Therapeutic Action of Ghrelin in a Mouse Model of Colitis

GASTROENTEROLOGY 2006;130:1707–1720

Therapeutic Action of Ghrelin in a Mouse Model of Colitis ELENA GONZALEZ–REY, ALEJO CHORNY, and MARIO DELGADO Institute of Parasitology and Biomedicine, CSIC, Granada, Spain

See CME Quiz, page 1903. Background & Aims: Ghrelin is a novel growth hormonereleasing peptide with potential endogenous anti-inflammatory activities ameliorating some pathologic inflammatory conditions. Crohn’s disease is a chronic debilitating disease characterized by severe T helper cell (Th)1-driven inflammation of the colon. The aim of this study was to investigate the therapeutic effect of ghrelin in a murine model of colitis. Methods: We examined the anti-inflammatory action of ghrelin in the colitis induced by intracolonic administration of trinitrobenzene sulfonic acid. Diverse clinical signs of the disease were evaluated, including weight loss, diarrhea, colitis, and histopathology. We also investigated the mechanisms involved in the potential therapeutic effect of ghrelin, such as inflammatory cytokines and chemokines, Th1-type response, and regulatory factors. Results: Ghrelin ameliorated significantly the clinical and histopathologic severity of the trinitrobenzene sulfonic acid-induced colitis; abrogating body weight loss, diarrhea, and inflammation; and increasing survival. The therapeutic effect was associated with down-regulation of both inflammatory and Th1-driven autoimmune response through the regulation of a wide spectrum of inflammatory mediators. In addition, a partial involvement of interluekin10/transforming growth factor-␤1-secreting regulatory T cells in this therapeutic effect was demonstrated. Importantly, the ghrelin treatment was therapeutically effective in established colitis and avoided the recurrence of the disease. Conclusions: Our data demonstrate novel antiinflammatory actions for ghrelin in the gastrointestinal tract, ie, the capacity to deactivate the intestinal inflammatory response and to restore mucosal immune tolerance at multiple levels. Consequently, ghrelin administration represents a novel possible therapeutic approach for the treatment of Crohn’s disease and other Th1-mediated inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis.

rohn’s disease (CD) is a chronic, idiopathic, and relapsing inflammatory bowel disease (IBD), characterized by dysfunction of mucosal T cells, altered cytokine production, and cellular inflammation that ultimately lead to damage of the distal small intestine and

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the colonic mucosa. These alterations result in a range of gastrointestinal and extraintestinal symptoms, ie, abdominal pain, rectal bleeding, diarrhea, weight loss, skin and eye disorders, and delayed growth and sexual maturation in children.1,2 Although its etiology remains unknown, there is circumstantial evidence to link CD to a failure of the mucosal immune system to attenuate the immune response to endogenous antigens.1 Several animal models of CD have been developed lately. Although many of these model incompletely resemble the human disease,3,4 the hapten-induced model of colonic inflammation in which 2,4,6-trinitrobenzene sulfonic acid (TNBS) is delivered intrarectally displays pathologic and clinical similarities to human CD and is used as a model system to test potential therapeutic agents.4 In this model, intestinal inflammation results from a covalent binding of the haptenizing agent to autologous host proteins with subsequent stimulation of a delayed-type hypersensitivity to TNBS-modified self-antigens. Both CD and TNBS-induced colitis are considered archetypal CD4 T helper (Th)1 cell-mediated autoimmune diseases in which activated Th1 cells promote an exaggerated macrophage and neutrophil infiltration and activation, giving rise to a prolonged severe transmural inflamed intestinal mucosa, characterized by uncontrolled production of inflammatory cytokines and chemokines.4 Inflammatory mediators such as cytokines (ie, interleukin [IL]12, interferon [IFN]-␥, and tumor necrosis factor [TNF]␣) and nitric oxide (free radical), produced by infiltrating cells and resident macrophages, play a critical role in colonic tissue destruction. Available therapies for CD based on immunosuppressive agents are not entirely Abbreviations used in this paper: BrdU, bromodeoxiuridine; CCR, chemokine receptor type CC; DSS, dextran sulfate sodium; GH, growth hormone; GHS-R, GH secretagogue receptor; IFN, interferon; IGF-I, insulin-like growth factor-I; IL, interleukin; IP-10, interferon-␥ inducible protein-10; LPMC, lamina propria mononuclear cells; mAb, monoclonal antibody; MCP, macrophage chemoattractant protein; MIF, macrophage migration inhibitory factor; MIP, macrophage inhibitory protein; MLN, mesenteric lymph nodes; MPO, myeloperoxidase; Th, T helper cells; TNBS, trinitrobenzene sulfonic acid; TNF, tumor necrosis factor; Treg, regulatory T cells. © 2006 by the American Gastroenterological Association Institute 0016-5085/06/$32.00 doi:10.1053/j.gastro.2006.01.041

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effective, are nonspecific, and have multiple adverse effects; and, in most cases, surgical resection is the ultimate alternative.2 This illustrates the need for novel therapeutic approaches that specifically modulate both components of the disease, ie, the inflammatory and Th1-driven responses. Ghrelin, a recently described endogenous ligand for the growth hormone secretagogue receptor (GHS-R), is a 28-amino acid acylated polypeptide produced principally by stomach cells.5 Ghrelin was initially identified as a potent circulating orexigen involved in the control of growth hormone (GH) secretion, energy expenditure, and adiposity.5–7 Recently, ghrelin and its receptors were detected in immune cells,8 indicating that the peptide may play a role in the immune system. Indeed, ghrelin has been described as an anti-inflammatory factor that protects from endotoxic shock by inhibiting the production of proinflammatory cytokines by activated monocytes and endothelial cells.8,9 Based on this wide antiinflammatory action, we here investigate the potential therapeutic effect of ghrelin in the murine model of TNBS-induced colitis. We report that treatment with ghrelin significantly reduces the clinical symptoms and pathology and that its therapeutic effect is associated with the down-regulation of both inflammatory and Th1-mediated immune responses.

Materials and Methods Induction of Colitis and Study Design Colitis was induced in Balb/c mice (6 – 8 weeks old, Jackson Laboratories, Bar Harbor, ME) as previously described.10 In brief, mice were lightly anesthetized with halothane, and a 3.5F catheter was inserted intrarectally 4 cm from the anus. To induce colitis, 100 ␮L of 3 mg TNBS (Sigma Chemical Co, St. Louis, MO) in 50% ethanol (to break the intestinal epithelial barrier) was slowly administered into the lumen via the catheter filled to a 1-mL syringe. Control mice received 50% ethanol alone (100 ␮L). Animals were treated intraperitoneally (IP) with medium or with different concentrations (0.05–2.0 nmol/mouse; 6 –250 ␮g/kg animal) of ghrelin (American Peptides Company, Sunnyvale, CA) 12 hours after TNBS instillation. To study the therapeutic effect of delayed administration of ghrelin on established colitis, ghrelin (2 nmol/mouse) was daily injected IP for 3 consecutive days starting 6 days after TNBS administration. To study the effect on disease recurrence, 1.5 mg TNBS was administered at days 0 and 9, and ghrelin (2 nmol/mouse) was injected IP once 12 hours after the first TNBS infusion. Some mice were injected intravenously (IV) with neutralizing anti-GH and/or anti– insulin-like growth factor (IGF)-I Abs (Preprotech, Rocky Hill, NJ) 2 and 24 hours after ghrelin administration at a dose of 200 ␮g/mouse/injection. Animals were monitored daily for appearance of diarrhea, body weight loss, and survival. Some

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animals were killed at the peak of the disease (day 3), blood samples were collected by cardiac puncture, and a segment of the colon (7 cm long) was excised for macroscopic damage evaluation and weighed. Tissue segments were immediately frozen in liquid nitrogen for histologic and immunohistologic studies, protein extraction, and cytokine determination, myeloperoxidase (MPO) activity measurement, and total RNA extraction. In another set of experiments, colon segments were used for the isolation of the lamina propria mononuclear cells (LPMC). Alternatively, acute colitis was induced in Balb/c mice by administration of 5% dextran sulfate sodium (DSS; M.W. 5000; Sigma) dissolved in the drinking water. Ghrelin (1 nmol/mouse/day) was injected IP at days 4 and 6. Development of colitis was assessed daily by measurement of drinking volume and body weight, evaluation of stool consistency, and detection of bloody stools. Disease severity was scored using a clinical disease activity index ranging from 0 to 4 that was calculated as previously described,11 using the following parameters: stool consistency, presence or absence of fecal blood, and weight loss. Mice were killed at day 8 and the colons processed for MPO activity. All experiments were performed according to the institutional guidelines for the care and use of laboratory animals in research and the approval of the local committee in the Consejo Superior de Investigaciones Cientificas.

Macroscopic and Microscopic Damage Evaluation Colons were examined under a dissecting microscope (⫻5) and graded for macroscopic lesions on a scale from 0 to 10 based on criteria reflecting inflammation (ie, hyperemia, thickening of the bowel, and extent of ulceration).12 Scores for stool consistency and rectal bleeding were assessed according to previously published procedures.13 For histopathologic analysis, a colon specimen from the middle part was fixed in 10% buffered formalin phosphate, embedded in sucrose, frozen in dry ice using optimal cutting temperature (OCT) compound and cryosectioned. Cross sections were stained with H&E, and inflammation was graded from 0 to 4 as follows in a blinded fashion: 0, no signs of inflammation; 1, low leukocyte infiltration; 2, moderate leukocyte infiltration; 3, high leukocyte infiltration, moderate fibrosis, high vascular density, thickening of the colon wall, moderate goblet cell loss, focal loss of crypts; and 4, transmural infiltrations, massive loss of goblet cells, extensive fibrosis, diffuse loss of crypts. For immunohistologic analysis, cryosections were blocked in 3% BSA/PBS for 30 minutes at room temperature, washed, and incubated with 5 ␮g/mL FITC- and PE-labeled anti-CD4, anti-CD11b, antiTLR4, or anti-TNF-␣ monoclonal antibodies (mAbs) for 1 hour at room temperature. Isotype immunoglobulin (Ig)G Abs were used as negative controls. After mounting, the stained sections were examined by dual immunofluorescent microscopy (Leica, Wetzlar, Germany).

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Cytokine and Hormone Determination For cytokine determination in colon mucosa, protein extracts were isolated by homogenization of colonic segments (50 mg tissue/mL) in 50 mmol/L Tris-HCl, pH 7.4, with 0.5 mmol/L DTT, and 10 ␮g/mL of a cocktail of proteinase inhibitors containing phenylmethylsulfonyl fluoride, pepstatin, and leupeptin (Sigma Chemical Co). Samples were centrifuged at 30,000g for 20 minutes and stored at ⫺80°C until cytokine determination. Cytokine, chemokine, GH, and IGF-I levels in the serum, colonic protein extracts, and culture supernatants were determined by specific sandwich ELISAs using capture/biotinylated detection Abs from BD Pharmingen (San Diego, CA) and Preprotech according to the manufactures’ recommendations. Serum amyloid A (SAA) levels were determined in serum samples by a murine ELISA kit (Tridelta Development, NJ). For intracellular analysis of cytokines in stimulated LPMC and MLN cells, 106 cells/mL were stimulated with phorbol myristate acetate (PMA; 10 ng/mL) plus ionomycin (20 ng/mL) for 8 hours in the presence of monensin. Cells were stained with PerCP-anti-CD4 mAbs for 30 minutes at 4°C, washed, fixed/saponin permeabilized with Cytofix/Cytoperm (Becton Dickinson), stained with 0.5 ␮g/ sample of FITC- and PE-conjugated anticytokine specific mAbs (BD Pharmingen), and analyzed on a FACScalibur flow cytometer (Becton Dickinson). To distinguish between monocyte/macrophage and T-cell sources, intracellular cytokine analysis was done exclusively in the PerCP-labeled CD4 T-cell population.

MPO Assay Neutrophil infiltration in the colon was monitored by measuring MPO activity as previously described.14 Briefly, colonic segments were homogenized at 50 mg/mL in phosphate buffer (50 mmol/L, pH 6.0) with 0.5% hexadecyltrimethylammonium bromide. Samples were frozen and thawed 3 times, centrifuged at 30,000g for 20 minutes. The supernatants were diluted 1:30 with assay buffer consisting in 50 mmol/L phosphate buffer, pH 6.0, with 0.167 mg/mL o-dianisidine (Sigma Chemical Co) and 0.0005% H2O2, and the colorimetric reaction was measured at 450 nm between 1 and 3 minutes in a spectrophotometer (Beckman Instruments, Irvine, CA). MPO activity per gram of wet tissue was calculated as the following: MPO activity共U⁄g wet tissue兲 ⫽ 共A450兲共13.5兲⁄tissue weight共g兲, where A450 is the change in the absorbance of 450 nm light from 1 to 3 minutes after the initiation of the reaction. The coefficient 13.5 was empirically determined such that 1 U MPO activity represents the amount of enzyme that will reduce 1 ␮mol peroxide/min.

Isolation and Culture of LPMC and Mesenteric Lymph Node Cells LPMC were isolated from freshly obtained colonic specimens using a modification of a described technique.10 In brief, colons were washed in calcium- and magnesium-free Hanks’ balanced salt solution (HBSS) medium and then

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treated with 1 mmol/L EDTA/phosphate-buffered saline (PBS) for 20 minutes to remove the epithelium. The tissue was then digested with type IV collagenase and DNase I (Sigma Chemical Co) for 20 minutes in a shaking incubator at 37°C. Released cells were layered on a 30%–70% Percoll gradient (Amersham Pharmacia, Uppsala, Sweden) and spun at 1800 rpm to obtain the leukocyte-enriched population at the 30%– 70% interface. Single-cell suspensions were obtained from mesenteric lymph node (MLN) freshly collected at the peak of the disease. MLN cells were enriched in T cells by incubating MLN cells in petri dishes for 2 hours at 37°C to remove nonadherent cells. LPMC and MLN cells were incubated in complete medium (RPMI-1640 supplemented with 100 U/mL penicillin/streptomycin, 2 mmol/L L-glutamine, 50 ␮mol/L 2-mercaptoethanol, and 10% heat-inactivated fetal calf serum) at a concentration of 106 cells/mL, in the absence (unstimulated) or presence of PMA (10 ng/mL) and Con A (2.5 ␮g/mL). Cell proliferation (expressed as A450) was evaluated in 96-well microtiter plates for 96 hours by using a cell proliferation assay (BrdU) from Roche Diagnostics GmbH (Mannheim, Germany). Cytokine/chemokine production in culture supernatants was determined after 48-hour culture as described above. Intracellular cytokine content was determined in stimulated cells as described previously.

Gene Expression Analysis Total RNA was isolated from colonic specimens by using Ultraspec RNA reagent (Biotecx, Houston, TX), and the messenger RNA (mRNA) expression of a variety of chemokines, chemokine receptors, cytokines, enzymes, and leukocyte markers was quantified using a GEArray focused DNA microarray for 96 inflammatory and autoimmune factors (Superarray Bioscience, Frederick, MD) following manufacturers’ recommendations. Briefly, total RNA (10 ␮g) was labeled with Biotin-16-UTP (Roche) using the TrueLabeling-AMP linear RNA amplification kit (Superarray Bioscience) and hybridized at 60°C overnight with the GEArray membrane containing oligonucleotide probes for different inflammatory and autoimmune factors. Membranes were washed at 60°C under high stringency conditions with SDS/SSC solutions, and specific signals were detected with a CCD camera image station by chemoluminescence detection using alkaline phosphatase-conjugated streptavidin and CPD-Star chemoluminescence substrate. Sample-to-sample variation in RNA loading was controlled by comparison with the housekeeping gene ␤-actin. In addition, the results obtained for the expression of key genes (TNF-␣, IFN-␥, IL-1␤, IL-10, and macrophage inhibitory protein [MIP]-2) were confirmed by real-time PCR.

Analysis of Foxp3 Expression by Flow Cytometry MLN cells (106 cells) were isolated in ice-cold RPMI complete medium and washed twice with PBS containing 0.1% sodium azide plus 2% heat-inactivated FCS (wash buffer). Cells were incubated with PerCP-anti-CD4 and FITCanti-CD25 mAbs (BD Pharmingen, 2.5 ␮g/mL final concen-

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Figure 1. Treatment with ghrelin protects against TNBS-induced colitis. Colitis was induced by intracolonic administration of TNBS (3 mg/mouse) in 50% ethanol. Mice were treated IP with ghrelin (2 nmol/mouse, or different doses in B) 12 hours after TNBS injection. Mice treated with 50% ethanol were used as controls. Clinical evolution and severity was monitored by body weight changes (A and B), colitis score (C), and survival (D). N ⫽ 12–18 mice/group. *P ⬍ .001 vs TNBS-treated mice.

tration) at 4°C for 1 hour. Isotype-matched Abs were used as controls, and IgG block (Sigma Chemical Co) was used to block the nonspecific binding to Fc receptors. After extensive washing, cells were fixed/saponin permeabilized with Cytofix/ Cytoperm solution (BD PharMingen) and incubated for 45 minutes at 4°C with PE-anti-Foxp3 mAb (0.5 ␮g/sample) diluted in PBS ⫹ 1% BSA ⫹ 0.5% saponin. After extensive washing, cells were analyzed on a FACScalibur flow cytometer.

Data Analysis All values are expressed as mean ⫾ SD of mice/experiment. The differences between groups were analyzed by Mann– Whitney U test and, if appropriate, by Kruskal–Wallis ANOVA test. Survival curves were analyzed by the Kaplan–Meier log-rank test. Changes in body weight were compared by use of the Wilcoxon matched-pair signed-rank test.

Results Treatment With Ghrelin Protects Against Colitis Development We investigated the potential therapeutic action of ghrelin in the TNBS model of colitis. Mice subjected to intrarectal administration of TNBS in 50% ethanol developed a severe illness characterized by bloody diarrhea, rectal prolapse, pancolitis accompanied by extensive wasting syndrome, and a profound and sustained weight loss resulting in a mortality of 60% (Figure 1A–D). Mice treated with ghrelin (2 nmol; 250 ␮g/kg) 12 hours after TNBS instil-

lation had a survival rate of 85%, rapidly recovered the lost body weight, and regained a healthy appearance similar to control mice treated with 50% ethanol alone (Figure 1A– D). The therapeutic effect of ghrelin was dose dependent, showing maximal effects at doses between 0.2 and 2 nmol (25 to 250 ␮g/kg) (Figure 1B). These effects were not due to differences in chow intake. Macroscopic examination of colons obtained 3 days after colitis induction showed striking hyperemia, inflammation, and necrosis, compared with control animals that only showed slight inflammation; in contrast, the colons of ghrelin-treated mice showed no signs of macroscopic inflammation (Figure 2A). Histologic examination of the distal colon of mice given TNBS showed a transmural inflammation involving all layers of the bowel wall, with a marked increase in the thickness of the muscular layer, adherence to surrounding tissues, patchy ulceration, epithelial cell loss, pronounced depletion of mucinproducing goblet cells, reduction of the density of the tubular glands, disseminate fibrosis, and focal loss of crypts (Figure 2B). Inflammatory cell infiltrate consisted of macrophages, lymphocytes, and neutrophils in the lamina propria (Figure 2B). Immunohistologic analysis revealed CD4 T cells, TNF-␣-producing cells, Toll-like receptor 4 (TLR4)-expressing inflammatory cells, and CD11b⫹ granulocytes/macrophages (Figure 2C). Neutrophil infiltration correlated with increased colonic MPO activity (Figure 2D). When mice were treated with ghrelin, these macroscopic

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Figure 2. Ghrelin prevents TNBS-induced pathology. Colitis was induced by intracolonic administration of TNBS (3 mg/mouse) in 50% ethanol. Mice were treated IP with ghrelin (2 nmol/mouse) 12 hours after TNBS injection. Mice treated with 50% ethanol were used as controls. (A) Macroscopic-damage score was determined at 3 days after TNBS administration. (B) Histopathologic analysis was determined in H&E-stained sections of colons obtained at day 3 of disease (original magnification, ⫻200). (C) Inflammatory infiltrates in the colons (day 3) were phenotypically characterized by immunostaining against TLR4, TNF-␣-producing cells, CD11b cells, or CD4 T cells. Isotype FITC-labeled IgG Ab was used as negative control (original magnification, ⫻200). (D) Colonic MPO activity was determined in the acute phase of the disease (day 3). N ⫽ 12–18 mice/group. *P ⬍ .001 vs TNBS-treated mice.

and histologic signs were much improved, with a significant reduction of inflammatory activity and neutrophil infiltration (Figure 2). Next, we wanted to examine whether ghrelin would be effective during the later phases of the disease when colitis is fully established. Administration of ghrelin on 3 consecutive days starting 6 days after disease onset rapidly reversed the lost body weight and macroscopic colon damage (Figure 3A). In addition, we investigated whether ghrelin was able to prevent recurrence of the disease. TNBS-treated mice reexposed on day 9 to a second dose of TNBS rapidly died (100% mortality) because of severe colitis, body weight loss, and colon inflammation (Figure 3B). In contrast, a single

administration of ghrelin 12 hours after the initial colitis induction conferred resistance to disease recurrence after a second administration of TNBS (Figure 3B). To exclude that the therapeutic effect of ghrelin was exclusive of the TNBS-induced colitis model, we investigated the effect of ghrelin in a model of acute colitis induced by the addition of DSS in drinking water. DSS administration was associated with significant clinical changes, including weight loss (starting on day 2), appearance of occult fecal blood (on day 3), and diarrhea (on day 4) (Figure 4). Treatment with ghrelin resulted in significant amelioration of colitis by day 8, as shown by decrease in colon shortening, improvement in stool consistency, and

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Figure 3. Treatment with ghrelin abrogates established colitis and reduces disease recurrence. (A) Established colitis. Colitis was induced by intracolonic administration of TNBS (1.5 mg/mouse) at days 0 and 6. Mice were treated daily for 3 consecutive days with ghrelin (2 nmol/mouse/day) starting 6 days after TNBS administration (arrow). Disease progression was assessed by body weight loss and macroscopic score at day 12. N ⫽ 8 mice/group. (B) Disease recurrence. Colitis was induced by intracolonic administration of TNBS (3 mg/mouse) at days 0 and 9 (arrows). Mice were treated IP with ghrelin (2 nmol/mouse) 12 hours after TNBS injection. Controls were given a second injection of ethanol at day 9. Disease progression was assessed by body weight loss, survival percentage, and macroscopic score at day 11. Numbers in parentheses represent daily mortality percentage after the second TNBS infusion. N ⫽ 8 –10 mice/group.

reduced rectal bleeding and MPO activity. Ghrelin administration resulted in a 60% reduction in the clinical disease activity index (Figure 4). Treatment With Ghrelin Reduces Systemic and Colonic Inflammatory Responses in Mice With TNBS-Induced Colitis We next evaluated the effect of ghrelin treatment on the production of inflammatory mediators that are mechanistically linked to TNBS-induced colitis. Ghrelin administration strikingly reduced protein and mRNA expression of inflammatory cytokines (TNF-␣, IFN-␥, IL-6, IL-1␣, IL-1␤, IL-12, IL-18, IL-17, IL-15, and macrophage migration inhibitory factor [MIF]), chemokines (Rantes, MIP-1␣, MIP-1␤, MIP-3␤, macrophage chemoattractant protein [MCP]-1, MCP-3, interferon-␥ inducible protein [IP]-10, and MIP-2) and chemokine receptors (CCR-1, CCR-2, CCR-3, CCR-5, and CCR-7) in the mucosa of colitic mice (Figure 5). In addition, colons of ghrelin-treated mice showed increased levels of the anti-inflammatory cytokine IL-10 and the chemokine receptors CCR-4 and CCR-8 (Figure 5). The decrease of inflammatory mediators could be a consequence of the diminished infiltration of inflammatory cells in the co-

lonic mucosa in the ghrelin-treated mice. However, LPMC isolated from ghrelin-treated mice produced decreased levels of the inflammatory cytokines TNF-␣ (0.87 ng/mL for TNBS vs 0.23 ng/mL for ghrelin), IL-6 (1.12 ng/mL for TNBS vs 0.31 ng/mL for ghrelin), and MIP-2 (1.43 ng/mL for TNBS vs 0.32 ng/mL for ghrelin) upon in vitro culture. This suggests that, in addition to the reduction of inflammatory infiltration, ghrelin deactivates the colonic inflammatory response. The broad anti-inflammatory activity of ghrelin in the colon was accompanied by down-regulation of the systemic inflammatory response implicated in colonic inflammation (Figure 5). Ghrelin decreased the TNBS-induced increase in the serum levels of the proinflammatory cytokines TNF-␣, IL-1␤, IL-6, and MIP-2 and of the serum amyloid A (SAA), an hepatic acute phase protein involved in inflammation-induced tissue damage.15 We next investigated the effect of ghrelin on the production of inflammatory mediators in established colitis. Administration of ghrelin 5 days after the TNBS infusion decreased the chronic levels of TNF-␣, IL-6, and IFN-␥ observed in colon extracts of colitic animals (Figure 6). In contrast, IL-10 levels were significantly increased (Figure 6).

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Figure 4. Ghrelin protects against established DSS-induced colitis. Colitis was induced by DSS intake. Ghrelin (2 nmol/mouse) was injected IP on 2 alternate days starting at day 4. Body weight loss, macroscopic appearance of the colon, colonic MPO activity, and disease activity index were determined 8 days after the induction of DSS colitis. N ⫽ 8 mice/group. *P ⬍ .001 vs DSS-treated mice.

These results suggest that ghrelin is able to turn off an established in vivo inflammatory response. Ghrelin Suppresses Th1 Cytokine Response, Stimulates IL-10/TGF-␤1 Production, and Induces Regulatory CD4ⴙCD25ⴙFoxp3ⴙ T cells in TNBS-Induced Colitis Although macrophages and neutrophils are the major sources of inflammatory mediators, CD4 T cells have a key role in the initiation and perpetuation of CD, producing IFN-␥, a potent inducer of the inflammatory response.4 In fact, CD and TNBS-induced colitis are

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considered Th1-type cell-mediated autoimmune diseases. High levels of Th1-type cytokines (eg, IFN-␥) are detected in the colon at the height of disease in both CD and TNBS-induced colitis, and conversely, neutralizing Th1-type, cytokine-specific antibodies ameliorate disease progression in the murine model.4,10,16 Therefore, we next determined the effect of in vivo administered ghrelin on the ability of LPMC and draining lymph node cells (mesenteric lymph nodes, MLN) to produce IFN-␥ and proliferate upon in vitro restimulation. Dual-color flow cytometry showed that the isolated LPMC and MLN cells consisted mostly of monocytes and CD4 T cells and that the cellular composition of LPMC isolated from ghrelin-treated and untreated colitic mice was similar, although the number recovered for each cell type was reduced in the ghrelin-treated mice (not shown). LPMC and MLN cells obtained from TNBS-treated mice proliferate more and produced significantly more IFN-␥ than control (ethanol treated) mice, and in vitro activation of these cells caused further cell expansion and increased IFN-␥ production (Figure 7A and B). In contrast, LPMC and MLN cells isolated from ghrelin-treated colitic animals proliferated less and produced significantly lower amounts of IFN-␥, even after stimulation with PMA/Con A (Figure 7A and B). In addition, the production of the regulatory cytokines IL-10 and TGF-␤1 was significantly increased in LPMC and MLN cells obtained from ghrelin-treated mice; in contrast, the Th2-type cytokine IL-4 was not significantly affected (Figure 7B). Thus, ghrelin administration not only reduces Th1 cytokine production in vivo, but it also abrogated the responsiveness of LPMC and draining MLN cells to subsequent in vitro stimulation. The diminished production of IFN-␥ and the capacity of LPMC and MLN cells to proliferate is specific to cells residing in the lamina propria environment or draining lymph nodes because splenocytes from ghrelin-treated and untreated TNBS mice show similar levels of proliferation and IFN-␥ production (not shown). Because decreased IFN-␥ production could be a consequence of either down-regulation of the IFN-␥ release or inhibition of Th1-cell differentiation, and increased IL-10 levels could be due to both macrophages and CD4 T cells, we used flow cytometry in sorted CD4 T cells from LPMC and MLN for intracellular expression of IFN-␥/IL-10. Ghrelin significantly decreased the number of IL-2/IFN-␥-producing Th1 cells and increased the number of IL-10-producing CD4 T cells in LPMN and MLN of TNBStreated mice (Figure 7C). Thus, in vivo administration of ghrelin to colitic mice regulates the generation/differentiation of autoreactive/inflammatory Th1 cells and of regulatory IL-10/TGF-␤-secreting T cells.

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Figure 5. Ghrelin decreases systemic and colonic inflammatory responses in the TNBS model of colitis. Colitis was induced by intracolonic administration of TNBS. Mice were treated IP with ghrelin (2 nmol/mouse) 12 hours after TNBS injection. Mice treated with ethanol alone were used as controls. Serum was collected, and protein extracts and total RNA were obtained from colons at the acute phase of the disease (day 3). (A) Cytokine/chemokine contents in protein extracts were determined by ELISA. N ⫽ 5 or 6 mice/group. (B) Gene expression of several inflammatory/autoimmune mediators was determined by using a microarray. Results are representative of 2 separate experiments (n ⫽ 4 or 5 mice/group). (C) Cytokine/chemokine and SAA contents in the serum were determined by ELISA. N ⫽ 5 or 6 mice/group. *P ⬍ .001 vs TNBS-treated mice.

Although IL-10 and TGF-␤ are factors involved in the function of regulatory T cells (Treg), Treg are not the only source of IL-10 and TGF-␤. To confirm that ghrelin induces the appearance of Treg in colitic mice, we determined the percentage of CD4⫹CD25⫹Foxp3⫹ T cells in MLN. The administration of ghrelin significantly increased the number of CD4⫹CD25⫹Foxp3⫹ T cells in MLN (Figure 8). Interestingly, the increased number of CD4⫹CD25⫹Foxp3⫹ T cells induced by ghrelin persisted for a long period of time, even following a second TNBS dose (Figure 8).

Evidence That GH and IGF-I Do Not Mediate the Effects of Ghrelin Ghrelin acts as a GH secretagogue, inducing the secretion of GH and IGF-I, 2 hormones with antiinflammatory properties in various pathologic conditions, including IBD.17,18 To investigate the possibility that ghrelin acts as an anti-inflammatory agent, at least partially, through the induction of GH/IGF-I, we first measured the levels of GH and IGF-I in serum after the administration of ghrelin. Although serum levels of GH

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Figure 6. Ghrelin decreased chronic inflammatory response in established colitis. Colitis was induced by intracolonic administration of TNBS (1.5 mg/mouse) at days 0 and 6. Mice were treated daily for 3 consecutive days with ghrelin (2 nmol/mouse/day) starting 6 days after TNBS administration (arrow). Protein extracts were obtained from colons on different phases of the disease, and the cytokine contents were determined by ELISA. N ⫽ 5 or 6 mice/group. *P ⬍ .001 vs TNBS-treated mice.

and IGF-I increased 30 minutes after ghrelin administration to TNBS-colitic mice, they returned to baseline levels thereafter (Figure 9A). In addition, in vivo blockage of GH and IGF-I with neutralizing antibodies did not reverse the therapeutic effect of ghrelin in TNBSinduced colitis (Figure 9B). Furthermore, ghrelin inhibited in vitro the production of IFN-␥ and TNF-␣ by stimulated MLN cells from TNBS-treated animals in the presence or absence of anti-GH and anti-IGF-1 Abs (Figure 9C). This suggests a direct effect of ghrelin on the inflammatory cells.

Discussion Ghrelin has received considerable attention for its effects on food intake and adiposity, in addition to its GH-releasing properties.5–7 Recently, it has been reported that ghrelin is a potent anti-inflammatory factor capable of down-regulating the production of proinflammatory cytokines (IL-1␤, IL-6, and TNF-␣) and chemokines (IL-8) by activated monocytes and endothelial cells.8,9 These anti-inflammatory effects could help explain the beneficial effect of ghrelin in several pathologic conditions in which the inflammatory response is exacerbated, including endotoxemia, pancreatitis, myocardial reperfusion injury, and cardiac cachexia.8,9,19 In the present study, we show for the first time that ghrelin provides a highly effective treatment for TNBS-induced colitis, a murine experimental model of CD. Our data demonstrate that a single injection of ghrelin (250 ␮g/

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kg) at the onset of the disease ameliorates the clinical and histopathologic severity of the wasting disease, abrogating body weight loss, diarrhea, and intestinal inflammation and reducing the high mortality caused by this syndrome. From a therapeutic point of view, it is important to take into account that the delayed treatment with ghrelin reduced colitis severity in animals with established disease and that an initial infusion of ghrelin prevented the recurrence of the disease following a second dose of TNBS. This therapeutic effect was not limited to TNBS colitis because ghrelin administration also ameliorated clinical signs in a model of established acute colitis induced by DSS. There are several potential mechanisms for the effect of ghrelin on the effector phase of TNBS colitis. CD and TNBS-induced colitis are characterized by transmural inflammation of the colon because of an IL-12-driven, Th1-cell-mediated response to TNBS-haptenated colonic proteins and/or crossreactive luminal antigens.4,16 Innate and acquired immune responses overlap during the effector phase of bowel inflammation, involving multiple inflammatory mediators. Ghrelin strongly reduced mucosal inflammation by down-regulating the production of a wide panel of mediators involved in the local and systemic inflammatory response. Chemokines are responsible for the mucosal infiltration and activation of various leukocyte populations, which contribute to colitis development.20 The fact that ghrelin treatment reduced the production of multiple chemokines could partially explain the absence of inflammatory infiltrates in the colonic mucosa of mice treated with ghrelin, being especially relevant for chemokines such as MIP-2 (chemotactic for neutrophils), IP-10 (for Th1 cells), and Rantes/MIP-1␣ (for macrophages and T cells), all involved in CD pathogenesis.20,21 Ghrelin has been previously reported to inhibit the production of the chemokine IL-8 and mononuclear cell adhesion in human vascular endothelial cells during inflammation.9 In addition to the regulation of cell recruitment to the lamina propria during colitis, ghrelin regulates inflammatory cell activation and cytokine production. Thus, ghrelin down-regulated the production of the proinflammatory/cytotoxic cytokines TNF-␣, IFN-␥, IL-6, IL-1␤, IL-12, IL-15, IL-17, IL-18, and MIF by mucosal immune cells and increased the levels of the anti-inflammatory cytokine IL-10. The decrease in inflammatory mediators could be the consequence of a diminished infiltration of inflammatory cells in the colonic mucosa in the ghrelintreated TNBS mice. However, the fact that LPMC isolated from ghrelin-treated mice produced lower levels of proinflammatory factors upon in vitro activation argues against this hypothesis. This suggests that, in addition to

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Figure 7. Ghrelin down-regulates Th1 cytokine response and stimulates IL-10 production in TNBS-induced colitis. Colitis was induced by intracolonic administration of TNBS, and mice were treated IP with ghrelin (2 nmol/mouse) 12 hours after TNBS injection. Mice treated with ethanol alone were used as controls. Mesenteric lymph node cells (MLN) and lamina propria mononuclear cells (LPMN) were isolated from the different experimental groups at the peak of the disease (day 3) and cultured with medium alone (unstimulated) or with PMA plus ConA (stimulated). (A) Proliferative response was determined after 4 days, culture as described in the Materials and Methods section. Cytokine contents in the supernatants were determined after 48-hour culture by ELISA. (B) Stimulated lymphocytes were analyzed for CD4 and intracellular cytokine expression by flow cytometry. Double staining for IFN-␥/IL-2 or IL-4/IL-10 expression was performed in gated CD4 T cells. Flow cytometry plots are representative of LPMC. The number of IFN-␥- and IL-10-expressing T cells relative to 104 CD4 T cells was determined (right panels). Data shown represent pooled values from 2 independent experiments (n ⫽ 5 or 6 mice/group/experiment). *P ⬍ .001 vs TNBS-treated mice.

the reduction in inflammatory infiltration, ghrelin deactivates mucosal inflammatory responses. A recent study performed with human cells demonstrated that monocytes are the main targets for the anti-inflammatory action of ghrelin.8 We have also found that ghrelin acts as a macrophage-deactivating factor by down-regulating the production of a wide array of inflammatory mediators (not shown). In fact, tissue macrophages were found to express the ghrelin receptor GHS-R only in inflammatory conditions (unpublished results, M.D. and E.G.–R., 2005). Therefore, it is plausible that deactivation of resident and infiltrating muco-

sal-activated macrophages could be a major mechanism involved in the anti-inflammatory action of ghrelin in colitis. However, because ghrelin receptors are ubiquitously expressed by monocytes/macrophages, B cells, and dendritic cells,8 the participation of other cells cannot be ruled out. In the colon, GHS-R is strongly expressed in mienteric plexus, nerve terminals, and cells scattered throughout the mucosa (ie, mast cells, macrophages, lymphocytes, and endocrine-like cells) but not in the muscle cells nor in epithelial cells (Dass et al22 and unpublished data). In addition, the effect of ghrelin is not limited to cells of colonic mucosa because part of its

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but not IL-4, in CD4 T cells from LPMC and MLN argues against the involvement of a predominant Th2 response. IL-10 is a signature cytokine for a subset of CD4 T cells that exert regulatory functions in the immune response.24 –27 IL-10-secreting Treg plays a key role in the control of self-antigen-reactive T cells and the induction of peripheral tolerance in vivo.24,25,27 Deletion of these regulatory cells results in the appearance of multiple autoimmune disorders, especially in the gastrointestinal tract.27–31 In addition to IL-10, ghrelin increased the production of TGF-␤1 by activated LPMC and MLN cells. TGF-␤1 is another mediFigure 8. Ghrelin induces the appearance of CD4⫹CD25⫹Foxp3⫹ T cells in TNBS-induced colitis. Colitis was induced by intracolonic administration of TNBS. Mice were treated IP with ghrelin (2 nmol/ mouse) 12 hours after TNBS injection. MLN cells were isolated from the different experimental groups at different times after TNBS infusion, and the percentage of CD4⫹CD25⫹Foxp3⫹ cells was determined by flow cytometry. Alternatively, a second TNBS dose (3 mg/mouse) was given at day 8 (arrow) to both untreated (solid circle) and ghrelin-treated (open circle) mice, and percentage of CD4⫹CD25⫹Foxp3⫹ cells was determined 2 days later. N ⫽ 5 mice/group.

effect is exerted in draining lymph nodes, mainly on GHS-R-bearing T cells and macrophages. The local anti-inflammatory action of ghrelin in the colon was reflected systemically. Of special consideration is the reduction by ghrelin of the acute phase protein SAA. Although its precise role during intestinal inflammation is unknown, SAA is used for clinical monitoring of CD and has been involved in tissue damage during several inflammatory conditions.15,23 At this point, the obligatory question is the following: how does ghrelin regulate such a wide spectrum of inflammatory mediators? The answer to this question could be found in the fact that ghrelin down-regulates the activation of the transcription factor nuclear factor␬B,9 a factor essential for the transcriptional activation of most of the inflammatory cytokines and chemokines. On the other hand, TNBS-induced colitis is a Th1mediated disease, requiring T-cell activation as a central initiating event that subsequently leads to macrophage recruitment and activation.4,16 Increased levels of Th1 cytokines (mainly IFN-␥ and TNF-␣) vs low levels of Th2type cytokines (IL-4, IL-5, and IL-10) are involved in chronic inflammation. Our results demonstrate that the expression of the Th1-type cytokines IFN-␥ and TNF-␣ in the colon is down-regulated in response to ghrelin. The inhibition of Th1 responses might be due to a direct action on LPMC and draining MLN cells because LPMC and MLN cells obtained from ghrelin-treated animals were refractory in vitro to Th1-cell restimulation. In contrast, ghrelin treatment increased the production of IL-10 in vivo. However, the fact that ghrelin increased IL-10 expression,

Figure 9. GH and IGF-I are not involved in the therapeutic effect of ghrelin. Colitis was induced by intracolonic administration of TNBS. Mice were treated IP with ghrelin (2 nmol/mouse) 12 hours after TNBS injection. (A) GH and IGF-I levels were determined at different times in serum. N ⫽ 4 mice/group. (B) Mice treated with TNBS and ghrelin were injected with neutralizing anti-GH and/or anti-IGF-I Abs (200 ␮g/mouse/dose) 2 hours and 24 hours after ghrelin administration. Disease severity was determined by body weight changes at day 3. N ⫽ 4 mice/group. (C) MLN cells (5 ⫻ 105 cells/mL) isolated from TNBS-treated mice were stimulated with PMA (10 ng/mL) plus ConA (2.5 ␮g/mL) in the absence (none) or presence of ghrelin (10-7 mol/L). Neutralizing anti-GH and/or anti-IGF-I Abs (20 ␮g/mL) were added to the cultures. After 48-hour culture, cytokines were determined in supernatants by ELISA. N ⫽ 3 experiments performed in duplicate.

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ator of Treg function.32 Our data could suggest that ghrelin favors the generation/activation of IL-10/TGF-␤-secreting Treg cells, a finding that correlates with the increase in the percentage of Foxp3-expressing CD4⫹CD25⫹ T cells in MLN, following ghrelin administration.24 –27 Ghrelin increased the colonic expression of CCR-4 and CCR-8, 2 chemokine receptors expressed by Treg.33–35 This might suggest that ghrelin favors the recruitment of Treg to the inflamed mucosa. However, we did not observe a significant increase in the expression of ligands for CCR4 and CCR8 (CCL17 and CCL1) in colon after ghrelin treatment. Further experiments are needed to elucidate the mechanisms involved in the potential generation of Treg by ghrelin in inflammatory conditions. The maintenance of tolerance by IL-10/TGF-␤-secreting T cells could be related to the resistance to disease recurrence observed in ghrelin-treated mice, mainly because the numbers of CD4⫹CD25⫹Foxp3⫹ T cells persist for a long period of time, even after the administration of a second TNBS dose. In vitro ghrelin affects T cells directly (Dixit et al8 and unpublished data, M.D. and E.G.–R., 2005). Although injection of ghrelin to TNBS-treated mice did not affect the expression of CD80, CD86, and CD40 in MLN CD11c⫹ dendritic cells (not shown), preliminary in vitro experiments from our laboratory showed that ghrelin inhibited the expression of these costimulatory molecules in activated bone marrow-derived dendritic cells. The improvement in colitis score seen in mice treated with ghrelin (approximately 80%) compares favorably with that achieved with other therapies, such as blocking of IL-12, TNF-␣, IL-6, or IFN-␥4,10,36 –38 The capacity of ghrelin to regulate a wide spectrum of inflammatory mediators, together with the suppression of Th1-type responses and the potential generation of Treg, might offer a therapeutic advantage over neutralizing antibodies directed against a single mediator. An additional advantage of ghrelin that should contribute to its therapeutic effect is the gastroprotective effect of ghrelin, especially for intestinal ulcerations.39 On the other hand, the fact that ghrelin acts as a GH secretagogue, inducing the secretion of GH and IGF-I, 2 hormones with antiinflammatory properties,17,18 raises the possibility that ghrelin mediates its in vivo anti-inflammatory action, at least partially, through the induction of the GH/IGF-I. However, ghrelin did not induce a sustained production of systemic GH/IGF-I along the disease, and in vivo neutralization of GH and IGF-I did not abrogate the therapeutic effect of ghrelin on colitis. In addition, the in vitro effect of ghrelin as a macrophage/monocyte-deactivating factor was independent of GH/IGF-I production (Dixit et al8 and present study), supporting a direct effect

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of ghrelin on the inflammatory response. Although these findings argue against a possible mediation of GH or IGF-I on the ghrelin effect, their involvement cannot be totally ruled out because the initial transient GH/IGF-I increase observed in ghrelin-treated animals could elicit an effect, such as modification of signal transduction pathways, that contributes to later effects. Of physiologic relevance is the observation that, in certain pathologic inflammatory states, such as endotoxemia, rheumatoid arthritis, and Helicobacter pylori infection, the secretion of ghrelin drops dramatically.40 – 43 The inhibition of ghrelin secretion during inflammatory processes might potentiate the ongoing inflammatory insult. A reduction in endogenous ghrelin was reported to contribute to the increased incidence of atherosclerosis in patients with obesity.44 Therefore, proposed interventions to decrease ghrelin levels or to block ghrelin receptors for treatment of obesity may result in an exacerbation of ongoing inflammatory insults or lead to immune disregulation. High levels of ghrelin have been found in serum of patients with active CD and ulcerative colitis, with normal levels during CD remission.45 The increased ghrelin levels during acute CD could be interpreted as an acute response to a gastrointestinal inflammatory insult, resulting in the overproduction of endogenous anti-inflammatory factors including ghrelin to counterbalance the ongoing inflammatory process. In summary, here, we describe novel anti-inflammatory actions of ghrelin in the gastrointestinal tract and identify ghrelin as a potent immunomodulatory factor capable of deactivating the intestinal inflammatory response and restoring mucosal immune tolerance at multiple levels. Consequently, the use of ghrelin or the activation of its signaling pathway represents a novel therapeutic approach for the treatment of CD, and possibly of other Th1-mediated inflammatory diseases such as rheumatoid arthritis and multiple sclerosis.

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Received July 18, 2005. Accepted January 11, 2006. Address requests for reprints to: Mario Delgado, MD, Instituto de Parasitologia y Biomedicina, CSIC, Avd. Conocimiento, PT Ciencias de la Salud, Granada 18100, Spain. e-mail: [email protected]; fax: (34) 958-181632. Supported by grants from the Spanish Ministry of Health (PI04/ 0674), the Ramon Areces Foundation, and fellowships from the Junta de Andalucia (to M.D. and E.G-R.) and the Spanish Ministry of Education and Science (to M.D.). The authors thank Dr D. Ganea from Temple University for advice and critical reading of the manuscript.