What the duct: Imaging ductular reaction spanning the fibrotic areas in primary sclerosing cholangitis (PSC)

1. Introduction

Primary sclerosing cholangitis (PSC) is a heterogenous and idiopathic type of chronic liver disease that targets the epithelial cells (i.e., cholangiocytes) lining the bile ducts in the liver [1,2]. The pathogenesis of PSC is undetermined, yet genetic and environmental factors are widely regarded as contributors to both the disease progression and outcome [1,3]. Additionally, 70–80% of PSC patients are also diagnosed with inflammatory bowel disease, indicating a role for gut-derived factors and microbiome dysregulation in PSC etiopathogenesis [1]. There are limited treatment options available for PSC patients, and approximately 40% of PSC patients require liver transplant, as it is one of the few viable options [1]. Cholangiocytes vary in size and function based on location in either small ducts or large ducts, and PSC is known to affect both small and large bile ducts and results in progressive biliary inflammation [1]. Following injury, small and large cholangiocytes proliferate and begin to synthesize and secrete a plethora of factors that influence the surrounding environment through paracrine interactions [4]. While biliary involvement in fibroinflammatory response during PSC is known, it is unclear of the full involvement of cholangiocytes in this process. Considering the unknown etiopathogenesis and lack of curative options, it is imperative that we understand the underlying biological processes driving PSC progression to find new targetable pathways.

One key hallmark of PSC is the presence of ductular reaction (DR), a phenomenon defined as the proliferation of reactive bile ducts, following liver injury, that are demarcated as misshapen, tortuous and lacking a lumen [3,5]. DR leads to the expansion of the biliary compartment composed of both cholangiocytes and liver progenitor cells, which is postulated to aid in wound-healing responses and parenchymal regeneration [6]. Conversely, the extent of DR is often directly related to degree of liver damage and patient’s health outcomes [5]. Moreover, DR is associated with liver fibrosis, another characteristic of PSC, due to the ability of DR cells to directly activate hepatic stellate cells (main fibrogenic cell of the liver) and induce fibrogenesis [5]. Therefore, it is unclear if DR is a protective or damaging mechanism during PSC progression. Periductular accumulation of collagen in PSC patients leads to narrowing segments of bile ducts called biliary strictures, which cause bile retention and further inflammation through increased toxic bile acid-induced damage [1,3]. Peribiliary fibrosis can evolve into bridging fibrosis, liver cirrhosis and end-stage liver disease, as well as put patients at higher risk for developing cholangiocarcinoma, which is a rare liver cancer arising from the biliary compartment [7].

Given that DR and biliary fibrosis play key roles in PSC progression, it is of great interest to study the interplay between proliferating cholangiocytes and fibrogenesis; thus, we developed a co-staining protocol to visually observe this interaction. The current cover of BBA Molecular Basis of Disease shows the expansion of DR within the fibrotic septa in a PSC patient sample. Immunohistochemical staining marked CK-19-positive cholangiocytes that were stained brown to highlight the bile ducts. Following chromogen development, Fast Green Sirius Red (FGSR) was utilized to counterstain the tissue green, in lieu of Hematoxylin, and also stain collagen deposits red. Therefore, the image showcases extensive DR located specifically within regions of bridging fibrosis, which may be an important indicator of disease progression.

Integrating FGSR counterstaining into the immunohistochemistry protocol allows us to study how different cell types interact with fibrotic tissues. In our case, we demonstrate that extensive DR happens in areas with elevated levels of liver fibrogenesis; this is of interest as it allows us to further study how these two factors coincide with one another. This approach can be utilized to identify the role of DR during fibrosis in other liver diseases. As well, others will be able to adapt this protocol into their own studies to identify the association of their cell of interest with fibrogenesis. For instance, staining of various immune cells could be used to image immune cell infiltration with liver fibrosis. Outside of the liver, this co-staining technique can be applied to other organs and tissues. Future applications of this protocol will allow for further studies on the association and location of nearly any cell types in respect to fibrosis.

2. Methods

Deidentified human liver tissue from patients diagnosed with late-stage PSC were collected by Dr. Burcin Esker at Indiana University School of Medicine following written informed consent and IRB approval. Liver samples were processed, embedded into paraffin blocks, and sectioned at 5 μm for staining. Immunohistochemistry staining of bile ducts using anti-CK-19 antibody (Abcam; Waltham, MA), Vector ImmPRESS^® HRP Reagent Kit and chromogen development using the Vector ImmPACT^® DAB substrate (Vector Laboratories; Burlingame, CA) was performed. FGSR staining solution is made by adding Picrosirius Red (0.1%) and Fast Green FCF (0.1%) to saturated picric acid (1.2% w/v), and slides are incubated in the pre-made solution for 30 min. We chose to use FGSR staining since it provides well-defined collagen fibers and the highest sensitivity for quantitative analysis compared to Sirius Red alone [8]. Lastly, slides are cleared, mounted, scanned using the Lecia Aperio AT2 system and imaged with the Lecia Aperio ImageScope software (Leica Biosystems; Buffalo Grove, IL).

Abbreviations:

CK-19

cytokeratin-19

DR

ductular reaction

FGSR

Fast Green Sirius Red

PSC

primary sclerosing cholangitis