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Open Access Publications from the University of California

Scholarly Works (8 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2023)

Solid organ transplantation is a lifesaving therapy for patients with end-stage organ failure. Although modern immunosuppressive strategies have allowed organ survival one-year post-transplants, chronic allograft rejection remains a main clinical challenge. Approximately 20% of recipients lose their graft within 5 years, and 50% within 10 years. Antibody-mediated rejection (AMR) described by the recipient production of donor-specific antibodies (DSA) remains the main risk factor contributing to late graft loss. DSA target human leukocyte antigen (HLA) class I and class II molecules present of the donor graft endothelium. DSA triggers strong alloimmune responses against allograft endothelial cells (ECs) leading to vascular injury and inflammation. Chronic inflammation induces vascular remodeling processes consisting of EC, smooth muscle cell (SMC), and myofibroblast proliferation leading to thickening of the intimal layer of vessels (neointima). This process ultimately leads to vessel occlusion, a condition termed transplant vasculopathy (TV) and specifically in heart transplant, cardiac allograft vasculopathy (CAV). Although macrophages remain a distinguishing feature of graft pathology in both AMR and CAV lesions, their precise phenotype and function in the context of HLA class I DSA remain poorly understood. Furthermore, there is a lack of research exploring the vascular characteristics of CAV-affected vessels in patients with DSA.In this dissertation, we utilized both in vitro and in vivo approaches to investigate the mechanisms by which HLA class I DSA activate EC signaling and the impact on monocyte-to-macrophage polarization and functions. Our findings revealed that crosslinking of anti-HLA I (IgG and F(ab')2) antibodies with HLA class I molecules (HLA I) forms a complex with TLR4. Signaling through TLR4 and the adaptor protein MyD88 triggers the release of Weibel-Palade bodies (WPbs) containing P-selectin. P-selectin surface expression mediates the capture of monocytes to ECs via interactions with monocyte P-selectin glycoprotein ligand-1 (PSGL-1). Second, we identified that anti-HLA I antibodies (IgG and F(ab')2) antibody-activated ECs induced the polarization of M2-like macrophages with distinct cytokine/chemokine secretion and transcriptomic expression. We further delineated that M2-macrophage polarization was enhanced via Fc-gamma receptor (FcR) interactions. For example, monocytes co-cultured with HLA I IgG-stimulated ECs differentiated into CD68+CD206+CD163+ macrophages, while monocytes co-cultured with HLA I F(ab’)2 only upregulated CD206. Thirdly, by inhibiting TLR4 signaling and PSGL-1-P-selectin interaction during monocyte transmigration across HLA I (IgG or F(ab’)2) antibody-activated ECs, we discovered that macrophage expression of CD206 and the secretion of matrix metalloproteinase-9 (MMP9) is regulated by P-selectin. We validated the expression of CD68+CD206+CD163+MMP9+ M2-like macrophages within CAV affected lesions of DSA+ rejected cardiac explants. Finally, using innovative spatial multi-omics techniques, we examined protein and transcriptomic expression in arterial regions of DSA+ CAV+ human rejected cardiac allografts. Our analysis revealed distinct profiles in regions with low and high neointima, encompassing differentially expressed proteins, transcripts, gene modules, and gene regulatory networks. Notably, low neointimal regions exhibited elevated inflammatory profiles, while high neointima regions demonstrated features associated with fibrotic and remodeling processes. These results suggested a speculative sequential time-frame of the arterial changes that may occur during CAV progression. This dissertation provides valuable insights into the immunological mechanisms driving AMR and CAV progression. The study highlights the role of HLA I DSA-activated ECs in regulating macrophage functions and uncovers molecular signatures of different arterial regions from DSA+ CAV+ rejected cardiac allografts. These findings offer a deeper understanding of AMR and CAV pathogenesis, suggesting potential therapeutic targets to prevent leukocyte infiltration and EC activation in rejecting grafts. Finally, this research contributes to the development of interventions for improved long-term graft survival in transplantation.

    Cover page: Investigating Macrophage Phenotype and Arterial Heterogeneity in Antibody-Mediated Rejection and Cardiac Allograft Vasculopathy
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2022)
    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 β4 to induce mTOR-dependent firm monocyte adhesion via ICAM-1 clustering on ECs, two processes implicated in Ab-mediated rejection.
      Cover page: Cross-Talk between HLA Class I and TLR4 Mediates P-Selectin Surface Expression and Monocyte Capture to Human Endothelial Cells.
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2024)
      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 TGFβ-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.
        Cover page: Spatial multiomics of arterial regions from cardiac allograft vasculopathy rejected grafts reveal novel insights into the pathogenesis of chronic antibody-mediated rejection.
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2023)
        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.
          Cover page: Immune Features of Disparate Liver Transplant Outcomes in Female Hispanics With Nonalcoholic Steatohepatitis.
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2022)
          Cardiac allograft vasculopathy (CAV) limits the long-term success of heart transplants. Generation of donor-specific antibodies (DSAs) is associated with increased incidence of CAV clinically, but mechanisms underlying development of this pathology remain poorly understood. Major histocompatibility complex-mismatched A/J cardiac allografts in B6.CCR5-/- recipients have been reported to undergo acute rejection with little T-cell infiltration, but intense deposition of C4d in large vessels and capillaries of the graft accompanied by high titers of DSA. This model was modified to investigate mechanisms of antibody-mediated CAV by transplanting A/J hearts to B6.CCR5-/- CD8-/- mice that were treated with low doses of anti-CD4 monoclonal antibody to decrease T-cell-mediated graft injury and promote antibody-mediated injury. Although the mild inhibition of CD4 T cells extended allograft survival, the grafts developed CAV with intense C4d deposition and macrophage infiltration by 14 days after transplantation. Development of CAV correlated with recipient DSA titers. Transcriptomic analysis of microdissected allograft arteries identified the Notch ligand Dll4 as the most elevated transcript in CAV, associated with high versus low titers of DSA. More importantly, these analyses revealed a differential expression of transcripts in the CAV lesions compared with the matched apical tissue that lacks large arteries. In conclusion, these findings report a novel model of antibody-mediated CAV with the potential to facilitate further understanding of the molecular mechanisms promoting development of CAV.
            Cover page: Molecular Signature of Antibody-Mediated Chronic Vasculopathy in Heart Allografts in a Novel Mouse Model
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2020)
            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.
              Cover page: Antibody‐induced vascular inflammation skews infiltrating macrophages to a novel remodeling phenotype in a model of transplant rejection
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2023)
              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.
                Cover page: Disulfide-HMGB1 signals through TLR4 and TLR9 to induce inflammatory macrophages capable of innate-adaptive crosstalk in human liver transplantation.
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2021)

                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.
                  Cover page: Disulfide High‐Mobility Group Box 1 Drives Ischemia‐Reperfusion Injury in Human Liver Transplantation
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