Eur J Nutr DOI 10.1007/s00394-014-0772-2
ORIGINAL CONTRIBUTION
Pulverized konjac glucomannan ameliorates oxazolone-induced colitis in mice Toshiko Onitake • Yoshitaka Ueno • Shinji Tanaka • Shintaro Sagami • Ryohei Hayashi Kenta Nagai • Michihiro Hide • Kazuaki Chayama
•
Received: 9 April 2014 / Accepted: 1 August 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose Pulverized konjac glucomannan (PKGM) is a natural biologically active compound extracted from konjac, a Japanese traditional food. In the present study, we investigated the role of PKGM in intestinal immunity in a mouse model of oxazolone (OXA)-induced colitis. Methods C57BL/6(B6) mice were fed PKGM or control food from 2 weeks before the induction of OXA colitis. Body weight change, colon length, and histological change in the colon were examined. The mononuclear cells were purified from colon and stimulated with PMA/ionomycin. The levels of TNF-a, interferon (IFN)-c, interleukin (IL)-4, and IL-13 from the supernatant were measured by ELISA. Results Oral administration of PKGM prevented the body weight loss and shortening of colon length associated with OXA-induced colitis. Histological analysis revealed that the colonic inflammation was improved by the administration of PKGM. The levels of IL-4 and IL-13, the critical inflammatory cytokines in OXA colitis, derived from mononuclear cells from the lamina propria of the colon were significantly suppressed by PKGM administration. PKGM-fed mice showed a significantly lower IL-4/IFN-c
ratio in the colonic lamina propria compared with that in control-fed mice. Fluorescence-activated cell sorting analysis revealed that natural killer (NK) 1.1? T cells in the liver were significantly decreased in PKGM-fed mice. Finally, the preventive role of PKGM in OXA-induced colitis was not observed in invariant natural killer T celldeficient mice. Conclusions PKGM ameliorated OXA-induced colitis in mice. This effect is associated with a decreased population of NK1.1? T cells and induction of Th1-polarized immune responses. Keywords Konjac glucomannan Oxazolone colitis IL-13 Th1/Th2 balance NKT cell Abbreviations AD Atopic dermatitis iNKT Invariant natural killer T KGM Konjac glucomannan LPMCs Lamina propria mononuclear cells OXA Oxazolone PKGM Pulverized konjac glucomannan UC Ulcerative colitis
T. Onitake S. Sagami R. Hayashi K. Nagai K. Chayama Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan Y. Ueno (&) S. Tanaka Department of Endoscopy, Hiroshima University Hospital, Hiroshima, Japan e-mail: [email protected] M. Hide Department of Dermatology, Integrated Health Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
Introduction Ulcerative colitis (UC) is a chronic inflammatory disorder of the colon, but its etiology is far from being understood in detail. Despite various treatments for UC, patients do not recover completely and frequently undergo repeat relapse and remission. Although the mechanisms underlying UC are not poorly understood, much of the chronic
123
Eur J Nutr
inflammation of the colon that characterizes this disease is thought to be due to inappropriate activation of the immune system [1–3]. Currently, patients with UC are usually treated with 5-aminosalicylic acid and/or corticosteroids. However, the prolonged use of corticosteroids may be limited by potential adverse effects, such as osteoporosis, fracture, infection, and cataracts. Various alternative pharmacological approaches have been put forward to decrease the dosage of steroids to decrease their side effects [4]. Konjac is a glucomannan-rich Japanese traditional food, which is derived from the tubers of Amorphophallus konjac, and has been consumed traditionally in Asia. Konjac glucomannan (KGM) is a highly viscous polysaccharide composed of glucose and mannose residues at a molar ratio of 1:1.6 joined through b1,4-glucosidic linkages. It has proved to be a health or dietary food due to its watersoluble fibers. Numerous clinical studies have been published on KGM, which ameliorates glycemia and other associated risk factors for coronary heart disease in type 2 diabetes [5] and delays the absorption of both cholesterol and triglyceride from the small intestine [6]. Moreover, KGM is effective on intestine by modulating cecal and fecal microflora in mice [7], and it inhibits carcinogenesis in rat [8]. However, the precise mechanisms underlying the immunologic effects of KGM remain largely unclear. Nutritional supplementation with highly viscous KGM is restricted on account of its low solubility, which requires long time intervals to attain peak viscosity. To circumvent this restriction, we prepared highly specific surface area of the KGM molecule that would be expected to possess some extent of these well-known physiological activities [9]. To investigate the immunomodulating potency of low-viscous KGM, several particle sizes of pulverized KGM (PKGM) were used in NC/Nga mice, a murine model for human AD [10]. Production of hyper-IgE and inflammatory cytokines was suppressed in the PKGM-fed mice, and AD-like skin symptoms were markedly suppressed by oral intake of PKGM [11, 12]. There are several studies on the effects of carbohydrates like inulin, galactomannan, and glucomannan in the form of KGM [13, 14]. So far, however, there have been no reports demonstrating the effect of PKGM on intestinal inflammation. Experimental animal models are indispensable for the accurate understanding of the pathogenesis of human diseases. Support for this view may be drawn from animal models of colitis, which consistently exhibit an imbalance of regulatory and inflammatory cytokines. Rectal instillation of the haptenating reagent oxazolone (OXA) leads to rapid development of colitis limited to the distal half of the colon. The histologic features and distribution of OXA colitis have characteristics that resemble those of UC [15]. The inflammation is characterized by increased secretion of
123
IL-4 and IL-5 and can be ameliorated by the administration of anti-IL-4 monoclonal antibodies [15]. Therefore, this model has been used to study the contribution of the Th2dependent immune response to intestinal inflammation. IL-13 seems to be a key pathogenic cytokine of OXA colitis because IL-13 neutralization prevents its development [16]. IL-13 is known as an inflammatory cytokine and is considered a growth factor for epithelial cells in asthma [17]. Recently, it has been reported that IL-13 is also critical for the pathogenesis of UC [18]. Therefore, IL-13 may be a candidate for the treatment of UC. IL-13 is produced by iNK Tcells in OXA colitis in mice [16] and by non-invariant NKT cells in UC [19]. In the present study, we report that dietary PKGM was effective in reducing inflammation in OXA colitis in mice. We show that PKGM reduced colonic inflammation and suppressed the mediators of both innate and adaptive immunity. Mechanistic studies carried out using mononuclear cells derived from intestinal lamina propria show that PKGM effectively regulates the function of effector T cells by shifting from a Th2- to a Th1-dependent immune response.
Materials and methods KGM powders Two kinds of low-viscous KGM powders, PA (PROPOLÒ; Shimizu Chemical Corporation, Hiroshima, Japan) and pulverized PA (PKGM) (Nishikawa Rubber Corporation, Hiroshima, Japan), were used in this study. The GM contents were measured to be approximately 99 % for PA and 97 % for PKGM. The mean particle sizes of the KGM powders were estimated to be about 300 lm for PA and 105 lm for PKGM. The peak velocity of the 1 % PA solutions reached more than 100,000 mPa s after 7 h at 25 °C, while that of PKGM solutions attained values of 32,000–35,000 mPa s within 0.5 h. Animals Female C57BL/6(B6) mice (6–8 weeks old) were purchased from CLEA JAPAN (Tokyo, Japan). Female Ja281deficient [Va14NKT cell-deficient (KO)] mice on B6 background (6–10 weeks old) were generated as described previously [20]. Mice were divided into eight groups in clear plastic cages with a mesh top under specific pathogen-free conditions, kept at room temperature in a controlled environment at 22 ± 2 °C (12-h dark/light cycle), and had free access to food and water. The food was formed to a typical murine solid feed which firmed KGM powders. Food and water were changed fresh weekly.
Eur J Nutr
Animal care was in compliance with the regulations of Hiroshima University. Feeding The mice were fed either with a commercial rodent diet, MF (Oriental Yeast, Tokyo, Japan), PA diet (MF containing 5 % (w/w) PA powder), or the PKGM diet (MF containing 5 % (w/w) PKGM powder) ad libitum. We started to presensitize the mice with OXA 2 weeks after the start of feeding with either diet. The food intake in the different experimental groups was similar because there was no difference in the body weights 2 weeks after the intake of each diet. Induction of colitis OXA colitis was induced as previously described, with minor modifications [15]. OXA (4-ethoxymethylene-2phenyl-2-OXA-5-one) was obtained from Sigma-Aldrich (St. Louis, MO). To presensitize the mice, a 1.5 9 1.5 cm field of the abdominal skin was shaved, and 200 ll of a 3 % solution of OXA in 100 % ethanol or 100 % ethanol alone was applied. Five days after presensitization, the mice underwent general anesthesia with isoflurane and were rechallenged intrarectally with 150 ll 1 % OXA in 50 % ethanol or, for the control-fed mice, 50 % ethanol alone. To ensure distribution of OXA within the entire colon and cecum, mice were held in a head-down vertical position for 60 s after the intrarectal injection. Mice were then monitored for the appearance of diarrhea, loss of body weight, and overall mortality.
damage affecting the submucosa as 3. The combined inflammatory and injury scores resulted in an overall score ranging from 0 (no changes) to 6 (severe inflammatory infiltrate and mucosal damage). Isolation and purification of mononuclear cells Mice were killed at 2 days after colitis induction, and hepatic mononuclear cells or lamina propria mononuclear cells (LPMCs) were isolated. Hepatic mononuclear cells were isolated by grinding the tissue in a Petri dish and filtering the cell suspension through a 40-lm mesh. Mononuclear cells were isolated using 44 and 66 % solutions of Percoll. LPMCs were isolated as previously described in detail [22, 23]. In brief, LPMCs were isolated after removal of epithelial cells by incubation of colon strips in Ca??Mg??-free Hanks’ balanced salt solution containing 1 mM EDTA (SigmaAldrich) for 15 min at 37 °C. Mononuclear cells were released by digesting the tissue in RPMI-1,640 medium (Sigma-Aldrich) supplemented with 150 U/ml collagenase (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and incubated for 1.5 h at 37 °C with stirring. Finally, leukocytes were separated from epithelial cells by centrifugation in 44 and 66 % solutions of Percoll (pH 8.5–9.5) (Sigma-Aldrich). Cell culture for cytokine production The cells were harvested and stimulated in vitro with 5 lg/ml of plate-bound anti-CD3 antibody (145-2C11) (BD Biosciences Pharmingen, San Diego, CA), 5 lg/ml of PMA ? ionomycin, or Con A for 48 h at 37 °C. Supernatants were collected and stored at -80 °C until further analysis.
Histological examination of colitis ELISA assays Colon samples were removed from mice at 5 days after induction of colitis. The colons were opened longitudinally and rolled concentrically. The tissues were then fixed in 10 % buffered formalin and embedded in paraffin. Paraffin sections were made and stained with hematoxylin and eosin. Histological examination was evaluated as previously described [21]. The degree of inflammation and epithelial injury on microscopic cross-section was graded semiquantitatively on a scale of 0–6. Inflammatory cell infiltration was scored from 0 to 3, and tissue damage was scored from 0 to 3. Occasional or no inflammatory infiltrate in the lamina propria was scored as 0, increased numbers of inflammatory cells restricted to the lamina propria as 1, inflammatory infiltrates reaching the submucosa as 2, and transmural inflammation as 3. The tissue damage subscore took into account epithelial lesions: No mucosal damage was scored as 0, focal crypt lesions as 1, surface mucosal erosions or focal ulcerations as 2, and extensive mucosal
Cytokine concentrations in culture supernatants were measured by ELISA kits according to the manufacturers’ instructions. IFN-c, TNF-a, and IL-4 were measured with OptEIA ELISA sets (BD Biosciences Pharmingen), and IL13 was measured with Quantikine M ELISA kit (R&D Systems, Minneapolis, MN). FACS analysis Cells were stained with antibodies to CD3 (17A2) and NK1.1 (NKR-P1B and NKR-PIC). Surface staining cells were analyzed using a fluorescence-activated cell sorting (FACS) Calibur system (BD Biosciences). Percentage values were calculated with CellQuest software (BD Biosciences) from the scatter plots, which were attained with propidium iodide (Sigma-Aldrich) staining after gating on living lymphocytes.
123
Eur J Nutr
Statistical analysis
Results
Data are expressed as mean ± SD of N observations. All values in the figures and text were analyzed with the Japanese version of JMP (SAS Institute Inc., Cary, NC, USA), and the data sets were examined with one-way ANOVA. Differences were considered significant at p \ 0.05.
Preventive role of dietary PKGM in OXA-induced colitis
% Body weight from day 0
A
To examine the role of PKGM in the development of OXA-induced colitis in vivo, we administrated the chow
B
5
0
Ethanol OXA
-5
OXA+PKGM
-10
-15
Ethanol OXA+PKGM OXA+PA OXA
0
OXA+PA
1
2
3
4
5
6
7
day
C 10
Colon length, cm
D
OXA
* 8
6
Ethanol
OXA
OXA
OXA+PA
+ PKGM
E
Ethanol
OXA+PKGM
OXA+ PA
6
*
5
Score
4 3 2 1 0 Ethanol
OXA
OXA
OXA+PA
+ PKGM
Fig. 1 Pulverized konjac glucomannan (PKGM)-fed mice are less susceptible than control or PA-fed mice to oxazolone (OXA)-induced colitis. a Body weight change in mice induced by OXA colitis. Mice were fed with PKGM, PA, or control diet for 2 weeks. OXA colitis was induced by sensitizing mice with OXA, followed by intrarectal administration of the hapten reagent after 5 days. Body weight was monitored after oxazolone rechallenge at the indicated time points. Mean value ± SEM from 2 representative experiments of 6 is shown. For this experiment, 5–8 control diet-fed, PKGM-fed, and PA-fed mice were used. *p \ 0.05, OXA ? control-fed group versus OXA ? PKGM-fed group. **p \ 0.05, OXA ? PA-fed group versus OXA ? PKGM-fed group. b Macroscopic changes in colons on day 3
123
after OXA administration. Representative photographs of dissected large intestine from ethanol-treated control (top), mice treated with OXA ? control (second row), OXA ? PKGM (third row), and OXA ? PA (bottom row) on day 3 after induction of colitis. c Colon length of mice measured on day 5 after OXA administration. Each bar represents 6 mice. *p \ 0.01, OXA ? control-fed group versus OXA ? PKGM-fed group. d Histological sections of colonic inflammation in control or PA-fed or PKGM-fed mice upon OXA administration. e Quantitative histopathological assessment of colitis activity. *p \ 0.05, OXA ? control-fed group versus OXA ? PKGM-fed group. Data represent mean value ± SD from 1 representative experiment of 5 mice for each group
Eur J Nutr
containing 5 % PKGM or 5 % PA to C57BL/6 mice for 14 days before OXA treatment. Two days after rectal administration of OXA, PKGM-fed mice had lost about 12 % of their initial body weight. However, PKGM-fed mice showed a significant improvement in body weight from day 4 to day 5 after 1 % OXA administration as indicated in Fig. 1a (p \ 0.05). PA-fed mice recovered their body weight loss later than PKGM-fed mice. Macroscopic examinations on day 5 after 1 % OXA administration revealed that shortening of colon length was significantly more inhibited in PKGM-fed mice than in control or PA-fed mice (Fig. 1b, c; p \ 0.05). The colons of control or PA-fed mice were more reddish due to bloody stool compared with those of PKGM-fed mice, suggesting that a less severe intestinal inflammation had occurred in the PKGM-fed mice (Fig. 1b). On histological examination of involved colon in the 1 % OXA-treated PKGM-fed mice, we observed a superficial inflammation characterized by the presence of epithelial cell loss, patchy ulceration, pronounced depletion of mucin-producing goblet cells, and a reduction in the density of the tubular glands. These histopathological changes in OXA-induced colitis were significantly less severe in the colon of PKGM-fed mice than in control and PA-fed mice on day 5 after 1 % OXA administration (Fig. 1d, e; p \ 0.05). These data suggest that treatment with dietary PKGM prevents intestinal inflammation induced by OXA. Cytokine production of PKGM-fed mice with oxazolone-induced colitis To investigate whether PKGM treatment has immunological modulations, we examined production of local cytokines. Pro-inflammatory cytokine TNF-a from the mononuclear cells of the colon was induced by OXA colitis, which was suppressed by the administration of PKGM (Fig. 2a, p \ 0.05). The mononuclear cells from colon of mice administered OXA produced IL-4 and IL-13, major Th2 cytokines, which were also significantly inhibited by feeding with PKGM (Fig. 2b, c; p \ 0.05). No change in the level of Th1 cytokine IFN-c from LPMCs was observed in OXA colitis LPMCs (Fig. 2d). When we compared the IL-4/IFN-c ratio, a significantly lower ratio was found in the PKGM-fed mice versus the control-fed mice in LPMCs (Fig. 2e). These data raise the possibility that PKGM has a capacity to suppress Th2-related but not Th1-related cytokines induced by OXA. Effect of PKGM treatment on NK1.1? T cell population To identify the target cells involved in the prevention OXA-induced colitis in PKGM-fed mice, we isolated
lymphocytes from liver and colon and investigated a surface antigen of the cells. Flow cytometric analysis revealed that the frequency of NK1.1? T cells significantly decreased in liver of the PKGM-fed mice compared with that observed in liver of control or PA-fed mice (Fig. 3; Table 1, p \ 0.05). There was no change in the population of NK1.1? CD3- cells or NK1.1- CD3? cells. There was no difference in the frequency of NK1.1? T cells in the colon between PKGM- and control-fed mice (data not shown). Therefore, iNK1.1 T cells in the liver were selectively decreased by treatment with dietary PKGM. PKGM does not improve OXA-induced colitis in iNKT cell-deficient mice Because PKGM administration reduced NK1.1? T cells in the liver of B6 mice, experiments were carried out to further define the immune compartment mediating PKGM actions in vivo. For this purpose, we tested PKGM in Ja281-deficient [Va14NKT cell-deficient (KO)] mice. Treatment of these mice with rectal application of OXA resulted in similar weight loss pattern (Fig. 4a) and colon shortening (Fig. 4b) between PKGM-fed mice and PA-fed mice because of massive inflammation of the colonic tissue (Fig. 4c, d). In this experimental model, PKGM was unable to reverse local signs of inflammation caused by OXA administration. Although the levels of TNF-a in the colon were significantly increased after OXA treatment (Fig. 5a), no increase was found in the levels of IL-4 or IL-13 in the colon (Fig. 5b, c). Moreover, IFN-c secretion was not induced by OXA treatment (Fig. 5d), and the Th1/Th2 ratio did not change by PKGM treatment (Fig. 5e). These data suggested that mechanisms other than induction of IL13 or skewing of the Th1/Th2 ratio may be involved in the intestinal inflammation produced in KO mice by OXA. These findings suggest that PKGM, at least partially, acts directly on iNKT cells in vivo.
Discussion In this study, we showed that PKGM was effective for the prevention of Th-2-mediated OXA colitis by skewing the Th1/Th2 balance from Th2 toward Th1. PKGM decreased the population of NK1.1? T cells. The protective effect of PKGM against OXA colitis was absent in the presence of iNKT cell deficiency. Konjac is known to be a healthy, traditional food in Japan and several other Asian countries. KGM is a polysaccharide, which is used as food in paste or coagulated form. KGM itself is a soluble dietary fiber, but the coagulated form of KGM is not water soluble and has strong elasticity. Thus, KGM is popular as a dietary food because
123
Eur J Nutr
A 1600
B
1400 1200
*
1200
IL-4, pg/ml
TNF-α, pg/ml
1400
1000 800 600
800 600 400
200
200 Ethanol
OXA
OXA
0
OXA+PA
*
1000
400
0
Ethanol
OXA
+ PKGM 700
D
*
3000
500
2500
400 300 200
Ethanol
OXA
OXA
OXA+PA
+ PKGM 0.7
1500 1000
0 Ethanol
OXA
OXA
OXA+ PA
+ PKGM
*
0.6
IL-4/ IFN-
2000
500
100
E
OXA+PA
3500
600
0
OXA
+ PKGM
IFN-γ,, pg/ml
C
IL-13, pg/ml
Fig. 2 Cytokine production from colonic lymphocytes of mice with oxazolone (OXA) colitis. Cytokine production by mononuclear cells of the colon from control, PA-fed, and pulverized konjac glucomannan (PKGM)-fed mice with OXAinduced colitis. Colonic mononuclear cells were stimulated with PMA/ ionomycin for 48 h, followed by analysis of culture supernatants using ELISA. Data represent mean values of 4–8 mice per group. a TNF-a, b IL-4, c IL13, d IFN-c, and e Th1/Th2 ratio. *p \ 0.05, OXA ? control-fed group versus OXA ? PKGM-fed group
0.5 0.4 0.3 0.2 0.1 0
Ethanol
OXA
OXA
OXA+PA
+ PKGM
of its very low caloric value and the large amount of dietary fiber it provides. KGM is not degradable by human digestive enzymes but is available to be partially converted to a fatty acid by the intestinal microflora [7]. Moreover, it has been reported that KGM blocks the absorption of cholesterol in the small intestine and maintains a lower plasma cholesterol level [6], and it is effective in protecting against diabetes because KGM suppresses the rapid absorption of glucose from the upper small intestine, thus sparing the secretion of insulin [24]. It is also reported that KGM has a therapeutic effect on human and mouse dermatitis. Oral intake of konjac
123
ceramide improves both skin symptoms in children with AD and allergen-specific IgE production with skewing of the cytokine pattern toward the Th1 type in allergenstimulated cultures [25]. Oral intake of konjac-derived glucosylceramide improved skin roughness caused by SDS treatment in mice [26]. The KGM that has been used until now, however, has high viscosity and low solubility that requires long time intervals to attain peak viscosity. Therefore, we prepared various particle sizes of low viscous and highly specific surface area of the KGM molecule that would be expected to possess the more effect of these well-known
Eur J Nutr
NK1.1
OXA
Ethanol 3.2
6.2
69.7
68.8
OXA+PKGM
OXA+PA
3.8
5.8
69.8
70.4
CD3 Fig. 3 Representative FACS pattern of liver lymphocytes. Mononuclear cells from liver were obtained and stained with anti-NK1.1 mAb and anti-CD3 mAb for evaluation by flow cytometry
physiological activities. Production of IL-4 and IFN-c in splenocytes of NC/Nga mice, an experimental model of human AD, was decreased in the PKGM-fed mice, and AD-like skin symptoms were markedly suppressed by oral intake of PKGM [11, 12]. Soluble PKGM suppressed IgE production in splenocytes of mice in vitro [27]. Both the Th1 and Th2 cytokines play important roles in AD [28], and Th2-specific chemokines are overproduced in NC/Nga mice [29]. OXA colitis is a well-known experimental model of human UC. Rectal administration of the hapten reagent OXA induces a severe colitis in mice that is characterized by weight reduction and diarrhea and which leads to high death rates [15]. Superficial inflammation includes epithelial cell loss and patchy ulceration as well as pronounced depletion of goblet cells accompanied by a mixed inflammatory infiltrate of lymphocytes and granulocytes. This inflammation is characterized by increased IL-4 and IL-13 secretion and can be ameliorated by the administration of anti-IL-4 or anti-IL-13 monoclonal antibodies [15]. In the present study, PKGM suppressed exacerbation of OXA colitis. PKGM but not PA specifically suppressed body weight loss, shortening of colon length, and histological
inflammation of the colon. PKGM and PA have been shown to have different immunological effects. Onishi et al. [9] showed that PKGM but not PA suppressed IL-4 and IFN-c from spleen cells in mice. We suppose that PKGM, with a smaller particle size than that of KGM, might gain its protective effect because it possesses a lower viscosity and has higher absorbance in the intestine, although the mechanisms are not fully understood. The exact mechanisms by which PKGM but not PA induces immunosuppression are also not completely understood. In the present study, we clearly showed that PKGM substantially reduced the inflammatory response. PKGM treatment decreased Th2 cytokine levels such as those of IL-4 and IL-13, but there was no change in levels of IFN-c, known as a Th1 cytokine. PKGM suppressed the levels of IL-4 production in ConA-stimulated mononuclear cells in vitro, indicating that the effect of PKGM on Th2 cells may directly affect Th2 differentiation rather than lymphocyte trafficking. The Th2/Th1 ratio, represented by the IL-4/IFN-c ratio, was significantly lower in the PKGMfed mice relative to the control and PA-fed mice. We also found that PKGM suppressed IL-13 from mononuclear cells in the colon. It is well known that IL-13 is a key cytokine in OXA-induced colitis [5] and is an important effector cytokine in human UC that impairs epithelial barrier function by affecting epithelial apoptosis, tight junctions, and restitution velocity [30]. Recent studies found that IFN-b-1a suppresses inflammation in human UC by inhibiting IL-13 production [31] and that colitis and intestinal inflammation in IL10-/- mice resulted from IL13Ra2-mediated attenuation of IL-13 activity [32]. In the present study, the reduction in IL-13 was already shown on day 2 after 1 % OXA administration, and we have proved that PKGM suppressed OXA colitis because of the decrease in IL-13 production. Moreover, we examined which subset of cells was influenced by PKGM in OXA-induced colitis. Interestingly, the population of NK1.1? T cells in the liver significantly decreased in the PKGM group. It has been shown that NK1.1-positive T cells are the main producers of IL-13. The reduction in IL-13 in the present study may be due to the decrease in NK1.1-positive T cells. The role of iNKT cells in intestinal inflammation
Table 1 Decrease in natural killer T (NKT) cell population from PKGM-fed mice with OXA-induced colitis Ethanol
OXA
CD3? NK1.1? (%)
3.24 ± 0.33
6.24 ± 2.04
CD3? NK1.1- (%)
69.69 ± 10.01
68.8 ± 4.78
?
?
OXA ? PKGM
OXA ? PA
3.78 ± 0.54*
5.81 ± 1.89
69.78 ± 5.89
70.4 ± 3.39 ?
2
The frequency of NK1.1 CD3 NKT cells was decreased in PKGM-fed but not PA-fed mice. However, NK1.1 CD3 NK cells were not affected by treatment with PKGM. The frequency of NKT cells is shown as a percentage of the total lymphocytes * p \ 0.05, OXA ? control-fed group versus OXA ? PKGM-fed group
123
Eur J Nutr
% Body weight from day 0
A
8 6 4 2 0 -2 -4 Ethanol OXA+PKGM OXA OXA+PA
-6 -8 -10 -12
0
1
2
3
4
5
6
7
day
Colon length, cm
B
C
10
Ethanol
OXA
8
6
Ethanol
OXA
OXA
OXA+PA
OXA+ PKGM
OXA+ PA
+ PKGM
D
6 5
*
Score
4 3 2 1 0
Ethanol
OXA
OXA
OXA+PA
+ PKGM Fig. 4 The preventive role of PKGM in OXA-induced colitis was not shown in invariant natural killer T cell-deficient (NKTKO) mice. NKTKO mice were treated with OXA alone or in combination with PKGM. Colitis was induced by OXA in NKTKO mice. a Body weight change, b shortening of colon length, c microscopic injury of
histological section, and d scores induced by intrarectal instillation of OXA. *p \ 0.05, ethanol group versus OXA ? control-fed group. Data represent the mean ? SD of 6 mice per group. This experiment was repeated three times
has been widely studied [33, 34], and the role of noninvariant NKT cells in the pathogenesis of human UC and OXA colitis in mice has been reported. Further investigation is required to reveal the mechanism by which PKGM decreases iNKT cells. To determine whether the immune compartment mediates the effect of PKGM in vivo, we examined the effect of PKGM in OXA colitis in iNKT cell-deficient mice. Interestingly, OXA colitis was also induced in iNKT cell-
deficient mice with local colonic TNF-a induction. Moreover, we found that neither IL-4 nor IL-13 was elevated in the colon of iNKT cell-deficient mice after OXA treatment. These data suggested that mechanisms other than induction of IL-13 or skewing of the Th1/Th2 ratio may be involved in the intestinal inflammation induced by OXA in KO mice. Finally, we also found that in comparison with control-fed mice, PKGM exerted no effective role in iNKT cell-deficient mice, strongly suggesting that the iNKT cell
123
Eur J Nutr
A
900
B
800
400
700 300
600
IL-4, pg/ml
TNF- , pg/ml
Fig. 5 Cytokine production by lymphocytes from invariant NKTKO mice with OXAinduced colitis and cytokine production by mononuclear cells of the colon from control, PA-fed, and PKGM-fed mice with OXA-induced colitis. Mononuclear cells were stimulated with PMA/ ionomycin for 48 h, followed by analysis of culture supernatants using ELISA. Data represent mean values of 5 mice per group. a TNF-a, b IL-4, c IL13, d IFN-c, and e Th1/Th2 ratio
500 400 300
200
100
200 100 0 Ethanol
OXA
OXA
0
OXA+PA
Ethanol
C
D
1200
800 600 400
2000
1200
800
400
200
Ethanol
OXA
OXA
OXA+PA
0
Ethanol
OXA
OXA
OXA+PA
+ PKGM
+ PKGM
E
OXA+PA
1600
IFN- , pg/ml
IL-13, pg/ml
1000
0
OXA
OXA
+ PKGM
+ PKGM
1.6 1.4
IL-4/ IFN-
1.2 1 0.8 0.6 0.4 0.2 0
Ethanol
OXA
OXA
OXA+PA
+ PKGM
population could be a target for this agent. These findings further suggest that PKGM, at least partially, acts directly on iNKT cells in vivo. In UC, the main producer of IL-13 is not iNKT cells but non-invariant NKT cells [19]. Further investigation will be required to elucidate whether both invariant and non-invariant NKT cells are influenced by PKGM.
OXA-induced colitis by targeting, among other mechanisms, lamina propria T cells. In light of the fact that therapeutic approaches for inhibiting T cell proliferation such as glucocorticoids, azathioprine/6-MP, and antiTNF-a antibodies are effectively used to treat UC, our results support the notion that PKGM might be added to the list of compounds that have utility in the treatment of UC.
Conclusion
Acknowledgments We thank the Shimizu Chemical Co. and Nishikawa Rubber Co. for the donation of the PA (PROPOLÒ) and PKGM, respectively. This work was carried out at the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University.
In summary, we have provided evidence that PKGM exerts anti-inflammatory activities in a mouse model of
123
Eur J Nutr
References 1. Abraham C, Cho JH (2009) Inflammatory bowel disease. N Engl J Med 361:2066–2078 2. Baumgart DC, Carding SR (2007) Inflammatory bowel disease: cause and immunobiology. Lancet 369:1627–1640 3. Xavier RJ, Podolsky DK (2007) Unravelling the pathogenesis of inflammatory bowel disease. Nature 448:427–434 4. Baumgart DC, Sandborn WJ (2007) Inflammatory bowel disease: clinical aspects and established and evolving therapies. Lancet 369:1641–1657 5. Vuksan V, Jenkins DJ, Spadafora P, Sievenpiper JL, Owen R, Vidgen E, Brighenti F, Josse R, Leiter LA, Bruce-Thompson C (1999) Konjac-mannan (glucomannan) improves glycemia and other associated risk factors for coronary heart disease in type 2 diabetes. A randomized controlled metabolic trial. Diabetes Care 22:913–919 6. Ebihara K, Shneeman BO (1989) Interaction of bile acids, phospholipids, cholesterol and triglyceride with dietary fibers in the small intestine of rats. J Nutr 119:1100–1106 7. Chen HL, Fan YH, Chen ME, Chan Y (2005) Unhydrolyzed and hydrolyzed konjac glucomannans modulated cecal and fecal microflora in Balb/c mice. Nutrition 21:1059–1064 8. Mizutani T, Mitsuoka T (1983) Effect of Konjac mannan on 1,2dimethylhydrazine-induced intestinal carcinogenesis in Fischer 344 rats. Cancer Lett 19:1–6 9. Onishi N, Kawamoto S, Nishimura M, Nakano T, Aki T, Shigeta S, Shimizu H, Hashimoto K, Ono K (2005) A new immunomodulatory function of low-viscous konjac glucomannan with a small particle size: its oral intake suppresses spontaneously occurring dermatitis in NC/Nga mice. Int Arch Allergy Immunol 136:258–265 10. Matsuda H, Watanabe N, Geba GP, Sperl J, Tsudzuki M, Hiroi J, Matsumoto M, Ushio H, Saito S, Askenase PW, Ra C (1997) Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC/Nga mice. Int Immunol 9:461–466 11. Onishi N, Kawamoto S, Nishimura M, Nakano T, Aki T, Shigeta S, Shimizu H, Hashimoto K, Ono K (2004) The ability of konjacglucomannan to suppress spontaneously occurring dermatitis in NC/ Nga mice depends upon the particle size. BioFactors 21:163–166 12. Onishi N, Kawamoto S, Ueda K, Yamanaka Y, Katayama A, Suzuki H, Aki T, Hashimoto K, Hide M, Ono K (2007) Dietary pulverized Konjac glucomannan prevents the development of allergic rhinitis-like symptoms and IgE response in mice. Biosci Biotechnol Biochem 71:2551–2556 13. Holma R, Juvonen P, Asmawi MZ, Vapaatalo H, Korpela R (2002) Galacto-oligosaccharides stimulate the growth of bifidobacteria but fail to attenuate inflammation in experimental colitis in rats. Scand J Gastroenterol 37:1042–1047 14. Hoentjen F, Welling GW, Harmsen HJ, Zhang X, Snart J, Tannock GW, Lien K, Churchill TA, Lupicki M, Dieleman LA (2005) Reduction of colitis by prebiotics in HLA-B27 transgenic rats is associated with microflora changes and immunomodulation. Inflamm Bowel Dis 11:977–985 15. Boirivant M, Fuss IJ, Chu A, Strober W (1998) Oxazolone colitis: a murine model of T helper cell type 2 colitis treatable with antibodies to interleukin 4. J Exp Med 188:1929–1939 16. Heller F, Fuss IJ, Nieuwenhuis EE, Blumberg RS, Strober W (2002) Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-producing NK-T cells. Immunity 17:629–638 17. Kuperman DA, Huang X, Koth LL, Chang GH, Dolganov GM, Zhu Z, Elias JA, Sheppard D, Erle DJ (2002) Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma. Nat Med 8:885–889
123
18. Fuss IJ, Strober W (2008) The role of IL-13 and NK T cells in experimental and human ulcerative colitis. Mucosal Immunol 1(Suppl 1):S31–S33 19. Fuss IJ, Heller F, Boirivant M, Leon F, Yoshida M, FichtnerFeigi S, Yang Z, Exley M, Kitani A, Blumberg RS, Mannon P, Strober W (2004) Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize and atypical Th2 response in ulcerative colitis. J Clin Invest 113:1490–1497 20. Kawano T, Cui J, Koezuka Y, Toura I, Kaneko Y, Sato H, Kondo E, Harada M, Koseki H, Nakayama T, Tanaka Y, Taniguchi M (1998) Natural killer-like nonspecific tumor cell lysis mediated by specific ligand-activated Va14 NKT cells. Proc Natl Acad Sci USA 95:5690–5693 21. Mudter J, Amoussina L, Schenk M, Yu J, Bru¨stle A, Weigmann B, Atreya R, Wirtz S, Becker C, Hoffman A, Atreya I, Biesterfeld S, Galle PR, Lehr HA, Rose-John S, Mueller C, Lohoff M, Neurath MF (2008) The transcription factor IFN regulatory factor-4 controls experimental colitis in mice via T cell-derived IL6. J Clin Invest 118:2415–2426 22. Okamoto S, Watanabe M, Yamazaki M, Yajima T, Hayashi T, Ishii H, Mukai M, Yamada T, Watanabe N, Jameson BA, Hibi T (1999) A synthetic mimetic CD4 is able to suppress disease in a rodent model of immune colitis. Eur J Immunol 29:355–366 23. Numata Y, Tazuma S, Ueno Y, Nishioka T, Hyogo H, Chayama K (2005) Therapeutic effect of repeated natural killer T cell stimulation in mouse cholangitis complicated by colitis. Dig Dis Sci 50:1844–1851 24. Doi K, Matsuura M, Kawara A, Baba S (1979) Treatment of diabetes with glucomannan (konjac mannan). Lancet 1:987–988 25. Kimata H (2006) Improvement of atopic dermatitis and reduction of skin allergic responses by oral intake of konjac ceramide. Pediatr Dermatol 23:386–389 26. Uchiyama T, Nakano Y, Ueda O, Mori H, Nakashima M, Noda A, Ishizaki C, Mizoguchi M (2008) Oral intake of glucosylceramide improves relatively higher level of transepidermal water loss in mice and healthy human subjects. J Health Sci 54:559–566 27. Suzuki H, Oomizu S, Yanase Y, Onishi N, Uchida K, Mihara S, Ono K, Kameyoshi Y, Hide M (2010) Hydrolyzed Konjac glucomannan suppresses IgE production in mice B cells. Int Arch Allergy Immunol 152:122–130 28. Spergel JM, Mizoguchi E, Oettgen H, Bhan AK, Geha RS (1999) Roles of TH1 and TH2 cytokines in a murine model of allergic dermatitis. J Clin Invest 103:1103–1111 29. Vestergaard C, Yoneyama H, Murai M, Nakamura K, Tamaki K, Terashima Y, Imai T, Yoshie O, Irimura T, Mizutani H, Matsushima K (1999) Overproduction of Th2-specific chemokines in NC/Nga mice exhibiting atopic dermatitis-like lesions. J Clin Invest 104:1097–1105 30. Heller F, Florian P, Bojarski C, Richter J, Christ M, Hillenbrand B, Mankertz J, Gitter AH, Bu¨rgel N, Fromm M, Zeitz M, Fuss I, Strober W, Schulzke JD (2005) Interleukin-13 is the key effector Th2 cytokine in ulcerative colitis that affects epithelial tight junctions, apoptosis, and cell restitution. Gastroenterology 129:550–564 31. Mannon PJ, Hornung RL, Yang Z, Yi C, Groden C, Friend J, Yao M, Strober W, Fuss IJ (2011) Suppression of inflammation in ulcerative colitis by interferon-b-1a is accompanied by inhibition of IL-13 production. Gut 60:449–455 32. Wilson MS, Ramalingam TR, Rivollier A, Shenderov K, Mentink-Kane MM, Madala SK, Cheever AW, Artis D, Kelsall BL, Wynn TA (2011) Colitis and intestinal inflammation in IL10-/mice results from IL-13Ra2-mediated attenuation of IL-13 activity. Gastroenterology 140:254–264
Eur J Nutr 33. Zeissig S, Kaser A, Dougan SK, Nieuwenhuis EE, Blumberg RS (2007) Roles of NKT cells in the digestive system. III. Role of NKT cells in intestinal immunity. Am J Physiol Gastrointest Liver Physiol 293:G1101–G1105
34. Wingender G, Kronenberg M (2008) Role of NKT cells in the digestive system. IV. The role of canonical natural killer T cells in mucosal immunity and inflammation. Am J Physiol Gastrointest Liver Physiol 294:G1–G8
123
ORIGINAL CONTRIBUTION
Pulverized konjac glucomannan ameliorates oxazolone-induced colitis in mice Toshiko Onitake • Yoshitaka Ueno • Shinji Tanaka • Shintaro Sagami • Ryohei Hayashi Kenta Nagai • Michihiro Hide • Kazuaki Chayama
•
Received: 9 April 2014 / Accepted: 1 August 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose Pulverized konjac glucomannan (PKGM) is a natural biologically active compound extracted from konjac, a Japanese traditional food. In the present study, we investigated the role of PKGM in intestinal immunity in a mouse model of oxazolone (OXA)-induced colitis. Methods C57BL/6(B6) mice were fed PKGM or control food from 2 weeks before the induction of OXA colitis. Body weight change, colon length, and histological change in the colon were examined. The mononuclear cells were purified from colon and stimulated with PMA/ionomycin. The levels of TNF-a, interferon (IFN)-c, interleukin (IL)-4, and IL-13 from the supernatant were measured by ELISA. Results Oral administration of PKGM prevented the body weight loss and shortening of colon length associated with OXA-induced colitis. Histological analysis revealed that the colonic inflammation was improved by the administration of PKGM. The levels of IL-4 and IL-13, the critical inflammatory cytokines in OXA colitis, derived from mononuclear cells from the lamina propria of the colon were significantly suppressed by PKGM administration. PKGM-fed mice showed a significantly lower IL-4/IFN-c
ratio in the colonic lamina propria compared with that in control-fed mice. Fluorescence-activated cell sorting analysis revealed that natural killer (NK) 1.1? T cells in the liver were significantly decreased in PKGM-fed mice. Finally, the preventive role of PKGM in OXA-induced colitis was not observed in invariant natural killer T celldeficient mice. Conclusions PKGM ameliorated OXA-induced colitis in mice. This effect is associated with a decreased population of NK1.1? T cells and induction of Th1-polarized immune responses. Keywords Konjac glucomannan Oxazolone colitis IL-13 Th1/Th2 balance NKT cell Abbreviations AD Atopic dermatitis iNKT Invariant natural killer T KGM Konjac glucomannan LPMCs Lamina propria mononuclear cells OXA Oxazolone PKGM Pulverized konjac glucomannan UC Ulcerative colitis
T. Onitake S. Sagami R. Hayashi K. Nagai K. Chayama Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan Y. Ueno (&) S. Tanaka Department of Endoscopy, Hiroshima University Hospital, Hiroshima, Japan e-mail: [email protected] M. Hide Department of Dermatology, Integrated Health Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
Introduction Ulcerative colitis (UC) is a chronic inflammatory disorder of the colon, but its etiology is far from being understood in detail. Despite various treatments for UC, patients do not recover completely and frequently undergo repeat relapse and remission. Although the mechanisms underlying UC are not poorly understood, much of the chronic
123
Eur J Nutr
inflammation of the colon that characterizes this disease is thought to be due to inappropriate activation of the immune system [1–3]. Currently, patients with UC are usually treated with 5-aminosalicylic acid and/or corticosteroids. However, the prolonged use of corticosteroids may be limited by potential adverse effects, such as osteoporosis, fracture, infection, and cataracts. Various alternative pharmacological approaches have been put forward to decrease the dosage of steroids to decrease their side effects [4]. Konjac is a glucomannan-rich Japanese traditional food, which is derived from the tubers of Amorphophallus konjac, and has been consumed traditionally in Asia. Konjac glucomannan (KGM) is a highly viscous polysaccharide composed of glucose and mannose residues at a molar ratio of 1:1.6 joined through b1,4-glucosidic linkages. It has proved to be a health or dietary food due to its watersoluble fibers. Numerous clinical studies have been published on KGM, which ameliorates glycemia and other associated risk factors for coronary heart disease in type 2 diabetes [5] and delays the absorption of both cholesterol and triglyceride from the small intestine [6]. Moreover, KGM is effective on intestine by modulating cecal and fecal microflora in mice [7], and it inhibits carcinogenesis in rat [8]. However, the precise mechanisms underlying the immunologic effects of KGM remain largely unclear. Nutritional supplementation with highly viscous KGM is restricted on account of its low solubility, which requires long time intervals to attain peak viscosity. To circumvent this restriction, we prepared highly specific surface area of the KGM molecule that would be expected to possess some extent of these well-known physiological activities [9]. To investigate the immunomodulating potency of low-viscous KGM, several particle sizes of pulverized KGM (PKGM) were used in NC/Nga mice, a murine model for human AD [10]. Production of hyper-IgE and inflammatory cytokines was suppressed in the PKGM-fed mice, and AD-like skin symptoms were markedly suppressed by oral intake of PKGM [11, 12]. There are several studies on the effects of carbohydrates like inulin, galactomannan, and glucomannan in the form of KGM [13, 14]. So far, however, there have been no reports demonstrating the effect of PKGM on intestinal inflammation. Experimental animal models are indispensable for the accurate understanding of the pathogenesis of human diseases. Support for this view may be drawn from animal models of colitis, which consistently exhibit an imbalance of regulatory and inflammatory cytokines. Rectal instillation of the haptenating reagent oxazolone (OXA) leads to rapid development of colitis limited to the distal half of the colon. The histologic features and distribution of OXA colitis have characteristics that resemble those of UC [15]. The inflammation is characterized by increased secretion of
123
IL-4 and IL-5 and can be ameliorated by the administration of anti-IL-4 monoclonal antibodies [15]. Therefore, this model has been used to study the contribution of the Th2dependent immune response to intestinal inflammation. IL-13 seems to be a key pathogenic cytokine of OXA colitis because IL-13 neutralization prevents its development [16]. IL-13 is known as an inflammatory cytokine and is considered a growth factor for epithelial cells in asthma [17]. Recently, it has been reported that IL-13 is also critical for the pathogenesis of UC [18]. Therefore, IL-13 may be a candidate for the treatment of UC. IL-13 is produced by iNK Tcells in OXA colitis in mice [16] and by non-invariant NKT cells in UC [19]. In the present study, we report that dietary PKGM was effective in reducing inflammation in OXA colitis in mice. We show that PKGM reduced colonic inflammation and suppressed the mediators of both innate and adaptive immunity. Mechanistic studies carried out using mononuclear cells derived from intestinal lamina propria show that PKGM effectively regulates the function of effector T cells by shifting from a Th2- to a Th1-dependent immune response.
Materials and methods KGM powders Two kinds of low-viscous KGM powders, PA (PROPOLÒ; Shimizu Chemical Corporation, Hiroshima, Japan) and pulverized PA (PKGM) (Nishikawa Rubber Corporation, Hiroshima, Japan), were used in this study. The GM contents were measured to be approximately 99 % for PA and 97 % for PKGM. The mean particle sizes of the KGM powders were estimated to be about 300 lm for PA and 105 lm for PKGM. The peak velocity of the 1 % PA solutions reached more than 100,000 mPa s after 7 h at 25 °C, while that of PKGM solutions attained values of 32,000–35,000 mPa s within 0.5 h. Animals Female C57BL/6(B6) mice (6–8 weeks old) were purchased from CLEA JAPAN (Tokyo, Japan). Female Ja281deficient [Va14NKT cell-deficient (KO)] mice on B6 background (6–10 weeks old) were generated as described previously [20]. Mice were divided into eight groups in clear plastic cages with a mesh top under specific pathogen-free conditions, kept at room temperature in a controlled environment at 22 ± 2 °C (12-h dark/light cycle), and had free access to food and water. The food was formed to a typical murine solid feed which firmed KGM powders. Food and water were changed fresh weekly.
Eur J Nutr
Animal care was in compliance with the regulations of Hiroshima University. Feeding The mice were fed either with a commercial rodent diet, MF (Oriental Yeast, Tokyo, Japan), PA diet (MF containing 5 % (w/w) PA powder), or the PKGM diet (MF containing 5 % (w/w) PKGM powder) ad libitum. We started to presensitize the mice with OXA 2 weeks after the start of feeding with either diet. The food intake in the different experimental groups was similar because there was no difference in the body weights 2 weeks after the intake of each diet. Induction of colitis OXA colitis was induced as previously described, with minor modifications [15]. OXA (4-ethoxymethylene-2phenyl-2-OXA-5-one) was obtained from Sigma-Aldrich (St. Louis, MO). To presensitize the mice, a 1.5 9 1.5 cm field of the abdominal skin was shaved, and 200 ll of a 3 % solution of OXA in 100 % ethanol or 100 % ethanol alone was applied. Five days after presensitization, the mice underwent general anesthesia with isoflurane and were rechallenged intrarectally with 150 ll 1 % OXA in 50 % ethanol or, for the control-fed mice, 50 % ethanol alone. To ensure distribution of OXA within the entire colon and cecum, mice were held in a head-down vertical position for 60 s after the intrarectal injection. Mice were then monitored for the appearance of diarrhea, loss of body weight, and overall mortality.
damage affecting the submucosa as 3. The combined inflammatory and injury scores resulted in an overall score ranging from 0 (no changes) to 6 (severe inflammatory infiltrate and mucosal damage). Isolation and purification of mononuclear cells Mice were killed at 2 days after colitis induction, and hepatic mononuclear cells or lamina propria mononuclear cells (LPMCs) were isolated. Hepatic mononuclear cells were isolated by grinding the tissue in a Petri dish and filtering the cell suspension through a 40-lm mesh. Mononuclear cells were isolated using 44 and 66 % solutions of Percoll. LPMCs were isolated as previously described in detail [22, 23]. In brief, LPMCs were isolated after removal of epithelial cells by incubation of colon strips in Ca??Mg??-free Hanks’ balanced salt solution containing 1 mM EDTA (SigmaAldrich) for 15 min at 37 °C. Mononuclear cells were released by digesting the tissue in RPMI-1,640 medium (Sigma-Aldrich) supplemented with 150 U/ml collagenase (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and incubated for 1.5 h at 37 °C with stirring. Finally, leukocytes were separated from epithelial cells by centrifugation in 44 and 66 % solutions of Percoll (pH 8.5–9.5) (Sigma-Aldrich). Cell culture for cytokine production The cells were harvested and stimulated in vitro with 5 lg/ml of plate-bound anti-CD3 antibody (145-2C11) (BD Biosciences Pharmingen, San Diego, CA), 5 lg/ml of PMA ? ionomycin, or Con A for 48 h at 37 °C. Supernatants were collected and stored at -80 °C until further analysis.
Histological examination of colitis ELISA assays Colon samples were removed from mice at 5 days after induction of colitis. The colons were opened longitudinally and rolled concentrically. The tissues were then fixed in 10 % buffered formalin and embedded in paraffin. Paraffin sections were made and stained with hematoxylin and eosin. Histological examination was evaluated as previously described [21]. The degree of inflammation and epithelial injury on microscopic cross-section was graded semiquantitatively on a scale of 0–6. Inflammatory cell infiltration was scored from 0 to 3, and tissue damage was scored from 0 to 3. Occasional or no inflammatory infiltrate in the lamina propria was scored as 0, increased numbers of inflammatory cells restricted to the lamina propria as 1, inflammatory infiltrates reaching the submucosa as 2, and transmural inflammation as 3. The tissue damage subscore took into account epithelial lesions: No mucosal damage was scored as 0, focal crypt lesions as 1, surface mucosal erosions or focal ulcerations as 2, and extensive mucosal
Cytokine concentrations in culture supernatants were measured by ELISA kits according to the manufacturers’ instructions. IFN-c, TNF-a, and IL-4 were measured with OptEIA ELISA sets (BD Biosciences Pharmingen), and IL13 was measured with Quantikine M ELISA kit (R&D Systems, Minneapolis, MN). FACS analysis Cells were stained with antibodies to CD3 (17A2) and NK1.1 (NKR-P1B and NKR-PIC). Surface staining cells were analyzed using a fluorescence-activated cell sorting (FACS) Calibur system (BD Biosciences). Percentage values were calculated with CellQuest software (BD Biosciences) from the scatter plots, which were attained with propidium iodide (Sigma-Aldrich) staining after gating on living lymphocytes.
123
Eur J Nutr
Statistical analysis
Results
Data are expressed as mean ± SD of N observations. All values in the figures and text were analyzed with the Japanese version of JMP (SAS Institute Inc., Cary, NC, USA), and the data sets were examined with one-way ANOVA. Differences were considered significant at p \ 0.05.
Preventive role of dietary PKGM in OXA-induced colitis
% Body weight from day 0
A
To examine the role of PKGM in the development of OXA-induced colitis in vivo, we administrated the chow
B
5
0
Ethanol OXA
-5
OXA+PKGM
-10
-15
Ethanol OXA+PKGM OXA+PA OXA
0
OXA+PA
1
2
3
4
5
6
7
day
C 10
Colon length, cm
D
OXA
* 8
6
Ethanol
OXA
OXA
OXA+PA
+ PKGM
E
Ethanol
OXA+PKGM
OXA+ PA
6
*
5
Score
4 3 2 1 0 Ethanol
OXA
OXA
OXA+PA
+ PKGM
Fig. 1 Pulverized konjac glucomannan (PKGM)-fed mice are less susceptible than control or PA-fed mice to oxazolone (OXA)-induced colitis. a Body weight change in mice induced by OXA colitis. Mice were fed with PKGM, PA, or control diet for 2 weeks. OXA colitis was induced by sensitizing mice with OXA, followed by intrarectal administration of the hapten reagent after 5 days. Body weight was monitored after oxazolone rechallenge at the indicated time points. Mean value ± SEM from 2 representative experiments of 6 is shown. For this experiment, 5–8 control diet-fed, PKGM-fed, and PA-fed mice were used. *p \ 0.05, OXA ? control-fed group versus OXA ? PKGM-fed group. **p \ 0.05, OXA ? PA-fed group versus OXA ? PKGM-fed group. b Macroscopic changes in colons on day 3
123
after OXA administration. Representative photographs of dissected large intestine from ethanol-treated control (top), mice treated with OXA ? control (second row), OXA ? PKGM (third row), and OXA ? PA (bottom row) on day 3 after induction of colitis. c Colon length of mice measured on day 5 after OXA administration. Each bar represents 6 mice. *p \ 0.01, OXA ? control-fed group versus OXA ? PKGM-fed group. d Histological sections of colonic inflammation in control or PA-fed or PKGM-fed mice upon OXA administration. e Quantitative histopathological assessment of colitis activity. *p \ 0.05, OXA ? control-fed group versus OXA ? PKGM-fed group. Data represent mean value ± SD from 1 representative experiment of 5 mice for each group
Eur J Nutr
containing 5 % PKGM or 5 % PA to C57BL/6 mice for 14 days before OXA treatment. Two days after rectal administration of OXA, PKGM-fed mice had lost about 12 % of their initial body weight. However, PKGM-fed mice showed a significant improvement in body weight from day 4 to day 5 after 1 % OXA administration as indicated in Fig. 1a (p \ 0.05). PA-fed mice recovered their body weight loss later than PKGM-fed mice. Macroscopic examinations on day 5 after 1 % OXA administration revealed that shortening of colon length was significantly more inhibited in PKGM-fed mice than in control or PA-fed mice (Fig. 1b, c; p \ 0.05). The colons of control or PA-fed mice were more reddish due to bloody stool compared with those of PKGM-fed mice, suggesting that a less severe intestinal inflammation had occurred in the PKGM-fed mice (Fig. 1b). On histological examination of involved colon in the 1 % OXA-treated PKGM-fed mice, we observed a superficial inflammation characterized by the presence of epithelial cell loss, patchy ulceration, pronounced depletion of mucin-producing goblet cells, and a reduction in the density of the tubular glands. These histopathological changes in OXA-induced colitis were significantly less severe in the colon of PKGM-fed mice than in control and PA-fed mice on day 5 after 1 % OXA administration (Fig. 1d, e; p \ 0.05). These data suggest that treatment with dietary PKGM prevents intestinal inflammation induced by OXA. Cytokine production of PKGM-fed mice with oxazolone-induced colitis To investigate whether PKGM treatment has immunological modulations, we examined production of local cytokines. Pro-inflammatory cytokine TNF-a from the mononuclear cells of the colon was induced by OXA colitis, which was suppressed by the administration of PKGM (Fig. 2a, p \ 0.05). The mononuclear cells from colon of mice administered OXA produced IL-4 and IL-13, major Th2 cytokines, which were also significantly inhibited by feeding with PKGM (Fig. 2b, c; p \ 0.05). No change in the level of Th1 cytokine IFN-c from LPMCs was observed in OXA colitis LPMCs (Fig. 2d). When we compared the IL-4/IFN-c ratio, a significantly lower ratio was found in the PKGM-fed mice versus the control-fed mice in LPMCs (Fig. 2e). These data raise the possibility that PKGM has a capacity to suppress Th2-related but not Th1-related cytokines induced by OXA. Effect of PKGM treatment on NK1.1? T cell population To identify the target cells involved in the prevention OXA-induced colitis in PKGM-fed mice, we isolated
lymphocytes from liver and colon and investigated a surface antigen of the cells. Flow cytometric analysis revealed that the frequency of NK1.1? T cells significantly decreased in liver of the PKGM-fed mice compared with that observed in liver of control or PA-fed mice (Fig. 3; Table 1, p \ 0.05). There was no change in the population of NK1.1? CD3- cells or NK1.1- CD3? cells. There was no difference in the frequency of NK1.1? T cells in the colon between PKGM- and control-fed mice (data not shown). Therefore, iNK1.1 T cells in the liver were selectively decreased by treatment with dietary PKGM. PKGM does not improve OXA-induced colitis in iNKT cell-deficient mice Because PKGM administration reduced NK1.1? T cells in the liver of B6 mice, experiments were carried out to further define the immune compartment mediating PKGM actions in vivo. For this purpose, we tested PKGM in Ja281-deficient [Va14NKT cell-deficient (KO)] mice. Treatment of these mice with rectal application of OXA resulted in similar weight loss pattern (Fig. 4a) and colon shortening (Fig. 4b) between PKGM-fed mice and PA-fed mice because of massive inflammation of the colonic tissue (Fig. 4c, d). In this experimental model, PKGM was unable to reverse local signs of inflammation caused by OXA administration. Although the levels of TNF-a in the colon were significantly increased after OXA treatment (Fig. 5a), no increase was found in the levels of IL-4 or IL-13 in the colon (Fig. 5b, c). Moreover, IFN-c secretion was not induced by OXA treatment (Fig. 5d), and the Th1/Th2 ratio did not change by PKGM treatment (Fig. 5e). These data suggested that mechanisms other than induction of IL13 or skewing of the Th1/Th2 ratio may be involved in the intestinal inflammation produced in KO mice by OXA. These findings suggest that PKGM, at least partially, acts directly on iNKT cells in vivo.
Discussion In this study, we showed that PKGM was effective for the prevention of Th-2-mediated OXA colitis by skewing the Th1/Th2 balance from Th2 toward Th1. PKGM decreased the population of NK1.1? T cells. The protective effect of PKGM against OXA colitis was absent in the presence of iNKT cell deficiency. Konjac is known to be a healthy, traditional food in Japan and several other Asian countries. KGM is a polysaccharide, which is used as food in paste or coagulated form. KGM itself is a soluble dietary fiber, but the coagulated form of KGM is not water soluble and has strong elasticity. Thus, KGM is popular as a dietary food because
123
Eur J Nutr
A 1600
B
1400 1200
*
1200
IL-4, pg/ml
TNF-α, pg/ml
1400
1000 800 600
800 600 400
200
200 Ethanol
OXA
OXA
0
OXA+PA
*
1000
400
0
Ethanol
OXA
+ PKGM 700
D
*
3000
500
2500
400 300 200
Ethanol
OXA
OXA
OXA+PA
+ PKGM 0.7
1500 1000
0 Ethanol
OXA
OXA
OXA+ PA
+ PKGM
*
0.6
IL-4/ IFN-
2000
500
100
E
OXA+PA
3500
600
0
OXA
+ PKGM
IFN-γ,, pg/ml
C
IL-13, pg/ml
Fig. 2 Cytokine production from colonic lymphocytes of mice with oxazolone (OXA) colitis. Cytokine production by mononuclear cells of the colon from control, PA-fed, and pulverized konjac glucomannan (PKGM)-fed mice with OXAinduced colitis. Colonic mononuclear cells were stimulated with PMA/ ionomycin for 48 h, followed by analysis of culture supernatants using ELISA. Data represent mean values of 4–8 mice per group. a TNF-a, b IL-4, c IL13, d IFN-c, and e Th1/Th2 ratio. *p \ 0.05, OXA ? control-fed group versus OXA ? PKGM-fed group
0.5 0.4 0.3 0.2 0.1 0
Ethanol
OXA
OXA
OXA+PA
+ PKGM
of its very low caloric value and the large amount of dietary fiber it provides. KGM is not degradable by human digestive enzymes but is available to be partially converted to a fatty acid by the intestinal microflora [7]. Moreover, it has been reported that KGM blocks the absorption of cholesterol in the small intestine and maintains a lower plasma cholesterol level [6], and it is effective in protecting against diabetes because KGM suppresses the rapid absorption of glucose from the upper small intestine, thus sparing the secretion of insulin [24]. It is also reported that KGM has a therapeutic effect on human and mouse dermatitis. Oral intake of konjac
123
ceramide improves both skin symptoms in children with AD and allergen-specific IgE production with skewing of the cytokine pattern toward the Th1 type in allergenstimulated cultures [25]. Oral intake of konjac-derived glucosylceramide improved skin roughness caused by SDS treatment in mice [26]. The KGM that has been used until now, however, has high viscosity and low solubility that requires long time intervals to attain peak viscosity. Therefore, we prepared various particle sizes of low viscous and highly specific surface area of the KGM molecule that would be expected to possess the more effect of these well-known
Eur J Nutr
NK1.1
OXA
Ethanol 3.2
6.2
69.7
68.8
OXA+PKGM
OXA+PA
3.8
5.8
69.8
70.4
CD3 Fig. 3 Representative FACS pattern of liver lymphocytes. Mononuclear cells from liver were obtained and stained with anti-NK1.1 mAb and anti-CD3 mAb for evaluation by flow cytometry
physiological activities. Production of IL-4 and IFN-c in splenocytes of NC/Nga mice, an experimental model of human AD, was decreased in the PKGM-fed mice, and AD-like skin symptoms were markedly suppressed by oral intake of PKGM [11, 12]. Soluble PKGM suppressed IgE production in splenocytes of mice in vitro [27]. Both the Th1 and Th2 cytokines play important roles in AD [28], and Th2-specific chemokines are overproduced in NC/Nga mice [29]. OXA colitis is a well-known experimental model of human UC. Rectal administration of the hapten reagent OXA induces a severe colitis in mice that is characterized by weight reduction and diarrhea and which leads to high death rates [15]. Superficial inflammation includes epithelial cell loss and patchy ulceration as well as pronounced depletion of goblet cells accompanied by a mixed inflammatory infiltrate of lymphocytes and granulocytes. This inflammation is characterized by increased IL-4 and IL-13 secretion and can be ameliorated by the administration of anti-IL-4 or anti-IL-13 monoclonal antibodies [15]. In the present study, PKGM suppressed exacerbation of OXA colitis. PKGM but not PA specifically suppressed body weight loss, shortening of colon length, and histological
inflammation of the colon. PKGM and PA have been shown to have different immunological effects. Onishi et al. [9] showed that PKGM but not PA suppressed IL-4 and IFN-c from spleen cells in mice. We suppose that PKGM, with a smaller particle size than that of KGM, might gain its protective effect because it possesses a lower viscosity and has higher absorbance in the intestine, although the mechanisms are not fully understood. The exact mechanisms by which PKGM but not PA induces immunosuppression are also not completely understood. In the present study, we clearly showed that PKGM substantially reduced the inflammatory response. PKGM treatment decreased Th2 cytokine levels such as those of IL-4 and IL-13, but there was no change in levels of IFN-c, known as a Th1 cytokine. PKGM suppressed the levels of IL-4 production in ConA-stimulated mononuclear cells in vitro, indicating that the effect of PKGM on Th2 cells may directly affect Th2 differentiation rather than lymphocyte trafficking. The Th2/Th1 ratio, represented by the IL-4/IFN-c ratio, was significantly lower in the PKGMfed mice relative to the control and PA-fed mice. We also found that PKGM suppressed IL-13 from mononuclear cells in the colon. It is well known that IL-13 is a key cytokine in OXA-induced colitis [5] and is an important effector cytokine in human UC that impairs epithelial barrier function by affecting epithelial apoptosis, tight junctions, and restitution velocity [30]. Recent studies found that IFN-b-1a suppresses inflammation in human UC by inhibiting IL-13 production [31] and that colitis and intestinal inflammation in IL10-/- mice resulted from IL13Ra2-mediated attenuation of IL-13 activity [32]. In the present study, the reduction in IL-13 was already shown on day 2 after 1 % OXA administration, and we have proved that PKGM suppressed OXA colitis because of the decrease in IL-13 production. Moreover, we examined which subset of cells was influenced by PKGM in OXA-induced colitis. Interestingly, the population of NK1.1? T cells in the liver significantly decreased in the PKGM group. It has been shown that NK1.1-positive T cells are the main producers of IL-13. The reduction in IL-13 in the present study may be due to the decrease in NK1.1-positive T cells. The role of iNKT cells in intestinal inflammation
Table 1 Decrease in natural killer T (NKT) cell population from PKGM-fed mice with OXA-induced colitis Ethanol
OXA
CD3? NK1.1? (%)
3.24 ± 0.33
6.24 ± 2.04
CD3? NK1.1- (%)
69.69 ± 10.01
68.8 ± 4.78
?
?
OXA ? PKGM
OXA ? PA
3.78 ± 0.54*
5.81 ± 1.89
69.78 ± 5.89
70.4 ± 3.39 ?
2
The frequency of NK1.1 CD3 NKT cells was decreased in PKGM-fed but not PA-fed mice. However, NK1.1 CD3 NK cells were not affected by treatment with PKGM. The frequency of NKT cells is shown as a percentage of the total lymphocytes * p \ 0.05, OXA ? control-fed group versus OXA ? PKGM-fed group
123
Eur J Nutr
% Body weight from day 0
A
8 6 4 2 0 -2 -4 Ethanol OXA+PKGM OXA OXA+PA
-6 -8 -10 -12
0
1
2
3
4
5
6
7
day
Colon length, cm
B
C
10
Ethanol
OXA
8
6
Ethanol
OXA
OXA
OXA+PA
OXA+ PKGM
OXA+ PA
+ PKGM
D
6 5
*
Score
4 3 2 1 0
Ethanol
OXA
OXA
OXA+PA
+ PKGM Fig. 4 The preventive role of PKGM in OXA-induced colitis was not shown in invariant natural killer T cell-deficient (NKTKO) mice. NKTKO mice were treated with OXA alone or in combination with PKGM. Colitis was induced by OXA in NKTKO mice. a Body weight change, b shortening of colon length, c microscopic injury of
histological section, and d scores induced by intrarectal instillation of OXA. *p \ 0.05, ethanol group versus OXA ? control-fed group. Data represent the mean ? SD of 6 mice per group. This experiment was repeated three times
has been widely studied [33, 34], and the role of noninvariant NKT cells in the pathogenesis of human UC and OXA colitis in mice has been reported. Further investigation is required to reveal the mechanism by which PKGM decreases iNKT cells. To determine whether the immune compartment mediates the effect of PKGM in vivo, we examined the effect of PKGM in OXA colitis in iNKT cell-deficient mice. Interestingly, OXA colitis was also induced in iNKT cell-
deficient mice with local colonic TNF-a induction. Moreover, we found that neither IL-4 nor IL-13 was elevated in the colon of iNKT cell-deficient mice after OXA treatment. These data suggested that mechanisms other than induction of IL-13 or skewing of the Th1/Th2 ratio may be involved in the intestinal inflammation induced by OXA in KO mice. Finally, we also found that in comparison with control-fed mice, PKGM exerted no effective role in iNKT cell-deficient mice, strongly suggesting that the iNKT cell
123
Eur J Nutr
A
900
B
800
400
700 300
600
IL-4, pg/ml
TNF- , pg/ml
Fig. 5 Cytokine production by lymphocytes from invariant NKTKO mice with OXAinduced colitis and cytokine production by mononuclear cells of the colon from control, PA-fed, and PKGM-fed mice with OXA-induced colitis. Mononuclear cells were stimulated with PMA/ ionomycin for 48 h, followed by analysis of culture supernatants using ELISA. Data represent mean values of 5 mice per group. a TNF-a, b IL-4, c IL13, d IFN-c, and e Th1/Th2 ratio
500 400 300
200
100
200 100 0 Ethanol
OXA
OXA
0
OXA+PA
Ethanol
C
D
1200
800 600 400
2000
1200
800
400
200
Ethanol
OXA
OXA
OXA+PA
0
Ethanol
OXA
OXA
OXA+PA
+ PKGM
+ PKGM
E
OXA+PA
1600
IFN- , pg/ml
IL-13, pg/ml
1000
0
OXA
OXA
+ PKGM
+ PKGM
1.6 1.4
IL-4/ IFN-
1.2 1 0.8 0.6 0.4 0.2 0
Ethanol
OXA
OXA
OXA+PA
+ PKGM
population could be a target for this agent. These findings further suggest that PKGM, at least partially, acts directly on iNKT cells in vivo. In UC, the main producer of IL-13 is not iNKT cells but non-invariant NKT cells [19]. Further investigation will be required to elucidate whether both invariant and non-invariant NKT cells are influenced by PKGM.
OXA-induced colitis by targeting, among other mechanisms, lamina propria T cells. In light of the fact that therapeutic approaches for inhibiting T cell proliferation such as glucocorticoids, azathioprine/6-MP, and antiTNF-a antibodies are effectively used to treat UC, our results support the notion that PKGM might be added to the list of compounds that have utility in the treatment of UC.
Conclusion
Acknowledgments We thank the Shimizu Chemical Co. and Nishikawa Rubber Co. for the donation of the PA (PROPOLÒ) and PKGM, respectively. This work was carried out at the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University.
In summary, we have provided evidence that PKGM exerts anti-inflammatory activities in a mouse model of
123
Eur J Nutr
References 1. Abraham C, Cho JH (2009) Inflammatory bowel disease. N Engl J Med 361:2066–2078 2. Baumgart DC, Carding SR (2007) Inflammatory bowel disease: cause and immunobiology. Lancet 369:1627–1640 3. Xavier RJ, Podolsky DK (2007) Unravelling the pathogenesis of inflammatory bowel disease. Nature 448:427–434 4. Baumgart DC, Sandborn WJ (2007) Inflammatory bowel disease: clinical aspects and established and evolving therapies. Lancet 369:1641–1657 5. Vuksan V, Jenkins DJ, Spadafora P, Sievenpiper JL, Owen R, Vidgen E, Brighenti F, Josse R, Leiter LA, Bruce-Thompson C (1999) Konjac-mannan (glucomannan) improves glycemia and other associated risk factors for coronary heart disease in type 2 diabetes. A randomized controlled metabolic trial. Diabetes Care 22:913–919 6. Ebihara K, Shneeman BO (1989) Interaction of bile acids, phospholipids, cholesterol and triglyceride with dietary fibers in the small intestine of rats. J Nutr 119:1100–1106 7. Chen HL, Fan YH, Chen ME, Chan Y (2005) Unhydrolyzed and hydrolyzed konjac glucomannans modulated cecal and fecal microflora in Balb/c mice. Nutrition 21:1059–1064 8. Mizutani T, Mitsuoka T (1983) Effect of Konjac mannan on 1,2dimethylhydrazine-induced intestinal carcinogenesis in Fischer 344 rats. Cancer Lett 19:1–6 9. Onishi N, Kawamoto S, Nishimura M, Nakano T, Aki T, Shigeta S, Shimizu H, Hashimoto K, Ono K (2005) A new immunomodulatory function of low-viscous konjac glucomannan with a small particle size: its oral intake suppresses spontaneously occurring dermatitis in NC/Nga mice. Int Arch Allergy Immunol 136:258–265 10. Matsuda H, Watanabe N, Geba GP, Sperl J, Tsudzuki M, Hiroi J, Matsumoto M, Ushio H, Saito S, Askenase PW, Ra C (1997) Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC/Nga mice. Int Immunol 9:461–466 11. Onishi N, Kawamoto S, Nishimura M, Nakano T, Aki T, Shigeta S, Shimizu H, Hashimoto K, Ono K (2004) The ability of konjacglucomannan to suppress spontaneously occurring dermatitis in NC/ Nga mice depends upon the particle size. BioFactors 21:163–166 12. Onishi N, Kawamoto S, Ueda K, Yamanaka Y, Katayama A, Suzuki H, Aki T, Hashimoto K, Hide M, Ono K (2007) Dietary pulverized Konjac glucomannan prevents the development of allergic rhinitis-like symptoms and IgE response in mice. Biosci Biotechnol Biochem 71:2551–2556 13. Holma R, Juvonen P, Asmawi MZ, Vapaatalo H, Korpela R (2002) Galacto-oligosaccharides stimulate the growth of bifidobacteria but fail to attenuate inflammation in experimental colitis in rats. Scand J Gastroenterol 37:1042–1047 14. Hoentjen F, Welling GW, Harmsen HJ, Zhang X, Snart J, Tannock GW, Lien K, Churchill TA, Lupicki M, Dieleman LA (2005) Reduction of colitis by prebiotics in HLA-B27 transgenic rats is associated with microflora changes and immunomodulation. Inflamm Bowel Dis 11:977–985 15. Boirivant M, Fuss IJ, Chu A, Strober W (1998) Oxazolone colitis: a murine model of T helper cell type 2 colitis treatable with antibodies to interleukin 4. J Exp Med 188:1929–1939 16. Heller F, Fuss IJ, Nieuwenhuis EE, Blumberg RS, Strober W (2002) Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-producing NK-T cells. Immunity 17:629–638 17. Kuperman DA, Huang X, Koth LL, Chang GH, Dolganov GM, Zhu Z, Elias JA, Sheppard D, Erle DJ (2002) Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma. Nat Med 8:885–889
123
18. Fuss IJ, Strober W (2008) The role of IL-13 and NK T cells in experimental and human ulcerative colitis. Mucosal Immunol 1(Suppl 1):S31–S33 19. Fuss IJ, Heller F, Boirivant M, Leon F, Yoshida M, FichtnerFeigi S, Yang Z, Exley M, Kitani A, Blumberg RS, Mannon P, Strober W (2004) Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize and atypical Th2 response in ulcerative colitis. J Clin Invest 113:1490–1497 20. Kawano T, Cui J, Koezuka Y, Toura I, Kaneko Y, Sato H, Kondo E, Harada M, Koseki H, Nakayama T, Tanaka Y, Taniguchi M (1998) Natural killer-like nonspecific tumor cell lysis mediated by specific ligand-activated Va14 NKT cells. Proc Natl Acad Sci USA 95:5690–5693 21. Mudter J, Amoussina L, Schenk M, Yu J, Bru¨stle A, Weigmann B, Atreya R, Wirtz S, Becker C, Hoffman A, Atreya I, Biesterfeld S, Galle PR, Lehr HA, Rose-John S, Mueller C, Lohoff M, Neurath MF (2008) The transcription factor IFN regulatory factor-4 controls experimental colitis in mice via T cell-derived IL6. J Clin Invest 118:2415–2426 22. Okamoto S, Watanabe M, Yamazaki M, Yajima T, Hayashi T, Ishii H, Mukai M, Yamada T, Watanabe N, Jameson BA, Hibi T (1999) A synthetic mimetic CD4 is able to suppress disease in a rodent model of immune colitis. Eur J Immunol 29:355–366 23. Numata Y, Tazuma S, Ueno Y, Nishioka T, Hyogo H, Chayama K (2005) Therapeutic effect of repeated natural killer T cell stimulation in mouse cholangitis complicated by colitis. Dig Dis Sci 50:1844–1851 24. Doi K, Matsuura M, Kawara A, Baba S (1979) Treatment of diabetes with glucomannan (konjac mannan). Lancet 1:987–988 25. Kimata H (2006) Improvement of atopic dermatitis and reduction of skin allergic responses by oral intake of konjac ceramide. Pediatr Dermatol 23:386–389 26. Uchiyama T, Nakano Y, Ueda O, Mori H, Nakashima M, Noda A, Ishizaki C, Mizoguchi M (2008) Oral intake of glucosylceramide improves relatively higher level of transepidermal water loss in mice and healthy human subjects. J Health Sci 54:559–566 27. Suzuki H, Oomizu S, Yanase Y, Onishi N, Uchida K, Mihara S, Ono K, Kameyoshi Y, Hide M (2010) Hydrolyzed Konjac glucomannan suppresses IgE production in mice B cells. Int Arch Allergy Immunol 152:122–130 28. Spergel JM, Mizoguchi E, Oettgen H, Bhan AK, Geha RS (1999) Roles of TH1 and TH2 cytokines in a murine model of allergic dermatitis. J Clin Invest 103:1103–1111 29. Vestergaard C, Yoneyama H, Murai M, Nakamura K, Tamaki K, Terashima Y, Imai T, Yoshie O, Irimura T, Mizutani H, Matsushima K (1999) Overproduction of Th2-specific chemokines in NC/Nga mice exhibiting atopic dermatitis-like lesions. J Clin Invest 104:1097–1105 30. Heller F, Florian P, Bojarski C, Richter J, Christ M, Hillenbrand B, Mankertz J, Gitter AH, Bu¨rgel N, Fromm M, Zeitz M, Fuss I, Strober W, Schulzke JD (2005) Interleukin-13 is the key effector Th2 cytokine in ulcerative colitis that affects epithelial tight junctions, apoptosis, and cell restitution. Gastroenterology 129:550–564 31. Mannon PJ, Hornung RL, Yang Z, Yi C, Groden C, Friend J, Yao M, Strober W, Fuss IJ (2011) Suppression of inflammation in ulcerative colitis by interferon-b-1a is accompanied by inhibition of IL-13 production. Gut 60:449–455 32. Wilson MS, Ramalingam TR, Rivollier A, Shenderov K, Mentink-Kane MM, Madala SK, Cheever AW, Artis D, Kelsall BL, Wynn TA (2011) Colitis and intestinal inflammation in IL10-/mice results from IL-13Ra2-mediated attenuation of IL-13 activity. Gastroenterology 140:254–264
Eur J Nutr 33. Zeissig S, Kaser A, Dougan SK, Nieuwenhuis EE, Blumberg RS (2007) Roles of NKT cells in the digestive system. III. Role of NKT cells in intestinal immunity. Am J Physiol Gastrointest Liver Physiol 293:G1101–G1105
34. Wingender G, Kronenberg M (2008) Role of NKT cells in the digestive system. IV. The role of canonical natural killer T cells in mucosal immunity and inflammation. Am J Physiol Gastrointest Liver Physiol 294:G1–G8
123
