Madeleine A. Vernon

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.

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)

Macrophages and Renal Fibrosis

Renal fibrosis is a key determinant of the progression of renal disease irrespective of the original cause and thus can be regarded as a final common pathway that dictates eventual outcome. The development of renal fibrosis involves many cellular and molecular mediators including leukocytes, myofibroblasts, cytokines, and growth factors, as well as metalloproteinases and their endogenous inhibitors. Study of experimental and human renal disease has shown the involvement of macrophages in renal fibrosis resulting from diverse disease processes. Recent work exploring the nature of both circulating monocytes and tissue macrophages has highlighted their multifaceted phenotype and this impacts their role in renal fibrosis in vivo. In this review we outline the key players in the fibrotic response of the injured kidney and discuss the role of monocytes and macrophages in renal scarring.

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.)

The induction of macrophage hemeoxygenase-1 is protective during acute kidney injury in aging mice

Aging is thought to be associated with a higher susceptibility to renal ischemia-reperfusion injury (IRI). To study whether defective induction of hemeoxygenase-1 (HO-1, a protective and anti-inflammatory enzyme) might contribute to this, we found that while 12-month-old mice had similar baseline renal function and HO-1 expression, the induction of HO-1 usually seen in ischemia-reperfusion was reduced. This was also associated with worsened renal function and acute tubular necrosis in the aged compared with young mice. In the older mice, heme arginate (HA) induced HO-1 in the cortex and medulla, significantly improved renal function, and reduced tissue injury. Cellular HO-1 induction in the medulla in response to injury or HA treatment was found to be interstitial rather than epithelial, as evidenced by its colocalization with macrophage markers. In vitro, HA treatment of primary macrophages resulted in marked HO-1 induction without impairment of classical activation pathways. Macrophage depletion, caused by diphtheria toxin treatment of 12-month-old CD11b-DTR transgenic animals, resulted in the loss of interstitial HO-1-positive cells and reversal of the protective phenotype of HA treatment. Thus, failure of HO-1 induction following renal IRI worsens structural and functional injury in older mice and represents a therapeutic target in the elderly. Hence, HO-1-positive renal macrophages mediate HA-induced protection in IRI.

A murine model of irreversible and reversible unilateral ureteric obstruction.

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive nephropathy induced by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). In the irreversible UUO model, the ureter may be obstructed for variable periods of time in order to induce increasingly severe renal inflammation and interstitial fibrotic scarring. In the reversible R-UUO model the ureter is obstructed to induce hydronephrosis, tubular dilation and inflammation. After a suitable period of time the ureteric obstruction is then surgically reversed by anastomosis of the severed previously obstructed ureter to the bladder in order to allow complete decompression of the kidney and restoration of urinary flow to the bladder. The irreversible UUO model has been used to investigate various aspects of renal inflammation and scarring including the pathogenesis of disease and the testing of potential anti-inflammatory or anti-fibrotic therapies. The more challenging model of R-UUO has been used by some investigators and does offer significant research potential as it allows the study of inflammatory and immune processes and tissue remodeling in an injured and scarred kidney following the removal of the injurious stimulus. As a result, the R-UUO model offers investigators the opportunity to explore the resolution of kidney inflammation together with key aspects of tissue repair. These experimental models are of relevance to human disease as patients often present with obstruction of the renal tract that requires decompression and are commonly left with significant residual kidney impairment that has no current treatment options and may lead to eventual end stage kidney failure.

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.

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.