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Resolution of Liver Fibrosis: Basic Mechanisms and Clinical Relevance

1 The University of Edinburgh/ Medical Research Council (MRC) Centre

for Inflammation Research, Queen’s Medical Research Institute, Edinburgh, United Kingdom Semin Liver Dis 2015;35:119–131.

Abstract

Keywords

► matrix degradation ► myofibroblast fate ► proresolution macrophage ► antifibrotic therapy

Address for correspondence Jonathan A. Fallowfield, BSc, BM, MRCP, PhD, The University of Edinburgh/ Medical Research Council (MRC) Centre for Inflammation Research, Queen’s Medical Research Institute, 47 Little France Crescent, Room W2.30, Edinburgh, EH16 4TJ, United Kingdom (e-mail: [email protected]).

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.

Clinical Relevance Resolution of liver fibrosis, and even cirrhosis, is a wellestablished phenomenon that has not only been observed in models of liver fibrosis in rodents,1,2 but also in patients with chronic liver disease of diverse etiologies after cure of the underlying disease.3 The successful treatment of hepatitis B,4 hepatitis C,5 or hepatitis D6 with antivirals has been shown to lead to reversal of fibrosis, even in some patients with biopsy-proven cirrhosis. Similarly, venesection in hemochromatosis,7 chelation of copper in Wilson’s disease,8 immunomodulatory therapy in autoimmune hepatitis,9 bariatric surgery in nonalcoholic steatohepatitis (NASH),10 bone

Issue Theme Liver Fibrosis; Guest Editors, Robert Schwabe, MD, and Ramon Bataller, MD, PhD

marrow transplantation in thalassaemia,11 and reversal of biliary obstruction12 can also promote fibrosis regression. This is also observed in patients with alcohol-related liver disease after prolonged abstinence, yet specific studies are lacking. But it is recent large-scale trials in chronic viral hepatitis that have shown the most compelling results in terms of remodeling of fibrosis,13–15 such that regression of cirrhosis is no longer considered a lofty ambition or point of contention, rather it is part of the vernacular of contemporary hepatology, a rational goal of treatment, and an advertising strap-line for the new generation of antiviral drugs and surgical therapies for NASH. Critically, regression of fibrosis also appears to correlate with improved clinical outcomes. For

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DOI http://dx.doi.org/ 10.1055/s-0035-1550057. ISSN 0272-8087.

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Prakash Ramachandran, MBChB (Hons), BSc (Hons), MRCP, PhD1 John P. Iredale, BM (Hons), FMedSci, FRCP, FRSE1 Jonathan A. Fallowfield, BSc (Hons), BM (Hons), MRCP, PhD1

Resolution of Liver Fibrosis: Basic Mechanisms and Clinical Relevance example, in patients with hepatitis C virus- (HCV-) related cirrhosis treated with antivirals, “regressors” had better 10year survival rates than “nonregressors” (100% vs. 74%)16 (see also “Liver Fibrosis in the Post-HCV Era” by Pinzani in this issue). Moreover, studies investigating histological–hemodynamic associations in liver fibrosis have demonstrated that semiquantitative (small nodule size, thick fibrotic bands) and quantitative (collagen proportionate area) biopsy characteristics correlate with clinically significant portal hypertension.17–19 These studies have significant pathophysiological and clinical implications. In particular, they focus attention on linking the underlying biology of fibrosis with clinical expression of disease and identify hepatic venous pressure gradient (HVPG; or better, a noninvasive biomarker for HVPG) as a suitable measure to stratify and monitor progression or regression of fibrosis. However, robust long-term longitudinal data tracking fibrosis regression after successful treatment of chronic viral hepatitis is limited. A recent study in patients with HCV used the FibroSure (FibroTest) serum biomarker assay and showed that virological cure was associated with a modest long-term impact on fibrosis.20 After 10 years, 49% of SVR individuals with advanced baseline fibrosis had a significant improvement, but the net reduction of cirrhosis prevalence was only 5%. Disappointingly, there was a residual 5% risk of hepatocellular carcinoma (HCC) despite viral clearance. Clinical outcome data will continue to emerge from ongoing interventional studies in individuals with hepatic fibrosis due to chronic hepatitis B or NASH. Clearly, treatment of the underlying disease may not be sufficient for a proportion of patients and it is likely that as yet undefined environmental and genetic factors will influence this. Therefore, further understanding of molecular events that drive fibrosis regression is needed and may provide additional means of reducing longterm complications of chronic liver disease. In addition to clinical endpoints, antifibrotic trials are likely to require surrogate endpoints that reflect clinically meaningful benefit within shorter observation periods—their definition and validation (probably on a disease-specific basis) remains a priority for the field (see also “Diagnosis of Liver Fibrosis: Present and Future” by Patel, Bedossa, and Castera in this issue).

The Fate of Hepatic Myofibroblasts in Liver Fibrosis Resolution Liver fibrosis becomes problematic, and clinically relevant, when dysregulated and excessive scarring occurs in response to chronic injury and leads to altered tissue function. Although it is clear that the potential for regression of established liver fibrosis exists, for this to occur there must be cessation of active scar production and also degradation of the extracellular matrix (ECM) that has already been deposited to restore normal tissue architecture. As described in detail elsewhere in this issue, the principal cell type responsible for ECM deposition in the damaged liver is the activated HSC,21,22 which transdifferentiates into a MF phenotype following liver injury. In addition to ECM synthesis, activated Seminars in Liver Disease

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HSC-MFs have a range of additional profibrogenic properties,23 including immunomodulatory functions and a possible role in amplifying hepatic injury.24 Furthermore, hepatic MFs are the principal source of tissue inhibitors of metalloproteinases (TIMPs), in particular TIMP1 and to a lesser extent TIMP2.25–28 Tissue inhibitors of metalloproteinases are secreted into the extracellular milieu and bind noncovalently to the active domain and inhibit the group of matrix-degrading enzymes known as matrix metalloproteinases (MMPs), which are potentially capable of breaking down a variety of ECM components including fibrillar collagens. Importantly, even during progressive hepatic fibrosis a range of MMPs are expressed in the liver,29–32 demonstrating the inherent potential for scar degradation. However, TIMP expression by hepatic MFs inhibits this hepatic MMP activity, preventing scar degradation and producing a microenvironment favoring ECM deposition.27,33 The development of animal models of reversible liver fibrosis has enabled more detailed study of the role and fate of the HSC-MF during fibrosis regression. In rats, bile duct ligation results in a dense biliary fibrosis, with a restoration of near normal liver architecture following biliojejunal anastamosis.2 Similarly, following chronic CCl4 injury in rats, advanced liver fibrosis was remodeled to levels close to uninjured liver after the cessation of injury.1 What became clear from these initial studies is that resolution of fibrosis was associated with a profound loss of MFs1,2 from the receding hepatic scar. In addition to the loss of the ECM producing cells with a potential proinflammatory role, a decline in the MF number also removes the source of TIMPs. This has been confirmed in in vivo models, where hepatic MMP levels remained relatively unchanged and loss of TIMP1 during fibrosis resolution altered the overall MMP-TIMP balance resulting in increased hepatic collagenase activity and net ECM degradation.1,34 Indeed, in a transgenic murine model with persistent hepatic overexpression of TIMP1, there was a failure of remodeling of fibrosis even after the cessation of injury.35 Hence, the loss of activated HSCs represents a critical step for matrix degradation to occur. This has led to several pivotal studies in rodent models of fibrosis resolution investigating the fate of MFs and underlying mechanisms that could potentially be exploited therapeutically (►Fig. 1). Although the relevance of these findings will need to be confirmed in human disease, paired liver biopsies before and after long-term lamivudine treatment in patients with chronic hepatitis B did indeed demonstrate a significant reduction in α-SMA staining compared with placebo, indicating that a change in MF number or activation status was associated with regression of fibrosis.4

Myofibroblast Apoptosis Initial studies investigating MF fate demonstrated clear evidence of programmed cell death, or apoptosis, in these cells during fibrosis resolution.1,2 Subsequently, work has focused on the pathways that regulate MF apoptosis in liver.36 It has become apparent that proinflammatory signaling, acting via NF-κB activation in HSC-MFs, results in expression of profibrotic genes and promotes resistance to apoptosis.25,37 TIMP1

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Fig. 1 Summary of the fate of hepatic myofibroblasts (MFs) during liver fibrosis regression. During fibrogenesis quiescent hepatic stellate cells (HSCs) become activated, adopting a MF phenotype and proliferate. During fibrosis regression these activated MFs can undergo apoptosis, induced by immune cell interactions, proapoptotic signals or loss of cell survival signals such as tissue inhibitor of metalloproteinase (TIMP). Activated MFs can also undergo senescence, induced by p53 signaling, IL-22, CCN1/CYR61, and as yet other undefined mechanisms. Recent data demonstrate that activated MFs can also revert to a quiescent HSC like phenotype, which is primed to respond to profibrogenic signals. The signals controlling this reversion process are undefined. Natural killer (NK) cells play an important role in killing activated and senescent MFs. (Adapted with permission from Pellicoro A, Ramachandran P, Iredale JP, Fallowfield JA. Liver fibrosis and repair: immune regulation of wound healing in a solid organ. Nat Rev Immunol 2014;14(3):181–194).

itself can act in an autocrine fashion on hepatic MFs to promote survival and inhibit apoptosis.38,39 Indeed, the interaction between activated HSC-MFs and ECM itself can modulate apoptosis, with collagen I binding promoting HSC activation while loss of integrin-mediated contact leads to HSC-MFs entering the apoptotic pathway.40 Further evidence for the importance of this interaction comes from hepatic injury in the r/r collagen mouse, in which collagen I is resistant to degradation. Liver fibrosis due to chronic CCl4 in these mice failed to resolve and activated HSC-MFs persisted adjacent to the scar tissue.41 A variety of signals have also been demonstrated to stimulate apoptosis in activated HSC-MFs. These include farnesoid X receptor (FXR or bile acid receptor), CEBP/β signaling, cannabinoid receptor 2 (CB2), adiponectin, and TRAIL (also known as TNFSF10).25 Nerve growth factor (NGF) can be released from infiltrating inflammatory cells or regenerating hepatocytes and is proapoptotic for activated HSC-MFs.42–44 Furthermore, direct cellular contact between macrophages and activated HSCs has been demonstrated to promote HSC death.45 The relative contribution of these various regulatory pathways in mediating HSC apoptosis in vivo remains to be elucidated, but it is likely that a complex cellular crosstalk exists that is impossible to recapitulate in vitro.

HSC-MFs in rodent and human liver fibrosis express senescence markers.46 Inhibition of cellular senescence in HSC-MFs using cell-specific p53 knockout mice resulted in increased HSC proliferation and exacerbated liver fibrosis in response to CCl4. Additionally, following the cessation of CCl4, p53 deficiency resulted in persistence of activated HSC-MFs and prevented scar resolution, suggesting that a HSC-MF senescence program is important in the removal of activated MFs during liver fibrosis resolution. Further, in vitro characterization of senescent hepatic MFs demonstrated that senescence resulted in reduced expression of ECM components, increased expression of MMPs, and upregulation of genes involved in immune surveillance, which could potentially aid the clearance of senescent HSC-MFs by NK cells.46 These findings have led authors to investigate the signals inducing MF senescence. CCN1/CYR61, an ECM-associated signaling protein, is one such molecule that has been shown to induce MF senescence and have an antifibrotic effect in vivo.47,48 Additionally, IL-22 can induce HSC-MF senescence and accelerate liver fibrosis resolution.49 Intriguingly, a recent report has suggested that atorvastatin treatment can induce HSC-MF senescence,50 consistent with studies suggesting an antifibrotic effect in humans.51 These findings clearly merit further study.

Myofibroblast Reversion Myofibroblast Senescence Senescence is a stable form of cell-cycle arrest, which has mostly been investigated in the context of malignant disease. Interestingly, it has now been identified that a proportion of

More recently, the development of transgenic mice has enabled lineage tracing of activated MFs and shown that they can also revert to a near-quiescent phenotype following the cessation of injury.52,53 Kisseleva et al utilized transgenic Seminars in Liver Disease

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Resolution of Liver Fibrosis: Basic Mechanisms and Clinical Relevance

Resolution of Liver Fibrosis: Basic Mechanisms and Clinical Relevance mice with fluorescently labeled cells that had expressed collagen I during their lifetime. Using this system, they induced liver fibrosis with CCl4 or alcohol followed by a period of fibrosis resolution. After protracted recovery, when no significant residual liver fibrosis was present, fluorescent quiescent HSCs were detected, indicating an origin from a previously activated MF population.52 Similarly, Troeger et al used transgenic mice to fluorescently label hepatic MFs during CCl4 liver injury and also identified fluorescent quiescent HSCs following fibrosis resolution. The authors estimated that 40% of HSCs derived from reversion to a quiescent phenotype,53 similar to the proportion identified by Kisseleva.52 Both groups also determined that reverted HSCs were not identical to pre-injury quiescent HSCs, with reverted HSCs remaining in a “primed” state and demonstrating an augmented response to further fibrogenic stimuli.52,53 Although the signals causing MF reversion rather than apoptosis and the factors responsible for the maintenance of this inactivated but primed HSC population remain to be elucidated, these findings may have major implications for human liver fibrosis, where periods of repetitive tissue injury are a common feature (e.g., in alcoholrelated liver disease).

Regulation of Fibrosis Resolution by Immune Cells As discussed, the activation and subsequent fate of HSC-MFs are key determinants of liver fibrosis progression and regression. There is now compelling evidence supporting the role of different immune cell populations in promoting fibrosis resolution.

The Role of the Macrophage The role of cells of the monocyte/macrophage lineage in hepatic fibrogenesis and fibrosis resolution has been most widely studied (►Fig. 2). This is perhaps unsurprising, given that a significant proportion of hepatic macrophages are found in very close proximity to MFs and scar tissue following chronic hepatic injury (so-called scar-associated macrophages [SAMs]).54–56 Investigators have used different macrophage depletion strategies in rodent models of liver fibrosis including gadolinium chloride,57 liposomal clodronate,58,59 or transgenic CD11b-DTR mice (where administration of diphtheria toxin results in depletion of CD11b-expressing macrophages),54 and consistently shown that removal of hepatic macrophages during ongoing injury results in reduced liver fibrosis. Macrophages are potent producers of proinflammatory and profibrotic mediators and this injury promoting effect may well be mediated through interactions with HSC-MFs. Specifically, macrophages can produce the archetypal profibrogenic cytokine TGFβ, which acts on MFs to promote activation and ECM synthesis.60,61 Additionally, they can express high levels of thrombospondin-1, a potent activator of TGFβ in vivo.56 Macrophages are also a potential source of other soluble mediators including TNF-α and IL-1β (which act on activated HSCs to induce NF-κB activity, promote survival, and increase liver fibrosis59), plateletSeminars in Liver Disease

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derived growth factor (which can induce MF proliferation), IL-4 and IL-13 (which can directly stimulate collagen synthesis in fibroblasts), CCL7 and CCL8 (which can recruit MFs), and a range of other chemokines that can promote further leucocyte migration and perpetuate the inflammatory response.61 Galectin-3 is a macrophage-derived lectin that has been shown to promote MF activation in both liver and kidney fibrosis models62,63 and whose inhibition has an antifibrotic effect.64 Such profibrogenic mediators are also produced by human monocytes/macrophages derived from patients with chronic liver disease.65–67 Despite this array of profibrogenic capabilities, intriguing data also describe a role for macrophages in the resolution of liver fibrosis (►Fig. 2). Depletion of macrophages during the resolution of liver fibrosis using the aforementioned CD11bDTR mice54 or MAFIA (CSF1R-FKB12) mice68 demonstrated a clear failure of matrix degradation. Similar observations have been made during the resolution of murine pulmonary fibrosis.69 The mechanisms underpinning these exciting findings are the subject of active investigation. What is clear is that macrophages, in particular SAMs, are a key source of matrix-degrading MMPs, including MMP1355 and MMP12.70 This macrophage MMP expression is upregulated by phagocytosis of dead cells,56,71 a major function of this cell type in inflamed tissue. Furthermore, macrophages are a potential source of molecules such as MMP956 and TRAIL,45 which can promote MF apoptosis. To understand the apparently dichotomous effects of macrophages in liver fibrosis, it is important to appreciate the potential heterogeneity of macrophages. It is now apparent that the traditional M1/M2 classification is wholly inadequate to describe in vivo macrophage phenotypes.56,72 Rather, a classification on the basis of ontogeny and function is more likely to be useful. In the liver, resident Kupffer cells make up the majority of hepatic macrophages during the steady state; indeed they are the largest macrophage pool in the entire body.73 A distinct population of hepatic monocyte-derived macrophages can also be identified.56,73–75 Although there is a longstanding debate as to the origin of these distinct populations, a recent series of lineage tracing murine models have demonstrated that under steady state, resident Kupffer cells derive from yolk sac macrophages and are maintained via local proliferation with no meaningful contribution from recruited monocytes.76–78 The precise function of these distinct resident macrophages following hepatic injury remains to be defined. However, following injury there is a massive expansion of the total hepatic macrophage pool, predominantly via recruitment of circulating monocytes.73 Further studies have identified a subset of this population, expressing high levels of the marker Ly6C and recruited via the CCR2-CCL2 axis, as being a principal driver of hepatic fibrogenesis.60,79,80 These Ly-6Chi monocyte-derived cells also have a profibrotic role in lung69 and renal injury.81 Our group went on to identify a population of Ly-6Clo hepatic monocyte-derived macrophages, termed the “restorative macrophage,” which was responsible for murine liver fibrosis resolution following chronic CCl4 injury.56 These cells,

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Fig. 2 Schematic showing the proposed role of macrophages in hepatic fibrogenesis and fibrosis regression. In response to hepatic injury, inflammatory monocytes are recruited to the liver and form profibrotic Ly-6Chi macrophages in the liver. Ly-6C hi macrophages can express proinflammatory cytokines (IL-1β and TNF-α), which promote hepatocellular damage and myofibroblast (MF) survival, chemokines that promote recruitment of inflammatory cells/monocytes (CCL2, CXCL10) and MFs (CCL7, CCL8), TGFβ and galectin-3 that promote activation of hepatic stellate cells (HSCs) into a MF phenotype, thrombospondin-1 (thbs1) that can activate latent TGFβ, and platelet-derived growth factor (PDGF) that promotes MF proliferation. Activated MFs proliferate and secrete extracellular matrix (resulting in scar formation) and TIMP1, which inhibits both matrix-degradation by matrix metalloproteinases (MMPs) and HSC apoptosis. Within the liver these profibrogenic macrophages then change phenotype, at least partly in response to phagocytosis of cellular debris. This results in the generation of Ly-6Clo restorative hepatic macrophages, which mediate fibrosis regression. These restorative macrophages upregulate opsonins and phagocytosis receptors, enhancing the clearance of cellular debris, and hence promoting the generation of further restorative macrophages. Ly-6C lo macrophages downregulate proinflammatory cytokine expression promoting MF apoptosis, may express proapoptotic mediators such as MMP9 and TRAIL, and upregulate matrix-degrading enzymes such as MMP12, MMP13, and MMP9, which in combination with falling TIMP1 levels, results in increased hepatic matrix degrading activity and hence scar degradation. Restorative macrophage can also express Arginase-1, anti-inflammatory receptors (CX3CR1), proresolution chemokines such as CXCL9 and growth factors such as IGF-1, which may promote hepatocyte regeneration. Vascular endothelial growth factor has a dual role, promoting monocyte recruitment but also proresolution features in hepatic macrophages. (Adapted with permission from Pellicoro A, Ramachandran P, Iredale JP, Fallowfield JA. Liver fibrosis and repair: immune regulation of wound healing in a solid organ. Nat Rev Immunol 2014;14(3):181–194.)

which accumulate maximally in livers at the time of scar degradation, are derived from a phenotypic switch of profibrogenic Ly-6Chi macrophages potentially induced by macrophage phagocytosis, resulting in downregulation of proinflammatory cytokines and chemokines and increased expression of MMPs and other antifibrotic genes such as CX3CR182,83 and arginase-1.61 The fact that the proresolution macrophage subset has the same origin as the profibrogenic subset is supported by data from transgenic CCR2 deficient mice, which although partially protected from liver fibrogenesis are also unable to degrade the accumulated scar.80 Recent findings by Baeck et al shed further light on this process.84 Using a Spiegelmer technique, they inhibited CCL2 in vivo during the resolution of murine liver fibrosis from either CCl4 or MCD diet (a murine model of NASH). This intervention caused a reduction in profibrogenic Ly-6Chi monocyte-derived macrophages and an increase in restor-

ative Ly-6Clo macrophages and resulted in accelerated fibrosis resolution.84 These findings mirror our own, where the administration of liposomes during fibrosis resolution induced phagocytic behavior and caused a very similar change in hepatic macrophage subsets with a consequent increase in matrix degradation.56 Hence, it seems to be the balance of profibrotic and restorative macrophages that governs the overall tissue phenotype and a greater understanding of the mechanisms controlling this could yield novel therapeutic targets. A recent insight into this has been provided by Yang et al.68 Vascular endothelial growth factor (VEGF) has previously been thought to be a profibrogenic molecule85 and inhibition of VEGF has been suggested as a potential therapy for fibrosis.86 However, blockade of VEGF during resolution from murine BDL or CCl4 liver fibrosis prevented fibrosis regression while overexpression of VEGF accelerated it.68 The beneficial effect of VEGF during fibrosis resolution seemed to Seminars in Liver Disease

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Resolution of Liver Fibrosis: Basic Mechanisms and Clinical Relevance

Resolution of Liver Fibrosis: Basic Mechanisms and Clinical Relevance be mediated via effects on sinusoidal endothelial permeability affecting monocyte recruitment and also direct stimulation of SAMs to produce MMP13 and the antifibrotic chemokine CXCL9.68,87 This apparent dual role of VEGF in fibrogenesis and fibrosis resolution resembles that seen with macrophages overall and highlights an important point when considering translational strategies: namely that caution needs to be exercised when identifying antifibrotic targets as these molecules may also have a key role in fibrosis resolution.88 What remains to be seen is how closely this murine macrophage heterogeneity relates to human disease. Similar to murine Kupffer cells, a population of “resident” macrophages can be identified in human cirrhotic liver as CD14l ° CD16
, while human hepatic monocyte-derived macrophages could be defined as CD14hiCD16
and CD14þCD16þ subsets.65,66 Interestingly, analogous to murine data on the restorative macrophage, CD14þCD16þ macrophages can derive from CD14hiCD16
cells and have a high phagocytic capacity,66 but this same population also expresses high levels of proinflammatory mediators and can directly activate HSCs.65,66 It may well be that further, as yet undefined, heterogeneity exists within these macrophage subpopulations in human liver.

Other Immune Cells Several other immune cell populations have been studied in relation to a role in hepatic fibrosis, albeit in less depth than monocytes/macrophages. We will briefly summarize the data suggesting an antifibrotic role for these cell types.

Dendritic Cells Dendritic cells (DCs) may mediate ECM degradation during liver fibrosis resolution, potentially via MMP9 expression.89 These findings merit further investigation, but a note of caution is that the distinction between macrophages and DCs is not always clear cut, particularly in the context of inflammation.90

Neutrophils

Despite being an early responder to hepatic injury,91 no clear role for neutrophils has been identified in hepatic fibrogenesis.92 Some authors have proposed a potential role for neutrophil-derived MMPs in fibrosis resolution,93 but it is likely that neutrophils are not present in livers in sufficient numbers for this to be a meaningful effect during fibrosis resolution.

Natural Killer Cells Natural killer (NK) cells have been studied in liver fibrosis and have been demonstrated to have an antifibrotic effect (►Fig. 1).94–96 This seems to be mediated via NK cell-mediated killing of activated HSCs as a result of IFN-γ expression and/or expression of death receptors or ligands such as NKG2D,94 TRAIL97 or FasL.98 Additionally, NK cells have a key role in the clearance of senescent HSCs, with NK cell depletion causing persistent senescent HSCs in vivo and a failure of scar resolution, while activation of NK cells accelerSeminars in Liver Disease

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ated removal of senescent HSCs and augmented fibrosis resolution.46

T Cells T cells play a central role in the adaptive immune response, with a variety of subtypes having differing immunomodulatory roles. T helper cells (TH cells) become activated by interactions with antigen presenting cells (APCs) and secrete cytokines that influence other immune cells. The cytokine profile expressed by the TH cells is dependent on the nature of the signaling from the APC. In liver fibrosis, the TH2 subtype, expressing IL-4, IL-5, IL-13, and IL-21, has been shown to be strongly profibrogenic.99 Conversely, a TH1 immune response is associated with IFN-γ and IL-12 expression, which have been shown to be antifibrotic.99 Thus, the relative balance of TH1 and TH2 cells may influence the degree of fibrosis progression.100 This is likely to be an overly simplistic view as our understanding of the complexities of T cell responses increases. Regulatory T cells (Treg) for example, are a specific subset that has been associated with an antifibrotic effect in animal models101 and human liver disease.102

γδ T Cells γδ T cells are a specific subpopulation of nonconventional T cells that share features of innate immune cells and conventional T cells, the liver being one of the richest sources of this subpopulation in the body. A recent study has identified that these γδ T cells, recruited to the liver via CCR6, have an antifibrotic effect potentially via the induction of HSC apoptosis in a FasL-dependent manner.103

Is Liver Fibrosis Always Reversible? Having discussed data demonstrating that liver fibrosis is reversible and some of the underlying mechanisms, a key question is whether all liver fibrosis is reversible, and if not, what factors limit ECM degradation? Critically, cirrhosis is not simply advanced fibrosis, but also encompasses architectural disruption, nodular hepatocyte regeneration, and vascular changes including angiogenesis. Although rodent models have been invaluable in defining the pathophysiology of liver fibrosis, it is worth noting that liver fibrosis is less reversible in humans, probably as a result of the dense fibrosis that typically develops over decades (rather than weeks in rodents) and the presence of prominent angioarchitectural changes such as vascularized septae that are probably irreversible.104 Indeed, the fibrotic response to chronic CCl4 in mice is weak and resolves very rapidly after withdrawal of the toxin. Outbred rats (e.g., Sprague-Dawley) exhibit a more robust fibrotic reaction and it is possible to generate cirrhosis.34 Chronic thioacetamide treatment in mice also appears to induce cirrhosis that does not reverse.105 In cirrhotic rats, although significant matrix remodeling occurred during recovery (particularly areas of recently deposited scar), fibrotic septae failed to completely regress even after 1 year, resulting in an attenuated macronodular pattern34 similar to that described in the human cirrhosis specimens by Wanless and colleagues.106 Close scrutiny of residual (compared

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with remodeled) ECM in this model showed that persistent scars were heavily crosslinked (potentially mediated by tissue transglutaminase or lysyl oxidase enzymes), rich in elastin, and were relatively hypocellular (lacking in MFs and inflammatory cells capable of secreting proteases).34 It is unlikely that any of these mechanisms operate in isolation. For example, tissue transglutaminase seems dispensable for ECM crosslinking in murine liver fibrosis,105 while lysyl oxidase has immunomodulatory functions in addition to effects on ECM crosslinking.107 It is likely that a combination of ECM modifications, in addition to physical factors (e.g., deeper areas of dense fibrous scars may simply be inaccessible to MMPs) confer resilience to established hepatic scar tissue. The differences between liver fibrosis in mice and men may partly explain why translation of efficacious preclinical antifibrotic candidate drugs (such as INFγ or glitazones) has not always proven to be as successful in humans.108,109 However, while entirely normal liver architecture may never be fully restored, even limited matrix remodeling may be sufficient to reduce portal pressure, risk of HCC, and improve patient outcomes. Susceptibility to fibrosis regression may also differ with etiology. Moreover, etiological variation in the pattern of fibrosis distribution (i.e., postnecrotic, biliary, centrilobular, or perisinusoidal) influences the degree of associated angiogenesis, which in turn may increase the rate of disease progression to cirrhosis and limit the potential for fibrosis regression.110 Interestingly, at the time of transplantation, cirrhotic livers were shown to have different ranges of collagen proportionate area (CPA), according to etiology (e.g., median CPA in alcohol-related cirrhosis 30% compared with 16.5% in HBV-related cirrhosis).111 However, although qualitative and quantitative differences have been identified in human biopsy samples, we need better tissue markers of regressing fibrosis and “the point of no return” to distinguish patients who may reverse fibrosis (e.g., in response to disease-specific or antifibrotic drug therapy) from those who probably will not.

Resolution of Fibrosis and Liver Regeneration In contrast to other adult tissues, the liver has a remarkable regenerative capacity—a phenomenon observed in all vertebrate organisms. However, epithelial cell proliferation is severely blunted in human chronic liver disease due to several mechanisms including hepatocyte cell cycle arrest, upregulated p21 expression, and senescence.112 As a result many patients with end-stage cirrhosis will require a liver transplant. For functional restitution to occur, resolution of liver fibrosis must also initiate a robust regenerative response. Studies have shown a critical link between ECM degradation and hepatic epithelial regeneration. In the aforementioned study by Issa et al, mice bearing a mutated collagen I gene (r/r mice), which confers resistance to collagenase degradation, demonstrated less fibrosis regression, but also a blunted hepatocyte regenerative response compared with wild-type controls.41 Furthermore, using extracted collagen I from each

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genotype as culture substrata, wild-type collagen I promoted hepatocyte proliferation via stimulation of integrin αvβ3.41 In advanced liver damage, hepatic regeneration can also occur through proliferation of a transit-amplifying population of “facultative” resident hepatic progenitor cells (HPCs), located within a stereotypical niche in close association with MFs and bone marrow (BM) derived macrophages.113 It was shown using r/r mice and MMP13 knockout mice that failure of ECM remodeling in experimental fibrosis hindered the ability of the liver to activate HPCs.114 HPCs are bipotential (i.e., can differentiate into hepatocytes or cholangiocytes) and subsequent HPC fate is determined by differential signaling cascades and underlying disease etiology.115 In fact, hepatic macrophages may also play a key role in epithelial regeneration, by expression of molecules such as TWEAK to promote HPC proliferation,116 Wnt3a to promote HPC differentiation to hepatocytes,115 and growth factors such as IGF-156 that can act directly on hepatocytes to stimulate proliferation and survival (►Fig. 2).117,118 Finally, the recent finding in mice that stimulation of 5-HT2B serotonin receptors on activated HSCs inhibited hepatocyte regeneration via increased TGFβ production119 indicates that, in addition to fibrosis resolution, exploring the relationship between cellular regeneration and fibrogenesis in the liver could also identify new therapeutic approaches as an alternative to liver transplantation.

Regression of Fibrosis: Diagnostic and Therapeutic Applications Our increasing knowledge of the underlying mechanisms and natural history of liver fibrosis/cirrhosis resolution offers opportunities to exploit these insights to develop new diagnostic tests to track disease regression and new treatments that directly promote liver repair.

Diagnostic Approaches for Monitoring Fibrosis Resolution As described by Patel et al in this issue, liver biopsy for the assessment of fibrosis has several limitations. These make it unsuitable for monitoring regression of disease in response to etiology-specific or antifibrotic treatments where large sample sizes and prolonged study duration would be required. Although currently available noninvasive serum and imaging (e.g., FibroScan) markers of hepatic fibrosis have demonstrated acceptable performance for the diagnosis of advanced versus no/mild fibrosis, they are unlikely to be accurate enough to evaluate the subtle changes expected from the pharmacological treatment of fibrosis, especially at earlier stages of disease. Furthermore, no consensus exists on what minimum change in serum marker level defines a clinically important effect and there are very limited data on the use of elastography techniques (transient or magnetic resonance imaging [MRI]) to monitor fibrosis regression. 120 The availability of a quantitative noninvasive serum or imaging biomarker, especially a dynamic measure of fibrogenesis or fibrolysis, would circumvent many of these issues. Seminars in Liver Disease

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Resolution of Liver Fibrosis: Basic Mechanisms and Clinical Relevance

Resolution of Liver Fibrosis: Basic Mechanisms and Clinical Relevance One approach that may hold promise is the detection of serum protein markers that reflect connective tissue formation and degradation (turnover). When specific MMPs degrade ECM components characteristic neo-epitopes are exposed at the site of degradation and these can be detected in serum by enzyme-linked immunosorbent assay (ELISA). Serum levels of specific ECM markers have been shown to correlate with levels of hepatic fibrosis in CCl4-treated rats.121 Furthermore, a combination of two serum ECM markers and the Model for End Stage Liver Disease (MELD) score correlated significantly with HVPG in patients with alcohol-related cirrhosis.122 These protein fingerprints, directly associated with ECM turnover, may change more rapidly than other measures of liver fibrosis (e.g., biopsy collagen, imaging stiffness) and thus could be utilized as early diagnostic, prognostic, and treatment-monitoring markers. Novel dynamic imaging methods that employ a small molecular ligand for ECM components or a cell-associated molecule coupled to a radioimaging or MRI agent could provide a sensitive indicator of change in fibrosis over the whole liver and permit early assessment of antifibrogenic effect in therapeutic trials. Gadolinium-based MRI probes targeted to collagen I123 or fibronectin124 have shown promise. The use of molecular tracers that bind to cell surface receptors overexpressed in the fibrotic liver (such as αvβ6 on activated cholangiocytes125 or serotonin receptors on HSCMFs126) is an attractive approach, but signal-to-noise ratio could be a limiting factor.

Therapeutic Application A comprehensive review of potential antifibrotic therapies is discussed in detail in the article “Antifibrotic Therapies in the

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Liver” by Mehal and Schuppan in this issue. A variety of promising drug therapies have been shown to directly target HSC-MFs or accumulated scar ECM (►Table 1). Combined development of drug-targeting methodology could enable an oncology-type approach to deliver potent drugs selectively to the fibrotic liver in a cell-specific manner. Alternatively, cell infusion approaches could potentially induce matrix degrading and/or pro-regenerative effects in chronic liver disease (►Table 1).127 There are some concerns regarding the use of autologous unsorted bone marrow cell (BMC) infusions for the treatment of liver fibrosis as the BMCs contain mesenchymal stem cells that can differentiate into MFs in certain settings. A better strategy might be to use macrophages, hematopoietic stem cells, or BM mononuclear cells. In particular, macrophages are biologically well-defined and can be generated in large numbers from blood monocytes. Injected autologous macrophages have been shown to be antifibrotic, anti-inflammatory, and stimulate liver regeneration in models of liver injury and fibrosis.128 Several hurdles remain with respect to clinical translation of emerging antifibrotic therapies. First, standard operating procedures for the best preclinical models for proof-ofconcept studies are long overdue. History indicates that inappropriate selection of preclinical models may lead to future disappointment in clinical trials.129 In addition, antifibrotic drug development is currently hindered by the long and usually asymptomatic natural history of most fibrotic liver diseases, varying clinical phenotypes that have not been defined (e.g., the small % of patients with nonalcoholic fatty liver disease who progress to NASH), lack of well-defined and validated endpoints (both for cirrhotic and noncirrhotic trial populations), and lack of data to support the Food and Drug

Table 1 Potential antifibrotic therapies that target fibrosis regression or liver regeneration Promote apoptosis, quiescence, or senescence of hepatic myofibroblasts • Sulfasalazine or sulfasalazine analogs130 • Cannabinoid receptor (CB1) antagonists131 • TIMP1 blockade using siRNA,132 neutralizing antibody133 or inactive MMP sump134,135 • Hepatic stellate cell-myofibroblast specific targeted therapies o Anti-EGFR single chain fragment variable antibody-TRAIL fusion protein136 o Vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone137 o Single chain antibody (C1–3) targeted gliotoxin138 • PPARγ agonism or derepression139 • FXR agonism140,141 • Atorvastatin50,51 Stimulate degradation of accumulated scar ECM • TIMP1 blockade using siRNA,132 neutralizing antibody133 or inactive MMP sump134,135 • MMP gene therapy142,143 • Halofuginone144 • Lysyl oxidase (LoxL2) inhibitors107 • Cell therapies o Autologous granulocyte colony stimulating factor (G-CSF) mobilized CD133þ bone marrow stem cells145 o Autologous CD14þ monocyte-derived macrophages146 Target the immune response • Blockade of CCL2/CCR284 • Enhance restorative macrophages by liposome administration56 • Modulation of CXCL9/CXCR3 axis87,147 Abbreviations: ECM, extracellular matrix; EGFR, epidermal growth factor receptor; FXR, farnesoid X receptor; MMP, Matrix metalloproteinase; PPAR, peroxisome proliferator-activated receptor. Seminars in Liver Disease

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Administration’s “reasonably likely to predict clinical benefit” standard of surrogate endpoints. Although trial populations with more advanced stage disease have higher clinical event rates (4%/y), such hard endpoints may not be suitable for some antifibrotic drug targets (e.g., metabolic therapies in NASH). Moreover, fibrosis in patients with established cirrhosis will inherently be less reversible. Until these issues are resolved, translation (and prioritization) of antifibrotic candidates will be challenging. In the future, selection and stratification of trial participants should be based on those most likely to progress/respond. The “ideal” antifibrotic treatment would be potent yet specific, easily administered, and safe/well-tolerated as most patients are likely to be asymptomatic. In particular, treatment in cirrhotic patients should not increase risk of HCC.

Flt3 HCC HCV HPC HSC-MF HVPG IGF-1 MCD MMP NASH NK cell SAM SVR TIMP TRAIL

Conclusion

TWEAK

As we have discussed above, there is now compelling evidence from patients and rodent models that significant remodeling of even relatively advanced liver fibrosis is possible with net matrix degradation, a loss of the activated MF physically or functionally, and restitution of more normal liver architecture. Very recent data suggests that macrophages, recruited in the inflammatory phase but remaining in situ, orchestrate the resolution of fibrosis and may directly influence HPC proliferation and promote their specification to hepatocytes, thereby repopulating organ parenchyma. These recent steps forward in our understanding of the resolution of liver fibrosis pave the way to the design of novel treatments based on drugs, small molecules, and arguably, cell-based therapies. At the same time, research into fibrogenesis and fibrosis resolution has for the first time generated a panoply of surrogate markers of fibrosis and fibrosis degradation, which will facilitate the future evaluation of these new treatments.

VEGF Wnt3a YFP

Abbreviations α-SMA ANIT BDL BM CCl4 CCL2 CCL7 CCL8 CCN1/CYR61 CCR2 CCR6 Cd11b Cd11c CX3CR1 CXCL9 DTR ECM FasL

α-smooth muscle actin alpha-naphthylisothiocyanate bile duct ligation bone marrow carbon tetrachloride chemokine (C-C motif) Ligand 2 (MCP-1) chemokine (C-C motif) ligand 7 (MCP-3) chemokine (C-C motif) ligand 8 (MCP-2) cysteine rich protein 61 chemokine (C-C motif) receptor 2 chemokine (C-C motif) receptor 6 integrin αm integrin αx chemokine (C-X3-C motif) receptor 1 (fractalkine receptor) chemokine (C-X-C motif) ligand 9 human diphtheria toxin receptor (DTR) extracellular matrix Fas ligand (TNF superfamily, member 6)

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FMS-like tyrosine kinase 3 hepatocellular carcinoma hepatitis C virus hepatic progenitor cell hepatic stellate cell-myofibroblast hepatic venous pressure gradient insulin-like growth factor 1 methionine choline deficient diet matrix metalloproteinase nonalcoholic steatohepatitis natural killer cell scar-associated macrophage sustained virological response tissue inhibitor of metalloproteinase tumor necrosis factor (ligand) superfamily, member 10 (Tnfsf10) tumor necrosis factor (ligand) superfamily, member 12 vascular endothelial growth factor drosophila melanogaster wingless 3a yellow fluorescent protein

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Resolution of Liver Fibrosis: Basic Mechanisms and Clinical Relevance

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