). Furthermore, there is compelling evidence in the literature indicating that leptin, like IL-6, can act as an ACTH secretagogue (44). Thus, the increased leptin levels in the Crh⫺/⫺ mice in our model could also contribute to the observed rise in circulating corticosterone. In addition to its function as a stress mediator and proinflammatory factor, CRH affects negatively feeding behavior when infused intracerebroventricularly (45). However, Crh⫺/⫺ mice have no apparent feeding phenotype under

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baseline conditions (13), whereas pronounced anorexia has been described in these mice after adrenalectomy (46), fasting (23), and in response to chronic stressors or inflammation (17, 47). In the current study, severe anorexia was apparent in TNBS-treated Crh⫺/⫺ mice (Fig. 4B) despite their reduced local inflammatory responses (Fig. 1). The reduced food intake of the Crh⫺/⫺ mice was reflected in the lack of upregulation of NPY and down-regulation of POMC expression in the hypothalamus, evident in the Crh⫹/⫹ mice 48 h after induction of colitis (Fig. 5). Previous immunoneutralization experiments have shown that both leptin (48) and IL-6 (49) are critical mediators of anorexia and weight loss associated with acute inflammation in rodents. Therefore, the observed persistent anorexia in the Crh⫺/⫺ mice (Fig. 4B) could be, at least in part, explained by a sustained up-regulation of leptin (Fig. 3C) and in particular IL-6 (Fig. 3B). The latter is considered the primary mediator of cachexia and hepatic acute-phase response to inflammation (50). Alternatively, the prolonged anorexia in the TNBS-treated Crh⫺/⫺ mice could be explained by a compensatory and brain-specific up-regulation of the CRH system during inflammation. Besides CRH, the CRH family of peptides includes urocortins (Ucn) I, II, and III, which bind to CRHR-1 and -2 with various affinities. Ucn II and III are exclusive ligands for CRHR2, whereas CRH and Ucn I bind to both receptors, although with different affinities (51). Anorectic effects have been associated with activation of either CRH receptor in several brain regions, some of them stress-independent (52–54). In our own studies, we detected a 40 –75% down-regulation of basal hypothalamic CRHR1 in the Crh⫺/⫺ mice (100 ⫾ 12 vs. 61 ⫾ 15 arbitrary units in Crh⫹/⫹ vs. Crh⫺/⫺ mice, respectively, P ⬍ 0.05), CRHR2 (100 ⫾ 12 vs. 36 ⫾ 5 arbitrary units, P ⬍ 0.01), and Ucn III (100 ⫾ 25 vs. 23.4 ⫾ 1.4 arbitrary units, P ⬍ 0.05) mRNA expression, as evaluated by real-time RT-PCR. Those levels remained largely unaffected by induction of colitis. The direction of these changes cannot explain the inflammation-related anorexia in the CRH-deficient mice. However, up-regulation of the same molecules in additional brain areas that have been implicated in the control of food intake cannot be entirely excluded (53). In conclusion, our results support a proinflammatory role for CRH in acute experimental colitis, but also reveal dissociation between local and systemic inflammatory responses in the Crh⫺/⫺ mice, underscoring the importance of CRH in homeostatic pathways that link inflammation and metabolism. Acknowledgments Received December 11, 2007. Accepted April 1, 2008. Address all correspondence and requests for reprints to: Katia Karalis, BRFAA, 4 Soranou Efessiou, Papagou, 115 27 Athens, Greece. E-mail: [email protected]. This work was supported by National Institutes of Health Grants DK47977 (to K.P.K.), P30DK40561 (pilot and feasibility project, to K.P.K.), and PO-1 DK 33506 (to C.P., K.P.K.). Disclosure Statement: The authors of this manuscript have nothing to declare.

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