Introduction Hepatic fibrosis is potentially reversible. Macrophages are heterogenous and have distinct roles in fibrogenesis and resolution. Studies have identified a pro-inflammatory Ly-6Chi macrophage subset in mediating fibrogenesis. However, little is known about the identity or phenotype of the restorative hepatic macrophage. Here we identify and characterise the macrophage subset mediating resolution of liver fibrosis following chronic injury.
Methods Liver fibrosis was induced in adult male mice by twice-weekly CCl4 injections for 4 weeks, followed by histological assessment or flow cytometry analysis at serial time points following cessation.
Results Following cessation of CCl4 distinct phases of injury and recovery could be identified: active inflammation and fibrogenesis (24 h), peak fibrosis (48–72 h), early resolution with dynamic loss of myofibroblasts and remodelling of majority of scar tissue (72–96 h) and late resolution with degradation of remainder of fibrosis (96–256 h).
Intrahepatic macrophage subsets demonstrated an increase in Ly-6Chi cells during inflammation and fibrogenesis. During resolution there is a rapid loss of these Ly-6Chi cells and emergence of Ly-6C intermediate and Ly-6Clo macrophage subsets. Using CD11B-DTR transgenic mice we depleted macrophages during early resolution, resulting in a failure to remodel hepatic scar. Critically, depletion was selective for the Ly-6Cint and Ly-6Clo liver macrophage subsets, and the degree of depletion of these cells correlated significantly with the amount of persistent fibrosis, thus confirming their role in scar resolution. Adoptive transfer and tracking experiments demonstrated that during early resolution the Ly-6Cint cells derive from Ly-6Chi circulating monocytes, indicating a phenotypic switch from pro-inflammatory to early pro-resolution macrophages. During late resolution Ly-6Clo monocytes are recruited to replenish resident ‘Kupffer’ cells.
Gene expression analysis on FACS sorted pro-resolution Ly-6Cint macrophages compared with pro-fibrotic Ly-6Chi macrophages demonstrated reduced expression of pro-inflammatory mediators such as IL-1α, IL-1β, IL-6, CXCL2 and MCP-1 and an increase in expression of matrix degrading enzymes such as MMP-12 and MMP-9.
Conclusion We have identified novel Ly-6Cint and Ly-6Clo intrahepatic macrophage subsets as central orchestrators in the resolution of liver fibrosis. Critically, during early resolution the Ly-6Cint cells derive from a phenotypic switch in pro-fibrotic Ly-6Chi cells, resulting in a change in macrophage gene expression from one promoting fibrogenesis to one favouring fibrosis resolution.
- liver fibrosis
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Competing interests None.
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