Figure 1.

Liver infiltrating Ly6C^hi monocytes, monocyte-derived macrophage (MoMF), and neutrophils spatially and temporally coincide at the inflammatory and resolution phases of acetaminophen-induced liver injury (AILI). (A) Hematoxylin and eosin (H&E) staining of liver sections at 12, 24, 48, and 72 h following AILI. Original magnification ×20. Bars, 100 µm. (B) Immunostaining for Ly6G and hematoxylin on paraffin-embedded liver sections at 24 and 48 h after AILI. Original magnifications ×20 (left image) and ×40 (right image). Bars, 100 µm (×20) and 85 µm (×40). (C) Immunofluorescence staining at 24 and 48 h after AILI in Cx3cr1^gfp/+ mice. At 48 h, 3D-reconstruction was performed of Z-stacks acquired from 20 µm thick slides. In green are infiltrating monocyte-derived cells and in red are neutrophils, which are stained for Ly6G. Original magnification ×20. Bars, 50 µm for 24 h and 20 µm for 48 h. (D) Flow cytometry-based definition of non-parenchymal Cx3cr1^gfp/+ liver cells isolated at 24 h (Ly6C^hi monocytes and neutrophils) or 72 h [MoMF and Kupffer cell (KC)] following AILI. (E) Flow cytometry-based analysis of the dynamics of neutrophils and liver-macrophage subsets at steady state (SS) and at different time points following AILI: Ly6G⁺ neutrophils (white), infiltrating Ly6C^hiCX₃CR1-GFP⁺ monocytes (black), resident CX₃CR1-GFP^neg/lo KC (red), and Ly6C^loCX₃CR1-GFP^hi MoMF (blue), presented as cell counts normalized per liver tissue mass in gram. Data are presented as mean ± SEM; n ≥ 5 mice for each time point. Experiments were repeated at least three times.