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Hepatic myofibroblast apoptosis is critical to resolution of liver fibrosis. We show that human hepatic myofibroblasts co-express p75NTR (p75 neurotrophin receptor) and sortilin, thus facilitating differential responses to mature and pro nerve growth factor (proNGF). Although mature NGF is proapoptotic, proNGF protects human hepatic myofibroblasts from apoptosis. Moreover, in recovery from experimental liver fibrosis, the decrease in proNGF parallels loss of hepatic myofibroblasts by apoptosis. Macrophage-derived matrix metalloproteinase 7 (MMP7) cleaves proNGF in a concentration-dependent manner, and its expression in the liver coincides with falling proNGF levels. To define the dominant effect of p75NTR-mediated events in experimental liver fibrosis, we have used a mouse lacking the p75NTR ligand-binding domain but expressing the intracellular domain. We show that absence of p75NTR ligand-mediated signals leads to significantly retarded architectural resolution and reduced hepatic myofibroblast loss by apoptosis. Lack of the ligand-competent p75NTR limits hepatocyte and oval cell proliferative capacity in vivo without preventing hepatic stellate cell transdifferentiation. Conclusion: NGF species have a differential effect on hepatic myofibroblast survival. Our data suggest that cleavage of proNGF by MMP7 during the early phase of recovery from liver fibrosis alters the pro/mature NGF balance to facilitate hepatic myofibroblast loss. Whereas fibrosis develops in the absence of p75NTR signaling, the dominant effects of loss of p75NTR ligand-mediated events are the retardation of liver fibrosis resolution via regulation of hepatic myofibroblast proliferation and apoptosis, and the reduction of hepatocyte and oval cell proliferation. (HEPATOLOGY 2009.)

Although macrophages are widely recognized to have a profibrotic role in inflammation, we have used a highly tractable CCl 4 -induced model of reversible hepatic fibrosis to identify and characterize the macrophage phenotype responsible for tissue remodeling: the hitherto elusive restorative macrophage. This CD11B hi F4/80 int Ly-6C lo macrophage subset was most abundant in livers during maximal fibrosis resolution and represented the principle matrix metalloproteinase (MMP) -expressing subset. Depletion of this population in CD11B promoter–diphtheria toxin receptor (CD11B-DTR) transgenic mice caused a failure of scar remodeling. Adoptive transfer and in situ labeling experiments showed that these restorative macrophages derive from recruited Ly-6C hi monocytes, a common origin with profibrotic Ly-6C hi macrophages, indicative of a phenotypic switch in vivo conferring proresolution properties. Microarray profiling of the Ly-6C lo subset, compared with Ly-6C hi macrophages, showed a phenotype outside the M1/M2 classification, with increased expression of MMPs, growth factors, and phagocytosis-related genes, including Mmp9, Mmp12, insulin-like growth factor 1 (Igf1), and Glycoprotein (transmembrane) nmb (Gpnmb). Confocal microscopy confirmed the postphagocytic nature of restorative macrophages. Furthermore, the restorative macrophage phenotype was recapitulated in vitro by the phagocytosis of cellular debris with associated activation of the ERK signaling cascade. Critically, induced phagocytic behavior in vivo, through administration of liposomes, increased restorative macrophage number and accelerated fibrosis resolution, offering a therapeutic strategy to this orphan pathological process.

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.