Angiotensin-converting enzyme-2(ACE2) expression in pediatric liver disease

Background: Angiotensin-converting enzyme-2 (ACE2) is a cell membrane protein that helps regulate blood pressure and inflammation. It has also gained recent notoriety as the receptor SARS-CoV-2 uses to invade human cells. While ACE2 has been shown to have moderate expression within the liver, prior research is mostly limited to animal models and the role of ACE2 in pediatric liver disease has not been discerned. Here we aim to analyze ACE2 expression in children with immune-mediated liver diseases, to better understand its evolving presence and utility in chronic liver disease as well as shed light on COVID-19's potential effects within the liver. Methods: Immunofluorescent staining of paraffin-fixed liver biopsy tissues of patients with autoimmune hepatitis and primary sclerosing cholangitis was done with ACE2-specific antibodies along with healthy controls. Cholangiocyte and nuclear antibody markers were concurrently added. ACE2 expression was analyzed by confocal microscopy. Computational deep learning-based segmentation models, StartDist and Cellpose, identified individual nuclei and cells within the tissue. Individual masks were generated for nuclei and cells using Fiji. Input from these masks in CellProfiler was used to quantify cellular, cytosolic, and nuclear mean IF intensity in samples. Additionally, state-of-the-art spatial transcriptomics technology was used to provide high throughput gene expression analysis in intact tissue section determining the cellular composition for ACE2 expression. Finally, an ACE2 enzyme-linked immunosorbent assay (ELISA) was used to quantify ACE2 serologic expression in patients and controls. Results: ACE2 showed clear and specific expression in all the liver biopsy specimens which were verified with nonspecific staining and negative controls. Computational deep machine learning effectively identified distinct cell and nuclear membranes, allowing for separate nuclear and cytosolic analysis. High ACE2 expression was evident at the apical surface of cholangiocytes for both patients and controls. Relatively lower expression was seen within the cytosol and nuclei of hepatocytes and hepatic lymphocytes. Children with immune-mediated liver diseases were found to have overall higher mean hepatic expression of ACE2 than controls (p<0.01). No significant differences were seen in serologic ACE2 expression. Spatial transcriptomics identified cell composition of ACE2 expressing spots containing antibody-secreting cells and was different as compared to the other spots. Conclusions: ACE2 expression is prevalent throughout the liver, with strong localization to cholangiocyte apical membranes as well as a presence within hepatocytes and immune cells. Machine learning can be used to rapidly identify hepatic cellular components for histologic analysis. ACE2 expression in the liver may be higher in pediatric immune-mediated liver disease. Future work will be directed to better understand the implications of hepatic ACE2 on COVID-19 presentation and clinical course.