Novel insight into mechanisms of cholestatic liver injury

• Citation: Woolbright BL, Jaeschke H. Novel insight into mechanisms of cholestatic liver injury. World J Gastroenterol 2012; 18(36): 4985-4993
• URL: https://www.wjgnet.com/1007-9327/full/v18/i36/4985.htm
• DOI: https://dx.doi.org/10.3748/wjg.v18.i36.4985

CHOLESTATIC LIVER INJURY - CLINICAL RELEVANCE

A reduction in bile flow (cholestasis) can result from gall stones, impingement from a local tumor, intrahepatic cholestasis of pregnancy, genetic deficiency in bile export proteins or in autoimmune disorders amongst other etiologies[1,2]. Cholestasis results in a dramatic increase in liver and serum bile acid levels that eventually lead to acute liver toxicity, proliferation of bile ducts, and fibrosis progressing to cirrhosis[3]. Neonatal disorders such as progressive familial intrahepatic cholestasis and biliary atresia are particularly difficult to treat effectively[4]. While surgical means can prevent disease progression in the case of gall stones or biliary atresia, there are a limited number of treatment options pharmacologically for diseases such as primary sclerosing cholangitis or primary biliary cirrhosis[5]. Ursodiol, a pharmaceutical form of ursodeoxycholic acid, is the current mainline defense against cholestasis, although its efficacy is limited in some circumstances[5,6].

While many of the etiologies of disease initiation are well described, the molecular mechanisms behind the early liver injury associated with cholestasis are extensively studied, but not well understood. A major hypothesis was based on the assumption that the buildup of bile acids in hepatocytes and blood results in hepatocellular apoptosis dependent upon bile acid concentrations[6,7]. While these studies on the direct toxicity of bile acids have been supported by many in vitro studies from multiple groups, recent methodology measuring the concentrations of individual bile acids after the onset of cholestasis in vivo has cast some doubt that bile acid levels may actually reach the levels necessary for cell toxicity[8-10]. A growing volume of work has instead focused on how sterile inflammation and innate immunity may result in the initial injury[11-15]. This review will discuss current and developing paradigms in the pathophysiology of early cholestatic liver injury, and provide insight into how recent advances in methodology may affect these theories.

DIRECT BILE ACID TOXICITY AS A MODEL FOR CHOLESTATIC LIVER INJURY

High concentrations of bile acids given to cultured hepatocytes in vitro[16] and bile acid-induced cholestasis[17], or bile duct ligation in vivo[18], have long been established as models of toxicity to hepatocytes. After the discovery that ursodeoxycholic acid could be used pharmacologically as a choleretic agent with protective effects against cholestasis[19], a renewed interest in the subject emerged. While previous papers had focused on the detergent properties of amphipathic bile acids[20,21] recent research resulted in a series of papers illustrating a detailed mechanism of how toxic bile acids or the respective bile salt, could induce apoptosis in primary human and rat hepatocytes[22-27]. Much of this work has spearheaded the current revival of direct bile acid toxicity as a model of cholestasis, or liver injury, in vitro and in vivo.

INFLAMMATORY LIVER INJURY AFTER BDL - A NOVEL HYPOTHESIS

If bile acids are not directly responsible for cholestatic liver injury in vivo, then what is the mechanism of injury, and what role do bile acids play in the pathophysiology and progression of the injury? Accumulation of neutrophils, largely around sites of cell injury, within 24 h after bile duct ligation has been reported numerous times[11,12,76,77]. In addition, an increase in macrophage accumulation is observed approximately one week post ligation[77,78]. Figure 1 shows typical histology and neutrophil extravasation after bile duct ligation in mice. H&E staining shows multiple areas of hepatic focal necrosis throughout the liver. Neutrophils are present around the site of injury as early as six hours post BDL, and peak around 48-72 h after BDL, which is also the peak of liver injury[8,11,77].

Figure 1 Sample liver sections from C57Bl/6 mice illustrating neutrophil accumulation after bile duct ligation. C57BL/6 mice were subjected to bile duct ligation and sacrificed 24 h later. Hematoxylin and eosin (H and E) stained sections showing focal necrosis at 100× and 400× magnification. Sections stained with an anti-neutrophil antibody. Neutrophils are present largely around areas of focal necrosis in this model.

Neutrophil accumulation has been directly associated with the injury, as deficiency of adhesion molecules such as ICAM-1 or CD18 reduced the number of neutrophils that extravasate into liver parenchyma, and drastically reduced liver injury[11,12]. A neutrophil is a particularly toxic leukocyte due to its capacity to produce superoxide via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and the potent oxidant hypochlorous acid via myeloperoxidase[79]. Neutrophil accumulation after bile duct ligation has been associated with an increase in myeloperoxidase activity[80] and intracellular chlorotyrosine adduct formation in hepatocytes[11,12,81]. This indicates that neutrophil-derived hypochlorous acid can cause an intracellular oxidant stress in hepatocytes, which is responsible for cell injury[82,83]. Inhibition of NADPH oxidase reduces neutrophil-mediated oxidant stress and protects against neutrophil cytotoxicity[84,85]. In addition, hepatic NADPH oxidases located in hepatic stellate cells (Nox1) and Kupffer cells (Nox2) contribute to BDL-induced fibrosis[86,87] but their contribution to the initial liver injury (bile infarcts) has not been determined.

Together these data strongly implicate neutrophils in the pathogenesis of early cholestatic liver injury. In support of this hypothesis is recent data that exposure of cultured hepatocytes to both toxic and non-toxic bile acids at levels that recapitulate the in vivo concentrations result in dramatic increases in chemotactic factors such as MIP-2 and mKC, and adhesion molecules such as ICAM-1, all factors that can be involved in hepatic neutrophil recruitment[15]. The inflammatory mediator induction is dependent on the activation of the transcription factor Egr-1 in bile duct-ligated mice and in bile acid-exposed hepatocytes[15,62]. Egr-1 expression is also increased in human patients with cholestatic liver disease and the hepatic Egr-1 levels correlate with interleukin-8 and ICAM-1 induction[15]. Therefore, we initially hypothesized that after BDL, bile leaks back into the parenchyma after rupture of bile ducts and hepatocytes are locally exposed to high concentrations of pro-inflammatory bile acids such as TCA, β-MC and TMC that initiate an inflammatory response[8,13,15]. Additionally these hepatocytes are exposed to low concentrations of cytotoxic bile acids such as DCA, TLCA, and GCDCA that may initiate a low level of mitochondrial dysfunction that stresses the hepatocytes, but is insufficient to directly kill the cells[8,29,37,38,41-43]. This bile acid-induced stress triggers the expression of ICAM-1, MIP-2, and mKC in hepatocytes[8,15]. The subsequent release of these CXC chemokines can activate neutrophils in circulation[88] and provide a chemotactic gradient for the extravasation of neutrophils, which then attack the stressed hepatocytes and cause cell death through reactive oxygen formation[11,12,81,82]. Given a dramatic enough increase in toxic bile acid concentrations by feeding e.g. a lithocholic acid-containing diet[89], it is possible to directly cause cell death via bile acid toxicity but independent of neutrophils (Woolbright and Jaeschke unpublished observation). However the in vivo bile acid concentrations in models such as BDL are insufficient to meet this threshold, and thus the neutrophil-mediated cell death is dominant.

APOPTOTIC AND NECROTIC CELL DEATH AFTER BDL

A central but controversial issue in the debate on the mechanisms of cell death in cholestatic liver disease is whether the injury occurs through apoptosis or necrosis[90]. This is a key point as the therapeutic options to prevent injury will progress in two very different directions depending on the answer. Necrosis is characterized by cellular swelling, membrane blebbing, DNA fragmentation and release of cellular components, whereas apoptosis is characterized by cellular shrinking, caspase activation, DNA fragmentation, chromatin condensation with initially no release of cellular contents[32]. These processes lead to dramatically different outcomes after cell death in regards to progression of disease with necrosis leading to increased inflammation and further aggravation by inflammatory cells[81,90,91].

Although there is no question that high concentrations of certain hydrophobic bile acids can cause apoptotic cell death in cultured rodent hepatocytes[22-27,34-42,67,68] as discussed earlier, the pathophysiological relevance of these observations for in vivo models, e.g., BDL, is questioned due to the fact that the pro-apoptotic bile acids do not achieve sufficiently high levels in vivo to directly cause cell death[8,9]. Thus, the evaluation of the mechanism of cell injury has to be based on the in vivo evidence. Early studies concluded that BDL-induced liver injury was caused by apoptosis mainly due to the positive terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay[92-94] which is clearly not specific for apoptotic cell death[30-32]. In addition, immunofluorescence was used to detect cells with active caspases 3/7[95] but this was not confirmed by others[96]. Additional arguments for apoptosis were the reduced BDL-induced liver injury in Fas receptor-deficient lpr mice[94] and after anti-sense treatment to reduce Bid expression[97]. However, lpr mice are protected due to the reduced inflammatory response[98] and genetically Bid-deficient mice did not show reduced liver injury[96]. In contrast, numerous studies did not find any morphological evidence of apoptosis[11,12,68,98-101] and no evidence of caspase activation (enzyme activity and/or active fragments) or immunohistochemical staining for cytokeratin 18 cleavage products or active caspase-3/7[96,98,99]. In addition, potent caspase inhibitors, which effectively eliminated caspase activation and cell death in Fas- or TNF-mediated apoptosis models[98,102], did not protect against BDL-induced liver injury[98]. The one study reporting reduced injury after a caspase inhibitor in the absence of relevant caspase activation[95] may be due to off-target effects on other proteases[103]. Likewise, the anti-sense knock-down of Bid gene expression may have caused compensatory protective gene activation, which may have attenuated BDL-induced liver injury[95]. Clearly, Bid-deficient mice were not protected against BDL-induced liver injury[96]. The cathepsin B knockout mouse also showed protection against apoptotic injury[92], although apoptosis was assessed entirely based on the TUNEL assay. While the reduction in injury was attributed to lack of cathepsin B, and thus a lack of apoptotic processing proteins, the paper also showed a significant reduction in the neutrophilic inflammatory response[92]. Thus, the reduced injury in cathepsin B-deficient mice may have been due to reduced inflammation rather than apoptotic cell death. Taken together, the preponderance of experimental evidence supports the conclusion that liver injury after BDL involves mainly necrotic cell death.

In conclusion, when using morphological criteria of apoptosis and caspase activation in combination with positive controls of apoptotic cell death in vivo, the overwhelming experimental evidence supports the conclusion that there is no relevant apoptotic cell death but almost exclusively necrosis after BDL. The main paradigm shift comes from the recognition that the well-known hepatotoxic bile acids never reach concentrations in the liver after BDL that would directly cause cell death. In contrast, the most common bile acids that reach close to mmol/L levels trigger inflammatory mediator formation, which initiates an inflammatory response and cell death caused by neutrophils through oxidant stress. Initial data from human patients with cholestatic liver disease appear to confirm several aspects of the mouse model. Thus, the focus of future research should be on evaluating the cell stress mechanisms in hepatocytes considering pathophysiologically relevant bile acid mixtures and on further characterizing the initiation of the inflammatory response after BDL. Understanding these early injury mechanisms may not only reveal therapeutic approaches to reduce liver injury but also the subsequent fibrosis.