Krishnan, Anuradha

Cholangiocyte pathophysiological activation was examined in a model of murine cholestasis. CX3CR1 + macrophages are recruited to the periportal region, likely mediated by cholangiocyte expression of CX3CL1. Cholangiocyte transcriptomics from cholestatic mice display activation of a “pathogen-induced cytokine storm” pathway, and exhibit activation of the transcription factor NF-Y. In human cholangiocytes, NF-Y promotes expression of the profibrogenic ligand TGFβ1. These observations provide insights into the cholestatic cholangiocyte pathobiology contributing to periductal inflammation and fibrosis.

Background: NASH is the progressive form of NAFLD characterized by lipotoxicity, hepatocyte injury, tissue inflammation, and fibrosis. Previously, Rho-associated protein kinase (ROCK) 1 has been implicated in lipotoxic signaling in hepatocytes in vitro and high-fat diet-induced lipogenesis in vivo. However, whether ROCK1 plays a role in liver inflammation and fibrosis during NASH is unclear. Here, we hypothesized that pathogenic activation of ROCK1 promotes murine NASH pathogenesis. Methods and Results: Patients with NASH had increased hepatic ROCK1 expression compared with patients with fatty liver. Similarly, hepatic ROCK1 levels and activity were increased in mice with NASH induced by a western-like diet that is high in fat, fructose, and cholesterol (FFC). Hepatocyte-specific ROCK1 knockout mice on the FFC diet displayed a decrease in liver steatosis, hepatic cell death, liver inflammation, and fibrosis compared with littermate FFC-fed controls. Mechanistically, these effects were associated with a significant attenuation of myeloid cell recruitment. Interestingly, myeloid cell-specific ROCK1 deletion did not affect NASH development in FFC-fed mice. To explore the therapeutic opportunities, mice with established NASH received ROCKi, a novel small molecule kinase inhibitor of ROCK1/2, which preferentially accumulates in liver tissue. ROCK inhibitor treatment ameliorated insulin resistance and decreased liver injury, inflammation, and fibrosis. Conclusions: Genetic or pharmacologic inhibition of ROCK1 activity attenuates murine NASH, suggesting that ROCK1 may be a therapeutic target for treating human NASH.

Background and Aims In cholestatic liver diseases, ductular reactive (DR) cells extend into the hepatic parenchyma and promote inflammation and fibrosis. We have previously observed that multidrug‐resistant 2 (Mdr2−/− ) double knockout (DKO) mice lacking tumor necrosis factor–related apoptosis‐inducing ligand receptor (Tr−/− ) display a more extensive ductular reaction and hepatic fibrosis compared to Mdr2−/− mice. This observation suggests that the magnitude of the DR‐cell population may be regulated by apoptosis. Approach and Results To examine this concept, we cultured epithelial cell adhesion molecule–positive reactive cholangioids (ERCs) obtained from wild‐type (WT), Tr−/− , Mdr2−/− and DKO mice. Single‐cell transcriptomics and immunostaining of both WT and DKO ERCs confirmed their DR‐cell phenotype. Moreover, DKO ERCs displayed a unique translational cluster with expression of chemokines, indicating a reactive state. Incubation with the myeloid cell leukemia 1 (MCL1) inhibitor S63845, a proapoptotic BH3‐mimetic therapy, significantly decreased DKO and Mdr2−/− ERC viability compared to WT. Intravenous administration of S63845 significantly reduced the DR‐cell population and markers of inflammation and liver fibrosis in Mdr2−/− and DKO mice. Furthermore, DKO mice treated with S63845 displayed a significant decrease in hepatic B lymphocytes compared to untreated mice as assessed by high‐definition mass cytometry by time‐of‐flight. Coculture of bone marrow–derived macrophages with ERCs from DKO mouse livers up‐regulated expression of the B cell–directed chemokine (C‐C motif) ligand 5. Finally, DR cells were noted to be primed for apoptosis with Bcl‐2 homologous antagonist/killer activation in vitro and in vivo in primary sclerosing cholangitis liver specimens. Conclusions DR cells appear to play a key role in recruiting immune cells to the liver to actively create an inflammatory and profibrogenic microenvironment. Pharmacologic targeting of MCL1 in a mouse model of chronic cholestasis reduces DR‐cell and B‐cell populations and hepatic fibrosis.

In mouse models of biliary tract diseases, macrophages are recruited to the periductal milieu and promote injury and cholestasis. Although cell necrosis with release of biomolecules termed damage-associated molecular patterns (DAMPs) promotes recruitment and activation of macrophages, necrosis was not observed in these studies. Because extracellular vesicles (EVs) are important in cell-to-cell communication, we postulated that activated cholangiocytes may release EVs containing DAMPs as cargo. Both the human (NHC) and mouse cholangiocyte (603B) cell lines display constitutive activation with mRNA expression of chemokines. Proteomic analysis revealed that EVs from both cell lines contained the DAMP S100A11, a ligand for the receptor for advanced glycation end products (RAGE). Bone marrow-derived macrophages (BMDM) incubated with EVs derived from the mouse 603B cell line increased mRNA expression of proinflammatory cytokines. Genetic or pharmacologic inhibition of RAGE reduced BMDM expression of proinflammatory cytokines treated with EVs. RAGE signaling resulted in activation of the canonical NF-κB pathway, and consistently, proinflammatory cytokine expression was blunted by the IKKα/β inhibitor TPCA-1 in BMDM incubated with EVs. We also demonstrated that primary mouse cholangiocyte-derived organoids express chemokines indicating cholangiocyte activation, release EVs containing S100A11, and stimulate proinflammatory cytokine expression in BMDM by a RAGE-dependent pathway. In conclusion, these observations identify a non-cell death mechanism for cellular release of DAMPs by activated cholangiocytes, namely by releasing DAMPs as EV cargo. These data also suggest RAGE inhibitors may be salutary in macrophage-associated inflammatory diseases of the bile ducts.

Prohibitin 1 (PHB1) is best known as a mitochondrial chaperone, and its role in cancer is conflicting. Mice lacking methionine adenosyltransferase α1 (MATα1) have lower PHB1 expression, and we reported that c‐MYC interacts directly with both proteins. Furthermore, c‐MYC and MATα1 exert opposing effects on liver cancer growth, prompting us to examine the interplay between PHB1, MATα1, and c‐MYC and PHB1's role in liver tumorigenesis. We found that PHB1 is highly expressed in normal hepatocytes and bile duct epithelial cells and down‐regulated in most human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). In HCC and CCA cells, PHB1 expression correlates inversely with growth. PHB1 and MAT1A positively regulate each other's expression, whereas PHB1 negatively regulates the expression of c‐MYC, MAFG, and c‐MAF. Both PHB1 and MATα1 heterodimerize with MAX, bind to the E‐box element, and repress E‐box promoter activity. PHB1 promoter contains a repressive E‐box element and is occupied mainly by MAX, MNT, and MATα1 in nonmalignant cholangiocytes and noncancerous tissues that switched to c‐MYC, c‐MAF, and MAFG in cancer cells and human HCC/CCA. All 8‐month‐old liver‐specific Phb1 knockout mice developed HCC, and one developed CCA. Five‐month‐old Phb1 heterozygotes, but not Phb1 flox mice, developed aberrant bile duct proliferation; and one developed CCA 3.5 months after left and median bile duct ligation. Phb1 heterozygotes had a more profound fall in the expression of glutathione synthetic enzymes and higher hepatic oxidative stress following left and median bile duct ligation. Conclusion: We have identified that PHB1, down‐regulated in most human HCC and CCA, heterodimerizes with MAX to repress the E‐box and positively regulates MAT1A while suppressing c‐MYC, MAFG, and c‐MAF expression; in mice, reduced PHB1 expression predisposes to the development of cholestasis‐induced CCA. (Hepatology 2017;65:1249‐1266).

c‐Myc induction drives cholestatic liver injury and cholangiocarcinoma (CCA) in mice, and induction of Maf proteins (MafG and c‐Maf) contributes to cholestatic liver injury, whereas S‐adenosylmethionine (SAMe) administration is protective. Here, we determined whether there is interplay between c‐Myc, Maf proteins, and methionine adenosyltransferase α1 (MATα1), which is responsible for SAMe biosynthesis in the liver. We used bile duct ligation (BDL) and lithocholic acid (LCA) treatment in mice as chronic cholestasis models, a murine CCA model, human CCA cell lines KMCH and Huh‐28, human liver cancer HepG2, and human CCA specimens to study gene and protein expression, protein‐protein interactions, molecular mechanisms, and functional outcomes. We found that c‐Myc, MATα1 (encoded by MAT1A), MafG, and c‐Maf interact with one another directly. MAT1A expression fell in hepatocytes and bile duct epithelial cells during chronic cholestasis and in murine and human CCA. The opposite occurred with c‐Myc, MafG, and c‐Maf expression. MATα1 interacts mainly with Mnt in normal liver, but this switches to c‐Maf, MafG, and c‐Myc in cholestatic livers and CCA. Promoter regions of these genes have E‐boxes that are bound by MATα1 and Mnt in normal liver and benign bile duct epithelial cells that switched to c‐Myc, c‐Maf, and MafG in cholestasis and CCA cells. E‐box positively regulates c‐Myc, MafG, and c‐Maf, but it negatively regulates MAT1A. MATα1 represses, whereas c‐Myc, MafG, and c‐Maf enhance, E‐box‐driven promoter activity. Knocking down MAT1A or overexpressing MafG or c‐Maf enhanced CCA growth and invasion in vivo. Conclusion: There is a novel interplay between MATα1, c‐Myc, and Maf proteins, and their deregulation during chronic cholestasis may facilitate CCA oncogenesis. (Hepatology 2016;64:439‐455)

HCV-specific neutralizing antibodies protect humanized mice from challenge and suppress established infections.