Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) deletion in myeloid cells augments cholestatic liver injury.

Ductular reactive (DR) cells exacerbate cholestatic liver injury and fibrosis. Herein, we posit that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) emanates from recruited macrophages and restrains DR cell expansion, thereby limiting cholestatic liver injury. Wild type (WT), Trailfl/fl and myeloid-specific Trail deleted (TrailΔmye) C57BL/6 mice were exposed to DDC diet-induced cholestatic liver injury, which induced hepatomegaly and liver injury as compared to control diet-fed mice. However, parameters of liver injury, fibrosis, and inflammation were all increased in the TrailΔmye mice as compared to the WT and Trailfl/fl mice. High dimensional mass cytometry indicated that cholestasis resulted in increased hepatic recruitment of subsets of macrophages and neutrophils in the TrailΔmye mice. Spatial transcriptomics analysis revealed that the PanCK+ cholangiocytes from TrailΔmye mice had increased expression of the known myeloid attractants S100a8, Cxcl5, Cx3cl1, and Cxcl1. Additionally, in situ hybridization of Cxcl1, a potent neutrophil chemoattractant, demonstrated an increased expression in CK19+ cholangiocytes of TrailΔmye mice. Collectively, these data suggest that TRAIL from myeloid cells, particularly macrophages, restrains a subset of DR cells (i.e., Cxcl1 positive cells), limiting liver inflammation and fibrosis. Reprogramming macrophages to express TRAIL may be salutary in cholestasis.

IL-17 Signaling in Primary Sclerosing Cholangitis Patient-Derived Organoids.

The pathogenesis of primary sclerosing cholangitis (PSC) is unclear, although studies implicate IL-17A as an inflammatory mediator in this disease. However, a direct assessment of IL-17 signaling in PSC cholangiocytes is lacking. In this study we aimed to investigate the response of PSC extrahepatic cholangiocyte organoids (ECO) to IL-17A stimulation. Cholangiocytes obtained from PSC and non-PSC patients by endoscopic retrograde cholangiography (ERC) were cultured as ECO. The ECO were treated with vehicle or IL-17A and assessed by transcriptomics, secretome analysis, and genome sequencing (GS). Unsupervised clustering of all integrated scRNA-seq data identified 8 cholangiocyte clusters which did not differ between PSC and non-PSC ECO. However, PSC ECO cells demonstrated a robust response to IL-17 treatment, noted by an increased number of differentially expressed genes (DEG) by transcriptomics, and more abundant chemokine and cytokine expression and secretion. After rigorous filtering, GS identified candidate somatic variants shared among PSC ECO from unrelated individuals. However, no candidate rare variants in genes regulating the IL-17 pathway were identified, but rare variants regulating the MAPK signaling pathway were present in all PSC ECO. In conclusion, PSC and non-PSC patient derived ECO respond differently to IL-17 stimulation implicating this pathway in the pathogenesis of PSC.

Targeted Apoptosis of Ductular Reactive Cells Reduces Hepatic Fibrosis in a Mouse Model of Cholestasis.

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.

The Spectrum of Reactive Cholangiocytes in Primary Sclerosing Cholangitis.

Cholangiocytes are the target of a group of chronic liver diseases termed the "cholangiopathies," in which cholangiocytes react to exogenous and endogenous insults, leading to disease initiation and progression. In primary sclerosing cholangitis (PSC), the focus of this review, the cholangiocyte response to genetic or environmental insults can lead to a heterogeneous response; that is, a subpopulation acquires a ductular reactive and proliferative phenotype, while another subpopulation undergoes senescence and growth arrest. Both ductular reactive cholangiocytes and senescent cholangiocytes can modify the periductal microenvironment through their ability to secrete various cytokines, chemokines, and growth factors, initiating and perpetuating inflammatory and profibrotic responses. This review discusses the similarities and differences, the interrelationships, and the potential pathogenic roles of these reactive proliferative and senescent cholangiocyte subpopulations in PSC.

The transcription factor ETS1 promotes apoptosis resistance of senescent cholangiocytes by epigenetically up-regulating the apoptosis suppressor BCL2L1.

Primary sclerosing cholangitis (PSC) is an idiopathic, progressive cholangiopathy. Cholangiocyte senescence is important in PSC pathogenesis, and we have previously reported that senescence is regulated by the transcription factor ETS proto-oncogene 1 (ETS1) and associated with overexpression of BCL2 like 1 (BCL2L1 or BCL-xL), an anti-apoptotic BCL2-family member. Here, we further explored the mechanisms regulating BCL-xL-mediated, apoptosis resistance in senescent cholangiocytes and uncovered that ETS1 and the histone acetyltransferase E1A-binding protein P300 (EP300 or p300) both promote BCL-xL transcription. Using immunofluorescence, we found that BCL-xL protein expression is increased both in cholangiocytes of livers from individuals with PSC and a mouse model of PSC. Using an in vitro model of lipopolysaccharide-induced senescence in normal human cholangiocytes (NHCs), we found increased BCL-xL mRNA and protein levels, and ChIP-PCRs indicated increased occupancy of ETS1, p300, and histone 3 Lys-27 acetylation (H3K27Ac) at the BCL-xL promoter. Using co-immunoprecipitation and proximity ligation assays, we further demonstrate that ETS1 and p300 physically interact in senescent but not control NHCs. Additionally, mutagenesis of predicted ETS1-binding sites within the BCL-xL promoter blocked luciferase reporter activity, and CRISPR/Cas9-mediated genetic deletion of ETS1 reduced senescence-associated BCL-xL expression. In senescent NHCs, TRAIL-mediated apoptosis was reduced ∼70%, and ETS1 deletion or RNAi-mediated BCL-xL suppression increased apoptosis. Overall, our results suggest that ETS1 and p300 promote senescent cholangiocyte resistance to apoptosis by modifying chromatin and inducing BCL-xL expression. These findings reveal ETS1 as a central regulator of both cholangiocyte senescence and the associated apoptosis-resistant phenotype.

Activated cholangiocytes release macrophage-polarizing extracellular vesicles bearing the DAMP S100A11.

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/ß 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.

Macrophages contribute to the pathogenesis of sclerosing cholangitis in mice.

BACKGROUND & AIMS: Macrophages contribute to liver disease, but their role in cholestatic liver injury, including primary sclerosing cholangitis (PSC), is unclear. We tested the hypothesis that macrophages contribute to the pathogenesis of, and are therapeutic targets for, PSC. METHODS: Immune cell profile, hepatic macrophage number, localization and polarization, fibrosis, and serum markers of liver injury and cholestasis were measured in an acute (intrabiliary injection of the inhibitor of apoptosis antagonist BV6) and chronic (Mdr2-/- mice) mouse model of sclerosing cholangitis (SC). Selected observations were confirmed in liver specimens from patients with PSC. Because of the known role of the CCR2/CCL2 axis in monocyte/macrophage chemotaxis, therapeutic effects of the CCR2/5 antagonist cenicriviroc (CVC), or genetic deletion of CCR2 (Ccr2-/- mice) were determined in BV6-injected mice. RESULTS: We found increased peribiliary pro-inflammatory (M1-like) and alternatively-activated (M2-like) monocyte-derived macrophages in PSC compared to normal livers. In both SC models, genetic profiling of liver immune cells identified a predominance of monocytes/macrophages; immunohistochemistry confirmed peribiliary monocyte-derived macrophage recruitment (M1>M2-polarized), which paralleled injury onset and was reversed upon resolution in acute SC mice. PSC, senescent and BV6-treated human cholangiocytes released monocyte chemoattractants (CCL2, IL-8) and macrophage-activating factors in vitro. Pharmacological inhibition of monocyte recruitment by CVC treatment or CCR2 genetic deletion attenuated macrophage accumulation, liver injury and fibrosis in acute SC. CONCLUSIONS: Peribiliary recruited macrophages are a feature of both PSC and acute and chronic murine SC models. Pharmacologic and genetic inhibition of peribiliary macrophage recruitment decreases liver injury and fibrosis in mouse SC. These observations suggest monocyte-derived macrophages contribute to the development of SC in mice and in PSC pathogenesis, and support their potential as a therapeutic target. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is an inflammatory liver disease which often progresses to liver failure. The cause of the disease is unclear and therapeutic options are limited. Therefore, we explored the role of white blood cells termed macrophages in PSC given their frequent contribution to other human inflammatory diseases. Our results implicate macrophages in PSC and PSC-like diseases in mice. More importantly, we found that pharmacologic inhibition of macrophage recruitment to the liver reduces PSC-like liver injury in the mouse. These exciting observations highlight potential new strategies to treat PSC.

Biliary tract instillation of a SMAC mimetic induces TRAIL-dependent acute sclerosing cholangitis-like injury in mice.

Primary sclerosing cholangitis (PSC) is a cholestatic liver disease of unknown etiopathogenesis characterized by fibrous cholangiopathy of large and small bile ducts. Systemic administration of a murine TNF-related apoptosis-inducing ligand (TRAIL) receptor agonist induces a sclerosing cholangitis injury in C57BL/6 mice, suggesting endogenous TRAIL may contribute to sclerosing cholangitis syndromes. Cellular inhibitor of apoptosis proteins (cIAP-1 and cIAP-2) are negative regulators of inflammation and TRAIL receptor signaling. We hypothesized that if endogenous TRAIL promotes sclerosing cholangitis, then cIAP depletion should also induce this biliary tract injury. Herein, we show that cIAP protein levels are reduced in the interlobular bile ducts of human PSC livers. Downregulation of cIAPs in normal human cholangiocytes in vitro by use of a SMAC mimetic (SM) induces moderate, ripoptosome-mediated apoptosis and RIP1-independent upregulation of proinflammatory cytokines and chemokines. Cytokine and chemokine expression was mediated by the non-canonical activation of NF-κB. To investigate whether downregulation of cIAPs is linked to generation of a PSC-like phenotype, an SM was directly instilled into the mouse biliary tree. Twelve hours after biliary instillation, TUNEL-positive cholangiocytes were identified; 5 days later, PSC-like changes were observed in the SM-treated mice, including a fibrous cholangiopathy of the interlobular bile ducts, portal inflammation, significant elevation of serum markers of cholestasis and cholangiographic evidence of intrahepatic biliary tract injury. In contrast, TRAIL and TRAIL-receptor deficient mice showed no sign of cholangiopathy following SM intrabiliary injection. We conclude that in vivo antagonism of cIAPs in mouse biliary epithelial cells is sufficient to trigger cholangiocytes apoptosis and a proinflammatory response resulting in a fibrous cholangiopathy resembling human sclerosing cholangitis. Therefore, downregulation of cIAPs in PSC cholangiocytes may contribute to the development of the disease. Our results also indicate that inhibition of TRAIL signaling pathways may be beneficial in the treatment of PSC.

Cellular inhibitor of apoptosis (cIAP)-mediated ubiquitination of phosphofurin acidic cluster sorting protein 2 (PACS-2) negatively regulates tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity.

Lysosomal membrane permeabilization is an essential step in TRAIL-induced apoptosis of liver cancer cell lines. TRAIL-induced lysosomal membrane permeabilization is mediated by the multifunctional sorting protein PACS-2 and repressed by the E3 ligases cIAP-1 and cIAP-2. Despite the opposing roles for PACS-2 and cIAPs in TRAIL-induced apoptosis, an interaction between these proteins has yet to be examined. Herein, we report that cIAP-1 and cIAP-2 confer TRAIL resistance to hepatobiliary cancer cell lines by reducing PACS-2 levels. Under basal conditions, PACS-2 underwent K48-linked poly-ubiquitination, resulting in PACS-2 proteasomal degradation. Biochemical assays showed cIAP-1 and cIAP-2 interacted with PACS-2 in vitro and co-immunoprecipitation studies demonstrated that the two cIAPs bound PACS-2 in vivo. More importantly, both cIAP-1 and cIAP-2 directly mediated PACS-2 ubiquitination in a cell-free assay. Single c-Iap-1 or c-Iap-2 gene knock-outs in mouse hepatocytes did not lead to PACS-2 accumulation. However, deletion of both cIAP-1 and cIAP-2 reduced PACS-2 ubiquitination, which increased PACS-2 levels and sensitized HuH-7 cells to TRAIL-induced lysosomal membrane permeabilization and apoptosis. Correspondingly, deletion of cIAPs sensitized wild-type, but not PACS-2-deficient hepatocarcinoma cells or Pacs-2-/- mouse hepatocytes to TRAIL-induced apoptosis. Together, these data suggest cIAPs constitutively downregulate PACS-2 by polyubiquitination and proteasomal degradation, thereby restraining TRAIL-induced killing of liver cancer cells.

Degradation of cIAPs contributes to hepatocyte lipoapoptosis.

Hepatocyte apoptosis is a hallmark of nonalcoholic steatohepatitis. We have previously observed that the saturated free fatty acids (FFAs) induce hepatocyte apoptosis in part via a death receptor 5 (DR5)-mediated signaling pathway. Cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) proteins are potent inhibitors of death receptor-mediated apoptosis. However, the role of the cIAPs in FFA-mediated hepatocyte apoptosis is unexplored. Our aim was to determine whether cIAPs are dysregulated during hepatocyte lipoapoptosis. cIAP proteins underwent rapid cellular elimination following treatment with the saturated FFAs palmitate (PA) and stearate. In contrast, PA did not decrease cIAP-1 and cIAP-2 mRNA expression in the cells. Degradation of cIAPs was dependent on their E3-ligase activity, suggesting that cIAPs undergo autoubiquitination that leads to proteasomal degradation. Huh-7 cells stably expressing shRNA targeting cIAP-1, but not cIAP-2, displayed enhanced sensitivity to PA-mediated apoptosis. Incubation with the SMAC mimetic JP1584, which induces rapid degradation of cIAPs, also enhanced PA-mediated apoptosis. Hepatocytes isolated from DR5 knockout mice exhibited reduced apoptosis following treatment with PA plus JP1584, implying that degradation of cIAPs sensitizes to DR5-mediated cell death pathways. A decrease of cIAP-1 was also observed in tissue from patients with nonalcoholic steatohepatitis compared with normal obese subjects. Collectively, these results implicate proteasomal degradation of cIAPs by FFA as a mechanism contributing to hepatocyte lipoapoptosis.

Unshackling caspase-7 for cancer therapy.

Numerous solid tumors and hematologic malignancies acquire resistance to apoptosis-inducing chemotherapeutic drugs by downregulating the key effector caspase-3. These cells rely on caspase-7 to execute the apoptotic program, yet binding with XIAP constitutively inhibits active caspase-7 (p19/p12-CASP7). In this issue, Lin et al. describe how a newly synthesized drug is able to disrupt the XIAP:p19/p12-CASP7 complex and induce apoptosis in caspase-3-deficient cancer cells in vitro and in vivo. As this compound appears to exhibit minimal toxicity on normal tissues, it may represent a promising therapeutic agent to help treat caspase-3-deficient tumors.

Apoptosis and necrosis in the liver.

Because of its unique function and anatomical location, the liver is exposed to a multitude of toxins and xenobiotics, including medications and alcohol, as well as to infection by hepatotropic viruses, and therefore, is highly susceptible to tissue injury. Cell death in the liver occurs mainly by apoptosis or necrosis, with apoptosis also being the physiologic route to eliminate damaged or infected cells and to maintain tissue homeostasis. Liver cells, especially hepatocytes and cholangiocytes, are particularly susceptible to death receptor-mediated apoptosis, given the ubiquitous expression of the death receptors in the organ. In a quite unique way, death receptor-induced apoptosis in these cells is mediated by both mitochondrial and lysosomal permeabilization. Signaling between the endoplasmic reticulum and the mitochondria promotes hepatocyte apoptosis in response to excessive free fatty acid generation during the metabolic syndrome. These cell death pathways are partially regulated by microRNAs. Necrosis in the liver is generally associated with acute injury (i.e., ischemia/reperfusion injury) and has been long considered an unregulated process. Recently, a new form of "programmed" necrosis (named necroptosis) has been described: the role of necroptosis in the liver has yet to be explored. However, the minimal expression of a key player in this process in the liver suggests this form of cell death may be uncommon in liver diseases. Because apoptosis is a key feature of so many diseases of the liver, therapeutic modulation of liver cell death holds promise. An updated overview of these concepts is given in this article.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein-induced lysosomal translocation of proapoptotic effectors is mediated by phosphofurin acidic cluster sorting protein-2 (PACS-2).

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of liver cancer cell lines requires death receptor-5 (DR5)-dependent permeabilization of lysosomal membranes. Ligated DR5 triggers recruitment of the proapoptotic proteins Bim and Bax to lysosomes, releasing cathepsin B into the cytosol where it mediates mitochondria membrane permeabilization and activation of executioner caspases. Despite the requirement for lysosome membrane permeabilization during TRAIL-induced apoptosis, little is known about the mechanism that controls recruitment of Bim and Bax to lysosomal membranes. Here we report that TRAIL induces recruitment of the multifunctional sorting protein phosphofurin acidic cluster sorting protein-2 (PACS-2) to DR5-positive endosomes in Huh-7 cells where it forms an immunoprecipitatable complex with Bim and Bax on lysosomal membranes. shRNA-targeted knockdown of PACS-2 prevents recruitment of Bim or Bax to lysosomes, blunting the TRAIL-induced lysosome membrane permeabilization. Consistent with the reduced lysosome membrane permeabilization, shRNA knockdown of PACS-2 in Huh-7 cells reduced TRAIL-induced apoptosis and increased clonogenic cell survival. The determination that recombinant PACS-2 bound Bim but not Bax in vitro and that shRNA knockdown of Bim blocked Bax recruitment to lysosomes suggests that TRAIL/DR5 triggers endosomal PACS-2 to recruit Bim and Bax to lysosomes to release cathepsin B and induce apoptosis. Together, these findings provide insight into the lysosomal pathway of apoptosis.