ABSTRACT
Cardiac allograft vasculopathy (CAV) causes late graft failure and mortality after heart transplantation. Donor-specific antibodies (DSAs) lead to chronic endothelial cell injury, inflammation, and arterial intimal thickening. In this study, GeoMx digital spatial profiling was used to analyze arterial areas of interest (AOIs) from CAV+DSA+ rejected cardiac allografts (N = 3; 22 AOIs total). AOIs were categorized based on CAV neointimal thickening and underwent whole transcriptome and protein profiling. By comparing our transcriptomic data with that of healthy control vessels of rapid autopsy myocardial tissue, we pinpointed specific pathways and transcripts indicative of heightened inflammatory profiles in CAV lesions. Moreover, we identified protein and transcriptomic signatures distinguishing CAV lesions exhibiting low and high neointimal lesions. AOIs with low neointima showed increased markers for activated inflammatory infiltrates, endothelial cell activation transcripts, and gene modules involved in metalloproteinase activation and TP53 regulation of caspases. Inflammatory and apoptotic proteins correlated with inflammatory modules in low neointima AOIs. High neointima AOIs exhibited elevated TGFß-regulated transcripts and modules enriched for platelet activation/aggregation. Proteins associated with growth factors/survival correlated with modules enriched for proliferation/repair in high neointima AOIs. Our findings reveal novel insight into immunological mechanisms mediating CAV pathogenesis.
Subject(s)
Graft Rejection , Heart Transplantation , Heart Transplantation/adverse effects , Graft Rejection/etiology , Graft Rejection/pathology , Graft Rejection/immunology , Humans , Male , Allografts , Isoantibodies/immunology , Middle Aged , Female , Transcriptome , Neointima/pathology , Neointima/immunology , Neointima/etiology , Graft Survival/immunology , Prognosis , Follow-Up Studies , Gene Expression Profiling , Biomarkers/metabolism , Tissue Donors , Vascular Diseases/etiology , Vascular Diseases/immunology , Vascular Diseases/pathology , MultiomicsABSTRACT
HLA donor-specific antibodies (DSA) elicit alloimmune responses against the graft vasculature, leading to endothelial cell (EC) activation and monocyte infiltration during antibody-mediated rejection (AMR). AMR promotes chronic inflammation and remodeling, leading to thickening of the arterial intima termed transplant vasculopathy or cardiac allograft vasculopathy (CAV) in heart transplants. Intragraft-recipient macrophages serve as a diagnostic marker in AMR; however, their polarization and function remain unclear. In this study, we utilized an in vitro Transwell coculture system to explore the mechanisms of monocyte-to-macrophage polarization induced by HLA I DSA-activated ECs. Anti-HLA I (IgG or F(ab')2) antibody-activated ECs induced the polarization of M2 macrophages with increased CD206 expression and MMP9 secretion. However, inhibition of TLR4 signaling or PSGL-1-P-selectin interactions significantly decreased both CD206 and MMP9. Monocyte adherence to Fc-P-selectin coated plates induced M2 macrophages with increased CD206 and MMP9. Moreover, Fc-receptor and IgG interactions synergistically enhanced active-MMP9 in conjunction with P-selectin. Transcriptomic analysis of arteries from DSA+CAV+ rejected cardiac allografts and multiplex-immunofluorescent staining illustrated the expression of CD68+CD206+CD163+MMP9+ M2 macrophages within the neointima of CAV-affected lesions. These findings reveal a novel mechanism linking HLA I antibody-activated endothelium to the generation of M2 macrophages which secrete vascular remodeling proteins contributing to AMR and CAV pathogenesis.
Subject(s)
Toll-Like Receptor 4 , Vascular Diseases , Humans , Matrix Metalloproteinase 9 , P-Selectin , Macrophages , Endothelium , HLA Antigens , Allografts , Immunoglobulin GABSTRACT
Donor-specific HLA Abs contribute to Ab-mediated rejection (AMR) by binding to HLA molecules on endothelial cells (ECs) and triggering intracellular signaling, leading to EC activation and leukocyte recruitment. The molecular mechanisms involving donor-specific HLA Ab-mediated EC activation and leukocyte recruitment remain incompletely understood. In this study, we determined whether TLRs act as coreceptors for HLA class I (HLA I) in ECs. We found that human aortic ECs express TLR3, TLR4, TLR6, and TLR10, but only TLR4 was detected on the EC surface. Consequently, we performed coimmunoprecipitation experiments to examine complex formation between HLA I and TLR4. Stimulation of human ECs with HLA Ab increased the amount of complex formation between HLA I and TLR4. Reciprocal coimmunoprecipitation with a TLR4 Ab confirmed that the crosslinking of HLA I increased complex formation between TLR4 and HLA I. Knockdown of TLR4 or MyD88 with small interfering RNAs inhibited HLA I Ab-stimulated P-selectin expression, von Willebrand factor release, and monocyte recruitment on ECs. Our results show that TLR4 is a novel coreceptor for HLA I to stimulate monocyte recruitment on activated ECs. Taken together with our previous published results, we propose that HLA I molecules form two separate signaling complexes at the EC surface, that is, with TLR4 to upregulate P-selectin surface expression and capture of monocytes to human ECs and integrin ß4 to induce mTOR-dependent firm monocyte adhesion via ICAM-1 clustering on ECs, two processes implicated in Ab-mediated rejection.
Subject(s)
Endothelial Cells , Intercellular Adhesion Molecule-1 , Cells, Cultured , Endothelium, Vascular/metabolism , HLA Antigens/metabolism , Humans , Integrin beta4/metabolism , Intercellular Adhesion Molecule-1/metabolism , Monocytes , Myeloid Differentiation Factor 88/metabolism , P-Selectin/metabolism , TOR Serine-Threonine Kinases/metabolism , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 4/metabolism , Toll-Like Receptor 6/metabolism , von Willebrand Factor/metabolismABSTRACT
Ischemia-reperfusion injury (IRI) during orthotopic liver transplantation (OLT) contributes to graft rejection and poor clinical outcomes. The disulfide form of high mobility group box 1 (diS-HMGB1), an intracellular protein released during OLT-IRI, induces pro-inflammatory macrophages. How diS-HMGB1 differentiates human monocytes into macrophages capable of activating adaptive immunity remains unknown. We investigated if diS-HMGB1 binds toll-like receptor (TLR) 4 and TLR9 to differentiate monocytes into pro-inflammatory macrophages that activate adaptive immunity and promote graft injury and dysfunction. Assessment of 106 clinical liver tissue and longitudinal blood samples revealed that OLT recipients were more likely to experience IRI and graft dysfunction with increased diS-HMGB1 released during reperfusion. Increased diS-HMGB1 concentration also correlated with TLR4/TLR9 activation, polarization of monocytes into pro-inflammatory macrophages, and production of anti-donor antibodies. In vitro, healthy volunteer monocytes stimulated with purified diS-HMGB1 had increased inflammatory cytokine secretion, antigen presentation machinery, and reactive oxygen species production. TLR4 inhibition primarily impeded cytokine/chemokine and costimulatory molecule programs, whereas TLR9 inhibition decreased HLA-DR and reactive oxygen species production. diS-HMGB1-polarized macrophages also showed increased capacity to present antigens and activate T memory cells. In murine OLT, diS-HMGB1 treatment potentiated ischemia-reperfusion-mediated hepatocellular injury, accompanied by increased serum alanine transaminase levels. This translational study identifies the diS-HMGB1/TLR4/TLR9 axis as potential therapeutic targets in OLT-IRI recipients.
Subject(s)
HMGB1 Protein , Liver Transplantation , Reperfusion Injury , Humans , Mice , Animals , Toll-Like Receptor 9/metabolism , HMGB1 Protein/metabolism , Toll-Like Receptor 4/metabolism , Reactive Oxygen Species/metabolism , Liver , Reperfusion Injury/metabolism , Macrophages , Cytokines/metabolism , Apoptosis , Mice, Inbred C57BLABSTRACT
BACKGROUND AND AIMS: Sterile inflammation is a major clinical concern during ischemia-reperfusion injury (IRI) triggered by traumatic events, including stroke, myocardial infarction, and solid organ transplantation. Despite high-mobility group box 1 (HMGB1) clearly being involved in sterile inflammation, its role is controversial because of a paucity of patient-focused research. APPROACH AND RESULTS: Here, we examined the role of HMGB1 oxidation states in human IRI following liver transplantation. Portal blood immediately following allograft reperfusion (liver flush; LF) had increased total HMGB1, but only LF from patients with histopathological IRI had increased disulfide-HMGB1 and induced Toll-like receptor 4-dependent tumor necrosis factor alpha production by macrophages. Disulfide HMGB1 levels increased concomitantly with IRI severity. IRI+ prereperfusion biopsies contained macrophages with hyperacetylated, lysosomal disulfide-HMGB1 that increased postreperfusion at sites of injury, paralleling increased histone acetyltransferase general transcription factor IIIC subunit 4 and decreased histone deacetylase 5 expression. Purified disulfide-HMGB1 or IRI+ blood stimulated further production of disulfide-HMGB1 and increased proinflammatory molecule and cytokine expression in macrophages through a positive feedback loop. CONCLUSIONS: These data identify disulfide-HMGB1 as a mechanistic biomarker of, and therapeutic target for, minimizing sterile inflammation during human liver IRI.
Subject(s)
HMGB1 Protein/metabolism , Liver Transplantation/adverse effects , Reperfusion Injury/etiology , Cytokines/metabolism , Disulfides/blood , Female , Fluorescent Antibody Technique , HMGB1 Protein/blood , Humans , Liver/metabolism , Male , Microscopy, Confocal , Middle Aged , Monocytes/metabolism , Reperfusion Injury/blood , Reperfusion Injury/metabolism , Tissue DonorsABSTRACT
HLA donor-specific antibodies (DSAs) binding to vascular endothelial cells of the allograft trigger inflammation, vessel injury, and antibody-mediated rejection (AMR). Accumulation of intragraft-recipient macrophages is a histological characteristic of AMR, which portends worse outcome. HLA class I (HLA I) DSAs enhance monocyte recruitment by activating endothelial cells and engaging FcγRs, but the DSA-activated donor endothelial influence on macrophage differentiation is unknown. In this study, we explored the consequence of DSA-activated endothelium on infiltrating monocyte differentiation. Here we show that cardiac allografts from murine recipients treated with MHC I DSA upregulated genes related to monocyte transmigration and Fc receptor stimulation. Human monocytes co-cultured with HLA I IgG-stimulated primary human endothelium promoted monocyte differentiation into CD68+ CD206+ CD163+ macrophages (M(HLA I IgG)), whereas HLA I F(ab')2 stimulated endothelium solely induced higher CD206 (M(HLA I F(ab')2 )). Both macrophage subtypes exhibited significant changes in discrete cytokines/chemokines and unique gene expression profiles. Cross-comparison of gene transcripts between murine DSA-treated cardiac allografts and human co-cultured macrophages identified overlapping genes. These findings uncover the role of HLA I DSA-activated endothelium in monocyte differentiation, and point to a novel, remodeling phenotype of infiltrating macrophages that may contribute to vascular injury.
Subject(s)
Endothelial Cells , Graft Rejection , Allografts , Animals , Graft Rejection/etiology , HLA Antigens , Humans , Inflammation/etiology , Isoantibodies , Macrophages , Mice , Phenotype , Tissue DonorsABSTRACT
OBJECTIVE AND BACKGROUND: Pattern recognition receptors (PRRs) on immune and parenchymal cells can detect danger-associated molecular patterns (DAMPs) released from cells damaged during ischemia-reperfusion injury (IRI), in heart attack or stroke settings, but also as an unavoidable consequence of solid organ transplantation. Despite IRI being a significant clinical problem across all solid organ transplants, there are limited therapeutics and patient-specific diagnostics currently available. METHODS: We screened portal blood samples obtained from 67 human liver transplant recipients both pre- [portal vein (PV) sample] and post-(liver flush; LF) reperfusion for their ability to activate a panel of PRRs, and analyzed this reactivity in relation to biopsy-proven IRI. RESULTS: PV samples from IRI+ orthotopic liver transplantation (OLT) patients (n = 35) decreased activation of hTLR4- and hTLR9-transfected cells, whereas PV from IRI- patients (n = 32) primarily increased hTLR7 and hNOD2 activation. LF samples from OLT-IRI patients significantly increased activation of hTLR4 and hTLR9 over IRI- LF. In addition, the change from baseline reactivity to hTLR4/9/NOD2 was significantly higher in IRI+ than IRI- OLT patients. CONCLUSIONS: These results demonstrate that TLR4/7/9 and NOD2 are involved in either promoting or attenuating hepatic IRI, and suggest a diagnostic screening of portal blood for reactivity to these PRRs might prove useful for prediction and/or therapeutic intervention in OLT patients before transplantation.
Subject(s)
Biomarkers/blood , Liver Transplantation , Nod2 Signaling Adaptor Protein/blood , Pattern Recognition, Automated , Precision Medicine , Reperfusion Injury/immunology , Reperfusion Injury/prevention & control , Toll-Like Receptor 4/blood , Female , Humans , Immunity, Innate , Male , Middle Aged , Nod2 Signaling Adaptor Protein/immunology , Signal Transduction , Toll-Like Receptor 4/immunologyABSTRACT
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.
Subject(s)
Heme Oxygenase-1/metabolism , Liver Transplantation/methods , Liver/pathology , Macrophages/metabolism , Neutrophils/immunology , Reperfusion Injury/prevention & control , Animals , Apoptosis , Humans , Liver/immunology , Liver/metabolism , Macrophages/cytology , Macrophages/immunology , Mice , Mice, Inbred C57BL , Reperfusion Injury/immunology , Reperfusion Injury/metabolism , Signal TransductionABSTRACT
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 (IL1ß), 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).
Subject(s)
Hepatocytes/drug effects , Liver Transplantation/adverse effects , Receptors, Glucocorticoid/metabolism , Relaxin/therapeutic use , Reperfusion Injury/prevention & control , Adolescent , Adult , Aged , Animals , Apoptosis/drug effects , Female , HMGB1 Protein/metabolism , Hepatocytes/metabolism , Humans , Liver/drug effects , Liver/metabolism , Liver Transplantation/mortality , Male , Mice, Inbred C57BL , Middle Aged , Oxidative Stress , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, G-Protein-Coupled/metabolism , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Relaxin/metabolism , Relaxin/pharmacology , Reperfusion Injury/etiology , Young AdultABSTRACT
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.
Subject(s)
Autophagy , Gene Expression Regulation , Heme Oxygenase-1/metabolism , Liver Transplantation , Reperfusion Injury/prevention & control , Sirtuin 1/metabolism , Animals , Apoptosis , Cells, Cultured , Cytoprotection , Graft Survival , Heme Oxygenase-1/administration & dosage , Heme Oxygenase-1/genetics , Humans , Macrophages/metabolism , Macrophages/pathology , Mice , Mice, Inbred C57BL , Signal Transduction , Sirtuin 1/geneticsABSTRACT
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.
Subject(s)
Endothelial Cells/metabolism , Graft Rejection/etiology , Heart Transplantation/adverse effects , Histocompatibility Antigens Class I/immunology , Intercellular Adhesion Molecule-1/metabolism , Monocytes/metabolism , TOR Serine-Threonine Kinases/metabolism , Animals , Cell Communication , Cells, Cultured , Endothelial Cells/immunology , Graft Rejection/metabolism , Graft Rejection/pathology , Humans , Isoantibodies/adverse effects , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Monocytes/immunologyABSTRACT
Evidence has suggested both a pathogenic and a protective role for the proinflammatory cytokine IFN-γ in experimental autoimmune encephalomyelitis (EAE). However, the mechanisms underlying the protective role of IFN-γ in EAE have not been fully resolved, particularly in the context of CNS antigen-presenting cells (APCs). In this study we examined the role of IFN-γ in myelin antigen uptake by CNS APCs during EAE. We found that myelin antigen colocalization with APCs was decreased substantially and that EAE was significantly more severe and showed a chronic-progressive course in IFN-γ knockout (IFN-γ-/-) or IFN-γ receptor knockout (IFN-γR-/-) mice as compared with WT animals. IFN-γ was a critical regulator of phagocytic/activating receptors on CNS APCs. Importantly, "free" myelin debris and lipid peroxidation activity at CNS lesions was increased in mice lacking IFN-γ signaling. Treatment with N-acetyl-l-cysteine, a potent antioxidant, abolished lipid peroxidation activity and ameliorated EAE in IFN-γ-signaling-deficient mice. Taken together the data suggest a protective role for IFN-γ in EAE by regulating the removal of myelin debris by CNS APCs and thereby limiting the substrate available for the generation of neurotoxic lipid peroxidation products.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/immunology , Interferon-gamma/immunology , Lipid Peroxidation/immunology , Myelin Sheath/immunology , Animals , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Apoptosis/genetics , Apoptosis/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cells, Cultured , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/metabolism , Flow Cytometry , Interferon-gamma/deficiency , Interferon-gamma/genetics , Lymphocyte Activation/immunology , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Myelin Sheath/metabolism , Phagocytosis/genetics , Phagocytosis/immunology , Receptors, Interferon/deficiency , Receptors, Interferon/genetics , Receptors, Interferon/immunology , Signal Transduction/genetics , Signal Transduction/immunology , Interferon gamma ReceptorABSTRACT
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.
Subject(s)
Heme Oxygenase-1/physiology , Inflammation/etiology , Liver/blood supply , Macrophages/physiology , Reperfusion Injury/etiology , Sirtuin 1/physiology , Tumor Suppressor Protein p53/physiology , Animals , Heme Oxygenase-1/analysis , Humans , Macrophage Activation , Mice , Mice, Inbred C57BLABSTRACT
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.
Subject(s)
End Stage Liver Disease/surgery , Graft Rejection/immunology , Liver Transplantation/adverse effects , Signal Transduction/immunology , Sirtuin 1/immunology , Adult , Allografts/immunology , Allografts/pathology , Animals , Biopsy , Cells, Cultured , Cytokines/immunology , Cytokines/metabolism , Disease Models, Animal , End Stage Liver Disease/mortality , Female , Follow-Up Studies , Graft Rejection/prevention & control , Graft Survival/immunology , Humans , Kaplan-Meier Estimate , Liver/immunology , Liver/pathology , Liver Transplantation/methods , Lymphocytes , Macrophages , Male , Mice , Mice, Inbred C57BL , Middle Aged , Reperfusion Injury/immunology , Resveratrol , Signal Transduction/drug effects , Sirtuin 1/metabolism , Stilbenes/pharmacology , Stilbenes/therapeutic use , Young AdultABSTRACT
Induction of experimental autoimmune encephalomyelitis (EAE) in susceptible animals requires reactivation of encephalitogenic CD4(+) T cells by APCs in the CNS. However, it has remained unresolved from where APCs in the CNS acquire myelin Ag for T cell activation and under which conditions, that is, whether only during EAE or also in the naive CNS. In this study, we investigated the kinetics of myelin Ag uptake by CNS APCs during EAE and in the naive CNS. Our results show that during EAE CX3CR1(+)CD11b(+) microglia were the first APCs in the CNS to contain myelin Ag upon induction of disease, albeit in very small numbers. Dendritic cells (DCs) arrived in the CNS in sizable numbers significantly later (day 5 postimmunization), without detectable myelin Ag, but acquired it by day 7 postimmunization. Furthermore, a sharp increase in neuroantigen-containing DCs coincided with the onset of EAE symptoms. Importantly, in naive mice a low but consistent number of microglia contained myelin Ag, suggesting release by oligodendrocytes under steady state conditions. Although microglia isolated from naive brain and spinal cord did not elicit a strong CD4(+) T cell response in vitro, myelin Ag-containing microglia may still play a local role in modulating encephalitogenic CD4(+) T cell responses in early EAE prior to the arrival of other professional APCs, such as DCs. Finally, newly arriving DCs in the CNS not yet loaded with myelin Ag before the onset of EAE may be a potential therapeutic target.
Subject(s)
Antigens/metabolism , Brain/metabolism , Encephalomyelitis, Autoimmune, Experimental/metabolism , Myelin Proteolipid Protein/pharmacokinetics , Myelin-Oligodendrocyte Glycoprotein/pharmacokinetics , Myeloid Cells/metabolism , Peptide Fragments/pharmacokinetics , Spinal Cord/metabolism , Adoptive Transfer , Animals , Antigen Presentation , Antigens/immunology , Autoantibodies/biosynthesis , Autoantibodies/immunology , Brain/immunology , Cell Movement , Dendritic Cells/immunology , Dendritic Cells/metabolism , Female , Freund's Adjuvant , Imaging, Three-Dimensional , Immunization , Injections, Intraperitoneal , Injections, Subcutaneous , Lymphocyte Activation , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Neurologic Mutants , Mice, Transgenic , Microglia/metabolism , Microscopy, Confocal , Myelin Basic Protein/analysis , Myelin Basic Protein/immunology , Myelin Proteolipid Protein/immunology , Myelin-Oligodendrocyte Glycoprotein/immunology , Peptide Fragments/immunology , Pertussis Toxin , Spinal Cord/immunologyABSTRACT
BACKGROUND: Ischemia-reperfusion injury (IRI) is a significant clinical concern in liver transplantation, with a key influence on short-term and long-term allograft and patient survival. Myeloid cells trigger and sustain tissue inflammation and damage associated with IRI, but the mechanisms regulating these activities are unknown. To address this, we investigated the molecular characteristics of intragraft myeloid cells present in biopsy-proven IRI- and IRI+ liver transplants. METHODS: RNA-sequencing was performed on 80 pre-reperfusion and post-reperfusion biopsies from 40 human recipients of liver transplantation (23 IRI+, 17 IRI-). We used transcriptional profiling and computational approaches to identify specific gene coexpression network modules correlated with functional subsets of MPO+, lysozyme+, and CD68+ myeloid cells quantified by immunohistochemistry on sequential sections from the same patient biopsies. RESULTS: A global molecular map showed gene signatures related to myeloid activation in all patients regardless of IRI status; however, myeloid cell subsets differed dramatically in their spatial morphology and associated gene signatures. IRI- recipients were found to have a natural corticosteroid production and response profile from pre-reperfusion to post-reperfusion, particularly among monocytes/macrophages. The pre-reperfusion signature of IRI+ recipients included acute inflammatory responses in neutrophils and increased translation of adaptive immune-related genes in monocytes/macrophages coupled with decreased glucocorticoid responses. Subsequent lymphocyte activation at post-reperfusion identified transcriptional programs associated with the transition to adaptive immunity found only among IRI+ recipients. CONCLUSIONS: Myeloid subset-specific genes and related signaling pathways provide targets for the development of therapeutic strategies aimed at limiting IRI in the clinical setting of liver transplantation.
Subject(s)
Liver Transplantation , Reperfusion Injury , Humans , Liver Transplantation/adverse effects , Reperfusion Injury/genetics , Leukocytes , Adaptive Immunity , Biopsy , InflammationABSTRACT
BACKGROUND: Pulmonary antibody-mediated rejection is still a challenging diagnosis as C4d immunostaining has poor sensitivity. Previous studies have indicated that the phosphorylated S6 ribosomal protein, a component of the mammalian target of rapamycin (mTOR) pathway, is correlated with de novo donor-specific antibodies in lung transplantation. The objective of this study was to evaluate the phosphorylation of S6 ribosomal protein as a surrogate for antibody-mediated rejection diagnosis in lung transplant patients. METHODS: This multicentre retrospective study analyzed transbronchial biopsies from 216 lung transplanted patients, 114 with antibody-mediated rejection and 102 without (19 with acute cellular rejection, 17 with ischemia/reperfusion injury, 18 with infection, and 48 without post-transplant complications). Immunohistochemistry was used to quantify phosphorylated S6 ribosomal protein expression in macrophages, endothelium, epithelium, and inter-pathologist agreement was assessed. RESULTS: Median phosphorylated S6 ribosomal protein expression values were higher in antibody-mediated rejection cases than in controls for all cell components, with the highest sensitivity in macrophages (0.9) and the highest specificity in endothelial expression (0.8). The difference was mainly significant in macrophages compared to other post-lung transplantation complications. Inter-pathologist agreement was moderate for macrophages and endothelium, with higher agreement when phosphorylated S6 ribosomal protein expression was dichotomized into positive/negative. The inclusion of phosphorylated S6 ribosomal protein in the diagnostic algorithm could have increased antibody-mediated rejection certainty levels by 25%. CONCLUSIONS: The study supports the role of the mTOR pathway in antibody-mediated rejection-related graft injury and suggests that tissue phosphorylation of S6 ribosomal protein could be a useful surrogate for a more accurate pathological diagnosis of lung antibody-mediated rejection.
Subject(s)
Antibodies , Ribosomal Proteins , Humans , Retrospective Studies , Lung/metabolism , Sirolimus , TOR Serine-Threonine Kinases/metabolismABSTRACT
Background: Nonalcoholic steatohepatitis (NASH) is a severe immune-mediated stage of nonalcoholic fatty liver disease that is rapidly becoming the most common etiology requiring liver transplantation (LT), with Hispanics bearing a disproportionate burden. This study aimed to uncover the underlying immune mechanisms of the disparities experienced by Hispanic patients undergoing LT for NASH. Methods: We enrolled 164 LT recipients in our institutional review board-approved study, 33 of whom presented with NASH as the primary etiology of LT (20%), with 16 self-reported as Hispanic (48%). We investigated the histopathology of prereperfusion and postreperfusion biopsies, clinical liver function tests, longitudinal soluble cytokines via 38-plex Luminex, and immune cell phenotypes generated by prereperfusion and postreperfusion blood using 14-color flow cytometry and enzyme-linked immunosorbent assay. Results: Hispanic LT recipients transplanted for NASH were disproportionately female (81%) and disproportionately suffered poor outcomes in the first year posttransplant, including rejection (26%) and death (38%). Clinically, we observed increased pro-inflammatory and apoptotic histopathological features in biopsies, increased AST/international normalized ratio early posttransplantation, and a higher incidence of presensitization to mismatched HLA antigens expressed by the donor allograft. Experimental investigations revealed that blood from female Hispanic NASH patients showed significantly increased levels of leukocyte-attracting chemokines, innate-to-adaptive switching cytokines and growth factors, HMGB1 release, and TLR4/TLR8/TLR9/NOD1 activation, and produced a pro-inflammatory, pro-apoptotic macrophage phenotype with reduced CD14/CD68/CD66a/TIM-3 and increased CD16/CD11b/HLA-DR/CD80. Conclusions: A personalized approach to reducing immunological risk factors is urgently needed for this endotype in Hispanics with NASH requiring LT, particularly in females.
ABSTRACT
BACKGROUND: Solid organ transplantation is the therapy of choice for many patients with end-stage organ failure; however, recipients must remain on lifelong immunosuppression, leaving them susceptible to infections and cancer. The study of transplant tolerance to prolong graft survival in the absence of immunosuppression has been restricted to recipients of living donor allografts; however, deceased donors significantly outnumber living donors. Mobilization of hematopoietic stem cells (HSCs) from the bone marrow to peripheral blood (PB) could allow PB-HSCs to be used to induce tolerance in deceased donor kidney recipients; however, a major concern is the well-known concomitant mobilization of immune cells into the liver. METHODS: We mobilized HSCs to the PD using a protocol of 2 doses of granulocyte colony-stimulating factor and 1 dose of plerixafor, followed by the collection of mobilized cells via apheresis in 3 deceased donors. The physiological, laboratory, and radiographic parameters were monitored throughout the procedure. Longitudinal biopsies were performed to assess the potential for ectopic liver mobilization. RESULTS: The use of both agents led to the successful mobilization of peripheral blood CD34+ cells, demonstrating the potential for use in transplant tolerance protocols. Increased immune cell trafficking into the liver was not observed, and apheresis of mobilized cells resulted in a uniform decrease in all liver leukocyte subsets. CONCLUSIONS: HSCs can be mobilized and collected from the PB of brain-dead donors. This new approach may facilitate the dissemination of immune tolerance trials beyond living-donor kidney transplantation to deceased-donor transplantation, without sacrificing the transplantability of the liver.
Subject(s)
Blood Component Removal , Hematopoietic Stem Cell Transplantation , Heterocyclic Compounds , Humans , Hematopoietic Stem Cell Mobilization/methods , Hematopoietic Stem Cells , Antigens, CD34/metabolism , Granulocyte Colony-Stimulating Factor/pharmacology , Living Donors , Hematopoietic Stem Cell Transplantation/adverse effectsABSTRACT
BACKGROUND: Per the ISHLT 2016 definition, a C4d-positive lung biopsy is required to meet criteria for definite antibody-mediated rejection (AMR). Unfortunately, C4d has poor sensitivity and specificity, and low inter-rater reliability. Phosphorylated S6 ribosomal protein (p-S6RP) expressed via the mTOR pathway has been shown to be a biomarker of AMR and correlates with donor-specific antibodies (DSA) in heart allografts. However, p-S6RP immunohistochemistry (IHC) in the setting of pulmonary AMR has yet to be evaluated. We sought to determine whether p-S6RP IHC performed on lung biopsies correlates with de novo DSA. METHODS: IHC for p-S6RP performed on 26 biopsies from lung transplant recipients with de novo HLA DSA (DSA+) and 28 biopsies from patients with no DSA (DSA-) were evaluated by 3 pathologists who independently scored the degree of alveolar macrophage and pneumocyte staining. Staining in ≥50% of the biopsy as determined by at least 2 pathologists was considered positive. RESULTS: Twenty-one (81%) DSA+ biopsies stained positive for p-S6RP in pneumocytes and 21 (81%) in macrophages. Six DSA- biopsies (21%) stained positive for p-S6RP in pneumocytes, 6 (21%) were positive in macrophages. Pneumocyte p-S6RP staining was 81% sensitive and 79% specific for DSA. Macrophage staining showed the same sensitivity and specificity but with lower inter-rater agreement (κ = 0.53 vs 0.68). CONCLUSIONS: This study demonstrates a positive relationship between de novo DSA and p-S6RP expression in pneumocytes and macrophages using IHC. p-S6RP is relatively sensitive and specific, and has superior inter-rater reliability compared to C4d.