Hirohide Ohnishi

Toll-Like Receptor 9 Promotes Steatohepatitis by Induction of Interleukin-1β in Mice

BACKGROUND & AIMS Development of nonalcoholic steatohepatitis (NASH) involves the innate immune system and is mediated by Kupffer cells and hepatic stellate cells (HSCs). Toll-like receptor 9 (TLR9) is a pattern recognition receptor that recognizes bacteria-derived cytosine phosphate guanine (CpG)-containing DNA and activates innate immunity. We investigated the role of TLR9 signaling and the inflammatory cytokine interleukin-1beta (IL-1beta) in steatohepatitis, fibrosis, and insulin resistance. METHODS Wild-type (WT), TLR9(-/-), IL-1 receptor (IL-1R)(-/-), and MyD88(-/-) mice were fed a choline-deficient amino acid-defined (CDAA) diet for 22 weeks and then assessed for steatohepatitis, fibrosis, and insulin resistance. Lipid accumulation and cell death were assessed in isolated hepatocytes. Kupffer cells and HSCs were isolated to assess inflammatory and fibrogenic responses, respectively. RESULTS The CDAA diet induced NASH in WT mice, characterized by steatosis, inflammation, fibrosis, and insulin resistance. TLR9(-/-) mice showed less steatohepatitis and liver fibrosis than WT mice. Among inflammatory cytokines, IL-1beta production was suppressed in TLR9(-/-) mice. Kupffer cells produced IL-1beta in response to CpG oligodeoxynucleotide. IL-1beta but not CpG-oligodeoxynucleotides, increased lipid accumulation in hepatocytes. Lipid accumulation in hepatocytes led to nuclear factor-kappaB inactivation, resulting in cell death in response to IL-1beta. IL-1beta induced fibrogenic responses in HSCs, including secretion of tissue inhibitor of metalloproteinase-1. IL-1R(-/-) mice had reduced steatohepatitis and fibrosis, compared with WT mice. Mice deficient in MyD88, an adaptor molecule for TLR9 and IL-1R signaling, also had reduced steatohepatitis and fibrosis. TLR9(-/-), IL-1R(-/-), and MyD88(-/-) mice had less insulin resistance than WT mice on the CDAA diet. CONCLUSIONS In a mouse model of NASH, TLR9 signaling induces production of IL-1beta by Kupffer cells, leading to steatosis, inflammation, and fibrosis.

Hepatic recruitment of macrophages promotes nonalcoholic steatohepatitis through CCR2

Inflammatory cell infiltration in the liver is a hallmark of nonalcoholic steatohepatitis (NASH). The chemokine-chemokine receptor interaction induces inflammatory cell recruitment. CC-chemokine receptor (CCR)2 is expressed on hepatic macrophages and hepatic stellate cells. This study aims to investigate the therapeutic potential of CCR2 to NASH. Twenty-two weeks on a choline-deficient amino acid-defined (CDAA) diet induced steatosis, inflammatory cell infiltration, and liver fibrosis with increased CCR2 and monocyte chemoattractant protein (MCP)-1 expression in the wild-type livers. The infiltrated macrophages expressed CD68, CCR2, and a marker of bone marrow-derived monocytes, Ly6C. CCR2(-/-) mice had less steatosis, inflammatory cell infiltration, and fibrosis, and hepatic macrophages expressing CD68 and Ly6C were decreased. Toll-like receptor (TLR)4(-/-), TLR9(-/-), and MyD88(-/-) mice had reduced hepatic macrophage infiltration with decreased MCP-1 and CCR2 expression because TLR signaling is a potent inducer of MCP-1. To assess the role of Kupffer cells at the onset of NASH, Kupffer cells were depleted by liposomal clodronate. The Kupffer cell depletion ameliorated steatohepatitis with a decrease in the MCP-1 expression and recruitment of Ly6C-expressing macrophages at the onset of NASH. Finally, to test the therapeutic potential of targeting CCR2, a CCR2 inhibitor was administered to mice on a CDAA diet. The pharmaceutical inhibition of CCR2 prevented infiltration of the Ly6C-positive macrophages, resulting in an inhibition of liver inflammation and fibrosis. We concluded that CCR2 and Kupffer cells contribute to the progression of NASH by recruiting bone marrow-derived monocytes.

Role of Toll-Like Receptors and Their Downstream Molecules in the Development of Nonalcoholic Fatty Liver Disease

Activation of innate immunity is associated with the development of liver disease, including non-alcoholic fatty liver disease (NAFLD). In the innate immune system, Toll-like receptors (TLRs) are sensors that recognize bacterial and viral components such as lipopolysaccharide, bacterial DNA, and peptidoglycan. Recent data have demonstrated that the liver is exposed to a high load of TLR ligands due to bacterial overgrowth and increased intestinal permeability in NAFLD. Upon stimulation by these TLR ligands, hepatic immune cells produce various mediators that are involved in host defense. On the other hand, these mediators alter lipid metabolism, insulin signaling, and cell survival. Indeed, some TLR-deficient mice demonstrate lesser degrees of NAFLD even though TLR ligands are increased. This paper will highlight the recent progress on the study of TLR signaling and their downstream molecules in the development of NAFLD.

Activin A is an autocrine activator of rat pancreatic stellate cells: potential therapeutic role of follistatin for pancreatic fibrosis

Background and aim: The present study was conducted to examine the effect of activin A on activation of rat pancreatic stellate cells (PSCs). Methods: PSCs were prepared from rat pancreas using collagenase digestion and centrifugation with Nycodenz gradient. Activation of PSCs was examined by determining smooth muscle actin expression with western blotting. The presence of activin A receptors in PSCs was investigated by reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunocytochemistry. Expression of activin A and transforming growth factor β (TGF-β) mRNA was examined by RT-PCR. Activin A and TGF-β peptide concentrations were examined with ELISA. Existence of activin A peptide in PSCs was investigated by immunocytochemistry. Collagen secretion was determined by Sirius red dye binding. Results: Activin A receptors I and IIa were present in PSCs. PSCs expressed activin A mRNA and secreted activin A. Activin A enhanced PSC activation and collagen secretion in a dose dependent manner. TGF-β and activin A increased each other’s secretion and mRNA expression of PSCs. Follistatin decreased TGF-β mRNA expression and TGF-β secretion of PSCs, and inhibited both PSC activation and collagen secretion. Conclusion: Activin A is an autocrine activator of PSCs. Follistatin can inhibit PSC activation and collagen secretion by blocking autocrined activin A and decreasing TGF-β expression and secretion of PSCs.

Eicosapentaenoic acid ameliorates steatohepatitis and hepatocellular carcinoma in hepatocyte-specific Pten-deficient mice☆

BACKGROUND/AIMS Eicosapentaenoic acid (EPA) has been known as a reagent for improving lipid metabolism and inflammation. Hepatocyte-specific Pten-deficient mice exhibit hepatic lesions analogous to non-alcoholic steatohepatitis (NASH). Therefore, we administered EPA to Pten-deficient mice to investigate the mechanisms of NASH. METHODS Pten-deficient mice were assigned to a control group fed with a standard chow or an EPA group fed with a 5% EPA-supplemented standard chow. At 40 weeks, livers from each group were processed to measure triglyceride content, gene expression analysis, Western blotting analysis, and histological examination. Level of serum reactive oxygen species (ROS) was also determined. Forty- and 76-week-old mice were used in tumor burden experiments. RESULTS EPA-ameliorated hepatic steatosis in Pten-deficient mice was based on decreased expression of AMPKalpha1-mediated SREBP-1c and increased PPARalpha expression. The EPA group exhibited less severe chronic hepatic inflammation compared to the control group, resulting from decreased ROS formation and a dramatically low ratio of arachidonic acid to EPA. Moreover, EPA inhibited development of hepatocellular carcinoma (HCC) in Pten-deficient mice based on an inhibition of MAPK activity and a low ratio of oleic to stealic acid, and a reduction in ROS formation. CONCLUSIONS EPA ameliorated steatohepatitis and development of HCC in Pten-deficient mice.

Existence of autocrine loop between interleukin‐6 and tranforming growth factor‐β1 in activated rat pancreatic stellate cells

Interleukin (IL)‐6 is a proinflammatory cytokine assumed to participate in pancreatic fibrosis by activating pancreatic stellate cells (PSCs). Autocrine TGF‐β1 is to central in PSC functional regulation. In this study, we examined IL‐6 secretion from culture‐activated rat PSCs and its regulatory mechanism. Activated PSCs express and secrete IL‐6. When anti‐TGF‐β1 neutralizing antibody was added in the culture medium, IL‐6 secretion from activated PSCs was inhibited, whereas exogenous TGF‐β1 added in the culture medium enhanced IL‐6 expression and secretion by PSCs in a dose dependent manner. Infection of PSCs with an adenovirus expressing dominant‐negative Smad2/3 attenuated basal and TGF‐β1‐stimulated IL‐6 expression and secretion of PSCs. We also demonstrated the reciprocal effect of PSCs‐secreted IL‐6 on autocrine TGF‐β1. Anti‐IL‐6 neutralizing antibody inhibited TGF‐β1 secretion from PSCs. Preincubation of cells with 10 nM PD98059, an extracellular signal‐regulated kinase (ERK)‐dependent pathway inhibitor, attenuated IL‐6‐enhanced TGF‐β1 expression and secretion of PSCs. In addition, IL‐6 activated ERK in PSCs. These data indicate the existence of autocrine loop between IL‐6 and TGF‐β1 through ERK‐ and Smad2/3‐dependent pathways in activated PSCs. J. Cell. Biochem.

Localization of p0071-interacting proteins, plakophilin-related armadillo-repeat protein-interacting protein (PAPIN) and ERBIN, in epithelial cells.

PAPIN has six PDZ domains and interacts with p0071, a catenin-related protein. Recent studies have revealed that catenins determine the subcellular localization of some PDZ proteins. We have examined whether the localization of PAPIN is determined by p0071 in epithelial cells. PAPIN was localized not only on the lateral membrane but also on the apical membrane, where p0071 was absent. The targeting to both membranes was mediated by the middle region of PAPIN and did not require the p0071-interacting PDZ domain. In cells that came into contact, PAPIN was diffusely distributed on the plasma membrane, while p0071 was concentrated at immature cell–cell contacts. When epithelial cells were exposed to the low concentration of calcium, p0071 was internalized, whereas PAPIN remained on the plasma membrane. We also confirmed that the interaction with p0071 was not essential for the membrane targeting of ERBIN, a recently identified p0071- and ErbB2-binding protein. PAPIN, p0071, and ERBIN formed a complex in 293T cells. Furthermore, ERBIN and ErbB2 were colocalized with PAPIN on the lateral membrane. These findings suggest that PAPIN, p0071, and ERBIN come to the cell–cell contacts independently and interact with each other on the lateral membrane.

Gastrointestinal stromal tumor in the jejunum: diagnosis and control of bleeding with electrocoagulation by using double-balloon enteroscopy.

A 43-year-old man presented with gastrointestinal bleeding. A tumor with central ulceration was observed in the jejunum, with the use of a new enteroscopy system called “double-balloon enteroscopy”. Bleeding after biopsy sampling of the tumor was controlled endoscopically by using electrocoagulation. Histological findings of the biopsy specimens were consistent with gastrointestinal stromal tumor, and this was surgically resected. Double-balloon enteroscopy was useful for the diagnosis as well as the control of bleeding in this patient.

Indian hedgehog promotes the migration of rat activated pancreatic stellate cells by increasing membrane type-1 matrix metalloproteinase on the plasma membrane

Indian hedgehog (Ihh) is a member of hedgehog peptides family that exerts diverse effects on multiple cellular functions. Since Ihh expression is elevated in the pancreas of chronic pancreatitis patients, Ihh has been assumed to participate in the chronic pancreatic injury, especially in pancreatic fibrosis. However, its function in pancreatic fibrosis is still unknown. We thus examined Ihh effects on rat activated pancreatic stellate cells (PSCs) that play a central role in pancreatic fibrosis. Activated PSCs express both patched‐1 and smoothened that are essential components of hedgehog receptor system. Ihh did not alter the PSC expression of collagen‐1 or α‐smooth muscle actin, a parameter of PSC transformation, or did not change PSC proliferation. However, Ihh enhanced PSC migration in both chemotactic and chemokinetic manners. Furthermore, Ihh increased the amount of membrane‐type 1 matrix metalloproteinase (MT1‐MMP) and altered its localization on the plasma membrane, which plays a stimulatory role in cellular migration. In addition, tissue inhibitor of metalloproteinase‐2 (TIMP‐2) attenuated Ihh‐stimulated PSC migration. Since most hedgehog intracellular signals are mediated by Gli‐1 transcription factor, we investigated its contribution to Ihh‐enhancement of PSC migration. Ihh induced Gli‐1 nuclear accumulation in PSCs, indicating that Ihh stimulates Gli‐1‐dependent signaling pathway in PSCs. Unexpectedly, however, adenovirus‐mediated Gli‐1 overexpression blocked the Ihh enhancement of both MT1‐MMP localization on the plasma membrane and PSC migration. Furthermore, reduction of Gli‐1 expression with RNA interference augmented Ihh‐stimulated PSC migration. These data indicate that Ihh promotes PSC migration by enhancing MT1‐MMP localization on the plasma membrane but is negatively regulated by Gli‐1. J. Cell. Physiol. 216: 38–46, 2008.

Helicobacter pylori eradication induces marked increase in H+/K+-adenosine triphosphatase expression without altering parietal cell number in human gastric mucosa

Background and aims: Gastric acid secretion is downregulated by Helicobacter pylori infection and upregulated after its eradication, but the mechanisms are still unclear. We examined the effects of H pylori eradication on the number of parietal cells and on expression of molecules functioning in acid secretion in the human gastric mucosa. Methods: We enrolled 111 consecutive men with chronic gastritis induced by H pylori. Biopsy specimens were endoscopically obtained before and 12 weeks after successful eradication of H pylori and parietal cell numbers were counted. mRNA expression levels of H+/K+-adenosine triphosphatase (H+/K+-ATPase), anion exchanger 2, M3 muscarinic receptor, intrinsic factor, and interleukin 1β were determined with a real time reverse transcriptase-polymerase chain reaction method. The severity of gastric atrophy was evaluated using the serum pepsinogen I/II ratio. Results: No significant difference was observed in parietal cell numbers before and after H pylori eradication. Median mRNA expression levels of H+/K+-ATPase in the gastric mucosa increased 250-fold after H pylori eradication accompanied by attenuation of interleukin 1β. A large increase in H+/K+-ATPase expression was observed even in patients with severe atrophic gastritis. In contrast, fold increases in mRNA expression levels, including intrinsic factor, anion exchanger 2, and M3 muscarinic receptor, after eradication therapy, were limited to 1.4, 2.3, and 2.5 times, respectively. Conclusions: In the absence of alteration of parietal cell number, gastric H+/K+-ATPase mRNA expression was markedly restored after successful H pylori eradication, suggesting a central role for the restoration of H+/K+-ATPase expression in gastric acid secretion recovery after H pylori eradication.