A. Kawaguchi

Omega‐3 fatty acids exacerbate DSS‐induced colitis through decreased adiponectin in colonic subepithelial myofibroblasts

Background: Although the immunoregulatory effects of &ohgr;‐3 fatty acid and adiponectin have been postulated, their role in intestinal inflammation is controversial. The aim of this study was to determine whether dietary fat intake influences activity of colonic inflammation through modulating this system. Methods: C57BL/6 mice received dextran sulfate sodium for induction of colitis. Mice were fed a control diet, &ohgr;‐3 fat‐rich diet, &ohgr;‐6 fat‐rich diet, or saturated fat‐rich diet. Some mice were administered a peroxisome proliferator activated receptor‐gamma; agonist, pioglitazone. Messenger RNA expression of adiponectin and its receptors were analyzed. Adiponectin expression in colonic mucosa of ulcerative colitis patients was also analyzed. Results: The receptors for adiponectin were found to be ubiquitously expressed in epithelial cells, intraepithelial lymphocytes, lamina proprial mononuclear cells, and subepithelial myofibroblasts from colonic tissue, but adiponectin was only expressed in myofibroblasts. Induction of colitis significantly decreased the expression of adiponectin in colonic mucosa. The &ohgr;‐3 fat diet group, but not the other fat diet groups, showed exacerbated colitis with a further decrease of adiponectin expression. Pioglitazone treatment ameliorated the level of decrease in adiponectin expression and improved colonic inflammation induced by the &ohgr;‐3 fat‐rich diet. In patients with ulcerative colitis, the expression level of adiponectin in colonic mucosa was also decreased compared with that in control mucosa. Conclusions: Adiponectin was found to be expressed in myofibroblasts. Adiponectin expression was significantly suppressed by induction of colitis, and aggravation of colitis after exposure to &ohgr;‐3 fat may be due to a further decrease in the expression level of adiponectin.

In vivo demonstration of T lymphocyte migration and amelioration of ileitis in intestinal mucosa of SAMP1/Yit mice by the inhibition of MAdCAM-1

The aetiology of Crohns disease (CD) remains unknown. Since SAMP1/Yit mice have been reported to develop CD‐like spontaneous enteric inflammation, such mice have been studied as an animal model of CD. In this study, using this model we examined T lymphocyte migration in microvessels of intestinal mucosa in vivo and the expression of adhesion molecules by immunohistochemistry. Fluorescence‐labelled T lymphocytes isolated from AKR/J (control) mice were injected into the tail veins of recipient mice, and T lymphocyte migration in the postcapillary venules of Peyers patches, submucosal microvessels, and villus capillaries of the terminal ileum was monitored using an intravital microscope. Adhesion of T lymphocytes was significantly increased in 35 week old SAMP1/Yit mice compared with that in AKR/J or 15 week old SAMP1/Yit mice. Immunohistochemical study showed increased infiltration of CD4, CD8 and β7‐integrin‐positive cells and increased expression of MAdCAM‐1 and VCAM‐1 in the terminal ileum of SAMP1/Yit mice. Antibodies against MAdCAM‐1 and VCAM‐1 significantly inhibited adhesion of T lymphocytes to microvessels of the terminal ileum, and anti‐MAdCAM‐1 antibody showed stronger suppressive effect than the anti‐VCAM‐1 antibody. Periodical administration of anti‐MAdCAM‐1 antibody twice a week for 7 weeks significantly ameliorated ileitis of SAMP1/Yit mice, but submucosal hypertrophy was not significantly suppressed. Anti‐VCAM‐1 antibody treatment failed to show significant resolution of ileitis. In addition, anti‐MAdCAM‐1 antibody treatment also attenuated established ileitis. The results demonstrate that, although MAdCAM‐1 and VCAM‐1 play an important role in T lymphocyte–endothelial cell interactions in SAMP1/Yit mice, MAdCAM‐1 may be a more appropriate target for therapeutic modulation of chronic ileitis.

Involvement of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the pathogenesis of granulomatous colitis in rats

Although increased expression of mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1) has been demonstrated in inflammatory sites of various diseases, its role in colitis remains unknown. In this study, we examined whether MAdCAM‐1 is involved in the pathogenesis of granulomatous colitis induced by peptidoglycan‐polysaccharide (PG‐PS). Experimental colitis was induced by intramural injection of PG‐PS to rat colon. After 3 weeks the colon was removed and the mucosal inflammation was assessed. The area of MAdCAM‐1‐positive venules and the subsets of infiltrating cells were determined in colonic mucosa by immunohistochemistry. In another experiment, monoclonal antibody against MAdCAM‐1 was administered intraperitoneally to examine its attenuating effect on colitis. The intramural injection of PG‐PS induced significant colonic inflammation with granuloma formation. The submucosa was drastically thickened with the infiltration of CD4 positive lymphocytes and ED‐1 positive macrophages. Intense MAdCAM‐1 expression was observed on endothelium of the submucosal venules in inflamed mucosa. Administration of anti‐MAdCAM‐1 antibody significantly attenuated the PG‐PS‐induced colonic damage and cell infiltration. Enhanced expression of MAdCAM‐1 was demonstrated in venular endothelium of the inflamed colon in PG‐PS‐induced colitis. The attenuating effect of anti‐MAdCAM‐1 suggests the importance of the MAdCAM‐1‐dependent process in the formation of chronic granulomatous colitis.

Omega-3 polyunsaturated fatty acids ameliorate the severity of ileitis in the senescence accelerated mice (SAM)P1/Yit mice model

Clinical studies using omega‐3 polyunsaturated fatty acids (ω3‐PUFA) to Crohns disease (CD) are conflicting. Beneficial effects of dietary ω3‐PUFA intake in various experimental inflammatory bowel disease (IBD) models have been reported. However, animal models of large intestinal inflammation have been used in all previous studies, and the effect of ω3 fat in an animal model of small intestinal inflammation has not been reported. We hypothesized that the effects of ω3 fat are different between large and small intestine. The aim of this study was to determine whether the direct effect of ω3 fat is beneficial for small intestinal inflammation. Senescence accelerated mice (SAM)P1/Yit mice showed remarkable inflammation of the terminal ileum spontaneously. The numbers of F4/80‐positive monocyte–macrophage cells as well as β7‐integrin‐positive lymphocytes in the intestinal mucosa were increased significantly compared with those in the control mice (AKR‐J mice). The area of mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1)‐positive vessels was also increased. The degree of expression levels of monocyte chemoattractant protein‐1 (MCP‐1), interleukin (IL)‐6 and interferon (IFN)‐γ mRNA were increased significantly compared with those in the control mice. The feeding of two different kinds of ω3 fat (fish‐oil‐rich and perilla‐oil‐rich diets) for 16 weeks to SAMP1/Yit mice ameliorated inflammation of the terminal ileum significantly. In both the ω3‐fat‐rich diet groups, enhanced infiltration of F4/80‐positive monocytes/macrophages in intestinal mucosa of SAMP1/Yit mice cells and the increased levels of MCP‐1, IL‐6 and IFN‐γ mRNA expression were ameliorated significantly compared with those in the control diet group. The results suggest that ω3 fat is beneficial for small intestinal inflammation by inhibition of monocyte recruitment to inflamed intestinal mucosa.

Trans fatty acids exacerbate dextran sodium sulphate-induced colitis by promoting the up-regulation of macrophage-derived proinflammatory cytokines involved in T helper 17 cell polarization

Numerous reports have shown that a diet containing large amounts of trans fatty acids (TFAs) is a major risk factor for metabolic disorders. Although recent studies have shown that TFAs promote intestinal inflammation, the underlying mechanisms are unknown. In this study, we examined the effects of dietary fat containing TFAs on dextran sodium sulphate (DSS)‐induced colitis. C57 BL/6 mice were fed a diet containing 1·3% TFAs (mainly C16:1, C18:1, C18:2, C20:1, C20:2 and C22:1), and then colitis was induced with 1·5% DSS. Colonic damage was assessed, and the mRNA levels of proinflammatory cytokines and major regulators of T cell differentiation were measured. The TFA diet reduced survival and exacerbated histological damage in mice administered DSS compared with those fed a TFA‐free diet. The TFA diet significantly elevated interleukin (IL)‐6, IL‐12p40, IL‐23p19 and retinoic acid‐related orphan receptor (ROR)γt mRNA levels in the colons of DSS‐treated animals. Moreover, IL‐17A mRNA levels were elevated significantly by the TFA diet, with or without DSS treatment. We also examined the expression of proinflammatory cytokines in lipopolysaccharide (LPS)‐stimulated RAW264.7 cells and peritoneal macrophages. These cells were exposed to TFAs (linoelaidic acid or elaidic acid) with or without LPS and the mRNA levels of various cytokines were measured. IL‐23p19 mRNA levels were increased significantly by TFAs in the absence of LPS. Cytokine expression was also higher in LPS‐stimulated cells exposed to TFAs than in unexposed LPS‐stimulated cells. Collectively, our results suggest that TFAs exacerbate colonic inflammation by promoting Th17 polarization and by up‐regulating the expression of proinflammatory cytokines in the inflamed colonic mucosa.

Endotoxin stimulates monocyte-endothelial cell interactions in mouse intestinal Peyer's patches and villus mucosa.

Although monocyte–endothelial cell interactions represent an initial step in controlling the recruitment of monocytes in inflamed tissues, their dynamic processes in microvessels of lymphoid (Peyers patches) and non‐lymphoid (villus) regions in gut‐associated lymphoid tissue remain poorly understood. We monitored the migration of fluorescence‐labelled monocytes derived from the spleen in intestinal microvessels with or without lipopolysaccharide (LPS) treatment and investigated the role of adhesion molecules, P‐selectin, vascular cell adhesion molecule (VCAM‐1) and intercellular adhesion molecule‐1 (ICAM‐1). In control mice, there were few interactions between infused monocytes and the endothelium of intestinal microvessels. The monocyte–endothelial interactions (both rolling and adhesion) were significantly increased in intestinal microvessels of LPS‐treated mice compared with those in controls. Anti‐P‐selectin monoclonal antibody (MoAb) significantly suppressed the LPS‐induced increase in monocyte rolling in postcapillary venules of Peyers patches and submucosal venules. Anti‐VCAM‐1 MoAbs significantly suppressed the LPS‐induced increase in monocyte adhesion to postcapillary venules (PCVs) of Peyers patches, submucosal venules, and villus capillaries. In contrast, anti‐ICAM‐1 MoAb significantly suppressed the number of adherent monocytes in PCV of Peyers patches but not in submucosal venules or villus capillaries. These observations demonstrated that LPS treatment resulted in a significant increase in recruitment of monocytes both in microvessels of lymphoid and non‐lymphoid regions and that P‐selectin, VCAM‐1 and ICAM‐1 appeared to play important roles in LPS‐induced interactions.

Interferon-α increases monocyte migration via platelet–monocyte interaction in murine intestinal microvessels

The aim of this study was to investigate the effect of interferon (IFN)‐α on recruitment of platelets and monocytes within the murine small intestinal venular endothelium. Monocytes were isolated from bone marrow of C57B6 mice. Platelets were collected from murine blood. Rolling and adhesion to submucosal microvessels in the small intestine were examined under an intravital fluorescence microscope after injection of fluorescein‐labelled monocytes or platelets. In some mice, IFN‐α (5 × 105U/kg) was administered intraperitoneally. After treatment with an antibody against P‐selectin, changes in monocyte and platelet migration were also investigated. Changes in monocyte migration under the condition of thrombocytopenia were also investigated. Platelets and monocytes interacted with murine intestinal microvessels, although only few platelets and monocytes showed migration behaviour. Intraperitoneal injection of IFN‐α enhanced the migration of both platelets and monocytes in the intestinal microvessels. Pretreatment with anti‐P‐selectin attenuated the increase in migration of platelets and monocytes induced by administration of IFN‐α. Thrombocytopenia decreased the rolling ratio of monocytes, suggesting that the effect of IFN‐α on migration was P‐selectin‐dependent, derived from both the endothelium of microvessels and platelets. The results of this study suggest that IFN‐α acts as a potent proinflammatory agent via its stimulatory effect on the endothelium–platelet–monocyte interaction in intestinal microvessels by a P‐selectin‐dependent mechanism.