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REVIEWS Novel therapeutic targets in primary biliary cirrhosis Jessica K. Dyson, Gideon M. Hirschfield, David H. Adams, Ulrich Beuers, Derek A. Mann, Keith D. Lindor and David E. J. Jones Abstract | Primary biliary cirrhosis (PBC) is a chronic immune-mediated liver disease characterized by progressive cholestasis, biliary fibrosis and eventually cirrhosis. It results in characteristic symptoms with marked effects on life quality. The advent of large patient cohorts has challenged the view of PBC as a benign condition treated effectively by the single licensed therapy—ursodeoxycholic acid (UDCA). UDCA nonresponse or under-response has a major bearing on outcome, substantially increasing the likelihood that liver transplantation will be required or that patients will die of the disease. In patients with high-risk, treatmentunresponsive or highly symptomatic disease the need for new treatment approaches is clear. Evolution in our understanding of disease mechanisms is rapidly leading to the advent of new and re-purposed therapeutic agents targeting key processes. Notable opportunities are offered by targeting what could be considered as the ‘upstream’ immune response, ‘midstream’ biliary injury and ‘downstream’ fibrotic processes. Combination therapy targeting several pathways or the development of novel agents addressing multiple components of the disease pathway might be required. Ultimately, PBC therapeutics will require a stratified approach to be adopted in practice. This Review provides a current perspective on potential approaches to PBC treatment, and highlights the challenges faced in evaluating and implementing those treatments. Dyson, J. K. et al. Nat. Rev. Gastroenterol. Hepatol. 12, 147–158 (2015); published online 3 February 2015; doi:10.1038/nrgastro.2015.12 Introduction Institute of Cellular Medicine, 3rd Floor William Leech Building, Medical School, Framlington Place, Newcastle University, Newcastle upon Tyne NE2 4HH, UK (J.K.D., D.A.M., D.E.J.J.). Centre for Liver Research, NIHR Biomedical Research Unit, University of Birmingham, Wolfson Drive, Birmingham B15 2TT, UK (G.M.H., D.H.A.). Department of Gastroenterology & Hepatology, Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, G4‑216, University of Amsterdam, PO Box 22600, NL‑1100 DD, Amsterdam, Netherlands (U.B.). College of Health Solutions, Arizona State University, 550 North 3rd Street, Phoenix, AZ 85004, USA (K.D.L.). Correspondence to: J.K.D. jessicadyson@ doctors.org.uk Primary biliary cirrhosis (PBC) is a chronic immunemediated liver disease characterized by damage to, and destruction of, the biliary epithelial cells (BECs) and small intrahepatic bile ducts, leading to progressive cholestasis, biliary fibrosis and eventually cirrhosis.1 PBC, which has a prevalence of up to 35 per 100,000 in Europe,2 affects patients through the development of cirrhosis with its attendant complications and risk of death, and through the development of characteristic symptoms including pruritus and fatigue with substantial effects on quality of life.3,4 PBC was, until the past 5 years or so, perceived to be a typically benign condition of those >50 years old, effectively treated by a single cheap and safe licensed therapy, ursodeoxycholic acid (UDCA). The advent of Competing interests G.M.H. is an investigator and/or consultant for Biotie Therapies, Bristol–Myers Squibb, Falk Pharma, FF Pharma, Gilead, GlaxoSmithKline, Intercept, Johnson & Johnson and NGM Biopharmaceuticals; he is a co-investigator for UK‑PBC, supported by a stratified medicine award from the UK Medical Research Council. U.B. has consultant agreements via the University of Amsterdam with Intercept and Novartis, and receives lectures fees from the Falk Foundation. D.A.M. is a consultant and collaborator with GlaxoSmithKline, Medimmune, Novartis and UCB. K.D.L. is an unpaid member of the advisory board for Intercept and Lumena and a paid consultant for Abbvie and Gilead. D.E.J.J. receives UK‑PBC research and trial funding from GlaxoSmithKline, Intercept and Lumena, and acts as a consultant via Newcastle University for Intercept, Johnson & Johnson and Novartis. The other authors declare no competing interests. national and international patient cohorts has, however, challenged this view,4 identifying substantial unmet clinical need,5 particularly in patients showing inadequate biochemical response to UDCA (which includes up to 30% of patients, rising to >50% in patients presenting under the age of 40 years4). Poor biochemical response to UDCA has a major effect on outcome, substantially increasing the risk of death or need for liver transplantation.6,7 In addition, UDCA treatment has little effect on the characteristic symptoms of PBC such as pruritus and fatigue.3,4 Awareness of unmet need has triggered marked interest in second-line therapy for PBC. This Review provides a current perspective on potential new approaches to treatment in PBC, and highlights some of the challenges we face in evaluating and effectively implementing those treatments. Current status of second-line therapy At present, no second-line therapies for patients with inadequate response to UDCA therapy have been approved. A number of agents have, however, undergone evaluation in patients stratified for nonresponse to UDCA. With the availability of new drugs on the horizon, the question of tailoring therapy to individual patients will become progressively more important. Three interlinked biological processes are at play in PBC (Figure 1), with drug development activity related to each. The initial pathogenic insult is conventionally thought to be targeted at BECs, and to be immunological NATURE REVIEWS | GASTROENTEROLOGY & HEPATOLOGY © 2015 Macmillan Publishers Limited. All rights reserved VOLUME 12 | MARCH 2015 | 147 REVIEWS Key points ■■ Primary biliary cirrhosis (PBC) leads to progressive cholestasis, biliary fibrosis and cirrhosis and characteristic symptoms with a marked effect on life quality ■■ Ursodeoxycholic acid (UDCA) nonresponse or under-response has a major effect on outcome, substantially increasing the likelihood that liver transplantation will be required, or that patients will die of the disease ■■ Patients with high-risk, treatment-unresponsive or highly symptomatic disease need new treatment approaches; as yet, no agents demonstrating disease‑modifying actions have been shown to improve systemic symptoms ■■ Excellent opportunities are offered by targeting the immune response, biliary injury and fibrotic processes ■■ Combination therapies with a stratified approach, in which specific treatments are targeted at the pathological processes at play, or single agents with multiple actions, are likely to be needed in nature. Broadly acting immunosuppressive therapies have, however, proved disappointing to date (at least when used in a non-stratified manner) and have not entered routine use (Supplementary Table 1 online). A number of these agents and approaches might warrant re-evaluation under the stratified therapy model that targets secondline therapies at UDCA nonresponders. Furthermore, interest in targeted immunosuppression has increased following the characterization of the role of immunoregulation in PBC risk8,9 and improved application of disease stratification,4,10 and a number of trials of biologic agents are underway. Approaches have included ustekinumab (NCT0138997311)—an antibody directed at the common IL‑12/IL‑23 p40 chain—underpinnedby evidence from genetic studies and others implicating the IL‑12 and IL‑23 pathways in PBC.12–14 Another agent that has been evaluated is NI‑08091—an anti-CXCL10 monoclonal antibody (NCT0143042915)—supported by evidence implicating the CXCR3/CXCL10 chemokine/receptor pair in T‑cell homing into the liver in PBC.16 A further agent potentially set for PBC studies (NCT0219336017) is FFP104, an anti-CD40 human monoclonal antibody; a role for the CD40/CD40L (CD154) interaction in T‑cell licensing and B‑cell activation has already been proven, as well as a parallel role for this interaction in BEC apoptosis.18 In addition to T‑cell-directed immunotherapy, B‑celltargeted therapy has undergone evaluation in the form of the anti-CD20 monoclonal antibody, rituximab (NCT00364819 19). Benefit, albeit very limited, has been shown in terms of improving liver biochemistry in patients who do not respond to UDCA.20,21 However, although the safety profile of rituximab in PBC trials seems benign,20,21 concern remains because of demonstration of disease deterioration in mouse models of PBC following anti-CD20 therapy,22 and descriptions of colitis in human trials in other disease settings.23 All biologic immunotherapy trials to date, however, have evaluated efficacy in patients who did not respond to UDCA therapy and who could thus be regarded as being in the second, cholestatic phase of the disease. The difficulty is that patients present with high alkaline phosphatase levels, implying that the phase of disease might not be well distinguished by biochemical markers. A critical question is the extent to which ongoing immune injury is present in such patients and, if so, whether it 148 | MARCH 2015 | VOLUME 12 has reached a level characterized by persistent epithelial and stromal cell activation that makes it more resistant to standard immunosuppression. This issue might explain the disappointing results seen with biologic agents to date. Alternatively, perhaps trials are not continued long enough to see an effect owing to the selected end point being alkaline phosphatase levels. As yet, no ‘diseasemodifying therapy’ (modification of the immune process in patients with the earliest evidence of immune injury and prior to UDCA use) has been attempted. Work is currently ongoing to try to identify biomarkers associated with immune injury risk in PBC to better target immunotherapy at potentially responsive patients. After early (or initiating) immune injury, the disease process seems to combine cholestasis and fibrosis. The relative role of each of these mechanisms at a given time in the course of the disease is unknown. The fact that UDCA seems to be effective and have a major anti cholestatic effect suggests that cholestasis has a central role in disease progression. Current second-line therapy evaluation in the areas of cholestasis and fibrosis is largely around two therapy types, farnesoid X receptor (FXR) agonists (in particular obeticholic acid, which has shown efficacy in both phase II and III evaluation)24,25 and peroxisome proliferator-activated receptor‑α (PPARα) agonists (bezafibrate and fenofibrate), which have shown efficacy in case reports and series, and which are currently undergoing phase III evaluation (NCT0165473126).27,28 The fibroblast growth factor‑19 (FGF‑19) analogue NGM282 is also undergoing evaluation (NCT0202640129) and is relevant given the evidence suggesting that FXR agonists act in part through FGF‑19 upregulation.30 Other ongoing trials in PBC are summarized in Table 1. Future directions in prognostic therapy Targeting the immune response If we are to move beyond current therapy, and those agents currently under evaluation, what are the opportunities for new target development, and how do they fit into our understanding of the stages of disease pathogenesis? PBC is undoubtedly a disease with an autoimmune component; abundant human and animal data have demonstrated the central importance of a highly specific and orchestrated immune response to BECs (Figure 2). Reactivity occurs at both the T‑cell and B‑cell level to highly conserved mitochondrial antigens, in particular the E2 component of the pyruvate dehydrogenase complex (PDC).1,31–33 With evermore sophisticated approaches to manipulating the immune system (such as antibody-mediated therapy, targeted inhibitors of cellular pathways relevant to immune signalling, as well as broader cell therapy approaches aimed at re-setting an imbalanced immunoregulatory axis) a renewed opportunity exists for future PBC therapy to be immune-focused (Figure 3). Such efforts are underpinned by an increasingly robust set of insights into the initial immune insults seen in patients with PBC, fundamentally revolving around a failure of immunoregulation. The PBC liver is heavily infiltrated by both CD4+ and CD8+ T cells that react with PDC‑E2, which can be isolated from biopsy samples.34,35 A predominant type I cytokine pattern with www.nature.com/nrgastro © 2015 Macmillan Publishers Limited. All rights reserved REVIEWS Interventions Immune injury Small intrahepatic bile duct Immunotherapy BEC Accumulation of bile acids Impaired bile flow Cholestasis Bile acid therapy Bile duct loss Epithelial-to-mesenchymal transition ? Ductopenia Antifibrotic therapy Fibrosis Cirrhosis Transplantation End-stage disease Figure 1 | Pathway for the current understanding of |PBC pathogenesis and Nature Reviews Gastroenterology & Hepatology potential targets for therapeutic intervention. A key upstream disease process is immune injury to the bile duct cells of the small intrahepatic bile ducts. Although autoreactive responses in PBC are characteristically directed against highly conserved self-antigens (in particular the dihydrolipoamide acetyltransferase [E2] component of pyruvate dehydrogenase complex), the potential for a secondary inflammatory response following BEC senescence is now emerging. Following initial immune injury to BECs, progressive bile duct loss is seen, which leads to impaired bile flow and retention of bile acids. The predominantly hydrophobic bile acid pool seen in PBC results in enhanced injury to BECs (hydrophobic bile acids are cytotoxic) leading to a self-sustaining cholestatic cycle of enhanced bile duct injury and thus progressive cholestasis. Ductopenia occurs as an end-stage of this process (potentially compounded by BEC replicative senescence and phenotypic change through a process of BEC epithelial to mesenchymal transition). Progressive cholestasis leads to fibrosis development and eventually cirrhosis and end-stage disease. Abbreviations: BEC, biliary epithelial cell; PBC, primary biliary cirrhosis. high levels of IFN‑γ, IL‑5, IL‑6, IL‑10, IL‑12 and IL‑15 in the blood and liver of patients with PBC has been demonstrated.14 The portal tracts in patients with PBC are rich in chemokines secreted by inflamed cholangiocytes, including CXCL10, CXCL9, CX3CL1 and CCL20, which are responsible for recruiting T effector cells that bear their cognate receptors. Type 1 T helper cells (TH1) and cytotoxic T cells express CXCR3 (for CXCL9 and CXCL10) and CX3CR1, and TH17 cells express CCR6.36 Although both T‑cell subsets seem to recognize similar sequences within the same epitope of PDC‑E2 it is believed that CD8+ T cells have a role in the initial degeneration and death of cholangiocytes that aberrantly express PDC‑E2.33,35,37 Moreover, an increase in specific cytotoxic T cells in the liver compared with the peripheral blood has been reported, which supports the role of these cytotoxic cells in the evolution of bile duct injury in PBC.33,35 Furthermore, animal models have highlighted how T cells and B cells might be relevant to liver injury (reviewed elsewhere38). On the basis of data from both animal models and human studies, a disease model has been developed in which a combination of environmental triggers39–42 in genetically predisposed hosts12,13,43,44 leads to uncontrolled immune activation targeted against the highly conserved mitochondrial antigens. The critical steps in the development of an effector cell response, including antigen presentation, T‑cell differentiation and proliferation and recruitment, are all under consideration as novel immune targets1 (Figure 3). In particular, pathway analysis of the genetic risk loci identified suggests the importance of the IL‑12 pathway and downstream JAK–STAT signalling. However, the early disappointing results of therapy targeting this pathway 45 suggest an ongoing need for more-detailed functional post-genomic studies, including consideration of the potential importance of epigenetic regulation. The specificity and efficacy of the immunomodulators being investigated must also be considered. The challenge in considering targeting of the immune response in PBC therefore lies not so much in generating a list of targets, or indeed products, as in trying to quantify the relevance of any particular pathway in diseased liver tissue. The efficacy of interventions must then be evaluated, assuming that at the time of trial entry a patient has sufficient ongoing immune-mediated liver injury to benefit (as opposed to progressive cholestatic or fibrotic disease). Without good markers of treatment efficacy that can provide an early and sensitive indicator of nonresponse to therapeutic intervention there is a risk that novel immunotherapeutic agents will fall foul of short-term evaluations in classically designed earlyphase trials that use biochemical end points such as alkaline phosphatase levels, which are of limited relevance to phases of the disease that are pre-cholestatic (as a number of such agents might already have done). Conceptually, specific targeting of the autoimmune response in PBC could occur at several levels. These different levels include depletion of culprit cell subsets (for example, anti-CD3 or alemtuzumab therapy), the prevention or modulation of T‑cell priming (such as interfering with CD80 signalling), modulation of the degree or nature of the resulting T‑cell response through the induction of tolerance (regulatory T cells or mesenchymal stem cell therapy or antigen-specific immunotherapy) and the prevention of recruitment of effector cells into the liver. The challenge for immunotherapy in PBC is the balance between efficacy of the immunomodulatory action on the disease and immune-compromise. NATURE REVIEWS | GASTROENTEROLOGY & HEPATOLOGY © 2015 Macmillan Publishers Limited. All rights reserved VOLUME 12 | MARCH 2015 | 149 REVIEWS Table 1 | Examples of active clinical trials in PBC Therapeutic target Agents Background Budesonide Phase III trial to compare the efficacy and tolerability of combination therapy with UDCA (12–16 mg/kg body weight daily) plus budesonide (9 mg daily) vs UDCA (12–16 mg/kg body weight daily) plus placebo Depending on ALT values 6 mg daily budesonide are allowed Study population is patients with PBC at risk of disease progression (NCT00746486150) Budesonide is a corticosteroid agent with high first-pass liver metabolism, allowing liver action with limited systemic adverse effects Biologic agents Phase IV trial of abatacept to determine if it is effective in patients with PBC who do not respond adequately to UDCA (NCT02078882151) Abatacept consists of a fusion protein of the extracellular domain of CTLA‑4 and human IgG1, which binds to CD80 and CD86 molecules on the APC and prevents the co-stimulatory signal being delivered to the T cell that is needed for an immune response152 Highly active antiretroviral therapy Trial of combination therapy with tenofovir– emtricitabine and Iopinavir–ritonavir in patients with PBC (NCT01614405153) Recruitment was limited owing to poor tolerance of lopinavir–ritonavir Some evidence suggests that PBC might occur as a result of a retroviral infection in patients who are more susceptible to disease than others Early studies with combination antiviral therapy showed some improvement in biochemistry and histology results A NOD.c3c4 mouse model of autoimmune biliary disease showed response to combination antiretroviral therapy154–157 Stem cells A phase I study of nonmyeloablative allogeneic haematopoietic stem cell transplantation in patients with PBC (NCT00393185158) was withdrawn prior to enrolment The study was designed to examine whether modulation of the immune system by treating patients with high dose cyclophosphamide, fludarabine and CAMPATH-1H, followed by return of blood stem cells that have been previously collected from a patient’s sibling would stop or reverse the disease The stem cell infusion was to restore blood production after treatment with cyclophosphamide, fludarabine and CAMPATH-1H, and to produce a normal immune system Phase I and II study to assess the safety and efficacy of umbilical cord mesenchymal stem cells in PBC (NCT01662973159) Umbilical cord mesenchymal stem cells have been used to treat several autoimmune diseases, such as immune thrombocytopenia, systemic lupus erythematosus and therapy-resistant rheumatoid arthritis Abbreviations: APC, antigen presenting cell; ALT, alanine aminotranferase; PBC, primary biliary cirrhosis. There is also the issue of when in the disease course such therapy should be given. Early, disease-modifying therapy would be the ideal solution, but this approach is not plausible when broadly acting agents are being considered owing to our current inability to predict which patients are at risk of clinically significant and rapid progression and in whom the benefits of such therapy would outweigh the risks. The challenge of this balance explains why depleting therapies have largely not been explored in PBC and why results with plausible agents such as ustekinumab have been disappointing (as they have perhaps been used too late in the disease process, or they need to be used in conjunction with therapies that also modulate cholestasis). Depleting therapies are most likely to be effective in early disease before it is clear that a patient is at high risk of progression (given that we lack predictive biomarkers at present). It is difficult to justify the use of expensive immunemodulating therapies that might have unpredictable longterm consequences in patients in whom the level of benefit is uncertain. Chemokine targeting therapy is challenging because of the redundancy of the chemokine networks and the potential difficulty of finding the correct dose to use (that is, at a level that can account for the degree of inflammatory activity). Tolerogenic therapy is potentially the most attractive option, particularly if detailed knowledge of the nature of the autoantigen in PBC could be used to 150 | MARCH 2015 | VOLUME 12 develop antigen-specific tolerogenic approaches. However, it should be noted that early attempts to use this approach in patients have proved unsuccessful, albeit not harmful.46 Targeting cholestasis Bile lies at the heart of PBC and its therapy, given the nature and identity of the targets for cellular injury, and the fact that the only proven drug therapy to date is itself a bile acid. A critical concept in disease pathogenesis is that injury to the bile duct, regardless of its original trigger, is cyclical in nature with cholestasis resulting from bile duct injury potentially causing further bile duct injury through the actions of retained hydrophobic bile acids.47–49 This process potentially provides both a mechanism for chronic bile duct injury and a therapeutic opportunity through breaking the cycle. Bile formation is regulated by a complex signalling network of transcriptional and post-transcriptional mechanisms in hepatocytes and cholangiocytes.50,51 Cholestasis might result from both hepatocellular and cholangiocellular secretory defects and obstructive bile duct lesions, and evidence suggests that both are at play in PBC. To date, the best studied anti-cholestatic agent in hepatocellular cholestasis remains UDCA.52–54 UDCA has potent anticholestatic and antiapoptotic properties and is enriched from 1–2% to ~40% of total bile acids in the www.nature.com/nrgastro © 2015 Macmillan Publishers Limited. All rights reserved REVIEWS Cytotoxic T cell Apoptosis PDC component/ epitope 2 Latent TGF-β 1 ROS Active TGF-β NK cell ? 3 Hydrophobic bile acid Bicarbonate transporter (e.g. AE2) ‘Bicarbonate umbrella’ Anti-PDC antibody Senescence Apoptosis EMT? Figure 2 | Mechanisms of BEC injury in PBC. (1) Immune-mediated injury via the Nature Reviews | Gastroenterology & Hepatology actions of cytotoxic T‑cells reactive with PDC derived epitopes. An alternative potential mechanism is through antibody-dependent cell cytotoxicity with antibody reactivity against PDC/PDC cross-reactive proteins expressed on the surface of BECs in PBC and mediated by natural killer and other effector cells. (2) Toxic effects of hydrophobic bile acids able to induce apoptosis of BECs owing to the loss of the protective ‘bicarbonate umbrella’. (3) BEC plasticity resulting from the local actions of TGF‑β converted from the latent to the active form and triggered by oxidative stress resulting from the actions of ROS and other entities. Pathways include senescence and EMT. The three mechanisms are not mutually exclusive, indeed they might be fully interdependent. Antigen or epitope generation for autoreactive immune responses might occur as a consequence of the BEC changes resulting from the hostile environment following failure of the bicarbonate umbrella. Furthermore, oxidative stress causing BEC plasticity might also result from the actions of hydrophobic bile acids in the hostile bile duct environment. All postulated mechanisms offer potential targets for novel therapy development. Abbreviations: BEC, biliary epithelial cell; EMT, epithelial to mesenchymal transition; NK, natural killer; PBC, primary biliary cirrhosis; PDC, pyruvate dehydrogenase complex; ROS, reactive oxygen species; TGF-β, transforming growth factor beta. bile of patients with PBC and healthy volunteers treated with therapeutic daily doses of 15 mg/kg body weight.55 How does UDCA exert its anticholestatic properties at the level of the hepatocyte? In contrast to hydrophobic bile acids of the type seen to predominate in PBC and thought to contribute substantially to bile duct injury, UDCA does not markedly affect transport protein expression in vivo at the transcriptional level to modulate transport capacity.54 The conjugates of UDCA were unravelled in the 1990s as calcium agonists56–58 and activators of protein kinase C (cPKCα59–61), ERK1, EKR2, p38 MAP kinase62–64 and integrins.65 The concept that these conjugates might enhance the secretory capacity of hepatocytes by stimulating vesicular exocytosis and, thereby, insertion of key carriers such as the bile salt export pump (BSEP)63,66 or the conjugate export pump (MRP2)67 into their target membranes was independently developed in healthy liver in the context of bile-acid-induced hepatocyte swelling 68 and in the cholestatic liver.69 Experimental models showed that taurine conjugates of UDCA exert choleretic effects by apical carrier insertion via a dual MAPKdependent and integrin-dependent mechanism in healthy liver 63,64 and anticholestatic effects by stimulation of impaired apical carrier insertion via mechanisms dependent on Ca2+-/type II inositol‑1,3,4-triphosphate receptor, cPKCα and PKA.66,67,70–72 Optimization of bile acid therapy in PBC by additional stimulation of signalling related to secretory mechanisms might be an attractive approach. In cholangiocellular and obstructive cholestasis in PBC the precise interplay of different mechanisms of the disease process is not understood. The model of a ‘biliary bicarb onate umbrella’ as a physiological protective mechanism of bile duct epithelia against the toxic effects of millimolar concentrations of glycine-conjugated bile acids in the human biliary tree has been proposed.73 Experimental confirmation of this hypothesis in vitro74 led to the speculation that defects in the biliary bicarbonate umbrella might have a critical role in the development of bile duct lesions in PBC as well as other fibrosing cholangiopathies.73,74 An emerging body of clinical and experimental evidence supports the concept that therapeutic stabilization of the bicarb onate umbrella of small intrahepatic bile ductules might be crucial in the treatment of PBC through anti-inflammatory and antifibrotic properties. UDCA stimulates biliary bicarbonate secretion in patients with PBC as well as in various experimental settings.54,75 Biliary bicarbonate secretion is stimulated by current treatment with UDCA (13–15 mg/kg daily) alone75 or in combination with obeticholic acid,76 the glucocorticoid receptor–pregnane X receptor (PXR) agonist budesonide (for which there is emerging evidence for clinical utility in some patients with early PBC77,78), or the C23-homologue of UDCA, nor-UDCA.79–81 Nor-UDCA is the strongest bicarbonate secretagogue so far identified in humans.79 In addition, the PPARα agonists might also stabilize the biliary bicarbonate umbrella of small intrahepatic bile ductules by enhancing expression of the apical phospholipid floppase ABCB4,82 thereby stimulating biliary phospholipid secretion82 and mixed micelle formation and lowering the levels of potentially toxic bile acid monomers in bile. These effects might, at least in part, explain the observed anticholestatic, anti-inflammatory and antifibrotic properties of these compounds in biliary diseases.1,8,51 Regardless of the cholestatic mechanism, a net result of cholestasis seems to be a cycle of BEC injury and a consequent proliferation of ductular cells. Critically, BEC phenotype changes can occur over time, with senescence and epithelial to mesenchymal transition seeming to occur as linked processes contributing to progressive ductopenia.83–86 Targeting senescence as a mechanism for BEC and eventual duct loss is an attractive and novel therapeutic approach. Notably, the process of senescence is not passive—it is characterized by an inflammatory ‘senescent secretome’ in vitro and in vivo.86,87 This observation adds another layer of complexity to the interplay between immune and cholestatic processes in PBC and the sequencing and combining of therapies. Targeting fibrosis As PBC evolves it is increasingly characterized by progressive deposition of fibrotic extracellular matrix and, ultimately, the development of cirrhosis. The fibrotic reaction in the cholangitic lesions of PBC has distinct features that are important to recognize, such as the loss of bile ducts, a profound ductular reaction, development of periportal hepatitis and the formation of predominantly NATURE REVIEWS | GASTROENTEROLOGY & HEPATOLOGY © 2015 Macmillan Publishers Limited. All rights reserved VOLUME 12 | MARCH 2015 | 151 REVIEWS IL-23 Inhibiting T cell co-stimulation p40 p19 Anti-IL-12/IL-23 TH17 IL-12 p40 p35 APC IL-2 TNF CD80 CD28 T cell p50 p65 IL-12R TYK2 JAK2 PKCθ MHC-II TCR P STAT4 P NKT cell NK cell JAK inhibitors M0 macrophage Cytotoxicity T H0 IFN-γR IFN-γ Maturation PDC-E2 Apoptotic bodies TNF IFN-γ IL-18 IL-18R TH1 JAK inhibitors JAK2 JAK1 P STAT1 STAT1 P CD4 CXCR3 CD8 CXCL10 CCR6 Anti-CXCL10 CCL20 TH17 TNF IL-6 IRAK1 IRAK4 p50 p65 Mθ macrophage CD8 BEC Myd88 IRF5 IRF5 P Recruitment TLRs B cell AMA TH17 Differentiation TH0 IL-23R Figure 3 | The immune response in PBC is multifaceted and involves both adaptive and innate components. Potential Nature Reviews | Gastroenterology & Hepatology novel therapeutic targets are shown in red. Breakdown in immune tolerance to PDC occurs as a consequence of molecular mimicry to bacterial antigen or environmentally modified self-antigen, or in response to exposure to self-PDC modified within BECs, with antigen presentation by activated dendritic cells and APCs and differentiation of the subsequent cellular immune response. Critical aspects to the subsequent immune response in PBC include: one, the phenotype of the resulting CD4+ T‑cell response (with evidence to support increased activity of the TH1 and TH17 compartments); two, the nature and processes responsible for the effector immune response targeting BEC (including the activity of CD8 + cells and more innate processes including the action of NK and NKT cells and macrophages); and three, the processes for recruitment of effector T‑cells into the liver including through the actions of chemokines. Potential targets for immunotherapy include through T‑cell–APC interaction (blockade of CD80 co-stimulation), alteration of differentiation pathways through targeting of IL‑12–IL‑23, and blockade of chemokine pathways responsible for recruitment of effector cells into the liver (CXCL10). Abbreviations: AMA, apical membrane antigen; APC, antigen presenting cell; BEC, biliary epithelial cell; NK, natural killer; NKT, natural killer T cell; PBC, primary biliary cirrhosis; PDC, pyruvate dehydrogenase complex; TCR, T cell receptor; TH1, type 1 T helper cell; TH17, type 17 T helper cell; TLR, Toll-like receptor. portal-to-portal fibrotic lesions. The current mechanistic description for the initiation of fibrosis in PBC is that T‑cell-mediated and cholestasis-driven ductular injury results in the release of a variety of fibrogenic mediators from damaged BECs.1 These mediators, including transforming growth factor (TGF)‑β1, connective tissue growth factor (CTGF), platelet-derived growth factor BB (PDGF-BB) and endothelin‑1, trigger the activation of neighbouring portal fibroblasts88 to adopt a myofibroblastic phenotype and secrete fibril-forming collagens. This process contrasts with the early pathological events for sinusoidal fibrosis that is typically associated with alcoholic liver disease and NAFLD. In these diseases, the cellular events responsible for triggering fibrosis involve activation of liver macrophages, which then stimulate the transdifferentiation of perisinusoidal hepatic stellate cells into fibrogenic myofibroblasts.89 Immunohistochemical analyses have also documented the contribution of hepatic stellate cells to the fibrotic process at the later cirrhotic stages of the disease.90 152 | MARCH 2015 | VOLUME 12 The distinct anatomical and immunological components of PBC pathology, coupled with differences in the phenotypes of portal fibroblasts and hepatic stellate cells must not be overlooked when considering the applic ability of emerging antifibrotic therapeutics for PBC. A potential problem with the translation of some of the emerging antifibrotics into the PBC arena is that their targets have been discovered and validated in preclinical systems that do not necessarily recapitulate molecular and cellular events that shape the portal-to-portal and periductular fibrosis of PBC, for example myofibroblasts derived from hepatic stellate cells in 2D culture models or rodent models of chronic liver damage.91 Most in vivo models do not recapitulate the fibrogenic mechanisms observed in PBC. Bile duct ligation in rodents is widely used as a model of cholestasis and is characterized by bile duct damage, a ductular reaction and initiation of fibrosis between portal tracts.92 However, this model is an acute model that lacks intrinsic immune-mediated biliary injury, and the role of portal fibroblasts as a source www.nature.com/nrgastro © 2015 Macmillan Publishers Limited. All rights reserved REVIEWS of myofibroblasts in this model is highly contested by lineage tracing studies.93,94 A second rodent model of cholestasis is the Mdr2 (Abcb4)–/– mouse, which spontaneously develops histological features of sclerosing cholangitis, including the onion-skin-like periductal fibrosis also seen in some patients with PBC.95 The spontaneous fibrogenic mechanism in these animals is triggered by regurgitation of bile into the portal tracts leading to portal inflammation and the activation of portal fibroblasts.96 Hence, mechanistically the model more closely resembles human primary sclerosing cholangitis, but has relevance to PBC when the aim is to validate therapeutics that target portal fibroblast-derived myofibroblasts. Of the emerging antifibrotics currently in clinical studies, inhibitors of the integrin αVβ6 and lysyl oxidase homolog 2 (LOXL2) probably have the best potential to ameliorate disease progression in PBC. αVβ6 is expressed during epithelial repair, in which it has a pivotal role in the local activation of TGF‑β1.97 Although the appearance of αVβ6 expression on hepatic epithelia is not disease specific it is a feature of surface bile duct epithelia and transitioning hepatocytes in PBC.98 Encouraging preclinical studies have reported a protective effect of αVβ6 inhibition in the bile duct ligation and Mdr2 (Abcb4)–/– models.99 A humanized monoclonal antibody, STX‑100, which targets αVβ6, has advanced to phase II trials in idiopathic pulmonary fibrosis (NCT01371305100) and chronic allograft nephropathy (NCT00878761 101).102 LOXL2 is an enzyme that promotes collagen and elastin cross-linking and in experimental models has been shown to be important for the progression and maturation of fibrosis.103 LOXL2 expression is enhanced in PBC as well as in other liver diseases, and there is great interest in the outcome of ongoing trials with anti-LOXL2 (GS‑6624) in NASH and primary sclerosing cholangitis (NCT01672879104 and NCT01672853,105 respectively). Targeting multiple stages of the disease process The ultimate approach is to personalize treatment to the patient and their disease activity and stage. This approach might require therapy with a single agent targeting multiple levels of the postulated disease process in PBC. Is this a realistic goal? Data suggest that the biological actions of bile acids are substantially more diverse than had originally been thought, with important proimmune and proinflammatory actions.106 This finding raises the possibility that the microenvironment of cholestasis promotes the autoimmune component of the disease as well as, potentially, generating neoepitopes that contribute to tolerance breakdown. UDCA, and to a greater extent the FXR-agonist bile acids, have antiinflammatory and immunomodulatory actions.106 It is unclear, however, whether the immunomodulatory capacity of bile acid therapy is sufficient to mitigate against the seemingly potent cytotoxic CD8 + auto reactive T‑cell response directed at BECs throughout the early stages of PBC.33 Ultimately, a combined approach of anti-immune and anticholestatic therapy might be needed to address the combined processes affecting BECs in PBC. The issue of the trigger for breakdown of immune tolerance to PDC and the resulting immune response targeted against BECs is highly germane to the issue of the overlap between effective treatment of cholestasis and immune-mediated injury. If cholestasis is a downstream, self-sustaining consequence of immune-mediated BEC injury then it is likely that effective anticholestatic therapy in isolation will be insufficient to ultimately prevent ductopenia as that therapy might be insufficient to prevent the independent immune injury. By contrast, if autoreactive BEC injury occurs as a consequence of a primary cholestatic injury causing altered PDC processing 107,108 then early effective targeting of cholestasis might alter the natural history of immune-mediated BEC injury. Carefully designed trials of anticholestatic therapy with appropriate immunological assessment will be essential to address this question. Agents with dual efficacy against cholestatic and fibrotic injury processes seem potentially more realistic. Nor-UDCA has, in addition to its potent choleretic and cholangiocyte healing actions,109 notable antifibrotic actions in mouse models of liver disease,110 whilst FXR agonists also seem to be potently antifibrotic.111 However, the apparent functional absence of FXR in human (as opposed to mouse) myofibroblasts argues against this mechanism of action in humans.112 Future directions in symptomatic therapy Symptoms of PBC, in particular pruritus and fatigue, are a major issue for patients. These symptoms are unresponsive to UDCA therapy and no data so far suggest that second-line prognostic therapies are any more likely to improve them (indeed obeticholic acid worsens pruritus).113 They are, therefore, important targets for specific therapies. Although pruritus can be effectively treated by bile acid sequestrants and rifampicin, such therapy is frequently poorly tolerated with substantial adverse effects,114 or lack of efficacy.2 No therapies are currently proven to improve PBC fatigue. Targeting pruritus Pruritus is common and has marked systemic effects, particularly when accompanied by sleep deprivation.52,115–117 The pruritogens in cholestasis have not been clearly identified so far, with the result that therapy is largely empirical (albeit supported by a reasonable trials evidence base, at least in the case of rifampicin). 118,119 Clinical observations suggest that pruritogens accumulate in the systemic circulation; severe pruritus is at least in part relieved after treatment with plasmapheresis, albumin dialysis (for example MARS®, Gambro, Sweden) or plasma separation and anion absorption.120,121 They are also secreted into bile—pruritus can be attenuated after oral administration of the anion exchange resin cholestyramine,122,123 or in the most severe cases, transiently relieved after nasobiliary drainage.124 Owing to these observations, trials of ileal apical sodium bile-acid transporter inhibitors are currently undergoing phase II evaluation, including LUM001 (NCT01904058125) and GSK2330672 (NCT01899703126). Pruritogens are also NATURE REVIEWS | GASTROENTEROLOGY & HEPATOLOGY © 2015 Macmillan Publishers Limited. All rights reserved VOLUME 12 | MARCH 2015 | 153 REVIEWS Secreted into bile Accumulation in systemic circulation Plasmapheresis Albumin dialysis Anion absorption Cholestyramine Nasobiliary drainage Pruritogens Naltrexone Sertraline Rifampicin Lysophosphatidic acid Affect the endogenous opioidergic and serotonergic systems Biotransformed in liver and/or gut Autotaxin Rifampicin Figure 4 | Therapy for pruritus is largely empirical. Clinical observations suggest Nature Reviews | Gastroenterology & Hepatology that potential pruritogens accumulate in the systemic circulation, are secreted into bile, are biotransformed in the liver and/or gut and affect the endogenous opioidergic and serotoninergic system. Various treatments target these different processes. Autotaxin is the enzyme that forms lysophosphatidic acid, a potent activator of itch neurons. Targeting the autotaxin–lysophosphatidic acid pathway might provide novel therapy for cholestatic pruritus. biotransformed in the liver and/or gut (rifampicin is a potent PXR agonist), and affect the endogenous opioid ergic and serotoninergic system—opioid antagonists (for example, naltrexone) and selective serotonin reuptake inhibitors (for example, sertraline) have moderate antipruritic activity 2,119,127 (Figure 4). Studies published in the past 3 years indicate that lysophosphatidic acid, a potent activator of itch neurons, and autotaxin (also known as ectonucleotide pyrophosphatase/phosphodiesterase 2), the enzyme that catalyses production of lysophosphatidic acid, might together represent a key element of the long sought pruritogenic signalling cascade in patients with cholestasis and itch.116,128,129 Serum levels of autotaxin, but no other pruri togen candidate (such as bile salts, endogenous opioids, histamine, or serotonin) studied so far, correlate with itch intensity.128,129 Notably, autotaxin serum level mirrors treatment response for therapeutic interventions such as colesevelam, rifampicin, nasobiliary drainage, or MARS® treatment.129 Rifampicin reduced autotaxin transcription in human liver-derived cell lines by PXR-dependent mechanisms, possibly partly explaining the strong antipruritic effect of rifampicin.129 Targeting the autotaxin and lysophosphatidic acid pathway offers considerable hope for novel therapy for PBC and other cholestatic pruritus. G-protein coupled bile acid receptor 1 (GPBAR1; also known as takeda G protein-coupled receptor 5 [TGR5]), might also have an interesting role. GPBAR1 is a G‑proteincoupled receptor that is activated by bile acids and important in the regulation of bile acid homeostasis and the 154 | MARCH 2015 | VOLUME 12 inflammatory response. Mouse models have shown that bile acids and a Gpabr1-selective agonist activate Gpabr1 on sensory nerves, stimulating the release of neuropeptides that transmit itch.130,131 Interestingly, high concentrations of unconjugated deoxycholic acid, a bile acid that is not or barely found in cholestasis, have been investigated in these mouse models. The findings might point towards neurosteroids being important, some of which are better ligands than most bile acids for GPBAR1. In models of primary sclerosing cholangitis, activation of GPBAR1 in BECs promotes bicarbonate secretion, triggers cell proliferation and prevents apoptotic cell death. Pharmacological activation of Gpbar1 in mice reduces hepatic and systemic inflammation but also provokes pruritus.132 Targeting fatigue Chronic fatigue is the most frequently reported symptom in PBC and markedly affects quality of life.133 Fatigue does not seem to improve substantially after liver transplantation, which raises the possibility that underlying processes are at least partially irreversible by the time advanced disease develops.134 Fatigue in PBC is inherently complex with both peripheral and central components. It is strongly associated, in its expression at least, with sleep disturbance (in particular daytime somnolence)115 and autonomic dysfunction,135 both of which are potential targets for fatigue mitigating approaches. Central fatigue in PBC is mirrored by fatigue seen, albeit typically at lower levels, in other human cholestatic diseases such as primary sclerosing cholangitis136 and in animal models, such as the bile duct ligation rodent in which the complex behavioural components of fatigue behaviour seen in patients with PBC (including loss of socialization, cognitive impairment, reduced activity and frustration) can be surprisingly effectively modelled.137–140 Human neurophysiology studies point to abnormalities in regulatory circuits that persist post-transplantation,141 along with structural white matter lesions and loss of cerebral autoregulation linked to autonomic dysfunction.142,143 Studies in cholestatic rodents point to direct inflammatory processes linked to monocyte activation and recruitment into the brain, which are directly reversible using biologic agents.139 Aggressive treatment of inflammatory pathways, and early aggressive modification of cholestasis to reduce the inflammatory drive are interesting therapeutic options. Tauroursodeoxcholic acid (TUDCA) in particular seems to be a potentially attractive agent because of proven neuroprotective actions through reduction of endoplasmic reticulum stress.144 Peripheral fatigue has an overtly energetic element with increased anaerobic metabolism in muscles seemingly linked to PDC dysfunction and the anti-PDC antibodies characteristic of PBC.145 B‑cell depletion therapy to address this metabolic fatigue in PBC is currently undergoing evaluation.146 Alternative potentially attractive approaches include targeting of PDC to normalize low PDC function (for example using drugs that target pyruvate dehydrogenase kinase, developed for the treatment of type 2 diabetes), and increasing the activity of the monocarboxylase transporters (in particular MCT4), which are responsible www.nature.com/nrgastro © 2015 Macmillan Publishers Limited. All rights reserved REVIEWS for lactate export from muscle, using either exercise therapy or exercise-mimetic approaches.147,148 Conclusions The majority of patients with PBC are older (>55 years)4 with mild disease and limited symptoms who respond well to UDCA (a cheap and well-tolerated therapy). The treatment challenge in this group is timely diagnosis and effective delivery of therapy rather than the development of new therapeutics. In the important minority of patients with high risk, treatment unresponsive or highly symptomatic disease the need for new treatment approaches is now clear and well accepted.149 In the setting of disease modification, evolution in our understanding of disease mechanisms is evolving rapidly into the advent of new and re-purposed therapeutics targeting key processes. Excellent opportunities are offered, in particular, by immunotherapeutic approaches targeting the upstream immune response postulated to initiate BEC injury, and by enhanced bile-acid therapies targeting the bile acid biosynthetic and feedback processes that drive ‘midstream’ injury to BECs. Targeting of the BEC homeostatic process to enhance recovery following initial injury of the ‘downstream’ fibrotic process are exciting if more distant potential approaches. The future challenge in PBC therapeutics will not be the existence of plausible treatment candidates, but the need to target therapies at appropriate patients in both 1. Hirschfield, G. M. & Gershwin, M. E. The immunobiology and pathophysiology of primary biliary cirrhosis. Annu. Rev. Pathol. 8, 303–330 (2013). 2. Griffiths, L., Dyson, J. K. & Jones, D. E. The new epidemiology of primary biliary cirrhosis. Semin. Liver Dis. 34, 318–328 (2014). 3. Mells, G. et al. 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