Haofeng Ji

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.

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.

The emerging role of T cell immunoglobulin mucin-1 in the mechanism of liver ischemia and reperfusion injury in the mouse.

The T cell immunoglobulin and mucin domain‐containing molecules (TIM) protein family, which is expressed by T cells, plays a crucial role in regulating host adaptive immunity and tolerance. However, its role in local inflammation, such as innate immunity‐dominated organ ischemia–reperfusion injury (IRI), remains unknown. Liver IRI occurs frequently after major hepatic resection or liver transplantation. Using an antagonistic anti–TIM‐1 antibody (Ab), we studied the role of TIM‐1 signaling in the model of partial warm liver ischemia followed by reperfusion. Anti–TIM‐1 Ab monotherapy ameliorated the hepatocellular damage and improved liver function due to IR, as compared with controls. Histological examination has revealed that anti–TIM‐1 Ab treatment decreased local neutrophil infiltration, inhibited sequestration of T lymphocytes, macrophages, TIM‐1 ligand–expressing TIM‐4+ cells, and reduced liver cell apoptosis. Intrahepatic neutrophil activity and induction of proinflammatory cytokines/chemokines were also reduced in the treatment group. In parallel in vitro studies, anti–TIM‐1 Ab suppressed interferon‐γ (IFN‐γ) production in concanavalin A (conA)–stimulated spleen T cells, and diminished tumor necrosis factor α (TNF‐α)/interleukin (IL)‐6 expression in a macrophage/spleen T cell coculture system. This is the first study to provide evidence for the novel role of TIM‐1 signaling in the mechanism of liver IRI. TIM‐1 regulates not only T for the role of cell activation but may also affect macrophage function in the local inflammation response. These results provide compelling data for further investigation of TIM‐1 pathway in the mechanism of IRI, to improve liver function, expand the organ donor pool, and improve the overall success of liver transplantation. (HEPATOLOGY 2010.)

Programmed death-1/B7-H1 negative costimulation protects mouse liver against ischemia and reperfusion injury.

Programmed death‐1 (PD‐1)/B7‐H1 costimulation acts as a negative regulator of host alloimmune responses. Although CD4 T cells mediate innate immunity‐dominated ischemia and reperfusion injury (IRI) in the liver, the underlying mechanisms remain to be elucidated. This study focused on the role of PD‐1/B7‐H1 negative signaling in liver IRI. We used an established mouse model of partial liver warm ischemia (90 minutes) followed by reperfusion (6 hours). Although disruption of PD‐1 signaling after anti–B7‐H1 monoclonal antibody treatment augmented hepatocellular damage, its stimulation following B7‐H1 immunoglobulin (B7‐H1Ig) fusion protected livers from IRI, as evidenced by low serum alanine aminotransferase levels and well‐preserved liver architecture. The therapeutic potential of B7‐H1 engagement was evident by diminished intrahepatic T lymphocyte, neutrophil, and macrophage infiltration/activation; reduced cell necrosis/apoptosis but enhanced anti‐necrotic/apoptotic Bcl‐2/Bcl‐xl; and decreased proinflammatory chemokine/cytokine gene expression in parallel with selectively increased interleukin (IL)‐10. Neutralization of IL‐10 re‐created liver IRI and rendered B7‐H1Ig–treated hosts susceptible to IRI. These findings were confirmed in T cell–macrophage in vitro coculture in which B7‐H1Ig diminished tumor necrosis factor‐α/IL‐6 levels in an IL‐10–dependent manner. Our novel findings document the essential role of the PD‐1/B7‐H1 pathway in liver IRI. Conclusion: This study is the first to demonstrate that stimulating PD‐1 signals ameliorated liver IRI by inhibiting T cell activation and Kupffer cell/macrophage function. Harnessing mechanisms of negative costimulation by PD‐1 upon T cell–Kupffer cell cross‐talk may be instrumental in the maintenance of hepatic homeostasis by minimizing organ damage and promoting IL‐10–dependent cytoprotection. (HEPATOLOGY 2010.)

Neuropeptide PACAP in mouse liver ischemia and reperfusion injury: Immunomodulation by the cAMP-PKA pathway†‡

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. (HEPATOLOGY 2013)

Small Interfering RNA Targeting Heme Oxygenase-1 (HO-1) Reinforces Liver Apoptosis Induced by Ischemia–Reperfusion Injury in Mice: HO-1 Is Necessary for Cytoprotection

We have shown that overexpression of heme oxygenase-1 (HO-1) prevents the liver inflammation response leading to ischemia and reperfusion injury (IRI). This study was designed to explore the precise function and mechanism of HO-1 cytoprotection in liver IRI by employing a small interfering RNA (siRNA) that effectively suppresses HO-1 expression both in vitro and in vivo. Using a partial lobar liver warm ischemia model, mice were injected with HO-1 siRNA/nonspecific control siRNA or Ad-HO-1/Ad-beta-gal. Those treated with HO-1 siRNA showed increased serum glutamic-oxaloacetic transaminase levels, significant liver edema, sinusoidal congestion/cytoplasmic vacuolization, and severe hepatocellular necrosis. In contrast, Ad-HO-1-pretreated animals revealed only minimal sinusoidal congestion without edema/vacuolization or necrosis. Administration of HO-1 siRNA significantly increased local neutrophil accumulation and the frequency of apoptotic cells. Mice treated with HO-1 siRNA were characterized by increased caspase-3 activity and reduced HO-1 expression, whereas those given Ad-HO-1 showed decreased caspase-3 activity and increased HO-1/Bcl-2/Bcl-x(L), data confirmed by use of an in vitro cell culture system. Thus, by using an siRNA approach this study confirms that HO-1 provides potent cytoprotection against hepatic IRI and regulates liver apoptosis. Indeed, siRNA provides a powerful tool with which to study gene function in a wide range of liver diseases.

β‐catenin regulates innate and adaptive immunity in mouse liver ischemia‐reperfusion injury

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 β‐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 β‐catenin activation by way of GSK‐3β phosphorylation. The activation of β‐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 β‐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 β‐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 β‐catenin to modulate DC maturation and function at the innate‐adaptive interface. Activation of β‐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 β‐catenin regulates DC function, our findings provide the rationale for novel therapeutic approaches to manage local inflammation and injury in IR‐stressed liver. (HEPATOLOGY 2013)

Adoptive Transfer of Ex Vivo HO-1 Modified Bone Marrow–derived Macrophages Prevents Liver Ischemia and Reperfusion Injury

Macrophages play a critical role in the pathophysiology of liver ischemia and reperfusion (IR) injury (IRI). However, macrophages that overexpress antioxidant heme oxygenase-1 (HO-1) may exert profound anti-inflammatory functions. This study explores the cytoprotective effects and mechanisms of ex vivo modified HO-1-expressing bone marrow-derived macrophages (BMDMs) in well-defined mouse model of liver warm ischemia followed by reperfusion. Adoptive transfer of Ad-HO-1-transduced macrophages prevented IR-induced hepatocellular damage, as evidenced by depressed serum glutamic-oxaloacetic transaminase (sGOT) levels and preserved liver histology (Suzuki scores), compared to Ad-beta-gal controls. This beneficial effect was reversed following concomitant treatment with HO-1 siRNA. Ad-HO-1-transfected macrophages significantly decreased local neutrophil accumulation, TNF-alpha/IL-1beta, IFN-gamma/E-selectin, and IP-10/MCP-1 expression, caspase-3 activity, and the frequency of apoptotic cells, as compared with controls. Unlike in controls, Ad-HO-1-transfected macrophages markedly increased hepatic expression of antiapoptotic Bcl-2/Bcl-xl and depressed caspase-3 activity. These results establish the precedent for a novel investigative tool and provide the rationale for a clinically attractive new strategy in which native macrophages can be transfected ex vivo with cytoprotective HO-1 and then infused, if needed, to prospective recipients exposed to hepatic IR-mediated local inflammation, such as during liver transplantation, resection, or trauma.

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

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. (Hepatology 2014;60:2051–2063)

Targeting TIM-1 on CD4 T Cells Depresses Macrophage Activation and Overcomes Ischemia-Reperfusion Injury in Mouse Orthotopic Liver Transplantation

Hepatic injury due to cold storage followed by reperfusion remains a major cause of morbidity and mortality after orthotopic liver transplantation (OLT). CD4 T cell TIM‐1 signaling costimulates a variety of immune responses in allograft recipients. This study analyzes mechanisms by which TIM‐1 affects liver ischemia‐reperfusion injury (IRI) in a murine model of prolonged cold storage followed by OLT. Livers from C57BL/6 mice, preserved at 4°C in the UW solution for 20 h, were transplanted to syngeneic recipients. There was an early (1 h) increased accumulation of TIM‐1+ activated CD4 T cells in the ischemic OLTs. Disruption of TIM‐1 signaling with a blocking mAb (RMT1–10) ameliorated liver damage, evidenced by reduced sALT levels and well‐preserved architecture. Unlike in controls, TIM‐1 blockade diminished OLT expression of Tbet/IFN‐γ, but amplified IL‐4/IL‐10/IL‐22; abolished neutrophil and macrophage infiltration/activation and inhibited NF‐κB while enhancing Bcl‐2/Bcl‐xl. Although adoptive transfer of CD4 T cells triggered liver damage in otherwise IR‐resistant RAG−/− mice, adjunctive TIM‐1 blockade reduced Tbet transcription and abolished macrophage activation, restoring homeostasis in IR‐stressed livers. Further, transfer of TIM‐1HiCD4+, but not TIM‐1LoCD4+ T cells, recreated liver IRI in RAG−/− mice. Thus, TIM‐1 expressing CD4 T cells are required in the mechanism of innate immune‐mediated hepatic IRI in OLTs.