Shunsuke Komoto

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)

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.

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

Background and Aim:  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.

Acyl-CoA:cholesterol acyltransferase 1 mediates liver fibrosis by regulating free cholesterol accumulation in hepatic stellate cells

BACKGROUND & AIMS Acyl-coenzyme A: cholesterol acyltransferase (ACAT) catalyzes the conversion of free cholesterol (FC) to cholesterol ester, which prevents excess accumulation of FC. We recently found that FC accumulation in hepatic stellate cells (HSCs) plays a role in progression of liver fibrosis, but the effect of ACAT1 on liver fibrosis has not been clarified. In this study, we aimed to define the role of ACAT1 in the pathogenesis of liver fibrosis. METHODS ACAT1-deficient and wild-type mice, or Toll-like receptor 4 (TLR4)(-/-)ACAT1(+/+) and TLR4(-/-)ACAT1(-/-) mice were subjected to bile duct ligation (BDL) for 3 weeks or were given carbon tetrachloride (CCl4) for 4 weeks to induce liver fibrosis. RESULTS ACAT1 was the major isozyme in mice and human primary HSCs, and ACAT2 was the major isozyme in mouse primary hepatocytes and Kupffer cells. ACAT1 deficiency significantly exaggerated liver fibrosis in the mouse models of liver fibrosis, without affecting the degree of hepatocellular injury or liver inflammation, including hepatocyte apoptosis or Kupffer cell activation. ACAT1 deficiency significantly increased FC levels in HSCs, augmenting TLR4 protein and downregulating expression of transforming growth factor-β (TGFβ) pseudoreceptor Bambi (bone morphogenetic protein and activin membrane-bound inhibitor), leading to sensitization of HSCs to TGFβ activation. Exacerbation of liver fibrosis by ACAT1 deficiency was dependent on FC accumulation-induced enhancement of TLR4 signaling. CONCLUSIONS ACAT1 deficiency exaggerates liver fibrosis mainly through enhanced FC accumulation in HSCs. Regulation of ACAT1 activities in HSCs could be a target for treatment of liver fibrosis.

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.

Exposure to fatty acids modulates interferon production by intraepithelial lymphocytes

Intraepithelial lymphocytes (IELs) play important roles in intestinal mucosal immunity. Although fatty acids are known to modulate the functions of immune effector cells, there has been no information about how fat exposure affects immunological function of IELs. In this study, we examined how fatty acids of various chain lengths modulate the production of interferon (IFN)-gamma by IELs stimulated with T-cell receptor (TCR) or interleukin (IL)-12/IL-18. IELs isolated from the small intestine of BALB/c mice were stimulated with plate-coated anti-CD3 monoclonal antibody (mAb) or IL-12/IL-18. They were coincubated in microtiter plates for 3 days with various concentrations of fatty acid micelles. We used arachidonic acid, linoleic acid, and oleic acid as long-chain fatty acids, and used octanoic acid as a medium-chain fatty acid. IFN-gamma in the supernatants were measured by ELISA, and the expression of IFN-gamma mRNA in IELs was determined by RT-PCR. Significant production of IFN-gamma from IELs was observed after anti-CD3 mAb stimulation. The combination of IL-12 and IL-18 induced significant levels of IFN-gamma production without TCR stimulation. Increased IFN-gamma mRNA was also observed after anti-CD3 or IL-12/IL-18 stimulation. Long-chain fatty acids dose-dependently inhibited the stimulated-IFN-gamma production at concentrations greater than 10 micro M, but the medium-chain fatty acid did not cause any significant changes in IFN-gamma production. IFN-gamma production from gammadelta IELs was very low compared with alphabeta IELs, however, both populations showed similar attenuating patterns when treated with long-chain fatty acids. There is a possibility that the exposure of IELs to intraluminal fatty acids significantly modifies the immune function of intestinal mucosa.

CXCL12 and CCL20 Play a Significant Role in Mucosal T-Lymphocyte Adherence to Intestinal Microvessels in Mice

Objective: Although it is known that the chemokines CXCL12 and CCL20 are expressed in the intestine, their contribution to lymphocyte homing has not been investigated in detail. The authors investigated whether the CXCL12‐CXCR4 and CCL20‐CCR6 systems are involved in T lymphocyte–endothelial interaction in microvessels of the small and large intestines.

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.

Increased expression of lipocalin-type-prostaglandin D synthase in ulcerative colitis and exacerbating role in murine colitis

The pathogenesis of ulcerative colitis (UC) is unclear, but enhancement of disease activity by usage of nonsteroidal anti-inflammatory drugs suggests involvement of prostanoid in its pathophysiology. However, biological effect of prostaglandin (PG) D(2) on intestinal inflammation remains unknown. We investigated the expression of enzymes for PGD(2) synthesis, prostaglandin D synthase (PGDS), and its relation to the activity of colitis in UC patients. The role of lipocalin-type PGDS (L-PGDS) using a murine colitis model was also assessed. Tissue samples were obtained by colonic biopsies from patients with UC. Expression levels of mRNAs for L-PGDS and hematopoietic-type PGDS were investigated by quantitative RT-PCR. COX-2 and L-PGDS expression was investigated by immunohistochemistry. Localization of L-PGDS expression was also determined by in situ hybridization. In experimental study, mice were treated with dextran sodium sulfate in the drinking water to induce colitis. The degree of colonic inflammation was compared with L-PGDS(-/-) mice and control mice. The level of L-PGDS mRNA expression was increased in UC patients in parallel with disease activity. Colocalization of L-PGDS and cyclooxygenase (COX) 2 was observed in lamina proprial infiltrating cells and muscularis mucosa in UC patients. The level of hematopoietic PGDS mRNA expression did not differ from control mucosa. Dextran sodium sulfate treatment to L-PGDS(-/-) mice showed lower disease activity than control mice. We reported for the first time the presence of L-PGDS in the COX-2-expressing cells in the mucosa of active UC patients and that only L-PGDS increased with disease activity. An animal model study suggests that PGD(2) derived from L-PGDS-expressing cells plays proinflammatory roles in colitis.