Satoshi Kuboki

Targeted Deletion of HIF-1α Gene in T Cells Prevents their Inhibition in Hypoxic Inflamed Tissues and Improves Septic Mice Survival

Background Sepsis patients may die either from an overwhelming systemic immune response and/or from an immunoparalysis-associated lack of anti-bacterial immune defence. We hypothesized that bacterial superantigen-activated T cells may be prevented from contribution into anti-bacterial response due to the inhibition of their effector functions by the hypoxia inducible transcription factor (HIF-1α) in inflamed and hypoxic areas. Methodology/Principal Findings Using the Cre-lox-P-system we generated mice with a T–cell targeted deletion of the HIF-1α gene and analysed them in an in vivo model of bacterial sepsis. We show that deletion of the HIF-1α gene leads to higher levels of pro-inflammatory cytokines, stronger anti-bacterial effects and much better survival of mice. These effects can be at least partially explained by significantly increased NF-κB activation in TCR activated HIF-1 α deficient T cells. Conclusions/Significance T cells can be recruited to powerfully contribute to anti-bacterial response if they are relieved from inhibition by HIF-1α in inflamed and hypoxic areas. Our experiments uncovered the before unappreciated reserve of anti-bacterial capacity of T cells and suggest novel therapeutic anti-pathogen strategies based on targeted deletion or inhibition of HIF-1 α in T cells.

Peroxiredoxin-6 protects against mitochondrial dysfunction and liver injury during ischemia-reperfusion in mice

Hepatic ischemia-reperfusion (I/R) injury is an important complication of liver surgery and transplantation. Mitochondrial function is central to this injury. To examine alterations in mitochondrial function during I/R, we assessed the mitochondrial proteome in C57Bl/6 mice. Proteomic analysis of liver mitochondria revealed 234 proteins with significantly altered expression after I/R. From these, 13 proteins with the greatest expression differences were identified. One of these proteins, peroxiredoxin-6 (Prdx6), has never before been described in mitochondria. In hepatocytes from sham-operated mice, Prdx6 expression was found exclusively in the cytoplasm. After ischemia or I/R, Prdx6 expression disappeared from the cytoplasm and appeared in the mitochondria, suggesting mitochondrial trafficking. To explore the functional role of Prdx6 in hepatic I/R injury, wild-type and Prdx6-knockout mice were subjected to I/R injury. Prdx6-knockout mice had significantly more hepatocellular injury compared with wild-type mice. Interestingly, the increased injury in Prdx6-knockout mice occurred despite reduced inflammation and was associated with increased mitochondrial generation of H(2)O(2) and dysfunction. The mitochondrial dysfunction appeared to be related to complex I of the electron transport chain. These data suggest that hepatocyte Prdx6 traffics to the mitochondria during I/R to limit mitochondrial dysfunction as a protective mechanism against hepatocellular injury.

Hepatocyte signaling through CXC chemokine receptor‐2 is detrimental to liver recovery after ischemia/reperfusion in mice

CXC chemokines and their receptor, CXC chemokine receptor‐2 (CXCR2), are important components of the hepatic inflammatory response to ischemia/reperfusion (I/R). However, direct effects of CXC chemokines on hepatocytes during this response have not been studied. Wild‐type and CXCR2−/− mice were subjected to 90 minutes of partial hepatic ischemia followed by up to 96 hours of reperfusion. CXCR2−/− mice had significantly less liver injury at all reperfusion times compared with wild‐type mice. Early neutrophil recruitment (12 hours) was diminished in CXCR2−/− mice, but within 24 hours it was the same as that of wild‐type mice. Hepatocyte proliferation and regeneration was accelerated in CXCR2−/− mice compared with wild‐type mice. These effects were associated with increased activation of nuclear factor κB and signal transducers and activators of transcription‐3, despite there being no difference in the expression of proliferative factors such as tumor necrosis factor α, interleukin‐6, and hepatocyte growth factor. To establish whether the accelerated proliferation and regeneration observed in CXCR2−/− mice was due to effects on hepatocytes rather than just a generalized decrease in acute inflammatory injury, mice were treated with the CXCR2 antagonist, SB225002, after neutrophil recruitment and injury were maximal (24 hours after reperfusion). SB225002 treatment increased hepatocyte proliferation and regeneration in a manner identical to that observed in CXCR2−/− mice. Treatment of primary wild‐type hepatocytes with macrophage inflammatory protein‐2 revealed that low concentrations protected against cell death, whereas high concentrations induced cell death. These effects were absent in hepatocytes from CXCR2−/− mice. Conclusion: Our data suggest that hepatocyte CXCR2 regulates proliferation and regeneration after I/R injury and reveal important differences in the role of this receptor in liver regeneration and repair induced under different conditions that may be related to ligand concentration. (HEPATOLOGY 2008.)

Age-dependent responses to hepatic ischemia/reperfusion injury

The current study explored the concept that adult and pediatric populations differ in their response to major injury. Male C57BL/6 mice of a “young adult” (8-12 weeks) or “mature adult” (12-13 months) age were subjected to partial hepatic ischemia and reperfusion. Mature adult mice displayed significantly more liver injury than young adult mice as assessed histologically and by serum levels of alanine aminotransferase. Interestingly, there was far less neutrophil accumulation in the livers of mature adult mice. However, liver-recruited neutrophils from mature adult mice had a higher activation state than those from young adult mice. Activation of the inflammatory transcription factor, NF-κB, was suppressed in whole livers from mature adult mice. In isolated liver cells, Kupffer cells showed no difference in NF-κB activation, but hepatocytes from mature adult mice had delayed NF-κB activation in response to TNF. Furthermore, isolated hepatocytes from young adult mice produced abundant amounts of the chemokine, macrophage inflammatory protein-2, whereas hepatocytes from mature adult mice produced little, if any macrophage inflammatory protein-2. Mature adult mice had much lower hepatic expression of the cytoprotective protein, heat shock protein 70, than did young adult mice. In contrast, serum heat shock protein 70 levels, which has been linked to subsequent tissue injury, were higher in mature adult mice than in young adult mice. These data suggest that there are multiple alterations at the cellular and molecular levels that contribute to enhanced postischemic liver injury in mature adult mice.

Activation of hepatocytes by extracellular heat shock protein 72

Heat shock protein (HSP) 72 is released by cells during stress and injury. HSP-72 also stimulates the release of cytokines in macrophages by binding to Toll-like receptors (TLR) 2 and 4. Circulating levels of HSP-72 increase during hepatic ischemia-reperfusion injury. The role of extracellular HSP-72 (eHSP-72) in the injury response to ischemia-reperfusion is unknown. Therefore, the objective of the present study was to determine whether eHSP-72 has any direct effects on hepatocytes. Primary mouse hepatocytes were treated with purified human recombinant HSP-72. Conditioned media were evaluated by ELISA for the cytokines, TNF-alpha, IL-6, and macrophage inflammatory protein 2 (MIP-2). Stimulation of hepatocytes with eHSP-72 did not induce production of TNFalpha or IL-6 but resulted in dose-dependent increases in MIP-2 production. To evaluate the pathway responsible for this response, expression of TLR2 and TLR4 was confirmed on hepatocytes by immunohistochemistry. Hepatocyte production of MIP-2 was significantly decreased in hepatocytes obtained from TLR2 or TLR4 knockout mice. MIP-2 production was found to be partially dependent on NF-kappaB because inhibition of NF-kappaB with Bay 11-7085 significantly decreased eHSP-72-induced MIP-2 production. Inhibitors of p38 mitogen-activated protein kinase or c-Jun NH(2)-terminal kinase had no effect on production of MIP-2 induced by eHSP-72. The data suggest that eHSP-72 binds to TLR2 and TLR4 on hepatocytes and signals through NF-kappaB to increase MIP-2 production. The fact that eHSP-72 did not increase TNF-alpha or IL-6 production may be indicative of a highly regulated signaling pathway downstream from TLR.

Distinct Contributions of CD4+ T Cell Subsets in Hepatic Ischemia/Reperfusion Injury

Helper T cells are known to mediate hepatic ischemia/reperfusion (I/R) injury. However, the precise mechanisms and subsets of CD4(+) T cells that contribute to this injury are still controversial. Therefore, we sought to determine the contributions of different CD4(+) T cell subsets during hepatic I/R injury. Wild-type, OT-II, or T cell receptor (TCR)-delta-deficient mice were subjected to 90 min of partial hepatic ischemia followed by 8 h of reperfusion. Additionally, wild-type mice were pretreated with anti-CD1d, -NK1.1, or -IL-2R-alpha antibodies before I/R injury. OT-II mice had diminished liver injury compared with wild-type mice, implicating that antigen-dependent activation of CD4(+) T cells through TCRs is involved in hepatic I/R injury. TCR-delta knockout mice had decreased hepatic neutrophil accumulation, suggesting that gammadelta T cells regulate neutrophil recruitment. We found that natural killer T (NKT) cells, but not NK cells, contribute to hepatic I/R injury via CD1d-dependent activation of their TCRs, as depletion of NKT cells by anti-CD1d antibody or depletion of both NKT cells and NK cells by anti-NK1.1 attenuated liver injury. Although regulatory T cells (Treg) are known to suppress T cell-dependent inflammation, depletion of Treg cells had little effect on hepatic I/R injury. The data suggest that antigen-dependent activation of CD4(+) T cells contributes to hepatic I/R injury. Among the subsets of CD4(+) T cells, it appears that gammadelta T cells contribute to neutrophil recruitment and that NKT cells directly injure the liver. In contrast, NK cells and Treg have little effects on hepatic I/R injury.

CXC chemokines play a critical role in liver injury, recovery, and regeneration.

BACKGROUND Hepatic ischemia/reperfusion (I/R) injury is a principal consideration of trauma, resectional liver surgery, and transplantation. Despite improvements in supportive care, hepatic I/R injury continues to negatively impact patient outcomes because of significant tissue damage and organ dysfunction. CXC chemokines have been implicated as key mediators in the deleterious inflammatory cascade after hepatic I/R and also as important, beneficial regulators of liver recovery and regeneration. As such, their potential to mediate both beneficial and detrimental effects on hepatocytes makes them a key target for therapy. Herein, we provide a review of the inflammatory mechanisms of hepatic I/R injury, with a focus on the divergent functions of CXC chemokines in this response compared with other liver insults, and offer an explanation of this apparent paradox. DATA SOURCES MEDLINE and PubMed. CONCLUSIONS CXC chemokines are key mediators of both the inflammatory response to hepatic I/R as well as the recovery from this injury. Their contrasting functions in the regeneration of liver mass after an ischemic insult indicates that therapeutic manipulation of these mediator pathways should differ depending on the surgical milieu.

Peroxisome proliferator‐activated receptor‐γ protects against hepatic ischemia/reperfusion injury in mice

The function of peroxisome proliferator‐activated receptor‐γ (PPARγ) in hepatic inflammation and injury is unclear. In this study, we sought to determine the role of PPARγ in hepatic ischemia/reperfusion injury in mice. Male mice were subjected to 90 minutes of partial hepatic ischemia followed by up to 8 hours of reperfusion. PPARγ was found to be constitutively activated in hepatocytes but not in nonparenchymal cells. Upon induction of ischemia, hepatic PPARγ activation rapidly decreased and remained suppressed throughout the 8‐hour reperfusion period. This reduced activation was not a result of decreased protein availability as hepatic nuclear PPARγ, retinoid X receptor‐α (RXRα), and PPARγ/RXRα heterodimer expression was maintained. Accompanying the decrease in PPARγ activation was a decrease in the expression of the natural ligand 15‐deoxy‐Delta12,14‐prostaglandin J2. This was associated with reduced interaction of PPARγ and the coactivator, p300. To determine whether PPARγ activation is hepatoprotective during hepatic ischemia/reperfusion injury, mice were treated with the PPARγ agonists, rosiglitazone and connecting peptide. These treatments increased PPARγ activation and reduced liver injury compared to untreated mice. Furthermore, PPARγ‐deficient mice had more liver injury after ischemia/reperfusion than their wild‐type counterparts. Conclusion: These data suggest that PPARγ is an important endogenous regulator of, and potential therapeutic target for, ischemic liver injury. (HEPATOLOGY 2007.)

Activation of Peroxisome Proliferator-Activated Receptor-γ During Hepatic Ischemia Is Age-Dependent

Hepatic ischemia/reperfusion injury is a complication of liver surgery, transplantation, and shock and is known to be age-dependent. Our laboratory has recently shown that peroxisome proliferator-activated receptor-gamma (PPARgamma) is down-regulated during hepatic ischemia and that this exacerbates injury. Here we examined whether activation of PPARgamma during ischemia was age-dependent. Male mice of different ages (young: 4-5 weeks; adult: 10-12 weeks; old: 10-12 months) were subjected to up to 90 min of hepatic ischemia. PPARgamma activation occurred throughout ischemia in young mice, whereas activation in adult and old mice was lost after 30 min. No significant differences were noted in PPARgamma ligand expression among the age groups. However, in young mice we observed a predominance of PPARgamma1 in the nucleus, whereas in old mice this isoform remained largely in the cytoplasm. Finally, the degree of PPARgamma activation was associated with autophagy in the liver, a mechanism of self-preservation. PPARgamma activation is prolonged in young mice as compared to older mice. This appears to be mediated by a selective retention of PPARgamma1 in the nucleus and is associated with increased autophagy. The data suggest that PPARgamma activation is an important component of the age-dependent response to hepatic ischemia/reperfusion injury.

Repeat pancreatectomy for pancreatic ductal cancer recurrence in the remnant pancreas after initial pancreatectomy: Is it worthwhile?

BACKGROUND The clinical implications of repeat completion pancreatectomy for recurrent pancreatic cancer in the remnant pancreas after initial pancreatectomy have not been clarified. We retrospectively analyzed our patients and evaluated the clinical implications of repeat pancreatectomy for isolated local recurrence in the remnant pancreas after initial resection for pancreatic cancer. METHODS One-hundred seventy patients who had recurrence of pancreatic cancer out of 326 patients who had initially undergone resection for pancreatic cancer were included in this study. Sixty-seven of 170 recurrent patients were diagnosed as having isolated local recurrence of pancreatic cancer. Eleven of these 67 patients with isolated local recurrence only in the remnant pancreas underwent repeat pancreatectomy. Characteristics and operative outcomes for these 11 patients with repeat pancreatectomy were analyzed and evaluated in comparison with other recurrent patients. RESULTS Among 170 patients with recurrence after initial resection for pancreatic cancer, the median survival time was 78.2 and 20.3 months after initial resection, in the repeat pancreatectomy group and the unresectable group, respectively (P < .001), and the 2- and 5-year survival probability rates after initial resection were 91%, and 82% vs 42%, and 13%, respectively. Among 67 patients with isolated local recurrence, the median survival time after repeat resection or diagnosis of recurrence was 25.0 and 9.3 months, and the 2- and 5-year survival probability rates after repeat resection or diagnosis of recurrence were and 61% and 46% vs 19% and 6.2% in the repeat pancreatectomy group and the unresectable group, respectively (P < .01). There was no difference in survivals between the unresectable isolated local recurrence group and the unresectable nonlocal recurrence group. CONCLUSION Repeat pancreatectomy might bring about beneficial effects on prognosis in selected patients with isolated local recurrence in the remnant pancreas after initial pancreatectomy for pancreatic cancer without increased operative morbidity or mortality.