Shinichiro Yokota

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.

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)

Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity

Significance Gene therapy has made significant strides in the treatment and even cure of single-gene defects. However, the maturation of this field will require more sophisticated vehicles capable of cell-selective delivery of large genetic payloads whose regulated expression will restore or enhance cellular functionality. High-capacity herpes simplex virus vectors have the potential to meet these challenges but have been limited by the need to preserve one particularly cytotoxic viral product, infected cell polypeptide 0, to maintain transgene transcriptional activity. Our study describes a vector design that solves this conundrum, thereby promising the near-term availability of viral vectors that can efficiently deliver large or multiple regulated transgenes to a diversity of cells without attendant cytotoxicity. The design of highly defective herpes simplex virus (HSV) vectors for transgene expression in nonneuronal cells in the absence of toxic viral-gene activity has been elusive. Here, we report that elements of the latency locus protect a nonviral promoter against silencing in primary human cells in the absence of any viral-gene expression. We identified a CTCF motif cluster 5′ to the latency promoter and a known long-term regulatory region as important elements for vigorous transgene expression from a vector that is functionally deleted for all five immediate-early genes and the 15-kb internal repeat region. We inserted a 16.5-kb expression cassette for full-length mouse dystrophin and report robust and durable expression in dystrophin-deficient muscle cells in vitro. Given the broad cell tropism of HSV, our design provides a nontoxic vector that can accommodate large transgene constructs for transduction of a wide variety of cells without vector integration, thereby filling an important void in the current arsenal of gene-therapy vectors.

CD39 deficiency in murine liver allografts promotes inflammatory injury and immune-mediated rejection.

Adenosine triphosphate (ATP), an essential metabolic energy source, is released following cell apoptosis or necrosis. It acts as a damage-associated molecule pattern to stimulate innate immune cells. The ectonucleotidase CD39 regulates immune activation by hydrolysis of extracellular ATP. We have shown previously that CD39 expression by donor livers helps protect syngeneic grafts with extended (24 hr) cold preservation time from ischemia reperfusion injury. Given its immune regulatory properties, we hypothesized that CD39 expression in donor livers might modulate transplant tolerance that occurs following mouse allogeneic liver transplantation (LTx). Livers from C57BL/6 (B6) wild-type (WT) or CD39 KO mice were transplanted into normal C3H recipients with minimal (approximately 1 hr) cold ischemia. Serum alanine aminotransferase levels at day 4 post LTx were significantly higher in animals given CD39KO compared with WT livers. Moreover, IFN-γ production by liver-infiltrating CD8+ T cells at day 4 was significantly higher in CD39KO than in WT grafts. Furthermore, splenic T cells from CD39KO liver recipients exhibited greater proliferative responses to donor alloantigens than those from mice given WT grafts. By contrast, there was a concomitant significant reduction in the frequency of regulatory T cells (Treg) in CD39KO than in WT livers. Whereas WT liver allografts survived > 100 days, no CD39KO grafts survived beyond 40 days (median survival time [MST]: WT: >100 days vs CD39KO: 8 days; p<0.01). In addition, soluble CD39 administration significantly prolonged CD39KO liver allograft survival (MST: 27.5 days). These novel data suggest that CD39 expression in liver allografts modulates tissue injury, inflammation, anti-donor effector T cell responses and Treg infiltration and can suppress transplant rejection.

IRF-1 Promotes Liver Transplant Ischemia/Reperfusion Injury via Hepatocyte IL-15/IL-15Rα Production

Ischemia and reperfusion (I/R) injury following liver transplantation (LTx) is an important problem that significantly impacts clinical outcomes. IFN regulatory factor-1 (IRF-1) is a nuclear transcription factor that plays a critical role in liver injury. Our objective was to determine the immunomodulatory role of IRF-1 during I/R injury following allogeneic LTx. IRF-1 was induced in liver grafts immediately after reperfusion in both human and mouse LTx. IRF-1 contributed significantly to I/R injury because IRF-1–knockout (KO) grafts displayed much less damage as assessed by serum alanine aminotransferase and histology. In vitro, IRF-1 regulated both constitutive and induced expression of IL-15, as well as IL-15Rα mRNA expression in murine hepatocytes and liver dendritic cells. Specific knockdown of IRF-1 in human primary hepatocytes gave similar results. In addition, we identified hepatocytes as the major producer of soluble IL-15/IL-15Rα complexes in the liver. IRF-1–KO livers had significantly reduced NK, NKT, and CD8+ T cell numbers, whereas rIL-15/IL-15Rα restored these immune cells, augmented cytotoxic effector molecules, promoted systemic inflammatory responses, and exacerbated liver injury in IRF-1–KO graft recipients. These results indicate that IRF-1 promotes LTx I/R injury via hepatocyte IL-15/IL-15Rα production and suggest that targeting IRF-1 and IL-15/IL-15Rα may be effective in reducing I/R injury associated with LTx.

DAP12 deficiency in liver allografts results in enhanced donor DC migration, augmented effector T cell responses and abrogation of transplant tolerance

Liver interstitial dendritic cells (DC) have been implicated in immune regulation and tolerance induction. We found that the transmembrane immuno‐adaptor DNAX‐activating protein of 12 kDa (DAP12) negatively regulated conventional liver myeloid (m) DC maturation and their in vivo migratory and T cell allostimulatory ability. Livers were transplanted from C57BL/6(H2b) (B6) WT or DAP12−/− mice into WT C3H (H2k) recipients. Donor mDC (H2‐Kb+CD11c+) were quantified in spleens by flow cytometry. Anti‐donor T cell reactivity was evaluated by ex vivo carboxyfluorescein diacetate succinimidyl ester‐mixed leukocyte reaction and delayed‐type hypersensitivity responses, while T effector and regulatory T cells were determined by flow analysis. A threefold to fourfold increase in donor‐derived DC was detected in spleens of DAP12−/− liver recipients compared with those given WT grafts. Moreover, pro‐inflammatory cytokine gene expression in the graft, interferon gamma (IFNγ) production by graft‐infiltrating CD8+ T cells and systemic levels of IFNγ were all elevated significantly in DAP12−/− liver recipients. DAP12−/− grafts also exhibited reduced incidences of CD4+Foxp3+ cells and enhanced CD8+ T cell IFNγ secretion in response to donor antigen challenge. Unlike WT grafts, DAP12−/− livers failed to induce tolerance and were rejected acutely. Thus, DAP12 expression in liver grafts regulates donor mDC migration to host lymphoid tissue, alloreactive T cell responses and transplant tolerance.

Regeneration and cell recruitment in an improved heterotopic auxiliary partial liver transplantation model in the rat

Background Auxiliary partial liver transplantation (APLT) in humans is a therapeutic modality used especially to treat liver failure in children or congenital metabolic disease. Animal models of APLT have helped to explore therapeutic options. Though many groups have suggested improvements, standardizing the surgical procedure has been challenging. Additionally, the question of whether graft livers are reconstituted by recipient-derived cells after transplantation has been controversial. The aim of this study was to improve experimental APLT in rats and to assess cell recruitment in the liver grafts. Methods To inhibit recipient liver regeneration and to promote graft regeneration, we treated recipients with retrorsine and added arterial anastomosis. Using green fluorescence protein transgenic rats as recipients, we examined liver resident cell recruitment within graft livers by immunofluorescence costaining. Results In the improved APLT model, we achieved well-regenerated grafts that could maintain regeneration for at least 4 weeks. Regarding the cell recruitment, there was no evidence of recipient-derived hepatocyte, cholangiocyte, or hepatic stellate cell recruitment into the graft. Macrophages/monocytes, however, were consistently recruited into the graft and increased over time, which might be related to inflammatory responses. Very few endothelial cells showed colocalization of markers. Conclusions We have successfully established an improved rat APLT model with arterial anastomosis as a standard technique. Using this model, we have characterized cell recruitment into the regenerating grafts.