Jonathan A. Fallowfield

Liver fibrosis and repair: immune regulation of wound healing in a solid organ

Fibrosis is a highly conserved and co-ordinated protective response to tissue injury. The interaction of multiple pathways, molecules and systems determines whether fibrosis is self-limiting and homeostatic, or whether it is uncontrolled and excessive. Immune cells have been identified as key players in this fibrotic cascade, with the capacity to exert either injury-inducing or repair-promoting effects. A multi-organ approach was recently suggested to identify the core and regulatory pathways in fibrosis, with the aim of integrating the wealth of information emerging from basic fibrosis research. In this Review, we focus on recent advances in liver fibrosis research as a paradigm for wound healing in solid organs and the role of the immune system in regulating and balancing this response.

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.

Therapeutic targets in liver fibrosis

Detailed analysis of the cellular and molecular mechanisms that mediate liver fibrosis has provided a framework for therapeutic approaches to prevent, slow down, or even reverse fibrosis and cirrhosis. A pivotal event in the development of liver fibrosis is the activation of quiescent hepatic stellate cells (HSCs) to scar-forming myofibroblast-like cells. Consequently, HSCs and the factors that regulate HSC activation, proliferation, and function represent important antifibrotic targets. Drugs currently licensed in the US and Europe for other indications target HSC-related components of the fibrotic cascade. Their deployment in the near future looks likely. Ultimately, treatment strategies for liver fibrosis may vary on an individual basis according to etiology, risk of fibrosis progression, and the prevailing pathogenic milieu, meaning that a multiagent approach could be required. The field continues to develop rapidly and starts to identify exciting potential targets in proof-of-concept preclinical studies. Despite this, no antifibrotics are currently licensed for use in humans. With epidemiological predictions for the future prevalence of viral, obesity-related, and alcohol-related cirrhosis painting an increasingly gloomy picture, and a shortfall in donors for liver transplantation, the clinical urgency for new therapies is high. There is growing interest from stakeholders keen to exploit the market potential for antifibrotics. However, the design of future trials for agents in the developmental pipeline will depend on strategies that enable equal patient stratification, techniques to reliably monitor changes in fibrosis over time, and the definition of clinically meaningful end points.

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)

Remodelling of extracellular matrix is a requirement for the hepatic progenitor cell response

Background and methods In advanced liver damage, hepatic regeneration can occur through proliferation of a resident hepatic progenitor cell (HPC) population. HPCs are located within a designated niche in close association with myofibroblasts and bone marrow (BM) derived macrophages. Extra-cellular matrix (ECM) laminin invariably surrounds HPCs, but the functional requirement of this matrix-cell association is untested in vivo. Using the collagen Iα1(r/r) mouse (r/r), which produces mutated collagen I resistant to matrix metalloproteinase degradation and has an exaggerated fibrotic response to liver injury, we test the relationship between collagen degradation, laminin deposition, and the HPC response. Results Chronic fibrotic carbon tetrachloride (CCl4) injury can induce a florid HPC response associated with dense laminin deposition. In the recovery phase after chronic CCl4 injury, r/r mice have a markedly attenuated HPC response compared to wild-types, together with persistence of collagen I and failure to deposit ECM laminin. Similar results were found in r/r mice given the choline-deficient ethionine supplemented diet, another model of the HPC response. In cross-over sex-mismatched BM transplantation (BMT) experiments between r/r mice and wild-types, the blunted HPC response of r/r mice was not rescued by wild-type BMT and likewise not conferred on to wild-type recipients by r/r BMT, demonstrating that the attenuated HPC response in r/r mice is a property intrinsic to the liver. Conclusion Failure of ECM remodelling after chronic fibrotic liver injury hinders the ability of the liver to activate HPCs. Laminin-progenitor cell interactions within the HPC niche are a critical for HPC mediated regeneration.

Relaxin modulates human and rat hepatic myofibroblast function and ameliorates portal hypertension in vivo.

Active myofibroblast (MF) contraction contributes significantly to the increased intrahepatic vascular resistance that is the primary cause of portal hypertension (PHT) in cirrhosis. We sought proof of concept for direct therapeutic targeting of the dynamic component of PHT and markers of MF activation using short‐term administration of the peptide hormone relaxin (RLN). We defined the portal hypotensive effect in rat models of sinusoidal PHT and the expression, activity, and function of the RLN‐receptor signaling axis in human liver MFs. The effects of RLN were studied after 8 and 16 weeks carbon tetrachloride intoxication, following bile duct ligation, and in tissue culture models. Hemodynamic changes were analyzed by direct cannulation, perivascular flowprobe, indocyanine green imaging, and functional magnetic resonance imaging. Serum and hepatic nitric oxide (NO) levels were determined by immunoassay. Hepatic inflammation was assessed by histology and serum markers and fibrosis by collagen proportionate area. Gene expression was analyzed by quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR) and western blotting and hepatic stellate cell (HSC)‐MF contractility by gel contraction assay. Increased expression of RLN receptor (RXFP1) was shown in HSC‐MFs and fibrotic liver diseases in both rats and humans. RLN induced a selective and significant reduction in portal pressure in pathologically distinct PHT models, through augmentation of intrahepatic NO signaling and a dramatic reduction in contractile filament expression in HSC‐MFs. Critical for translation, RLN did not induce systemic hypotension even in advanced cirrhosis models. Portal blood flow and hepatic oxygenation were increased by RLN in early cirrhosis. Treatment of human HSC‐MFs with RLN inhibited contractility and induced an antifibrogenic phenotype in an RXFP1‐dependent manner. Conclusion: We identified RXFP1 as a potential new therapeutic target for PHT and MF activation status. (Hepatology 2014;59:1492‐1504)

Coffee consumption and health: umbrella review of meta-analyses of multiple health outcomes

Objectives To evaluate the existing evidence for associations between coffee consumption and multiple health outcomes. Design Umbrella review of the evidence across meta-analyses of observational and interventional studies of coffee consumption and any health outcome. Data sources PubMed, Embase, CINAHL, Cochrane Database of Systematic Reviews, and screening of references. Eligibility criteria for selecting studies Meta-analyses of both observational and interventional studies that examined the associations between coffee consumption and any health outcome in any adult population in all countries and all settings. Studies of genetic polymorphisms for coffee metabolism were excluded. Results The umbrella review identified 201 meta-analyses of observational research with 67 unique health outcomes and 17 meta-analyses of interventional research with nine unique outcomes. Coffee consumption was more often associated with benefit than harm for a range of health outcomes across exposures including high versus low, any versus none, and one extra cup a day. There was evidence of a non-linear association between consumption and some outcomes, with summary estimates indicating largest relative risk reduction at intakes of three to four cups a day versus none, including all cause mortality (relative risk 0.83, 95% confidence interval 0.83 to 0.88), cardiovascular mortality (0.81, 0.72 to 0.90), and cardiovascular disease (0.85, 0.80 to 0.90). High versus low consumption was associated with an 18% lower risk of incident cancer (0.82, 0.74 to 0.89). Consumption was also associated with a lower risk of several specific cancers and neurological, metabolic, and liver conditions. Harmful associations were largely nullified by adequate adjustment for smoking, except in pregnancy, where high versus low/no consumption was associated with low birth weight (odds ratio 1.31, 95% confidence interval 1.03 to 1.67), preterm birth in the first (1.22, 1.00 to 1.49) and second (1.12, 1.02 to 1.22) trimester, and pregnancy loss (1.46, 1.06 to 1.99). There was also an association between coffee drinking and risk of fracture in women but not in men. Conclusion Coffee consumption seems generally safe within usual levels of intake, with summary estimates indicating largest risk reduction for various health outcomes at three to four cups a day, and more likely to benefit health than harm. Robust randomised controlled trials are needed to understand whether the observed associations are causal. Importantly, outside of pregnancy, existing evidence suggests that coffee could be tested as an intervention without significant risk of causing harm. Women at increased risk of fracture should possibly be excluded.

Resolution of Liver Fibrosis: Basic Mechanisms and Clinical Relevance

With evidence from a large number of animal models and clinical trials, it is now beyond debate that liver fibrosis and even cirrhosis are potentially reversible if the underlying cause can be successfully eliminated. However, in a significant proportion of patients cure of the underlying disease may not result in fibrosis regression or a significant reduction of the risk for hepatocellular carcinoma development. Understanding of the mechanistic pathways and regulatory factors that characterize matrix remodeling and architectural repair during fibrosis regression may provide therapeutic approaches to induce or accelerate regression as well as novel diagnostic tools. Recent seminal observations have determined that in resolving liver fibrosis a significant proportion of hepatic stellate cell-myofibroblasts (HSC-MFs) can revert to a near quiescent phenotype. Hepatic macrophages derived from inflammatory monocytes may contribute to fibrosis resolution through an in situ phenotypic switch mediated by phagocytosis. Emerging therapeutic approaches include deletion or inactivation of HSC-MFs, modulation of macrophage activity and autologous cell infusion therapies. Novel noninvasive diagnostic tests such as serum and imaging markers responsive to extracellular matrix degradation are being developed to evaluate the clinical efficacy of antifibrotic interventions.

Systematic review with meta‐analysis: coffee consumption and the risk of cirrhosis

Liver cirrhosis is a large burden on global health, causing over one million deaths per year. Observational studies have reported an inverse association between coffee and cirrhosis.

Models and mechanisms of fibrosis resolution

Liver fibrosis and its end stage, cirrhosis, represent the final common pathway of virtually all chronic liver diseases. As our understanding of the pathogenesis of liver fibrosis has progressed, it has become evident that the liver provides a useful generic model of inflammation and repair, demonstrating interplay between the epithelial, inflammatory, myofibroblast and extracellular matrix components of the mammalian wound healing response. In this review, the paradigm that liver fibrosis is a potentially reversible process-demonstrating both fibrosis (scarring) and resolution with remodeling and restitution of normal or near-normal tissue architecture-will be explored. The remarkable progress in unraveling the complexities of liver fibrosis has been due to developments in technologies including the isolation of discrete liver cell populations which have facilitated studies of their behavior in tissue culture and in vivo. More recently, animal models that mimic chronic liver diseases have been established. These models are tractable and can be applied in gene knockout and transgenic mice. This article will highlight recent studies that reveal key mechanisms mediating the regression of liver fibrosis which have derived from the use of such complementary animal and human model systems and describe how our greater understanding of this dynamic process is likely to inform the development of directed and effective anti-fibrotic approaches.