Abstract
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 Cx3cr1(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 TGFβ1. Consistent with this observation, Tgfβ1 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.NEW & NOTEWORTHY 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 TGFβ1. These observations provide insights into the cholestatic cholangiocyte pathobiology contributing to periductal inflammation and fibrosis.