Ryota Hokari

Increased expression of an inducible isoform of nitric oxide synthase and the formation of peroxynitrite in colonic mucosa of patients with active ulcerative colitis

Background—Increased production of reactive metabolites of oxygen and nitrogen has been implicated in chronic inflammation of the gut. The object of this study was to examine the magnitude and location of nitric oxide synthase (NOS) activity and peroxynitrite formation in the colonic mucosa of patients with ulcerative colitis in relation to the degree of inflammation. Subjects—Thirty three patients with active ulcerative colitis (17 with mild or moderate inflammation, 16 with severe inflammation). Methods—Inducible NOS activity was determined in the colonic mucosa by measuring the conversion ofl-arginine to citrulline in the absence of calcium. The localisation of NOS and nitrotyrosine immunoreactivity was assessed immunohistochemically using the labelled streptavidin biotin method. Results—Inducible NOS activity increased in parallell with the degree of inflammation of the mucosa. Expression of inducible NOS was found not only in the lamina propria, but also in the surface of the epithelium. Peroxynitrite formation as assessed by nitrotyrosine staining was frequently observed in the lamina propria of actively inflamed mucosa. Conclusions—Nitric oxide and peroxynitrite formation may play an important role in causing irreversible cellular injury to the colonic mucosa in patients with active ulcerative colitis.

Increased Nitric Oxide Production and Inducible Nitric Oxide Synthase Activity in Colonic Mucosa of Patients with Active Ulcerative Colitis and Crohn's Disease

It is postulated that an enhanced production ofnitric oxide by inflamed intestine plays a role in thepathophysiology of active inflammatory bowel disease. Inthis study, systemic NOx concentrations and colonic nitric oxide synthase activity weredetermined in patients with ulcerative colitis orCrohns disease. The relationship between these twoparameters and disease activity, as well as differences in nitric oxide synthase activity betweenulcerative colitis and Crohns disease, were areas ofspecific focus. Patients with active ulcerative colitisand Crohns disease had significantly elevated plasma NOx concentrations; a positivecorrelation was found between NOx values andinducible nitric oxide synthase activities in the activemucosa of these patients. In active ulcerative colitis,levels of inducible nitric oxide synthase were significantlyelevated in both normal and inflamed mucosa, althoughinducible nitric oxide synthase activity was higher inthe latter. These colonic inducible nitric oxidesynthase activities correlated well with the results ofendoscopic and histologic grading of inflammation. Therewas no increase in constitutive nitric oxide synthaseactivity in patients with active ulcerative colitis. However, constitutive nitric oxidesynthase activity was significantly increased in theinflamed mucosa in patients with Crohns disease. InCrohns disease, elevated inducible nitric oxidesynthase activity was found in both normal and inflamedmucosa, with no significant difference between thetissues. Such differences in nitric oxide production inthe colonic mucosa possibly reflect the significant differences in the pathophysiology andcharacteristic clinical features between ulcerativecolitis and Crohns disease.

Free cholesterol accumulation in hepatic stellate cells: Mechanism of liver fibrosis aggravation in nonalcoholic steatohepatitis in mice

Although nonalcoholic steatohepatitis (NASH) is associated with hypercholesterolemia, the underlying mechanisms of this association have not been clarified. We aimed to elucidate the precise role of cholesterol in the pathophysiology of NASH. C57BL/6 mice were fed a control, high‐cholesterol (HC), methionine‐choline‐deficient (MCD), or MCD+HC diet for 12 weeks or a control, HC, high‐fat (HF), or HF+HC diet for 24 weeks. Increased cholesterol intake accelerated liver fibrosis in both the mouse models without affecting the degree of hepatocellular injury or Kupffer cell activation. The major causes of the accelerated liver fibrosis involved free cholesterol (FC) accumulation in hepatic stellate cells (HSCs), which increased Toll‐like receptor 4 protein (TLR4) levels through suppression of the endosomal‐lysosomal degradation pathway of TLR4, and thereby sensitized the cells to transforming growth factor (TGF)β‐induced activation by down‐regulating the expression of bone morphogenetic protein and activin membrane‐bound inhibitor. Mammalian‐cell cholesterol levels are regulated by way of a feedback mechanism mediated by sterol regulatory element‐binding protein 2 (SREBP2), maintaining cellular cholesterol homeostasis. Nevertheless, HSCs were sensitive to FC accumulation because the high intracellular expression ratio of SREBP cleavage‐activating protein (Scap) to insulin‐induced gene (Insig) disrupted the SREBP2‐mediated feedback regulation of cholesterol homeostasis in these cells. HSC activation subsequently enhanced the disruption of the feedback system by Insig‐1 down‐regulation. In addition, the suppression of peroxisome proliferator‐activated receptor γ signaling accompanying HSC activation enhanced both SREBP2 and microRNA‐33a signaling. Consequently, FC accumulation in HSCs increased and further sensitized these cells to TGFβ‐induced activation in a vicious cycle, leading to exaggerated liver fibrosis in NASH. Conclusion: These characteristic mechanisms of FC accumulation in HSCs are potential targets to treat liver fibrosis in liver diseases including NASH. (Hepatology 2014;58:154–169)

A High-Cholesterol Diet Exacerbates Liver Fibrosis in Mice via Accumulation of Free Cholesterol in Hepatic Stellate Cells

BACKGROUND & AIMS Some studies have indicated that dietary cholesterol has a role in the progression of liver fibrosis. We investigated the mechanisms by which dietary cholesterol might contribute to hepatic fibrogenesis. METHODS C57BL/6 mice were fed a high-cholesterol diet or a control diet for 4 weeks; liver fibrosis then was induced by bile-duct ligation or carbon tetrachloride administration. Hepatic stellate cells (HSCs) were isolated from mice fed high-cholesterol diets or from Niemann-Pick type C1-deficient mice, which accumulate intracellular free cholesterol. RESULTS After bile-duct ligation or carbon tetrachloride administration, mice fed high-cholesterol diets had significant increases in liver fibrosis and activation of HSCs compared with mice fed control diets. There were no significant differences in the degree of hepatocellular injury or liver inflammation, including hepatocyte apoptosis or Kupffer cell activation, between mice fed high-cholesterol or control diets. Levels of free cholesterol were much higher in HSCs from mice fed high-cholesterol diets than those fed control diets. In cultured HSCs, accumulation of free cholesterol in HSCs increased levels of Toll-like receptor 4 (TLR4), leading to down-regulation of bone morphogenetic protein and activin membrane-bound inhibitor (a pseudoreceptor for transforming growth factor [TGF]β); the HSCs became sensitized to TGFβ-induced activation. Liver fibrosis was not aggravated by the high-cholesterol diet in C3H/HeJ mice, which express a mutant form of TLR4; HSCs that express mutant TLR4 were not activated by accumulation of free cholesterol. CONCLUSIONS Dietary cholesterol aggravates liver fibrosis because free cholesterol accumulates in HSCs, leading to increased TLR4 signaling, down-regulation of bone morphogenetic protein and activin membrane-bound inhibitor, and sensitization of HSC to TGFβ. This pathway might be targeted by antifibrotic therapies.

Reduced sensitivity of inducible nitric oxide synthase-deficient mice to chronic colitis.

Abstract Background: Overproduction of nitric oxide by the inducible form of nitric oxide synthase (iNOS) has been implicated in colitis. Different authors have postulated both toxic and protective effects of nitric oxide (NO) in the pathophysiology of active inflammation. The objective of this study was to examine the role of iNOS in experimental chronic colitis using iNOS-deficient mice. Methods: For induction of colitis, mice received three cycles of 2% of dextran sodium sulfate (DSS) (M.W. 40,000) treatment in drinking water. The degree of colonic inflammation, leukocyte infiltration, and the expression of cell adhesion molecules were determined. INOS expression and nitrotyrosine were also determined by immunohistochemistry. Results: After DSS treatment, a moderate colitis with marked cell infiltration was observed. Intense expression of iNOS was observed on infiltrating cells as well as on the colonic mucosal epithelium in these animals. In the iNOS-deficient mice, tissue damage was significantly diminished. No iNOS or nitrotyrosine staining was found in iNOS-deficient mice. The number of infiltrating cells and the expression of mucosal adressin cell adhesion molecule-1 were significantly attenuated in the DSS-treated colon of iNOS-deficient mice. Conclusion: Induction of iNOS seems to act as a critical toxic effector molecule in the pathogenesis of chronic colonic inflammation.

Modulation of intestinal immune system by dietary fat intake : Relevance to Crohn's disease

Gut‐associated lymphoid tissue is the major inductive site of the mucosal immune system, which is functionally independent of the systemic immune system. Both the amount and type of dietary fat modulate intestinal immune function. Absorption of long‐chain fatty acids stimulates lymphocyte flux and lymphocyte blastogenesis in intestinal lymphatics. Long‐chain fatty acid absorption also significantly enhances migration of T lymphocytes to Peyers patches, possibly due to up‐regulation of adhesion molecules, such as α4‐integrin and L‐selectin. Lipoproteins are involved in stimulation of lymphocyte function by both receptor‐dependent and independent mechanisms. However, unsaturated fatty acids at higher concentrations have a suppressive effect on cell‐mediated immunity via eicosanoid release, receptor affinity changes or interactions with intracellular signal transduction. Fat absorption also influences various other cells in the intestinal mucosa: increased cytokine release from intestinal epithelial cells follows long‐chain fatty acid absorption. In Crohns disease, elemental diets and total parenteral nutrition often induce remission, possibly by reducing antigenic load on activated immune cells in the intestine and, thus, down‐regulating hyperreactive CD4 cells. Dietary oleic acid supplements caused an immunological reversal effect in the intestinal immune system of animals fed an elemental diet. An excess of long‐chain fatty acids in an elemental diet, therefore, may negate its beneficial effect on gut‐associated lymphoid tissues in Crohns disease. In contrast, supplemental dietary fish oil apparently tends to prevent relapse of Crohns disease. Because dietary fat intake is closely associated with immunological function of the intestinal mucosa, careful manipulation of dietary fat can be important in management of this disease.

Propionibacterium freudenreichii component 1.4‐dihydroxy‐2‐naphthoic acid (DHNA) attenuates dextran sodium sulphate induced colitis by modulation of bacterial flora and lymphocyte homing

Background and aim: 1.4-Dihydroxy-2-naphthoic acid (DHNA), a bifidogenic growth stimulator from Propionibacterium freudenreichii, is thought to have a beneficial effect as a prebiotic; however, its in vivo effect on intestinal inflammation remains unknown. The aim of this study was to determine whether oral administration of DHNA can ameliorate dextran sodium sulphate (DSS) induced colitis and to determine the possible underlying mechanisms. Method: Colitis was induced in mice by treatment with 2.0% DSS for seven days. DHNA (0.6 or 2.0 mg/kg) was given in drinking water prior to (preventive study) or after (therapeutic study) DSS administration. Colonic damage was histologically scored, and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expression and β7 positive cell infiltration were determined by immunohistochemistry. mRNA levels of proinflammatory cytokines (interleukin (IL)-1β, IL-6 and tumour necrosis factor α (TNF-α)) were determined by quantitative real time polymerase chain reaction. In addition, bacterial flora in the caecum, concentrations of short chain acids, and luminal pH were examined. Results: DHNA improved survival rate and histological damage score in mice administered DSS in both the preventive and therapeutic studies. DHNA significantly attenuated the enhanced expression of MAdCAM-1, the increased β7 positive cell number, and the increased mRNA levels of IL-1β, IL-6, and TNF-α in DSS treated colon. In addition, the decreased number of Lactobacillus and Enterobacteriaceae induced by DSS was recovered by DHNA. Preventive effects on decrease in butyrate concentration and decrease in pH level in mice administered DSS were also observed in the DHNA preventive study. Conclusion: DHNA, a novel type of prebiotic, attenuates colonic inflammation not only by balancing intestinal bacterial flora but also by suppressing lymphocyte infiltration through reduction of MAdCAM-1.

Blockade of PSGL-1 attenuates CD14+ monocytic cell recruitment in intestinal mucosa and ameliorates ileitis in SAMP1/Yit mice.

The pathogenesis of Crohn’s disease (CD) is not known. However, monocytes and macrophages are thought to play important roles in the development of mucosal inflammation. Therefore, in this study, we examined the role of monocyte‐endothelial cell interactions in senescence‐accelerated mouse P1 (SAMP1)/Yit mice, a murine model of spontaneous ileitis. Fluorescence‐labeled CD14+ monocytic cells isolated from the spleen and mesenteric lymph nodes of AKR/J (control) mice were injected into the tail veins of recipient (AKR/J and SAMP1/Yit) mice, and migration in the postcapillary venules (PCV) of Peyer’s patches, submucosal venules, and villus microvessels of the terminal ileum was monitored by using an intravital microscope. Rolling and adhesion of CD14+ monocytic cells in the PCV of Peyer’s patches and microvessels of the terminal ileum were increased in SAMP1/Yit mice. An imunohistochemical study showed increased expression of P‐selectin glycoprotein‐1 (PSGL‐1), P‐selectin, and vascular cell adhesion molecule‐1 in the terminal ileum of SAMP1/Yit mice. Antibodies against these three adhesion molecules significantly inhibited adhesion of CD14+ monocytic cells to the PCV of Peyer’s patches and microvessels of the terminal ileum, treatment with an anti‐PSGL‐1 monoclonal antibody (mAb) showing the strongest suppressive effect. Anti‐PSGL‐1 mAb also attenuated T cell adhesion in microvessels of intestinal mucosa. In addition, periodical administration of an anti‐PSGL‐1 mAb for 7 weeks significantly ameliorated ileitis of SAMP1/Yit mice. The results suggest that PSGL‐1‐P‐selectin interaction plays an important role in monocyte‐endothelial cell interactions and the development of ileitis in a murine model of CD and that the blockade of this adhesion molecule may be a novel strategy for treating CD.

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.