Shinya Ueki

Inhibition of Kupffer cell‐mediated early proinflammatory response with carbon monoxide in transplant‐induced hepatic ischemia/reperfusion injury in rats

Proinflammatory responses play critical roles in hepatic ischemia/reperfusion (I/R) injury associating with liver transplantation (LTx), and carbon monoxide (CO) can effectively down‐regulate them. Using wild‐type (WT) to enhanced green fluorescent protein (EGFP)‐transgenic rat LTx with 18‐hour cold preservation in University of Wisconsin solution, this study analyzed the relative contribution of donor and host cells during early posttransplantation period and elucidated the mechanism of hepatic protection by CO. CO inhibited hepatic I/R injury and reduced peak alanine aminotransferase levels at 24 hours and hepatic necrosis at 48 hours. Abundant EGFP+ host cells were found in untreated WT liver grafts at 1 hour and included nucleated CD45+ leukocytes (myeloid, T, B, and natural killer cells) and EGFP+ platelet‐like depositions in the sinusoids. However, reverse transcription polymerase chain reaction (RT‐PCR) analysis of isolated graft nonparenchymal cells (NPCs) revealed that I/R injury‐induced proinflammatory mediators [for example, tumor necrosis factor alpha (TNF‐α), interleukin‐6 (IL‐6), and inducible nitric oxide synthase (iNOS)] were not up‐regulated in purified CD45+ cells of donor or host origin. Instead, TNF‐α and IL‐6 messenger RNA (mRNA) elevation was exclusively seen in isolated CD68+ cells, whereas iNOS mRNA up‐regulation was seen in hepatocytes. Nearly all CD68+ cells at 1 hour after LTx were EGFP− donor Kupffer cells, and CO efficiently inhibited TNF‐α and IL‐6 up‐regulation in the CD68+ Kupffer cell fraction. When graft Kupffer cells were inactivated with gadolinium chloride, activation of inflammatory mediators in liver grafts was significantly inhibited. Furthermore, in vitro rat primary Kupffer cell culture also showed significant down‐regulation of lipopolysaccharide (LPS)‐induced inflammatory responses by CO. Conclusion: These results indicate that CO ameliorates hepatic I/R injury by down‐regulating graft Kupffer cells in early postreperfusion period. The study also suggests that different cell populations play diverse roles by up‐regulating distinctive sets of mediators in the acute phase of hepatic I/R injury. (HEPATOLOGY 2008;48:1608–1620.)

Critical role of interferon regulatory factor‐1 in murine liver transplant ischemia reperfusion injury

Interferon regulatory factor‐1 (IRF‐1) is a transcription factor that regulates gene expression during immunity. We hypothesized that IRF‐1 plays a pivotal role in liver transplant (LTx) ischemia/reperfusion (I/R) injury. Mouse orthotopic LTx was conducted after 24 hours cold storage in University of Wisconsin (UW) solution in wildtype (WT) C57BL/6 and IRF‐1 knockout (KO) mice. IRF‐1 deficiency in liver grafts, but not in recipients, resulted in significant reduction of hepatocyte apoptosis and liver injury, as well as improved survival. IRF‐1 mRNA up‐regulation was typically seen in graft hepatocytes in WT→WT LTx. Deficiency of IRF‐1 signaling in graft resulted in significantly reduced messenger RNA (mRNA) levels for death ligands and death receptors in hepatocytes, as well as decreased caspase‐8 activities, indicating that IRF‐1 mediates death ligand‐induced hepatocyte death. Further, a smaller but significant IRF‐1 mRNA up‐regulation was seen in WT graft nonparenchymal cells (NPC) and associated with interferon gamma (IFN‐γ) mRNA up‐regulation exclusively in NPC. IFN‐γ mRNA was significantly reduced in IRF‐1 KO graft. Thus, IRF‐1 in graft hepatocytes and NPC has distinct effects in hepatic I/R injury. However, LTx with chimeric liver grafts showed that grafts lacking hepatocellular IRF‐1 had better protection compared with those lacking IRF‐1 in NPC. The study identifies a critical role for IRF‐1 in liver transplant I/R injury. (HEPATOLOGY 2010.)

Ex vivo Application of Carbon Monoxide in UW Solution Prevents Transplant-Induced Renal Ischemia/Reperfusion Injury in Pigs

I/R injury is a major deleterious factor of successful kidney transplantation (KTx). Carbon monoxide (CO) is an endogenous gaseous regulatory molecule, and exogenously delivered CO in low concentrations provides potent cytoprotection. This study evaluated efficacies of CO exposure to excised kidney grafts to inhibit I/R injury in the pig KTx model. Porcine kidneys were stored for 48 h in control UW or UW supplemented with CO (CO‐UW) and autotransplanted in a 14‐day follow‐up study. In the control UW group, animal survival was 80% (4/5) with peak serum creatinine levels of 12.0 ± 5.1 mg/dL. CO‐UW showed potent protection, and peak creatinine levels were reduced to 6.9 ± 1.4 mg/dL with 100% (5/5) survival without any noticeable adverse event or abnormal COHb value. Control grafts at 14 days showed significant tubular damages, focal fibrotic changes and numerous infiltrates. The CO‐UW group showed significantly less severe histopathological changes with less TGF‐β and p‐Smad3 expression. Grafts in CO‐UW also showed significantly lower early mRNA levels for proinflammatory cytokines and less lipid peroxidation. CO in UW provides significant protection against renal I/R injury in the porcine KTx model. Ex vivo exposure of kidney grafts to CO during cold storage may therefore be a safe strategy to reduce I/R injury.

LIVER GRAFT EXPOSURE TO CARBON MONOXIDE DURING COLD STORAGE PROTECTS SINUSOIDAL ENDOTHELIAL CELLS AND AMELIORATES REPERFUSION INJURY IN RATS

Hepatic ischemia/reperfusion (I/R) injury significantly influences short‐term and long‐term outcomes after liver transplantation (LTx). The critical step initiating the injury is known to include sinusoidal endothelial cell (SEC) alteration during the cold preservation period. As carbon monoxide (CO) has potent cytoprotective functions on vascular endothelial cells, this study examined if CO treatment of excised liver grafts during cold storage could protect SECs and ameliorate hepatic I/R injury. Rat liver grafts were preserved in University of Wisconsin (UW) solution containing 5% CO (CO‐UW solution) for 18 to 24 hours and were transplanted into syngeneic Lewis rats. After 18 hours of cold preservation, SEC damage was evident with propidium iodide (PI) nuclear staining on SECs, and the frequency of PI+ SECs was significantly lower in grafts stored in CO‐UW solution versus those stored in control UW solution. SEC protection with CO was associated with decreased intercellular cell adhesion molecule translocation and less matrix metalloproteinase release during cold preservation. After LTx with 18 hours of cold preservation, serum alanine aminotransferase levels and hepatic necrosis were significantly less in the CO‐UW group than in the control UW group. With 24 hours of cold storage, 35% (7/20) survived with control UW solution, whereas the survival with CO‐UW solution improved to 80% (8/10). These beneficial effects of CO‐UW solution were associated with a significant reduction of neutrophil extravasation, down‐regulation of hepatic messenger RNA for tumor necrosis factor alpha and intercellular cell adhesion molecule 1, and less hepatic extracellular signal‐regulated kinase activation. Liver grafts from Kupffer cell–depleted donors or pseudogerm‐free donors showed less SEC death during cold preservation, and CO‐UW solution further reduced SEC death. In conclusion, CO delivery to excised liver grafts during cold preservation efficiently ameliorates SEC damage and hepatic I/R injury. Liver Transpl 15:1458–1468, 2009.

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;)

Carbon monoxide inhibits apoptosis during cold storage and protects kidney grafts donated after cardiac death

Ischemia/reperfusion (I/R) injury remains as a serious deleterious factor in kidney transplantation (KTx). We hypothesized that carbon monoxide (CO), an endogenous potent cytoprotective molecule, inhibits hypothermia‐induced apoptosis of kidney grafts. Using the rat KTx model mimicking the conditions of donation after cardiac death (DCD) as well as nontransplantable human kidney grafts, this study examined effects of CO in preservation solution in improving the quality of marginal kidney grafts. After cardiac cessation, rat kidneys underwent 40 min warm ischemia (WI) and 24 h cold storage (CS) in control UW or UW containing CO (CO‐UW). At the end of CS, kidney grafts in control UW markedly increased mitochondrial porin release into the cytosol and resulted in increased cleaved caspase‐3 and PARP expression. In contrast, grafts in CO‐UW had significantly reduced mitochondrial breakdown and caspase pathway activation. After KTx, recipient survival significantly improved with CO‐UW with less TUNEL+ cells and reduced mRNA upregulation for proinflammatory mediators (IL‐6, TNF‐α, iNOS). Furthermore, when nontransplantable human kidney grafts were stored in CO‐UW for 24 h, graft PARP expression, TUNEL+ cells, and proinflammatory mediators were less than those in control UW. CO in UW inhibited hypothermia‐induced apoptosis and significantly improved kidney graft function and outcomes of KTx.

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.

The loss of renal dendritic cells and activation of host adaptive immunity are long-term effects of ischemia/reperfusion injury following syngeneic kidney transplantation

Ischemia/reperfusion injury associated with kidney transplantation induces profound acute injury, influences early graft function and affects long-term graft outcomes. To determine whether renal dendritic cells play any role during initial innate ischemia/reperfusion injury and the subsequent development of adaptive immune responses, we studied the behavior and function of renal graft and host infiltrating dendritic cells during early and late phases of renal ischemia/reperfusion injury. Wild type to GFP-transgenic rat kidney transplantation was performed with and without 24 hours cold storage. Ischemia/reperfusion injury in cold stored grafts resulted in histopathological changes of interstitial fibrosis and tubular atrophy by 10 weeks accompanied by upregulation of mRNAs of mediators of interstitial fibrosis and inflammation. In normal rat kidneys we identified two populations of renal dendritic cells, predominant CD103−CD11b/c+ and minor CD103+CD11b/c+ cells. After transplantation without cold storage, grafts maintained CD103− but not CD103+ GFP-negative renal dendritic cells for 10 weeks. In contrast, both cell subsets disappeared from cold stored grafts, which associated with a significant GFP-expressing host CD11b/c+ cell infiltration that included CD103+ dendritic cells with a TNF-α producing phenotype. These changes in graft/host dendritic cell populations were associated with progressive infiltration of host CD4+ T cells with effector/effector-memory phenotypes and IFN-γ secretion. Thus, renal graft ischemia/reperfusion injury causes graft dendritic cell loss and was associated with progressive host dendritic cell and T cell recruitment. Renal resident dendritic cells might function as a protective regulatory network.

Orthotopic mouse liver transplantation to study liver biology and allograft tolerance

Orthotopic liver transplantation in the mouse is a powerful research tool that has led to important mechanistic insights into the regulation of hepatic injury, liver immunopathology, and transplant tolerance. However, it is a technically demanding surgical procedure. Setup of the orthotopic liver transplantation model comprises three main stages: surgery on the donor mouse; back-table preparation of the liver graft; and transplant of the liver into the recipient mouse. In this protocol, we describe our procedure in stepwise detail to allow efficient completion of both the donor and recipient operations. The protocol can result in consistently high technical success rates when performed by personnel experienced in the protocol. The technique can be completed in ∼2–3 h when performed by an individual who is well practiced in performing mouse transplantation in accordance with this protocol. We have achieved a perioperative survival rate close to 100%.