Primary sclerosing cholangitis (PSC) is an incurable, fibroinflammatory biliary disease for which there is no effective pharmacotherapy. We recently reported cholangiocyte senescence as an important phenotype in PSC while others showed that portal macrophages accumulate in PSC. Unfortunately, our ability to explore cholangiocyte senescence and macrophage accumulation has been hampered by limited in vitro models. Thus, our aim was to develop and characterize a three dimensional model (3D) of normal and diseased bile ducts (cholangioids) starting with normal human cholangiocytes (NHC), senescent NHC (NHC-sen), and cholangiocytes from PSC patients. In 3D culture, NHCs formed spheroids of ~5000 cells with a central lumen of ~150 μm. By confocal microscopy and western blot, cholangioids retained expression of cholangiocyte proteins (cytokeratin 7/19) and markers of epithelial polarity (secretin receptor and GM130). Cholangioids are functionally active, and upon secretin stimulation, luminal size increased by ~ 80%. Cholangioids exposed to hydrogen peroxide exhibited cellular senescence (SA-βGal and γH2A.x expression) and the senescence associated secretory phenotype (SASP; increased IL-6, p21, β;-Gal and p16 expression). Furthermore, cholangioids derived from NHC-sen or PSC patients were smaller and had slower growth than the controls. When co-cultured with THP-1 macrophages, the number of macrophages associated with NHC-sen or PSC cholangioids was 5 to7-fold greater compared to co-culture with non-senescent NHC. We observed that NHC-sen and PSC cholangioids release greater numbers of extracellular vesicles (ECVs) compared to controls. Moreover, conditioned media from NHC-sen cholangioids resulted in an ~2-fold increase in macrophage migration. In summary, we developed a method to normal and diseased cholangioids, characterized them morphologically and functionally, showed that they can be induced to senescence and SASP, and demonstrated both ECV release and macrophage attraction. This novel model mimics several features of PSC and thus will be useful for studying the pathogenesis of PSC and potentially identifying new therapeutic targets.

Objectives Biliary atresia is a rapidly progressive form of biliary fibrosis affecting neonates. We previously reported that primary cilia on the intrahepatic cholangiocytes of patients with both syndromic and non-syndromic biliary atresia were structurally abnormal. Our objective was to determine whether extrahepatic cholangiocytes in human biliary atesia, intrahepatic and extrahepatic cholangiocytes of rhesus rotavirus (RRV)-infected neonatal mice, and RRV-infected primary neonatal extrahepatic cholangiocytes also demonstrate ciliary abnormalities. Methods The livers of neonatal BALB/c mice injected with RRV that developed jaundice, human extra-hepatic bile duct samples obtained at time of hepatoportoenterostomy, and RRV-infected primary neonatal cholangiocytes were stained with antibodies against acetylated alpha tubulin to identify primary cilia. Results Extrahepatic cholangiocytes from RRV-treated mice demonstrated minimal loss of primary cilia at day 3 but almost complete loss at day 8 and partial loss at day 12. No changes were seen in mouse intrahepatic bile ducts at any of the time points. In the human biliary atresia samples, primary cilia were almost completely absent from extrahepatic duct cholangiocytes. There were, however, abundant cilia in the peri-biliary glands adjacent to extrahepatic ducts in the biliary atresia sample. Cilia in RRV-infected primary neonatal cholangiocytes were significantly decreased compared to control. Conclusions Primary cilia are selectively lost from neonatal extrahepatic but not intrahepatic cholangiocytes after RRV infection in BALB/c mice. The cilia are also decreased in rhesus rotavirus-infected primary cholangiocytes and the extrahepatic ducts from human biliary atresia patients. This suggests that ciliary abnormalities are part of the pathophysiology of biliary atresia.

Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory cholangiopathy (disease of the bile ducts) of unknown pathogenesis. We reported that cholangiocyte senescence features prominently in PSC and that neuroblastoma RAS viral oncogene homolog (NRAS) is activated in PSC cholangiocytes. Additionally, persistent microbial insult ( LPSs) induces cyclin-dependent kinase inhibitor 2A (CDKN2A/p16) expression and senescence in cultured cholangiocytes in an NRAS-dependent manner. However, the molecular mechanisms involved in LPS-induced cholangiocyte senescence and NRAS-dependent regulation of CDKN2A remain unclear. Using our senescence model, we found that LPS-induced expression coincided with a 4.5-fold increase in () mRNA, suggesting that ETS1 is involved in regulating This idea was confirmed by RNAi-mediated suppression or genetic deletion of ETS1, which blocked CDKN2A expression and reduced cholangiocyte senescence. Furthermore, site-directed mutagenesis of a predicted ETS-binding site within the promoter abolished luciferase reporter activity. Pharmacological inhibition of RAS/MAPK reduced ETS1 and CDKN2A protein expression and promoter-driven luciferase activity by ∼50%. In contrast, constitutively active NRAS expression induced ETS1 and CDKN2A protein expression, whereas ETS1 RNAi blocked this increase. Chromatin immunoprecipitation-PCR detected increased ETS1 and histone 3 lysine 4 trimethylation (H3K4Me3) at the promoter following LPS-induced senescence. Additionally, phospho-ETS1 expression was increased in cholangiocytes of human PSC livers and in the () mouse model of PSC. These data pinpoint ETS1 and H3K4Me3 as key transcriptional regulators in NRAS-induced expression of , and this regulatory axis may therefore represent a potential therapeutic target for PSC treatment.

Autophagy is increased in PLD cholangiocytes, contributes to hepatic cystogenesis, and represents a potential therapeutic target for disease treatment. (Hepatology 2018;67:1088-1108).

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The cholangiopathies are a group of liver diseases resulting from different etiologies but with the cholangiocyte as the primary target. As a group, the cholangiopathies result in significant morbidity and mortality and represent one of the main indications for liver transplant in both children and adults. Contributing to this situation is the absence of a thorough understanding of their pathogenesis and a lack of adequate diagnostic and prognostic biomarkers. MicroRNAs are small non-coding RNAs that modify gene expression post-transcriptionally. They have been implicated in the pathogenesis of many diseases, including the cholangiopathies. Thus, in this review we provide an overview of the literature on miRNAs in the cholangiopathies and discuss future research directions.

A number of cell culture approaches have been described for maintenance of primary hepatocytes. Forming hepatocytes into 3D spheroids is one well accepted method for extending epithelial phenotype of these cells. Our lab has previously observed enhanced function of 2D (monolayer) hepatocyte cultures in microfluidic devices due to increased production of several hepato-inductive growth factors, including hepatocyte growth factor (HGF). In the present study we wanted to test a hypothesis that placing hepatocytes spheroids (3D) into microfluidic devices will also result in enhanced phenotype and function. To test this hypothesis, we fabricated devices with small and large volumes. Both types of devices included a microstructured floor containing arrays of pyramidal wells to promote assembly of single hepatocytes into spheroids with individual diameter of ~100 µm. The hepatocyte spheroids were found to be more functional in low volume compared to large volume devices. Importantly, high functionality of spheroid cultures correlated with elevated levels of HGF secretion. While decay of hepatic function (albumin secretion) was observed over the course three weeks, this behavior could be abrogated by inhibiting TGF-β1 signaling. With TGF-β1 inhibitor, microfluidic hepatocyte spheroid cultures maintained high and stable levels of albumin synthesis over the course of four weeks. To further highlight utility of this culture platform for liver disease modeling, we carried out alcohol injury experiments in microfluidic devices and tested protective effects of interleukin (IL)-22 - a potential therapy for alcoholic hepatitis.

Human immunodeficiency virus type 1 (HIV-1) infection confers an increased risk for the development of many cancers. Although the incidences of acquired immunodeficiency syndrome (AIDS)-defining malignancies have declined since the advent of antiretroviral therapy (ART), a number of non-AIDS-defining cancers appear more common in HIV-1-infected individuals relative to the general population. ART-treated HIV-1-infected subjects are also aging, leading to an increased cancer burden in these populations. However, longevity alone is not sufficient to explain these epidemiologic trends. A causative link between HIV-1-induced immune suppression and elevated cancer risk is well defined in certain malignancies; however, the direct role of HIV-1 replication products in oncogenesis remains unclear. Nevertheless, it is evident that cooperation between HIV-1 and co-infecting viruses in targeting immune compartments as well as nonimmune microenvironments can regulate both the development and progression of cancer. Treating cancer in HIV-1-infected patients remains challenging due to drug interactions, compounded side effects and intensified immunosuppression from chemotherapy and/or radiation. While survival of HIV-1-infected patients with certain cancers now rivals that of their uninfected counterparts, a better understanding of HIV-1-induced oncogenesis, viral mechanisms of immune perturbation, nonimmune microenvironmental abnormalities and outcomes of therapy will provide the basis for better diagnosis and management of cancer.