Novel phenotypical and functional sub‐classification of liver macrophages highlights changes in population dynamics in experimental mouse models

Liver macrophages are critical components of systemic immune system defense mechanisms. F4/80 high Kupffer cells (KCs) are the predominant liver‐resident macrophages and the first immune cells to contact pathogens entering the liver. F4/80 low monocyte‐derived macrophages (MoMφs) are essential macrophages that modulate liver immune functions. Here we report a novel method of identifying subpopulations of these two populations using traditional flow cytometry and examine each subpopulation for its putative roles in the pathogenesis of an experimental non‐alcoholic steatohepatitis model. Using male C57BL/6 mice, we isolated and analyzed liver non‐parenchymal cells by flow cytometry. We identified F4/80 high and F4/80 low macrophage populations and characterized subpopulations using uniform manifold approximation and projection. We identified three subpopulations in F4/80 high macrophages: CD163(+) KCs, CD163(−) KCs, and liver capsular macrophages. CD163(+) KCs had higher phagocytic and bactericidal activities and more complex cellular structures than CD163(−) KCs. We also identified four subpopulations of F4/80 low MoMφs based on Ly6C and MHC class II expression: infiltrating monocytes, pro‐inflammatory MoMφs, Ly6C(−) monocytes, and conventional dendritic cells. CCR2 knock‐out mice expressed lower levels of these monocyte‐derived cells, and the count varied by subpopulation. In high‐fat‐ and cholesterol‐diet‐fed mice, only one subpopulation, pro‐inflammatory MoMφs, significantly increased in count. This indicates that changes to this subpopulation is the first step in the progression to non‐alcoholic steatohepatitis. The community can use our novel subpopulation and gating strategy to better understand complex immunological mechanisms in various liver disorders through detailed analysis of these subpopulations.