Atsushi Kawaguchi

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)

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.

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.

Expression of bone morphogenetic proteins in colon carcinoma with heterotopic ossification.

Here we report the case of a 50‐year‐old woman with adenocarcinoma of the colon, showing heterotopic ossification. The patient was referred to our hospital for investigation of anemia secondary to occult gastrointestinal blood loss. By colonoscopy, an irregular polypoid mass was found in the ascending colon. A biopsy of the lesion revealed moderately to poorly differentiated adenocarcinoma with heterotopic ossification. A right hemicolectomy was done and revealed areas of heterotopic bone within the tumor, but no ossification was evident in the metastatic lesions within the mesenteric lymph nodes. The formation of heterotopic bone in gastrointestinal tumors is rare and its exact mechanism is unknown. Immunohistochemical localization of bone morphogenetic proteins (BMP), known to be primary inducers of new bone formation, was determined. BMP‐5 and ‐6 were prominent in the cytoplasm of tumor cells, and they stained weakly in osteoblast‐like cells adjacent to newly formed bone. Cytoplasmic staining for BMP‐2 and ‐4 was weak in tumor cells, osteoblast‐like cells, and stromal fibroblast cells. BMP may play an important role in heterotopic ossification in colon adenocarcinoma.

Changes in regulatory molecules for lymphangiogenesis in intestinal lymphangiectasia with enteric protein loss

Background and Aim:u2002 Vascular endothelial growth factor receptor 3 (VEGFR3) and LYVE‐1 are specifically expressed in the endothelium of the lymphatic systems. VEGF‐C, D, FOXC2, Prox 1, and SOX18 are known to play central roles in lymphatic development. We investigated the expression of regulatory molecules for lymphangiogenesis in the duodenal mucosa of idiopathic intestinal lymphangiectasia.

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.

Anti-inflammatory effects of the genus Bifidobacterium on macrophages by modification of phospho-IκB and SOCS gene expression.

Although beneficial roles of probiotics for inflammatory bowel diseases have been reported, their direct action on immune cells has not been elucidated. In this study, we investigated how three species of Bifidobacterium and Enterococcus faecalis differentially modulate production of cytokines from lipopolysaccharide (LPS)‐stimulated macrophages in vitro using RAW264.7 cells. The mRNA levels of proinflammatory cytokines were remarkably increased after exposure to LPS, E. faecalis alone and LPS combined with E. faecalis. In contrast, IL‐10 mRNA levels were significantly decreased after exposure to E. faecalis compared with exposure to Bifidobacterium species. When cells were exposed to Bifidobacterium species combined with LPS, mRNA levels of IL12p40 were decreased by co‐culture with B. breve and B. longum, IL‐1β mRNA levels were decreased by B. breve and B. adorescentis and TNF‐α mRNA levels were decreased by B. adolescentis compared with LPS alone. The three species of Bifidobacterium significantly inhibited phosphorylation of IκB‐α induced by LPS. The mRNA levels of SOCS1 and SOCS3 were increased by exposure to LPS alone; however, the mRNA levels of SOCS1 or SOCS3 were increased more by exposure to Bifidobacterium species combined with LPS. Conversely, E. faecalis combined with LPS induced significantly lower levels of SOCS mRNA than those induced by Bifidobacterium species combined with LPS. These results indicated that certain species of genus Bifidobacterium could negatively modulate mRNA levels of proinflammatory cytokines produced from LPS‐stimulated RAW264.7 cells, which is possibly related to inhibition of IκB‐α phosphorylation and stimulation of SOCS signalling.

Role of nociceptin/orphanin FQ (Noc/oFQ) in murine experimental colitis.

Nociceptin/orphanin (Noc/oFQ), endogenous agonist for nociceptin receptor (NOR), is thought to be a stimulator of neurogenic inflammation. We investigated the possible role of Noc/oFQ in the development of colitis using NOR-deficient mice treated with dextran sulfate sodium (DSS). Colitis was significantly improved in NOR-deficient mice against wild-type mice. Expression level of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and infiltrating cells also significantly decreased in NOR-deficient mice against wild-type mice. Nociceptin expression increased in wild-type mice after DSS treatment. These results suggest stimulation by Noc/oFQ deteriorates colonic inflammation via up-regulation of adhesion molecule.

Differential expression of CCR5 and CRTH2 on infiltrated cells in colonic mucosa of patients with ulcerative colitis

Background and Aim:u2002 The pathogenesis of ulcerative colitis (UC) is unclear, but abnormal infiltration of T lymphocytes in the colonic mucosa has been implicated in the mucosal tissue damage. The abnormal cytokine production because of a T helper (h)1/Th2 imbalance may play an important role in continuing inflammation in the colonic mucosa. In the present study, the expression of chemokine receptor 5 (CCR5) as a Th1 marker and a chemoattractant receptor‐homologs molecule expressed on Th2 cells (CRTH2) were investigated in order to analyze impaired Th1/Th2 responses in the colonic mucosa of UC patients.

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.