Dynamic Cholangiocyte Responses in a Murine Model of Reversible Cholestasis: Macrophage Remodeling and NF-Y Mediated TGFβ1 Expression.

During cholestasis, cholangiocytes become activated, promoting macrophage-associated periductal infiltration and fibrosis. The cholangiocyte specific mechanisms responsible for these processes are unclear. To gain insight into the cholangiocyte signaling mechanisms contributing to these pathophysiologic processes, mice were fed a DDC diet for 10 days to induce liver injury and then switched to a chow diet to permit recovery, designated as R days. Profiling of isolated intrahepatic leukocytes by mass spectrometry revealed an abundant CX3CR1⁺ macrophage population on the DDC diet which declined during the recovery period. This observation was confirmed using Cx3cr1^GFP mice. Next, cholangiocytes were isolated from control, DDC, and R15 mice, and RNA-seq performed. Cholangiocyte CX3CL1 expression, the cognate ligand for CX3CR1, increased in DDC fed mice and returned to basal values by R15, implicating cholangiocytes in CX3CR1⁺ macrophage recruitment. Ingenuity pathway analysis (IPA) of the RNAseq data revealed upregulation of the pathogen induced cytokine storm pathway in cholangiocytes activated from DDC fed mice, and resolution of this pathway in R15 isolated cholangiocytes. SCENIC regulon analysis identified that NF-Y, a transcription factor complex, was activated only on the DDC diet, but not in control or R15 mice. Finally, siRNA targeted suppression of NF-YA in normal human cholangiocytes (NHC) reduced cholangiocyte expression of the profibrogenic ligand TGFβ1. Consistent with this observation, Tgfβ1 was increased in cholangiocytes from DDC fed animals which returned to control values at day R15. Collectively, these observations provide mechanistic insights into cholangiocyte pathobiology during cholestasis.