Masaaki Higashiyama

Umami receptor activation increases duodenal bicarbonate secretion via glucagon-like peptide-2 release in rats

Luminal nutrient chemosensing during meal ingestion is mediated by intestinal endocrine cells, which regulate secretion and motility via the release of gut hormones. We have reported that luminal coperfusion of l-Glu and IMP, common condiments providing the umami or proteinaceous taste, synergistically increases duodenal bicarbonate secretion (DBS) possibly via taste receptor heterodimers, taste receptor type 1, member 1 (T1R1)/R3. We hypothesized that glucose-dependent insulinotropic peptide (GIP) or glucagon-like peptide (GLP) is released by duodenal perfusion with l-Glu/IMP. We measured DBS with pH and CO2 electrodes through a perfused rat duodenal loop in vivo. GIP, exendin (Ex)-4 (GLP-1 receptor agonist), or GLP-2 was intravenously infused (0.01–1 nmol/kg/h). l-Glu (10 mM) and IMP (0.1 mM) were luminally perfused with or without bolus intravenous injection (3 or 30 nmol/kg) of the receptor antagonists Pro3GIP, Ex-3(9-39), or GLP-2(3-33). GIP or GLP-2 infusion dose-dependently increased DBS, whereas Ex-4 infusion gradually decreased DBS. Luminal perfusion of l-Glu/IMP increased DBS, with no effect of Pro3GIP or Ex-3(9-39), whereas GLP-2(3-33) inhibited l-Glu/IMP-induced DBS. Vasoactive intestinal peptide (VIP)(6–28) intravenously or NG-nitro-l-arginine methyl ester coperfusion inhibited the effect of l-Glu/IMP. Perfusion of l-Glu/IMP increased portal venous concentrations of GLP-2, followed by a delayed increase of GLP-1, with no effect on GIP release. GLP-1/2 and T1R1/R3 were expressed in duodenal endocrine-like cells. These results suggest that luminal l-Glu/IMP-induced DBS is mediated via GLP-2 release and receptor activation followed by VIP and nitric oxide release. Because GLP-1 is insulinotropic and GLP-2 is intestinotrophic, umami receptor activation may have additional benefits in glucose metabolism and duodenal mucosal protection and regeneration.

Expression of PD-1, PD-L1, and PD-L2 in the liver in autoimmune liver diseases

OBJECTIVES:PD-L1 (also B7-H1) and PD-L2 (also B7-DC) are ligands for programmed death-1 (PD-1), which is a member of the CD28/B7 superfamily of costimulatory molecules and plays an inhibitory role on the periphery. Impaired regulation of this system may cause disruption to self-tolerance leading to autoimmunity; however, the role of these molecules in the liver is unknown. Therefore, we examined the expression of PD-1, PD-L1, and PD-L2 in the liver in autoimmune liver diseases.METHODS:We examined the liver expression of these molecules in autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) with no previous medical treatment using immunohistochemical staining and real-time PCR, and compared with chronic hepatitis type C (CHC) as a control.RESULTS:Although PD-1, PD-L1, and PD-L2 were expressed in the liver in AIH, PBC, as well as CHC, the expressions were relatively lower in PBC. In AIH, despite more severe inflammation than in CHC, the expression of these molecules was not greater than in CHC, and when compared with the relative expression of PD-L1, PD-L2 was lower in AIH. PD-L1 and PD-L2 expressions were well correlated with the level of IFN-γ; however, relatively decreased induction for PD-L1 and PD-L2 by IFN-γ was observed in AIH or PBC than in CHC.CONCLUSION:Modulation of PD-1/PD-L1 and PD-L2 systems may play a role in the development of autoimmune liver diseases.

HIF-1 in T cells ameliorated dextran sodium sulfate-induced murine colitis

HIF‐1 is active in hypoxia, such as inflamed mucosa, and HIF‐1 in epithelium has been reported to control inflamed mucosa in IBD models. Although T cells play an important role for pathogenesis of IBD, the function of HIF‐1 in T cells remains to be elucidated. We aimed to clarify the function of HIF‐1 in T cells in IBD with focus on the balance between Treg and Teff. Double immunohistochemistry of colonic mucosa in IBD patients showed that HIF‐1 was expressed in T cells infiltrating the inflamed mucosa, suggesting that HIF‐1 in T cells is involved in the pathogenesis. DSS administration to T cell‐specific HIF‐1α KO mice showed more severe colonic inflammation than control mice with the up‐regulation of Th1 and Th17. Hypoxic stimulation in vitro increased Treg activation in WT T cells but not in HIF‐1‐deleted T cells. In contrast, hypoxic stimulation increased Th17 activation, and the degree was higher in HIF‐1‐deleted cells than in control cells. These results show that hypoxia controls intestinal inflammation by regulating cytokine balance in a HIF‐1‐dependent manner, suggesting that strengthening HIF‐1 induction in T cells at the sites of inflammation might be a therapeutic strategy for IBD regulation.

Prevalence of serum celiac antibody in patients with IBD in Japan

BackgroundAlthough the incidence of inflammatory bowel diseases (IBD) in Japan has increased, the prevalence of celiac disease is considered very low with the lowest genetic disposition. IBD is reported as the most common comorbidity because of the high positive rate of serological celiac markers. The aim of this study was to examine the current incidence of celiac disease, especially in IBD patients in Japan, where both wheat consumption and incidence of IBD have increased.MethodsA total of 172 patients with IBD and 190 controls in Japan were screened for serum antibody of tissue transglutaminase and deaminated gliadin peptide. In sero-positive patients, HLA testing and upper gastrointestinal endoscopy with duodenal biopsy was performed. Some of the sero-positive patients started a gluten-restricted or unrestricted diet, and serological change was determined.ResultsThe positivity of both serum antibodies was significantly higher in IBD and correlated with disease activity. However, no biopsy-defined or HLA-defined true celiac disease was found. A decrease in serum antibody titers was observed with a gluten-restricted diet.ConclusionsDespite the increased incidence of IBD and high positivity for serum celiac antibody in Japanese IBD patients, no true-positive celiac disease was noted, suggesting the presence of gluten intolerance in these populations.

Involvement of autotaxin/lysophospholipase D expression in intestinal vessels in aggravation of intestinal damage through lymphocyte migration

Lysophosphatidic acid (LPA) has a critical role in lymphocyte migration to secondary lymphoid organs. Autotaxin (ATX)/lysophospholipase D, in the vascular endothelium, is the main enzyme involved in LPA production. Whether ATX is involved in pathological lymphocyte migration to the inflamed mucosa has not been studied. We investigated the involvement of ATX in inflammatory bowel disease patients and two murine models of colitis. Tissue samples were obtained by intestinal biopsies from patients with Crohn’s disease and those with ulcerative colitis with informed consent. ATX immunoreactivity was colocalized with MAdCAM-1-positive high-endothelial-like vessels, close to sites of lymphocyte infiltration. Enhanced expression of ATX mRNA was observed in the inflamed mucosa from Crohn’s disease and ulcerative colitis patients. ATX mRNA expression level was remarkably higher in the actively inflamed mucosa than in the quiescent mucosa in the same patient. In the T-cell-transferred mouse model, ATX mRNA expression level gradually increased as colitis developed. In the dextran sodium sulfate mouse model, the expression level was considerably higher in colonic mucosa of chronically developed colitis than in colonic mucosa of acute colitis. Administration of an ATX inhibitor, bithionol, remarkably decreased lymphocyte migration to the intestine and ameliorated both dextran sodium sulfate-induced colitis and CD4-induced ileocolitis. In transwell assays, administration of bithionol or 1-bromo-3(s)-hydroxy-4-(palmitoyloxy) butylphosphonate (BrP-LPA) significantly decreased transmigration of splenocytes through high-endothelial-like vessels induced by TNF-α. We conclude that enhanced expression of ATX in the active mucosa has been implicated in the pathophysiology of inflammatory bowel disease through enhancing aberrant lymphocyte migration to the inflamed mucosa.

Physiological stress exacerbates murine colitis by enhancing proinflammatory cytokine expression that is dependent on IL-18

Psychological stress is an environmental factor considered to be a precipitating factor of inflammatory bowel disease. Interleukin (IL)-18 plays a role in stress-induced aggravation in some diseases. The aim of this study was to establish a model of murine colitis exacerbated by psychological stress and to clarify the role of IL-18 in this model. Male C57Bl/6 mice and IL-18(-/-) mice were used for this study. The mice received dextran sulfate sodium (DSS) for induction of colitis. Some mice were exposed to psychological stress using a communication box. Body weight, colonic length, and histological inflammation were measured for assessment of colitis. Tumor necrosis factor (TNF)-α and IL-18 expression in the colon and IL-18 expression in the adrenal gland were analyzed using real-time PCR. The effect of anti-IL-18 antibody was also investigated. Effects of TNF-α and IL-18 on cytokine expressions were studied using the colonic epithelial cell line LS174T. Induction of psychological stress in DSS-treated wild-type mice significantly exacerbated colitis with enhanced expression of proinflammatory cytokines and IL-18. However, induction of psychological stress in DSS-treated IL-18(-/-) mice did not aggravate colitis compared with that in the IL-18(-/-) group given only DSS treatment. Stress-induced aggravation of colitis was ameliorated significantly by anti-IL-18 antibody treatment. IL-18 did not enhance TNF-α-induced expression of intercellular adhesion molecule-1 or IL-8 in LS174T. We established a model of colitis exacerbated by psychological stress. Psychological stress enhanced IL-18 expression and plays a proinflammatory role in stress-induced aggravation of colitis.

P-Selectin-Dependent Monocyte Recruitment Through Platelet Interaction in Intestinal Microvessels of LPS-Treated Mice

Background: Although platelets or monocytes are thought to be involved in intestinal inflammation, there has been no report on whether platelets can modulate monocyte recruitment in intestinal microvessels. The objective of this study was to determine whether blockade of platelet adhesion attenuates monocyte recruitment in inflamed murine intestinal microvessels. Methods: Monocytes and platelet‐rich plasma were obtained from C57B6/J mice. Interaction of monocytes and platelets with intestinal microvessels was observed under an intravital microscope. Lipopolysaccharide (LPS) was administered intraperitoneally. The effects of anti‐P‐selectin or anti‐platelets antibody treatments or phosphodiesterase (PDE) inhibitors (PDE‐3 and PDE‐2/4 inhibitor) treatments were also studied. Results: LPS‐treatment increased the rolling and adhesion of both platelets and monocytes. Pretreatment with an anti‐P‐selectin antibody inhibited the increased platelet adhesion to venular walls and also attenuated the monocyte adhesion. A PDE‐2/4 inhibitor (ibuzilast) also ameliorated both platelet and monocyte adhesion. A PDE‐3 inhibitor (cilostazol) ameliorated only monocyte adhesion without directly affecting the adhesion of platelets to microvessels. Conclusions: We observed inhibition of platelets adhesion attenuated monocytes recruitment in intestinal microvessels. Attenuation of LPS induced monocyte adhesion by a specific PDE‐3 inhibitor suggests that P‐selectin on activated platelets may play an important role through monocyte and platelet interaction.

Dietary lipids and sweeteners regulate glucagon-like peptide-2 secretion

Glucagon-like peptide-2 (GLP-2) is a potent intestinal growth factor derived from enteroendocrine L cells. Although food intake is known to increase GLP-2 secretion, its regulatory mechanisms are largely unknown as a result of its very short half-life in venules. The aims of this study were to compare the effects of luminal nutrients on the stimulation of GLP-2 secretion in vivo using lymph samples and to clarify the involvement of the sweet taste receptor in this process in vitro. Lymph samples were collected from the thoracic duct after bolus administration of dietary lipids or sweetening agents into the duodenum of rats. Human enteroendocrine NCI-H716 cells were also used to compare the effects of various nutrients on GLP-2 secretion. GLP-2 concentrations were measured by ELISA in vivo and in vitro. GLP-2 secretion was enhanced by polyunsaturated fatty acid- and monounsaturated fatty acid-rich dietary oils, dietary carbohydrates, and some kinds of sweeteners in rats; this effect was reproduced in NCI-H716 cells using α-linolenic acid (αLA), glucose, and sweeteners. GLP-2 secretion induced by sweetening agents was inhibited by lactisole, a sweetness-antagonizing inhibitor of T1R3. In contrast, lactisole was unable to inhibit GLP-2 secretion induced by αLA alone. Our results suggested that fatty acid- and sweetener-induced GLP-2 secretion may be mediated by two different pathways, with the sweet taste receptor involved in the regulation of the latter.

Toll-like receptor (TLR) 2 agonists ameliorate indomethacin-induced murine ileitis by suppressing the TLR4 signaling.

Few drugs have been found satisfactory in the treatment of nonsteroidal anti‐inflammatory drugs (NSAIDs)‐induced enteropathy. Toll‐like receptor (TLR) 4 and aberrant leukocyte migration to the intestinal mucosa are reported to be involved in the pathology of intestinal enteropathy and TLR2 agonists have been found to evoke hyposensitivity to TLR4 stimulation in vitro. In this study, we investigated whether and how lipoarabinomannan (LAM) or lipoteichoic acid (LTA), TLR2 agonists, attenuated indomethacin (IND)‐induced intestinal damage.

Cilostazol, a specific PDE-3 inhibitor, ameliorates chronic ileitis via suppression of interaction of platelets with monocytes.

Excessive migration of monocytes to a site of intestinal inflammation contributes to tissue damage in Crohns disease. It is known that cilostazol, a specific phosphodiesterase-3 (PDE-3) inhibitor of platelets, decreases monocyte recruitment to intestinal mucosa through suppression of platelet-monocyte interactions. The objective of this study was to clarify whether cilostazol ameliorates murine ileitis by suppression of monocyte migration. Significant inflammation was induced in the ileum of SAMP1/Yit mice at 23 wk of age after piroxicam treatment for 3 wk. Weight of the terminal ileum of mice was significantly greater with inflammatory cell infiltration in SAMP1/Yit mice than in control mice (AKR-J). Treatment of SAMP1/Yit mice with cilostazol-containing food (200 ppm) for 3 wk significantly attenuated the increase in intestinal weight and the histological changes, including invasion of F4/80-positive macrophages. A significant increase in migration of monocytes and platelets to microvessels of the ileal mucosa was observed in SAMP/Yit mice in vivo by using an intravital fluorescence microscope. Pretreatment with cilostazol significantly attenuated the increased migration of monocytes, possibly through suppression of platelet-monocyte interactions. In conclusion, a PDE-3 inhibitor ameliorates murine ileitis through attenuating migration of monocytes to the intestinal mucosa, suggesting a potential usefulness of antiplatelet drugs for treatment of Crohns disease.