Development of intestinal inflammation in double IL-10-and leptin ...

Development of intestinal inflammation in double IL-10- and leptin-deficient mice Britta Siegmund,* Joseph A. Sennello,† Hans A. Lehr,‡ Arvind Batra,* Inka Fedke,* Martin Zeitz,* and Giamila Fantuzzi†,1 *Charite´ Universitaetsmedizin Berlin, Campus Benjamin Franklin, Germany; †Department of Medicine, University of Colorado Health Sciences Center, Denver; and ‡Institute of Pathology, University of Mainz, Germany

Abstract: Leptin-deficient (ob/ob) mice are resistant in different models of autoimmunity and inflammation, suggesting that leptin regulates immunity and inflammation. To investigate whether leptin deficiency modulates the spontaneous intestinal inflammation observed in interleukin (IL)-10-deficient mice, double IL-10- and leptin-deficient [IL10 knockout (KO) ob/ob] mice were generated and compared with single IL-10 KO mice for colitis severity. Body weight in IL-10 KO ob/ob mice was significantly reduced compared with that of ob/ob mice. However, when compared with wild-type or IL-10 KO mice, IL-10 KO ob/ob mice were still markedly obese. IL-10 KO and IL-10 KO ob/ob mice developed colitis with a comparable timecourse and severity in terms of macroscopic and histologic scores. Likewise, production of interferon-␥, IL-6, and IL-13 from colon cultures and splenocytes did not differ among these two groups. Conversely, rates of apoptosis were higher in lamina propria lymphocytes obtained from the colon of IL-10 KO ob/ob compared with IL-10 KO mice. In conclusion, although leptin deficiency has been associated with resistance in models of autoimmunity and inflammation induced by exogenous stimuli, leptin appears not to play a significant role in the spontaneous colitis of IL-10 KO mice, although it modulates survival of intestinal lymphocytes. J. Leukoc. Biol. 76: 782–786; 2004. Key Words: obesity 䡠 cytokines 䡠 colon 䡠 leptin

INTRODUCTION Leptin was discovered for its effects in the regulation of food intake and metabolism [1]. However, leptin also modulates immune and inflammatory responses, presumably by interacting with functional leptin receptors expressed by many types of immune cells. Although adipocytes are quantitatively the most important source of leptin, a variety of other cell types express this protein. The immune-modulating effects of leptin include protection of T cells from apoptosis, as well as modulation of T cell proliferation and activation, macrophage phagocytosis, and expression of adhesion molecules [2]. 782

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Leptin deficiency—in humans and mice—is associated with reduced production of several cytokines [3–5]. The pattern of cytokines regulated by leptin seems to differ with the type of stimulus used. For example, leptin-deficient mice produce low levels of tumor necrosis factor ␣ (TNF-␣) and interleukin (IL)-18 but not interferon-␥ (IFN-␥) or IL-12, following administration of concanavalin A (Con A), whereas in the same mice, injection of endotoxin leads to production of low levels of IL-10 and IL-1 receptor antagonist but not TNF-␣ or IL-1␤ [6, 7]. Mice deficient for production of leptin (ob/ob mice) are obese and resistant in a variety of experimental models of inflammation or autoimmunity induced by administration of exogenous substances [5, 8, 9]. In particular, ob/ob mice are resistant to intestinal inflammation induced by dextran sulfate sodium or trinitrobenzene sulfonic acid to experimental autoimmune encephalomyelitis induced by immunization with myelin basic protein peptides and to arthritis induced by administration of methylated bovine serum albumin [8 –10]. In these models, resistance to disease in the absence of leptin is associated with reduced cytokine production and in the two latter models, with a shift from a T helper cell type 1 (Th1) to a Th2 pattern of cytokine responses. In humans, a possible role for leptin in regulating intestinal inflammation is suggested by data indicating an increased expression of leptin mRNA in the mesenteric adipose tissue obtained from patients with inflammatory bowel disease [11] Despite these important observations, the role of leptin in regulating inflammatory or autoimmune responses developing spontaneously as a result of alterations in the immune system—a situation that more closely reflects human autoimmune disease— has not been studied in great detail. The only published study about this topic demonstrates that in the nonobese diabetic (NOD) model of type I diabetes, administration of exogenous leptin accelerates destruction of insulin-producing ␤ cells and increases production of IFN-␥ [12]. IL-10-deficient [IL-10 knockout (KO)] mice spontaneously develop chronic intestinal inflammation, which is mediated by CD4⫹ T cells and is associated with enhanced Th1 responses in the early course of disease, and Th2 cytokines progressively increase later on [13, 14]. As colitis in IL-10 KO mice is

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Correspondence: University of Colorado, Department of Medicine, Box B168, 4200 East Ninth Ave., Denver, CO 80262. E-mail: [email protected] Received April 16, 2004; revised May 27, 2004; accepted June 13, 2004; doi: 10.1189/jlb.0404239.

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dependent on the very cell types and cytokines that are thought to be regulated by leptin, we generated leptin-deficient IL-10 KO mice (IL-10 KO ob/ob) to evaluate the role of leptin in a model of spontaneously developing inflammation. Our results indicate that leptin does not play an important role in modulating inflammation in the IL-10 KO model.

Cytokine measurements IFN-␥, IL-6, and IL-13 were measured using specific enzyme-linked immunosorbent assay (BD PharMingen and R&D Systems, Minneapolis, MN).

Flow cytometry Flow cytometry followed routine procedures using 1 ⫻ 105 cells per sample. Staining with Annexin V and propidium iodide (PI; BD PharMingen) was used for evaluation of the rate of apoptosis in LPL. Surface-marker expression was evaluated using antibodies from BD PharMingen and Caltag (S. San Francisco, CA). Analysis was conducted on a FACSCalibur (BD PharMingen) using the CellQuest analysis program (BD PharMingen).

MATERIALS AND METHODS Generation of IL-10 KO ob/ob mice Animal studies were approved by the animal committees of the University of Colorado Health Sciences Center (Denver) and the regional animal study committee of Berlin, Germany. C57BL/6J IL-10 KO ob/ob mice were generated by crossing heterozygote ob/⫹ mice (B6.V-Lepob/J) with IL-10 KO mice (B6.129P2-Il10tm1Cgn/J) on a C57BL6 background obtained from The Jackson Laboratories (Bar Harbor, ME). The progeny was genotyped for IL-10 deficiency using primers 5⬘-gTg ggT gCA gTT ATT gTC TTC CCg-3⬘, 5⬘-gCC TTC AgT ATA AAA ggg ggA CC-3⬘, and 5⬘-CCT gCg TgC AAT CCA TCT Tg-3⬘. This primer combination leads to generation of a 200-bp band for the wild-type (WT) IL-10 allele and of a 450-bp for the IL-10-deficient allele. Mice were genotyped for the single-base mutation in the leptin gene as described [15]. IL-10 ⫹/– ob/⫹ mice were selected and mated. The expected genotype ratios were obtained in the progeny. IL-10 KO and IL-10 KO ob/ob littermates were compared with each other and with their WT (IL-10 ⫹/⫹ or ⫹/⫺) and ob/ob (IL-10 ⫹/⫹ or ⫹/⫺) littermates.

Statistical analysis

Clinical and histological assessment of colitis

IL-10 deficiency is associated with reduced body weight [17], whereas leptin deficiency leads to marked obesity [1]. Body weight was evaluated in IL-10 KO and IL-10 KO ob/ob mice and compared with that of their respective WT littermates. As shown in Figure 1, IL-10 KO ob/ob mice were significantly heavier than IL-10 KO mice at all time-points examined, indicating that lack of leptin leads to obesity even in the context of IL-10 deficiency. However, the body weight of IL-10 KO ob/ob mice was 10 –20% lower than that of ob/ob mice, a percentage difference comparable with that observed between IL-10 KO and WT mice. The observation that IL-10 KO ob/ob mice are obese confirms that these mice are indeed leptindeficient, as also demonstrated by measurement of serum leptin levels, which are undetectable in ob/ob and IL-10 KO ob/ob mice (data not shown).

Mice were weighed weekly and monitored for appearance of diarrhea and blood in the stools. A disease scoring system was used as follows: Appearance of diarrhea was scored as 0 ⫽ well-formed pellets, 2 ⫽ pasty and semi-formed stools that did not adhere to the anus, 4 ⫽ liquid stools that did adhere to the anus. Appearance of blood in the stools was scored as 0 ⫽ no blood using hemoccult (Beckman Coulter, Palo Alto, CA), 2 ⫽ positive hemoccult, 4 ⫽ gross bleeding. Development of rectal prolapse was scored as 0 ⫽ no prolapse, 2 ⫽ prolapse evident only during defecation, 4 ⫽ prolapse evident at all times. The total scores given for diarrhea, stool blood, and prolapse were added and divided by 3, for a maximal disease score of 4. Postmortem, the entire colon was excised, and a 1-cm segment of the transverse colon was fixed in 10% buffered formalin for histological analysis. Paraffin sections were stained with hematoxylin/eosin. Four to six colon rings were obtained from each 1 cm colon segment and were thus available for histological examination. Histological scoring was performed in a blinded manner by a pathologist (H. A. Lehr) as a combined score of inflammatory cell infiltration (0 –3) and tissue damage (0 –3) as described [16].

Data are expressed as mean ⫾ SEM. Statistical significance of differences between treatment and control groups was determined by factorial ANOVA. Statistical analyses were performed using the XLStat software (Addinsoft, Brooklyn, NY).

RESULTS Body weight in IL-10 KO and IL-10 KO ob/ob mice

Colon 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 cut into strips of ⬃1 cm2 and placed in 24 flat-bottom well culture plates containing 1 ml RPMI supplemented with penicillin and streptomycin. Strips were incubated at 37°C for 24 h. Culture supernatants were then harvested and assayed for cytokines.

Preparation of intraepithelial (IEL) and lamina propria (LPL) cells IEL and LPL were isolated from the small intestine and the colon as described previously [10].

Preparation and culture of spleen cells Single-cell suspensions of splenocytes were prepared by passing cells through a 100-␮M strainer. Splenocytes were cultured for 24 h at 2.5 ⫻ 106/ml in 96-well plates in the presence of Con A (1 ␮g/ml) or plate-bound anti-CD3ε antibodies (BD PharMingen, San Diego, CA) at 1 ␮g/ml with or without soluble anti-CD28 antibodies at 1 ␮g/ml.

Fig. 1. Body weight (BW) in IL-10 KO and IL-10 KO ob/ob mice. WT (n⫽16), IL-10 KO (n⫽16), ob/ob (n⫽10), and IL-10 KO ob/ob (n⫽9) mice were weighed weekly starting at an age of 4 weeks. P ⬍ 0.001 for WT versus IL-10 KO and ob/ob versus IL-10 KO ob/ob at all time-points; P ⬍ 0.0001 for WT and IL-10 KO versus ob/ob or IL-10 KO ob/ob at all time-points. Data are mean ⫾ SEM.

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Fig. 2. Clinical disease and histologic score in IL-10 KO and IL-10 KO ob/ob mice. (A) Clinical disease score was calculated weekly for IL-10 KO (n⫽16) and IL-10 KO ob/ob (n⫽12) mice. Differences between the two groups did not reach statistical significance. (B) Histologic score was calculated at week 16. Data are mean ⫾ SEM. ***, P ⬍ 0.001, versus WT or ob/ob mice. Differences between IL-10 KO and IL-10 KO ob/ob mice did not reach statistical significance.

Development of colitis in IL-10 KO and IL-10 KO ob/ob mice IL-10 KO and IL-10 KO ob/ob mice were evaluated weekly starting at week 4 of age, and a clinical disease score was calculated as described in Materials and Methods. No significant differences in disease score were observed between IL-10 KO and IL-10 KO ob/ob mice at any of the time-points examined (Fig. 2A). At week 16, mice were killed and their colon examined histologically for signs of inflammation. As shown in Figure 2B, colonic inflammation—as quantified by a histologic score— did not differ between IL-10 KO and IL-10 KO ob/ob mice.

cells was observed in IEL and LPL populations. However, consistent with a role for leptin in modulating T cell death, a higher rate of apoptotic LPL was present in the colon of IL-10 KO ob/ob mice compared with IL-10 KO mice (Fig. 4).

DISCUSSION In the present report, we demonstrate that leptin deficiency does not alter development of intestinal inflammation in IL-10 KO mice.

Cytokine production Colonic production of IFN-␥ and IL-6 was evaluated from unstimulated colon cultures in IL-10 KO and IL-10 KO ob/ob mice at 16 weeks of age. As shown in Figure 3, no significant difference in production of these two cytokines was observed. Furthermore, splenocytes obtained from IL-10 KO and IL-10 KO ob/ob mice were stimulated with anti-CD3 and anti-CD28, and production of IFN-␥, IL-6, and IL-13 was evaluated. Again, no significant difference was observed for production of any of these cytokines (Table 1). Similarly, LPL obtained from the colon of IL-10 KO and IL-10 KO ob/ob mice produced similar levels of IFN-␥ when stimulated with Con A or platebound anti-CD3 (Table 2).

Characterization of intestinal mononuclear cells As leptin can modulate lymphocyte functions [2], IEL and LPL were isolated from the colon and small intestine of IL-10 KO and IL-10 KO ob/ob mice at 16 weeks of age to evaluate the presence of possible differences. In IL-10 KO and IL-10 KO ob/ob mice, the total number of cells recovered from the colon and the small intestine was strikingly increased compared with cells obtained from WT or ob/ob mice. No significant differences were observed between IL-10 KO and IL-10 KO ob/ob mice in the numbers of cells recovered (data not shown). Similarly, expression of the surface markers T cell receptor (TCR)␣␤, TCR␥␦, CD4, CD8␣, and CD8␤ did not differ in IEL and LPL obtained from the colon or the small intestine of IL-10 KO and IL-10 KO ob/ob mice (data not shown), although a trend toward a lower percentage of double-positive CD4 CD8␣ 784


Fig. 3. Cytokine production from colon cultures. Colon cultures of tissue obtained at week 16 from WT, ob/ob, IL-10 KO, and IL-10 KO ob/ob mice were performed. Levels of IFN-␥ and IL-6 were measured in the supernatant. Data are mean ⫾ SEM of five mice per group. *, P ⬍ 0.05; ***, P ⬍ 0.001, versus WT or ob/ob mice. Differences between IL-10 KO and IL-10 KO ob/ob mice did not reach statistical significance.


TABLE 1. Cytokine Production in Splenocytes Obtained from IL-10 KO and IL-10 KO ob/ob Mice

IFN-␥ IL-6 IL-13

IL-10 KO

IL-10 KO ob/ob

17.79 ⫾ 1.22 3.89 ⫾ 0.54 0.42 ⫾ 0.14

11.55 ⫾ 4.69 3.12 ⫾ 1.25 0.63 ⫾ 0.04

Splenocytes were stimulated with anti-CD3 and anti-CD28. Data are mean ⫾ SEM and are expressed as ng/ml. n ⫽ 6 for IL-10 KO, and n ⫽ 3 for IL-10 KO ob/ob. Differences between IL-10 KO and IL-10 KO ob/ob mice did not reach statistical significance for any of the cytokines measured.

Despite developing severe intestinal inflammation, IL-10 KO ob/ob mice are still markedly obese, suggesting that obesity secondary to leptin deficiency does not require the presence of IL-10. However, similar to IL-10 KO mice, body weight is significantly reduced in IL-10 KO ob/ob mice compared with their littermates not carrying a deletion of the IL-10 gene. As reduced body weight in IL-10 KO mice is a consequence of intestinal inflammation [14], these data indicate that diseaseassociated anorexia and body weight loss are not leptin-dependent. These results are in agreement with previous reports indicating that administration of endotoxin, TNF-␣, or IL-1 can induce anorexia and body weight loss in leptin-deficient and leptin receptor-deficient animals [18, 19]. Leptin is considered an important regulator of production of several cytokines, particularly IFN-␥ [3, 8 –10]. Reduced cytokine production in ob/ob mice is generally associated with disease amelioration, making it difficult to dissociate a possible primary effect of leptin on cytokine production from a secondary reduction as a result of development of less severe disease. In the present report, we demonstrate that production of the cytokines IFN-␥, IL-6, and IL-13 is not altered in IL-10 KO ob/ob mice, demonstrating that the presence of bioactive leptin is not an absolute requirement for these cytokines to be synthesized. These data are in agreement with previous reports demonstrating that the effect of leptin on cytokine production is variable depending on the type of stimulus used [5, 6, 10, 20]. IL-10 KO ob/ob mice develop intestinal inflammation to a similar degree to that observed in their IL-10 KO littermates in terms of clinical disease score and histologic examination of the colon. This observation is fundamentally different from previous data, indicating that ob/ob mice are protected in models of intestinal inflammation induced by administration of exogenous stimuli [10, 21] and that leptin receptor deficiency on T lymphocytes delays disease development in TABLE 2. IFN-␥ Production in LPL Obtained from IL-10 KO and IL-10 KO ob/ob Mice

Con A Anti-CD3

IL-10 KO

IL-10 KO ob/ob

3.37 ⫾ 1.51 3.09 ⫾ 1.53

4.15 ⫾ 1.96 3.11 ⫾ 1.32

LPL were stimulated with Con A or anti-CD3. Data are mean ⫾ SEM and are expressed as ng/ml (n ⫽ 3). Differences between IL-10 KO and IL-10 KO ob/ob mice did not reach statistical significance for any of the cytokines measured.

Fig. 4. High rates of apoptosis in LPL obtained from IL-10 KO ob/ob mice. Colonic LPL were isolated at week 16 from IL-10 KO and IL-10 KO ob/ob mice. Rates of apoptosis were measured by flow cytometry using Annexin V/PI staining. A representative figure obtained from one IL-10 KO and one IL-10 KO ob/ob mouse is shown. A total of three mice per group was evaluated.

the model of colitis induced by transfer of CD4⫹ CD45RBhigh splenocytes into scid recipients [22]. Our present data thus contrast with the concept that ob/ob mice are protected in various models of autoimmune disease and inflammatory conditions [5, 8 –10]. A critical difference between our present report and those previous studies resides in the requirement of exogenous substances or manipulation of cell populations: In IL-10 KO mice, intestinal inflammation develops spontaneously, as the results of “spontaneous” breaking of tolerance toward the enteric flora, whereas each of the alternative models studied to date requires external manipulations in the form of immunization, administration of exogenous substances, or transfer of cells to preactivate the immune system and eventually induce disease. Although similar cell types and soluble mediators are involved in development of autoimmunity and inflammation in each of the above-mentioned models and in the IL-10 KO mouse, our data suggest that fundamentally different mechanisms trigger disease in the two types of systems. In this context, it is important to note that colitis in IL-10 KO mice results from an unopposed immune and inflammatory response to the normal enteric flora [23]. Leptin and IL-10 use signal transducer and activator of transcription 3 (STAT-3) as a critical signal-transduction pathway [10, 24]. Although IL-10-induced activation of STAT-3 in macrophages generates anti-inflammatory effects [24], the same signaling pathway leads to proinflammatory activities

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when leptin is the inducer [25]. Our results indicate that in the absence of leptin and IL-10, the net effect of the lack of the anti-inflammatory mechanisms and subsequent lack of tolerance to colonic bacetria mediated by IL-10 is predominant and cannot effectively be counter-regulated by the absence of leptin. It should be noted that the data presented herein on the IL-10 KO mouse model of spontaneous colitis may not necessarily be transferred to other models of spontaneous colitis in which the specific balance of inflammatory mediators may well leave a critical role for leptin, as suggested in previous studies in NOD mice [12]. Of interest, IL-10 seems to play a pro- rather than antipathogenic role early in disease development in the NOD mouse model of diabetes, indicating a possible inter-relationship between the effects of leptin and IL-10 in regulating immune responses [26]. In the clinical situation, a possible role for leptin in participating in the regulation of intestinal inflammation is suggested by studies showing increased leptin expression in mesenteric adipose tissue obtained from patients affected by Crohn’s disease or ulcerative colitis compared with a control population [11]. However, as the primary pathogenic cause of inflammatory bowel disease is still largely unknown, only more extensive clinical trials in patients affected by Crohn’s disease or ulcerative colitis will eventually indicate whether and to what extent leptin contributes to intestinal inflammation.

ACKNOWLEDGMENTS This work has been supported by grants from the Broad Medical Research Program of the Eli and Edythe L. Broad Foundation, the Crohn’s and Colitis Foundation of America, National Institutes of Health DK061483 (to G. F.), and the Emmy-Noether program (DFG SI 749/3-1) of the Deutsche Forschungsgemeinschaft (to B. S.). The excellent technical assistance of Claudia Braun and Antonietta Valentino is greatly appreciated.

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