Michel Fausther

Advances in cholangiocyte immunobiology

Cholangiocytes, or bile duct epithelia, were once thought to be the simple lining of the conduit system comprising the intra- and extrahepatic bile ducts. Growing experimental evidence demonstrated that cholangiocytes are in fact the first line of defense of the biliary system against foreign substances. Experimental advances in recent years have unveiled previously unknown roles of cholangiocytes in both innate and adaptive immune responses. Cholangiocytes can release inflammatory modulators in a regulated fashion. Moreover, they express specialized pattern-recognizing molecules that identify microbial components and activate intracellular signaling cascades leading to a variety of downstream responses. The cytokines secreted by cholangiocytes, in conjunction with the adhesion molecules expressed on their surface, play a role in recruitment, localization, and modulation of immune responses in the liver and biliary tract. Cholangiocyte survival and function is further modulated by cytokines and inflammatory mediators secreted by immune cells and cholangiocytes themselves. Because cholangiocytes act as professional APCs via expression of major histocompatibility complex antigens and secrete antimicrobial peptides in bile, their role in response to biliary infection is critical. Finally, because cholangiocytes release mediators critical to myofibroblastic differentiation of portal fibroblasts and hepatic stellate cells, cholangiocytes may be essential in the pathogenesis of biliary cirrhosis.

Localization of plasma membrane bound NTPDases in the murine reproductive tract.

Extracellular nucleotides might influence aspects of the biology of reproduction in that ATP affects smooth muscle contraction, participates in steroidogenesis and spermatogenesis, and also regulates transepithelial transport, as in oviducts. Activation of cellular nucleotide purinergic receptors is influenced by four plasma membrane-bound members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, namely NTPDase1, NTPDase2, NTPDase3, and NTPDase8 that differ in their ecto-enzymatic properties. The purpose of this study was to characterize the expression profile of the membrane-bound NTPDases in the murine female and male reproductive tracts by immunological techniques (immunolabelling, Western blotting) and by enzymatic assays, in situ and on tissue homogenates. Other than the expected expression on vascular endothelial and smooth muscle cells, NTPDase1 was also detected in Sertoli cells and interstitial macrophages in testes, in ovarian granulosa cells, and in apical cells from epididymal epithelium. NTPDase2 was largely expressed by cells in the connective tissue; NTPDase3 in secretory epithelia, and finally, NTPDase8 was not detected in any of the tissues studied here. In addition, NTPDase6 was putatively detected in Golgi-phase acrosome vesicles of round spermatids. This descriptive study suggests close regulation of extracellular nucleotide levels in the genital tract by NTPDases that may impact specific biological functions.

Contribution of Myofibroblasts of Different Origins to Liver Fibrosis

The most common cause of liver failure is cirrhosis, due to progressive liver fibrosis and other architectural changes in the liver. Fibrosis occurs after liver injury or stress and results directly from an imbalance between the processes of extracellular matrix synthesis (fibrogenesis) and degradation (fibrolysis). Although research studies have identified several promising targets at the molecular level, current therapies to prevent and treat hepatic fibrosis in patients have only shown limited success. It is well established that liver myofibroblasts (MFs) are the primary effector cells responsible for the extensive extracellular matrix accumulation and scar formation observed during hepatic fibrosis, in both clinical and experimental settings. Thus, as the major fibrogenic cells implicated in wound healing and tissue repair response, liver MFs could represent excellent targets for antifibrotic therapies. Still, the exact nature and identities of liver MFs precursors have yet to be resolved, and their relative contribution to hepatic fibrosis to be determined. The goal of this review is to examine the relative importance of liver MF precursors in the pathogenesis of liver fibrosis.

Characterization of a monoclonal antibody as the first specific inhibitor of human NTP diphosphohydrolase‐3

The study and therapeutic modulation of purinergic signaling is hindered by a lack of specific inhibitors for NTP diphosphohydrolases (NTPDases), which are the terminating enzymes for these processes. In addition, little is known of the NTPDase protein structural elements that affect enzymatic activity and which could be used as targets for inhibitor design. In the present study, we report the first inhibitory monoclonal antibodies specific for an NTPDase, namely human NTPDase3 (EC 3.6.1.5), as assessed by ELISA, western blotting, flow cytometry, immunohistochemistry and inhibition assays. Antibody recognition of NTPDase3 is greatly attenuated by denaturation with SDS, and abolished by reducing agents, indicating the significance of the native conformation and the disulfide bonds for epitope recognition. Using site‐directed chemical cleavage, the SDS‐resistant parts of the epitope were located in two fragments of the C‐terminal lobe of NTPDase3 (i.e. Leu220–Cys347 and Cys347–Pro485), which are both required for antibody binding. Additional site‐directed mutagenesis revealed the importance of Ser297 and the fifth disulfide bond (Cys399–Cys422) for antibody binding, indicating that the discontinuous inhibitory epitope is located on the extracellular C‐terminal lobe of NTPDase3. These antibodies inhibit recombinant NTPDase3 by 60–90%, depending on the conditions. More importantly, they also efficiently inhibit the NTPDase3 expressed in insulin secreting human pancreatic islet cells in situ. Because insulin secretion is modulated by extracellular ATP and purinergic receptors, this finding suggests the potential application of these inhibitory antibodies for the study and control of insulin secretion.

Extracellular vesicles carry microRNA-195 to intrahepatic cholangiocarcinoma and improve survival in a rat model

The cancer microenvironment plays a central role in cancer development, growth, and homeostasis. This paradigm suggests that cancer fibroblasts support cancers, probably in response to stimuli received from the cancer cells. We aimed at investigating whether extracellular vesicles (EVs) can shuttle microRNA (miR) species between cancer‐associated fibroblasts (CAFs) and cancer cells. To this end, we extracted EVs according to published protocols. EVs were studied for their miR content by quantitative reverse‐transcription polymerase chain reaction. EVs were transfected with select miR species and utilized in vitro as well as in vivo in a rat model of cholangiocarcinoma (CCA). We found that miR‐195 is down‐regulated in CCA cells, as well as in adjoining fibroblasts. Furthermore, we report that EVs shuttle miR‐195 from fibroblasts to cancer cells. Last, we show that fibroblast‐derived EVs, loaded with miR‐195, can be administered in a rat model of CCA, concentrate within the tumor, decrease the size of cancers, and improve survival of treated rats. Conclusion: EVs play a salient role in trafficking miR species between cancer cells and CAFs in human CCA. Understanding of these mechanisms may allow devising of novel therapeutics. (Hepatology 2017;65:501‐514).

High expression and activity of ecto-5′-nucleotidase/CD73 in the male murine reproductive tract

Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, steroidogenesis, and maintenance of fluid composition. Interestingly, adenosine might act as a key capacitative effector for mammalian spermatozoa to acquire the capacity for fertilisation. Extracellular nucleotide levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family regroups the most abundant and effective enzymes to hydrolyse ATP and ADP to AMP in physiological conditions. In the male reproductive tract three members of this family have been indentified: NTPDase1, NTPDase2 and NTPDase3 (Martín-Satué et al. in Histochem Cell Biol 131:615–628, 2009). The purpose of the present study was to characterize in the male reproductive tract the expression profile of the main enzyme responsible for the generation of adenosine from AMP, namely the ecto-5′-nucleotidase (CD73). The enzyme was identified by immunological techniques and by in situ enzymatic assays, including inhibition experiments with α,β-methylene-ADP, a specific CD73 inhibitor. High levels of ecto-5′-nucleotidase were detected in testes in association with both germinal and somatic cells, in smooth muscle cells throughout the tract, in secretory epithelia from exocrine glands, and remarkably, in principal cells of epididymis, where co-localization with NTPDase3 was found. The relevance of this co-expression on nucleotide hydrolysis in these cells directly involved in the control of sperm fluid composition was addressed biochemically. This study suggests close regulation of extracellular nucleoside and nucleotide levels in the genital tract by ecto-5′-nucleotidase that, in concurrence with NTPDases, may impact male fertility.

Nucleotide receptors control IL-8/CXCL8 and MCP-1/CCL2 secretions as well as proliferation in human glioma cells

Glioma cells release cytokines to stimulate inflammation that facilitates cell proliferation. Here, we show that Lipopolysaccharide (LPS) treatment could induce glioma cells to proliferate and this process was dependent on nucleotide receptor activation as well as interleukin-8 (IL-8/CXCL8) secretion. We observed that extracellular nucleotides controlled IL-8/CXCL8 and monocyte chemoattractant protein 1 (MCP-1/CCL2) release by U251MG and U87MG human glioma cell lines via P2X7 and P2Y6 receptor activation. The LPS-induced release of these cytokines was also modulated by purinergic receptor activation since IL-8 and MCP-1 release was decreased by the nucleotide scavenger apyrase as well as by the pharmacological P2Y6 receptor antagonists suramin and MRS2578. In agreement with these observations, the knockdown of P2Y6 expression decreased LPS-induced IL-8 release as well as the spontaneous release of IL-8 and MCP-1, suggesting an endogenous basal release of nucleotides. Moreover, high millimolar concentrations of ATP increased IL-8 and MCP-1 release by the glioma cells stimulated with suboptimal LPS concentration which were blocked by P2X7 and P2Y6 antagonists. Altogether, these data suggest that extracellular nucleotides control glioma growth via P2 receptor-dependent IL-8 and MCP-1 secretions.

Cystic fibrosis remodels the regulation of purinergic signaling by NTPDase1 (CD39) and NTPDase3

Airway defenses are regulated by a complex purinergic signaling network located on the epithelial surfaces, where ATP stimulates the clearance of mucin and pathogens. The present study shows that the obstructive disease cystic fibrosis (CF) affects the activity, expression, and tissue distribution of two ectonucleotidases found critical for the regulation of ATP on airway surfaces: NTPDase1 and NTPDase3. Functional polarities and mRNA expression levels were determined on primary cultures of human bronchial epithelial (HBE) cells from healthy donors and CF patients. The in vitro model of the disease was completed by exposing CF HBE cultures for 4 days to supernatant of the mucopurulent material (SMM) collected from the airways of CF patients. We report that NTPDase1 and NTPDase3 are coexpressed on HBE cultures, where they regulate physiological and excess nucleotide concentrations, respectively. In aseptic conditions, CF epithelia exhibit >50% lower NTPDase1 activity, protein, and mRNA levels than normal epithelia, whereas these parameters are threefold higher for NTPDase3. Exposure to SMM induced opposite polarity shifts of the two NTPDases on both normal and CF epithelia, apical NTPDase1 being mobilized to basolateral surfaces and bilateral NTPDase3 to the apical surface. Their immunolocalization in human tissue revealed that NTPDase1 is expressed in epithelial, inflammatory, and endothelial cells, whereas NTPDase3 is restricted to epithelial cells. Furthermore, the SMM-exposed CF HBE cultures reproduced the impact of the disease on their in vivo distribution. This study provides evidence that an extensive remodeling of the enzymatic network regulating clearance occurs in the airways of CF patients.

Expression of Ecto-ATPase NTPDase2 in Human Dental Pulp

Dental pulpal nerve fibers express ionotropic adenosine triphosphate (ATP) receptors, suggesting that ATP signaling participates in the process of dental nociception. In this study, we investigated if the principal enzymes responsible for extracellular ATP hydrolysis, namely, nucleoside triphosphate diphosphohydrolases (NTPDases), are present in human dental pulp. Immunohistochemical and immunofluorescence experiments showed that NTPDase2 was predominantly expressed in pulpal nerve bundles, Raschkow’s nerve plexus, and in the odontoblast layer. NTPDase2 was expressed in pulpal Schwann cells, with processes accompanying the nerve fibers and projecting into the odontoblast layer. Odontoblasts expressed the gap junction protein, connexin43, which can form transmembrane hemichannels for ATP release. NTPDase2 was localized close to connexin43 within the odontoblast layer. These findings provide evidence for the existence of an apparatus for ATP release and degradation in human dental pulp, consistent with the involvement of ATP signaling in the process of dentin sensitivity and dental pain.

How does coffee prevent liver fibrosis? biological plausibility for recent epidemiological observations

The published epidemiological data demonstrating an inverse relationship between coffee (and potentially other caffeinated beverage) consumption and liver fibrosis and its downstream complications are weighty and rapidly accumulating. Several excellent recent reviews examine this evidence in great detail (1–3), and the overwhelming conclusion is that this inverse relationship is real – coffee drinking reduces liver fibrosis. Among the strongest studies to support this observation are the findings that, after adjustment for confounders, individuals in the highest quintile of caffeine consumption had less than one third the risk of ALT elevation of those in the lowest quintile (odds ratio (OR) 0.31, 95% CI 0.16–0.61) (4) and, perhaps more importantly, advanced liver fibrosis from chronic liver diseases of various etiologies is associated with reduced coffee and total caffeine consumption (5) with one study showing that the odds of having cirrhosis decreased with increasing daily consumption of coffee in a step-wise manner from an OR of 0.47 (95% CI 0.20–1.10) for patients consuming 1 cup of coffee per day to an OR of 0.16 (95% CI 0.05–0.50) for patients consuming 4 cups per day, compared to lifetime abstainers as the reference (OR 1.0) (6). Demonstrating the clinical significance of coffee consumption, Freedman and colleagues found that among patients with advanced fibrosis, those who consumed no coffee had a risk of hepatic decompensation or hepatocellular carcinoma (HCC) of 11.1 per 100 patient-years compared to just 6.3 per 100 patient-years in those consuming ≥ 3 cups of coffee per day, with no beneficial effect seen with tea or other sources of caffeine (7). Coffee consumption has also been shown to be associated with a lower risk of fatty liver disease (8), metabolic syndrome (9), and ultimately hepatocellular carcinoma (10). As a clinician or scientist interested in the pathogenesis of liver fibrosis, one may very well ask whether these findings are of great value.