Shoichi Kageyama

Macrophage heme oxygenase-1-SIRT1-p53 axis regulates sterile inflammation in liver ischemia-reperfusion injury

BACKGROUND & AIMS Hepatic ischemia-reperfusion injury (IRI), characterized by exogenous antigen-independent local inflammation and hepatocellular death, represents a risk factor for acute and chronic rejection in liver transplantation. We aimed to investigate the molecular communication involved in the mechanism of liver IRI. METHODS We analyzed human liver transplants, primary murine macrophage cell cultures and IR-stressed livers in myeloid-specific heme oxygenase-1 (HO-1) gene mutant mice, for anti-inflammatory and cytoprotective functions of macrophage-specific HO-1/SIRT1 (sirtuin 1)/p53 (tumor suppressor protein) signaling. RESULTS Decreased HO-1 expression in human post-reperfusion liver transplant biopsies correlated with a deterioration in hepatocellular function (serum ALT; p<0.05) and inferior patient survival (p<0.05). In the low HO-1 liver transplant biopsy group, SIRT1/Arf (alternative reading frame)/p53/MDM2 (murine double minute 2) expression levels decreased (p<0.05) while cleaved caspase 3 and frequency of TUNEL+cells simultaneously increased (p<0.05). Immunofluorescence showed macrophages were the principal source of HO-1 in human and mouse IR-stressed livers. In vitro macrophage cultures revealed that HO-1 induction positively regulated SIRT1 signaling, whereas SIRT1-induced Arf inhibited ubiquitinating activity of MDM2 against p53, which in turn attenuated macrophage activation. In a murine model of hepatic warm IRI, myeloid-specific HO-1 deletion lacked SIRT1/p53, exacerbated liver inflammation and IR-hepatocellular death, whereas adjunctive SIRT1 activation restored p53 signaling and rescued livers from IR-damage. CONCLUSION This bench-to-bedside study identifies a new class of macrophages activated via the HO-1-SIRT1-p53 signaling axis in the mechanism of hepatic sterile inflammation. This mechanism could be a target for novel therapeutic strategies in liver transplant recipients. LAY SUMMARY Post-transplant low macrophage HO-1 expression in human liver transplants correlates with reduced hepatocellular function and survival. HO-1 regulates macrophage activation via the SIRT1-p53 signaling network and regulates hepatocellular death in liver ischemia-reperfusion injury. Thus targeting this pathway in liver transplant recipients could be of therapeutic benefit.

Heme oxygenase-1 regulates sirtuin-1–autophagy pathway in liver transplantation: From mouse to human

Liver ischemia–reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although graft autophagy is essential for resistance against hepatic IRI, its significance in clinical OLT remains unknown. Despite recent data identifying heme oxygenase‐1 (HO‐1) as a putative autophagy inducer, its role in OLT and interactions with sirtuin‐1 (SIRT1), a key autophagy regulator, have not been studied. We aimed to examine HO‐1–mediated autophagy induction in human OLT and in a murine OLT model with extended (20 hours) cold storage, as well as to analyze the requirement for SIRT1 in autophagy regulation by HO‐1. Fifty‐one hepatic biopsy specimens from OLT patients were collected under an institutional review board protocol 2 hours after portal reperfusion, followed by Western blot analyses. High HO‐1 levels correlated with well‐preserved hepatocellular function and enhanced SIRT1/LC3B expression. In mice, HO‐1 overexpression by genetically modified HO‐1 macrophage therapy was accompanied by decreased OLT damage and increased SIRT1/LC3B expression, whereas adjunctive inhibition of SIRT1 signaling diminished HO‐1–mediated hepatoprotection and autophagy induction. Our translational study confirms the clinical relevance of HO‐1 cytoprotection and identifies SIRT1‐mediated autophagy pathway as a new essential regulator of HO‐1 function in IR‐stressed OLT.

Sirtuin 1 Attenuates Inflammation and Hepatocellular Damage in Liver Transplant Ischemia-Reperfusion: From Mouse-to-Human

Hepatic ischemia/reperfusion injury (IRI), an inevitable antigen‐independent inflammation response in cadaveric liver transplantation, correlates with poor early graft function, rejection episodes, and contributes to donor organ shortage. Sirtuin 1 (SIRT1) is a histone deacetylase that may regulate inflammatory cell activity and manage liver function in IRI, though its functional role and clinical relevance remains to be elucidated. We investigated the efficacy of SIRT1 activation in a murine liver IRI model and verified the concept of putative SIRT1‐mediated hepatoprotection in clinical liver transplantation. In the experimental arm, mice were subjected to 90 minutes of liver partial warm ischemia followed by 6 hours of reperfusion with or without adjunctive SIRT1 activation in vivo (resveratrol [Res]). In parallel, bone marrow–derived macrophage (BMDM) or spleen lymphocyte cultures were treated with Res. In the clinical arm, liver biopsies from 21 adult primary liver transplant patients (2 hours after reperfusion) were divided into “low” (n = 11) versus “high” (n = 10) SIRT1 expression groups, assessed by Western blots. Treatment with Res attenuated murine liver IRI while up‐regulating SIRT1, suppressing leukocyte infiltration, and decreasing proinflammatory cytokine programs. SIRT1 silencing (small interfering RNA) in BMDM cultures enhanced inflammatory cytokine programs, whereas addition of Res decreased proinflammatory response in a SIRT1‐dependent manner. In addition, Res decreased interferon γ production in liver‐infiltrating and spleen lymphocyte cultures. Human liver transplants with high SIRT1 levels showed improved hepatocellular function and superior survival (P = 0.04), accompanied by lower proinflammatory cytokine profile. In conclusion, our translational study is the first to identify SIRT1 as a regulator of hepatocellular function in human liver transplant recipients under ischemia/reperfusion stress. By targeting innate and adaptive immune activation, manipulation of SIRT1 signaling should be considered as a novel means to combat inflammation in liver transplantation. Liver Transplantation 23 1282–1293 2017 AASLD.

Serelaxin induces Notch1 signaling and alleviates hepatocellular damage in orthotopic liver transplantation

Liver ischemia‐reperfusion injury (IRI) represents a risk factor for early graft dysfunction and an obstacle to expanding donor pool in orthotopic liver transplantation (OLT). We have reported on the crucial role of macrophage Notch1 signaling in mouse warm hepatic IRI model. However, its clinical relevance or therapeutic potential remain unknown. Here, we used Serelaxin (SER), to verify Notch1 induction and putative hepatoprotective function in ischemia‐reperfusion–stressed OLT. C57BL/6 mouse livers subjected to extended (18‐hour) cold storage were transplanted to syngeneic recipients. SER treatment at reperfusion ameliorated IRI, improved post‐OLT survival, decreased neutrophil/macrophage infiltration, and suppressed proinflammatory cytokine programs, while simultaneously increasing Notch intracellular domain (NICD) and hairy and enhancer of split 1 (Hes1) target genes. In bone marrow–derived macrophage cultures, SER suppressed proinflammatory while enhancing antiinflammatory gene expression concomitantly with increased NICD and Hes1. Hepatic biopsies from 21 adult primary liver transplant patients (2 hours postreperfusion) were divided into low‐NICD (n = 11) and high‐NICD (n = 10) expression groups (western blots). Consistent with our murine findings, human livers characterized by high NICD were relatively IRI resistant, as shown by serum alanine aminotransferase (ALT) levels at day 1 post‐OLT. Our study documents the efficacy of SER–Notch1 signaling in mouse OLT and highlights the protective function of Notch1 in liver transplant patients.

A soluble form of PSGL‐1 requires Nrf2 signaling to protect liver transplant endothelial cells against ischemia‐reperfusion injury

Liver endothelial cell (LEC) damage is essential in the pathogenesis of ischemia–reperfusion injury (IRI) in transplant recipients. We analyzed the mechanism of LEC resistance against IRI by using a novel recombinant soluble form of P selectin glycoprotein ligand 1, tandem P selectin glycoprotein ligand immunoglobulin (TSGL‐Ig), in a mouse model of hepatic cold preservation (4°C in University of Wisconsin solution for 20 h) and syngeneic orthotopic liver transplantation (OLT). Unlike controls, TSGL‐Ig protected orthotopic liver transplants against ischemia–reperfusion (IR) stress, shown by depressed serum alanine aminotransferase levels, well‐preserved hepatic architecture, and improved survival (42% vs. 92%). TSGL‐Ig suppressed neutrophil/macrophage sequestration and proinflammatory cytokine/chemokine programs in OLT. Treatment with TSGL‐Ig mitigated LEC activation (P and E selectin, VCAM‐1 and intercellular adhesion molecule 1 expression). In parallel in vitro studies, TSGL‐Ig diminished cellular damage in H2O2‐stressed LEC cultures (lactic acid dehydrogenase and alanine aminotransferase levels). Increased thioredoxin, glutamate‐cysteine ligase, NAD(P)H quinone dehydrogenase 1, and hypoxia‐inducible factor 1α expression, along with transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2), implied that TSGL‐Ig exerts antioxidant functions in IR‐stressed OLT and H2O2‐stressed LECs. Indeed, Nrf2‐deficient livers suffered fulminant IRI compared with WT despite concomitant TSGL‐Ig therapy. Thus, TSGL‐Ig is not only acting as a competitive antagonist blocking leukocyte migration into IR‐stressed liver, but it may also act directly as an agonist stimulating Nrf2‐mediated cytoprotection in LECs. This study supports the role of P selectin signaling in hepatic homeostasis in OLT, with broad implications for tissue damage conditions.

Recombinant relaxin protects liver transplants from ischemia damage by hepatocyte glucocorticoid receptor: From bench‐to‐bedside

Hepatic ischemia‐reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and acute/chronic rejection as well as a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although glucocorticoid receptor (GR) signaling may enhance cytoprotective programs, clinical use of glucocorticoid is limited because of adverse effects, whereas clinical relevance of GR‐facilitated cytoprotection in OLT remains unknown. We aimed to evaluate the significance of hepatic GR in clinical OLT and verify the impact of recombinant human relaxin (rhRLX), which may function as a GR agonist in a tissue/disease‐specific manner. Fifty‐one OLT patients were recruited under an institutional research board (IRB) protocol. Liver biopsies were collected after cold storage (presurgery) and 2 hours postreperfusion (before abdominal closure), followed by western blotting–assisted hepatic analyses. Forty‐three percent of OLTs failed to increase GR perioperatively under surgical stress. Post‐/pre‐GR ratios at postoperative day 1 correlated negatively with serum aspartate aminotransferase (AST)/cleaved caspase‐3 and positively with B‐cell lymphoma‐extra large (Bcl‐xL)/B‐cell lymphoma 2 (Bcl‐2) levels. In a murine OLT model with extended (18‐hour) cold storage, treatment with rhRLX ameliorated ischemia‐reperfusion (IR) damage and improved survival while up‐regulating hepatocyte GR and Bcl‐xL/Bcl‐2 expression in OLT. rhRLX‐induced GR suppressed hepatocyte high‐mobility group box 1 (HMGB1) translocation/release, accompanied by decreased Toll‐like receptor 4 (TLR4)/receptor for advanced glycation end products (RAGE), suppressed interleukin 1 beta (IL1β), chemokine (C‐C motif) ligand 2 (CCL2), C‐X‐C motif chemokine (CXCL)10, tumor necrosis factor alpha (TNFα), CXCL1, and CXCL2 levels, and attenuated neutrophil/macrophage accumulation in OLT. Inhibition of GR in hepatocyte culture and in OLT diminished rhRLX‐mediated cytoprotection. Conclusion: This translational study underscores the role of rhRLX‐GR signaling as a regulator of hepatocellular protection against IR stress in OLT. In the context of a recent phase III clinical trial demonstrating positive outcomes of rhRLX in patients with acute heart failure, studies on rhRLX for the management of IRI in OLT recipients are warranted. (Hepatology 2018;68:258‐273).

Outside-in HLA class I signaling regulates ICAM-1 clustering and endothelial cell-monocyte interactions via mTOR in transplant antibody-mediated rejection

Antibody‐mediated rejection (AMR) resulting in transplant allograft vasculopathy (TAV) is the major obstacle for long‐term survival of solid organ transplants. AMR is caused by donor‐specific antibodies to HLA, which contribute to TAV by initiating outside‐in signaling transduction pathways that elicit monocyte recruitment to activated endothelium. Mechanistic target of rapamycin (mTOR) inhibitors can attenuate TAV; therefore, we sought to understand the mechanistic underpinnings of mTOR signaling in HLA class I Ab–mediated endothelial cell activation and monocyte recruitment. We used an in vitro model to assess monocyte binding to HLA I Ab–activated endothelial cells and found mTOR inhibition reduced ezrin/radixin/moesin (ERM) phosphorylation, intercellular adhesion molecule 1 (ICAM‐1) clustering, and monocyte firm adhesion to HLA I Ab–activated endothelium. Further, in a mouse model of AMR, in which C57BL/6. RAG1−/− recipients of BALB/c cardiac allografts were passively transferred with donor‐specific MHC I antibodies, mTOR inhibition significantly reduced vascular injury, ERM phosphorylation, and macrophage infiltration of the allograft. Taken together, these studies indicate mTOR inhibition suppresses ERM phosphorylation in endothelial cells, which impedes ICAM‐1 clustering in response to HLA class I Ab and prevents macrophage infiltration into cardiac allografts. These findings indicate a novel therapeutic application for mTOR inhibitors to disrupt endothelial cell‐monocyte interactions during AMR.

Recipient HO-1 inducibility is essential for posttransplant hepatic HO-1 expression and graft protection: From bench-to-bedside

By documenting potent antioxidative and anti‐inflammatory functions, preclinical studies encourage heme oxygenase‐1 (HO‐1)‐inducing regimens in clinical orthotopic liver transplantation (OLT). We aimed to determine the importance of recipient‐derived HO‐1 in murine and human OLTs. Hepatic biopsies from 51 OLT patients were screened for HO‐1 expression (Western blots) prior to put‐in (basal) and post reperfusion (stressed) and correlated with the hepatocellular function. In parallel, livers from HO‐1 proficient mice (WT; C57/BL6), subjected to ex vivo cold storage (18 hour), were transplanted to syngeneic myeloid HO‐1 deficient (mHO‐1 KO) or FLOX (control) hosts, and sampled postreperfusion (6 hour). In human OLT, posttransplant but not pretransplant HO‐1 expression correlated negatively with ALT levels (P = .0178). High posttransplant but not pretransplant HO‐1 expression trended with improved OLT survival. Compared with controls, livers transplanted into mHO‐1 KO recipient mice had decreased HO‐1 levels, exacerbated hepatic damage/frequency of TUNEL+ cells, increased mRNA levels coding for TNFα/CXCL1/CXCL2/CXCL10, higher frequency of Ly6G+/4HN+ neutrophils; and enhanced MPO activity. Peritoneal neutrophils from mHO‐1 KO mice exhibited higher CellRox+ ratio and increased TNFα/CXCL1/CXCL2/CXCL10 expression. By demonstrating the importance of posttransplant recipient HO‐1 phenotype in hepatic macrophage/neutrophil regulation and function, this translational study identifies recipient HO‐1 inducibility as a novel biomarker of ischemic stress resistance in OLT.