Human Lung Transplant Recipients Research Articles

Evaluating longitudinal cytomegalovirus-specific humoral immune responses and association with DNAemia risk in seropositive lung transplant recipients

BackgroundCytomegalovirus (CMV) is the most common viral infection among lung transplant recipients and is associated with chronic lung allograft dysfunction. There is a need for better therapeutics as well as biomarkers to enable effective stratification of CMV seropositive patient risk for developing CMV DNAemia to inform prophylaxis duration. MethodsCMV-specific IgG binding and functional responses were evaluated in a discovery cohort of longitudinal plasma samples from 51 CMV seropositive human lung transplant recipients, collected as part of the CTOT-20 and CTOT-22 consortium studies. Pre-transplant plasma from an additional 43 CMV seropositive lung transplant recipients were evaluated as a validation cohort. ResultsIn the discovery cohort with longitudinal samples, pre-transplant plasma IgG binding to CMV surface glycoproteins gH/gL, gH/gL/gO, and pentameric complex (PC), as well as neutralization of CMV in epithelial cells are associated with increased risk of CMV DNAemia post-prophylaxis. However, these results were not confirmed by the validation cohort. ConclusionWhile quantification of pre-transplant CMV-specific antibody responses showed association with DNAemia in the discovery cohort, additional clinical variables and/or known risk factors for CMV, such as patient CMV-specific T-cell responses, may need to be considered in combination with humoral immunity to effectively stratify risk of CMV DNAemia.

Regadenoson Reduces Soluble Receptor for Advanced Glycation End-Products in Lung Recipients

BackgroundThe selective adenosine A2A receptor (A2AR) agonist regadenoson reduces inflammation due to lung ischemia-reperfusion injury (IRI). The objective of this study was to investigate molecular and cellular mechanisms by which regadenoson reduces IRI in lung transplant recipients. MethodsFourteen human lung transplant recipients were infused for 12 hours with regadenoson and 7 more served as untreated controls. Plasma levels of high mobility group box 1 and its soluble receptor for advanced glycation end-products (sRAGE) were measured by Luminex. Matrix metalloproteinase (MMP) 2 and 9 were measured by gelatin zymography. Tissue inhibitor of metalloproteinase 1 was measured by mass spectroscopy. A2AR expression on leukocytes was analyzed by flow cytometry. MMP-9–mediated cleavage of RAGE was evaluated using cultured macrophages in vitro. ResultsRegadenoson treatment during lung transplantation significantly reduced levels of MMP-9 (P < .05), but not MMP-2, and elevated levels of tissue inhibitor of metalloproteinase 1 (P < .05), an endogenous selective inhibitor of MMP-9. Regadenoson infusion significantly reduced plasma levels of sRAGE (P < .05) during lung reperfusion compared with control subjects. A2AR expression was highest on invariant natural killer T cells and higher on monocytes than other circulating immune cells (P < .05). The shedding of RAGE from cultured monocytes/macrophages was increased by MMP-9 stimulation and reduced by an MMP inhibitor or by A2AR agonists, regadenoson or ATL146e. ConclusionsIn vivo and in vitro studies suggest that A2AR activation reduces sRAGE in part by inhibiting MMP-9 production by monocytes/macrophages. These results suggest a novel molecular mechanism by which A2AR agonists reduce primary graft dysfunction.

The mitigating effect of exogenous carbon monoxide on chronic allograft rejection and fibrosis post-lung transplantation.

Small airway inflammation and fibrosis or bronchiolitis obliterans (BO) is the predominant presentation of chronic lung allograft dysfunction (CLAD) post-lung transplantation. Carbon monoxide (CO) is a critical endogenous signaling transducer with known anti-inflammatory and anti-fibrotic effects but its therapeutic potential in CLAD remains to be fully elucidated. Here we investigate the effect of inhaled CO in modulating chronic lung allograft rejection pathology in a murine orthotopic lung transplant model of BO (B6D2F1/J→DBA/2J). Additionally, the effects of CO on the activated phenotype of mesenchymal cells isolated from human lung transplant recipients with CLAD were studied. Murine lung allografts treated with CO (250 ppm×30 minutes twice daily from days 7 to 40 post-transplantation) demonstrated decreased immune cell infiltration, fibrosis, and airway obliteration by flow cytometry, trichrome staining, and morphometric analysis, respectively. Decreased total collagen, with levels comparable to isografts, was noted in CO-treated allografts by quantitative hydroxyproline assay. In vitro, CO (250 ppm×16h) was effective in reversing the fibrotic phenotype of human CLAD mesenchymal cells with decreased collagen I and β-catenin expression as well as an inhibitory effect on ERK1/2 MAPK, and mTORC1/2 signaling. Sildenafil, a phosphodiesterase 5 inhibitor, partially mimicked the effects of CO on CLAD mesenchymal cells and was partially effective in decreasing collagen deposition in murine allografts, suggesting the contribution of cGMP-dependent and -independent mechanisms in mediating the effect of CO. These results suggest a potential role for CO in alleviating allograft fibrosis and mitigating chronic rejection pathology post-lung transplant.

Extracellular Vesicles Mediate Immune Responses to Tissue-Associated Self-Antigens: Role in Solid Organ Transplantations.

Transplantation is a treatment option for patients diagnosed with end-stage organ diseases; however, long-term graft survival is affected by rejection of the transplanted organ by immune and nonimmune responses. Several studies have demonstrated that both acute and chronic rejection can occur after transplantation of kidney, heart, and lungs. A strong correlation has been reported between de novo synthesis of donor-specific antibodies (HLA-DSAs) and development of both acute and chronic rejection; however, some transplant recipients with chronic rejection do not have detectable HLA-DSAs. Studies of sera from such patients demonstrate that immune responses to tissue-associated antigens (TaAgs) may also play an important role in the development of chronic rejection, either alone or in combination with HLA-DSAs. The synergistic effect between HLA-DSAs and antibodies to TaAgs is being established, but the underlying mechanism is yet to be defined. We hypothesize that HLA-DSAs damage the transplanted donor organ resulting in stress and leading to the release of extracellular vesicles, which contribute to chronic rejection. These vesicles express both donor human leukocyte antigen (HLA) and non-HLA TaAgs, which can activate antigen-presenting cells and lead to immune responses and development of antibodies to both donor HLA and non-HLA tissue-associated Ags. Extracellular vesicles (EVs) are released by cells under many circumstances due to both physiological and pathological conditions. Primarily employing clinical specimens obtained from human lung transplant recipients undergoing acute or chronic rejection, our group has demonstrated that circulating extracellular vesicles display both mismatched donor HLA molecules and lung-associated Ags (collagen-V and K-alpha 1 tubulin). This review focuses on recent studies demonstrating an important role of antibodies to tissue-associated Ags in the rejection of transplanted organs, particularly chronic rejection. We will also discuss the important role of extracellular vesicles released from transplanted organs in cross-talk between alloimmunity and autoimmunity to tissue-associated Ags after solid organ transplantation.

A decline in club cell secretory proteins in lung transplantation is associated with release of natural killer cells exosomes leading to chronic rejection

In human lung transplant recipients, a decline in club cell secretory protein (CCSP) in bronchoalveolar lavage fluid has been associated with chronic lung allograft dysfunction (CLAD) as well as the induction of exosomes and immune responses that lead to CLAD. However, the mechanisms by which CCSP decline contributes to CLAD remain unknown. To define the mechanisms leading to CCSP decline and chronic rejection, we employed two mouse models: 1) chronic rejection after orthotopic single lung transplantation and 2) anti-major histocompatibility complex (MHC) class I-induced obliterative airway disease. In the chronic rejection mouse model, we detected circulating exosomes with donor MHC (H2b) and lung self-antigens and also development of antibodies to H2b and lung self-antigens and then a decline in CCSP. Furthermore, DBA2 mice that received injections of these exosomes developed antibodies to donor MHC and lung self-antigens. In the chronic rejection mouse model, natural killer (NK) and CD8 T cells were the predominant graft-infiltrating cells on day 14 of rejection followed by exosomes containing NK cell-associated and cytotoxic molecules on day 14 and 28. When NK cells were depleted, exosomes with NK cell-associated and cytotoxic molecules as well as fibrosis decreased. Induction of exosomes led to immune responses to donor MHC and lung self-antigens, resulting in CCSP decline, leading to NK cell infiltration and release of exosomes from NK cells. These results suggest a novel role for exosomes derived from NK cells in the pathogenesis of chronic lung allograft rejection.

The role of miRNA-155 in the immunopathogenesis of obliterative airway disease in mice induced by circulating exosomes from human lung transplant recipients with chronic lung allograft dysfunction

Human lung transplant recipients undergoing rejection induce circulatory exosomes with lung self-antigens (SAgs), K-alpha 1 Tubulin and Collagen V, and immunization of C57BL/6 mice with exosomes induced obliterative airway disease (HEI-OAD). We analyzed whether exosomes with SAgs induced immunity in microRNA-155 knockout mice (miR-155KO), as microRNA-155 is an immune regulator. C57BL/6 and miR-155KO were immunized with exosomes from stable or chronic rejection (bronchiolitis obliterans syndrome (BOS) and on day 30, induction of exosomes, antibodies (Abs) to SAgs and cellular immunity were determined. C57BL/6 immunized with exosomes from BOS developed OAD. These immunized animals also developed Abs to SAgs and increased frequency of SAg-specific IFNγ and IL17- producing cells. In contrast, Abs to SAgs did not develop in miR-155KO and there was reduction in frequency of cells producing IL10. Upregulation of suppressor of cytokine signaling for lung inflammation was also noted resulting in abrogation of induction of exosomes with SAgs OAD.

Circulating exosomes from human lung transplant recipients with respiratory viral infections contain nucleic acids potential to activate innate immune signaling (cGAS/STING and RIG-1 Pathways)

Abstract Exosomes are 50–200nm size vesicles released by cells into the body fluids. We reported the presence of circulating exosomes with lung self-antigens (SAgs) (Collagen-V,K-α Tubulin) and donor HLA in lung transplant recipients (LTxRs) undergoing rejection. Respiratory viral infections (RVI) is a known risk factor for development of chronic lung allograft dysfunction (CLAD) post lung transplant. We postulated immune mechanism by which RVI increases risk for CLAD is by induction of exosomes with SAgs containing viral DNA/RNA capable of activating innate immune signaling via DNA/RNA sensing. Exosomes were isolated using ultracentrifugation. Exosomal DNA and RNA were used to generate libraries using Kapa Biosystem’s kit. Illumina 2×150bp pair-end reads were checked on FastQC, aligned to human/viral genome from CHIP seeker Database. Role of exosomes inducing cell signaling in human airway epithelial cells (KCC266) and Hep2 were analyzed by incubating exosomes from LTxRs with RVI or stable. Viral nucleic acid sequences were higher in exosomes from LTxRs with RVI while comparison with human genome identified the presence of DNA sequences specific to defensins, GTPase, apoptotic cleavage, and NMDA receptor in exosomes from RVI. We observed upregulation of proteins associated with cGAS/STING and RIG1 in KCC266 and Hep2 cells (STING; 1.5, cGAS; 1.9, pIRF3; 2.5, pTBK; 3.1, MAVS; 1.7 and IFNβ; 3.8 fold respectively) incubated with exosomes from LTxRs with RVI. We conclude that LTxRs with RVI leads to induction of circulating exosomes having unique viral nucleic acid sequences capable of inducing signaling. This can lead to activation of innate immune signaling resulting in adaptive immune responses to viral and donor antigens resulting in CLAD.

Circulating Exosomes from Human Lung Transplant Recipients with Rejection Induces IL-6 by Rab27A Signaling and Promotes Epithelial Mesenchymal Transition of Airway Epithelial Cells In Vitro

Exosomes are small membrane vesicles that regulate intracellular function and cell-to-cell communication. Our report has shown circulating exosomes are induced during lung allograft rejection (Am J Transplant, 2017; 17:474-484). Rab27, a member of the Rab subfamily of GTPases, is essential for exosome secretion. The aim of this study is to determine the role of Rab27A signaling in human bronchial epithelial cells (BEAS-2B) stimulated with exosomes from human lung transplant recipients (LTxRs) diagnosed with acute rejection, bronchiolitis obliterans syndrome (BOS), or stable. Exosomes were isolated using ultracentrifugation and 100µg of exosomes were added to BEAS-2B and incubated for 24hrs. Supernatants collected were analyzed for IL-6 using luminex platform. Epithelial mesenchymal transformation was determined using vimentin and α-SMA by Western blot. Protein band in western blot was normalized with β-Actin and densitometry was performed using Image-J software. Supernatants of BEAS-2B cells, stimulated with BOS-exosome, showed significantly higher production of IL-6 (2927, 4592 MFI p=<0.01) as compared to stable-exosome. Rab27A siRNA knockdown decreased the production of IL-6 (2998 MFI vs 1307 MFI p=<0.01) in supernatants. Further, Rab27A knockdown significantly reduced fibronectin protein in BOS (50% reduction) exosome treated BEAS-2B cells compared to stable (p=<0.01). Vimentin and α-SMA were significantly higher (p=<0.01) in BOS-exosome treated BEAS-2B cells compared to stable-exosome. Rab27A signaling plays an important role in the production of pro-inflammatory cytokine IL-6 by airway epithelial cells following incubation with exosomes isolated from LTxRs with rejection. In addition, Rab27A signaling plays a role in exosome mediated upregulation of fibronectin protein production resulting in epithelia mesenchymal transition of the airway epithelial cells.

Circulating Exosomes from Human Lung Transplant Recipients Having Respiratory Viral Infections Contain Nucleic Acids and Activate Signaling Pathways CGAS/STING and RIG-1

PurposeExosomes are membrane bound vesicles are released by cells into body fluids. Our laboratory demonstrated the presence of circulating exosomes with lung self-antigens (Collagen-V and K-α Tubulin) and donor HLA in lung transplant recipients (LTxRs) undergoing rejection. Since respiratory viral infections (RVI) is a risk factor for development of chronic lung allograft dysfunction (CLAD) post lung transplant, we postulated that RVI can lead to induction of exosomes with self-antigens containing viral DNA/RNA capable of activating innate immune signaling via cGAS/STING and RIG1 pathways, a mechanism leading to immune activation resulting in CLAD.MethodsExosomes were isolated using ultracentrifugation. Size (50-200nm) was determined using nanosight. DNA and RNA were isolated using kits and quantified on the Nanodrop. Libraries were generated using Kapa Biosystem's library kit. The raw Illumina 2x150bp pair-end reads were checked on FastQC and were aligned to the human and viral genome build from CHIPseeker Database. Validation was done using antibodies and primers for respiratory syncytial virus, coronavirus, and rhinovirus. To determine the role of exosomes to induce cell signaling and endoplasmic stress (ER), airway epithelial cells (KCC266), and Hep2 cells were incubated with exosomes from LTxRs with RVI or stable.ResultsViral nucleic acid sequences were noted in higher levels in exosomes from LTxRs with RVI in comparison to stable. Comparison with human genome identified the presence of DNA sequences specific to defensins, GTPase, apoptotic cleavage, and NMDA receptor in RVI LTxRs. Further, we identified upregulation of proteins associated with cGAS/STING and RIG1 (MAVS, MDA5, IFNβ) and ER stress (PERK, ATF4 and BiP) in KCC266 and Hep2 cells incubated with exosomes from LTxRs with RVI, but not stable. In contrast, exosomes from stable LTxRs had 91 sequences for MAP kinase and cell death signaling pathways.ConclusionWe conclude that LTxRs diagnosed with RVI leads to induction of circulating exosomes having unique viral nucleic acid sequences capable of inducing signaling and ER stress. This can lead to activate innate immune signaling via cGAS/STING and RIG1 pathways resulting in immune responses to viral and donor antigens resulting in CLAD.

Proteomics Analysis of Circulating Extracellular Vesicles Isolated from Plasma of Human Lung Transplant Recipients

We demonstrated induction of circulating exosomes in lung transplant recipients (LTxRs) with acute/chronic rejection (bronchiolitis obliterans syndrome [BOS]) containing donor human leukocyte antigen and lung self-antigens (SAgs) (Kα-1 Tubulin, Collagen V), costimulatory molecules (CD80, CD86), transcription factors (NFkB, 20S proteasome) (Am J Transplant 17(2):474-484, 2017). In the current study, we identified unique proteomic signatures in circulating extracellular vesicles (EVs) that can differentiate human LTxRs with acute rejection (AR), BOS, respiratory viral infection (RVI), and stable. EVs were isolated using ultracentrifugation from LTxRs plasma (stable, AR, BOS or RVI) following transplant. EVs were characterized for the presence of lung SAgs (Kα-1 Tubulin, Collagen V) and size (50-200nm) was determined using NanoSight. EV protein cargoes were prepared for shotgun proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified unique proteins (2 AR, 24 BOS, 4 RVI, 8 stable cohort) using LC-MS/MS. Differential analysis of the proteins from AR, BOS and RVI to stable yielded significant unique proteins (p-value <.05, fold-change >2) in each clinical cohort (27 AR, 2 BOS, 2 RVI). EVs from AR contained proteins involved in immunoglobulin, complement regulation, innate and adaptive immune response pathways. EVs from BOS revealed enrichment of immunoglobulin receptors and carboxypeptidase N catalytic chain. EVs from RVI were enriched for macrophage stimulating factor. Our results demonstrate unique signatures in acute/chronic rejection and RVI highlighting complex immune mechanisms underlying lung allograft rejection. Presence of complement proteins and immunoglobulins in circulating EVs from AR can be helpful in differentiating cellular rejection with a mixed phenotype (cellular and humoral). We identified immunoglobulins and complement proteins in BOS in support of significance of Abs in the pathogenesis of BOS. Macrophage stimulating factor contained in EVs specific to RVI can be useful for the differential diagnosis of RVI from AR. To conclude, proteomic signatures of circulating EVs from LTxRs provided insights into immunological mechanisms at play in graft rejection and RVI leading to increased risk for chronic lung allograft dysfunction.

Mitochondrial DNA Stimulates TLR9-Dependent Neutrophil Extracellular Trap Formation in Primary Graft Dysfunction.

The immune system is designed to robustly respond to pathogenic stimuli but to be tolerant to endogenous ligands to not trigger autoimmunity. Here, we studied an endogenous damage-associated molecular pattern, mitochondrial DNA (mtDNA), during primary graft dysfunction (PGD) after lung transplantation. We hypothesized that cell-free mtDNA released during lung ischemia-reperfusion triggers neutrophil extracellular trap (NET) formation via TLR9 signaling. We found that mtDNA increases in the BAL fluid of experimental PGD (prolonged cold ischemia followed by orthotopic lung transplantation) and not in control transplants with minimal warm ischemia. The adoptive transfer of mtDNA into the minimal warm ischemia graft immediately before lung anastomosis induces NET formation and lung injury. TLR9 deficiency in neutrophils prevents mtDNA-induced NETs, and TLR9 deficiency in either the lung donor or recipient decreases NET formation and lung injury in the PGD model. Compared with human lung transplant recipients without PGD, severe PGD was associated with high levels of BAL mtDNA and NETs, with evidence of relative deficiency in DNaseI. We conclude that mtDNA released during lung ischemia-reperfusion triggers TLR9-dependent NET formation and drives lung injury. In PGD, DNaseI therapy has a potential dual benefit of neutralizing a major NET trigger (mtDNA) in addition to dismantling pathogenic NETs.

Circulating Exosomes from Lung Transplant Recipients Diagnosed with Bronchiolitis Obliterans Syndrome Induces Immune Responses in C57BL/6 Mice Leading to Obliterative Airway Disease and miRNA 155 Plays an Essential Role in Immune Activation

Purpose MicroRNAs (miRNAs) are single-strand RNAs that are 18-25 nucleotides in length. Genes of miRNAs suppress newly synthesized proteins by causing miRNA degradation or inhibiting miRNA translation into proteins. MicroRNA-155 (miR155) has also been implicated as a positive regulator of inflammatory cytokine production especially IL17. miR155 is a pivotal regulator of the immune system and we determined the role of miR155 in immune activation of C57BL/6 and miR155 knock out (KO) mice leading to development of Abs and induction of exosomes with lung self-antigens (SAgs) following immunization with circulating exosomes isolated from human lung transplant recipients (LTxRs). Methods C57BL/6 and miR155 KO mice were immunized with exosomes isolated from stable and bronchiolitis obliterans syndrome (BOS) without Freund's adjuvant (20ug/injection, at 1,6,15 and 21 days). After last injection, mice were sacrificed, lungs were analyzed by H&E and trichrome staining. Blood samples collected at days 2,7,16 and 26, were analyzed for the development of Abs to K-alpha 1 Tubulin (KA1T), Collagen V (Col-V) and Col-I (control) by ELISA. Results Cellular immune responses to KA1T, Col-V, and Col-I were enumerated using ELISPOT. Circulating exosomes were isolated and analyzed for lung SAgs by western blot. Our results demonstrate development of Abs to lung SAgs with increased frequencies of IFN-γ and IL-17-producing cells with reduction of IL-10 producing cells (stable vs BOS) against lung-SAgs-following immunization of wild type C57BL/6 animals with BOS exosomes but not exosomes from stable LTxRs. Histological analysis of lungs demonstrated obliterative airway disease (OAD) following immunization with exosomes isolated from BOS but not stable. In contrast, immunization of miR155 KO mice did not result in Abs to lung SAgs, cellular immune response to lung SAgs, nor induction of circulating exosomes with lung SAgs (control vs stable and BOS). These animals also did not develop OAD lesions in the lungs. Conclusion We conclude that circulating exosomes plays a pivotal role in immune activation leading to chronic rejection and miR155 exerts an obligatory role in the pathogenesis of chronic rejection following human lung transplantation.

Prolongation of allograft survival by passenger donor regulatory T cells.

Tissue resident lymphocytes are present within many organs, and are presumably transferred at transplantation, but their impact on host immunity is unclear. Here, we examine whether transferred donor natural regulatory CD4 T cells (nT‐regs) inhibit host alloimmunity and prolong allograft survival. Transfer of donor‐strain lymphocytes was first assessed by identifying circulating donor‐derived CD4 T cells in 21 consecutive human lung transplant recipients, with 3 patterns of chimerism apparent: transient, intermediate, and persistent (detectable for up to 6 weeks, 6 months, and beyond 1 year, respectively). The potential for transfer of donor nT‐regs was then confirmed by analysis of leukocyte filters recovered from ex vivo normothermic perfusion circuits of human kidneys retrieved for transplantation. Finally, in a murine model of cardiac allograft vasculopathy, depletion of donor CD4 nT‐regs before organ recovery resulted in markedly accelerated heart allograft rejection and augmented host effector antibody responses. Conversely, adoptive transfer or purified donor‐strain nT‐regs inhibited host humoral immunity and prolonged allograft survival, and more effectively so than following administration of recipient nT‐regs. In summary, following transplantation, passenger donor‐strain nT‐regs can inhibit host adaptive immune responses and prolong allograft survival. Isolated donor‐derived nT‐regs may hold potential as a cellular therapy to improve transplant outcomes.

Spectrum of chronic lung allograft pathology in a mouse minor-mismatched orthotopic lung transplant model.

Chronic lung allograft dysfunction (CLAD) is a fatal condition that limits survival after lung transplantation (LTx). The pathological hallmark of CLAD is obliterative bronchiolitis (OB). A subset of patients present with a more aggressive CLAD phenotype, called restrictive allograft syndrome (RAS), characterized by lung parenchymal fibrosis (PF). The mouse orthotopic single LTx model has proven relevant to the mechanistic study of allograft injury. The minor-alloantigen-mismatched strain combination using C57BL/10(B10) donors and C57BL/6(B6) recipients reportedly leads to OB. Recognizing that OB severity is a spectrum that may coexist with other pathologies, including PF, we aimed to characterize and quantify pathologic features of CLAD in this model. Left LTx was performed in the following combinations: B10→B6, B6→B10, B6→B6. Four weeks posttransplant, blinded pathologic semi-quantitative assessment showed that OB was present in 66% of B10→B6 and 30% of B6→B10 grafts. Most mice with OB also had PF with a pattern of pleuroparenchymal fibroelastosis, reminiscent of human RAS-related pathology. Grading of pathologic changes demonstrated variable severity of airway fibrosis, PF, acute rejection, vascular fibrosis, and epithelial changes, similar to those seen in human CLAD. These assessments can make the murine LTx model a more useful tool for further mechanistic studies of CLAD pathogenesis.

Identification of extracellular vesicle proteins in circulating exosomes from human lung transplant recipients

Abstract Molecular mechanisms involved in rejection following human lung transplantation (LTx) are not well understood. We identified proteomic signatures of clinical outcomes in circulating extracellular vesicles (EVs) isolated from human lung transplant recipients (LTxRs) who was diagnosed with chronic rejection (Bronchiolitis Obliterans Syndrome (BOS)), acute rejection (AR), respiratory viral infection requiring intervention (RVI) or stable following transplantation. Differential analysis revealed one protein unique to AR (Skin-specific protein 32), four unique to RVI (Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2, Transmembrane 9 superfamily member 2, Ras-related C3 botulinum toxin substrate 2, and EH domain-containing protein) and two unique to stable (Coagulation factor X, and N-acetylmuramoyl-L-alanine amidase). Comparison of each rejection group to stable identified 128, 180, and 216 significantly differentially expressed proteins (p-value&amp;lt;0.05, fold-change&amp;gt;2) in BOS, AR, and RVI respectively. Although no unique signatures were found in BOS, an increased enrichment of immune processes such as antigen processing and presentation of exogenous peptide antigen via MHC class I and Fc receptor signaling pathway were observed. Functional enrichment analysis (Gene Ontology) associated differentially expressed EV proteins in AR and RVI conditions with wound repair processes. These diverse signatures detected in each condition highlight complex immune mechanisms underlying the pathophysiology of rejection following LTx. Future studies are needed to validate these signatures in larger LTxR cohorts in order to promote early clinical diagnosis and predict outcome.

Role of exosomes in rejection following human lung transplantation – an in vitro analysis using human airway epithelial cells

Abstract Studies have demonstrated that exosomes expressing donor human leukocyte antigen (HLA) and lung self-antigens (SAg), Collagen V (Col-V) and Kα1 Tubulin (Kα1T), can be detected in the circulation of human lung transplant recipients (LTxR) who developed antibodies (Ab) to donor HLA and diagnosed with rejection However, the mechanisms by which Ab mediate induction of exosomes into the circulation from airway epithelial cells (AEC) remains unclear. Ab ligation to HLA could enhance PERK-mediated pathways involved in endoplasmic reticular (ER) stress-dependent exosome release. The goal of this study is to determine the mechanism by which Ab induce exosomes from AEC. Towards this we employed human AEC, ligated antigens by Ab specific to HLA (HiPRA) and lung SAg (rabbit anti-Col-V or Kα1T) and the role of ER stress signaling molecule in exosomes release were determined. Effect of exosome inhibition by GW4869 was also analyzed. AEC were incubated with HiPRA or Ab to SAg for 24hr in both normoxia and hypoxia (0.1% O2) conditions. AEC incubated with HiPRA or Ab to SAg demonstrated induction of exosomes containing lung SAg, Col-V and Kα1T. Incubation with HiPRA and Ab (20μg/ml) to SAg also demonstrated induction of exosomes containing lung SAg, MHC class II, CD80, CD86. Treatment with GW4869, exosome inhibitor (20μM) reduced the exosomes and SAg. Effect of HiPRA and Ab to SAg on stress kinase in both normoxic and hypoxic conditions and influence of GW4869 in these pathways will be delineated. Identification of stress kinase and exosome inhibition will provide novel results for Ab mediated upregulation of stress leading to exosome release from the transplanted organ resulting in immune responses thus increasing the risk for chronic rejection following LTx.

Magnetisation transfer as a biomarker for chronic airway fibrosis in a mouse lung transplantation model

BackgroundChronic airway fibrosis (CAF) is the most prevalent complication in human lung transplant recipients. The aim of the study is to evaluate magnetisation transfer (MT) as a biomarker of developing CAF of lung transplants in a mouse model.MethodsLung transplantation was performed in 48 mice, applying major or minor histocompatibility mismatches between strains for the induction of CAF. MT measurements were performed in vivo with systematic variation of off-resonance frequencies and flip angle of the MT prepulse. MT ratios (MTRs) were compared for lungs showing CAF and without CAF.ResultsSeven out of 24 animals (29%) showed a pattern of CAF at histology. All mice developing CAF also showed signs of acute rejection, whereas none of the lungs showed signs of other post-transplant complications. After lung transplantation, pulmonary infiltration was a frequent finding (14 out of 24) exhibiting a higher MTR (24.8% ± 4.5%) compared to well-ventilated lungs (12.3% ± 6.9%, p = 0.001) at 8000 Hz off-resonance frequency, 3000° flip angle. In infiltrated lung tissue exhibiting CAF, lower MTR values (21.8% ± 5.7%) were found compared to infiltrated lungs showing signs of acute rejection alone (26.5% ± 2.9%, p = 0.028), at 8000 Hz, 3000° flip angle. The highest MTR values were observed at 3000° flip angle, using a 1000 Hz off-resonance frequency.ConclusionMTR might serve as a tool for the detection of CAF in infiltrated lung tissue.

Chronic Airway Fibrosis in Orthotopic Mouse Lung Transplantation Models-An Experimental Reappraisal.

Several mouse lung transplantation (Tx) models have been proposed for the study of chronic airway fibrosis (CAF), the most prevalent complication seen in human lung transplant recipients, termed chronic lung allograft dysfunction. Alternatively, it has been called for to establish an experimental animal model for restrictive allograft syndrome, another phenotype of chronic lung allograft dysfunction. However, these mouse transplant models exhibit significant heterogeneity in consistency and reproducibility. We therefore aimed at reevaluating current available models. Four different Tx combinations were used that manifest CAF: 2 minor antigen-mismatched Tx combinations (MINOR, donor: C57BL/10, recipient: C57BL/6J); or MINOR-N using recipient C57BL/6N, major histocompatibility antigen-mismatched immunosuppressed Tx (MAJOR, donor: BALB/c, recipient: C57BL/6J), and syngeneic Tx (donor and recipient: C57BL/6J) as control. The recipients were harvested and analyzed at week 8. Oxygenation, histology, reverse transcription polymerase chain reaction, and magnetic resonance imaging were performed to analyze outcome of those models. The most prominent manifestation of CAF, thickest subepithelial fibrotic changes, worst oxygenation, and the most severe acute rejection were detected in the MAJOR group compared with all other (P < 0.05). Gene expressions of TNF-α and TGF-β1 were higher, and IL-10 was lower in the MAJOR group. Immunohistochemistry found pleuroparenchymal fibrotic change in both the MAJOR and MINOR-J groups. We propose the major mismatch model under mild immunosuppression as the most suitable model for studying posttransplant CAF, and both the major and minor mismatch models for the restrictive phenotype.

(218) - Down Regulation of microRNA-21 Is Significantly Associated with Signaling and Activation of the Toll-Like Receptor in Human Lung Transplant Recipients with Primary Graft Dysfunction

(218) - Down Regulation of microRNA-21 Is Significantly Associated with Signaling and Activation of the Toll-Like Receptor in Human Lung Transplant Recipients with Primary Graft Dysfunction

(407) - Mitochondria Release Leads from Human Lung Transplant Recipients Promotes Primary Graft Dysfunction

(407) - Mitochondria Release Leads from Human Lung Transplant Recipients Promotes Primary Graft Dysfunction