Genetic deletion or pharmacologic inhibition of the Nlrp3 inflammasome did not ameliorate experimental NASH.

It has been postulated that inflammasomes, in particular the NLRP3 (NLR family pyrin domain containing 3) inflammasome, mediate the necroinflammation and fibrosis that characterize nonalcoholic steatohepatitis (NASH) by engaging innate immune responses. We aimed to investigate the impact of genetic deletion or pharmacologic inhibition of the NLRP3 inflammasome on experimental steatohepatitis. Global Nlrp3 KO (expected to inhibit the NLRP3 inflammasome) or Casp1 KO (expected to inhibit all inflammasomes) mice were compared to wild type controls after 6 months on a high-fat, high-cholesterol (HFHC, 1% cholesterol) diet known to induce fibrosing steatohepatitis. Additionally, wildtype mice on a HFHC diet (0.75% or 0.5% cholesterol) for 6 months were either treated or not treated with an oral, pharmacologic inhibitor of Nlrp3 (MCC950) that was delivered in the drinking water (0.3 mg/ml). We found that genetic deletion or pharmacologic inhibition of the NLRP3 inflammasome did not ameliorate any of the histological components of fibrosing NASH in HFHC-fed mice. Collectively, these results do not support NLRP3 inhibition as a potential target for human NASH.

Gallbladder epithelial cells that engraft in mouse liver can differentiate into hepatocyte-like cells.

We tested the hypothesis that well-differentiated gallbladder epithelial cells (GBECs) are capable of engrafting and surviving in murine liver and acquire phenotypic characteristics of hepatocytes. GBECs isolated from transgenic mice that constitutively express green fluorescent protein (GFP) were either cultured before transplantation or transplanted immediately following isolation. Recipient mice with severe-combined immunodeficiency underwent retrorsine treatment and either partial hepatectomy before transplantation or carbon tetrachloride treatment following transplantation. From 1 to 4 months following transplantation, the livers of recipient mice contained discrete colonies of GFP(+) cells. Most GFP(+) cells surrounded vesicles, were epithelial cell-like in morphology, and expressed the biliary epithelial markers cytokeratin 19 and carbonic anhydrase IV. Subpopulations of GFP(+) cells resembled hepatocytes morphologically and expressed the hepatocyte-specific markers connexin-32 and hepatic nuclear factor-4alpha, but not cytokeratin 19 or carbonic anhydrase IV. At 4 months, cells in GFP(+) colonies were not actively proliferating as determined by proliferating cell nuclear antigen expression. Thus, GBECs are capable of engrafting and surviving in damaged mouse livers, and some can differentiate into cells with hepatocyte-like features. These findings suggest that environmental cues in the recipient liver are sufficient to allow a subpopulation of donor GBECs to differentiate into hepatocyte-like cells in the absence of exogenous transcriptional reprogramming. GBECs might be used as donor cells in a cell transplantation approach for the treatment of liver disease.

Peripheral amyloid-beta levels regulate amyloid-beta clearance from the central nervous system.

Amyloid-beta (Abeta) is cleared from the brain by both proteolytic digestion and transport across the blood-brain-barrier into the peripheral circulatory system. To investigate the role peripheral Abeta levels play in regulating Abeta brain clearance, we measured the clearance of [125I]-Abeta(1-40) injected into the brains of liver-ligated rats that allowed peripheral Abeta levels to be maintained at elevated levels for approximately one hour with/without a single peripheral bolus of unlabeled Abeta(1-40). We found that elevating peripheral Abetalevels significantly decreased [125I]-Abeta(1-40) brain clearance, thus supporting the hypothesis that peripheral Abeta levels regulate Abeta clearance from the central nervous system.

Effects of lipopolysaccharide on platelet-derived growth factor isoform and receptor expression in cultured rat common bile duct fibroblasts and cholangiocytes.

BACKGROUND AND AIM: Little is known about the role of platelet-derived growth factor (PDGF) in biliary fibrosis in the setting of bacterial colonization of the biliary tree. We therefore sought to investigate whether exposure to bacterial lipopolysaccharide (LPS) alters PDGF isoform and receptor expression in cultured rat common bile duct fibroblasts (CBDF) and normal rat cholangiocytes (NRC). METHODS: Collagen content in cells and media was assessed by colorimetric assay and gel electrophoresis. mRNA levels of PDGF-A and -B, and PDGF-Receptors (PDGF-R) alpha and beta were measured by relative quantitative real-time PCR. Protein levels of PDGF-AA, AB and BB were measured by ELISA, and PDGF-Ralpha and PDGF-Rbeta by Western blot. RESULTS: In CBDF, LPS increased total soluble collagen synthesis and secretion. PDGF-Ralpha and beta mRNA and protein were also increased by LPS treatment in CBDF. Lipopolysaccharide treatment elicited an increase in PDGF-A and -B mRNA levels in CBDF. In NRC, levels of PDGF-AmRNAincreased in a dose-dependent fashion following LPS treatment, whereas PDGF-B mRNA showed no response. PDGF-AA secretion was higher by CBDF than by NRC. PDGF-BB levels were also higher in CBDF than in NRC. While PDGF-BB levels did not respond to LPS treatment in CBDF, there was a dosedependent response of this isoform to LPS in NRC. Intracellular and secreted PDGF-AB increased with LPS treatment in NRC. CONCLUSIONS: These results support a model in which chronic bacterial colonization of the biliary tree induces fibrosis through PDGF-dependent mechanisms.

Myofibroblasts are responsible for the desmoplastic reaction surrounding human pancreatic carcinomas.

BACKGROUND: The cell type responsible for the desmoplastic reaction surrounding human pancreatic carcinoma is unknown. Hepatic stellate cells, which activate to a myofibroblast-like form, are responsible for collagen deposition in cirrhosis and around hepatocellular carcinomas. Recently, pancreatic stellate cells have been described and implicated in the fibrosis of chronic pancreatitis. We sought to determine whether these cells are responsible for the scirrhous reaction surrounding pancreatic adenocarcinomas. METHODS: Archival formalin-fixed, paraffin-embedded pancreatic tissues from 10 patients undergoing pancreaticoduodenectomy for ductal adenocarcinoma and from 2 patients with pancreatic islet cell tumors were examined immunohistochemically for alpha-smooth muscle actin (alpha-SMA), smooth muscle myosin heavy chain (SMMHC), procollagen I, collagen IV, and endothelial cell markers, von Willebrand factor and cluster of differentiation 31. RESULTS: In non-neoplastic areas, staining for alpha-SMA and SMMHC was confined to interlobular septal regions. In contrast, the desmoplastic reaction surrounding all 10 pancreatic adenocarcinoma specimens displayed intense interstitial staining for alpha-SMA, SMMHC, and collagen IV but no staining for von Willebrand factor and cluster of differentiation 31. Procollagen I staining localized intracellularly to fibroblast-shaped cells within this alpha-SMA/SMMHC-positive scirrhous region. Islet cell tumors demonstrated an increase in alpha-SMA staining, although this was not as marked as in ductal adenocarcinomas. CONCLUSIONS: A massive increase in myofibroblast activity, compatible with the activation of stellate cells, is associated with the deposition of collagen types I and IV in the desmoplastic reaction around pancreatic adenocarcinomas.

Expression of cytokine and chemokine mRNA and secretion of tumor necrosis factor-alpha by gallbladder epithelial cells: response to bacterial lipopolysaccharides.

BACKGROUND: In addition to immune cells, many other cell types are known to produce cytokines. Cultured normal mouse gallbladder epithelial cells, used as a model system for gallbladder epithelium, were examined for their ability to express the mRNA of various cytokines and chemokines in response to bacterial lipopolysaccharide. The synthesis and secretion of the tumor necrosis factor-alpha (TNF-alpha) protein by these cells was also measured. RESULTS: Untreated mouse gallbladder cells expressed mRNA for TNF-alpha, RANTES, and macrophage inflammatory protein-2 (MIP-2). Upon treatment with lipopolysaccharide, these cells now produced mRNA for Interleukin-1beta (IL-1beta), IL-6, monocyte chemoattractant protein-1 (MCP-1), and showed increased expression of TNF-alpha and MIP-2 mRNA. Untreated mouse gallbladder cells did not synthesize TNF-alpha protein; however, they did synthesize and secrete TNF-alpha upon treatment with lipopolysaccharide. METHODS: Cells were treated with lipopolysaccharides from 3 strains of bacteria. Qualitative and semi-quantitative RT-PCR, using cytokine or chemokine-specific primers, was used to measure mRNA levels of TNFalpha, IL-1beta, IL-6, IL-10, KC, RANTES, MCP-1, and MIP-2. TNF-alpha protein was measured by immunoassays. CONCLUSION: This research demonstrates that gallbladder epithelial cells in response to lipopolysaccharide exposure can alter their cytokine and chemokine RNA expression pattern and can synthesize and secrete TNFalpha protein. This suggests a mechanism whereby gallbladder epithelial cells in vivo may mediate gallbladder secretory function, inflammation and diseases in an autocrine/paracrine fashion by producing and secreting cytokines and/or chemokines during sepsis.

Murine gallbladder epithelial cells can differentiate into hepatocyte-like cells in vitro.

We determined whether extrahepatic biliary epithelial cells can differentiate into cells with phenotypic features of hepatocytes. Gallbladders were removed from transgenic mice expressing hepatocyte-specific beta-galactosidase (beta-Gal) and cultured under standard conditions and under experimental conditions designed to induce differentiation into a hepatocyte-like phenotype. Gallbladder epithelial cells (GBEC) cultured under standard conditions exhibited no beta-Gal activity. beta-Gal expression was prominent in 50% of cells cultured under experimental conditions. Similar morphological changes were observed in GBEC from green fluorescent protein transgenic mice cultured under experimental conditions. These cells showed higher levels of mRNA for genes expressed in hepatocytes, but not in GBEC, including aldolase B, albumin, hepatocyte nuclear factor-4alpha, aldehyde dehydrogenase 1, and glutamine synthetase, and they synthesized bile acids. Additional functional evidence of a hepatocyte-like phenotype included LDL uptake and enhanced benzodiazepine metabolism. Connexin-32 expression was evident in murine hepatocytes and in cells cultured under experimental conditions, but not in cells cultured under standard conditions. Notch 1, 2, and 3 and Notch ligand Jagged 1 mRNAs were downregulated in these cells compared with cells cultured under standard conditions. CD34, alpha-fetoprotein, and Sca-1 mRNA were not expressed in cells cultured under standard conditions, suggesting that the hepatocyte-like cells did not arise from hematopoietic stem cells or oval cells. These results point to future avenues for investigation into the potential use of GBEC in the treatment of liver disease.

Paclitaxel interrupts TGF-beta1 signaling between gallbladder epithelial cells and myofibroblasts.

BACKGROUND: The cellular and molecular mechanisms of fibrogenesis in the extrahepatic biliary epithelium are not known. Transforming growth factor-beta1 (TGF-beta1) is a cytokine implicated in signaling pathways that mediate collagen formation. An observation that paclitaxel (PT), applied topically into the rat common bile duct, inhibited stricture formation led us to hypothesize that PT's effects might be due to interruption of TGF-beta1 signaling between biliary epithelial cells and subepithelial myofibroblasts. MATERIALS AND METHODS: We tested this hypothesis using an in vitro cell-culture model in which murine gallbladder epithelial cells (GBEC) are cultured separately or cocultured with human gallbladder myofibroblasts (GBMF). RESULTS: Exposure to Escherichia coli lipopolysaccharide (LPS) enhanced TGF-beta1 mRNA expression and stimulated TGF-beta1 protein secretion into both apical and basolateral compartments in GBEC. This effect was more prominent with basolateral secretion and was also more pronounced in the coculture system. In GBMF, collagen I mRNA expression and protein secretion were stimulated by treatment with LPS or TGF-beta1. GBMF also expressed TGF-beta1 mRNA, whose levels were enhanced by exposure to either LPS or exogenous TGF-beta1. PT inhibited LPS-induced TGF-beta1 mRNA expression and protein secretion in GBEC in both culture systems. Tumor necrosis factor-alpha mRNA expression and protein secretion were not affected by PT in GBEC, demonstrating that the effects were specific for TGF-beta1. PT also inhibited LPS- and TGF-beta1-induced collagen I mRNA expression and protein secretion in GBMF. CONCLUSIONS: These findings support a model in which GBEC communicate with subepithelial GBMF via TGF-beta1, leading to collagen deposition and fibrosis, and in which GBMF possess autocrine mechanisms involving TGF-beta1 that could regulate collagen production. PT inhibits these fibrogenic pathways.