Background and Aims
Lipopolysaccharides (LPS) is increased in nonalcoholic fatty liver disease (NAFLD), but its relationship with liver inflammation is not defined.
Approach and Results
We studied ...Escherichia coli LPS in patients with biopsy‐proven NAFLD, 25 simple steatosis (nonalcoholic fatty liver) and 25 nonalcoholic steatohepatitis (NASH), and in mice with diet‐induced NASH. NASH patients had higher serum LPS and hepatocytes LPS localization than controls, which was correlated with serum zonulin and phosphorylated nuclear factor‐κB expression. Toll‐like receptor 4 positive (TLR4+) macrophages were higher in NASH than simple steatosis or controls and correlated with serum LPS. NASH biopsies showed a higher CD61+ platelets, and most of them were TLR4+. TLR4+ platelets correlated with serum LPS values. In mice with NASH, LPS serum levels and LPS hepatocyte localization were increased compared with control mice and associated with nuclear factor‐κB activation. Mice on aspirin developed lower fibrosis and extent compared with untreated ones. Treatment with TLR4 inhibitor resulted in lower liver inflammation in mice with NASH.
Conclusions
In NAFLD, Escherichia coli LPS may increase liver damage by inducing macrophage and platelet activation through the TLR4 pathway.
Cholangiocarcinoma (CCA) includes a cluster of highly heterogeneous biliary malignant tumours that can arise at any point of the biliary tree. Their incidence is increasing globally, currently ...accounting for ~15% of all primary liver cancers and ~3% of gastrointestinal malignancies. The silent presentation of these tumours combined with their highly aggressive nature and refractoriness to chemotherapy contribute to their alarming mortality, representing ~2% of all cancer-related deaths worldwide yearly. The current diagnosis of CCA by non-invasive approaches is not accurate enough, and histological confirmation is necessary. Furthermore, the high heterogeneity of CCAs at the genomic, epigenetic and molecular levels severely compromises the efficacy of the available therapies. In the past decade, increasing efforts have been made to understand the complexity of these tumours and to develop new diagnostic tools and therapies that might help to improve patient outcomes. In this expert Consensus Statement, which is endorsed by the European Network for the Study of Cholangiocarcinoma, we aim to summarize and critically discuss the latest advances in CCA, mostly focusing on classification, cells of origin, genetic and epigenetic abnormalities, molecular alterations, biomarker discovery and treatments. Furthermore, the horizon of CCA for the next decade from 2020 onwards is highlighted.
Background and Aims
Cholangiocarcinoma (CCA) is a very aggressive cancer showing the presence of high cancer stem cells (CSCs). Doublecortin‐like kinase1 (DCLK1) has been demonstrated as a CSC marker ...in different gastroenterological solid tumors. Our aim was to evaluate in vitro the expression and the biological function of DCLK1 in intrahepatic CCA (iCCA) and perihilar CCA (pCCA).
Approach and Results
Specimens surgically resected of human CCA were enzymatically digested, submitted to immunosorting for specific CSC markers (LGR5 leucine‐rich repeat‐containing G protein‐coupled receptor, CD clusters of differentiation 90, EpCAM epithelial cell adhesion molecule, CD133, and CD13), and primary cell cultures were prepared. DCLK1 expression was analyzed in CCA cell cultures by real‐time quantitative PCR, western blot, and immunofluorescence. Functional studies have been performed by evaluating the effects of selective DCLK1 inhibitor (LRRK2‐IN‐1) on cell proliferation (MTS 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium assay, cell population doubling time), apoptosis, and colony formation capacity. DCLK1 was investigated in situ by immunohistochemistry and real‐time quantitative PCR. DCLK1 serum concentration was analyzed by enzyme‐linked immunosorbent assay. We describe DCLK1 in CCA with an increased gene and protein DCLK1 expression in pCCALGR5+ and in iCCACD133+ cells compared with unsorted cells. LRRK2‐IN‐1 showed an anti‐proliferative effect in a dose‐dependent manner. LRRK2‐IN‐1 markedly impaired cell proliferation, induced apoptosis, and decreased colony formation capacity and colony size in both iCCA and pCCA compared with the untreated cells. In situ analysis confirmed that DCLK1 is present only in tumors, and not in healthy tissue. Interestingly, DCLK1 was detected in the human serum samples of patients with iCCA (high), pCCA (high), HCC (low), and cirrhosis (low), but it was almost undetectable in healthy controls.
Conclusions
DCLK1 characterizes a specific CSC subpopulation of iCCACD133+ and pCCALGR5+, and its inhibition exerts anti‐neoplastic effects in primary CCA cell cultures. Human DCLK1 serum might represent a serum biomarker for the early CCA diagnosis.
The "Gut-Liver Axis" refers to the physiological bidirectional interplay between the gut and its microbiota and the liver which, in health, occurs thanks to a condition of immune tolerance. In recent ...years, several studies have shown that, in case of a change in gut bacterial homeostasis or impairment of intestinal barrier functions, cholangiocytes, which are the epithelial cells lining the bile ducts, activate innate immune responses against gut-derived microorganisms or bacterial products that reach the liver via enterohepatic circulation. Intestinal dysbiosis or impaired intestinal barrier functions cause cholangiocytes to be exposed to an increasing amount of microorganisms that can reactivate inflammatory responses, thus inducing the onset of liver fibrosis. The present review focuses on the role of the gut-liver axis in the pathogenesis of cholangiopathies.
Cholangiopathies, such as primary sclerosing cholangitis, biliary atresia, and cholangiocarcinoma, have limited experimental models. Not only cholangiocytes but also other hepatic cells including ...hepatic stellate cells and macrophages are involved in the pathophysiology of cholangiopathies, and these hepatic cells orchestrate the coordinated response against diseased conditions. Classic two‐dimensional monolayer cell cultures do not resemble intercellular cell‐to‐cell interaction and communication; however, three‐dimensional cell culture systems, such as organoids and spheroids, can mimic cellular interaction and architecture between hepatic cells. Previous studies have demonstrated the generation of hepatic or biliary organoids/spheroids using various cell sources including pluripotent stem cells, hepatic progenitor cells, primary cells from liver biopsies, and immortalized cell lines. Gene manipulation, such as transfection and transduction can be performed in organoids, and established organoids have functional characteristics which can be suitable for drug screening. This review summarizes current methodologies for organoid/spheroid formation and a potential for three‐dimensional hepatic cell cultures as in vitro models of cholangiopathies.
Primary biliary cholangitis is an autoimmune disease affecting the interlobular bile ducts. Limited information is available on its epidemiology and treatment in Italy.
To describe primary biliary ...cholangitis epidemiology and investigate treatment patterns for Italian patients with this disease.
Electronic medical records from 900 general practitioners (part of the QuintilesIMS™ Longitudinal Patient Databases) were examined. Demographics were compared with those from the Italian National Institute of Statistics dataset. The International Classification of Diseases, Ninth Revision, biliary cirrhosis code 571.6 was used for diagnosis, and data on comorbidities, concomitant medications, medical examinations, specialist referrals, and treatments were collected.
This dataset was representative of the Italian population. Point prevalence of primary biliary cholangitis was calculated as 27.90 per 100,000 and incidence as 5.31 per 100,000 inhabitants/year. Some associations between the disease and comorbidities were sex specific. The most common laboratory assays requested were for liver enzymes, and the majority of patients were not referred to a specialist. Ursodeoxycholic acid was the most common therapy.
This can be used as a benchmark for monitoring and identifying unmet needs to improve treatment in Italy.
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A therapeutically challenging subset of cells, termed cancer stem cells (CSCs) are responsible for cholangiocarcinoma (CCA) clinical severity. Presence of tumor-associated macrophages ...(TAMs) has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche.
CCA cells were cultured in 3D conditions to generate spheres. CCA sphere analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed. The in vitro and in vivo effect of CCA spheres on macrophage precursors was tested after culturing healthy donor cluster of differentiation (CD)14+ with CCA-sphere conditioned medium.
CCA spheres engrafted in 100% of transplanted mice and revealed a significant 20.3-fold increase in tumor-initiating fraction (p=0.0011) and a sustained tumorigenic potential through diverse xenograft-generations. Moreover, CCA spheres were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. Next, fluorescence-activated cell sorting analysis showed that in the presence of CCA sphere conditioned medium, CD14+ macrophages expressed key markers (CD68, CD115, human leukocyte antigen-D related, CD206) indicating that CCA sphere conditioned medium was a strong macrophage-activator. Gene expression profile of CCA sphere activated macrophages revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated macrophages from CCA resections recapitulated a similar molecular phenotype of in vitro-educated macrophages. Consistent with invasive features, the largest CD163+ set was found in the tumor front of human CCA specimens (n=23) and correlated with a high level of serum cancer antigen 19.9 (n=17). Among mediators released by CCA spheres, only interleukin (IL)13, IL34 and osteoactivin were detected and further confirmed in CCA patient sera (n=12). Surprisingly, a significant association of IL13, IL34 and osteoactivin with sphere stem-like genes was provided by a CCA database (n=104). In vitro combination of IL13, IL34, osteoactivin was responsible for macrophage-differentiation and invasion, as well as for in vivo tumor-promoting effect.
CCA-CSCs molded a specific subset of stem-like associated macrophages thus providing a rationale for a synergistic therapeutic strategy for CCA-disease.
Immune plasticity represents an important hallmark of tumor outcome. Since cancer stem cells are able to manipulate stromal cells to their needs, a better definition of the key dysregulated immune subtypes responsible for cooperating in supporting tumor initiation may facilitate the development of new therapeutic approaches. Considering that human cholangiocarcinoma represents a clinical emergency, it is essential to move to predictive models in order to understand the adaptive process of macrophage component (imprinting, polarization and maintenance) engaged by tumor stem-like compartment.
Hepatic progenitor cells (HPCs) play a major role in liver repair and regeneration. We evaluated HPC involvement in pediatric nonalcoholic fatty liver disease (pNAFLD). Thirty biopsies of consecutive ...children and adolescents with untreated NAFLD (19 with nonalcoholic steatohepatitis NASH and 11 without NASH) were studied using immunohistochemistry and immunofluorescence. HPCs and HPC‐expressing adipokines (e.g., adiponectin, resistin, and glucagon‐like peptide 1 GLP‐1) were counted and correlated with steatosis, inflammation, hepatocyte ballooning, fibrosis, and NAFLD activity score (NAS). The HPC compartment was expanded in pNAFLD, especially in children with NASH, and was independently associated with degree of fibrosis (r = 0.303; P = 0.033). NASH livers were also characterized by increased hepatocyte apoptosis, cell‐cycle arrest, and an expanded pool of intermediate hepatocytes. Adiponectin expression in HPCs of pNAFLD patients was down‐regulated with respect to the healthy liver, and this expression was inversely correlated with NAS score (r = −0.792; P < 0.001) and steatosis (r = −0.769; P < 0.001). Resistin expression in HPCs increased in pNAFLD and was related to degree of fibrosis (r = 0.432; P < 0.05). GLP‐1 was overexpressed in HPCs of pNAFLD patients, and GLP‐1 expression was related to degree of steatosis (r = 0.577; P < 0.05) and NAS (r = 0.594; P < 0.01). Conclusions: HPC activation is involved in the response of the liver to oxidative stress in pNAFLD and is correlated with fibrosis and the progression toward NASH. HPCs express adiponectin, resistin, and GLP‐1, which become available to resident liver cells and are strongly associated with the severity of NAFLD. These results may have important pathophysiological implications in the modulation of hepatic insulin resistance and the progression of liver injury. (HEPATOLOGY 2012;56:2142–2153)
Background & Aims Primary sclerosing cholangitis (PSC) is characterised by fibro-stenosing strictures involving extrahepatic and/or large intrahepatic bile ducts. Mechanisms leading to bile duct ...injury are poorly understood. We aimed to study the biliary tree stem cell compartment located in peribiliary glands of extrahepatic and large intrahepatic bile ducts and its role in the pathogenesis of biliary fibrosis in PSC. Methods Specimens containing extrahepatic or large intrahepatic bile ducts were obtained from normal liver (n = 6), liver explants from patients with PSC (n = 11), and primary biliary cirrhosis (n = 6). Specimens were processed for histology, immunohistochemistry and immunofluorescence. Results In PSC samples, progressive hyperplasia and mucinous metaplasia of peribiliary glands were observed in large ducts with fibrosis, but not in inflamed ducts without fibrosis. Peribiliary gland hyperplasia was associated with progressive biliary fibrosis and the occurrence of dysplastic lesions. Hyperplasia of peribiliary glands was determined by the expansion of biliary tree stem cells, which sprouted towards the surface epithelium. In PSC, peribiliary glands and myofibroblasts displayed enhanced expression of Hedgehog pathway components. Peribiliary glands in ducts with onion skin-like fibrosis expressed epithelial-to-mesenchymal transition traits associated with components of Hedgehog pathway, markers of senescence and autophagy. Conclusions The biliary tree stem cell compartment is activated in PSC, its activation contributes to biliary fibrosis, and is sustained by the Hedgehog pathway. Our findings suggest a key role for peribiliary glands in the progression of bile duct lesions in PSC and could explain the associated high risk of cholangiocarcinoma.
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