Ian N. Hines

Contribution of Gut Bacteria to Liver Pathobiology

Emerging evidence suggests a strong interaction between the gut microbiota and health and disease. The interactions of the gut microbiota and the liver have only recently been investigated in detail. Receiving approximately 70% of its blood supply from the intestinal venous outflow, the liver represents the first line of defense against gut-derived antigens and is equipped with a broad array of immune cells (i.e., macrophages, lymphocytes, natural killer cells, and dendritic cells) to accomplish this function. In the setting of tissue injury, whereby the liver is otherwise damaged (e.g., viral infection, toxin exposure, ischemic tissue damage, etc.), these same immune cell populations and their interactions with the infiltrating gut bacteria likely contribute to and promote these pathologies. The following paper will highlight recent studies investigating the relationship between the gut microbiota, liver biology, and pathobiology. Defining these connections will likely provide new targets for therapy or prevention of a wide variety of acute and chronic liver pathologies.

Susceptibility of murine periportal hepatocytes to hypoxia‐reoxygenation: Role for NO and Kupffer cell–derived oxidants

Ischemia/reperfusion (I/R) is an important problem in liver resection and transplantation that is associated with hepatocellular dysfunction and injury. This study was designed to investigate whether a difference in hepatocyte susceptibility occurs in the periportal (PP) and/or perivenous (PV) zones in response to hypoxia/reoxygenation (H/R), and to delineate the mechanisms underlying this susceptibility. H/R was induced in an in situ perfused mouse liver model with deoxygenated Krebs‐Henseleit buffer followed by oxygenated buffer. Selective destruction of PP or PV sites was achieved by digitonin perfusion into the portal or inferior vena cava, and was confirmed by histological evaluations and zone‐specific enzymes. Hepatocellular injury was assessed by alanine aminotransferase (ALT) release. In whole liver, H/R significantly increased perfusate ALT. H/R of PP‐enriched zones caused ALT release that was similar to that of whole liver (80 + 10 vs. 70 + 12 U/mg protein), consistent with significant PP hepatocyte injury. Minimal ALT release occurred in PV zones (10 + 5 U/mg protein). Administration of N‐acetyl L‐cysteine or a chimeric superoxide dismutase (SOD)—SOD2/3, a genetically engineered SOD—abrogated ALT release in H/R‐perfused PP zones, implicating a role for superoxide (O  2− ). This elevated ALT release was attenuated by gadolinium chloride pretreatment, indicating that Kupffer cells are the O  2− source. Enzymatic inhibition of cellular nitric oxide synthase (NOS) or genetic depletion of endothelial nitric oxide synthase (eNOS) aggravated hypoxia injury while exogenous NO and inducible nitric oxide synthase (iNOS) deficiency abolished reoxygenation injury. In conclusion, PP hepatocytes are more vulnerable to H/R; this injury is mediated directly or indirectly by Kupffer cell derived O  2− and is limited by eNOS‐derived NO. (HEPATOLOGY 2004;39:1544–1552.)

Sexual dimorphism in reduced-size liver ischemia and reperfusion injury in mice: Role of endothelial cell nitric oxide synthase

We have recently reported that female mice are protected to a much greater extent from the injurious effects of reduced-size liver ischemia and reperfusion (RSL+I/R) than are males by an estrogen-dependent mechanism. The objective of this study was to examine the possibility that the protective effect observed in female mice depends on the up-regulation and/or activation of endothelial cell NO synthase (eNOS). Anesthetized female and male wild-type or eNOS-deficient C57BL/6 mice were subjected to 70% liver ischemia for 45 min followed by resection of the remaining 30% nonischemic lobes and reperfusion of ischemic tissue. Survival was monitored daily, whereas liver injury was quantified by using serum alanine aminotransferase determinations and histopathology. Hepatic eNOS mRNA, protein, and enzymatic activity were determined in male and female mice subjected to RSL+I/R. We found that liver injury was reduced and survival increased in female mice compared with males. This protective effect correlated with significant increases in hepatic eNOS message levels and enzyme activity but not protein expression compared with males subjected to the surgery. Furthermore, Nω-nitro-l-arginine methyl ester-treated or eNOS-deficient female mice responded to RSL+I/R with dramatic increases in liver injury and 100% mortality within 2 days of surgery. Finally, we found that pravastatin pretreatment significantly attenuated hepatocellular injury and increased survival of male mice, which was associated with enhanced expression of eNOS message. We conclude that the protective effect afforded female mice is due to the activation of hepatic eNOS activity and enhanced NO production.

Superoxide and Post-Ischemic Liver Injury: Potential Therapeutic Target for Liver Transplantation

Cessation of blood flow to the liver is required during liver transplantation and resectional surgery. A growing body of experimental evidence suggests that restoration of blood flow to the ischemic liver initiates hepatocellular injury which may lead, in some cases, to severe liver injury and graft failure. A large number of studies have implicated reactive oxygen species as potential mediators of post-ischemic tissue injury. Recent developments in genetic engineering as well as chemical modeling, have allowed for the production of novel free radical scavengers including mutated forms of superoxide dismutase (SOD) and low molecular weight SOD mimics with extended circulating half-lives and/or significant membrane permeabilitys. Application of these newly developed free radical scavengers show promising results in animal models of liver I/R and may become powerful tools in the treatment of post-ischemic liver injury that occurs in liver transplantation.

Extrahepatic cells contribute to the progenitor/stem cell response following reduced-size liver transplantation in mice.

The extent to which extrahepatic cells participate in liver regeneration following transplantation is not known. Either full-size or reduced-size livers from wild-type mice were implanted into green fluorescent protein–positive (GFP+) transgenic recipient mice to determine whether regenerated liver contained host-derived GFP+ hepatic cells. After reduced-size liver transplantation, GFP+ cells were localized to the portal zone of the liver lobule. Interestingly, GFP+ cells stained for CD117, a marker for progenitor cells, beginning 2 days after transplantation. A significant number of GFP+ CD117+ cells were identified in donor livers after 28 days. GFP+ cells comprised nearly 9% of the donor liver 28 days after reduced-size liver transplant. Moreover, GFP+ cells also expressed the hepatic progenitor cell marker A6 and novel marker hepatic-specific antigen (HSA), as well as stem cell antigen-1 (Sca-1). Interestingly, some GFP− cells also were stained for CD117 and A6, suggesting that both extrahepatic and intrahepatic stem cells were present and may have contributed to the regenerative response under these conditions. Reduced-size liver transplantation using GFP+ transgenic mice supports the hypothesis that recipient-derived progenitor cells are present and may contribute to liver regeneration following transplantation.

Role of hedgehog signalling in bile ductular cells

Acute and chronic liver pathologies represent an important source of morbidity and mortality. Understanding the mechanisms of cell survival, repair and proliferation are critical to the development of future therapies for the treatment or prevention of liver disease of various aetiologies. Studies by Omenetti and colleagues published in this issue of Gut ( see page 1275 ) identified increased hedgehog (Hh) signalling within the livers of bile duct ligated (BDL) rats.1 In these studies, sonic hedgehog (Shh) and Indian hedgehog (Ihh) were found to be increased during bile duct ligation in periportal epithelial cells expressing pan-cytokeratin, representing potential liver progenitor cell populations. When cholestasis was corrected, Shh expression and down-stream signalling progressively declined in a manner similar to platelet-derived growth factor expression which accompanied the resolution of tissue injury. Further, these studies identified the ability of Shh to promote survival of biliary epithelial cells possibly mediated through inhibition of caspase activity. The authors conclude from these studies that Hh signalling may represent an important protective factor within the damaged liver and may promote the survival of small periportal epithelial cells, potential hepatic progenitor cells. Indeed, these findings are a continuation of previous work by this group which demonstrated the presence of Hh signalling components in hepatic epithelial progenitor cells in patients with primary biliary cirrhosis.2 These studies are interesting …