The hepatic crown-like structure in NASH: a nexus between monocyte-derived macrophages, hepatic stellate cells, and tissue fibrosis Sabine Daemen1,2, Li He1,2, Mandy M. Chan1,3, Brian Finck1,2, Joel D. Schilling1,2,3 1Washington University School of Medicine, St. Louis, MO; 2Department of Internal Medicine; 3Department of Pathology and Immunology Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis and hepatocyte injury. In response to liver damage macrophage composition changes dramatically and is characterized by a reduction in resident Kupffer cell (KC) number and recruitment of monocyte-derived macrophages (MdMs). One subset of infiltrating MdMs turns on expression of KC-specific genes and these cells serve to replenish the resident macrophage pool (i.e. Mo-KCs). In addition to Mo-KCs, we identified two populations of Trem2-expressing recruited MdMs using single cell RNA sequencing, flow cytometry and immunofluorescence. One macrophage subset expresses Cx3cr1 and Ccr2 and the second subset expresses Cd63, Cd9, and Gpnmb, which are markers previously ascribed to lipid-associated macrophages (LAMs) in obese adipose tissue. We therefore refer to these macrophage subsets as hepatic C-LAMs and LAMs, respectively. To gain insight into the function of these MdM populations in NASH pathogenesis we fed wild type and Ccr2 deficient mice a NASH-inducing diet for 4 months. In Ccr2 knockout (KO) animals, the C-LAMs failed to appear in the NASH liver and the number of macrophages expressing LAM markers was reduced. Confocal imaging revealed specific homing of C-LAMs and LAMs to macrophage aggregates referred to as hepatic crown-like structures (hCLS) and these structures were dramatically reduced in Ccr2 KO mice. Somewhat surprisingly, the loss of hCLS in Ccr2 KO mice was associated with increased liver fibrosis despite a similar degree of liver steatosis, arguing that hCLS may protect against further tissue injury. Within hCLS recruited macrophages formed tight interactions with activated hepatic stellate cells, identified by desmin and a-smooth muscle actin staining. Loss of Ccr2 did not impact stellate cell accumulation with diet stress but did lead to decreased stellate cell activation. Together these findings illustrate the pro- and anti-fibrotic nature of recruited MdMs in NASH and suggest that hCLS are dynamically involved in tissue remodeling. To understand the influence of macrophages on stellate cell biology we employed an ex vivo primary culture system, which revealed that macrophages isolated from NASH livers specifically alter the proliferation and organization of primary stellate cells compared to bone marrow-derived macrophages. Together these data indicate that recruited MdMs play a unique role(s) in the regulation of stellate cell biology and liver fibrosis during NASH.