Zhaoli Sun

Reactive oxygen species are essential mediators in antigen presentation by Kupffer cells

Kupffer cells (KC) act as APC in the liver and play a major role in the clearance of gut‐derived antigens and pathogens entering the liver with portal venous blood. Antigen presentation by KC has been implicated in regulation of the local and systemic immune responses. In this study, modulation of KC antigen presentation by antioxidants and the role of reactive oxygen species (ROS) as essential mediators of antigen presentation in KC were investigated. Co‐culture of KC with ovalbumin (OVA) antigens resulted in upstream intracellular endogenous ROS generation and increased expression of MHC class II and costimulator molecules, and consequent OVA‐specific CD4+ T‐cell proliferation in response to antigen presentation by KC. Scavenging of KC ROS by antioxidants, or blocking of KC ROS generation by specific inhibitors of NADPH oxidase and/or xanthine oxidase, or by specific inhibitors of the mitochondrial electron transport chain, significantly decreased OVA‐specific T‐cell proliferation in response to antigen presentation by KC. Increased expression of MHC class II and costimulatory molecules in KC pulsed with OVA antigens was blocked by inhibiting ROS generation enzymatically. Intracellular endogenous ROS generation during antigen processing may therefore provide essential secondary signalling for KC antigen presentation.

Stem cell mobilization is life saving in an animal model of acute liver failure.

Objective:No therapy except liver transplantation currently exists for patients with acute liver failure (ALF). The aim of this study was to determine whether pharmacologic mobilization of endogenous hematopoietic stem cells (HSCs) can aid in liver repair and improve survival in an animal model of ALF. Methods:Rodents were treated with a single near-lethal intraperitoneal injection of carbon tetrachloride (CCl4). After 12 hours, animals were randomized to receive plerixafor and granulocyte colony-stimulating factor (G-CSF), agents known to mobilize marrow-derived stem cells, or saline vehicle injection. Mice were observed for survival, and serial assessment of liver injury by serum transaminase measurements, and histologic analysis was performed. Results:In our ALF model, 7-day survival after injection of CCl4 was 25%. Administration of plerixafor and G-CSF following CCl4 resulted in 87% survival (n = 8, P < 0.05). On serial histopathologic analysis, animals treated with plerixafor and G-CSF demonstrated less hepatic injury compared with control animals. Evaluation of peripheral blood demonstrated an increase in circulating HSCs in response to plerixafor and G-CSF, and immunostaining suggested the infiltration of HSCs into the hepatic parenchyma after stem cell mobilization. Conclusions:Our results suggest a possible new treatment strategy for patients with ALF, a group for whom either liver transplantation or death is frequently the outcome. Pharmacologic agents that mobilize HSCs may lead to an infiltration of the injured liver with cells that may participate in or expedite liver regeneration. This therapy has the potential to avert liver transplantation in some patients with ALF and may be of benefit in a wide variety of medical and surgical patients with liver injury.

Interleukin-6 is an important mediator for mitochondrial DNA repair after alcoholic liver injury in mice.

We investigated the hypothesis that a prominent effect of chronic ethanol consumption is mitochondrial DNA (mtDNA) injury and compared this injury in IL‐6 knockout (KO) and wild‐type (WT) mice. Ethanol feeding for 4 weeks resulted in steatosis and oxidative mtDNA damage (8‐OHdG) in both IL‐6KO and WT mice. However, the WT mice were able to repair the injury by increased production of mtDNA repair enzymes (OGG‐1, Neil 1) and check point (p21, p53) proteins and avoid the mtDNA mutations. By contrast the IL‐6 KO mice were unable to repair mtDNA resulting in deletions and diminished transcription of the mtDNA encoded protein cytochrome c oxidase subunit‐I (COI). The mitochondrial injury was reflected by decreased membrane potential, reduced levels of ATP, and apoptosis‐inducing factor (AIF)‐induced apoptosis. Conclusion: IL‐6 plays a critical role in allowing the liver to recover from significant mtDNA oxidation caused by alcohol. The data suggests that IL‐6 activates mtDNA repair enzymes and induces cell cycle arrest allowing time for mtDNA repair. (HEPATOLOGY 2010;)

Cellular GFP Toxicity and Immunogenicity: Potential Confounders in in Vivo Cell Tracking Experiments

Green Fluorescent protein (GFP), used as a cellular tag, provides researchers with a valuable method of measuring gene expression and cell tracking. However, there is evidence to suggest that the immunogenicity and cytotoxicity of GFP potentially confounds the interpretation of in vivo experimental data. Studies have shown that GFP expression can deteriorate over time as GFP tagged cells are prone to death. Therefore, the cells that were originally marked with GFP do not survive and cannot be accurately traced over time. This review will present current evidence for the immunogenicity and cytotoxicity of GFP in in vivo studies by characterizing these responses.

Hepatoprotective versus Oncogenic Functions of STAT3 in Liver Tumorigenesis

Aberrantly hyperactivated STAT3 has been found in human liver cancers as an oncogene; however, STAT3 has also been shown to exert hepatoprotective effects during liver injury. The balancing act that STAT3 plays between hepatoprotection and liver tumorigenesis remains poorly defined. In this study, the diethylnitrosamine (DEN)-induced liver tumor model and the chronic carbon tetrachloride (CCl(4))-induced liver fibrosis model were both used to investigate the role of STAT3 in liver tumorigenesis. Hepatocyte-specific STAT3 knockout mice were resistant to liver tumorigenesis induced by a single DEN injection, whose tumorigenesis was associated with minimal chronic liver inflammation, injury, and fibrosis. In contrast, long-term CCl(4) treatment resulted in severe hepatic oxidative damage, inflammation, and fibrosis but rarely induced liver tumor formation in wild-type mice. Despite the oncogenic function of STAT3 in DEN-induced liver tumor, hepatocyte-specific STAT3 knockout mice were more susceptible to liver tumorigenesis after 16 weeks of CCl(4) injection, which was associated with higher levels of liver injury, inflammation, fibrosis, and oxidative DNA damage compared with wild-type mice. These findings suggest that the hepatoprotective feature of STAT3 prevents hepatic damage and fibrosis under the condition of persistent inflammatory stress, consequently suppressing injury-driven liver tumor initiation. Once liver tumor cells have developed, STAT3 likely acts as an oncogenic factor to promote tumor growth.

Oxidative mitochondrial DNA damage and deletion in hepatocytes of rejecting liver allografts in rats: Role of TNF‐α

An orthotopic liver transplant model in the rat was used to evaluate the role of tumor necrosis factor alpha (TNF‐α) in liver transplant rejection. There were significantly increased levels of TNF‐α mRNA and parallel increases in 8‐hydroxy‐2′ deoxyguanosine (8‐OHdG) indicative of oxidative DNA damage present 7 to 12 days after transplantation. Cells staining positively for 8‐OHdG were localized to the cytoplasm of hepatocytes adjacent to the TNF‐α expressing inflammatory cells in the portal areas or in patches surrounded by inflammatory cells in the hepatic sinusoids. Significantly more cells staining for 8‐OHdG were found in the allogeneic grafts that were strongly rejected than in the syngeneic controls or in the grafts placed in species that accepted the allograft permanently after a rejection episode. TUNEL reactivity lagged 2 days behind peak reactivity for 8‐OHdG. On day 12 after transplantation, many cells stained for both 8‐OHdG and TUNEL, indicating that the cells suffering oxidative DNA injury were undergoing apoptosis or death. Oxidative injury resulted in mtDNA deletion consisting of 4,834 base‐pairs. Studies of hepatocytes cultured from normal rats displayed dose‐dependent relationships between TNF‐α concentration and 8‐OHdG and mtDNA mutation. Repetitive intraperitoneal injection of Enbrel, a TNF receptor blocker, significantly decreased hepatocyte 8‐OHdG levels and the frequency of deleted mtDNA while greatly extending graft survival time. In conclusion, the data presented implicate TNF‐α as being capable of causing oxidative DNA damage and mtDNA mutation in hepatocytes. (HEPATOLOGY 2005;42:208–215.)

Over-Expression of AIF-1 in Liver Allografts and Peripheral Blood Correlates with Acute Rejection after Transplantation in Rats

Early and accurate detection of acute cellular rejection (ACR) is important in the management of liver allograft recipients. We hypothesized that expression of allograft inflammatory factor (AIF)‐1 would be associated with liver allograft rejection as previous studies have shown that a relationship exists between kidney and heart transplantation. Indeed using rat orthotopic transplant models we found that the expression of allograft inflammatory factor‐1 (AIF‐1) can be detected in both allograft and peripheral blood leukocytes with peak levels detected 7 days following liver transplantation. Interestingly, AIF‐1 expression increased 2‐fold in acutely rejecting liver allografts compared to chronically accepted livers on days 5, 7 and 10 after transplantation. AIF‐1 expression in peripheral blood leukocytes was also significantly greater in the rejection model than in the acceptance model. Flow cytometric analysis of peripheral blood leukocytes demonstrated that AIF‐1 was expressed in ED2‐positive cells, a marker for Kupffer cells. In vitro studies showed that AIF‐1 expression in Kupffer cells was up‐regulated by coculture with Th1 cytokines. However, neither LPS nor Escherichia coli (E. coli) administration had an affect on AIF‐1 expression. These data indicate that high levels of AIF‐1 expression reflect aggressive liver allograft rejection and suggest a role for monitoring AIF‐1 in peripheral blood leukocytes as a monitor for increased immunosuppression.

Activation of innate immunity (NK/IFN-γ) in rat allogeneic liver transplantation: contribution to liver injury and suppression of hepatocyte proliferation

Liver transplantation is presently the only curative treatment for patients with end-stage liver disease. However, the mechanisms underlying liver injury and hepatocyte proliferation posttransplantation remain obscure. In this investigation, liver injury and hepatocyte proliferation in syngeneic and allogeneic animal models were compared. Male Lewis and Dark Agouti (DA) rats were subjected to orthotopic liver transplantation (OLT). Rat OLT was performed in syngeneic (Lewis-Lewis) and allogeneic (Lewis-DA or DA-Lewis) animal models. Allogeneic liver grafts exhibited greater injury and cellular apoptosis than syngeneic grafts but less hepatocyte proliferation after OLT. Expression of IFN-gamma mRNA and activation of the downstream signal transducer and activator of transcription 1 (STAT1) and genes (interferon regulatory factor-1 and cyclin-dependent kinase inhibitor p21(CDKN1A)) were also greater in the allogeneic grafts compared with the syngeneic grafts. In contrast, STAT3 activation was lower in the allogeneic grafts. Furthermore, in the allogeneic grafts, depletion of natural killer (NK) cells decreased IFN-gamma/STAT1 activation but enhanced hepatocyte proliferation. These findings suggest that, compared with syngeneic transplantation, innate immunity (NK/IFN-gamma) is activated after allogeneic transplantation, which likely contributes to liver injury and inhibits hepatocyte proliferation.

Mobilization of Host Stem Cells Enables Long Term Liver Transplant Acceptance in a Strongly Rejecting Rat Strain Combination

Careful examination of liver, kidney and heart transplants in human recipients has revealed small numbers of host bone marrow derived stem cells in the graft. If the limited recipient repopulation of a donor graft that is currently observed could be facilitated, it is possible that conversion to a predominantly host phenotype would permit long‐term graft function without immunosuppression. We proposed to “engineer” repopulation after transplant in a strain combination (dark agouti [DA] to Lewis green fluorescent protein+[LEW GFP+]) which rejects liver grafts strongly, a model that more closely resembles the situation in humans. Treatment on days 0, 1, 2, 3 and 7 after transplantation with low‐dose (0.1 mg/kg) tacrolimus (T) designed to blunt rejection combined with plerixafor (P) to mobilize host stem cells resulted in greater than 180 days graft survival with extensive albeit spotty conversion of a small (50%) DA graft to the recipient LEW GFP+ genotype. Subsequent skin grafting revealed donor‐specific graft prolongation. The T plus P treatment resulted in higher levels of Lin‐Thy1+CD34+CD133+ stem cells and Foxp3+ regulatory T cells in the blood and liver at day 7. Thus, pharmacological mobilization of host stem cells sustains liver allografts by two mechanisms: repopulation of injured donor cells and regulation of the immune response.

Recruitment of host progenitor cells in rat liver transplants.

Despite major histocompatibility complex incompatibility, liver transplants from Lewis rats to dark agouti (DA) rats survive indefinitely without immunosuppression, and the studies we report sought the mechanism(s) responsible for this. At 1 year, most of the liver reacted positively to host anti‐DA antibody. When small (50%) grafts were transplanted, recruitment was more rapid because most of the organ assumed the host phenotype at 3 months. After transplantation, the Y chromosome was detected in the hepatocytes of XX to XY grafts by both in situ hybridization and polymerase chain reaction. Further, livers from transgenic Lewis rats carrying strong green fluorescent protein (GFP) markers lost the marker with time after transplantation to DA, GFP‐negative hosts. Few liver cells contained the Y chromosome in syngeneic XX to XY liver grafts or when the hosts of Lewis XX to DA XY allografts were treated with cyclosporine A at 10 mg/kg/day. This dosage also impeded enlargement of the liver at 10 days. Using GFP‐positive XX Lewis donors transplanted to GFP‐negative XY DA hosts, we found little Y DNA in GFP‐positive cells at 10 days. Host‐derived OV‐6–positive and c‐kit–positive, albumin‐positive cells were present at 3‐10 days, but cells with the CD34 marker were less common and some clearly still had the donor phenotype at 10 days. Cells positive for chemokine cysteine‐X‐cysteine receptor‐4 increased with time and were abundant 1 month after transplantation. We conclude: (1) extrahepatic cells can differentiate into liver tissues; (2) regenerative stimuli accelerate stem cell recruitment; (3) both regeneration and recruitment are impeded by cyclosporine A immunosuppression, and (4) donor GFP‐positive cells contained little host Y chromosome after transplantation, suggesting that cell fusion was uncommon and, therefore, unlikely to be the mechanism leading to the changes in genotype and phenotype we observed. (HEPATOLOGY 2008.)