A shift in paradigm towards human biology‐based systems for cholestatic‐liver diseases

Abstract

Cholestatic‐liver diseases (CLDs) arise from diverse causes ranging from genetic factors to drug‐induced cholestasis. The so‐called diseases of civilization (obesity, diabetes, metabolic disorders, non‐alcoholic liver disease, cardiovascular diseases, etc.) are intricately implicated in liver and gall bladder diseases. Although CLDs have been extensively studied, there seem to be important gaps in the understanding of human disease. Despite the fact that many animal models exist and substantial clinical data are available, translation of this knowledge towards therapy has been disappointingly limited. Recent advances in liver cell culture such as in vivo‐like 3D cultivation of human primary hepatic cells, human induced pluripotent stem cell‐derived hepatocytes; and cutting‐edge analytical techniques such as ‘omics’ technologies and high‐content screenings could play a decisive role in deeper mechanistic understanding of CLDs. This Topical Review proposes a roadmap to human biology‐based research using omics technologies providing quantitative information on mechanisms in an adverse outcome/disease pathway framework. With modern sensitive tools, a shift in paradigm in human disease research seems timely and even inevitable to overcome species barriers in translation.

Abbreviations

ABC

ATP‐binding cassette

AOP

adverse outcome pathway

BA

bile acids

BDL

bile duct ligation

BSEP

bile salt export pump

CAR

constitutive androstane/active receptor

CLD

cholestatic‐liver disease

FXR

farnesoid X receptor

hiPSC

human induced pluripotent stem cells

NTCP

Na⁺‐dependent taurocholate cotransporting polypeptide

OATP

organic anion transporting polypeptide

PBC

primary biliary cholangitis

PSC

primary sclerosing cholangitis

PXR

pregnane X receptor

Introduction

Liver and gall bladder diseases are very common all over the world, posing a significant health burden worldwide (Shaheen et al. 2006; Williams, 2006). It is now known that diseases related to modern lifestyle such as obesity, diabetes, non‐alcoholic fatty liver disease and other nutritional/metabolic disorders are either the cause or the consequences of liver and gall bladder diseases (Fig. 1). Liver is a very complex vital organ performing a diverse range of metabolic functions including the regulation of carbohydrate metabolism; lipid synthesis and secretion of plasma lipoproteins; cholesterol metabolism; synthesis and secretion of bile salts; digestion; storage of nutrients, vitamins and minerals; synthesis and secretion of serum albumin, clotting factors, enzymes and other proteins; ammonia detoxification through urea and glutamine formation; and biotransformation/detoxification of drugs and other xenobiotics. Liver disorders can result from various insults such as infections, drugs, toxins, ischaemia and autoimmune disorders. Persisting disturbances in liver functions due to resulting hepatocellular injury lead to chronic liver disease(s). The diverse functions of liver are performed by parenchymal (hepatocytes) and non‐parenchymal cells (mainly Kupffer cells, stellate cells, sinusoidal endothelial cells and biliary epithelial cells) communicating and working together. The liver parenchyma accounts for approximately 60% of total liver mass with non‐parenchymal cells making up the rest.

Figure 1.

Diseases of civilization are usually a cause or consequence of liver and gall bladder diseases

Biliary epithelial cells or cholangiocytes constitute around 5% of liver cells (Sirica et al. 2008). These form an intricate network of tiny channels (bile canaliculi) that merge to form the bile ducts channeling and collecting bile from liver lobules for storage in the gall bladder and subsequent intestinal secretion. Cholangiocytes that line the bile ducts have secretory functions and those that line the smaller bile ducts and canaliculi play roles in inflammatory and proliferative responses.

Bile mainly comprises the bile acids (BAs), which are the end products of cholesterol metabolism. From cholesterol to BAs, there are 17 energy consuming enzymatic reactions (Russell, 2009); and hence a very efficient and controlled recycling system for BAs exists in humans. About 95% of BAs are reabsorbed through the enterohepatic circulation. The functional BA pool is maintained by an extended system of transporters (Thomas et al. 2008) as shown in Fig. 2. As hepatocytes in the liver are organized over the sinusoids, specific hepatic transporters are expressed at the polarized membranes (Table 1). These transporters are involved in adaptive response to BAs overload and accumulation e.g. in disease conditions such as cholestasis. BAs, having hormonal functions; exert effects via the nuclear receptors (Fig. 3) in the regulation of a variety of metabolic effects – including glucose, lipid and energy metabolism (Watanabe et al. 2006; Lefebvre et al. 2009; Wei et al. 2009; Torres et al. 2012; Li & Chiang, 2015); cholesterol uptake, metabolism and secretion (De Fabiani et al. 2003); xenobiotic metabolism (Hofmann & Hagey, 2008; Zollner & Trauner, 2009); endocrine (Houten et al. 2006; Keitel et al. 2008) and immunological signalling (Ishizawa et al. 2008; Makishima et al. 2002) – and have antimicrobial effects in the digestive tract (Begley et al. 2005; Kurdi et al. 2006).

Figure 2. Bile acid transport system in hepatocytes .

After bile acid (BA) synthesis in hepatocytes, BAs are mono‐conjugated and are excreted through the bile salt export pump (BSEP) into the bile canaliculi. The divalent BAs are excreted by the multidrug resistance‐associated protein 2 (MRP2) and the multidrug export pump (MDR). These bile acids are secreted as bile into the ileum where they may be further conjugated and metabolized. BAs are then recycled into the liver via the portal vein, being taken up mainly by the Na⁺–taurocholate co‐transporting polypeptide (NTCP) and to a lesser extent by the organic anion transporter proteins (OATP1 and OATP4). During bile acid overload or cholestasis, BAs can be secreted into the systemic circulation through MRP3 and MRP4 and also to some extent via OATP2 and OST‐α and ‐β. Figure adapted from Thomas et al. (2008) with permission from Macmillan Publishers Ltd.

Table 1.

Bile acid transporters in human hepatocytes

Uptake	Canalicular efflux (apical side)	Basolateral efflux (basolateral side)
NTCP (SLC10A1)	BSEP (ABCB11)	MRP3 (ABCC3)
OATP1A2 (SLCO1A2; OATPA)	MRP2 (ABCC2)	MRP4 (ABCC4)
OATP1B1 (SLCO1B1; OATP2)	MDR1 (ABCB1)	OSTα/OSTβ
OATP1B3 (SLCO1B3; OATP8)	BCRP (ABCG2)
	MDR3 (ABCB4)
	ABCG5/ABCG8

Abbreviations, where an asterisk represents a number: ABCB, ATP‐binding cassette sub‐family B member*; ABCC, ATP‐binding cassette sub‐family C member*; ABCG, ATP‐binding cassette sub‐family G member*; BCRP, breast cancer resistance protein; BSEP, bile salt export pump; MDR, multidrug‐resistance*; MRP, multidrug resistance protein*; OATP, organic anion‐transporting protein*; OST, organic solute transporter (α or β); SLC: solute carrier*; SLCO, solute carrier organic anion*.

Figure 3. Bile acids, via their direct effects on nuclear receptors (FXR, PXR, CAR and PPARα), not only regulate their own synthesis, metabolism and clearance, but also play a significant role in glucose, lipid and cholesterol metabolism and xenobiotic (phase 0‐III) metabolism .

PPARα, peroxisome proliferator‐activated receptor α. SHP, small heterodimer partner; UGT, uridine 5'‐diphospo‐glucuronosyltransferase; SULTs, sulfotransferases.

Cholestatic‐liver diseases (CLDs), also called hepatobiliary diseases, include a range of clinical, biochemical and histological manifestations due to cholestasis. Cholestasis is derived from Greek chole meaning bile and stasis meaning halting/stopping. Cholestasis refers to obstruction of bile flow leading to the accumulation of bile in the liver and spillage of bile components into the systemic circulation. Cholestasis often involves inflammatory and autoimmune processes affecting the intrahepatic or extrahepatic biliary tree.

CLDs are diverse in occurrence; some are common in children, others in adults; and some occur predominately in men while others in women (Carbone et al. 2013). The underlying causes are also diverse including congenital disease, genetic predisposition, drug‐induced cholestasis and infections (Table 2). Up to 30% cases of drug‐induced liver injury are associated with cholestasis with a mortality rate of 8% (Bjornsson & Olsson, 2005). Cholestasis is often chronic in nature and ultimately leads to fibrosis and liver cirrhosis. In the case of autoimmune CLDs, in some patients especially those with primary sclerosing cholangitis (PSC; see Table 2), the risk for developing cholangiocarcinoma is high (Bergquist & von Seth, 2015).

Table 2.

Cholestatic liver diseases

Disorder	Explanation
Progressive familial intrahepatic cholestasis (PFIC)	Genetic disorders associated with mutations in the genes ATP8B1 (PFIC type 1), ABCB11 (PFIC type 2) and ABCB4 (PFIC type 3). These genes encode trans‐membrane transporters involved in the transport of aminophospholipids (FIC1), bile salts (BSEP) and phosphatidylcholine (MDR3), respectively.
Biliary atresia	An idiopathic inflammatory disorder characterized by absence of lumen in part or all of extrahepatic biliary tract and often affecting the intrahepatic bile ducts. It is the most frequent cause of chronic cholestasis in infants and children and the reason for liver transplantation.
Alagille syndrome	A congenital deficiency in interlobular bile ducts associated with mutations in human JAG1 encoding a ligand in the NOTCH signalling pathway. This autosomal dominant multi‐system disorder varies greatly in clinical phenotype even in monozygotic twins.
Arthrogryposis, renal dysfunction and cholestasis syndrome (ARC)	Genetic disorder associated with mutations in genes VPS33 and VIPAR (in some patients). VPS33B‐VIPAR complex plays a role in apical‐basolateral polarity in liver and kidney.
Primary biliary cirrhosis (PBC)	An autoimmune disease characterized by chronic small bile duct cholangitis occurring predominantly in women above 40 years of age. The disease is associated with an autoantibody against the mitochondrial pyruvate dehydrogenase and/or the E3 binding proteins. In severe cases, liver transplantation is the only lifesaving option.
Primary sclerosing cholangitis (PSC)	A chronic cholestatic syndrome of unknown aetiology characterized by progressive fibrosis of the biliary tree. It occurs predominantly in men with an average onset age of 40 years. It is a premalignant disease of the biliary tract and may lead to cholangiocarcinoma. Currently, liver transplantation is the only lifesaving option for patients with end stage PSC with 5 year post‐transplant survival of 75–85%.
Cholangiocarcinoma (CC)	Malignant hepatobiliary cancer arising from the cholangiocytes often diagnosed at a late stage with very poor prognosis.
Intrahepatic cholestasis of pregnancy (ICP)	Reversible cholestasis during pregnancy with incidence ranging from 0.1 to 15 %. The multifactorial pathogenesis includes genetic, hormonal and environmental factors. Mutations in the gene ABCB4 encoding the MDR3 transporter protein are reported as a major cause, in combination with hormones like oestrogen and progesterone.
Cholestasis associated with total parenteral nutrition (TPN)	TPN is administered in cases of intestinal failure and is associated with cholestasis. TNP‐cholestasis is common in children and infants due to prematurity and short bowel length. The cause is excess of glucose and lipids in the parenteral nutrition. The high glucose levels result in increased insulin release, which in turn activates fatty acid synthesis and inhibits fatty acid breakdown leading to steatosis and cholestasis.
Cholestasis associated with infections	Infections such as cytomegalovirus, rubella and syphilis may cause cholangitis leading to chronic cholestasis. Inflammation of the gut can result in leaking of bacterial endotoxins and bacterial contamination of the biliary tree leading to cholestasis.
Drug‐induced cholestasis	Drugs may interfere with bile acid synthesis, metabolism and transport leading to cholestasis. Although usually reversible upon withdrawal of the drug, drugs may cause chronic cholestasis resulting in hepatobiliary damage requiring liver transplantation.

Therapeutic options for CLDs are still very limited. Many mechanistic studies are based on animal data. On the other hand, substantial clinical data are also available, but the clinical translation of this knowledge in therapy has been disappointingly limited. This is partly due to the lack of validation of animal results in humans and the lack of integrated use of clinical data in the understanding of the disease.

This underlines the urgent need to understand the mechanistic details of physiological functions and pathobiology of the liver in humans and to apply this knowledge in diagnosis and therapy. Taking the example of cholestatic‐liver diseases, this Topical Review brings into focus the limitations of animal models and proposes a roadmap to a human biology‐based research paradigm using modern ‘omics’ technology, advanced in vitro human cell models with better physiological resemblance to in vivo and other scientific tools within an overarching disease pathway framework rooted in systems biology.

From animal models to human disease

Animal models have been and are still used in the mechanistic investigations of pathobiology of human CLDs. The general reasons for the use of animals (especially rodents) are usually the low costs, ease of breeding and the possibility of genetic manipulation in mice. The other advantage is the fact that effects can be monitored/measured in intact whole organisms allowing studies on inter‐organ effects as well as overall biological homeostasis. Animal models allow experiments that cannot be conducted in humans for ethical reasons. The possibility of genetic manipulation resulted in an increase in the number of animal models for the study of CLDs. Besides genetic modifications such as knock‐out animals, e.g. mdr2 ^−/− mice, other CLD animal models are established by dietary manipulation, chemical treatment, xenobiotic induction, bile duct ligation (BDL), immunization and infections. Bile duct injury and cholestasis can be provoked by the use of the chemicals such as 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine or α‐naphthylisothiocyanate in rodents and these models have been widely applied for the study of xenobiotic‐induced cholangiopathies. Detailed characteristics and limitations of animal models of hepatobiliary disease are described in recent reviews (Ueno et al. 2010; Osterreicher & Trauner, 2012; Halilbasic et al. 2013; Liu et al. 2013; Pollheimer et al. 2014). Although animal studies have provided mechanistic insights into CLDs, it is clear that direct extrapolation of animal data to human physiology is very challenging. The major reasons for this are species‐specific physiological differences in the gut and the liver, the BA pool composition and characteristics, the circadian rhythms and feeding behaviour, diet, and immunological and CYP 450 systems, in addition to differences in the onset and progression of cholestasis.

Human BAs are more hydrophobic (Heuman, 1989) and therefore more toxic. Elevated serum BAs in BDL mice are mouse specific and do not correspond to the elevated bile acids (such as chenodeoxycholic acid and cholic acid) in humans. Therefore, direct cytotoxic effect of BAs in the mouse BDL model is of very limited relevance (Zhang et al. 2012). Abcb11 ^−/− (mouse gene for bile salt export pump; BSEP) knock‐out mice show a very mild cholestatic phenotype as compared to humans (Wang et al. 2001). This is mainly due to compensatory up‐regulation of an ATP‐binding cassette (ABC) transporter, called mdr1 or abcb1a (Lam et al. 2005). In patients, such compensatory expression of MDR1 or ABCB1 (Keitel et al. 2005) is not observed. In mice the BAs can be excreted via other canalicular transporters and therefore BSEP transporter inhibition results in only mild cholestasis (Wang et al. 2001; Lam et al. 2005). In humans mutations in ABCB11 or inhibition of BSEP, via for example drugs, may lead to very serious consequences. As for another ABC transporter, the multidrug resistance associated protein (MDR2), heterozygous mice (mdr2 ^−/+) do not develop cholestatic liver injury, whereas heterozygous mutations in humans are reported to cause cholestasis (Jacquemin & Hadchouel, 1999). In addition, rats have very high basolateral bile salt efflux, which protects them from hepatic injury (Jemnitz et al. 2010). Human organic anion transporting polypeptides (OATPs) belonging to the SLC (solute carrier) family and located at the basolateral membranes of the hepatocytes are electrogenic and facilitate diffusion of bile salts down their electrochemical gradients (Martinez‐Becerra et al. 2011), whereas the rat oatp1a1 is electroneutral, suggesting that different members of the OATP family have different mechanisms of action. Moreover, significant species‐dependent differences in the inhibition of Na⁺‐dependent taurocholate cotransporting polypeptide (NTCP), another SLC transporter, have been reported explaining the lack of hepatotoxicity of the model drug bosentan in rats (Leslie et al. 2007). Nuclear receptors play an essential role in BA homeostasis by controlling the synthesis, metabolism and transport of BAs. Transcriptional regulation playing a key role in development of CLDs is different in rodents and other model organisms as compared to humans, e.g. feed‐forward regulation of CYP7A1 via the liver X‐receptor is limited to rodents (Goodwin et al. 2003).

Significant differences in the inflammatory responses between rats and humans (Seok et al. 2013) are also reported. Immune responses are different in rodents than in humans (Oertelt et al. 2006; Khanna & Burrows, 2011). In humans, anti‐mitochondrial antibodies are shown to play an essential role in the pathogenesis of primary biliary cirrhosis (Kaplan & Gershwin, 2005). In animal models, immunity to mitochondrial antigens is not sufficient to elicit hepatobiliary injury (Poupon et al. 2000). In humans, immunoglobulin (Ig) A binds to the polymeric Ig receptors located on the basolateral membranes of cholangiocytes and has a protective role in biliary mucosal immune defence (Mantis & Forbes, 2010). Mice do not have polymeric IgA receptors (Oertelt et al. 2006) and the immune mechanisms are different. Until now, no established animal model shows all the attributes of the two human auto‐immune diseases, namely primary biliary cholangitis (PBC) and PSC (Pollheimer et al. 2014; Tsuneyama et al. 2012).

Another very important consideration is that drug metabolism and elimination are different in animals and humans (Martignoni et al. 2006), not only in CYP 450‐mediated biotransformation but also in Phase II metabolism (conjugation reactions) and transport of the drug metabolites (elimination) into the bile. BAs cause apoptotic injury to liver parenchyma in rodents whereas in humans BAs induce necrosis providing further evidence that the mechanisms of obstructive cholestasis in humans are different from animals (Woolbright et al. 2015).

In addition, diet (fundamentally different in humans, an omnivore, from rodents, which are ganivores) plays a role in defining the gut microbiota of different species (Karasov et al. 2011). The most abundant bacterial genus in humans Bifidobacterium, does not colonize rodent gut and therefore such animal models are of very low relevance to humans (Pang et al. 2007). The gut microbiota plays an important role in BA homeostasis (Jones et al. 2014), energy regulation and metabolism (Nieuwdorp et al. 2014), immunity (Mann et al. 2013) and pathogenesis (Bourzac, 2014). Intestinal inflammation and immune response are modulated by gut microbiota and show significant differences between humans and rodents (Mann et al. 2013).

Summarizing, although a variety of animal models are available, significant species–specific differences in liver immunology and biliary physiology exist and these lead to differences in pathogenesis and progression of CLDs in humans as compared with animals. Species–specific differences often pose an insurmountable challenge in the translation of animal results into clinical practice (Pound & Bracken, 2014). It is most likely that studies with experimental animal models will continue but the 21st century scientific goal should be a move towards human systems, as explained below, in order to surmount the inevitable species barriers.

The new paradigm: understanding human organ physiology and disease pathways

Advances in cell culture methods and analytical techniques are now allowing study of disease mechanisms in vitro using human derived cells such as primary hepatocytes, genetically modified human cell lines and human induced pluripotent cell (hiPSC)‐derived liver models. These are expected to play a major role in the study of human CLDs and screening of therapeutic agents.

Human‐specific models and tools: advances in liver cell culture and techniques

In vitro studies are traditionally based on monolayer cultures of cells where the 3D architecture of the tissue is lost. In in vivo liver, cell‐to‐cell contacts and communication across the extracellular matrix is ensured within a three‐dimensional arrangement. The extracellular matrix regulates cell morphology and gene expression in vivo (Bissell et al. 1982; Le Beyec et al. 2007). A three‐dimensional environment influences the epigenetic plasticity of the cells (Spencer et al. 2007; Xu et al. 2007). Conventional 2D hepatic cultures rapidly lose liver‐like functionality (Paine & Andreakos, 2004; Godoy et al. 2013) leading to poor concordance between experimental in vitro data and in vivo data, especially with respect to xenobiotic metabolism and transporter activities. Using growth factors supplemented medium, primary hepatocytes can be maintained viable and functional for longer periods of time (Mueller et al. 2012). Primary human hepatocytes although offering the advantage of providing a palette of genetic backgrounds, are limited in their availability; disease aetiology and therapy of donors; and viability. As such, cell lines are still used in drug development and screenings (Gomez‐Lechon et al. 2014). However, hepatic cell line(s) with functional hepatocytes and co‐cultures will be essential in the study of human disease pathogenesis.

The differentiated human HepaRG cells, consisting of hepatocytes and biliary like cells, are commonly used in drug uptake, metabolism and elimination studies due to their primary hepatocyte‐like metabolic competence and transporter activities (Kanebratt and Andersson, 2008 a,b). HepaRG 3D cultures show a network of bile canaliculi and harbour functional apical and basolateral transporters (Guillouzo et al. 2007; Gunness et al. 2013; Klein, 2015; Mueller et al. 2014). This cell line alone or in co‐culture with other non‐parenchymal cells could be an important tool in the study of hepatobiliary diseases.

Other very significant progress in the area of hiPSCs (Asgari et al. 2010; Schwartz et al. 2014) is making the application of patient‐ and disease‐specific hiPS cells a reality (Ghodsizadeh et al. 2010; Siller et al. 2013; McCracken et al. 2014). These in vitro models are also expected to be useful in the screening of compounds for personalized therapy.

Much development effort is underway for high throughput generation of the 3D cultures as aggregates (Gevaert et al. 2014), micro‐patterned co‐cultures (Khetani & Bhatia, 2008) and 3D printing (Billiet et al. 2014). High‐content platforms are designed and are already in use in drug development for screening compounds (Bale et al. 2014; Tolosa et al. 2014). At the same time, highly advanced imaging and other techniques (including automated methods for assessing multiple readouts such as cell viability, shape of the nuclei, cell area, mitochondrial membrane potential, phospholipid accumulation, cytoskeleton integrity and apoptosis) are playing an important role in the study of biological pathways (Ramaiahgari et al. 2014; Sirenko et al. 2014). Such an advanced technology has opened up a great opportunity to study human disease in vitro as it enables analysis of biochemical and metabolic activities of living cells in functional tissue and organ contexts, at the same time allowing high‐resolution real‐time imaging (Bhatia & Ingber, 2014). These high content and high throughput platforms are already changing the toxicity screening paradigm (Patlewicz et al. 2013) and allowing pathway‐based in vitro‐only safety assessment (Adeleye et al. 2014; Kleensang et al. 2014).

Omics technologies for understanding disease pathobiology

Recent advances in technology along with high‐throughput, high‐content analyses and great leaps in computational power have played a key role in slowly but surely shifting the paradigm of understanding organ physiology, disease, target and biomarker identification, development of therapeutics and toxicology from the traditional reductionist approaches to a more holistic approach. In the past decade, the omics technologies have provided tremendous opportunities in discovery biology especially in the understanding of human disease.

A number of genome‐wide association studies (GWAS) on CLDs have appeared during the last few years and have been excellently reviewed (Suter et al. 2004; Krawczyk et al. 2010; Mullenbach & Lammert, 2011; Mells et al. 2013). These studies have provided important insights into the pathogenesis of hereditary (such as progressive familial intrahepatic cholestasis types I–III), autoimmune (PBC and PSC) as well as drug‐induced cholestasis. Genome‐wide association studies have also provided evidence of genetic heterogeneity of PBC and PSC based on ethnicity. Environmental factors and the epigenome are reported to have a profound impact on the development and progression of polygenic CLDs (Krawczyk et al. 2010; Mells et al. 2013). Trimethylation of the histone H3K4 is reported to be essential for the activation of the BSEP, NTCP and MRP2 genes by nuclear receptors (Ananthanarayanan et al. 2011). In the case of PBC, sex‐dependent epigenetic factors have also been reported (Selmi et al. 2004). An excellent review on the influence of epigenetic factors in bile acid homeostasis was recently published (Smith et al. 2013).

The bile proteome is becoming a major focus of interest due to its relatively easier application in a clinical set‐up and the possibility of discovering biomarkers for biliary disease (Farina et al. 2014). Bile proteomics was effectively applied to distinguish patients with PSC and cholangiocarcinoma (Lankisch et al. 2011). In another recent study, bile proteomics was used to recognize benign from the malignant biliary strictures in patients (Navaneethan et al. 2015). Liver biopsies can also be used for proteomics analysis for protein biomarkers for perturbed biological function. The lysosomal‐associated membrane protein‐2 from liver samples from PBC patients was identified as a marker for the prognosis of the disease (Wang et al. 2013).

Metabolomics includes a comprehensive qualitative and quantitative analysis of low molecular weight metabolites in a cell, organ or whole organism (Fiehn, 2002). Metabolomics sums up all the upstream effects of genomics, transcriptomics and proteomics; and represents the actual phenotype (Mueller et al. 2012; Klein and Heinzle, 2012; Ramirez et al. 2013). Metabolomics was used to distinguish between patients of PBC and PSC (Trottier et al. 2012) and has been valuable in defining a ‘core metabolomics phenotype’ for hepatobiliary diseases (Beyoglu & Idle, 2013).

Systems biology tools are no doubt expanding the understanding of human physiology and disease. However, there are yet unmet requirements for a wide application of the omics technologies. The most important limitations of these methods are the complexity of the methods and analytical techniques, standardization and validation. Omics technologies often generate huge datasets for which powerful bioinformatics tools are needed. Public databases are needed to store and exchange information and facilitate integrated systems analysis. Computational models incorporating mechanistic information as well as genetic information are required for prediction. Systems biology tools are aimed at very early indication of perturbation patterns in physiology that may lead to adverse outcomes/diseases. As such, many detected changes may not be biologically or pathologically relevant. However, integration of multi‐omics on different scales using mathematical models will allow identification of patterns of gene transcripts, proteins and metabolites and link them to an adverse outcome/disease pathway. In addition, omics technologies provide biomarkers at different levels of biological organization that are often non‐specific. It is expected that a set of biomarkers will provide better and more reliable information than single biomarkers. Finally, the successful implementation of systems biology tools in healthcare and industry is still a challenge.

In future, it is expected that human in vitro models based on human liver cells and patient‐specific iPSC‐derived hepatocytes will be preferentially used. These will yield rich human‐relevant information at different levels of omics (Fig. 4) not only for understanding the disease but also for identification of the most suitable therapeutic options under specific conditions.

Figure 4. Advanced human in vitro models, e.g. primary cells or those derived from hiPSC maintained in 3D, will provide high content and data‐rich ‘omics’ information and biomarkers for diagnosis; and can be used in screening for novel therapy options .

3D hepatic organoid by Daniel Mueller and Patrina Gunness; fluorescence image taken at the Karolinska Institute, Stockholm, Sweden.

Adverse outcome/disease pathway(s)

The concept of the adverse outcome pathway (AOP) was recently developed in the field of risk assessment for chemicals (Landesmann et al. 2013) and ecotoxicology (Ankley et al. 2010). An AOP is aimed at describing the link between a molecular initiating event (e.g. receptor binding) and an adverse outcome (e.g. cholestasis), with a number of intermediary ‘key’ events (Garcia‐Reyero, 2015). AOPs have been described for skin sensitization, liver cholestasis, liver steatosis and fibrosis (OECD, 2012; Vinken et al. 2013; Willett et al. 2014). Recently, an AOP for drug‐induced cholestasis has been described with BSEP inhibition as the molecular initiating event (Vinken et al. 2013). Inhibition of BSEP should result in BA accumulation, which leads to inflammation and activation of nuclear receptors farnesoid X receptor (FXR), pregnane X receptor (PXR) and constitutive androstane/active receptor (CAR). These in turn lead to adaptive changes in BA transporters and metabolism. However, persisting stress and BA load results in altered liver functions and ultimately cholestasis.

Components of the system including mechanistic details could be described in detail using a pathway framework as it is based on measurable changes in the biological state (Villeneuve et al. 2014 a,b). It is highly recommended for an AOP to have direct human relevance, and an AOP based on only animal data is not sufficient. Biological systems are highly complex and interconnected in addition to being very robust, showing adaptive response to stress stimuli. Biological processes are non‐linear and highly ‘wired’ together with feed‐back loops and cross regulation. An AOP should not only give information about the structure of the system but also provide clues to the dynamics of the system by temporal description of the biological processes. Although the idea of the AOP originated in the field of toxicology, a broader application to describing disease mechanisms is possible as a framework for the organization and linking of biological information at different biological levels (cells, tissues, organs).

Roadmap for cholestatic‐liver diseases research and perspectives for clinical translation and personalized medicine

Although toxicology is embracing technology and benefiting from a systems biology approach, research on disease is lagging behind despite the availability of human clinical data at the genome, proteome and metabolome levels. CLDs result from a diverse range of hepatobiliary dysfunction with causes ranging from genetic predisposition to life style. Various pathways are involved in liver injury and repair. Mechanistic understanding of these pathways will provide biomarkers for diagnosis and targets for new therapies. Integrated data analysis from traditional histopathology to transcriptomics, proteomics and metabolomics is expected to improve the understanding of the pathogenesis of the CLDs in humans. Systems analysis would allow the prediction of risk for developing these diseases. In addition, these methods could also be applied for monitoring disease progression and therapy success.

In a disease pathway framework, the effects at molecular, cellular and tissue levels should provide novel targets for therapy. Systems information can also be derived at the organ and organism levels allowing reliable and early diagnosis as well as guiding symptomatic therapy (Fig. 5).

Figure 5. An adverse outcome/disease pathway framework to scrutinize systems information (from ‘omics’) for the identification of novel targets for therapy and biomarkers for early diagnosis at different levels of biological organization .

NAFLD, non‐alcoholic fatty liver disease; NASH, non‐alcoholic steatohepatitis; ALT, alanine transaminase; AST, aspartate transaminase; γ‐GT‐gamma glutamyl transferase. An up arrow indicates increase and down arrow indicates decrease.

Currently, the therapeutic options especially for inflammatory cholestasis are very limited. Ursodeoxycholic acid is the only approved treatment for PBC, with little effectiveness in PSC. Immunosuppressive drugs are generally ineffective. The lack of therapeutic options is mainly due to the gaps in the understanding of human disease. A systems approach to the understanding of the pathogenesis of cholestasis is expected to provide opportunities for therapeutic interventions, as new targets could be discovered. The understanding of the genetic causes and the possibility of sequencing genomes could play a role in the prediction of an individual's risk of developing a CLD. Knowledge of the underlying causes will certainly help in the assessment of the prognosis of the disease. Temporal monitoring of the system (patient) will provide insights into the dynamics of the disease guiding the therapy.

Conclusion

CLDs are complex and although manifest as cholestasis resulting from perturbed bile acid homeostasis, they are intertwined with glucose, lipid and energy metabolism as well as the immune response of the patient. Such complex pathogenesis requires a systems understanding leaning on new technologies. Although animal studies have advanced our knowledge of CLDs, there has not been significant clinical translation of that knowledge in the treatment or prevention of these diseases. There is a huge amount of clinical and animal data available on cholestasis. Modern in vitro methods based on human cells (and co‐cultures) maintained in in vivo‐like conditions provide an invaluable tool for the investigation and validation of human‐relevant mechanisms involved in the development of cholestasis and its progression. Omics technologies and computational modelling will enhance the knowledge and allow prediction. The shift in paradigm towards a human‐relevant systems approach to the understanding of cholestasis seems essential to bring a breakthrough that will pave the way for new therapeutic options for CLDs and eventually personalized therapy.

Additional information

Competing interests

There are no conflicts of interest.

Funding

Humane Society International (HSI) provided support for the writing of this article. The author is a researcher working on the SEURAT‐1 NOTOX project funded by the European Community's Seventh Framework Programme (FP7/2007‐2013) under grant agreement N° 267038 and Cosmetics Europe.

Biography

Fozia Noor graduated summa cum laude from Heidelberg University at the Institute of Pharmacy and Molecular Biotechnology obtaining her PhD with Nils Metzler‐Nolte. She joined Elmar Heinzle's group at the Biochemical Engineering Institute of Saarland University where she is currently finalizing her Habilitation as a group leader of cell culture and systems toxicology laboratory. Her research focuses on the development and application of in vitro methods including 3D cultivation systems of liver and heart for toxicological and mechanistic studies in combination with in vitro metabolomics.