John Blanchard

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.

Interleukin-37 reduces liver inflammatory injury via effects on hepatocytes and non-parenchymal cells.

Background and Aim:  The purpose of the present study was to determine the effects of interleukin‐37 (IL‐37) on liver cells and on liver inflammation induced by hepatic ischemia/reperfusion (I/R).

Interleukin-33 is hepatoprotective during liver ischemia/reperfusion in mice.

Interleukin (IL)‐33 is a recently identified member of the IL‐1 family that binds to the receptor, ST2L. In the current study, we sought to determine whether IL‐33 is an important regulator in the hepatic response to ischemia/reperfusion (I/R). Male C57BL/6 mice were subjected to 90 minutes of partial hepatic ischemia, followed by up to 8 hours of reperfusion. Some mice received recombinant IL‐33 (IL‐33) intraperitoneally (IP) before surgery or anti‐ST2 antibody IP at the time of reperfusion. Primary hepatocytes and Kupffer cells were isolated and treated with IL‐33 to assess the effects of IL‐33 on inflammatory cytokine production. Primary hepatocytes were treated with IL‐33 to assess the effects of IL‐33 on mediators of cell survival in hepatocytes. IL‐33 protein expression increased within 4 hours after reperfusion and remained elevated for up to 8 hours. ST2L protein expression was detected in healthy liver and was up‐regulated within 1 hour and peaked at 4 hours after I/R. ST2L was primarily expressed by hepatocytes, with little to no expression by Kupffer cells. IL‐33 significantly reduced hepatocellular injury and liver neutrophil accumulation at 1 and 8 hours after reperfusion. In addition, IL‐33 treatment increased liver activation of nuclear factor kappa light‐chain enhancer of activated B cells (NF‐κB), p38 mitogen‐activated protein kinase (MAPK), cyclin D1, and B‐cell lymphoma 2 (Bcl‐2), but reduced serum levels of CXC chemokines. In vitro experiments demonstrated that IL‐33 significantly reduced hepatocyte cell death as a result of increased NF‐κB activation and Bcl‐2 expression in hepatocytes. Conclusion: The data suggest that IL‐33 is an important endogenous regulator of hepatic I/R injury. It appears that IL‐33 has direct protective effects on hepatocytes, associated with the activation of NF‐κB, p38 MAPK, cyclin D1, and Bcl‐2 that limits liver injury and reduces the stimulus for inflammation. (HEPATOLOGY 2012)

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.

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.

CXC chemokine receptor‐1 is expressed by hepatocytes and regulates liver recovery after hepatic ischemia/reperfusion injury

CXC chemokines mediate hepatic inflammation and injury following ischemia/reperfusion (I/R). More recently, signaling through CXC chemokine receptor‐2 (CXCR2) was shown to delay liver recovery and repair after I/R injury. The chemokine receptor CXCR1 shares ligands with CXCR2, yet nothing is known about its potential role in liver pathology. In the present study, we examined the role of CXCR1 in the injury and recovery responses to I/R using a murine model. CXCR1 expression was undetectable in livers of sham‐operated mice. However, after ischemia CXCR1 expression increased 24 hours after reperfusion and was maximal after 96 hours of reperfusion. CXCR1 expression was localized largely to hepatocytes. In order to assess the function of CXCR1, CXCR2−/− mice were treated with the CXCR1/CXCR2 antagonist, repertaxin. Prophylactic treatment with repertaxin had no effect on acute inflammation or liver injury. However, when repertaxin was administered 24 hours postreperfusion there was a significant increase in hepatocellular injury and a delay in recovery compared to control‐treated mice. CXCR1−/− mice also demonstrated delayed recovery and regeneration after I/R when compared to wild‐type mice. In vitro, hepatocytes from CXCR2−/− mice that were stimulated to express CXCR1 showed increased proliferation in response to ligand. Hepatocyte proliferation was decreased in CXCR1−/− mice in vivo. Conclusion: This is the first report to show that CXCR1 expression is induced in hepatocytes after injury. Furthermore, the data suggest that CXCR1 has divergent effects from CXCR2 and appears to facilitate repair and regenerative responses after I/R injury. (HEPATOLOGY 2011)

The peptidyl-prolyl isomerase, Pin1, facilitates NF-κB binding in hepatocytes and protects against hepatic ischemia/reperfusion injury

BACKGROUND/AIMS Our previous work suggested an important role for the peptidyl-prolyl isomerase, Pin1, in hepatic NF-kappaB activation and liver injury during ischemia/reperfusion (I/R). In this study, we sought to determine the function of Pin1 in the injury response to hepatic I/R. METHODS Wild-type and Pin1(-/-) mice were subjected to partial hepatic I/R. In addition, hepatocytes and Kupffer cells were isolated from these mice. RESULTS Pin1(-/-) mice had reduced hepatic NF-kappaB activation and more liver injury after I/R than wild-type mice. The increased injury was not a result of enhanced inflammation as Pin1(-/-) mice had the same level of proinflammatory cytokine production and less neutrophil accumulation in the liver. The reduced NF-kappaB activation was not a result of a defect in nuclear translocation of NF-kappaB. In fact, hepatic nuclear p65 protein expression was higher in Pin1(-/-) mice than wild-type mice. This suggests that Pin1 is important for NF-kappaB-DNA binding. This effect was specific to hepatocytes as isolated Kupffer cells from wild-type and Pin1(-/-) mice were identical in their activation of NF-kappaB and production of cytokines after stimulation. In contrast, hepatocytes stimulated with TNFalpha had greatly reduced NF-kappaB activation, reduced production of the CXC chemokine, MIP-2, and increased cell death. CONCLUSIONS These data suggest that Pin1 is a critical regulator of NF-kappaB activation in hepatocytes and its role in these cells appears to confer direct protective effects.

Receptor activator of nuclear factor‐κB ligand (RANKL) protects against hepatic ischemia/reperfusion injury in mice

The transcription factor nuclear factor kappaB (NF‐κB) plays diverse roles in the acute injury response to hepatic ischemia/reperfusion (I/R). Activation of NF‐κB in Kupffer cells promotes inflammation through cytokine expression, whereas activation in hepatocytes may be cell protective. The interaction of receptor activator of NF‐κB (RANK) and its ligand (RANKL) promotes NF‐κB activation; however, this ligand‐receptor system has not been studied in acute liver injury. In the current study, we sought to determine if RANK and RANKL were important in the hepatic response to I/R. Mice were subjected to partial hepatic ischemia followed by reperfusion. In some experiments, mice received recombinant RANKL or neutralizing antibodies to RANKL 1 hour prior to surgery or at reperfusion to assess the role of RANK/RANKL signaling during I/R injury. RANK was constitutively expressed in the liver and was not altered by I/R. RANK was strongly expressed in hepatocytes and very weakly expressed in Kupffer cells. Serum RANKL concentrations increased after I/R and peaked 4 hours after reperfusion. Serum levels of osteoprotegerin (OPG), a decoy receptor for RANKL, steadily increased over the 8‐hour period of reperfusion. Treatment with RANKL, before ischemia or at reperfusion, increased hepatocyte NF‐κB activation and significantly reduced liver injury. These beneficial effects occurred without any effect on cytokine expression or liver inflammation. Treatment with anti‐RANKL antibodies had no effect on liver I/R injury. Conclusion: During the course of injury, endogenous OPG appears to suppress the effects of RANKL. However, exogenous administration of RANKL, given either prophylactically or postinjury, reduces liver injury in a manner associated with increased hepatocyte NF‐κB activation. The data suggest that RANK/RANKL may be a viable therapeutic target in acute liver injury. (Hepatology 2012)