The NF-Y Transcription Factor Complex as a Regulator of the Cholangiocyte Proliferation-Associated Phenotype in a Model of Murine Cholestasis

Ductular reactive (DR) cells proliferate and contribute to liver injury during cholestasis by promoting myeloid cell derived inflammation. How DR cells promote this inflammation is unclear, and therefore, our aim was to gain insight into the DR cell-initiated signaling mechanisms contributing to this pathophysiologic process. 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 Cx3cr1GFP mice to more specifically distinguish this recruited macrophage population. CX3CL1, the ligand for CX3CR1, in DR cells was expressed by cholangiocytes from DDC fed mice. Next, cholangiocytes were isolated from control, DDC and R15 mice, and RNA-seq performed. IPA pathway analysis revealed activation 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, normal human cholangiocytes (NHC) were treated with an siRNA of NF-YA and chemokine expression assessed by PCR. Expression of CX3CL1 and other chemokines decreased following transfection of NHC cells with siRNA targeting NF-YA as assessed by NanoString analysis. Collectively, these observations implicate NF-Y as a participant transcription factor in the proliferation-associated secretory phenotype. Overall design: Wildtype C57BL/6 male mice, between 14-16 weeks of age, were fed the chow diet (control group), DDC diet for 10 days (DDC group), or DDC diet for 10 days and then transferred to a chow diet for 15 days (R15 group). Cholangiocytes were isolated from the livers and sequenced.