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OP14 Identification and characterisation of a Novel Ly-6Cintermediate intrahepatic macrophage population which mediates the resolution of liver fibrosis, is induced by phagocytosis and can be manipulated therapeutically in vivo

Introduction Macrophages are critical for the progression and resolution of hepatic fibrosis. Studies have identified Ly-6Chi macrophages as the pro-fibrogenic subset in mice. However, the identity of the pro-resolution hepatic macrophage population is unknown. Aim We aimed to identify and characterise the macrophage population mediating the resolution of hepatic fibrosis. Method We established a model of reversible murine hepatic fibrosis by administering 4 weeks of CCl4, followed by tissue harvests at serial timepoints after the final dose. Results Histological analysis identified maximal fibrosis resolution between 72 and 96 h after the final CCl4 dose. Flow cytometry of hepatic macrophages showed that during maximal fibrosis resolution there was a loss of pro-fibrotic Ly-6Chi macrophages and large increase in a Ly-6Cintermediate macrophage population, which were the most numerous macrophage subset identified at any timepoint during fibrogenesis and recovery. Using CD11B-DTR mice, macrophages were depleted during the rapid resolution phase, resulting in a failure to remodel the hepatic scar. Critically, this depletion strategy selectively ablated the Ly-6Cint subset, the degree of depletion correlating significantly with the amount of persistent fibrosis. A series of bone marrow transplantation, adoptive transfer and in situ labelling experiments identified that the pro-resolution Ly-6Cint macrophage population derives from recruitment of Ly-6Chi monocytes, a common origin to the pro-fibrotic Ly-6Chi macrophages, indicative of a phenotypic switch in situ. Microarray profiling of FACS sorted Ly-6Cint macrophages in comparison to pro-fibrotic Ly-6Chi macrophages, demonstrated a novel phenotype outside the M1/M2 macrophage paradigm, with down regulation of pro-inflammatory and pro-fibrotic genes, upregulation of matrix degrading enzymes and enrichment for phagocytosis related pathways. Confocal microscopy indicated that the Ly-6Cint population contained more intracellular apoptotic debris, confirming the post-phagocytic nature of these cells. Feeding primary murine macrophages with hepatocyte debris in vitro induced a similar phenotypic switch to that seen in vivo. Furthermore, this phagocytosis-induced switch could be modelled by feeding macrophages with liposomes in vitro. Critically, systemic administration of liposomes to mice during maximal fibrosis resolution increased the number of hepatic Ly-6Cint macrophages and accelerated the resolution of fibrosis. Conclusion In summary, we have identified the specific Ly-6Cint macrophage subset which mediates the resolution of hepatic fibrosis. Extensive characterisation demonstrated this macrophage phenotype is produced by the phagocytosis of dead cells and thus can be manipulated in vivo by the induction of phagocytic behaviour with a beneficial effect on fibrosis resolution.