O2196: Single nuclear RNA sequencing elucidates a longitudinal progression of ductular reaction in the liver after massive small bowel resection

Background: Intestinal failure associated liver disease (IFALD) is a highly morbid condition in patients with short gut syndrome (SGS). The proliferative expansion of biliary epithelial cells (BEC) in response to liver injury, known as ductular reaction (DR), is observed in IFALD patients; however, the timing and contribution to pathology is relatively unknown. Here we describe the temporal evolution of ΒΕCs at single nuclear resolution (snRNAseq) in a preclinical murine model of SGS.

Methods: Male and female mice (n=200) were randomized to receive 75% small bowel resection (SBR) or sham operations. They were further randomized into 2-, 5-, 10-, and 26-week postoperative cohorts. DR was determined by liver immunohistochemical staining for CK19. Single nuclear suspensions were prepared in a subset of male livers from 2-, 10-, and 26-week cohorts and sequenced. Seurat preprocessing, harmony integration, weighted gene co-expression network analysis (WGCNA), Kyoto Encyclopedia Genes and Genomes (KEGG) over representation analysis (ORA) and CellChat analysis were all conducted in R. BECs were identified by expression of canonical markers EpCam and CK19.

Results: SBR mice in both sexes exhibit a similar liver injury pattern progression from early steatosis (2 weeks) followed by steatohepatitis (10 weeks) and cirrhosis (26 weeks) (Figure 1). snRNAseq demonstrated emergence of a relatively large population of BEC’s at 10 weeks in SBR livers (Figure 2a-b). This population increase was verified by CK-19 IHC showing increased interlobular staining at later time points (Figure 1). To understand the function of this expanding population of BECs, WGCNA was performed on BECs resulting in 48 color modules (Figure 2c). A correlation analysis of module eigenvalues revealed two modules of interest. MEblack is positively correlated with SBR at all postoperative timepoints (r=0.78 (p < 0.001) whereas MEpink is negatively correlated to SBR at 2 weeks (-0.27, p=0.3), weakly correlated at 10 weeks (0.41, p=0.09) and strongly correlated at 26 weeks postoperatively (0.66, p=0.003) (Figure 2d). KEGG ORA of MEblack shows enrichment of gene sets related to cancer and the MAPK signaling pathway (Figure 2e); whereas the KEGG ORA of MEpink demonstrates enrichment of gene sets related to the extracellular matrix modulation and PI3K-Akt and TNF signaling pathways (Figure 2f). Cellchat analysis between SBR BECs at 26 and 2 weeks show upregulation of BEC ligands Lama4, Col4a6, and Pdgfc at 26 weeks with cellular communications with stellate cells and Kupffer cells (Figure 2g). 2-week SBR BECs show upregulation of ligands SPP1, Ptpm, and Cadm1 with primarily Kupffer cell communication.

Conclusion: In a longitudinal surgical model of IFALD, DR is evident as early as five weeks postoperatively and corresponds to an expansion of BECs that undergo progressive transcriptional changes and output signaling towards stellate and Kupffer cells. Future work includes analyzing dynamic transcription factor changes.