Stephen N. Hartland

Differential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis

Although macrophages are widely recognized to have a profibrotic role in inflammation, we have used a highly tractable CCl4-induced model of reversible hepatic fibrosis to identify and characterize the macrophage phenotype responsible for tissue remodeling: the hitherto elusive restorative macrophage. This CD11Bhi F4/80int Ly-6Clo 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-6Chi monocytes, a common origin with profibrotic Ly-6Chi macrophages, indicative of a phenotypic switch in vivo conferring proresolution properties. Microarray profiling of the Ly-6Clo subset, compared with Ly-6Chi 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.

Macrophage therapy for murine liver fibrosis recruits host effector cells improving fibrosis, regeneration, and function.

Clinical studies of bone marrow (BM) cell therapy for liver cirrhosis are under way but the mechanisms of benefit remain undefined. Cells of the monocyte‐macrophage lineage have key roles in the development and resolution of liver fibrosis. Therefore, we tested the therapeutic effects of these cells on murine liver fibrosis. Advanced liver fibrosis was induced in female mice by chronic administration of carbon tetrachloride. Unmanipulated, syngeneic macrophages, their specific BM precursors, or unfractionated BM cells were delivered during liver injury. Mediators of inflammation, fibrosis, and regeneration were measured. Donor cells were tracked by sex‐mismatch and green fluorescent protein expression. BM‐derived macrophage (BMM) delivery resulted in early chemokine up‐regulation with hepatic recruitment of endogenous macrophages and neutrophils. These cells delivered matrix metalloproteinases‐13 and ‐9, respectively, into the hepatic scar. The effector cell infiltrate was accompanied by increased levels of the antiinflammatory cytokine interleukin 10. A reduction in hepatic myofibroblasts was followed by reduced fibrosis detected 4 weeks after macrophage infusion. Serum albumin levels were elevated at this time. Up‐ regulation of the liver progenitor cell mitogen tumor necrosis factor‐like weak inducer of apoptosis (TWEAK) preceded expansion of the progenitor cell compartment. Increased expression of colony stimulating factor‐1, insulin‐like growth factor‐1, and vascular endothelial growth factor also followed BMM delivery. In contrast to the effects of differentiated macrophages, liver fibrosis was not significantly altered by the application of macrophage precursors and was exacerbated by whole BM. Conclusion: Macrophage cell therapy improves clinically relevant parameters in experimental chronic liver injury. Paracrine signaling to endogenous cells amplifies the effect. The benefits from this single, defined cell type suggest clinical potential. (HEPATOLOGY 2011;)

Elastin accumulation is regulated at the level of degradation by macrophage metalloelastase (MMP‐12) during experimental liver fibrosis

Elastin has been linked to maturity of liver fibrosis. To date, the regulation of elastin secretion and its degradation in liver fibrosis has not been characterized. The aim of this work was to define elastin accumulation and the role of the paradigm elastase macrophage metalloelastase (MMP‐12) in its turnover during fibrosis. Liver fibrosis was induced by either intraperitoneal injections of carbon tetrachloride (CCl4) for up to 12 weeks (rat and mouse) or oral administration of thioacetamide (TAA) for 1 year (mouse). Elastin synthesis, deposition, and degradation were investigated by immunohistochemistry, quantitative polymerase chain reaction (qPCR), western blotting, and casein zymography. The regulation of MMP‐12 elastin degradation was defined mechanistically using CD11b‐DTR and MMP‐12 knockout mice. In a CCl4 model of fibrosis in rat, elastin deposition was significantly increased only in advanced fibrosis. Tropoelastin expression increased with duration of injury. MMP‐12 protein levels were only modestly changed and in coimmunoprecipitation experiments MMP‐12 was bound in greater quantities to its inhibitor TIMP‐1 in advanced versus early fibrosis. Immunohistochemistry and macrophage depletion experiments indicated that macrophages were the sole source of MMP‐12. Exposure of CCl4 in MMP‐12−/− mice led to a similar degree of overall fibrosis compared to wildtype (WT) but increased perisinusoidal elastin. Conversely, oral administration of TAA caused both higher elastin accumulation and higher fibrosis in MMP‐12−/− mice compared with WT. Conclusion: Elastin is regulated at the level of degradation during liver fibrosis. Macrophage‐derived MMP‐12 regulates elastin degradation even in progressive experimental liver fibrosis. These observations have important implications for the design of antifibrotic therapies. (HEPATOLOGY 2012;55:1965–1975)

p75 neurotrophin receptor signaling regulates hepatic myofibroblast proliferation and apoptosis in recovery from rodent liver fibrosis

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.)

Active matrix metalloproteinase-2 promotes apoptosis of hepatic stellate cells via the cleavage of cellular N-cadherin

Background and Aims: Hepatic stellate cells (HSC) are known to synthesise excess matrix that characterises liver fibrosis and cirrhosis. Activated HSC express the matrix‐degrading matrix metalloproteinase enzymes (MMPs) and their tissue inhibitors (TIMPs). During spontaneous recovery from experimental liver fibrosis, the expression of TIMP‐1 declines and hepatic collagenolytic activity increases. This is accompanied by HSC apoptosis. In this study, we examine a potential mechanism whereby MMP activity might induce HSC apoptosis by cleaving N‐cadherin at the cell surface.

TGFbeta induces apoptosis and EMT in primary mouse hepatocytes independently of p53 , p21 Cip1 or Rb status

BackgroundTGFβ has pleiotropic effects that range from regulation of proliferation and apoptosis to morphological changes and epithelial-mesenchymal transition (EMT). Some evidence suggests that these effects may be interconnected. We have recently reported that P53, P21Cip1 and pRB, three critical regulators of the G1/S transition are variably involved in TGFβ-induced cell cycle arrest in hepatocytes. As these proteins are also involved in the regulation of apoptosis in many circumstances, we investigated their contribution to other relevant TGFβ-induced effects, namely apoptosis and EMT, and examined how the various processes were interrelated.MethodsPrimary mouse hepatocytes deficient in p53, p21 and/or Rb, singly or in combination were treated with TGFβ for 24 to 96 hours. Apoptosis was quantified according to morphology and by immunostaining for cleaved-capsase 3. Epithelial and mesenchymal marker expression was studied using immunocytochemistry and real time PCR.ResultsWe found that TGFβ similarly induced morphological changes regardless of genotype and independently of proliferation index or sensitivity to inhibition of proliferation by TGFβ. Morphological changes were accompanied by decrease in E-cadherin and increased Snail expression but the mesenchymal markers (N-cadherin, SMAα and Vimentin) studied remained unchanged. TGFβ induced high levels of apoptosis in p53-/-, Rb-/-, p21cip1-/- and control hepatocytes although with slight differences in kinetics. This was unrelated to proliferation or changes in morphology and loss of cell-cell adhesion. However, hepatocytes deficient in both p53 and p21cip1were less sensitive to TGFβ-induced apoptosis.ConclusionAlthough p53, p21Cip1 and pRb are well known regulators of both proliferation and apoptosis in response to a multitude of stresses, we conclude that they are critical for TGFβ-driven inhibition of hepatocytes proliferation, but only slightly modulate TGFβ-induced apoptosis. This effect may depend on other parameters such as proliferation and the presence of other regulatory proteins as suggested by the consequences of p53, p21Cip1 double deficiency. Similarly, p53, p21Cip1 and pRB deficiency had no effect on the morphological changes and loss of cell adhesion which is thought to be critical for metastasis. This indicates that possible association of these genes with metastasis potential would be unlikely to involve TGFβ-induced EMT.

LY-6C intermediate and LY-6C LO intrahepatic macrophage subsets orchestrate resolution of hepatic fibrosis following chronic injury

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.

OP07 Macrophage cell therapy causes the hepatic recruitment of host effector cells and improves structure and function in a murine model of chronic liver disease

Introduction Bone marrow (BM) cell populations have a number of roles in the development and resolution of chronic liver disease. Clinical trials of BM cell therapy have already begun. These have generally employed mixed cell populations often enriched for adult stem cells. Such cells may have a range of phenotypically diverse progeny. The identification of a defined cell type with beneficial effect will provide the basis of rational and predictable therapy. We have previously shown that macrophages are key mediators of scar remodelling. Iterative injury with carbon tetrachloride (CCl4) results in a well characterised model of murine hepatic fibrosis. Aim We sought to determine whether bone marrow derived macrophages (BMMs) could be used as cell therapy for liver fibrosis. Method Liver fibrosis was induced in female C57/Bl6 mice by 12 weeks i.p. carbon tetrachloride (CCl4). Macrophages were derived from the bone marrow of age-matched syngeneic mice cultured for 7 days under low adherence conditions in macrophage colony stimulating factor conditioned media. 8 weeks into the CCl4 injury protocol, mice received either 106 BMMs via the hepatic portal vein (n=8) or control medium (n=8). Serum was analysed for albumin and livers were analysed for mediators of inflammation, fibrosis and regeneration. To track donor cells, male (C57Bl/6) or transgenic green fluorescent protein+ (CBA) BMMs were delivered to strain-matched fibrotic wild type mice. Results BMMs were 88% F4/80+/CD11b+, possessed characteristic morphologic and phenotypic features, and expressed the chemokines MCP-1, MIP-1α and MIP-2. At 12 weeks, C57Bl/6 mice receiving the macrophage injection had 32% less fibrosis (mean±SEM: 2.5±0.4 vs 3.7±0.3%, p<0.05) and higher serum albumin levels (46±2.6 vs 39.9±0.86 g/l, p=0.05). Significant improvements in fibrosis and serum albumin were also demonstrated in CBA mice. Donor macrophages transiently engrafted the scar increasing hepatic levels of macrophage (MCP-1), and neutrophil (MIP-1α, MIP-2 and KC) chemoattractants (p<0.05). This enhanced recruitment of host macrophages and neutrophils to the hepatic scar areas with associated increases in MMP-13 and MMP-9 (p<0.05). A 60% reduction in myofibroblast staining (p<0.05) followed. The early influx of host leukocytes was accompanied by a 346% increase in hepatic levels of the anti-inflammatory cytokine IL-10. Donor BMMs expressed high levels of the progenitor cell mitogen TWEAK. Macrophage recipients upregulated hepatic TWEAK by 216% with a 40% increase in the number of liver progenitor cells (p<0.05). Hepatocyte proliferation was not significantly affected. Conclusion BMM therapy decreases fibrosis and increases regeneration improving clinically meaningful parameters of chronic liver disease in this model. The actions of the donor BMMs are amplified through paracrine signalling to numerically greater endogenous cell populations. Importantly, these effects are mediated by a single differentiated donor cell type, bringing clarity to the cause-effect relationship.