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 3,5-diethoxycarbonyl-1,4-dihydro-collidine (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 that declined during the recovery period. This observation was confirmed using <i>Cx3cr1</i>^GFP mice. Next, cholangiocytes were isolated from control, DDC, and R15 mice, and RNA sequencing (RNAseq) was 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 <i>TGFβ1</i>. Consistent with this observation, <i>Tgfβ1</i> was increased in cholangiocytes from DDC-fed animals that returned to control values at day R15. Collectively, these observations provide mechanistic insights into cholangiocyte pathobiology during cholestasis.<b>NEW & NOTEWORTHY</b> Cholangiocyte pathophysiological activation was examined in a model of murine cholestasis. CX3CR1⁺ macrophages are recruited to the periportal region, likely mediated by cholangiocyte expression of CX3CL1. Cholangiocyte transcriptomics from cholestatic mice display activation of a "pathogen-induced cytokine storm" pathway, and exhibit activation of the transcription factor NF-Y. In human cholangiocytes, NF-Y promotes expression of the profibrogenic ligand <i>TGFβ1</i>. These observations provide insights into the cholestatic cholangiocyte pathobiology contributing to periductal inflammation and fibrosis.