Shoko Kimura

A novel mouse model of depletion of stellate cells clarifies their role in ischemia/reperfusion- and endotoxin-induced acute liver injury

BACKGROUND & AIMS Hepatic stellate cells (HSCs) that express glial fibrillary acidic protein (GFAP) are located between the sinusoidal endothelial cells and hepatocytes. HSCs are activated during liver injury and cause hepatic fibrosis by producing excessive extracellular matrix. HSCs also produce many growth factors, chemokines and cytokines, and thus may play an important role in acute liver injury. However, this function has not been clarified due to unavailability of a model, in which HSCs are depleted from the normal liver. METHODS We treated mice expressing HSV-thymidine kinase under the GFAP promoter (GFAP-Tg) with 3 consecutive (3 days apart) CCl4 (0.16 μl/g; ip) injections to stimulate HSCs to enter the cell cycle and proliferate. This was followed by 10-day ganciclovir (40 μg/g/day; ip) treatment, which is expected to eliminate actively proliferating HSCs. Mice were then subjected to hepatic ischemia/reperfusion (I/R) or endotoxin treatment. RESULTS CCl4/ganciclovir treatment caused depletion of the majority of HSCs (about 64-72%), while the liver recovered from the initial CCl4-induced injury (confirmed by histology, serum ALT and neutrophil infiltration). The magnitude of hepatic injury due to I/R or endotoxemia (determined by histopathology and serum ALT) was lower in HSC-depleted mice. Their hepatic expression of TNF-α, neutrophil chemoattractant CXCL1 and endothelin-A receptor also was significantly lower than the control mice. CONCLUSIONS HSCs play an important role both in I/R- and endotoxin-induced acute hepatocyte injury, with TNF-α and endothelin-1 as important mediators of these effects.

Notch-Nrf2 Axis: Regulation of Nrf2 Gene Expression and Cytoprotection by Notch Signaling

ABSTRACT The Notch signaling pathway enables regulation and control of development, differentiation, and homeostasis through cell-cell communication. Our investigation shows that Notch signaling directly activates the Nrf2 stress adaptive response pathway through recruitment of the Notch intracellular domain (NICD) transcriptosome to a conserved Rbpjκ site in the promoter of Nrf2. Stimulation of Notch signaling through Notch ligand expression in cells and by overexpression of the NICD in RosaNICD/−::AlbCre mice in vivo induces expression of Nrf2 and its target genes. Continuous and transient NICD expression in the liver produces a Notch-dependent cytoprotective response through direct transcriptional activation of Nrf2 signaling to rescue mice from acute acetaminophen toxicity. This response can be reversed upon genetic disruption of Nrf2. Morphological studies showed that the characteristic phenotype of high-density intrahepatic bile ducts and enlarged liver in RosaNICD/−::AlbCre mice could be at least partially reversed after Nrf2 disruption. Furthermore, the liver and bile duct phenotypes could be recapitulated with constitutive activation of Nrf2 signaling in Keap1F/F::AlbCre mice. It appears that Notch-to-Nrf2 signaling is another important determinant in liver development and function and promotes cell-cell cytoprotective signaling responses.

Selective Expansion of Allogeneic Regulatory T Cells by Hepatic Stellate Cells: Role of Endotoxin and Implications for Allograft Tolerance

Hepatic stellate cells (HSCs) may play an important role in hepatic immune regulation by producing numerous cytokines/chemokines and expressing Ag-presenting and T cell coregulatory molecules. Due to disruption of the endothelial barrier during cold-ischemic storage and reperfusion of liver grafts, HSCs can interact directly with cells of the immune system. Endotoxin (LPS), levels of which increase in liver diseases and transplantation, stimulates the synthesis of many mediators by HSCs. We hypothesized that LPS-stimulated HSCs might promote hepatic tolerogenicity by influencing naturally occurring immunosuppressive CD4+CD25+Foxp3+ regulatory T cells (Tregs). Following their portal venous infusion, allogeneic CD4+ T cells, including Tregs, were found closely associated with HSCs, and this association increased in LPS-treated livers. In vitro, both unstimulated and LPS-stimulated HSCs upregulated Fas (CD95) expression on conventional CD4+ T cells and induced their apoptosis in a Fas/Fas ligand-dependent manner. By contrast, HSCs induced Treg proliferation, which required cell–cell contact and was MHC class II-dependent. This effect was augmented when HSCs were pretreated with LPS. LPS increased the expression of MHC class II, CD80, and CD86 and stimulated the production of IL-1α, IL-1β, IL-6, IL-10 and TNF-α by HSCs. Interestingly, production of IL-1α, IL-1β, IL-6, and TNF-α was strongly inhibited, but that of IL-10 enhanced in LPS-pretreated HSC/Treg cocultures. Adoptively transferred allogeneic HSCs migrated to the secondary lymphoid tissues and induced Treg expansion in lymph nodes. These data implicate endotoxin-stimulated HSCs as important immune regulators in liver transplantation by inducing selective expansion of tolerance-promoting Tregs and reducing inflammation and alloimmunity.

CD39 expression by hepatic myeloid dendritic cells attenuates inflammation in liver transplant ischemia‐reperfusion injury in mice

Hepatic innate immune cells, in particular, interstitial dendritic cells (DCs), regulate inflammatory responses and may promote inherent liver tolerogenicity. After tissue injury, adenosine triphosphate (ATP) is released and acts as a damage‐associated molecular pattern that activates innate immune cells by pattern recognition receptors. CD39 (ectonucleoside triphosphate diphosphohydrolase‐1) rapidly hydrolyzes extracellular ATP to maintain physiological levels. We hypothesized that CD39 expression on liver DCs might contribute to regulation of their innate immune functions. Mouse liver conventional myeloid DCs (mDCs) were hyporesponsive to ATP, compared with their splenic counterparts. This disparity was ascribed to more efficient hydrolysis of ATP by higher expression of CD39 on liver mDCs. Human liver mDCs expressed greater levels of CD39 than those from peripheral blood. The comparatively high expression of CD39 on liver mDCs correlated strongly with both ATP hydrolysis and adenosine production. Notably, CD39−/− mouse liver mDCs exhibited a more mature phenotype, greater responsiveness to Toll‐like receptor 4 ligation, and stronger proinflammatory and immunostimulatory activity than wild‐type (WT) liver mDCs. To investigate the role of CD39 on liver mDCs in vivo, we performed orthotopic liver transplantation with extended cold preservation using CD39−/− or WT donor mouse livers. Compared to WT liver grafts, CD39−/− grafts exhibited enhanced interstitial DC activation, elevated proinflammatory cytokine levels, and more‐severe tissue injury. Moreover, portal venous delivery of WT, but not CD39−/− liver mDCs, to donor livers immediately post‐transplant exerted a protective effect against graft injury in CD39−/− to CD39−/− liver transplantation. Conclusions: These data reveal that CD39 expression on conventional liver mDCs limits their proinflammatory activity and confers protective properties on these important innate immune cells against liver transplant ischemia/reperfusion injury. (Hepatology 2013; 58:2163–2175)

Gut Bacteria Drive Kupffer Cell Expansion via MAMP-Mediated ICAM-1 Induction on Sinusoidal Endothelium and Influence Preservation-Reperfusion Injury after Orthotopic Liver Transplantation

Bacteria in the gut microbiome shed microbial-associated molecule patterns (MAMPs) into the portal venous circulation, where they augment various aspects of systemic immunity via low-level stimulation. Because the liver is immediately downstream of the intestines, we proposed that gut-derived MAMPs shape liver immunity and affect Kupffer cell (KC) phenotype. Germ-free (GF), antibiotic-treated (AVMN), and conventional (CL) mice were used to study KC development, function, and response to the significant stress of cold storage, reperfusion, and orthotopic transplantation. We found that a cocktail of physiologically active MAMPs translocate into the portal circulation, with flagellin (Toll-like receptor 5 ligand) being the most plentiful and capable of promoting hepatic monocyte influx in GF mice. In MAMP-deficient GF or AVMN livers, KCs are lower in numbers, have higher phagocytic activity, and have lower major histocompatibility complex II expression. MAMP-containing CL livers harbor significantly increased KC numbers via induction of intercellular adhesion molecule 1 on liver sinusoidal endothelium. These CL KCs have a primed yet expected phenotype, with increased major histocompatibility complex class II and lower phagocytic activity that increases susceptibility to liver preservation/reperfusion injury after orthotopic transplantation. The KC number, functional activity, and maturational status are directly related to the concentration of gut-derived MAMPs and can be significantly reduced by broad-spectrum antibiotics, thereby affecting susceptibility to injury.

Roles of dendritic cells in murine hepatic warm and liver transplantation‐induced cold ischemia/reperfusion injury

Dendritic cells (DCs) induce and regulate both innate and adaptive immune responses; however, their in vivo functional importance in hepatic ischemia/reperfusion (IR) injury is perplexing. We hypothesized that liver‐resident DC and locally recruited blood‐borne DC might have distinctive roles in hepatic IR injury. We tested this hypothesis by using DC‐deficient, fms‐like tyrosine kinase 3 ligand (Flt3L) knockout (KO) mice in hepatic warm (70% partial clamping for 60 minutes) and cold IR injury (liver transplant [LTx] with 24‐hour cold storage). Flt3L KO liver and lymphoid organs contained virtually no CD11c+F4/80− DC. Hepatic warm IR injury was significantly lower in Flt3L KO than in wildtype (WT) mice with lower alanine aminotransferase (ALT) levels, reduced hepatic necrosis, and lower neutrophil infiltration. Hepatic messenger RNA (mRNA) and protein levels for inflammatory cytokines (tumor necrosis factor alpha [TNFα], interleukin [IL]‐6) and chemokines (CCL2, CXCL2) were also significantly lower in Flt3L KO than in WT mice, indicating that lack of both liver‐resident and blood‐borne DC ameliorated hepatic warm IR injury. Adoptive transfer of splenic or hepatic WT DC into Flt3L KO or WT mice increased hepatic warm IR injury, suggesting injurious roles of DC infusion. When Flt3L KO liver was transplanted into WT mice, ALT levels were significantly higher than in WT to WT LTx, with enhanced hepatic necrosis and neutrophil infiltration, indicating a protective role of liver‐resident DC. Conclusion: Using both warm and cold hepatic IR models, this study suggests differential roles of liver‐resident versus blood‐borne DC, and points to the importance of the local microenvironment in determining DC function during hepatic IR injury. (HEPATOLOGY 2013;57:1585–1596)

Hepatic B7 homolog 1 expression is essential for controlling cold ischemia/reperfusion injury after mouse liver transplantation†‡

Ischemia/reperfusion (I/R) injury remains a key risk factor significantly affecting morbidity and mortality after liver transplantation (LT). B7 homolog 1 (B7‐H1), a recently identified member of the B7 family, is known to play important roles in regulating local immune responses. We hypothesized that B7‐H1 plays crucial roles during innate immune responses induced by hepatic I/R injury, and using B7‐H1 knockout (KO) liver grafts, we tested this hypothesis in the mouse LT model with 24 hours of cold storage. Cold I/R injury in wild type (WT)‐to‐WT LT enhanced constitutive B7‐H1 expression on dendritic cells and sinusoidal endothelial cells and promptly induced B7‐H1 on hepatocytes. When B7‐H1 KO liver grafts were transplanted into WT recipients, serum alanine aminotransferase (ALT) and graft necrosis levels were significantly higher than those after WT‐to‐WT LT. Augmented tissue injury in B7‐H1 KO grafts was associated with increased frequencies and absolute numbers of graft CD3+ T cells (particularly CD8+ T cells). B7‐H1 KO grafts had significantly fewer annexin V+ CD8+ T cells, and this indicated a failure to delete infiltrating CD8+ T cells. To evaluate the relative contributions of parenchymal cell and bone marrow–derived cell (BMDC) B7‐H1 expression, we generated and transplanted into WT recipients chimeric liver grafts lacking B7‐H1 on parenchymal cells or BMDCs. A selective B7‐H1 deficiency on parenchymal cells or BMDCs resulted in similar levels of ALT and liver injury, and this suggested that parenchymal cell and BMDC B7‐H1 expression was involved in liver damage control. Human livers up‐regulated B7‐H1 expression after LT. Conclusion: The study demonstrates that graft tissue expression of B7‐H1 plays a critical role in regulating inflammatory responses during LT‐induced hepatic I/R injury, and negative coregulatory signals may have an important function in hepatic innate immune responses. (HEPATOLOGY 2011;)

Plasmacytoid dendritic cell‐derived IFN‐α promotes murine liver ischemia/reperfusion injury by induction of hepatocyte IRF‐1

Plasmacytoid dendritic cells (pDC) constitute the bodys principal source of type I interferon (IFN) and are comparatively abundant in the liver. Among various cytokines implicated in liver ischemia and reperfusion (I/R) injury, type I IFNs have been described recently as playing an essential role in its pathogenesis. Moreover, type I IFNs have been shown to up‐regulate hepatocyte expression of IFN regulatory factor 1 (IRF‐1), a key transcription factor that regulates apoptosis and induces liver damage after I/R. Our aim was to ascertain the capacity of IFN‐α released by liver pDC to induce liver damage through hepatic IRF‐1 up‐regulation after I/R injury. Our findings show that liver pDC mature and produce IFN‐α in response to liver I/R. Liver pDC isolated after I/R induced elevated levels of IRF‐1 production by hepatocytes compared with liver pDC isolated from sham‐operated mice. Notably, hepatic IRF‐1 expression was reduced significantly by neutralizing IFN‐α. In vivo, IFN‐α neutralization protected the liver from I/R injury by reducing hepatocyte apoptosis. This was associated with impaired expression of IRF‐1 and proapoptotic molecules such as Fas ligand, its receptor (Fas) and death receptor 5, which are regulated by IRF‐1. Furthermore, pDC‐depleted mice failed to up‐regulate hepatic IFN‐α and displayed less liver injury associated with reduced levels of hepatic interleukin (IL)‐6, tumor necrosis factor‐α, and hepatocyte apoptosis after I/R compared with controls. Conclusion: these data support the hypothesis that IFN‐α derived from liver pDC plays a key role in the pathogenesis of liver I/R injury by enhancing apoptosis as a consequence of induction of hepatocyte IRF‐1 expression. (Hepatology 2014;60:267–277)

Interferon regulatory factor-2 is protective against hepatic ischemia-reperfusion injury

Interferon regulatory factor (IRF)-1 is a nuclear transcription factor that induces inflammatory cytokine mediators and contributes to hepatic ischemia-reperfusion (I/R) injury. No strategies to mitigate IRF1-mediated liver damage exist. IRF2 is a structurally similar endogenous protein that competes with IRF1 for DNA binding sites in IRF-responsive target genes and acts as a competitive inhibitor. However, the role of IRF2 in hepatic injury during hypoxic or inflammatory conditions is unknown. We hypothesize that IRF2 overexpression may mitigate IRF1-mediated I/R damage. Endogenous IRF2 is basally expressed in normal livers and is mildly increased by ischemia alone. Overexpression of IRF2 protects against hepatic warm I/R injury. Furthermore, we demonstrate that IRF2 overexpression limits production of IRF1-dependent proinflammatory genes, such as IL-12, IFNβ, and inducible nitric oxide synthase, even in the presence of IRF1 induction. Additionally, isograft liver transplantation with IRF2 heterozygote knockout (IRF2(+/-)) donor grafts that have reduced endogenous IRF2 levels results in worse injury following cold I/R during murine orthotopic liver transplantation. These findings indicate that endogenous intrahepatic IRF2 protein is protective, because the IRF2-deficient liver donor grafts exhibited increased liver damage compared with the wild-type donor grafts. In summary, IRF2 overexpression protects against I/R injury by decreasing IRF1-dependent injury and may represent a novel therapeutic strategy.

Use of carbon monoxide in minimizing ischemia/reperfusion injury in transplantation

Although carbon monoxide (CO) is known to be toxic because of its ability to interfere with oxygen delivery at high concentrations, mammalian cells endogenously generate CO primarily via the catalysis of heme by heme oxygenases. Recent findings have indicated that heme oxygenases and generation of CO serve as a key mechanism to maintain the integrity of the physiological function of organs and supported the development of a new paradigm that CO, at low concentrations, functions as a signaling molecule in the body and exerts significant cytoprotection. Consequently, exogenously delivered CO has been shown to mediate potent protection in various injury models through its anti-inflammatory, vasodilating, and antiapoptotic functions. Ischemia/reperfusion (I/R) injury associated with organ transplantation is one of the major deleterious factors limiting the success of transplantation. Ischemia/reperfusion injury is a complex cascade of interconnected events involving cell damage, apoptosis, vigorous inflammatory responses, microcirculation disturbance, and thrombogenesis. Carbon monoxide has a great potential in minimizing I/R injury. This review will provide an overview of the basic physiology of CO, preclinical studies examining efficacy of CO in I/R injury models, and possible protective mechanisms. Carbon monoxide could be developed to be a valuable therapeutic molecule in minimizing I/R injury in transplantation.