Inflammation: Cause or consequence of chronic cholestatic liver injury

Journal Pre-proof Inflammation: Cause or consequence of chronic cholestatic liver injury Benjamin L. Woolbright PII:

S0278-6915(20)30020-X

DOI:

https://doi.org/10.1016/j.fct.2020.111133

Reference:

FCT 111133

To appear in:

Food and Chemical Toxicology

Received Date: 22 October 2019 Revised Date:

4 December 2019

Accepted Date: 14 January 2020

Please cite this article as: Woolbright, B.L., Inflammation: Cause or consequence of chronic cholestatic liver injury, Food and Chemical Toxicology (2020), doi: https://doi.org/10.1016/j.fct.2020.111133. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Author Contributions: Dr. Woolbright is wholely responsible for the entirety of this manuscript.

Inflammation: Cause or Consequence of Chronic Cholestatic Liver Injury Benjamin L. Woolbright Department of Urology, University of Kansas Medical Center, Kansas City, KS, USA.

Keywords: primary biliary cholangitis, primary sclerosing cholangitis, neutrophil, biliary atresia, apoptosis, T-cell, B-cell, bile acid

Correspondence: Benjamin L. Woolbright, PhD Department of Urology Kansas University Medical Center 3901 Rainbow Boulevard Kansas City, KS 66160 Email: [email protected]

Abstract: Cholestasis is a result of obstruction of the biliary tracts. It is a common cause of liver pathology after exposure to toxic xenobiotics and during numerous other liver diseases. Accumulation of bile acids in the liver is thought to be a major driver of liver injury during cholestasis and can lead to eventual liver fibrosis and cirrhosis. As such, current therapy in the field of chronic liver diseases with prominent cholestasis relies heavily on increasing choleresis to limit accumulation of bile acids. Many of these same diseases also present with autoimmunity before the onset of cholestasis though, indicating the inflammation may be an initiating component of the pathology. Moreover, cytotoxic inflammatory mediators accumulate during cholestasis and can propagate liver injury. Anti-inflammatory biologics and small molecules have largely failed clinical trials in these diseases though and as such, targeting inflammation as a means to address cholestatic liver injury remains debatable. The purpose of this review is to understand the different roles that inflammation can play during cholestatic liver injury and attempt to define how new therapeutic targets that limit or control inflammation may be beneficial for patients with chronic cholestatic liver disease.

Introduction: Cholestasis is the result of a reduction in biliary flow, commonly due to either 1) physical obstruction of the biliary tract, 2) loss or narrowing of the biliary ducts due to stricture or cell death, or 3) altered bile acid transport at the apical membrane of hepatocytes (1, 2). During cholestasis, liver cell death is driven by both bile acid overload, and inflammatory processes that propagate the injury. These inflammatory processes also activate hepatic stellate cells (HSCs) resulting in deposition of collagen, liver fibrosis, and eventually cirrhosis. Consensus mechanisms have been difficult to establish in the field due to differences in interpretation of results, laboratory models in use, and etiologic differences in origin of cholestasis and subsequent pathology. Understanding mechanisms that mediate cholestasis and subsequently cholestatic liver injury is imperative to generating useful therapeutic options. Chronic cholestasis is a primary pathological component of a diverse array of diseases including primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC), and biliary atresia (BA), and during numerous obstructive pathologies that block the common bile duct. A number of drug toxicities and xenobiotic induced toxicities present with pathology that phenotypically mirrors these diseases, suggesting xenobiotics may also contribute to common cholestatic diseases (3). These data are supported by a number of xenobiotic induced cholangitis models (3). The etiology of what initiates chronic cholestatic disorders remains poorly understood in some cases; however, some (PSC/PBC/BA) of these diseases are thought to contain an autoimmune component that precipitates the injury by damaging the biliary tracts (4). In contrast, acute cholestasis such as with the bile duct ligation (BDL) model in the mouse may result from increased biliary pressure due to bile acid retention during extrahepatic obstruction (5, 6). In part due to lack of established mechanisms of injury, therapeutic development in these areas has largely lagged behind other liver diseases, with some success found when cholestasis is alleviated (7, 8). Ursodeoxycholic acid (UDCA) is the primary treatment for both PSC and PBC and is thought to work through enhanced choleresis of bile; however, many patients are

refractory to treatment (9, 10). Obeticholic acid, a bile acid analogue, can improve biochemical outcomes in patients with PBC, but unfortunately carries high rates of pruritis (8, 11). Bezafibrate, a pan-agonist of the peroxisome proliferator activating receptor (PPAR) protein family, may be the first breakthrough in many years as it improves treatment of UDCA refractory PBC and early treatment with UDCA and fibrates may actually prevent progression to cirrhosis (7, 12). Similarly, norUDCA, a UDCA analogue with increased choleretic properties, improved patient biochemical cholestasis in patients with PSC (13). In spite of these recent advances, many patients remain refractory to UDCA and more importantly, helping later stage patients remains difficult without liver transplantation. Development of novel medical interventions that can either treat or reverse the course of advanced cholestatic diseases remains the primary need in the field. Liver inflammation is prominent in laboratory models of cholestasis and in human patients. Inflammation and the presence of auto-antibodies can predate the initiation of biochemical cholestasis in PBC, indicating the initiating etiology of the disease is primarily an immune disorder (14). However, even in the disease state most associated with autoantibody formation, PBC, only approximately 16% of patients with newly diagnosed anti-mitochondrial-antibodies (AMAs) go on to get PBC, indicating AMAs alone are likely not sufficient (14). Moreover, clinical trials targeting the prominent uncontrolled inflammation and autoimmunity observed in many of these diseases have been only modestly effective (15, 16). Further complicating things, conflicting data are present in the literature with regards to the role of inflammation and how specific inflammatory mediators participate in the injury process. The purpose of this review is to critically assess the role of inflammation as both an initiator of cholestatic hepatobiliary damage, and as a driver of hepatobiliary damage during established cholestasis. This review will attempt to define areas of need to move the field forward in addition to providing an understanding of the diverse roles inflammation plays in the development and propagation of cholestatic liver injury.

Cholestatic liver injury Initiation of cholestasis and the role of bile acids: Bile acids are a group of steroid-hormone like signaling molecules and digestive aids synthesized in hepatocytes, and one of the primary components of bile along with bilirubin and a mixed population of lipids (17-19). Normal bile flow involves synthesis of bile acids in hepatocytes, followed by excretion into the bile and then enterohepatic circulation through the gut and back to the liver. Reductions or loss of bile flow results in alterations in the disposition and retention of bile acids (1, 2). Altered disposition includes accumulation of bile acids in hepatocytes, which activates the farnesoid X receptor (FXR) (17, 19). FXR functionally alters expression of numerous genes involved in bile acid synthesis, transport and uptake (17, 19). Primarily, this results in downregulation of bile acid synthesizing genes and induces upregulation of apical and canalicular export transporters to increase bile acid export and help remove bile acids from the body (17, 19-22). Bile acids are then removed either by the urine or feces which reduces systemic levels and alleviates overall cholestasis. During cases of heavy obstruction, these processes cannot accommodate the liver, and liver injury occurs. Bile acid induced hepatocyte injury: Early injury in cholestasis is dependent in part upon accumulation of bile acids. Because of their amphipathic nature, localized high concentrations of bile acids can unequivocally kill hepatocytes and can potentially kill cholangiocytes under pathophysiological conditions (23-25). At the same time, recent evidence indicates bile acids themselves may be pro-inflammatory molecules that can either drive inflammation or reduce inflammation in a context dependent manner yielding a dual role for bile acids during cholestasis (26-28). The mechanisms associated with bile acids induced hepatic cell death have been studied extensively (1, 2, 29, 30). In cancer cell lines that exogenously express sodium taurocholate co-

transporting polypeptide (NTCP) required for bile acid uptake, and in rat isolated hepatocytes, bile acids such glycochenodeoxycholate induce cellular apoptosis at concentrations consistent with serum bile acid levels in cholestatic patients (25, 31, 32). This is dependent on caspase mediated mitochondrial apoptosis and upregulation of Death Receptor 5 (33, 34). In contrast, primary human and primary murine hepatocytes are relatively more resistant to bile acid induced injury in vitro and do not undergo the same cell death mechanisms as rat hepatocytes (24, 35). Similarly, human patients with cholestatic diseases do not undergo considerable levels of apoptosis when measured using the M30/M65 assays that evaluate cleavage of keratin-18 to determine apoptotic cell death versus total cell death (24). These differences may be in part due to differences in bile acid metabolism. These differences are especially prominent in the mouse, where bile acids are preferentially conjugated to less toxic taurine in favor of more toxic glycine yielding a less toxic bile acid milieu (36). Anchoring these data, serum bile acid levels more than 100-fold higher than the upper limit of normal in a patient with genetic sodium taurocholate cotransporting polypeptide mutation, and serum levels more than 10 fold higher than the upper limit of normal in mice/patients given the NTCP inhibitor myrcludex B failed to demonstrate any significant liver injury (37-40). As such, it is unlikely that exposure to serum bile acid concentrations is the source of hepatic injury. Instead, hepatocytes likely only undergo bile acid induced liver injury when major accumulation of bile acids occurs intrahepatically or when hepatocytes are acutely exposed to very high concentrations of bile acids such as during biliary leakage after an infarction of the biliary tracts. Recent work indicates that bile acid induced necrosis may still play a prominent role in early cholestasis though. Backflow of bile acids into the liver results in rupture of the small cholagioles near the apical surface of the hepatocyte, and releases much higher concentrations of bile acids into the local hepatic parenchyma in multiple murine models (6, 41, 42). As total bile acid concentrations are commonly greater than 10mM in bile, infarction of the biliary tracts results in

acute exposure to very high concentrations of bile acids. High concentrations of bile acids can kill hepatocytes through multiple mechanisms including disruption of the plasma membrane, induction of oxidative stress, and direct damage to the mitochondria (43-45). In humans, this results in necrosis, which is supported by elevated levels of uncleaved keratin-18 in serum, elevated levels of parameters such as ALT/AST, and histological necrosis (6, 24). Cellular necrosis induces a generalized inflammatory response through multiple mechanisms and thus bile acid induced hepatocyte injury precipitates an inflammatory response in part through tissue necrosis. Evidence from animal models supports the idea that this inflammation can provoke further hepatocyte damage. Bile acid induced cholangiocyte injury: The idea that bile acids directly induce cholangiocyte apoptosis remains controversial. Cholangiocytes are normally protected against bile acid induced injury through secretion of bicarbonate that maintain a physiological protective pH gradient (46-48). Isolated cholangiocytes apparently lose this protective measure as levels of bile acids such as chenodeoxycholic acid (CDCA) induce mitochondrial damage and cell death at concentrations considerably below their normal biliary levels (49). This protective layer is regulated by adenylate cyclase, a conserved biosensor for bicarbonate that maintains this gradient in vivo (23). Evidence of apoptosis of cholangiocytes in vivo is minimal in contrast to the significant evidence in favor of biliary cell loss through alternate mechanisms. While apoptosis of cholangiocytes has been reported via both TUNEL staining and the presence of the caspase cleaved M30 K18 antigen, levels of both analytes are relatively nominal outside of very severe disease (50, 51). In contrast, increased recognition of the senescence associated secretory phenotype in cholangiocytes is consistent with both cellular loss and secretion of proinflammatory molecules such as IL-8 (52, 53). T-cell mediated cell death remains a likely contributor as well (see section T-cells) and as such, the mechanisms controlling biliary cell loss during cholestasis are dependent upon multiple factors. Additionally, biliary bile acid levels

during PBC do not elevate appreciably during cholestasis indicating that there is no substantial increase in bile acid concentration that might initiate an injury (54). Finally, proliferation of cholangiocytes occurs in a bile acid dependent fashion and is likely a normal physiological response to increased biliary load (55). More research is needed in this area to understand how alterations in cholangiocyte physiology initiate susceptibility to bile acid induced cell death, and whether or not this occurs in vivo in patients. At face value, the idea that cholangiocytes are highly resistant to bile acid induced injury/cholestais stands in stark contrast to the increasingly well characterized apical rupture of cholangioles; however, models of bile acid overload indicate rupture may be largely a function of increased biliary pressure (5, 42). These data are anchored by studies using BDL in FXR-/- mice where it was noted that 1) biliary outflow increases only minimally upon bile duct ligation in FXR/-

animals due to lack of a compensatory response and 2) FXR-/- animals do not have the normal

infarction phenotype, but instead have diffuse liver injury, a major phenotypic shift in the pattern of their pathology (56). Current research indicates that bile acid induced hepatobiliary injury is likely limited to very specific circumstances in vivo. While cholestasis undoubtedly causes major alterations in bile acid disposition and metabolism, under pathophysiological conditions, these differences may not fully account for the observed pathology. Bile acid induced injury is a precipitating factor for hepatic injury and provoke an immune response supporting the idea that inflammation is in part a consequence of cholestasis.

Inflammation during cholestasis: Inflammation during cholestasis: Nearly every form of cholestatic liver injury presents with substantial inflammation in both murine models and in human patients (1, 2). Inflammation is a

normal response to hepatic injury and critical to proper wound healing. In contrast, unresolved inflammation can provoke liver injury by damaging local tissue through the release of soluble mediators and deleterious factors. How inflammation differentially affects the many models of cholestatic injury is a major source of how we understand these diseases today. As a brief primer, common cholestatic models and a brief description of what they model is noted in Table 1.

Neutrophils: Neutrophils densely infiltrate the liver throughout most cholestatic diseases (57). In the BDL model, after the initial insult from hepatic infarction of the biliary tree, neutrophils are recruited to the site of injury as early as six hours after obstruction (BDL), significantly before other populations in the liver such as B-cells or T-cells (58, 59). Neutrophils are also noted to infiltrate the liver in xenobiotic induced models such as with either alpha-napthylthioisocyanate (ANIT) induced liver injury (60), or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) induced liver injury (61). Neutrophil accumulation occurs early after bile acid overload induced by lithocholic acid feeding (41, 42). Neutrophils mediate a substantial portion of BDL induced liver injury as knockout of adhesion molecules required for neutrophil mediated liver injury prevents both neutrophil activation, extravasation, and BDL induced liver injury (62, 63). Similar results were found in ANIT induced liver injury (60), and in mdr2-/- mice (64), but not in lithocholic acid induced liver injury (41). This is likely due to direct lithocholic acid mediated cell death (41, 42). Neutrophil induced liver injury primarily occurs through release of toxic reactive oxygen species by neutrophils (65, 66). Neutrophil mediated liver injury is associated with chlorination of tyrosine by hypochlorous acid produced from neutrophils (67). This is highly likely to be dependent on firm adhesion between neutrophils and hepatocytes as knockout of adhesion factors is sufficient to reduce injury (62, 68). Both neutrophil accumulation and the characteristic

chlorotyrosine marks occur proximal to areas of biliary infarction (67). As such, there appears to be a pathophysiological connection between rupture of the biliary tracts, leakage of bile acids, activation of a pro-inflammatory signal, recruitment of neutrophils and provocation of liver injury. Neutrophil recruitment can be directly linked to bile acid accumulation (26, 27). Bile acids upregulate expression of multiple pro-inflammatory cytokines and adhesion factors such as macrophage inhibitory protein-2 (MIP-2), CXC chemokine ligand-1 (CXCL1), intercellular adhesion molecule-1 (ICAM-1) and more (26, 27). This occurs through a poorly understood mechanism dependent on cellular kinases such as c-Jun N-terminal kinase (JNK) and the transcription factor Egr1 (69, 70). In addition to a bile acid/Egr-1 dependent axis, there is also a bile acid/-IL-17/IL-23 dependent mechanism that is potentially independent of Egr-1 but responsive to bile acids (71-73). Depletion of IL-17A (71) is protective against BDL induced liver injury as is depletion of CD18 or ICAM-1 (62, 63), depletion of IL-1 receptor A (74), depletion of Egr-1 (69), depletion of plasminogen activator inhibitor-1 (75), depletion of Na/H exchanger regulatory factor 1 (68), and depletion of Fas receptor (76). The generally accepted mechanism for protection in these models is reduction in neutrophil recruitment or function. The consistency present between these studies is strong evidence that neutrophils are major mediators of hepatocyte damage during obstructive cholestatic liver disease through provocation of further liver damage after the initial biliary infarct. These data and those in the preceding paragraph support the idea that inflammation is contextually both a cause and a consequence of liver injury during cholestasis. Direct evidence that neutrophils in particular can also kill cholangiocytes in addition to hepatocytes is missing. In contrast, it is well understood that reactive cholangiocytes produce significant quantities of inflammatory cytokines such as interleukin-8 that potently recruits neutrophils (52). Moreover, portal inflammation is a signature aspect of most chronic cholestatic diseases setting the field for the potential of neutrophil mediated cholangiocyte injury (77, 78).

Novel studies are needed that are designed to directly assess whether neutrophils actively damage cholangiocytes, or reduce the proliferation of cholangiocytes which would potentially weaken local cholangioles and predispose towards infarction and further damage. While BDL is probably the most commonly used model of obstructive cholestasis, it does not accurately recapitulate many of the most critical factors of PBC/PSC/BA, especially the autoimmune components. Unfortunately, the importance of neutrophils has not been tested in many of the recently developed models that also feature auto-immunity despite noted increases near the portal tracts (77). However, if neutrophils were critically involved in the cholestatic process itself, it could be assumed that blocking neutrophils accumulation or activation would also limit biochemical markers of cholestasis such as alkaline phosphatase (ALP), bilirubin, or serum bile acid levels (as defined in (79)), potentially through ablation of cholangiocyte injury or through a direct effect on choleresis itself. Likewise, if the inflammatory process was a primary driver of fibrosis, blocking inflammation should consistently reduce fibrosis that occurs after 10-14 days of BDL. Notably, this does not always occur with interventions designed to prevent neutrophil mediated liver damage. For example, loss of IL-17 or loss of Na/H exchanger regulatory factor 1, both of which result in protection against BDL induced liver injury through reduced neutrophil accumulation/function, reduces hepatocyte injury significantly but has no effect on ALP/serum bile acids (68, 71). Similarly, blockade of neutrophil mediated hepatocyte injury does not always prevent disease progression to fibrosis (71); although there are notable cases where mice are protected from both hepatocyte cell death and fibrosis via anti-inflammatory interventions directed against neutrophils (80). Given the totality of these data there clearly exists evidence in favor of neutrophil mediated hepatocyte cell death; in contrast, limited evidence exists that indicates neutrophils damage or kill cholangiocytes although this has not been widely studied. Interventions that prevent neutrophil mediated cell death have a mixed response with regards to progression of injury such

as development of fibrosis. Bile acids induce expression of inflammatory proteins and thus inflammation in many murine models appears to be a consequence of cholestasis that drives hepatocyte injury. B-cells: High levels of serum auto-antibodies against self-proteins is widely reported in chronic cholestatic diseases (4, 81). B cells are the functional producers of antibodies and thus the presence of auto-antibodies is indicative of B cell dysfunction and loss of B cell tolerance. The importance of autoantibodies in PBC, PSC and BA are highly variable and disparate with regards to their prognostic or diagnostic capacity (81). Over 90% of PBC patients have autoantibodies, typically against components of the pyruvate dehydrogenase (PDH) complex including PDH-E2 and PDH-E1α; however, these numbers are generally far lower than in PSC and BA (4, 82). The functional role of autoantibodies in biliary pathology remains a major area of interest in cholangitis (4, 81). Experiments demonstrating loss of B cells using a monoclonal antibody against CD20 that depletes B-cells demonstrated many of the anticipated effects, such as reduction in the amount of AMAs against the PDH complex, but paradoxically did not consistently reduce autoimmune cholangitis and led to more colonic inflammation in one study, and led to increased cholangitis in another study (83-85). Similar results were found after bile duct ligation (86). Thus B-cells may be responsible for antibody production but also have secondary roles that limit cellular damage, inflammation, and fibrosis. As such, therapeutic interventions targeting B-cells may be unlikely to benefit later stage disease when substantial biliary damage has already occurred. Perhaps the most critical finding is the presence of anti-mitochondrial antibodies in the absence of serum indicators of biochemical cholestasis (defined by ALP>1.5XULN), in the case of PBC (4, 14, 81). These data strongly support the argument that inflammation precedes cholestasis in patients with PBC and argues in favor of the idea that inflammation is the initiating step due to loss of B-cell tolerance. To the best of the author’s knowledge, it is not understood if this

extends to PSC or BA. One of the major questions remaining in the field is whether or not autoantibodies directly precipitate the biliary inflammation, and perhaps more importantly, can this be used therapeutically to prevent further disruption of the biliary tracts. If AMA induced inflammation could be identified in patients with early PBC, significant progress might be possible through targeted intervention of the resulting cholangitis. Better functional biomarkers of early PBC/PSC are sorely needed. T-Cells and T-cell mediators: Auto-antibody generation results in a T-cell immune response. This has been demonstrated in a number of xenobiotic induced murine models of cholangitis and is likely amplified by direct release of cytokines from stressed cholangioytes (52). The mechanism includes both hyperactivation of CD4+ and CD8+ T-cells (87, 88) in addition to suppression of immunoregulatory T-Reg cells that control T-cell action (89-91). T-cells damage or kill cholangiocytes in a perforin and granzyme B dependent fashion consistent with their normal cytotoxic effectors in a murine rotavirus infection aimed at recapitulating BA (92). Activation of T-cells extends to natural-killer T-cells as cholangiocytes can present antigens to NKT cells and pharmacological or genetic blockade of NKT cell function reduced pathology in models of xenobiotic induced biliary cholangitis (77). Somewhat surprisingly, there is increasing evidence that training of T-cells in cholangitis largely occurs in the gut followed by migration to the liver (93). Gnotobiotic mice generated using fecal transfer from patients with PSC indicated the presence of Klebsiella in PSC feces increased immunogenicity of T-cells that migrated to the liver, and thus increased Th17 mediated liver injury after DDC exposure (94). More studies aimed at understanding gut microbiome/tolerance in the context of PSC are imperative, especially given the high rates of concurrent autoimmune colitis in patients with PSC. Altering the microbiome through fecal transplantation or use of pro/prebiotics may be a means of retraining T-cells away from a damaging phenotype and promoting T-Reg formation and immune control.

The resultant destruction of local cholangiocytes after loss of T-cell tolerance is thought to provoke the subsequent cholestasis in these diseases. This is supported by consistent evidence that: 1) cholangiocytes present antigens to T-cells, especially CD8+ cells, resulting in biliary inflammation, and 2) removal of T-cell mediated cholangitis through intervention against the auto-immune component or enhanced T-reg activity/presence reduces cholangiocyte and hepatic injury, reduces biliary inflammation, and reduces disease progression (87, 89-91, 9597). T-cell mediated cholangiocyte injury in response to loss of B-cell/T-cell tolerance is thus the basis for the idea that inflammation precedes cholestasis during these diseases, and inflammation is the initiating cause of cholestatic injury in these diseases. An important growing trend in therapy is the use of checkpoint inhibition. Checkpoint inhibitors function by removing inhibitory signals that prevent T-cells from activating upon cell recognition. Not surprisingly, T-cell mediated tissue injury is a noted possible side effect due to overactivation of T-cells (98). Recent studies indicate that treatment with nivolumab and other checkpoint inhibitors can result in unintentional destructive cholangitis in the liver (99). These data support the idea that dysregulation of T-cell mediated normal immunity is the primary precipitating factor for destructive cholangitis as these patients have no incidence of cholestasis before onset of treatment. Xenobiotic exposure has been proposed as a potential participating or precipitating factor in autoimmune cholangitis and has widely been used in murine models (Table 1). Idiopathic drug induced liver injury after xenobiotic exposure is also thought to be related to dysregulation of T-cells and loss of tolerance (100). As such, future research aimed at understanding the intersection between xenobiotic exposure, T-cell biology and checkpoint inhibition may reveal novel mechanisms through which the liver loses normal immune tolerance and cholangitis/cholestatic liver injury is initiated. In contrast to models of primary cholangitis, suppression of the checkpoint protein Programmed Cell Death Protein-1 (PD-1) is protective against hepatocyte damage in the BDL model as

evidenced by reduced histological necrosis and reduced serum ALT levels (101). This protection likely extends to the biliary tracts as PD-1-/- animals also had reduced ALP and bilirubin levels indicative of reduced cholestasis (101). Knockout of PD-1 should increase T-cell mediated damage and thus these data contrast prior data indicating T-cell activation is critical for biliary damage, but may be explained by effects on T-regs due to global PD-1 knockout (101). Alternately, in the absence of auto-immunity as such with BDL, T-cells may function differently further complicating treatment paradigms as targeting T-cell function may paradoxically exacerbate overall immunity in the liver despite reducing associated cholangitis.

Macrophages: Significant interaction occurs between macrophages and bile acids themselves that accumulate during cholestasis. Kupffer cells are endogenously present in the liver and typically represent the first line against inflammation in the innate inflammatory system. Macrophages express the G-protein coupled bile acid receptor (TGR5) which acts as a receptor for bile acids (28). Activation of the receptor results in an anti-inflammatory phenotype in macrophages that suppresses inflammation (28). Conflicting prior results indicate that selective loss of Kupffer cells using gadolinium chloride prevents BDL induced liver injury and fibrosis; however, this may be due to prior stimulation of other cell types to remove the dead macrophages (80). In contrast, removal of Kupffer cells using clodronated liposomes demonstrated the opposite effect and BDL was worsened (102). Similar results were acquired with the analogous ANIT model wherein Kupffer cell depletion provoked injury (103). While both gadolinium chloride and clodronated liposome treatments are likely to have other effects, the idea that depletion of macrophages removes an anti-inflammatory mediator, due to the presence of TGR5:bile acid interactions, indicates macrophages are likely protective against cholestatic liver injury, similar to what is observed with B cells. Thus, general blockade of inflammation may not be a rational approach. The potential for a second mechanism of

protection in macrophages was recently described when it was noted that loss of macrophage production of Wnts also resulted in higher levels of DDC induced liver injury (61). While the endogenous Kupffer cell population may be protective, recruited monocytes often take on highly disparate phenotypes towards either pro-inflammatory status or anti-inflamamtory status (104). Pro-inflammatory macrophage/monocyte recruitment is observed in both human patients and in murine models of cholangitis (105). These appear to be M1 (pro-inflammatory) type macrophages as prevention of macrophage recruitment lead to a reduced phenotype in multiple murine cholangitits models (105, 106). The disparity between results obtained in models of cholangitis versus models of direct obstructive cholestasis again underscore the need for understanding inflammation in a context dependent fashion, especially in light of the fact that many diseases with prominent cholangitis eventually proceed to highly obstructive cholestasis. Recruited monocytes may exacerbate biliary inflammation, but at the same time, endogenous macrophages may control innate immunity in the liver. Dichotomies such as these may explain the lack of success of many clinical trials in the area and argue in favor of targeted therapies designed to block specific interactions.

Is inflammation a cause or consequence of chronic cholestatic liver injury? Current data suggest the answer to the question proposed in this review is highly nuanced and context dependent. Targeting inflammation to alleviate these diseases will likely require precision medicine approaches and a more refined understanding of the underlying mechanisms. Data favoring inflammation as a cause or consequence of cholestasis is listed in Table 2.

Inflammation as a cause of cholestasis: In models of auto-immune cholangitis, inflammation in the form of T-cell/B-cell loss of immune tolerance initiates cholestasis via destruction of the biliary tracts and thus inflammation is most likely a cause of cholestasis (4, 14). It is uncertain what precipitates the formation of self-antibodies, but their presence coincides with the presence of biliary cholangitis in both humans and patients and seems to precede increases in biomarkers of cholestasis. Unfortunately, their presence alone does not necessarily indicate the patient has active or advanced disease and thus clinical disease staging remains imperative. As such, there is a need for more diagnostic and predictive biomarkers that can identify early disease in patients. Subsequent to biliary inflammation, patients and mice develop substantial hepatic cholestasis leading to cell death of hepatocytes, hepatic fibrosis, and eventually liver cirrhosis. Advanced disease can also lead to a highly obstructed cholestasis, wherein additional inflammatory processes may develop, including neutrophil mediated cell death that would be a consequence of cholestasis. Moreover, significant overlap may exist between the loss of tolerance induced by some xenobiotics, and the loss of tolerance induced by checkpoint inhibitors, and thus this area may help understanding of what initiates loss of tolerance.

Inflammation as a consequence of cholestasis: Current data indicate that inflammation during obstructive cholestasis is primarily a consequence of cholestasis, and a driver of further hepatic injury. Recent studies demonstrate a direct linkage between bile acid accumulation, bile acid signaling in hepatocytes, and subsequent inflammation that provokes the injury. Upon the initiation of cholestasis, the inflammatory milieu that infiltrates the liver largely occurs after the development of hepatic infarcts indicative of prior cholestasis (1, 26, 27, 64, 65). The idea that inflammation, particularly neutrophils, provokes hepatocyte cell death during cholestasis has

now been repeated by multiple groups using a multitude of interventions. Conflicting data exists regarding whether or not limiting inflammation can reduce disease progression in the form of hepatic fibrosis and eventual cirrhosis. Novel studies are needed in this area to fundamentally determine whether blockage of neutrophil function can alleviate hepatocyte cell death or cholangiocyte cell death during obstructive cholestasis in the presence of biliary cholangitis and auto-immunity.

Targeting inflammation in cholestasis Clinical trials targeting inflammation are currently active in multiple cholestatic disease states. Because endoscopy and surgery are so efficient at ameliorating disease in chronic obstructive disorders such as those induced by gallstones, many of these trials are in more complex disease states such as autoimmune cholangitis i.e. PBC/PSC. Previous trials have had limited success in this area. Targeted therapies such as ustekinumab, an IL-12 inhibitor, rituximab, a CD20 inhibitor, and abatacept, a fusion protein of IgG1 and cytotoxic T-lymphocyte associated protein-4, have failed in UDCA refractory patients (15, 16, 107). These patients had advanced disease already refractory to standard of care. Targeting patients with earlier stage disease may be more fruitful, as patients refractory to UDCA may already have such substantial cholangiocyte damage, such that blocking further inflammation does not allow for restoration of a functional biliary tract. As the primary outcome in many of these states is a measure of biliary injury (alkaline phosphatase levels), increased focus should be placed on whether or not these therapeutics reduce hepatocyte damage (alkaline/aspartate aminotransferase) levels as well. Some trials have reported positive results when blocking inflammation. Combination therapy with budesonide, an anti-inflammatory corticosteroid, improved UDCA therapy in a small trial (108). This study attempted to recruit earlier stage patients; however, identification of these

patients remains a problem, putting further impetus on development of early biomarkers of liver disease in PSC/PBC. The relative rarity of the disease in the case of PSC and BA further complicates matters. Fecal transplantation proved effective in a pilot trial of patients with PSC as it was safe, and some patients had reduced ALP values of up to 50% (109). Future studies aimed at defining microbial pathobionts and replacing these with a healthy and diverse microbiome may be a safe and effective therapeutic route. Agonism of bile acid receptors has potential to effect inflammation and potentially reduce injury during cholestasis. A recent subset trial using obeticholic acid, an FXR agonist, demonstrated histological improvements including reduced inflammation in patients with PBC (110). This correlates with prior results from a larger trial indicating obeticholic acid improved serum parameters of biochemical cholestasis (11). Similarly, agonism of TGR5 also is protective against bile acid overload.(111) Notably, a dual FXR/TGR5 agonist has been reported to reduce cholangiopathy in the mdr2-/- mouse model and thus dual agonists may be an even more effective route (112). While obeticholic acid has already shown promising results clinically, other agonists of TGR5 and FXR may be necessary as obeticholic acid treatment results in untenable pruritis in some patients (11). Continued development of bile acid receptor agonists and understanding their role in inflammation should help development of future drugs as well as yield novel, testable hypotheses for future clinical studies. It should be noted that despite failures with prior trials, modulating inflammation remains a viable and scientifically reasonable means for addressing chronic cholestatic liver injury. While previous trials have addressed specific inflammatory mediators found to be critical in many disease states, the inflammatory milieu during cholestasis is likely fundamentally different. For example, multiple studies have indicated that macrophages are highly protective, further complicating generalized anti-inflammatory treatments. Moreover, inhibition of mediators such as IL-17 provided promising results in vivo in murine models but little is understood about the

role of IL-17 in human patients. Finally, with the diverse inflammatory profile of these diseases, single agents may not be sufficient. The field needs a better understanding of the broad inflammatory contribution to cholestasis to continue to develop and test targeted therapies. Conclusions: In summary, deleterious inflammation is likely both a cause and a consequence of cholestasis. Teasing out differences and the mediators of these differences is likely to improve our ability to target therapy to cholestatic diseases. Consistently, controlling inflammation through T-Reg or macrophage populations or other anti-inflammatory populations seems to reduce cholestasis associated pathology whether with regards to cell death, inflammation or fibrosis. Therapeutics aimed at improving T-reg differentiation/development or training myeloid cells towards a proresolution phenotype through interactions between the gut and lymphocytes may be the most fruitful means for alleviating hepatobiliary injury. Disclosures: The author has nothing to disclose Funding: The author is funded by an American Urological Association Research Scholar Award.

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Table 1: Common Mouse Models of Cholestasis

Model

Clinical parallel/clinical correlate

Features

obstructive cholestasis

biliary infarcts, inflammation, fibrosis/cirrhosis cholemic nephropathy

bile duct ligation

Methodology surgical obstruction of common bile duct

alphanapthylisothiocyanate

xenobiotic administration

obstructive cholestasis

biliary infarcts, Inflammation/fibrosis, biliary inflammation

bile acid overload (bile acid feeding)

bile acid administration in feed

No clinical correlate

diverse effects depending on which bile acid is used

3,5-diethoxycarbonyl1,4-dihydrocollidine administration

xenobiotic administration

biliary inflammation/ PSC

Inflammation/fibrosis

2-octynoic acid-BSA/2ocytnoic acid-OVA

xenobiotic administration

PBC

AMAs, lymphocytic infiltrates, fibrosis

NOD.c3.c4 mice

genetic alteration

PBC

lymphocytic infiltrates biliary infarcts/fibrosis

dominant negative transforming growth

genetic alteration

PBC

lymphocytic infiltrate, AMAs

mdr2 knockout

genetic alteration

PSC/PBC without AMA

neutrophil infiltrate, fibrosis, cirrhosis

BA

lymphocyte mediated cell death, neutrophil/lymphocyte accumulation, fibrosis

Rotavirus infection

viral infection

Table 1: Common mouse models of cholestasis. A list of common mouse models, their methodology of induction, clinical parallels and features that are reproducible and also mimic clinical correlates. PBC – primary biliary cirrhosis, PSC – primary sclerosing cholangitis, BA – biliary atresia.

Table 2: Inflammation as a cause or consequence of cholestasis Finding Data Supporting Cause of Injury Autoimmune antibody formation noted in many chronic cholestatic disease prior to biochemical cholestasis Cholangiocyte inflammation noted in multiple disease - loss of cholangiocytes Coincidence of IBD in PSC - autoimmune injury in multiple tissues Presencee of Klebsiella in PSC microbiota - PSC microbiota induces susceptibilty to injury Data Supporting Consequence of Injury Bile acids capable of directly killing cholangiocytes/hepatocytes Bile acids induce cytokine formation and secretion UDCA remains the primary treatment in most diseases BDL/ANIT induced injury dependent on neutrophil accumulation Chlorotyrosine adducts in hepatocytes indicate neutrophil specific protein release Abatacerpt and other biologics targetting T-cell function/activity have largely failed clinical trials Conflicting Data Interventions against inflammation still leave significant rises in ALT and do not always affect fibrosis Inconsistent results with B cells depletion Conflicting data on role of macrophages/monocytes in cholestasis

Table 2: Inflammation as cause or consequence of cholestasis. UDCA – ursodeoxycholic acid, ALT - Alanine aminotransferase, PSC – primary sclerosing cholangitis, IBD – inflammatory bowel disease, BDL – bile duct ligation, ANIT – alpha-napthylthioisocyanate

Highlights: Pathological and xenobiotic induced cholestasis remain major problems Accumulation of bile acids provokes liver injury which enhances inflammation Autoimmune cholestasis is precipitated by biliary inflammation Inflammation remains a major drug target in cholestasis Novel studies are needed to identify critical inflammatory mediators in patients.

Declaration of interests X The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: