Gene expression profiling in biliary epithelial cells of primary biliary cirrhosis using laser capture microdissection and cDNA microarray

doi:10.1016/j.trsl.2006.04.007

Translational Research

Volume 148, Issue 3, September 2006, Pages 103-113

https://doi.org/10.1016/j.trsl.2006.04.007 Get rights and content

Primary biliary cirrhosis (PBC) is a chronic, cholestatic liver disease characterized by progressive destruction of interlobular bile ducts that leads to biliary cirrhosis. To elucidate the etiology of PBC, the gene expression profile in biliary epithelial cells (BECs) was analyzed. Liver specimens of 5 PBC, 3 chronic hepatitis C (CHC), and 3 normal subjects were obtained. BECs were selectively collected by laser capture microdissection (LCM), RNA were obtained by extraction and amplification with T7 RNA polymerase, and a cDNA microarray analysis was performed. The following genes exhibited increased expression in BEC of PBC, as compared with CHC or normal subjects: human leukocyte antigen DQ alpha 1 (HLA-DQA-1), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and vascular cell adhesion molecule 1 (VCAM-1). The immunohistochemistry for HLA-DQA-1, CEACAM1, TRAIL, and VCAM-1 confirmed these results. Furthermore, two-way cluster analysis showed that the gene expression profiling in BEC of PBC were categorized into a separate cluster, distinct from CHC or normal subjects. Conclusions: The gene expression profiling in BEC of PBC differed from those of CHC and normal subjects, and the genes concerning local immune response, such as HLA-DQA-1, CEACAM1, TRAIL, and VCAM-1, exhibited increased expression, indicating that they were involved in the development of bile duct injury.

Section snippets

Subjects

Liver specimens were obtained from 5 PBC patients (the PBC group) and 3 CHC patients (the CHC group) by laparoscopic liver biopsy. Liver specimens were also obtained from 3 normal subjects (the normal group), who were living donors of liver transplantation, by intraoperative needle biopsy. All subjects were admitted to Okayama University Hospital of Medicine and Dentistry, and liver biopsy was performed for the diagnosis of liver diseases. The study was conducted in accordance with the

Clinical, serological, and histopathological profile of the subjects

Clinical, serological, and histopathological data of the 5 PBC patients, the 3 CHC patients, and the 3 normal subjects are shown in Table I. Histological diagnoses of the PBC patients were stage 1/2/3 in 1/2/2 patients, respectively, according to Scheuer’s and Ludwig’s classifications.18, 19 Serum liver enzymes, that is, AST, ALT, ALP, and γ-GTP, were elevated in all PBC patients, and T-Bil was elevated in the 2 PBC patients. AMA and AMA-M2 were positive in all PBC patients. ANAs were positive

Discussion

To elucidate the etiology of PBC, it seems necessary to analyze the alterations of gene expression in BEC, the target cells that are mainly disordered in PBC. It was hypothesized that combining recently developed methods—LCM, T7-based RNA amplification, and cDNA microarray—would make it possible to analyze the gene expression in the selectively isolated BEC.

The first method, LCM, enabled identification and selective collection of a particular cell under a microscope. In the authors’ experiment,

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Cholangiocyte death in ductopenic cholestatic cholangiopathies: Mechanistic basis and emerging therapeutic strategies
2019, Life Sciences
Citation Excerpt :
Regarding the TRAILR/TRAIL system, human cholangiocytes constitutively express TRAIL receptor type 2 (TRAILR2). In ductopenic cholangiopathies such as PSC and PBC, expression of this receptor is increased [28,119,122], an effect likely induced by the bile salts accumulated during the cholestatic process [123], via activation of the nuclear factor Sp1 by the c-Jun N-terminal kinase (JNK)-dependent pathway [124]. Serum levels of soluble TRAIL are also elevated in patients with PBC [119,125].
Among hepatic diseases, cholestatic ductopenic cholangiopathies are poorly studied, and they are rarely given the importance they deserve, especially considering their high incidence in clinical practice. Although cholestatic ductopenic cholangiopathies have different etiologies and pathogenesis, all have the same target (the cholangiocyte) and similar mechanistic basis of cell death. Cholestatic cholangiopathies are characterized, predominantly, by obstructive or functional damage in the biliary epithelium, resulting in an imbalance between proliferation and cholangiocellular death; this leads to the progressive disappearance of bile ducts, as has been shown to occur in primary sclerosing cholangitis, primary biliary cholangitis, low-phospholipid-associated cholelithiasis syndrome, cystic fibrosis-related liver disease, and drug-induced ductopenia, among other biliary disorders. This review summarizes the features of the more common ductopenic syndromes and the cellular mechanisms involved in cholengiocellular death, with focus on the main forms of cholangiocyte death described so far, namely apoptosis, autophagy, necrosis, and necroptosis. It also emphasizes the importance to study in depth the molecular mechanisms of cholengiocyte death to make possible to counteract them with therapeutic purposes. These therapeutic strategies are limited in number and efficacy at present, and this is why it is important to find complementary, safe strategies to stimulate cholangiocellular proliferation in order favor bile duct replenishment as well. Successful in finding appropriate treatments would prevent the patient from having liver transplantation as the only therapeutic alternative.
Increased sensitivity of Treg cells from patients with PBC to low dose IL-12 drives their differentiation into IFN-γ secreting cells
2018, Journal of Autoimmunity
Citation Excerpt :
Transfer of CD4 but not CD8 T cells from ARE-Del−/− mice to B6/Rag1−/− mice is able to induce moderate portal inflammation and mild parenchymal inflammation, further implying an important role of CD4 T cells in the pathological progression of the disease. In human PBC, gene expression analysis on liver biopsy tissues has also identified IFNγ signalling being significantly detectable in both early and late stages of disease [28–30] and recent GWAS studies have revealed several single nucleotide variants enriched in CD4 T cell signalling, such as the IL12-JAK-STAT4 signalling pathway [31]. Loss of type I IFN receptor (IFN-a/β) receptor signalling using double knock out ARE-Del−/−Ifnr1−/− mice has been shown to reduce liver pathology, abrogate sex bias and correct germinal centre (sites implicated in loss of tolerance) abnormalities [32].
IL-12 is a pro-inflammatory cytokine that induces the production of interferon-γ (IFNγ) and favours the differentiation of T helper 1 (Th1) cells. In the presence of IL-12 human Treg cells acquire a Th1-like phenotype with reduced suppressive activity in vitro. Primary biliary cholangitis (PBC) is an autoimmune cholestatic liver disease characterised by high Th1 and Th17 infiltrating cells, reduced frequencies of Treg cells, and a genetic association with IL-12 signalling. Herein, we sought to evaluate the IL-12 signalling pathway in PBC pathology, by studying human samples from patients with PBC, alongside those with primary Sjögren’s syndrome (pSS)(autoimmune disease with IL-12 signalling gene association), primary sclerosing cholangitis (PSC) (cholestatic liver disease without IL-12 gene association) and healthy individuals. Our data revealed that TLR stimulation of PBC (n = 17) and pSS monocytes (n = 6) resulted in significant induction of IL12A mRNA (p < 0.05, p < 0.01, respectively) compared to PSC monocytes (n = 13) and at similar levels to HC monocytes (n = 8). PSC monocytes expressed significantly less IL-12p70 (108 pg/ml, mean) and IL-23 (358 pg/ml) compared to HC (458 pg/ml and 951 pg/ml, respectively) (p < 0.01, p < 0.05). Treg cells from patients with PBC (n = 16) and pSS (n = 3) but not PSC (n = 10) and HC (n = 8) responded to low dose (10 ng/ml) IL-12 stimulation by significant upregulation of IFNγ (mean 277 and 254 pg/ml, respectively) compared to PSC and HC Treg cells (mean 22 and 77 pg/ml, respectively)(p < 0.05). This effect was mediated by the rapid and strong phosphorylation of STAT4 on Treg cells from patients with PBC and pSS (p < 0.05) but not PSC and HC. In the liver of patients with PBC (n = 7) a significantly higher proportion of IL-12Rβ2⁺Tregs (16% on average) was detected (p < 0.05) compared to other liver disease controls (5%)(n = 18) which also showed ex vivo high IFNG and TBET expression. CONCLUSION: Our data show an increased sensitivity of PBC and pSS Treg cells to low dose IL-12 stimulation, providing ongoing support for the importance of the IL12-IL-12Rβ2-STAT4 pathway on Treg cells in disease pathogenesis and potentially treatment.
miR-425 regulates inflammatory cytokine production in CD4<sup>+</sup> T cells via N-Ras upregulation in primary biliary cholangitis
2017, Journal of Hepatology
Citation Excerpt :
In PBC, differentially expressed miRNAs have been identified in liver tissue, biliary epithelium, serum, and peripheral blood mononuclear cells (PBMCs) [19–22]. In addition, mRNA profiling studies in PBC have also been reported [7,23,24]. However, no studies have yet analysed miRNA or mRNA expression profiles in CD4+ T cells isolated from PBC patients.
Primary biliary cholangitis (PBC) is an autoimmune liver disease of unknown pathogenesis. Consequently, therapeutic targets for PBC have yet to be identified. CD4⁺ T cells play a pivotal role in immunological dysfunction observed in PBC, and therefore, microRNA (miRNA) and mRNA expression were analysed in CD4⁺ T cells, to investigate PBC pathogenesis and identify novel therapeutic targets.
Integral miRNA and mRNA analysis of 14 PBC patients and ten healthy controls was carried out using microarray and quantitative real-time polymerase chain reaction (qRT-PCR), with gene set enrichment analysis. The functional analyses of miRNA were then assessed using reporter and miRNA-overexpression assays.
The integral analysis of miRNA and mRNA identified four significantly downregulated miRNAs (miR-181a, -181b, -374b, and -425) related to the T cell receptor (TCR) signalling pathway in CD4⁺ T cells of PBC. N-Ras, a regulator of the TCR signalling pathway, was found to be targeted by all four identified miRNAs. In addition, in vitro assays confirmed that decreased miR-425 strongly induced inflammatory cytokines (interleukin [IL]-2 and interferon [IFN]-γ) via N-Ras upregulation in the TCR signalling pathway.
The decreased expression of four miRNAs that dysregulate TCR signalling in PBC CD4⁺ T cells was identified. miR-425 was demonstrated as an inflammatory regulator of PBC via N-Ras upregulation. Therefore, the restoration of decreased miR-425 or the suppression of N-Ras may be a promising immunotherapeutic strategy against PBC.
Primary biliary cholangitis (PBC) is an autoimmune liver disease, but the causes are unknown. MicroRNAs are molecules known to regulate biological signals. In this study, four microRNAs were identified as being decreased in PBC patients, leading to activation of T cell receptor signalling pathways, involved in inflammation. One particular target, N-Ras, could be an attractive and novel immunotherapeutic option for PBC.
Microarray data are deposited in GEO (GEO accession: GSE93172).
The immunogenetics of primary biliary cirrhosis: A comprehensive review
2015, Journal of Autoimmunity
Citation Excerpt :
Importantly, CD4+ [11] and CD8+ [12] T cells specific to mitochondrial auto-antigens have been demonstrated in the peripheral blood, livers and liver-draining lymph nodes of affected patients, while not detected in either healthy controls or patients with other liver diseases. Both MHC class I and II proteins are also expressed on BECs of PBC patients and thought to present antigen to cytotoxic CD8+ and helper CD4+ T cells, respectively [13–15]. The cytokine signature associated with PBC is also indicative of immune system activation with a Th1/Th17 bias.
Primary biliary cirrhosis (PBC), a classic autoimmune liver disease, is characterised by a progressive T cell predominant lymphocytic cholangitis, and a serologic pattern of reactivity in the form of specific anti-mitochondrial antibodies (AMA). CD4+ T cells are particularly implicated by PBC's cytokine signature, the presence of CD4+ T cells specific to mitochondrial auto-antigens, the expression of MHC II on injured biliary epithelial cells, and PBC's coincidence with other similar T cell mediated autoimmune conditions. CD4+ T cells are also central to current animal models of PBC, and their transfer typically also transfers disease. The importance of genetic risk to developing PBC is evidenced by a much higher concordance rate in monozygotic than dizygotic twins, increased AMA rates in asymptomatic relatives, and disproportionate rates of disease in siblings of PBC patients, PBC family members and certain genetically defined populations. Recently, high-throughput genetic studies have greatly expanded our understanding of the gene variants underpinning risk for PBC development, so linking genetics and immunology. Here we summarize genetic association data that has emerged from large scale genome-wide association studies and discuss the evidence for the potential functional significance of the individual genes and pathways identified; we particularly highlight associations in the IL-12-STAT4-Th1 pathway. HLA associations and epigenetic effects are specifically considered and individual variants are linked to clinical phenotypes where data exist. We also consider why there is a gap between calculated genetic risk and clinical data: so-called missing heritability, and how immunogenetic observations are being translated to novel therapies. Ultimately whilst genetic risk factors will only account for a proportion of disease risk, ongoing efforts to refine associations and understand biologic links to disease pathways are hoped to drive more rational therapy for patients.
Anatomy and Physiology of the Biliary Epithelium
2010, Comprehensive Toxicology, Second Edition
The biliary tree of the liver is a system of ducts that transport bile out of the liver to the duodenum. The ducts are lined by epithelial cells (cholangiocytes) which actively modify bile by secreting or absorbing solutes and water using transporters, exchangers, channels, and receptors, and by processes such as endocytosis, exocytosis, and pinocytosis. Cholangiocyte function is regulated by hormones, neurotransmitters, growth factors, bile acids, osmosensors, and mechanoreceptors. In man, bile is stored and concentrated in the gallbladder for secretion during digestion. In numerous vertebrates, however, a gallbladder is lacking. Thus, storage/concentration of biliary constituents is not a requirement for normal biliary function. Cholangiocyte physiology is studied in vivo using bile-duct cannulated animals and in vitro using freshly isolated cholangiocytes, cholangiocyte monolayers, cell lines, and isolated bile duct units. Cholangiocytes express phase I and II enzymes plus cholangiocyte-specific proteins. Additionally, receptors and transport proteins are differentially expressed by cholangiocytes from large versus small ducts.
Liver architecture and volume is restored following moderate injury or resection via a cascade of cytokines and growth factors that induces proliferation of all liver cell types. In contrast, a stem cell compartment (oval or progenitor cells) begins to proliferate following massive or chronic liver injury, which induces a variable ‘ductular reaction’ correlated with the degree of inflammation and fibrosis. Diseases of the biliary tree (cholangiopathies) are collectively termed ‘vanishing bile duct syndromes,’ due to progressive destruction of intra- and extrahepatic bile ducts. Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are the most prevalent immune-mediated cholangiopathies. Proposed etiology of PBC is formation of ‘neo-antigens’ (chemically modified lipoyl domains of mitochondrial matrix proteins) that elicit immunogenic autoepitopes via chronic low-level protein turnover (i.e., breakdown of tolerance). New knockout mouse models suggest that PSC is caused by elevations of cytotoxic bile acids in ductular bile and/or leaky tight junctions (TJ) leading to cholangiocyte injury, portal tract inflammation, and peribiliary fibrosis. Proposed etiologies for drug-induced cholangiocyte injury are direct cytotoxicity by drugs or their reactive metabolites, or initiation of an immune-mediated (drug hypersensitivity) response that targets cholangiocytes. Genetic and environmental risk factors identified for PBC and PSC include certain human leukocyte antigen (HLA) alleles, urban living, and proximity to superfund toxic waste sites (i.e., environmental toxicants); risk factors for drug-induced cholangiopathies include HLA alleles, gender, age, dose, number of courses of therapy, and concomitant viral infection. Although knowledge of cholangiocyte physiology and function has been considerably improved in the last 10 years, critical insight into the mechanisms by which cholangiopathies occur continues to elude investigators.
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Original articleGene expression profiling in biliary epithelial cells of primary biliary cirrhosis using laser capture microdissection and cDNA microarray

Section snippets

Subjects

Clinical, serological, and histopathological profile of the subjects

Discussion

Primary biliary cirrhosis

Primary biliary cirrhosisan orchestrated immune response against epithelial cells

Immunol Rev

Aberrant expression of HLA-DR antigens on bileduct epithelium in primary biliary cirrhosisrelevance to pathogenesis

Lancet

Increased expression of intercellular adhesion molecule 1 on bile ducts in primary biliary cirrhosis and primary sclerosing cholangitis

Hepatology

Bile duct epithelia as target cells in primary biliary cirrhosis and primary sclerosing cholangitis

Virchows Arch

Expression of co-stimulatory factor B7-2 on the intrahepatic bile ducts in primary biliary cirrhosis and primary sclerosing cholangitisan immunohistochemical study

J Pathol

Immunohistochemical analysis of adhesion molecules in the micro-environment of portal tracts in relation to aberrant expression of PDC-E 2 and HLA-DR on the bile ducts in primary biliary cirrhosis

J Pathol

Immunohistochemical analysis of cell-matrix adhesion molecules and their ligands in the portal tracts of primary biliary cirrhosis

J Pathol

Identification of novel molecules and pathogenic pathways in primary biliary cirrhosiscDNA array analysis of intrahepatic differential gene expression

Gut

Genomic analysis of differentially expressed genes in liver and biliary epithelial cells of patients with primary biliary cirrhosis

J Autoimmun

Quantitative monitoring of gene expression patterns with a complementary DNA microarray

Science

Gene expressionprofiles of laser-captured adjacent neuronal subtypes

Nat Med

Laser capture microdissection-generated target sample for high density oligonucleotide array hybridization

BioTechniques

Laser capture micro-dissection

Science (Wash. DC)

Laser capture dissectionmolecular analysis of tissue

Science (Wash. DC)

Amplified RNA synthesized from limited quantities of heterogeneous cDNA

Proc Natl Acad Sci USA

Analysis of gene expression in single live neurons

Proc Natl Acad Sci USA

Original article
Gene expression profiling in biliary epithelial cells of primary biliary cirrhosis using laser capture microdissection and cDNA microarray