Initial objectives

·          Define the relationship between graft inflammation, cholangiocyte senescence and induction of epithelial to mesenchymal (EMT) transition in liver tissue.

·          Define the conditions necessary to produce EMT (and mesenchymal to epithelial transition: MET) in primary human cholangiocytes (HBEC)

·          Characterise two human cholangiocyte cell lines (MMNK-1 and H69) and assess their ability to model EMT in monolayer and 3-dimensional (3-D) culture

·          Define the contribution of S100A4 protein in the initiation and progression of EMT in cholangiocytes

·          Initiate the study of differentiation of intragraft T cells to a regulatory phenotype in order to define their function more fully

Progress to date

Following on from our paper in Hepatology¹ in which we observed the progression of EMT in biopsies of post-transplant recurrent primary biliary cirrhosis, in January 2008 we published a paper in Laboratory Investigation² defining the conditions necessary to produce EMT (and MET) in HBEC. In both studies the protein S100A4 was used as a definitive marker of early EMT.

More recently we have characterised both MMNK-1 and H69 human cholangiocyte cell lines in terms of epithelial markers and their ability to undergo EMT under the same conditions required by HBEC (Fig. 1A). Both cell lines have been used successfully to form 3-D structures in matrigel. Addition of relevant growth factors caused disintegration of the 3-D structures and formation of elongated cells, indicating EMT. A protein array revealed that the cholangiocyte cell lines secrete chemokines capable of attracting T cells, in particular, high levels of MCP-1. We have used confocal microscopy to observe invasion of 3-D structures by T cells expressing the αEβ7 integrin, CD103 (Fig. 1B). A recent paper³ demonstrated how chemokines can enhance the potential of CD103⁺ cells to bind E-cadherin on graft epithelium.

Gene silencing technology was used to study the possible mechanism by which S100A4 initiates EMT in cholangiocytes (H69). S100A4 knockdown clones were generated in which S100A4 mRNA downregulation was significant (real-time PCR) and invasive capacity of the cells was reduced by 75-90% when compared with wild type cells and cells transfected with non-targeting control sequences (Fig. 1C). Monolayers of transfected clones retained epithelial markers following stimulation with TGF-β, unlike the wild type and non-targeting controls, which underwent EMT. MMP-9 and fibronectin expression were unaffected by knockdown of S100A4. These results confirm that S100A4 is involved in releasing cholangiocytes from the epithelial layer and the acquisition of cell motility.

Triple labelling of liver transplant tissue sections to detect senescence markers or T cells alongside cytokeratin 19 and S100A4 has revealed cells undergoing EMT within ductular structures associated with senescent cells (Fig. 1E). Graft-invading T cells are often associated with S100A4⁺ cholangiocytes (Fig. 1F). Immunohistochemical studies in 50 liver transplant biopsies are currently ongoing.

We previously reported that allospecific CD103⁺ T cells co-express the FOXP3 transcription factor, suggesting that some of the T cells observed within bile ducts might have a regulatory phenotype. Preliminary immunohistochemistry in liver transplant sections reinforces this suggestion (Fig. 1G).

Figure 1. A) Monolayer cultures of the H69 human cholangiocyte cell line unstimulated (control) and stimulated with TGF-β1, EGF, TGF-β1/EGF and Alk5 inhibitor pre-treatment with subsequent stimulation as indicated above each column. Epithelial and mesenchymal markers (indicated to the left of each row) were subsequently detected by immunocytochemistry and visualised with confocal microscopy. Results show morphological changes and loss of E-cadherin, ZO-1 and cytokeratin-7 in stimulated cells and gain of the mesenchymal marker, vimentin, in stimulated cells. These results are particularly pronounced in cells stimulated with TGF-β1/EGF. Pre-treatment with Alk5 inhibitor prevents the changes. B) 3-D branching structures of H69 incubated with MOLT16 T cells; E-cadherin (FITC, green) and T cells (red), illustrates the attraction between cholangiocytes and T cells. The inset shows an xz section through a ‘branch’ and confirms that the T cells invade the epithelial structure. C) The graph is representative of the results of an in vitro invasion assay carried out using S100A4 shRNA transfectants (selected ‘knockdown’ clones cl10-2,24,34,35), wild type H69 cells (WT) and non-targeting control cells (c1). Each bar represents the total number of cells transmigrating through matrigel-coated filters (8 µm pore size) during a 24 hour incubation period. Triple immunohistochemical labelling of post-transplant liver biopsy sections: E) Cyan arrows indicate p21 positive epithelial nuclei lying adjacent to an S100A4 positive epithelial cell (black arrow). Both cells are within a ductular structure (40X magnification). F) A focus of CD8⁺ T cells surrounding and invading a bile duct. The white arrow indicates an S100A4 positive epithelial cell lying adjacent to CD8⁺ T cells (grey arrow: 20X magnification). G) A FOXP3⁺ intraepithelial T cell (grey arrow) lies adjacent to an S100A4 positive bile duct epithelial cell. FOXP3⁺ cells are also present in the surrounding accumulation of immune cells.