Selective deletion of ENTPD1/CD39 in macrophages exacerbates biliary fibrosis in a mouse model of sclerosing cholangitis.

Purinergic signaling is important in the activation and differentiation of macrophages, which play divergent roles in the pathophysiology of liver fibrosis. The ectonucleotidase CD39 is known to modulate the immunoregulatory phenotype of macrophages, but whether this specifically impacts cholestatic liver injury is unknown. Here, we investigated the role of macrophage-expressed CD39 on the development of biliary injury and fibrosis in a mouse model of sclerosing cholangitis. Myeloid-specific CD39-deficient mice (LysMCreCd39fl/fl) were generated. Global CD39 null (Cd39-/-), wild-type (WT), LysMCreCd39fl/fl, and Cd39fl/fl control mice were exposed to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to induce biliary fibrosis. Hepatic hydroxyproline levels, liver histology, immunohistochemistry, mRNA expression levels, and serum biochemistry were then assessed. Following 3 weeks of DDC-feeding, Cd39-/- mice exhibited more severe fibrosis, when compared to WT mice as reflected by morphology and increased liver collagen content. Myeloid-specific CD39 deletion in LysMCreCd39fl/fl mice recapitulated the phenotype of global Cd39-/-, after exposure to DDC, and resulted in similar worsening of liver fibrosis when compared to Cd39fl/fl control animals. Further, DDC-treated LysMCreCd39fl/fl mice exhibited elevated serum levels of transaminases and total bilirubin, as well as increased hepatic expression of the profibrogenic genes Tgf-ß1, Tnf-α, and α-Sma. However, no clear differences were observed in the expression of macrophage-elaborated specific cytokines between LysMCreCd39fl/fl and Cd39fl/fl animals subjected to biliary injury. Our results in the DDC-induced biliary type liver fibrosis model suggest that loss of CD39 expression on myeloid cells largely accounts for the exacerbated sclerosing cholangitis in global CD39 knockouts. These findings indicate that macrophage expressed CD39 protects from biliary liver injury and fibrosis and support a potential therapeutic target for human hepatobiliary diseases.

Distinct roles of ecto-nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) in liver regeneration and fibrosis.

Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) are cell surface-located transmembrane ecto-enzymes of the CD39 superfamily which regulate inflammation and tissue repair by catalyzing the phosphohydrolysis of extracellular nucleotides and modulating purinergic signaling. In the liver, NTPDase2 is reportedly expressed on portal fibroblasts, but its functional role in regulating tissue regeneration and fibrosis is incompletely understood. Here, we studied the role of NTPDase2 in several models of liver injury using global knockout mice. Liver regeneration and severity of fibrosis were analyzed at different time points after exposure to carbon tetrachloride (CCl4) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or partial hepatectomy in C57BL/6 wild-type and globally NTPDase2-deficient (Entpd2 null) mice. After chronic CCl4 intoxication, Entpd2 null mice exhibit significantly more severe liver fibrosis, as assessed by collagen content and histology. In contrast, deletion of NTPDase2 does not have a substantial effect on biliary-type fibrosis in the setting of DDC feeding. In injured livers, NTPDase2 expression extends from the portal areas to fibrotic septae in pan-lobular (CCl4-induced) liver fibrosis; the same pattern was observed, albeit to a lesser extent in biliary-type (DDC-induced) fibrosis. Liver regeneration after partial hepatectomy is not substantively impaired in global Entpd2 null mice. NTPDase2 protects from liver fibrosis resulting from hepatocellular injury induced by CCl4. In contrast, Entpd2 deletion does not significantly impact fibrosis secondary to DDC injury or liver regeneration after partial hepatectomy. Our observations highlight mechanisms relating to purinergic signaling in the liver and indicate possible therapeutic avenues and new cellular targets to test in the management of hepatic fibrosis.

Generation of a murine hepatic angiosarcoma cell line and reproducible mouse tumor model.

Hepatic angiosarcoma (AS) is a rare and highly aggressive tumor of endothelial origin with dismal prognosis. Studies of the molecular biology of AS and treatment options are limited as animal models are rare. We have previously shown that inducible knockout of Notch1 in mice leads to spontaneous formation of hepatic AS. The aims of this study were to: (1) establish and characterize a cell line derived from this murine AS, (2) identify molecular pathways involved in the pathogenesis and potential therapeutic targets, and (3) generate a tumor transplantation model. AS cells retained specific endothelial properties such as tube formation activity, as well as expression of CD31 and Von Willebrand factor. However, electron microscopy analysis revealed signs of dedifferentiation with loss of fenestrae and loss of contact inhibition. Microarray and pathway analysis showed substantial changes in gene expression and revealed activation of the Myc pathway. Exposing the AS cells to sorafenib reduced migration, filopodia dynamics, and cell proliferation but did not induce apoptosis. In addition, sorafenib suppressed ERK phosphorylation and expression of cyclin D2. Injection of AS cells into NOD/SCID mice resulted in formation of undifferentiated tumors, confirming the tumorigenic potential of these cells. In summary, we established and characterized a murine model of spontaneous AS formation and hepatic AS cell lines as a useful in vitro tool. Our data demonstrate antitumor activity of sorafenib in AS cells with potent inhibition of migration, filopodia formation, and cell proliferation, supporting further evaluation of sorafenib as a novel treatment strategy. In addition, AS cell transplantation provides a subcutaneous tumor model useful for in vivo preclinical drug testing.

Downregulation of the endothelial genes Notch1 and ephrinB2 in patients with nodular regenerative hyperplasia.

BACKGROUND & AIMS: Nodular regenerative hyperplasia (NRH) is a rare liver disease characterized by small regenerative nodules without fibrosis and can cause portal hypertension. Aetiology and pathogenesis of NRH remain unclear. We have recently shown that Notch1 knockout induces NRH with portal hypertension through vascular remodelling in mice. The aim of this study was to analyse histological and clinical data of NRH patients and to explore if the endothelial pathways identified in our NRH mouse model are also regulated in human NRH. METHODS: Patients were identified retrospectively from the pathology database. Clinical and laboratory patient data were retrieved. mRNA expression was measured in liver biopsies from a subset of NRH patients. RESULTS: Diagnosis of NRH was confirmed in needle biopsies of 51 patients, including 31 patients with grade 1, 12 patients with grade 2 and 8 patients with grade 3 NRH. Grade 3 nodularity significantly correlated with the presence of portal hypertension: 50% of the patients with grade 3 NRH vs. 6.5% with grade 1 (P = 0.0105). mRNA expression analysis in liver biopsies from 14 NRH patients and in primary human sinusoidal endothelial cells revealed downregulation of identical genes as in the murine NRH model, which are implicated in vascular differentiation: Notch1, delta-like 4 (Dll4) and ephrinB2. CONCLUSIONS: In this large NRH needle biopsy cohort, we demonstrated that advanced nodularity correlates with presence of portal hypertension. Downregulation of the endothelial signalling pathways Dll4/Notch1 and ephrinB2/EphB4 supports the hypothesis that human NRH is caused by a sinusoidal injury providing first insights into the molecular pathogenesis of this liver condition.

Disruption of Notch1 induces vascular remodeling, intussusceptive angiogenesis, and angiosarcomas in livers of mice.

BACKGROUND & AIMS: Notch signaling mediates embryonic vascular development and normal vascular remodeling; Notch1 knockout mice develop nodular regenerative hyperplasia (NRH). The pathogenesis of NRH is unclear, but has been associated with vascular injury in the liver sinusoids in clinical studies. We investigated the role of Notch1 signaling in liver sinusoidal endothelial cells (LSECs). METHODS: We studied MxCre Notch1(lox/lox) mice (conditional knockout mice without tissue-specific disruption of Notch1); mice with hepatocyte-specific knockout were created by crossing Notch1(lox/lox) with AlbCre(+/-) mice. Portal vein pressure was measured; morphology of the hepatic vasculature was assessed by histologic and scanning electron microscopy analyses. We performed functional and expression analyses of isolated liver cells. RESULTS: MxCre-induced knockout of Notch1 led to NRH, in the absence of fibrosis, with a persistent increase in proliferation of LSECs. Notch1 deletion led to de-differentiation, vascular remodeling of the hepatic sinusoidal microvasculature, intussusceptive angiogenesis, and dysregulation of ephrinB2/EphB4 and endothelial tyrosine kinase. Time-course experiments revealed that vascular changes preceded node transformation. MxCre Notch1(lox/lox) mice had reduced endothelial fenestrae and developed portal hypertension and hepatic angiosarcoma over time. In contrast, mice with hepatocyte-specific disruption of Notch1 had a normal phenotype. CONCLUSIONS: Notch1 signaling is required for vascular homeostasis of hepatic sinusoids; it maintains quiescence and differentiation of LSECs in adult mice. Disruption of Notch1 signaling in LSECs leads to spontaneous formation of angiosarcoma, indicating its role as a tumor suppressor in the liver endothelium.

ELIMINATE: a PCR record-based macroelimination project for systematic recall of HCV-RNA-positive persons in Austria.

BACKGROUND AND AIMS: Micro-elimination projects targeted to specific hepatitis C virus (HCV) risk populations have been successful. Systematic identification of persons with HCV viremia, regardless of risk group, based on already available laboratory records may represent an effective macroelimination approach to achieve global HCV elimination. METHODS: Persons with a last positive HCV-RNA PCR result between 2008-2020 in the reference virology laboratories in eastern Austria were identified. First, (i) we described their demographic characteristics, (ii) we systematically recalled persons to the respective centers and (iii) started antiviral treatment if HCV-RNA viremia was confirmed, and (iv) recorded sustained virologic response (SVR). This interim report includes the preliminary results from 8 participating centers. RESULTS: During the study period 22,682 persons underwent HCV-RNA PCR testing, 11,216 (49.4%) were positive at any point in time, and 6006 (26.5%) showed detectable HCV-RNA at the last PCR test, suggesting ongoing HCV viremia. At the time of this interim report, 2546/6006 HCV-RNA PCR(+) persons were evaluated: 443/2546 (17.4%) had died, 852/2546 (33.5%) had invalid contact data, and 547/2546 (21.5%) had achieved SVR between data retrieval and recall. Contact could be established in 236/704 (33.5%) of the remaining target population with 97/236 (41.1%) presenting at the clinic for treatment evaluation. Ultimately, 71/236 (30.1%) started antiviral treatment and SVR was documented in 47/71 (66.2%). CONCLUSION: This ELIMINATE project based on systematic assessment of HCV-RNA PCR-records, identified 6006 persons with potential persisting HCV viremia. Invalid contact data and missed visits for treatment evaluation were the main barriers towards HCV elimination within this project. Importantly, many subjects with HCV viremia lost to follow-up were successfully linked to care and started antiviral treatment.

Author Correction: NADH oxidase-dependent CD39 expression by CD8+ T cells modulates interferon gamma responses via generation of adenosine.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Control of tumor-associated macrophages and T cells in glioblastoma via AHR and CD39.

In the version of this article initially published, author Alexandre Prat's surname was misspelled. The error has been corrected in the HTML and PDF versions of the article.

Control of tumor-associated macrophages and T cells in glioblastoma via AHR and CD39.

Tumor-associated macrophages (TAMs) play an important role in the immune response to cancer, but the mechanisms by which the tumor microenvironment controls TAMs and T cell immunity are not completely understood. Here we report that kynurenine produced by glioblastoma cells activates aryl hydrocarbon receptor (AHR) in TAMs to modulate their function and T cell immunity. AHR promotes CCR2 expression, driving TAM recruitment in response to CCL2. AHR also drives the expression of KLF4 and suppresses NF-κB activation in TAMs. Finally, AHR drives the expression of the ectonucleotidase CD39 in TAMs, which promotes CD8+ T cell dysfunction by producing adenosine in cooperation with CD73. In humans, the expression of AHR and CD39 was highest in grade 4 glioma, and high AHR expression was associated with poor prognosis. In summary, AHR and CD39 expressed in TAMs participate in the regulation of the immune response in glioblastoma and constitute potential targets for immunotherapy.

The ectonucleotidase ENTPD1/CD39 limits biliary injury and fibrosis in mouse models of sclerosing cholangitis.

The pathogenesis of primary sclerosing cholangitis (PSC) and the mechanistic link to inflammatory bowel disease remain ill-defined. Ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1)/clusters of differentiation (CD) 39, the dominant purinergic ecto-enzyme, modulates intestinal inflammation. Here, we have explored the role of CD39 in biliary injury and fibrosis. The impact of CD39 deletion on disease severity was studied in multidrug resistance protein 2 (Mdr2)-/- and 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse models of sclerosing cholangitis and biliary fibrosis. Antibody-mediated CD8+ T-cell depletion, selective gut decontamination, experimental colitis, and administration of stable adenosine triphosphate (ATP) agonist were performed. Retinoic acid-induced gut imprinting on T cells was studied in vitro. Over half of Mdr2-/-;CD39-/- double mutants, expected by Mendelian genetics, died in utero. Compared to Mdr2-/-;CD39+/+, surviving Mdr2-/-;CD39-/- mice demonstrated exacerbated liver injury, fibrosis, and ductular reaction. CD39 deficiency led to a selective increase in hepatic CD8+ T cells and integrin α4ß7, a T-cell gut-tropism receptor. CD8+ cell depletion in Mdr2-/-;CD39-/- mice diminished hepatobiliary injury and fibrosis. Treatment with antibiotics attenuated, whereas dextran sulfate sodium-induced colitis exacerbated, liver fibrosis in Mdr2-/- mice. Colonic administration of αß-ATP into CD39-sufficient Mdr2-/- mice triggered hepatic CD8+ cell influx and recapitulated the severe phenotype observed in Mdr2-/-;CD39-/- mice. In vitro, addition of ATP promoted the retinoic acid-induced imprinting of gut-homing integrin α4ß7 on naive CD8+ cells. CD39 expression was relatively low in human normal or PSC livers but abundantly present on immune cells of the colon and further up-regulated in samples of patients with inflammatory bowel disease. Conclusion: CD39 deletion promotes biliary injury and fibrosis through gut-imprinted CD8+ T cells. Pharmacological modulation of purinergic signaling may represent a promising approach for the treatment of PSC. (Hepatology Communications 2017;1:957-972).

NADH oxidase-dependent CD39 expression by CD8(+) T cells modulates interferon gamma responses via generation of adenosine.

Interferon gamma (IFNγ)-producing CD8(+) T cells (Tc1) play important roles in immunological disease. We now report that CD3/CD28-mediated stimulation of CD8(+) T cells to generate Tc1 cells, not only increases IFNγ production but also boosts the generation of reactive oxygen species (ROS) and augments expression of CD39. Inhibition of NADPH oxidases or knockdown of gp91phox in CD8(+) T cells abrogates ROS generation, which in turn modulates JNK and NFκB signalling with decreases in both IFNγ levels and CD39 expression. CD39(+)CD8(+) T cells substantially inhibit IFNγ production by CD39(-)CD8(+) T cells via the paracrine generation of adenosine, which is operational via adenosine type 2A receptors. Increases in numbers of CD39(+)CD8(+) T cells and associated enhancements in ROS signal transduction are noted in cells from patients with Crohn's disease. Our findings provide insights into Tc1-mediated IFNγ responses and ROS generation and link these pathways to CD39/adenosine-mediated effects in immunological disease.