Liver-derived signals sequentially reprogram myeloid enhancers to initiate and maintain Kupffer cell identity (RNA-Seq)

Tissue environment plays a powerful role in establishing and maintaining the distinct phenotypes of resident macrophages, but the underlying molecular mechanisms remain poorly understood. Here, we characterized transcriptomic and epigenetic changes in repopulating liver macrophages following acute Kupffer cell depletion as a means to infer signaling pathways and transcription factors that promote Kupffer cell differentiation. We obtain evidence that combinatorial interactions of the Notch ligand DLL4 and transforming growth factor-b (TGF-?) family ligands produced by sinusoidal endothelial cells and endogenous LXR ligands were required for the induction and maintenance of Kupffer cell identity. DLL4 regulation of the Notch transcriptional effector RBPJ activated poised enhancers to rapidly induce LXR? and other Kupffer cell lineage-determining factors. These factors in turn reprogrammed the repopulating liver macrophage enhancer landscape to converge on that of the original resident Kupffer cells. Collectively, these findings provide a framework for understanding how macrophage progenitor cells acquire tissue-specific phenotypes. Overall design: RNA-seq for Blood Ly6C high monocytes from C57BL/6J mice; RLMs (repopulating liver macrophages) from Clec4f-Cre/DTR mice; Kupffer cells from Clec4f-Cre/DTR mice, Nr1h3 flox/flox mice, Nr1h3 flox/flox Clec4f-cre+/– mice, Smad4 flox/flox mice, or Smad4 flox/flox Clec4f-Cre+/– mice; BMDMs (bone marow-derived macrophages) from C57BL/6J mice, Smad4 flox/flox mice, or Smad4 flox/flox LysM-cre+/– mice stimulated with or without DLL4, TGF-ß, DAPT, and/or DMHCA. RNA-seq for RLMs (repopulating liver macrophages) from Clec4f-Cre/DTR mice treated with or without LY411575 or antibodies for DLL1 and DLL4; bone marrow monocytes stimulated with or without DLL4, TGF-beta, and/or desmosterol.