“CHOP”ing intestinal stem cells on way to cholestatic liver injury
Authors: Akanksha Sharma, Chandrashekhar R. Gandhi
Journal: Hepatology (2017)

Potential conflict of interest: Nothing to report. Supported by VA Merit Review Award 1IO1BX001174‐01 and Department of Defense W81XWH‐14. See Article on Page 1441 Chronic hepatobiliary diseases, including idiopathic primary sclerosing cholangitis (PSC), are characterized by persistent inflammation and fibrosis. The close association between PSC and inflammatory bowel disease (IBD) exemplifies the importance of gut‐liver axis, evidenced by 5.0%‐7.5% of individuals with IBD eventually developing PSC and around 70%‐80% of patients with PSC developing IBD.1 Although intestinal bacterial overgrowth and translocation are characteristic features of chronic liver disease,3 with an important role of microbial products in fibrosis,6 the mechanisms of intestinal injury are largely unknown. In this issue of Hepatology, Liu and coworkers8 report that endoplasmic reticulum (ER) stress‐induced intestinal epithelial injury and permeability drives bacterial translocation and progression of bile duct ligation (BDL)‐induced cholestatic liver damage to fibrosis. ER stress is a physiological response of a cell to certain stimuli causing accumulation of unfolded or misfolded proteins in the ER lumen (known as unfolded protein response). This triggers a series of transcriptional and translational events to restore ER and cellular homeostasis. However, persistent or overactivated ER stress results in increased expression of a transcription factor, CCAAT/enhancer‐binding protein homologous protein (CHOP), a negative cell growth regulator and apoptosis promoter.9 ER stress and CHOP have been shown to play an important role in gastrointestinal disorders and cholestatic liver injury.10 However, Liu et al. found that hepatic ER stress and increase in CHOP expression lasted for 3 days and the innate immune system was activated at a later time post‐BDL in mice. This suggested that hepatic ER stress may not be a cause for inflammation and fibrosis in the BDL mouse. Thus, CHOP's role in intestinal epithelial cell death and impairment of mucosal barrier function, and resistance of CHOP‐knockout mice to develop BDL‐induced liver fibrosis, hinted that CHOP activity in the gut could be critical in cholestatic liver injury and fibrosis. Indeed, Liu et al. found that CHOP‐induced intestinal epithelial cell apoptosis, suppression of the proliferation and differentiation of intestinal stem cells (ISCs), disruption of tight junctions, and consequent bacterial translocation promoted BDL‐induced hepatic inflammation (characterized by monocyte‐derived CD11b+/F4/80+ macrophages) and fibrosis. These effects of BDL were strongly prevented in CHOP‐knockout mice, and their survival rate also improved compared to the wild‐type (WT) mice. To confirm the role of ER stress in BDL‐induced intestinal epithelial injury, Liu et al. performed in vitro experiments with intestinal epithelial organoids from WT and CHOP‐knockout mice. In addition to being larger in size than the organoids from WT mice, organoids from CHOP‐knockout mice had longer and greater number of crypts than those from WT mice. They also observed that ER stress inducer thapsigargin inhibited formation of the organoids, and endotoxin (lipopolysaccharide; LPS) caused much greater shedding of differentiated epithelial cells and destruction of the crypts; these effects were mitigated in CHOP‐knockout organoids. These interesting findings, however, do not entirely explain intestinal epithelial cell injury given that both epithelial cells and their precursor stem cells are continually exposed to LPS produced by the gut‐resident Gram‐negative bacteria. Furthermore, gut permeability post‐BDL is increased similarly in control and antibiotics‐treated mice.3 Whether the signals that induce the epithelial cell injury originate from the cholestatic liver, within the gut itself, or both, and precise identification of these mechanisms will be important to better understand this pathophysiology. The time course of innate immune activation both in the liver and intestine coincided with bacterial translocation in the mesenteric lymph nodes (MLNs) at 5 days post‐BDL. These results are somewhat different from an earlier report from Schnabl's group,3 showing significant bacterial translocation to MLNs as well as increased circulating LPS and LPS‐binding protein, and the presence of bacterial DNA in the whole blood of mice within a day post‐BDL. Nevertheless, Liu et al. prove their point about the importance of ER stress in the intestinal injury upon BDL by showing mitigation of bacterial translocation and activation of innate immune system in CHOP‐knockout mice. At the cellular level, BDL increased expression of Toll‐like receptor (TLR)2, TLR4, CD14, interleukin (IL)‐18, and IL‐1β in Kupffer cells and hepatocytes, but not in hepatic stellate cells (HSCs). Although these findings explain inflammatory response of Kupffer cells and hepatocytes to LPS and other bacterial products, it is rather surprising to see their unaltered expression in HSCs. It has been shown by others that LPS increases CD14 and TLR4 expression in HSCs, and instigates strong inflammatory response,11 and LPS‐TLR4 interaction is critical to fibrosis development.6 To determine the clinical relevance of the findings in BDL mice, Liu et al. examined human tissues of patients with PSC and active hepatitis virus C (HCV) infection. They found increased hepatic expression of the genes responsive to microbial products, including CD14, TLR2, and TLR4, in WT BDL mice and human PSC. Significantly lower elevation in expression of these genes in CHOP‐knockout mice post‐BDL confirmed that they are resistant to liver damage and fibrosis. Furthermore, duodenal biopsy samples of patients with HCV‐induced cirrhosis also had increased expression of ER stress‐related activating transcription factor‐4, glucose‐regulated protein 78, X‐box binding protein‐1, and CHOP. From these data, increased ER stress in the gut appears to be a critical mechanism of progression of chronic liver disease, regardless of the etiology. The findings of this study thus provide a convincing argument for CHOP‐induced intestinal injury and permeability as a cause for hepatic inflammation and fibrosis. However, because ER stress is a physiological process that regulates cell growth, it is conceivable that longer intestine, longer crypt structures and their increased number, and up‐regulated stem cell markers in the CHOP‐knockout mouse might be a part of such a response. Global CHOP deletion may also affect the physiology of liver and other organs. Whether deletion of CHOP also alters gut microbiota is another possibility worthy of examination. Perhaps, cell/organ‐specific deletion of CHOP may provide even more conclusive evidence. It should also be noted that PSC can occur in the absence of IBD, and, in many instances, PSC is diagnosed well before the development of IBD,2 although this does not necessarily mean that the intestinal permeability is not compromised. Also, whereas the heterogeneous PSC progresses slowly to end‐stage liver disease, the mouse BDL model represents a very rapidly developing pathology with high mortality within 2‐3 weeks. Nevertheless, in the absence of an animal model that truly recapitulates human PSC, the findings of Liu et al. put forth a novel mechanism of the gut‐liver axis that drives cholestatic liver injury and fibrosis (Fig. 1).Figure 1: During the initiation of cholestasis, there is increased ER stress, and CHOP‐induced apoptosis of hepatocytes. During this phase, activation of the innate immune system in the intestine (with signals derived possibly from the liver and/or triggered endogenously) induces ER stress and increases CHOP expression. CHOP then induces epithelial cell apoptosis and concomitantly inhibits their renewal from ISCs. These effects and loss of tight junctions result in increased gut permeability, causing microbial and microbial product(s) (e.g., LPS) translocation, leading to activation of the innate immune system and inflammation in the cholestatic liver. The inflammatory environment and continued liver injury cause activation of portal myofibroblasts (Pfb) and HSCs, the major cell type(s) of BDL (PSC)‐induced fibrosis. Author names in bold designate shared co‐first authorship.