Transcriptional factors PU.1 and CEBP/α drive direct reprogramming fibroblasts into tissue resident macrophages

Abstract

Abstract

Macrophages are highly plastic innate immune cells whose identities are established by lineage-determining transcription factors and tissue-specific enhancer landscapes. The ETS family transcription factor PU.1 and the CCAAT/enhancer-binding protein C/EBPα are core regulators of myeloid differentiation and macrophage fate. Previous studies have shown that enforced expression of these factors can directly convert fibroblasts and lymphoid progenitors into macrophage-like cells. However, the epigenomic properties of fibroblast-derived macrophage-like cells, particularly their relationship to tissue-resident macrophage (TRM) enhancer programs, remain incompletely understood. In this study, we established a doxycycline-inducible (Tet-On) PU.1/C-EBPα overexpression system in NIH-3T3 fibroblasts and characterized their direct conversion into macrophage-like cells. Induction of PU.1 and C/EBPα led to marked morphological remodeling, acquisition of macrophage surface markers CD11b and F4/80, and inflammatory responsiveness to lipopolysaccharide stimulation, as demonstrated by increased TNF-α and IL-1β expression. Furthermore, by integrating enhancer-centered chromatin profiling strategies with published TRM datasets, reprogrammed fibroblasts were shown to acquire macrophage-associated and TRM-like enhancer features. Super-enhancer mapping identified Hivep3 as a candidate transcription factor linked to TRM regulatory programs. Collectively, these findings demonstrate that inducible PU.1/C-EBPα expression is sufficient to generate functional macrophage-like cells from fibroblasts and partially recapitulate TRM enhancer architecture. This platform provides a controllable experimental system for dissecting macrophage gene regulatory networks and for identifying transcriptional regulators of tissue-resident macrophage identity.