PACAP neuropeptide promotes Hepatocellular Protection via CREB-KLF4 dependent autophagy in mouse liver Ischemia Reperfusion Injury.

Organ ischemia reperfusion injury (IRI), associated with acute hepatocyte death, remains an unresolved problem in clinical orthotopic liver transplantation (OLT). Autophagy, an intracellular self-digesting progress, is responsible for cell reprograming required to regain post-stress homeostasis. Methods: Here, we analyzed the cytoprotective mechanism of pituitary adenylate cyclase-activating polypeptide (PACAP)-promoted hepatocellular autophagy in a clinically relevant mouse model of extended hepatic cold storage (4 °C UW solution for 20 h) followed by syngeneic OLT. Results: In contrast to 41.7% of liver graft failure by day 7 post-transplant in control group, PACAP treatment significantly improved graft survival (91.7% by day 14), and promoted autophagy-associated regeneration programs in OLT. In parallel in vitro studies, PACAP-enhanced autophagy ameliorated cellular damage (LDH/ALT levels), and diminished necrosis in H2O2-stressed primary hepatocytes. Interestingly, PACAP not only induced nuclear cAMP response element-binding protein (CREB), but also triggered reprogramming factor Kruppel-like factor 4 (KLF4) expression in IR-stressed OLT. Indeed, CREB inhibition attenuated hepatic autophagy and recreated hepatocellular injury in otherwise PACAP-protected livers. Furthermore, CREB inhibition suppressed PACAP-induced KLF4 expression, whereas KLF4 blockade abolished PACAP-promoted autophagy and neutralized PACAP-mediated hepatoprotection both in vivo and in vitro. Conclusion: Current study documents the essential neural regulation of PACAP-promoted autophagy in hepatocellular homeostasis in OLT, which provides the emerging therapeutic principle to combat hepatic IRI in OLT.

Pituitary Adenylate Cyclase-activating Polypeptides Prevent Hepatocyte Damage by Promoting Yes-associated Protein in Liver Ischemia-Reperfusion Injury.

BACKGROUND: Hepatic ischemia-reperfusion injury (IRI) is a severe complication in liver transplantation, hepatectomy, and hemorrhagic shock. As neuropeptides transmit the regulatory signal between nervous and immune systems communication, our previous study documented that pituitary adenylate cyclase-activating polypeptides (PACAP) depressed hepatic Toll-like receptor 4 immune response in liver IRI. METHODS: Here, we focused on how PACAP suppressed hepatocellular damage and enhanced hepatocyte regeneration in a murine model of partial liver warm IRI. RESULTS: Yes-associated protein (YAP), a cellular modulator of tissue regeneration, was readily induced in wild type (WT) mouse IR-livers. As its induction was failed in PACAP-deficient livers, PACAP supplement enhanced YAP expression in WT mouse and promoted its nuclear translocation and downstream antioxidative/regenerative genes expression both in vivo and in vitro. Further, verteporfin, a YAP transcriptional inhibitor, abolished PACAP-mediated hepatoprotection significantly. Meanwhile, blockade of protein kinase A (PKA)-CRE-binding protein (CREB) signaling recreated liver damage in PACAP-protected liver as well as impeded stimulation on YAP and its downstream gene expressions. Consistently, inhibition of PKA-CREB decreased PACAP-promoted YAP expression in primary hepatocytes culture, and made them vulnerable to H2O2 stress in vitro. In addition, lysophosphatidic acid, another Hippo pathway inhibitor, failed to affect PACAP-mediated hepatoprotection or hepatocellular YAP induction. This implies that PACAP regulated YAP through PKA-CREB pathway at the transcriptional level rather than canonical hippo pathway. CONCLUSIONS: Our study discovered the neural modulation of PACAP-YAP axis in hepatic cytoprotection and homeostasis in liver IRI. These reveal a novel insight of neuropeptide PACAP in combating liver IRI in clinical patients.

Activation of YAP attenuates hepatic damage and fibrosis in liver ischemia-reperfusion injury.

BACKGROUND & AIMS: Hepatic ischemia-reperfusion injury (IRI) is a major complication of hemorrhagic shock, liver resection and transplantation. YAP, a key downstream effector of the Hippo pathway, is essential for determining cell fate and maintaining homeostasis in the liver. We aimed to elucidate its role in IRI. METHODS: The role of YAP/Hippo signaling was systematically studied in biopsy specimens from 60 patients after orthotopic liver transplantation (OLT), and in a mouse model of liver warm IRI. Human biopsy specimens were collected after 2-10 h of cold storage and 3 h post-reperfusion, before being screened by western blot. In the mouse model, the role of YAP was probed by activating or inhibiting YAP prior to ischemia-reperfusion. RESULTS: In human biopsies, high post-OLT YAP expression was correlated with well-preserved histology and improved hepatocellular function at postoperative day 1-7. In mice, the ischemia insult (90 min) triggered intrinsic hepatic YAP expression, which peaked at 1-6 h of reperfusion. Activation of YAP protected the liver against IR-stress, by promoting regenerative and anti-oxidative gene induction, while diminishing oxidative stress, necrosis/apoptosis and the innate inflammatory response. Inhibition of YAP aggravated hepatic IRI and suppressed repair/anti-oxidative genes. In mouse hepatocyte cultures, activating YAP prevented hypoxia-reoxygenation induced stress. Interestingly, YAP activation suppressed extracellular matrix synthesis and diminished hepatic stellate cell (HSC) activation, whereas YAP inhibition significantly delayed hepatic repair, potentiated HSC activation, and enhanced liver fibrosis at 7 days post-IRI. Notably, YAP activation failed to protect Nrf2-deficient livers against IR-mediated damage, leading to extensive fibrosis. CONCLUSION: Our novel findings document the crucial role of YAP in IR-mediated hepatocellular damage and liver fibrogenesis, providing evidence of a potential therapeutic target for the management of sterile liver inflammation in transplant recipients. LAY SUMMARY: In the clinical arm, graft YAP expression negatively correlated with liver function and tissue damage after human liver transplantation. YAP activation attenuated hepatocellular oxidative stress and diminished the innate immune response in mouse livers following ischemia-reperfusion injury. In the mouse model, YAP inhibited hepatic stellate cell activation, and abolished injury-mediated fibrogenesis up to 7 days after the ischemic insult.

Vertical Sleeve Gastrectomy Attenuates the Progression of Non-Alcoholic Steatohepatitis in Mice on a High-Fat High-Cholesterol Diet.

OBJECTIVE: To determine whether vertical sleeve gastrectomy (VSG) attenuates fibrosis in mice on a high-fat high-cholesterol (HFHC) diet. BACKGROUND: Bariatric surgery mitigates non-alcoholic steatohepatitis in 85-90% of obese patients. While animal models demonstrate similar results on a high-fat diet, none have observed the effects of bariatric surgery on a combined HFHC diet. METHODS: Mice on a HFHC diet were used to confirm the development of hepatic fibrosis at 8 (n = 15) and 24 (n = 15) weeks. A separate cohort of mice on a HFHC diet for 12 weeks was subjected to either VSG (n = 18) or sham (n = 12) operations and remained on a HFHC diet for an additional 20 weeks. Changes in weight, dyslipidemia, and the development of steatosis and fibrosis were documented. Serum was obtained for bile acid analysis by liquid chromatography and mass spectrometry, while hepatic gene expression by RT-PCR was performed to evaluate intrahepatic lipid metabolism. RESULTS: Hepatic steatosis and fibrosis developed after 8 weeks on the HFHC diet. After VSG, mice demonstrated a sustained decrease in weight with a significant decrease in fibrosis compared to sham mice. Serum total cholesterol, HDL, and LDL were significantly reduced following surgery, while serum bile acids were significantly elevated. Intra-hepatic cholesterol excretion was not upregulated based on hepatic gene expression of CYP7A1 and ABCG5/8. CONCLUSIONS: VSG attenuates the development of hepatic fibrosis in diet-induced obese mice, presumably through enhancement of cholesterol elimination at the intestinal level.

Inhibition of Cyclin-dependent Kinase 2 Signaling Prevents Liver Ischemia and Reperfusion Injury.

BACKGROUND: Liver ischemia and reperfusion injury (IRI) is a major complication of liver transplant, hepatectomy, and hemorrhagic shock. The cyclin-dependent kinase 2 (CDK2) acts as a pivotal regulator of cell cycle and proliferation. METHODS: This study evaluated the modulation and therapeutic potential of CDK2 inhibition in a mouse model of partial liver warm IRI. RESULTS: Liver IR-triggered intrinsic CDK2 expression, peaking by 0.5 hour of reperfusion and maintaining a high-level throughout 1 to 24 hours. Roscovitine, a specific CDK2 inhibitor, prevented liver IR-mediated damage with abolished serum alanine aminotransferase levels and reserved liver pathology. CDK2 inhibition-mediated liver protection was accompanied by decreased macrophage/neutrophil infiltration, diminished hepatocyte apoptosis, abolished toll like receptor 4 signaling and downstream gene inductions (C-X-C motif ligand-10, Tumor necrosis factor-α, interleukin-1ß, and interleukin-6), yet augmented interleukin-10 expression. In vitro, CDK2 inhibition by Roscovitine suppressed macrophage TLR4 activation and further depressed downstream inflammatory signaling (myeloid differentiation factor 88, interferon regulatory transcription factor 3, p38, c-Jun N-terminal kinase, and extracellular-regulated kinase). CONCLUSIONS: Our novel findings revealed the critical role of CDK2 in hepatic cytoprotection and homeostasis against liver IRI. As CDK2 inhibition regulated local immune response and prevented hepatocyte death, this study provided the evidence for new treatment approaches to combat IRI in liver transplant.

Hepatic Ischemic Preconditioning Alleviates Ischemia-Reperfusion Injury by Decreasing TIM4 Expression.

Ischemia-reperfusion injury (IRI) of the liver is a primary cause of post-liver-surgery complications and ischemic preconditioning (IPC) has been verified to protect against ischemia-reperfusion injury. TIM-4 activation plays an important role in macrophage mediated hepatic IRI. This study aimed to determine whether IPC protects against hepatic IRI through inhibiting TIM-4 activation. In this study, a model of warm liver ischemia (90 min) and reperfusion for 6 h was used. Mice were subjected to ischemia-reperfusion injury with or without ischemic preconditioning and TIM4 blocking antibody. Western blot was determined to detect the expression of TIM4 protein and mitochondrial apoptosis-related protein expression. Liver function was evaluated using the level of alanine transaminase (ALT) and aspartate transaminase (AST), cell apoptosis and pathological examination. We found that compared with the control group, ischemic preconditioning reduced IRI by decreasing hepatocyte apoptosis, ALT, AST, CD68 and CD3 positive cells, tissue myeloperoxidase activity(MPO), and downregulating TIM-4 expression. TIM4 blocking could reduce CD68 and CD3 positive cells in liver. Furthermore, activated monocytes transfusion significantly abolished the protect effect of IPC with increased hepatocyte apoptosis, ALT, AST, CD68 and CD3 positive cells while TIM-4 knockdown monocytes lost this effect. These results suggested that IPC protects against hepatic IRI by downregulating TIM-4 and indicated TIM-4 would be a novel therapeutic target to minimize IRI.

TIM-1 attenuates the protection of ischemic preconditioning for ischemia reperfusion injury in liver transplantation.

Ischemic preconditioning (IPC) has been introduced to protect grafts against ischemic reperfusion injury (IRI) during liver transplantation (LT) in recent years. However, the underlying molecular mechanisms of IPC are not fully understood. We aimed to confirm whether the efficacy of IPC is dependent on T cell Immunoglobulin and Mucin domain-containing molecules-1 (TIM-1). Quantitative real-time reverse transcription PCR and western blotting were used to detect the expression of genes of interest. Graft function was assessed using the levels of alanine transaminase (ALT) and aspartate transaminase (AST), percentage of apoptosis cells and pathological examination. IPC treatment alleviated graft function after ischemic reperfusion. AST, ALT, CD68, CD3 positive cells and tissue myeloperoxidase activity were decreased significantly by IPC. IPC decreased the expressions of the cytokines and chemokines. Compared with the IRI group, TIM-1 expression and TIM-1 positive cells were inhibited significantly in the IPC group. TIM-1 blockage abolished the protective effect of IPC on IRI damage. IPC could not further improve graft function and decrease the sequestration of immune cells after blocking TIM-1 signaling. IPC is a convenient therapeutic strategy against IRI during LT. The benefit of IPC depends on TIM-1 signaling.

Negative CD4 + TIM-3 signaling confers resistance against cold preservation damage in mouse liver transplantation.

Ischemia-reperfusion injury (IRI), an innate immunity-driven local inflammation, remains the major problem in clinical organ transplantation. T cell immunoglobulin and mucin domain (TIM-3)-Galectin-9 (Gal-9) signaling regulates CD4+ Th1 immune responses. Here, we explored TIM-3-Gal-9 function in a clinically relevant murine model of hepatic cold storage and orthotopic liver transplantation (OLT). C57BL/6 livers, preserved for 20 h at 4°C in UW solution, were transplanted to syngeneic mouse recipients. Up-regulation of TIM-3 on OLT-infiltrating activated CD4+ T cells was observed in the early IRI phase (1 h). By 6 h of reperfusion, OLTs in recipients treated with a blocking anti-TIM-3 Ab were characterized by: (1) enhanced hepatocellular damage (sALT levels, liver Suzuki's histological score); (2) polarized cell infiltrate towards Th1/Th17-type phenotype; (3) depressed T cell exhaustion markers (PD-1, LAG3); and (4) elevated neutrophil and macrophage infiltration/activation. In parallel studies, adoptive transfer of CD4+ T cells from naïve WT, but not from TIM-3 Tg donors, readily recreated OLT damage in otherwise IR-resistant RAG(-/-) test recipients. Furthermore, pre-treatment of mice with rGal-9 promoted hepatoprotection against preservation-association liver damage, accompanied by enhanced TIM-3 expression in OLTs. Thus, CD4+ T cell-dependent "negative" TIM-3 costimulation is essential for hepatic homeostasis and resistance against IR stress in OLTs.

Recipient T cell TIM-3 and hepatocyte galectin-9 signalling protects mouse liver transplants against ischemia-reperfusion injury.

BACKGROUND & AIMS: By binding to T cell immunoglobulin mucin-3 (TIM-3) on activated Th1 cells, galectin-9 (Gal-9) negatively regulates Th1-type alloimmunity. Although T cells contribute to hepatic ischemia-reperfusion injury (IRI), it is unknown whether negative T cell-dependent TIM-3 co-stimulation may rescue IR-stressed orthotopic liver transplants from innate immunity-driven inflammation. METHODS: We used wild type (WT) and TIM-3 transgenic (Tg) mice (C57BL/6) as liver donors and recipients in a clinically-relevant model of hepatic cold storage (20 h at 4°C in UW solution) and syngeneic orthotopic liver transplantation (OLT). RESULTS: Orthotopic liver transplants in WT or TIM-3Tg→TIM-3Tg groups were resistant against IR-stress, evidenced by preserved hepatocellular function (serum ALT levels) and liver architecture (Suzuki's score). In contrast, orthotopic liver transplants in WT or TIM-3Tg→WT groups were susceptible to IRI. TIM-3 induction in circulating CD4+ T cells of the recipient: (1) depressed T-bet/IFN-γ, while amplifying GATA3 and IL-4/IL-10 expression in orthotopic liver transplants; (2) promoted T cell exhaustion (PD-1, LAG-3) phenotype; and (3) depressed neutrophil and macrophage infiltration/function in orthotopic liver transplants. In parallel studies, we documented for the first time that Gal-9, a natural TIM-3 ligand, was produced primarily by and released from IR-stressed hepatocytes, both in vivo and in vitro. Moreover, exogenous recombinant Gal-9 (rGal-9) potentiated liver resistance against IRI by depressing T cell activation and promoting apoptosis of CD4+ T cells. CONCLUSIONS: Harnessing TIM-3/Gal-9 signalling at the T cell-hepatocyte interface facilitates homeostasis in IR-stressed orthotopic liver transplants. Enhancing anti-oxidant hepatocyte Gal-9 potentiates liver IR-resistance. Negative regulation by recipient TIM-3+CD4+ cells provides evidence for cytoprotective functions of a discrete T cell subset, which should be spared when applying T cell-targeted immunosuppression in transplant recipients.

T-cell immunoglobulin and mucin domain 4 (TIM-4) signaling in innate immune-mediated liver ischemia-reperfusion injury.

UNLABELLED: Hepatic ischemia-reperfusion injury (IRI), an innate immunity-driven inflammation response, occurs in multiple clinical settings including liver resection, transplantation, trauma, and shock. T-cell immunoglobulin and mucin (TIM)-4, the only TIM protein not expressed on T cells, is found on macrophages and dendritic cells. The regulatory function of macrophage TIM-4 in the engulfment of apoptotic/necrotic bodies in innate immunity-mediated disease states remains unknown. This study focuses on the putative role of TIM-4 signaling in a model of liver warm ischemia (90 minutes) and reperfusion. The ischemia insult triggered TIM-4 expression by stressed hepatocellular phosphatidylserine (PS) presentation, peaking at 6 hours of reperfusion, and coinciding with the maximal hepatocellular damage. TIM-4-deficient or wild-type WT mice treated with antagonistic TIM-4 monoclonal antibody (mAb) were resistant against liver IRI, evidenced by diminished serum alanine aminotransferase (sALT) levels and well-preserved hepatic architecture. Liver hepatoprotection rendered by TIM-4 deficiency was accompanied by diminished macrophage infiltration/chemoattraction, phagocytosis, and activation of Toll-like receptor (TLR)2/4/9-dependent signaling. Correlating with in vivo kinetics, the peak of TIM-4 induction in lipopolysaccharide (LPS)-activated bone marrow derived-macrophages (BMM) was detected in 6-hour cultures. To mimic liver IRI, we employed hydrogen peroxide-necrotic hepatocytes, which readily present PS. Indeed, necrotic hepatocytes were efficiently captured/engulfed by WT (TIM-4+) but not by TIM-4-deficient BMM. Finally, in a newly established model of liver IRI, adoptive transfer of WT but not TIM-4-deficient BMM readily recreated local inflammation response/hepatocellular damage in the CD11b-DTR mouse system. CONCLUSION: These findings document the importance of macrophage-specific TIM-4 activation in the mechanism of hepatic IRI. Macrophage TIM-4 may represent a therapeutic target to minimize innate inflammatory responses in IR-stressed organs.

KEAP1-NRF2 complex in ischemia-induced hepatocellular damage of mouse liver transplants.

BACKGROUND & AIMS: The Keap1-Nrf2 signaling pathway regulates host cell defense responses against oxidative stress and maintains the cellular redox balance. METHODS: We investigated the function/molecular mechanisms by which Keap1-Nrf2 complex may influence liver ischemia/reperfusion injury (IRI) in a mouse model of hepatic cold storage (20h at 4°C) followed by orthotopic liver transplantation (OLT). RESULTS: The Keap1 hepatocyte-specific knockout (HKO) in the donor liver ameliorated post-transplant IRI, evidenced by improved hepatocellular function and OLT outcomes (Keap1 HKO→Keap1 HKO; 100% survival), as compared with controls (WT→WT; 50% survival; p<0.01). By contrast, donor liver Nrf2 deficiency exacerbated IRI in transplant recipients (Nrf2 KO→Nrf2 KO; 40% survival). Ablation of Keap1 signaling reduced macrophage/neutrophil trafficking, pro-inflammatory cytokine programs, and hepatocellular necrosis/apoptosis, while simultaneously promoting anti-apoptotic functions in OLTs. At the molecular level, Keap1 HKO increased Nrf2 levels, stimulated Akt phosphorylation, and enhanced expression of anti-oxidant Trx1, HIF-1α, and HO-1. Pretreatment of liver donors with PI3K inhibitor (LY294002) disrupted Akt/HIF-1A signaling and recreated hepatocellular damage in otherwise IR-resistant Keap1 HKO transplants. In parallel in vitro studies, hydrogen peroxide-stressed Keap1-deficient hepatocytes were characterized by enhanced expression of Nrf2, Trx1, and Akt phosphorylation, in association with decreased release of lactate dehydrogenase (LDH) in cell culture supernatants. CONCLUSIONS: Keap1-Nrf2 complex prevents oxidative injury in IR-stressed OLTs through Keap1 signaling, which negatively regulates Nrf2 pathway. Activation of Nrf2 induces Trx1 and promotes PI3K/Akt, crucial for HIF-1α activity. HIF-1α-mediated overexpression of HO-1/Cyclin D1 facilitates cytoprotection by limiting hepatic inflammatory responses, and hepatocellular necrosis/apoptosis in a PI3K-dependent manner.

Vasoactive intestinal peptide attenuates liver ischemia/reperfusion injury in mice via the cyclic adenosine monophosphate-protein kinase a pathway.

Hepatic ischemia/reperfusion injury (IRI), an exogenous, antigen-independent, local inflammation response, occurs in multiple clinical settings, including liver transplantation, hepatic resection, trauma, and shock. The nervous system maintains extensive crosstalk with the immune system through neuropeptide and peptide hormone networks. This study examined the function and therapeutic potential of the vasoactive intestinal peptide (VIP) neuropeptide in a murine model of liver warm ischemia (90 minutes) followed by reperfusion. Liver ischemia/reperfusion (IR) triggered an induction of gene expression of intrinsic VIP; this peaked at 24 hours of reperfusion and coincided with a hepatic self-healing phase. Treatment with the VIP neuropeptide protected livers from IRI; this was evidenced by diminished serum alanine aminotransferase levels and well-preserved tissue architecture and was associated with elevated intracellular cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling. The hepatocellular protection rendered by VIP was accompanied by diminished neutrophil/macrophage infiltration and activation, reduced hepatocyte necrosis/apoptosis, and increased hepatic interleukin-10 (IL-10) expression. Strikingly, PKA inhibition restored liver damage in otherwise IR-resistant VIP-treated mice. In vitro, VIP not only diminished macrophage tumor necrosis factor α/IL-6/IL-12 expression in a PKA-dependent manner but also prevented necrosis/apoptosis in primary mouse hepatocyte cultures. In conclusion, our findings document the importance of VIP neuropeptide-mediated cAMP-PKA signaling in hepatic homeostasis and cytoprotection in vivo. Because the enhancement of neural modulation differentially regulates local inflammation and prevents hepatocyte death, these results provide the rationale for novel approaches to managing liver IRI in transplant patients.

Neuropeptide PACAP in mouse liver ischemia and reperfusion injury: immunomodulation by the cAMP-PKA pathway.

UNLABELLED: Hepatic ischemia and reperfusion injury (IRI), an exogenous antigen-independent local inflammation response, occurs in multiple clinical settings, including liver transplantation, hepatic resection, trauma, and shock. The immune system and the nervous system maintain extensive communication and mount a variety of integrated responses to danger signals through intricate chemical messengers. This study examined the function and potential therapeutic potential of neuropeptide pituitary adenylate cyclase-activating polypeptides (PACAP) in a murine model of partial liver "warm" ischemia (90 minutes) followed by reperfusion. Liver IRI readily triggered the expression of intrinsic PACAP and its receptors, whereas the hepatocellular damage was exacerbated in PACAP-deficient mice. Conversely, PACAP27, or PACAP38 peptide monotherapy, which elevates intracellular cyclic adenosine monophosphate/protein kinase A (cAMP-PKA) signaling, protected livers from IRI, as evidenced by diminished serum alanine aminotransferase levels and well-preserved tissue architecture. The liver protection rendered by PACAP peptides was accompanied by diminished neutrophil/macrophage infiltration and activation, reduced hepatocyte necrosis/apoptosis, and selectively augmented hepatic interleukin (IL)-10 expression. Strikingly, PKA inhibition readily restored liver damage in otherwise IR-resistant, PACAP-conditioned mice. In vitro, PACAP treatment not only diminished macrophage tumor necrosis factor alpha/IL-6/IL-12 levels in a PKA-dependent manner, but also prevented necrosis and apoptosis in primary mouse hepatocyte cultures. CONCLUSION: Our novel findings document the importance of PACAP-mediated cAMP-PKA signaling in hepatic homeostasis and cytoprotection in vivo. Because the enhancement of neural modulation differentially regulates local inflammation and prevents hepatocyte death, these results provide the rationale for novel approaches to manage liver inflammation and IRI in transplant patients.

ß-catenin regulates innate and adaptive immunity in mouse liver ischemia-reperfusion injury.

UNLABELLED: Dendritic cells (DCs) are critical mediators of immune responses that integrate signals from the innate immune system to orchestrate adaptive host immunity. This study was designed to investigate the role and molecular mechanisms of STAT3-induced ß-catenin in the regulation of DC function and inflammatory responses in vitro and in vivo. STAT3 induction in lipopolysaccharide (LPS)-stimulated mouse bone marrow-derived DCs (BMDCs) triggered ß-catenin activation by way of GSK-3ß phosphorylation. The activation of ß-catenin inhibited phosphatase and tensin homolog delete on chromosome 10 (PTEN) and promoted the phosphoinositide 3-kinase (PI3K)/Akt pathway, which in turn down-regulated DC maturation and function. In contrast, knockdown of ß-catenin increased PTEN/TLR4 (Toll-like receptor 4), interferon regulatory factor-3 (IRF3), nuclear factor kappa B (NF-κB) activity, and proinflammatory cytokine programs in response to LPS stimulation. In a mouse model of warm liver ischemia and reperfusion injury (IRI), disruption of ß-catenin signaling increased the hepatocellular damage, enhanced hepatic DC maturation/function, and PTEN/TLR4 local inflammation in vivo. CONCLUSION: These findings underscore the role of ß-catenin to modulate DC maturation and function at the innate-adaptive interface. Activation of ß-catenin triggered PI3K/Akt, which in turn inhibited TLR4-driven inflammatory response in a negative feedback regulatory mechanism. By identifying the molecular pathways by which ß-catenin regulates DC function, our findings provide the rationale for novel therapeutic approaches to manage local inflammation and injury in IR-stressed liver.

Activation of cyclic adenosine monophosphate-dependent protein kinase a signaling prevents liver ischemia/reperfusion injury in mice.

Hepatic ischemia/reperfusion injury (IRI) occurs in multiple clinical settings, including liver transplantation. The cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) pathway inhibits hepatocellular apoptosis and regulates toll-like receptor 4-triggered inflammation responses in vitro. Here we examined the function and therapeutic potential of cAMP-PKA activation in a murine (C57/BL6) model of liver warm ischemia (90 minutes) followed by reperfusion. Liver IRI triggered cAMP-PKA activation, whereas the administration of its specific inhibitor, H89, exacerbated hepatocellular damage. Conversely, forskolin therapy, which activates PKA by elevating cAMP levels, protected livers from IRI; this was evidenced by diminished serum alanine aminotransferase levels and well-preserved tissue architecture. Liver protection due to cAMP-PKA stimulation was accompanied by diminished neutrophil and macrophage infiltration/activation, reduced hepatocyte necrosis/apoptosis, and increased cAMP response element-binding protein (CREB) expression and augmented interleukin-10 (IL-10) expression. The neutralization of IL-10 restored liver damage in otherwise ischemia/reperfusion-resistant, forskolin-treated mice. In vitro, cAMP-PKA activation diminished macrophage tumor necrosis factor α, IL-6, and IL-12 in an IL-10-dependent manner and prevented necrosis/apoptosis in primary mouse hepatocyte cultures. Our novel findings in a mouse model of liver IRI document the importance of cAMP-PKA signaling in hepatic homeostasis and cytoprotection in vivo. The activation of cAMP-PKA signaling differentially regulates local inflammation and prevents hepatocyte death, and this provides a rationale for novel therapeutic approaches to combating liver IRI in transplant recipients.

HO-1-STAT3 axis in mouse liver ischemia/reperfusion injury: regulation of TLR4 innate responses through PI3K/PTEN signaling.

BACKGROUND & AIMS: Signal transducer and activator of transcription 3 (STAT3), a key mediator of anti-inflammatory cytokine signaling, is essential for heme oxygenase-1 (HO-1)-induced cytoprotection. The phosphoinositide 3-kinase (PI3K)/phosphatase and tensin homolog delete on chromosome 10 (PTEN) pathways regulate diverse innate immune responses. This study was designed to investigate the role of STAT3 in the regulation of PI3K/PTEN cascade after HO-1 induction in a mouse model of innate immune-dominated liver ischemia/reperfusion injury (IRI). METHODS: Partial warm ischemia was produced in the left and middle hepatic lobes of C57BL/6 mice for 90 min, followed by 6h of reperfusion. RESULTS: Mice subjected to Ad-HO-1 transfer were resistant to liver IRI, and this cytoprotective effect correlated with increased intrahepatic PI3K/Akt and diminished PTEN expression. In contrast, mice undergoing adjunctive Ad-HO-1 treatment and STAT3 knockdown (siRNA) remained susceptible to IR-mediated local inflammatory response and hepatocellular damage. Consistent with decreased cell apoptosis and inhibited TLR4 expression after PI3K/Akt activation, treatment with specific PI3k inhibitor increased local inflammation and recreated liver IRI despite Ad-HO-1 gene transfer. Parallel in vitro studies with bone marrow derived-macrophages have confirmed that HO-1-STAT3 axis-induced PI3K/Akt negatively regulated PTEN expression in TLR4-dependent fashion. CONCLUSIONS: These findings underscore the role of HO-1 induced STAT3 in modulating PI3K/PTEN in liver IRI cascade. Activating PI3K/Akt provides negative feedback mechanism for TLR4-driven inflammation. Identifying molecular pathways of STAT3 modulation in the innate immune system provides the rationale for novel therapeutic approaches for the management of liver inflammation and IRI in transplant patients.

Alloreactive CD8 T-cell primed/memory responses and accelerated graft rejection in B-cell-deficient sensitized mice.

BACKGROUND: The sensitized patients can develop an accelerated form of graft rejection mediated by humoral and T-cell-mediated responses, which are resistant to currently used immunosuppression. METHODS AND RESULTS: In our model of fulminant cardiac allograft rejection in sensitized hosts, groups of wild-type (WT) and B-cell-deficient (BKO) mice (B6) were challenged with skin grafts (B/c). Alloreactive CD8 T effector (Teff) activation and T memory (Tmem) differentiation during a 60-day follow-up period were reduced in the absence of B-cell help. The expression of interleukin (IL)-2Rα, IL-7Rα, and IL-15Rα, which support/program CD8 Teff/Tmem expansion, differentiation, and survival, were selectively decreased in BKO hosts. Unlike in WT, in vivo cytotoxic activity analysis of alloreactive Tmem recall response has revealed decreased donor-type (B/c) but not third-party (C3H) cell lysis in sensitized BKO hosts. However, such impaired allo-Ag specific Tmem recall function was insufficient to markedly prolong cardiac allograft survival in sensitized BKO recipients. Indeed, despite quantitative and statistically significant differences between both animal groups, the biological impact of decreased CD8 Teff/Tmem activation and function in the sensitization phase was marginal. Indeed, cardiac allografts underwent fulminant rejection in sensitized BKO, albeit with somewhat delayed kinetics. Interestingly, unlike in naïve counterparts, the rejection cascade remained CD154 blockade-resistant, evidenced by comparable kinetics, and intra-graft cytokine gene profiles in MR1 monoclonal antibody-treated sensitized WT and BKO hosts. CONCLUSION: Although B cells were important for optimal alloreactive CD8 Teff/Tmem function in the sensitization phase, the fulminant rejection of cardiac allografts was B-cell-independent, and CD154 blockade-resistant, as in WT hosts.

Native macrophages genetically modified to express heme oxygenase 1 protect rat liver transplants from ischemia/reperfusion injury.

We investigated whether native macrophages overexpressing heme oxygenase 1 (HO-1) could protect rat orthotopic liver transplant (OLT) against cold ischemia/reperfusion injury (IRI). Livers from Sprague-Dawley rats were stored at 4°C in University of Wisconsin solution for 24 hours, and then they were transplanted into syngeneic recipients. Bone marrow-derived macrophages (BMMs) that were transfected ex vivo with heme oxygenase 1 adenovirus (Ad-HO-1), ß-galactosidase adenovirus (Ad-ß-gal), or HO-1 small interfering RNA (siRNA) were infused directly into the OLT before reperfusion. Controls were OLT conditioned with unmodified or scrambled siRNA-transfected cells. The transfer of Ad-HO-1/BMMs increased the survival of OLT to 100% (versus 40%-50% for controls) and decreased serum alanine aminotransferase levels and histological features of hepatocellular damage. In contrast, an infusion of macrophages transfected with HO-1 siRNA/Ad-ß-gal failed to affect IRI. Gene therapy-induced HO-1 suppressed toll-like receptor 4 expression, decreased expression of proinflammatory tumor necrosis factor α, interleukin-1ß, monocyte chemoattractant protein 1, and chemokine (C-X-C motif) ligand 10, and attenuated endothelial intercellular cell adhesion molecule 1 expression with resultant diminished OLT leukocyte sequestration. Although Ad-HO-1/BMMs decreased the frequency of apoptotic cells positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and ameliorated caspase-3 activity, the expression of interleukin-10 and antiapoptotic B cell lymphoma 2/B cell lymphoma extra large increased in well-functioning OLT. Thus, the transfer of native macrophages transfected ex vivo with HO-1 can rescue rat iso-OLT from IRI. Our study validates a novel and clinically attractive concept: native macrophages transfected ex vivo with the antioxidant HO-1 can be applied at the time of transplantation to mitigate otherwise damaging antigen-independent liver inflammation and injury resulting from the peritransplant harvesting insult. If this new, refined strategy is proven to be effective in allo-OLT recipients, it should be considered in clinical settings to increase the supply of usable donor organs and ultimately improve the overall success of liver transplantation.