Characterization of macrophages in healthy and diseased livers in mice

Healthy livers contain many resident macrophages named Kupffer cells (KCs), which are partially replaced by infiltrating monocyte-derived macrophages (MoMFs) during acute or chronic liver injury. Despite extensive research, understanding macrophage heterogeneity, spatial distribution, and interactions with other cells within the liver remains challenging. We performed single-cell RNA sequencing to investigate liver macrophage gene expression in naïve and Concanavalin A (ConA)-treated mice. MoMF clusters expanded following ConA treatment, while KCs remained stable. Macrophages were divided into distinct subtypes, including C1q+ MoMFs, with differential expression of genes like Trem2, Spp1, Fabp5 and Gpnmb. Newly recruited C1q- MoMFs expressed high levels of Lyz and Ccr2, while Itgax (Cd11c)+ MoMFs expressed endothelin converting enzyme 1 (Ece1), a gene encoding ECE1 enzyme that activates endothelin to promote hepatic stellate cell contraction and necrotic lesion resolution. By immunostaining analysis of the proteins encoded by these signature genes, we identified several populations of MoMFs that were mainly located surrounding the necrotic lesion area and expressed various proteins that are involved dead cell debris clearance. Naïve mice and mice treated with ConA for 72 or 96 hours were anesthetized, and their livers were perfused and digested using a 0.075% collagenase solution (Worthington, Lakewood, NJ). Liver samples were homogenized, filtered through 70 μm cell strainers, and centrifuged at 400g through a Percoll density gradient (40% and 70%).