Leptin: A Pivotal Mediator of Intestinal Inflammation in Mice

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1 GASTROENTEROLOGY 2002;122: Leptin: A Pivotal Mediator of Intestinal Inflammation in Mice BRITTA SIEGMUND,* HANS ANTON LEHR, and GIAMILA FANTUZZI* *Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado; and Institute of Pathology, University of Mainz, Mainz, Germany Background & Aims: In addition to acting as a regulator of food intake and energy expenditure, leptin can also modulate immune and inflammatory responses. The role of leptin in intestinal inflammation is the focus of the present study. Methods: Acute and chronic colitis were induced in leptin-deficient ob/ob or wild-type (WT) mice using dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS). The severity of colitis was evaluated, and possible mechanisms were studied. Results: Leptin directly stimulates intraepithelial lymphocytes (IELs) and lamina propria mononuclear cells (LPMCs). In the DSS acute model, ob/ob mice exhibited a 72% reduction of colitis severity and spontaneous release of proinflammatory cytokines from the colon compared with WT mice. Replacement of leptin in ob/ob mice converted disease resistance to susceptibility, indicating that leptin deficiency, not obesity, accounts for the resistance to acute DSS-induced colitis. During chronic DSS-induced colitis and TNBS-induced colitis, in addition to reduced disease severity, ob/ob mice exhibited a significant attenuation in intestinal inflammation, accompanied by reduced production of cytokines and chemokines. When compared with WT mice, CD8 IELs of ob/ob mice were reduced in number as well as in their ability to synthesize interferon gamma. In addition, LPMCs of ob/ob mice showed increased apoptosis in untreated as well as DSS- or TNBS-treated mice. Phosphorylation of signal transducer and activator of transcription 3 and induction of cyclooxygenase 2 were absent in the colon of DSS-fed ob/ob mice. Conclusions: These results showthat leptin represents a functional link between the endocrine and immune systems. The primary role of leptin, the 16-kilodalton product of the ob gene, is considered to be its inhibitory effect on appetite. However, both leptin-deficient (ob/ob) and leptin receptor deficient (db/db) mice are not merely obese. These mice also develop a complex syndrome characterized by abnormal reproductive function, hormonal imbalances, alterations in the hematopoietic system, and, in particular, a dysfunctional immune system. 1 Leptin-deficient obese mice exhibit immune defects similar to those observed in starved animals and malnourished humans, both associated with low circulating leptin concentrations, as well as in leptin-deficient humans. 2 4 There is long-standing evidence that ob/ob and db/db mice have reduced T-cell function. 5 Leptin promotes T-cell activity in vitro, particularly the responsiveness of naive T-cell subsets. 6 Furthermore, a shift to a predominantly Th1 response is observed when leptin is added to T cells from leptin-deficient mice. However, this phenomenon is not observed in cells from db/db mice, indicating that this effect is mediated by the leptin receptor isoform principally involved in signal transduction (OB-Rb). 6 These in vitro findings are supported by data showing that leptin replacement in vivo can prevent the starvation-induced suppression of T-cell responsiveness in normal mice. 6 In fact, these results and other studies provide strong evidence that leptin, in addition to acting as a satiety factor, shares functional as well as structural similarities with members of the long-chain helical cytokines, suggesting that leptin should be classified as a cytokine. 7,8 These properties of leptin and the fact that leptin is present in nanogram-per-milliliter concentrations in the circulation lead to the intriguing question whether endogenous leptin levels affect inflammatory conditions. Furthermore, leptin could be involved in autoimmune diseases, in which the balance between Th1 and Th2 responses in favor of the Th1 cell subset seems to be of importance. 9,10 The long form of the OB receptor (OB-Rb) was initially described in the hypothalamus but is also present in several other tissues, in particular CD4 and CD8 lymphocytes, colon epithelial cells, endothelial cells, and platelets. 1,11 The OB-Rb cytosolic domain includes bind- Abbreviations used in this paper: COX, cyclooxygenase; DSS, dextran sulfate sodium; IFN, interferon; IL, interleukin; LPMC, lamina propria mononuclear cell; MIP, macrophage inflammatory protein; MPO, myeloperoxidase; OB-Rb, long form of the leptin receptor; STAT, signal transducer and activator of transcription; TNBS, trinitrobenzene sulfonic acid; TNF, tumor necrosis factor; WT, wild-type by the American Gastroenterological Association /02/$35.00 doi: /gast

2 2012 SIEGMUND ET AL. GASTROENTEROLOGY Vol. 122, No. 7 ing motifs associated with the activation of the Janusactivated kinase-signal transducer and activator of transcription (STAT) pathway. 1 Leptin activates STAT-1, STAT-3, and STAT-5. 1 Of particular interest for the present study is STAT-3 activation because hyperactivation of STAT-3, but not STAT-1 or STAT-5, results in severe colitis. 12 In addition, increased levels of phosphorylated STAT-3 were detected in colon samples from patients with Crohn s disease and ulcerative colitis as well as in mice after dextran sulfate sodium (DSS)- induced colitis. 12 Intestinal inflammation is associated with increased production of interleukin (IL)-12, tumor necrosis factor (TNF)-, interferon (IFN)-, and IL In addition, suppression of Th1 responses in the treatment of inflammatory bowel disease is effective in both animals and humans; in animals, antibodies against IL-12 or IL-18, both Th1-inducing cytokines, reduce the severity of colitis, and in patients with Crohn s disease, treatment with anti TNF- results in significant improvement of the severity of clinical colitis. 10,22 To study the role of leptin in intestinal inflammation, the DSS and trinitrobenzene sulfonic acid (TNBS) models were chosen. DSS is believed to cause colitis by interfering with the intestinal epithelial cell barrier function and by inducing regional inflammation within the colon through the up-regulation of both Th1 and Th2 cytokines as well as other inflammatory mediators. 23 The hapten TNBS induces distal colitis that is regulated by complex interactions among various functional subsets of CD4 T cells; TNBS-induced colitis is T cell dependent. 24,25 In the present study, ob/ob and WT mice were first examined in the acute DSS model, with and without leptin replacement. A macroscopic score and colon cytokine release were evaluated. Second, to study a more chronic inflammation, a 30-day time course of DSS was chosen and disease was evaluated by macroscopic and histologic scores as well as cytokine and chemokine release in the colon. Third, the model of TNBS-induced colitis as a model of T-cell dependent colitis was studied to further evaluate the effect of leptin on T cells during intestinal inflammation. In addition, differences in lamina propria mononuclear cells (LPMCs) and intraepithelial lymphocytes (IELs) were analyzed along with potential signaling pathways. Materials and Methods Reagents Recombinant murine leptin was purchased from R&D Systems, Inc. (Minneapolis, MN). RPMI and fetal calf serum were obtained from Life Technologies Inc. (Grand Island, NY). Concanavalin A (con A; type IV-S) was from Sigma Chemical Co. (St. Louis, MO). Mice Animal protocols were approved by the animal studies committee of the University of Colorado Health Sciences Center. Six- to 8-week-old female leptin-deficient (C57BL/6J ob/ob) mice and their wild-type (WT) lean littermates were obtained from Jackson Laboratory (Bar Harbor, ME). The animals were housed at controlled temperature with light-dark cycles, fed standard mice chow pellets, had access to tap water from bottles, and were acclimatized before being studied. At the end of an experimental period, the mice were killed by cervical dislocation under isoflurane anesthesia (Fort Dodge, Iowa City, IA). Induction of Acute DSS-Induced Colitis Mice were fed 3.5% DSS (mol wt, 40 kilodaltons; ICN, Aurora, OH) dissolved in sterile, distilled water ad libitum for 5 days followed by 5 days of normal drinking water, resulting in a 10-day experimental period. 26 Induction of Chronic DSS-Induced Colitis Mice were fed 2.5% DSS dissolved in sterile, distilled water ad libitum for 5 days followed by 5 days of normal drinking water. This cycle was repeated 3 times, resulting in a 30-day experimental period. Control mice were fed tap water (without DSS). The drinking amount per mouse per day was evaluated and found to be equal in each DSS-fed group. Induction of TNBS-Induced Colitis Mice were given a solution of TNBS in phosphatebuffered saline (Research Organics, Cleveland, OH) mixed with an equal volume of ethanol for a final concentration of 2% TNBS in 50% ethanol. Concentrations of TNBS and experimental time schedule used were as described previously in detail. 27 Leptin Replacement For leptin replacement, either ob/ob or WT mice received intraperitoneal injections of murine leptin (1 g/g initial body weight twice daily) or saline for a total of 10 days starting 5 days before the beginning of DSS exposure using the acute time course. Clinical Assessment of Colitis Body weight, the presence of occult or gross blood per rectum, and stool consistency were determined daily. These parameters were assessed by trained individuals blinded to the treatment groups. 26 Baseline body weight, stool consistency, and rectal bleeding were determined before the initiation of DSS or TNBS exposure. Weight change during the experimental time course was calculated as change in percent of the

3 June 2002 LEPTIN IN INTESTINAL INFLAMMATION 2013 weight at baseline. For stool consistency, 0 points were assigned for well-formed pellets, 2 points for pasty and semiformed stools that did not adhere to the anus, and 4 points for liquid stools that did adhere to the anus. Bleeding was scored 0 for no blood using Hemoccult (Beckman Coulter, Palo Alto, CA), 2 points for positive Hemoccult, and 4 points for gross bleeding. The mice were killed by cervical dislocation; the entire colon was removed from the cecum to the anus, and the colon length was measured as a marker of inflammation. Histologic Scoring The entire colon was excised postmortem, and a 1-cm segment of the transverse colon was fixed in 10% buffered formalin for histologic analysis. Paraffin sections were stained with H&E. Four to 6 colon rings were obtained from each 1-cm colon segment and were thus available for histologic examination. Histologic scoring was performed in a blinded fashion by a pathologist (H.A.L.) as a combined score of inflammatory cell infiltration (0 3) and tissue damage (0 3). 26,28 Briefly, the presence of occasional inflammatory cells in the lamina propria was scored as 0, increased numbers of inflammatory cells in the lamina propria as 1, confluence of inflammatory cells extending into the submucosa as 2, and transmural extension of the infiltrate as 3. For tissue damage, no mucosal damage was scored as 0, lymphoepithelial lesions were scored as 1, surface mucosal erosion or focal ulceration scored as 2, and extensive mucosal damage and extension into deeper structures of the bowel wall as 3. The combined histologic score ranged from 0 (no changes) to 6 (extensive cell infiltration and tissue damage). Colon Organ Culture A segment of the colon was removed, cut open longitudinally, and washed in phosphate-buffered saline containing penicillin and streptomycin. The colon was then further cut into strips of approximately 1 cm 2 and placed in 24 flat bottom-well culture plates containing 1 ml of fresh RPMI 1640 supplemented with penicillin and streptomycin. Strips were incubated at 37 C for 24 hours. Culture supernatants were harvested and assayed for cytokines. Protein concentration of the supernatant was quantified using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Myeloperoxidase Assay Myeloperoxidase (MPO) activity (kinetic assay) was measured in snap-frozen colon samples as described previously in detail. 29,30 IEL and LPMC Preparation and Cultivation Mice were killed by isoflurane inhalation and cervical dislocation, and the colon was trimmed of fat, mesenteric tissue, and Peyer s patches. IELs and LPMCs were isolated as described previously with minor modifications. 31 Briefly, the colon was opened longitudinally and cut into 5-mm crosswise pieces. The tissue was incubated in calcium- and magnesiumfree Hank s balanced salt solution containing 1 mmol/l EDTA for 30 minutes under vigorous vortexing at room temperature; this step was repeated once. Supernatants of the EDTA steps were collected and purified by a discontinuous 44/67% Percoll gradient (Sigma Chemical Co.) for 20 minutes at 600g. The remaining tissue was washed twice and subsequently incubated for 60 minutes at 37 C in calcium- and magnesium-free Hank s balanced salt solution supplemented with 5% fetal calf serum and 100 U/mL of collagenases type II and VIII and 300 U/mL hyaluronidase (Sigma Chemical Co.). Cells were separated from tissue debris by filtration through a 100- m cell strainer (BD Pharmingen, San Diego, CA) followed by purification through a discontinuous 44/67% Percoll gradient (Sigma Chemical Co.) for 20 minutes at 600g. Cytokine Measurements Murine IL-18, macrophage inflammatory protein (MIP)- 1, and TNF- levels were measured using an electrochemiluminescence method as described previously. 32,33 Electrochemiluminescence assays were established for murine IL-1 (antibodies and standard from Pierce Endogen, Rockford, IL), IL-10, and MIP-2 (antibodies and standard from R&D Systems, Inc.) as described previously. 32,33 The range of quantification is 20 pg/ml to 10 ng/ml for either TNF-, IL-18, IL-1, IL-10, or MIP-1 and 20 pg/ml to 2 ng/ml for MIP-2. IFN- and IL-6 were measured using a specific enzyme-linked immunosorbent assay (BD Pharmingen). FlowCytometry The isolated IELs and LPMCs were washed twice in staining buffer consisting of phosphate-buffered saline supplemented with 1% fetal calf serum and 0.1% azide. Flow cytometry followed routine procedures using cells per sample. To measure the expression of CD4 and CD8, cells were labeled with either a fluorescein isothiocyanate or PE-labeled antibody (Becton Dickinson, San Diego, CA). Flow-cytometric analysis was conducted on a FACSCalibur (Becton Dickinson) and analyzed using the CELLQUEST analysis program (Becton Dickinson). Intracellular IFN- Staining Using FlowCytometry The mice were killed, and IELs were immediately isolated as previously described. The cells were resuspended at a concentration of /ml and cultured at 37 C inthe presence of 1 L of GolgiPlug per milliliter of cell culture (Becton Dickinson) on CD3 -coated 96 flat bottom-well plates. After 4 hours, cells were harvested and washed once in staining buffer. Fifty microliters of cells ( /ml) were used for surface marker staining for 30 minutes. Cells were washed twice in staining buffer and then resuspended in Cytofix/Cytoperm solution (Becton Dickinson) and incubated for 20 minutes at 4 C. Cells were subsequently washed using Perm/Wash (Becton Dickinson), resuspended in Perm/Wash,

4 2014 SIEGMUND ET AL. GASTROENTEROLOGY Vol. 122, No. 7 and stained for IFN- for 30 minutes at 4 C. Cells were washed and resuspended in staining buffer before flow-cytometric analysis. Analysis of Apoptosis by Annexin Binding Exposure of phosphatidylserine at the outer plasma cell membrane of apoptotic cells was quantified by surface annexin V staining. Briefly, cells were washed in staining buffer, resuspended in 200 L binding buffer (10 mmol/l HEPES, ph 7.4, 2.5 mmol/l CaCl 2, and 140 mmol/l NaCl), and incubated with 0.5 g/ml annexin V/fluorescein isothiocyanate (Becton Dickinson) and 5 g/ml propidium iodide for 15 minutes in the dark. Cells were washed again, resuspended in binding buffer, and analyzed using flow cytometry as previously described. Western Blot Two-centimeter segments of the colon transversum were homogenized in radioimmunoprecipitation assay buffer, and Western blot analysis was performed as described previously in detail. 30 A rabbit anti STAT-3 (1:1000; New England Biolabs Inc., Beverly, MA), a rabbit anti STAT-3YP (1:1000; New England Biolabs Inc.), or a rabbit anti cyclooxygenase (COX)-2 antibody (1:1000; Cayman Chemical Company, Ann Arbor, MI) were used. In Vitro Cell Culture and Immunoprecipitation IELs and LPMCs were isolated as previously described and stimulated at cells/ml (6 mio cells per condition) with either IL-6 (10 ng/ml) or leptin (100 ng/ml) (both from Peprotech, Rocky Hills, NJ) for 15 minutes. The stimulation was terminated by addition of 50 L ice-cold lysis buffer containing 50 mmol/l EDTA. Cells were lysed on ice as previously described. 34 Cell debris was removed by centrifugation for 15 minutes at 14,000 rpm, and the supernatants were immunoprecipitated with anti STAT-3 antiserum (R&D Systems) as described previously. 34 The immunoprecipitates were washed 3 times with lysis buffer and then eluted from the beads by boiling the beads in the sample buffer. Samples were subsequently analyzed by Western blot analysis as previously described for detection of total STAT-3 or by using a monoclonal mouse anti-phosphotyrosine antibody (Zymed Laboratories Inc., South San Francisco, CA) for detection of phosphorylated STAT-3. Statistical Analysis The data are expressed as mean SEM. Statistical significance of differences between treatment and control groups was determined by factorial analysis of variance and a Bonferroni Dunn procedure as a post-hoc test. Statistical analyses were performed using Stat-View 4.51 software (Abacus Concepts, Calabasas, CA). Results Leptin-Stimulated LPMCs and IELs In Vitro To evaluate the direct stimulatory effect of leptin on the LPMC and IEL population, the following experiments were conducted. LPMCs were isolated from the colons of healthy C57BL/6 mice and cultured in the absence or presence of leptin (100 ng/ml) or con A (0.5 g/ml; Figure 1A). Stimulation with leptin significantly increased IL-18 concentration but did not affect TNF-. However, when stimulated with con A, a significant increase in both IL-18 and TNF- levels was observed. The combination of leptin and con A stimulation did not result in a further increase of TNF- or IL-18 levels (data not shown). In a second set of experiments, LPMCs and IELs isolated from WT mice were stimulated with either IL-6 (10 ng/ml) as positive control or leptin (100 ng/ml) for 15 minutes to evaluate the ability of leptin to directly induce STAT-3 phosphorylation. This is of significance, because the binding of leptin to the signal-transducing long isoform of the Figure 1. Direct stimulatory effect of leptin on LPMCs and IELs in vitro. LPMCs and IELs were isolated from the colons of healthy C57BL/6 mice. (A) LPMCs were stimulated for 24 hours in the presence or absence of leptin (100 ng/ml) or con A (0.5 g/ml) as described in Materials and Methods. TNF- and IL-18 concentrations were determined by electrochemiluminescence. Bars represent means SEM, n 4. ***P vs. control. (B) LPMCs or IELs were stimulated for 15 minutes with either IL-6 or leptin. Immunoprecipitation for STAT-3 and subsequent Western blotting for phosphotyrosine was performed as described in Materials and Methods.

5 June 2002 LEPTIN IN INTESTINAL INFLAMMATION 2015 leptin receptor (OB-Rb) results in STAT-3 activation. 35 As shown in Figure 1B, stimulation with either leptin or IL-6 leads to tyrosine phosphorylation of STAT-3 in the IEL and LPMC population. Effective Leptin Replacement in ob/ob Mice To investigate the function of endogenous leptin levels in intestinal inflammation, leptin-deficient ob/ob mice were used because no leptin-blocking agent is currently available. To verify that any difference in ob/ob compared with WT mice was due to leptin deficiency and not to obesity itself, leptin was administered to ob/ob mice to test for reversibility of the observed result. To evaluate the efficacy of leptin administration on the cellularity of lymphoid organs and on cytokine production, ob/ob mice were injected for 10 days with either saline or leptin followed by 5 days of observation. This schedule was consistent with the experimental schedule for the induction of acute colitis. Spleen and thymus cell counts in saline-injected ob/ob mice were significantly lower compared with the cell count in leptin-replaced ob/ob mice. The cell numbers in ob/ob mice receiving leptin replacement were comparable to WT mice (data not shown). To evaluate the effect of leptin replacement on cytokine production, splenocytes were isolated from WT or ob/ob mice and stimulated in vitro with con A for 48 hours. Significantly lower levels of IFN- and TNF- were observed in cultures of splenocytes obtained from saline-injected ob/ob mice when compared with WT mice. Leptin replacement in ob/ob mice completely restored IFN- and TNF- synthesis (data not shown as a figure). These data are in agreement with previously published results. 6,36 Macroscopic Evaluation of Acute DSS-Induced Colitis Using the leptin replacement schedule previously described, the role of leptin in acute DSS-induced colitis was examined. Rectal bleeding and stool consistency scores were determined daily. As shown in Figure 2A and B, when compared with DSS-fed WT mice, DSS-exposed ob/ob mice exhibited significantly less bleeding and had a lower stool consistency score starting at day 4 of DSS exposure and continuing until the end of the experiment on day 10. In addition, DSS-exposed ob/ob mice did not show histologic signs of inflammation, as indicated by a lower histologic score (Figure 2B). Leptin replacement in DSS-exposed ob/ob mice resulted in the full development of colitis severity, as evaluated by the bleeding and stool consistency scores and the histologic score compared with DSS-fed WT mice. Bleeding and stool consistency Figure 2. Evaluation of DSS-induced colitis and effect of leptin replacement. Ob/ob or WT mice were injected with leptin starting 5 days before the beginning of the 5-day DSS exposure, followed by a 5-day observation period. (A) Rectal bleeding and stool consistency were evaluated daily as described in Materials and Methods (ranging from 0 [healthy] to 4 [maximal disease activity]). Non DSS-fed ob/ob and WT mice with or without leptin replacement are summarized as controls, because there were no differences between these experimental conditions. (B) At the end of the experimental period, the histologic score was determined as described in detail in Materials and Methods. Scores are mean SEM; n 5 mice per group. *P 0.05, **P 0.01, ***P vs. DSS-fed ob/ob leptin-replaced, DSS-fed WT, and DSS-fed WT leptin-replaced mice. (C) Colon homogenate from the different experimental groups was evaluated for STAT-3 by Western blot as described in Materials and Methods.

6 2016 SIEGMUND ET AL. GASTROENTEROLOGY Vol. 122, No. 7 scores did not increase in non DSS-exposed mice, regardless of leptin administration (each score 0.5). As previously described, 26,37 the degree of reduction in colon length correlated with the severity of the clinical score at day 10. DSS-fed WT mice exhibited a 14.3% reduction in colon length compared with the non-dss WT control group. A further reduction in colon length (20.9%) was observed in DSS-fed WT mice that received leptin. In contrast, DSS-fed ob/ob mice did not develop colon shortening compared with non-dss ob/ob controls. However, leptin replacement resulted in an 18.3% reduction of colon length in response to DSS in ob/ob mice. Administration of leptin to non-dss ob/ob or WT mice did not affect colon length. As an additional parameter to evaluate the efficacy of leptin reconstitution in DSS-treated ob/ob mice, we determined STAT-3 phosphorylation in the colon homogenate of the different experimental groups (Figure 2C). In agreement with the other results, STAT-3 phosphorylation occurred in DSS-exposed WT and leptin-restored ob/ob mice, whereas no activation could be observed in DSS-exposed ob/ob mice and in non-dss control mice. Evaluation of Cytokine Production in Organ Cultures To evaluate the production of proinflammatory and anti-inflammatory cytokines in the acute DSS model and the effect of leptin replacement, colon segments from the different experimental groups were cultured for 24 hours and the spontaneous release of IL-18, TNF-, IFN-, and IL-10 was determined in the supernatant (Table 1). In DSS-fed WT mice, IL-18, TNF-, and IFN- levels were significantly elevated compared with control WT mice. Administration of leptin in this group did not further increase IL-18 and TNF- release. In contrast, DSS exposure in ob/ob mice did not result in a significant increase of IL-18, TNF-, and IFN-. However, leptin replacement in DSS-fed ob/ob mice converted cytokine production to levels comparable to DSS-fed WT mice. No significant differences could be detected in the different experimental non-dss groups. In contrast to the proinflammatory cytokines described, IL-10 levels were not influenced by injections of leptin. However, independently of leptin administration, IL-10 concentrations were significantly higher in DSS-exposed WT mice compared with DSS-exposed ob/ob mice. This difference was even more pronounced in the non-dss control groups, where significantly higher concentrations of IL-10 were detected in WT controls, with or without administration of leptin, compared with ob/ob controls with or without leptin replacement. Macroscopic and Histologic Evaluation of Chronic DSS-Induced Colitis The results from the acute DSS model show that leptin replacement in ob/ob mice converted disease resistance to susceptibility, indicating that leptin deficiency is responsible for resistance to acute DSSinduced colitis in these mice. To evaluate the role of leptin in intestinal inflammation in more detail, chronic inflammation was induced. To assess severity of colitis during the 30-day time course, weight, rectal bleeding, and diarrhea were determined every other day (Figure 3). DSS exposure in WT mice resulted in a 4% weight loss, whereas the non-dss WT mice showed a 13% weight increase. In contrast, DSSexposed and non-dss ob/ob mice presented with a 33% and 37% weight increase, respectively, over the 30- Table 1. Cytokines in Colon Culture Supernatant After Acute DSS Time Course, With or Without Leptin Replacement IL-18 (pg/mg protein) TNF- (pg/mg protein) IFN- (pg/mg protein) IL-10 (pg/mg protein) Control WT a ob/ob Leptin WT a ob/ob DSS WT a a b ob/ob DSS leptin WT a a b b ob/ob a a b NOTE. Values are means SEM, 5 mice for the DSS-fed groups and 3 mice for the non DSS-exposed groups. a P 0.01 vs. DSS-fed ob/ob mice. b P 0.05 vs. DSS-fed ob/ob mice.

7 June 2002 LEPTIN IN INTESTINAL INFLAMMATION 2017 Figure 3. Clinical score during chronic DSS treatment in ob/ob mice. WT and ob/ob mice were exposed to 2.5% DSS in drinking water for 5 days followed by 5 days of normal drinking water. This cycle was repeated 3 times, resulting in a 30-day experimental period. Body weight, stool consistency, and rectal bleeding were evaluated as described in Materials and Methods. Scores represent mean SEM; n 5 mice per treatment group. *P 0.05, **P 0.01, and ***P vs. DSS ob/ob mice. day period. In addition, ob/ob mice did not develop diarrhea, whereas the DSS-exposed WT mice exhibited diarrhea during the entire experimental period starting from day 5. DSS-exposed ob/ob and WT mice developed bleeding starting from day 5. However, bleeding in the ob/ob mice was significantly less severe than in the WT mice. Histologic examinations of colon sections from DSS-exposed WT mice showed multiple erosive lesions and extensive inflammatory cell infiltration composed mainly of macrophages, lymphocytes, neutrophils, and occasional eosinophils (Figure 4A). In some colon sections, particularly in advanced lesions, regeneration of epithelium was noted in the mucosa. In DSS-fed ob/ob mice, none of these lesions could be found. The histologic score, assessing the extent of inflammatory cell infiltration (0 3) and the degree of tissue damage (0 3), was determined in transversing colon, because it could be shown previously that the most severe lesions in this model occur in the transversing and descending colon. 38 After 30 days, a histologic severity score of was reached in the DSS-exposed WT mice, whereas ob/ob mice did not present a noteworthy inflammatory response to administration of DSS. The histologic severity score of DSSfed ob/ob mice ( ) was not significantly elevated when compared with untreated WT ( ) and ob/ob ( ) mice (Figure 4B). Colon Cytokine and Chemokine Levels in Chronic DSS-Induced Colitis Cytokine release in the model of chronic DSSinduced colitis was determined in the supernatant of colon cultures (Figure 5). Each of the 5 proinflammatory cytokines measured (namely, IFN-, TNF-, IL-18, IL- 1, and IL-6) was significantly lower in the supernatant of DSS-exposed ob/ob mice compared with DSS-exposed WT mice. A significant increase in IL-10 release in DSS-fed WT mice compared with non-dss controls was observed. However, DSS-exposed ob/ob mice did not show a significant increase of this anti-inflammatory cytokine. To explore the involvement of chemokines, MIP-2 and MIP-1 were determined in the same supernatants (Figure 6). Both chemokines were significantly elevated in DSS-fed WT mice. However, DSS-fed ob/ob mice did not show any increase. Furthermore, to evaluate neutrophil infiltration, MPO activity was measured in colon sections. MPO activity was elevated in the colon of DSS-fed WT mice, whereas no increase could be shown in tissue of DSS-fed ob/ob mice compared with non-dss controls (Figure 6). Characterization of IELs and LPMCs in WT and ob/ob Mice by FlowCytometry Different mechanisms can be responsible for the resistance to DSS of ob/ob mice. To determine whether alterations in cell populations and cell responsiveness in

8 2018 SIEGMUND ET AL. GASTROENTEROLOGY Vol. 122, No. 7 WT mice were IFN- positive, only 2.2% of the CD8 IELs of DSS-exposed ob/ob mice produced IFN- (Figure 7A). The evaluation of the LPMC population showed differences between WT and ob/ob mice regarding apoptosis (Figure 7B). Marked differences were observed when comparing apoptosis in non-dss WT and ob/ob mice; 56% 5% of the LPMCs were annexin V in ob/ob mice vs. 21% 2% in WT mice (Figure 7B). After DSS exposure, 51% 4% of LPMCs from ob/ob mice were annexin V compared with 17% 5% in WT mice. STAT-3 Activation and COX-2 Induction in Chronic DSS-Induced Colitis in WT and ob/ob Mice Among the STAT family members, phosphorylated STAT-3 is strongly positive in patients with ulcerative colitis and Crohn s disease as well as in DSSinduced colitis. 12 Activation of the long form of the leptin receptor, OB-Rb, the only isoform of the leptin receptor known to transmit intracellular signaling, results in activation of STAT Western blot analysis was performed for STAT-3 tyrosine phosphorylation in colon sections of DSS-exposed and unexposed WT and Figure 4. Histologic score after chronic DSS time course. At day 30, the end of the chronic DSS time course, a section of the transversing colon was taken for histologic analysis as described in Materials and Methods. (A) Histologic sections of DSS-fed WT and ob/ob mice with increasing magnifications. Because no significant difference between DSS-fed ob/ob and non-dss controls could be detected, the controls are not shown. (B) Mean SEM of the histologic score for 5 sections per treatment group. ***P vs. DSS WT mice. ob/ob mice contribute to the observed differences, CD4 and CD8 IELs were compared in non DSS-exposed WT and ob/ob mice. WT mice presented with an expected 73% CD8 cells. However, ob/ob mice had a 50% reduction in CD8 IELs. Similarly, WT mice had 26% CD8 and 27% CD4 cells in the LPMCs, whereas ob/ob mice had only 15% CD8 and 34% CD4 cells (data not shown). Furthermore, when IELs were isolated from DSS- and non DSS-exposed mice and restimulated in vitro with CD3 for 4 hours followed by staining for intracellular IFN-, these differences became even more striking. Although 7.1% of the CD8 IELs of DSS-fed Figure 5. Spontaneous cytokine production in chronic DSS colon culture supernatant. WT and ob/ob mice were exposed to 2.5% DSS for 5 days followed by 5 days of normal drinking water. This cycle was repeated 3 times, resulting in a 30-day experimental period. Pieces of colon (1 cm 2 ) were cultured for 24 hours as described in Materials and Methods, and cytokines were measured in the culture supernatant. Bars are mean SEM; n 5. *P 0.05, **P 0.01, and ***P vs. DSS WT mice.

9 June 2002 LEPTIN IN INTESTINAL INFLAMMATION 2019 ob/ob mice (Figure 8). Although similar amounts of total (phosphorylated plus nonphosphorylated) STAT-3 were detected in DSS-exposed and unexposed WT and ob/ob mice, phosphorylated STAT-3 could only be measured in DSS-exposed WT mice. COX-2 expression has been associated with protection during intestinal inflammation, 40,41 and macrophages from ob/ob mice show increased COX-2 expression. 42 Therefore, COX-2 levels were evaluated in colon samples. DSS-exposed WT mice showed a marked increase of COX-2, but no increase could be observed in DSS-exposed ob/ob mice (Figure 8C). Figure 6. Chemokines and MPO levels in chronic DSS-induced colitis. WT and ob/ob mice were exposed to the chronic DSS experimental course as described in Materials and Methods. Spontaneous production of MIP-2 and MIP-1 in colon culture supernatant was determined by electrochemiluminescence. MPO activity was measured in colon homogenates as described in Materials and Methods. Means SEM are shown; n 5. ***P vs. DSS WT mice. ob/ob Mice Are Resistant to TNBS-Induced Colitis To further evaluate the effect of leptin on intestinal T-cell populations and the possible implication in intestinal inflammation, experiments with TNBS-induced colitis were conducted. In each parameter analyzed, ob/ob mice showed resistance against TNBS-induced colitis. As shown in Figure 9A, TNBS-treated WT mice showed a 10% weight loss at day 10 compared with a 12% weight gain in the TNBS-treated ob/ob mice. This resistance could be confirmed when the histologic score was analyzed. In fact, TNBS-treated WT mice showed a significant increase in histologic score, whereas TNBS treatment in ob/ob mice did not result in colonic inflammation ( in TNBS-treated WT mice compared with in TNBS-treated ob/ob mice; Figure 9B). In addition, although no lethality was observed in ob/ob Figure 7. Flow-cytometric analysis of IELs after chronic DSS time course. WT and ob/ob mice were either left untreated or were exposed to 2.5% DSS in drinking water for 5 days followed by 5 days of normal drinking water. This cycle was repeated 3 times, resulting in a 30-day experimental period. IELs and LPMCs were subsequently isolated as described in Materials and Methods. (A) The isolated IELs were restimulated with CD3 in vitro for 4 hours and then stained for intracellular IFN- expression as described in detail in Materials and Methods. (B) The isolated LPMCs were stained for annexin V and propidium iodide as described in detail in Materials and Methods. The analysis shown is representative of 4 independent stainings performed per experimental group.

10 2020 SIEGMUND ET AL. GASTROENTEROLOGY Vol. 122, No. 7 production of the chemokine MIP-2 and the Th1 cytokine IFN- was detected in the supernatant of colon cultures obtained from TNBS-treated WT mice, whereas no significant increase could be observed in TNBStreated ob/ob mice (Figure 9D). In the population of LPMCs, a significant increase of apoptosis in the TNBStreated ob/ob mice compared with TNBS-treated WT mice could be detected (36.5% 2.5% vs. 11.1% 3.2%, respectively; Figure 9E). Figure 8. Lack of STAT-3 phosphorylation and COX-2 expression in the colons of DSS-treated ob/ob mice. WT and ob/ob mice were either left untreated or were exposed to 2.5% DSS in drinking water for 5 days followed by 5 days of normal drinking water. This cycle was repeated 3 times, resulting in a 30-day experimental period. At the end of the experiment, a colon section was homogenized in radioimmunoprecipitation assay buffer. Samples were analyzed by Western blot with antibodies specific for either (A) total STAT-3, (B) tyrosine phosphorylated STAT-3, or (C) COX-2. mice during the experimental course, a significant death rate was noted in TNBS-treated WT mice (Figure 9C). No lethality was observed in ethanol-treated WT and ob/ob control mice (not shown). A significant increase in Discussion In the present study, leptin was identified as a critical mediator of intestinal inflammation. Leptin-deficient ob/ob mice were found to be resistant to acute and chronic intestinal inflammation induced by DSS as well as to colitis induced by rectal administration of TNBS. In IELs and LPMCs, leptin stimulation resulted in STAT-3 activation. In addition, stimulation of LPMCs with leptin induced IL-18 production, indicating a direct effect of leptin on these cell populations. The resistance to DSS of ob/ob mice was due to leptin deficiency itself and not to obesity, because a short course of leptin replacement converted disease resistance into susceptibil- Figure 9. ob/ob mice are protected against TNBS-induced colitis. TNBS was administered twice, at days 1 and 7, as described in Materials and Methods. (A) Percentage weight change in the different TNBStreated and untreated groups. (B) Histologic score, evaluated as described in Materials and Methods. (C) Survival rate comparing TNBS-treated WT and ob/ob mice. (D) IFN- and MIP-2 release in the supernatant of colon cultures at the end of the experiment. (E) The isolated LPMCs were stained for annexin V and propidium iodide as described in detail in Materials and Methods. The analysis shown is representative of 4 independent stainings performed per experimental group. Results are mean SEM; n 10 per experimental group; **P 0.01 and ***P vs. TNBS WT mice.

11 June 2002 LEPTIN IN INTESTINAL INFLAMMATION 2021 ity. Levels of proinflammatory cytokines and chemokines as well as neutrophil infiltration into the colonic tissue were significantly suppressed in ob/ob compared with WT mice. Analysis of the IEL population showed a decreased number of CD8 cells in ob/ob mice and reduced production of IFN- by this cell population. In LPMCs of ob/ob mice, increased apoptosis was observed compared with WT mice. Furthermore, phosphorylation of STAT-3 and induction of COX-2 were detected only in DSS-exposed WT mice, whereas no activation occurred in ob/ob mice. In addition, the significant protection of ob/ob mice against TNBS-induced colitis emphasizes the immunomodulatory activity of leptin on intestinal lymphocyte populations. Administration of leptin to ob/ob mice converted resistance to DSS into susceptibility by affecting several parameters. The macroscopic scores for stool consistency, rectal bleeding, and colon length, which are reliable parameters in this model, 26,37,38 indicated that leptin replacement in ob/ob mice led to disease severity comparable to WT mice. Furthermore, colon cytokine production was nearly completely restored by leptin administration in ob/ob mice. Except for production of IFN- from colon cultures, administration of leptin did not significantly aggravate severity of disease in WT mice. This is in contrast with a report showing that leptin administration to WT mice in a model of experimental autoimmune encephalomyelitis exacerbated disease. 43,44 A possible explanation for these contrasting findings may reside in the less important role played by IFN- in DSS versus experimental autoimmune encephalomyelitis. Previous studies have shown that, although IFN- contributes to intestinal inflammation, blockade of IFN- alone is not sufficient to prevent disease. 45,46 The total colon culture system used in the present study permits the evaluation of a broad spectrum of cytokines spontaneously produced by the whole intestine under inflammatory conditions. 12,40 Using these cultures, markedly reduced production of proinflammatory cytokines in ob/ob compared with WT mice was observed in both the DSS and TNBS models. An almost complete suppression of IFN-, TNF-, and IL-1 release was observed in DSS-fed ob/ob mice. Ample evidence exists that each of these cytokines contributes to intestinal inflammation. 23,26,47,48 In particular, blockade of TNF- by infliximab, a chimeric anti TNF- antibody, is effective in the treatment of corticosteroid-refractory Crohn s disease. 10 Levels of IL-18 were also markedly reduced in the supernatant of colon cultures obtained from ob/ob mice. Although the IL-18 assay does not distinguish between the inactive precursor and the active mature form of IL-18, release of IL-18 primarily occurs as the mature form. 33 The present results indicate that reduced levels of bioactive IL-18 are produced by the colon of DSS-fed ob/ob mice. We could recently show that suppression of IL-18 results in an amelioration of DSSinduced colitis. 20 Furthermore, IL-18 synthesis could be localized to the colon epithelium, 20 which is in agreement with earlier studies analyzing colon biopsy specimens from patients with Crohn s disease. 17 Because epithelial cells are important producers of IL-18, which can up-regulate production of both TNF- and IFN-, 49 leptin may act on epithelial cells by increasing IL-18 levels. In the present report, we also showed that leptin directly induces IL-18 production in LPMCs obtained from WT mice. It is important to note that both colon epithelial cells and T lymphocytes express the signal transducing isoform of the leptin receptor, 6,11,50 therefore indicating that leptin may directly influence cytokine production in both cell types. IL-10 exerts protective effects in intestinal inflammation, as indicated by the observation that IL-10 deficient mice develop spontaneous enterocolitis. 55 IL-10 levels are significantly up-regulated in the colons of WT mice receiving DSS long-term. 56 Although a slight increase in IL-10 levels was observed in colon cultures of DSS-exposed ob/ob mice, IL-10 unlikely contributes to the protection of ob/ob mice from intestinal inflammation. Production of the chemokines MIP-2 and MIP-1 was almost completely suppressed in the colons of DSS-fed ob/ob mice compared with WT mice as well as in mice treated with TNBS. Accordingly, neutrophilic infiltrate, as evaluated by MPO activity, was detectable in WT but not in ob/ob mice receiving DSS. We and others have previously shown that ob/ob mice have normal neutrophil counts. 36 Therefore, neutrophils are most likely prevented from migrating into the colon because of reduced chemokine production, as shown in Figure 6. Because chemokine receptor deficient mice are protected from DSS-induced colitis, 57 these results point to an additional mechanism by which leptin deficiency is associated with disease resistance. Previous reports have shown reduced T-cell numbers in ob/ob mice. 1 However, the effect of leptin deficiency on IEL and LPMC populations remains unknown. Although the goal of the present study was to investigate the role of leptin in intestinal inflammation rather than in a specifically T-cell dependent model, T lymphocytes may contribute to DSS-induced colitis. Despite the dem-

12 2022 SIEGMUND ET AL. GASTROENTEROLOGY Vol. 122, No. 7 onstration that the disease can be induced by acute administration of DSS in SCID mice, 58 other studies provide evidence that cyclosporine and FK506 are protective in the acute DSS model, suggesting that T cells participate in disease development. 59,60 Furthermore, specific T-cell subsets exert protective effects following acute administration of DSS. 61 Activation of T cells has been consistently described in chronic DSS-induced colitis. 23 In the present study, we found that ob/ob mice have reduced CD8 cells in the IEL and LPMC population. These cells also produced significantly less IFN- in response to DSS. The evaluation of IEL and LPMC populations suggests that alterations in T-cell distribution and/or activation could contribute to disease resistance in ob/ob mice. This is further emphasized by the observation that ob/ob mice are also resistant against TNBS-induced colitis, which is believed to be T cell mediated. 25 The administration of anti IL-12 and anti IL-6R antibodies, which is protective in models of colitis, affects survival of CD4 T cells mediating inflammation (reviewed in Neurath et al. 19 ). In humans, normal LPMCs exhibit a high susceptibility to Fas-mediated apoptosis, whereas Crohn s disease is associated with resistance of LPMCs against multiple apoptotic pathways. 19 It was previously shown that leptin deficiency results in increased thymocyte apoptosis. 3 In the present report, we show that increased levels of apoptosis are observed in the LPMC population of ob/ob mice compared with WT mice in DSS-induced colitis. Similar results were obtained using the TNBS model. Reduced concentrations of IFN- in the ob/ob mice can contribute to reduced T-cell survival when comparing the sick mice, because IFN- can act as a protective factor against T-cell apoptosis. 62 However, leptin is clearly expressing an independent survival function on LPMCs, because healthy ob/ob mice already show a significant increase of apoptosis compared with healthy WT animals. This is not surprising because leptin is known to enhance phytohemagglutinin- and con A induced proliferation of human T lymphocytes. 50 Similar to IL-6, a role for STAT-3 activation is likely in the proliferative and antiapoptotic effects of leptin. 63 Because apoptosis helps to eliminate potentially autoreactive T cells in the colon, 19 the high levels of cell death observed in ob/ob mice could participate in resistance to intestinal inflammation. The data obtained strongly suggest that the resistances observed in the DSS and TNBS models are mediated through the T cell modifying effects of leptin. However, we cannot exclude that endocrine or epithelial mechanisms are also contributing to the results observed. COX-2 expression and consequent production of prostaglandin E 2 have been associated with protection in various models of intestinal inflammation. 40,41 Because macrophages from ob/ob mice show increased COX-2 expression, 42 levels of this enzyme were compared in DSS-fed WT and ob/ob mice. Although high expression of COX-2 was detectable in the colons of WT mice receiving DSS, no up-regulation of COX-2 was observed in ob/ob mice. These data argue against a relevant antiinflammatory role of COX-2 under the conditions of our experiment. Similar to IL-6, leptin activates STAT-3. Atreya et al. and Yamamoto et al. reported that administration of IL-6R monoclonal antibodies suppresses colitis induced by transfer of CD45Rb high CD4 T cells into SCID mice. 28,64 In addition, Suzuki et al. showed that IL-6 deficient mice develop less severe colitis induced by DSS. 12 STAT-3 probably plays both positive and negative roles in the development of inflammation. In STAT- 3 deficient T cells, IL-6 induced T-cell proliferation is impaired due to the lack of IL-6 mediated prevention of apoptosis. 63 In contrast, suppression of epithelial apoptosis and delayed mammary gland involution is observed in mice with a conditional knockout for STAT-3 in the mammary gland. 65 These data suggest that STAT-3 plays different, sometimes opposite roles in the apoptosis, proliferation, and function of different cell types. However, phosphorylated STAT-3 could be shown in colon biopsy specimens of patients with ulcerative colitis and Crohn s disease as well as in colon samples of mice after DSS-induced colitis, suggesting an involvement for this transcription factor in the inflammatory response. 12 In our study, colonic tissue from DSS-fed WT mice showed a significant increase in phosphorylated STAT-3, whereas phosphorylation of STAT-3 was not detected in the colons of DSS-exposed ob/ob mice. In addition, the in vitro data with IELs and LPMCs, showing the ability of leptin to directly stimulate STAT-3 phosphorylation, support the hypothesis that leptin acts as a significant immunomodulatory factor on T cells in the intestine. The lack of STAT-3 phosphorylation was associated with a marked reduction in IL-6 production from colon cultures of ob/ob mice. Therefore, the question remains whether leptin is directly responsible for STAT-3 phosphorylation during DSS-induced inflammation or whether the lack of STAT-3 phosphorylation observed in leptindeficient mice is an indirect consequence of the lack of inflammation and IL-6 production in these mice.

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