Background and Aims
Immune-mediated bile duct injury is the primary histological feature of autoimmune cholestatic liver diseases. Macrophages, the most abundant immun... More
Background and Aims
Immune-mediated bile duct injury is the primary histological feature of autoimmune cholestatic liver diseases. Macrophages, the most abundant immune cell population in the liver, have been postulated to play a critical role in biliary repair. However, it is unclear whether activated macrophages interact with injured biliary epithelial cells.
Methods
We evaluated the expression of insulin-like growth factor-binding protein 4 (IGFBP4) in primary monocytes, MDMΦ, serum, and liver tissue sections from a total of 292 samples from PBC, PSC, and healthy controls using RNA-sequencing, ELISA, and immunohistochemistry analysis. The signal pathways involved in the effect of IGFBP4 in human intrahepatic biliary epithelial cells were examined by phospho-kinase arrays.
Results
Herein we demonstrate a role for insulin-like growth factor binding protein 4 (IGFBP4) in the interaction of macrophages and biliary cells. Importantly, the serum levels of IGFBP4 are significantly increased in PBC and negatively correlate with bilirubin levels. Furthermore, immunohistochemistry revealed an increase in IGFBP4 positive cells located not only in the periductal area but also around the portal tract in the PBC liver. In vitro study indicated that IGFBP4 protected biliary cells from bile salt-induced cell injury and promoted biliary cell proliferation, which was associated with reduced expression of the bile acid receptor TGR5, bile acid efflux transporter SLCO3A1, and activation of the GSK-3β/β-catenin signalling pathway.
Conclusions
These data highlight that IGFBP4 not only serves as a potential biomarker for PBC but also plays a protective role against bile duct injury.
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Shotgun proteomics is a powerful analytic method to characterize complex protein mixtures in combination with multi-dimensional liquid chromatography-tandem mass spectrom... More
Shotgun proteomics is a powerful analytic method to characterize complex protein mixtures in combination with multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). We have used this platform for proteomic characterization of apoptotic bodies in efforts to define the complex protein mixtures found in primary cultures of human intrahepatic biliary epithelial cells (HiBEC), human renal proximal tubular epithelial cells, human bronchial epithelial cells, isolated intrahepatic biliary epithelial cells from explanted primary biliary cirrhosis (PBC) and control liver, using a total of 24 individual samples. Further, as additional controls and for purposes of comparison, proteomic signatures were also obtained from intact cells and apoptotic bodies. The data obtained from LC-MS/MS, combined with database searches and protein assembly algorithms, allowed us to address significant differences in protein spectral counts and identify unique pathways that may be a component to the induction of the signature inflammatory cytokine response against BECs, including the Notch signaling pathway, IL8, IL6, CXCR2 and integrin signaling. Indeed there are 11 proteins that localize specifically to apoptotic bodies of HiBEC and 8 proteins that were specifically absent in HiBEC apoptotic bodies. In conclusion, proteomic analysis of BECs from PBC liver compared to normal liver are significantly different, suggesting that an immunological attack affects the repertoire of proteins expressed and that such cells should be thought of as living in an environment undergoing continuous selection secondary to an innate and adaptive immune response, reflecting an almost “Darwinian” bias. Less
Background & Aims: Proliferating cholangiocytes secrete and respond to neuroendocrine hormones including secretin. We investigated whether secretin secreted by S cells an... More
Background & Aims: Proliferating cholangiocytes secrete and respond to neuroendocrine hormones including secretin. We investigated whether secretin secreted by S cells and cholangiocytes stimulates biliary proliferation in mice. Methods: Cholestasis was induced in secretin knockout (Sct−/−) and wild-type (control) mice by bile-duct ligation (BDL). At days 3 and 7 after BDL, control and Sct−/− mice received tail-vein injections of morpholinos against microRNA 125b or let7a. One week later, liver tissues and cholangiocytes were collected; immunohistochemical, immnoblot, luciferase reporter and real-time PCR assays were performed. Intrahepatic bile duct mass (IBDM) and proliferation were measured. Secretin secretion was measured in conditioned media from cholangiocytes and S cells, and in serum and bile. Results: Secretin secretion was increased in supernatants from cholangiocytes and S cells and in serum and bile following BDL in control mice. BDL Sct−/− mice had lower IBDM, reduced proliferation, and reduced production of vascular endothelial growth factor A (VEGFA) and nerve growth factor (NGF) compared with BDL control. BDL and control mice given morpholinos against microRNA 125b or let7a had increased IBDM. Livers of mice given morpholinos against microRNA 125b had increased expression of VEGFA while those treated with morpholinos against microRNA let7a had increased expression of NGF. Secretin regulated VEGF and NGF expression that negatively correlated with microRNA 125b and let7a levels in liver tissue. Conclusions: Following liver injury, secretin produced by cholangiocytes and S cells reduces microRNA 125b and let7a levels, resulting in upregulation of VEGF and NGF. Modulation of cholangiocyte expression of secretin could be a therapeutic approach for biliary diseases. Keywords: Biliary epithelium, cAMP, gastrointestinal hormones, heterogeneity Less
The phagocytic clearance of apoptotic cells is critical for tissue homeostasis; a number of non-professional phagocytic cells, including epithelial cells, can both take u... More
The phagocytic clearance of apoptotic cells is critical for tissue homeostasis; a number of non-professional phagocytic cells, including epithelial cells, can both take up and process apoptotic bodies, including the release of anti-inflammatory mediators. These observations are particularly important in the case of human intrahepatic biliary cells (HiBEC), because such cells are themselves a target of destruction in primary biliary cirrhosis, the human autoimmune disease. To address the apoptotic ability of HiBECs, we have focused on their ability to phagocytize apoptotic blebs from autologous HiBECs. In this study we report that HiBEC cells demonstrate phagocytic function from autologous HiBEC peers accompanied by up-regulation of the chemokines CCL2 [monocyte chemotactic protein-1 (MCP-1)] and CXCL8 [interleukin (IL)-8]. In particular, HiBEC cells express the phagocytosis-related receptor phosphatidylserine receptors (PSR), implying that HiBECs function through the ‘eat-me’ signal phosphatidylserine expressed by apoptotic cells. Indeed, although HiBEC cells acquire antigen-presenting cell (APC) function, they do not change the expression of classic APC function surface markers after engulfment of blebs, both with and without the presence of Toll-like receptor (TLR) stimulation. These results are important not only for understanding of the normal physiological function of HiBECs, but also explain the inflammatory potential and reduced clearance of HiBEC cells following the inflammatory cascade in primary biliary cirrhosis. Keywords: apoptosis, chemokine, intrahepatic biliary epithelial cells, phagocytosis Less
Background. Mucin 5AC (MUC5AC) overproduction plays important roles in stone formation and recurrence of hepatolithiasis. We aim to investigate the involved mechanism and... More
Background. Mucin 5AC (MUC5AC) overproduction plays important roles in stone formation and recurrence of hepatolithiasis. We aim to investigate the involved mechanism and the potential target to block this process. Methods. 42 bile duct samples from hepatolithiasis and 15 normal bile duct samples from hemangioma patients were collected for detecting MUC5AC expression by immunohistochemistry. MUC5AC and phosphoepidermal growth factor receptor (pEGFR) expressions in human intrahepatic biliary epithelial cells (HIBECs) cultured with or without lipopolysaccharide (LPS) were detected by real-time PCR and western blot analysis. Transforming growth factor-α (TGF-α) secretion in HIBECs was detected by ELISA. Results. MUC5AC was overexpressed in bile ducts of hepatolithiasis samples compared with bile ducts from hemangioma samples. LPS upregulated MUC5AC expression in HIBECs. LPS promoted EGFR activation, and inhibiting EGFR activation by AG1478 significantly decreased LPS-induced MUC5AC overexpression in HIBECs. Moreover, LPS increased TGF-α secretion, and inhibiting tumor necrosis factor-α converting enzyme (TACE), which has been implicated in ectodomain cleavage of TGF-α, significantly inhibited LPS-induced EGFR activation and subsequent MUC5AC overexpression in HIBECs. Conclusion. Our results suggested that LPS increases MUC5AC expression through the TACE/TGF-α/EGFR pathway in HIBECs. This new finding might give light to the prevention of stone formation and recurrence of hepatolithiasis. Less
MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma ... More
MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC. Less
Epigenetic changes are associated with the regulation of transcription of key cell regulatory genes [micro RNAs (miRNAs)] during different types of liver injury. This stu... More
Epigenetic changes are associated with the regulation of transcription of key cell regulatory genes [micro RNAs (miRNAs)] during different types of liver injury. This study evaluated the role of methylation-associated miRNA, miR-34a, in alcoholic liver diseases. We identified that ethanol feeding for 4 weeks significantly up-regulated 0.8% of known miRNA compared with controls, including miR-34a. Treatment of normal human hepatocytes (N-Heps) and cholangiocytes [human intrahepatic biliary epithelial cells (HiBECs)] with ethanol and lipopolysaccharide induced a significant increase of miR-34a expression. Overexpression of miR-34a decreased ethanol-induced apoptosis in both N-Heps and HiBECs. In support of the concept that the 5′-promoter region of miR-34a was noted to be embedded within a CpG island, the expression level of miR-34a was significantly increased after demethylation treatment in N-Heps and HiBECs. By methylation-specific PCR, we confirmed that miR-34a activation is associated with ethanol-linked hypomethylation of the miR-34a promoter. A combination of bioinformatics, dual-luciferase reporter assay, mass spectrometry, and Western blot analysis revealed that caspase-2 and sirtuin 1 are the direct targets of miR-34a. Furthermore, modulation of miR-34a also altered expression of matrix metalloproteases 1 and 2, the mediators involved in hepatic remodeling during alcoholic liver fibrosis. These findings provide the basis for an exciting field in which the epigenomic microRNAs of hepatic cells may be manipulated with potential therapeutic benefits in human alcoholic liver diseases. Less
Functional pluripotent characteristics have been observed in specific subpopulations of hepatic cells that express some of the known cholangiocyte markers. Although evide... More
Functional pluripotent characteristics have been observed in specific subpopulations of hepatic cells that express some of the known cholangiocyte markers. Although evidence indicates that specific cytokines, granulocyte-macrophage colony stimulating factors (GM-CSF) and stem cell factor (SCF) may be candidate treatments for liver injury, the role of these cytokines in intrahepatic biliary epithelium remodeling is unknown. Thus, our aim was to characterize the specific cytokines that regulate the remodeling potentials of cholangiocytes after 70% partial hepatectomy (PH). The expression of the cytokines and their downstream signaling molecules was studied in rats after 70% PH by immunoblots, and in small and large murine cholangiocyte cultures (SMCCs and LMCCs) by immunocytochemistry and real-time PCR. There was a significant and stable increase in SCF and GM-CSF levels until 7 days after PH. Real-time PCR analysis revealed significant increases of key remodeling molecules, such as S100A4 and miR-181b after SCF plus GM-CSF administration in SMCCs. SMCCs produced significant amounts of soluble and bound SCF and GM-CSF in response to TGF-β. When SMCCs were incubated with TGF-β plus anti–SCF and GM-CSF antibodies, there was a significant decrease in S100A4 expression. Furthermore, treatment of SMCCs with SCF + GM-CSF significantly increased matrix metalloproteinases (MMP-2 and MMP-9) mRNA as well as miR-181b expression along with a reduction of metalloproteinase inhibitor 3 (TIMP-3). The levels of MMP-2, MMP-9 and miR-181b were also up-regulated in rat liver and isolated cholangiocytes after PH. CONCLUSION Our data suggest that altered expression of SCF and GM-CSF following PH can contribute to biliary remodeling (for example post-transplantation) by functional deregulation of activity of key signaling intermediates involved in cell expansion and multipotent differentiation. Less
A major enigma of primary biliary cirrhosis (PBC) is the selective targeting of biliary cells. Our laboratory has reported that following apoptosis human intrahepatic bil... More
A major enigma of primary biliary cirrhosis (PBC) is the selective targeting of biliary cells. Our laboratory has reported that following apoptosis human intrahepatic biliary epithelial cells (HiBEC) translocate the E2 subunit of the pyruvate dehydrogenase complex immunologically intact into apoptotic bodies, forming an apotope. However, the cell type and specificity of this reaction has not been fully defined. To address this issue we have investigated whether PDC-E2, BCOADC-E2, OGDC-E2, four additional inner mitochondrial enzymes and four nuclear antigens remain immunologically intact with respect to post-apoptotic translocation in HiBEC and 3 additional control epithelial cells. We report that all three 2-oxo acid dehydrogenase enzymes share the ability to remain intact within the apotope of HiBEC. Interestingly the E2 subunit of the branched chain 2-oxo acid dehydrogenase complex also remained intact in the other cell types tested. We extended the data using 95 AMA+ and 19 AMA- PBC and 76 control sera for reactivity against the 7 mitochondrial proteins studied herein and also the ability of AMA- sera to react with HIBEC apotopes. Sera from 3/95 AMA+ sera, but none of the controls, reacted with 2, 4-dienoyl CoA reductase 1 (DECR1), an enzyme also present intact only in the HiBEC apotope; DECR1 has not been previously associated with any autoimmune disease. Finally the specificity of HIBEC apotope reactivity was confined to AMA+ sera. In conclusion, we submit that the biliary specificity of PBC is secondary to the unique processes of biliary apoptosis. Less
Primary biliary cirrhosis (PBC) is characterized by antimitochondrial antibodies (AMA), directed to the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Notwi... More
Primary biliary cirrhosis (PBC) is characterized by antimitochondrial antibodies (AMA), directed to the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Notwithstanding the presence of mitochondria in virtually all nucleated cells, the destruction in PBC is limited to small intrahepatic bile ducts. The reasons for this tissue specificity remain unknown, although biliary epithelial cells (BEC) uniquely preserve the PDC-E2 epitope following apoptosis. Notably, PBC recurs in an allogeneic transplanted liver, suggesting generic rather than host-PBC-specific susceptibility of BEC. We used cultured human intrahepatic BEC (HIBEC) and other well-characterized cell lines, including, HeLa, CaCo-2 cells, and non transformed human keratinocytes and bronchial epithelial cells (BrEpC), to determine the integrity and specific localization of PDC-E2 during induced apoptosis. All cell lines, both before and after apoptosis, were tested with sera from patients with PBC (n=30), other autoimmune liver and rheumatic diseases (n=20), and healthy individuals (n=20), a mouse monoclonal antibody against PDC-E2, and AMA with an IgA isotype. PDC-E2 was found to localize unmodified within apoptotic blebs of HIBEC, but not within blebs of various other cell lineages studied. The fact that AMA- containing sera reacted with PDC-E2 on apoptotic BEC without a requirement for permeabilization suggests that the autoantigen is accessible to the immune system during apoptosis. In conclusion, our data indicate that the tissue (cholangiocyte) specificity of the autoimmune injury in PBC is a consequence of the unique characteristics of HIBEC during apoptosis and can be explained by exposure to the immune system of intact immunoreactive PDC-E2 within apoptotic blebs. Less
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