Single-cell RNA sequencing identifies a unique ZEB2-Regulated Clec4d⁺ macrophage state contributing to early...

Macrophage-mediated inflammation plays an important role in determining early-phase cardiac hypertrophy, yet the underlying mechanisms and key regulatory factors remain unclear. We dissected macrophage heterogeneity by analyzing 25,328 single cells from cardiac tissues of Transverse aortic constriction (TAC)-induced hypertrophic mouse models and identified inflammatory regulators through differential expression, regulatory network, and trajectory analyses. The functional roles and regulatory mechanisms were further investigated using in vitro macrophage inflammatory models and ChIP-seq. We characterized a specific Clec4d⁺ macrophage state that existed during homeostasis, expanded significantly post-injury, acquired a pro-inflammatory phenotype, and identified the transcription factor ZEB2 as a regulator of its inflammatory activation. In vitro experiments demonstrated that ZEB2 knockdown reduced the expression of inflammatory factors IL-6 and TNF-α in macrophages, whereas ZEB2 overexpression exacerbated the macrophage-mediated inflammation. Mechanistically, ZEB2 binds to the Unc93b1 promoter and enhances Unc93b1 transcription during the early stage of cardiac hypertrophy. In summary, Clec4d⁺ macrophages play a crucial role in early cardiac inflammatory responses, and ZEB2 emerges as a potential diagnostic biomarker and therapeutic target, offering a theoretical basis for preventing and treating pathological cardiac hypertrophy.