BET-bromodomain and EZH2 inhibitor-treated chronic GVHD mice have blunted germinal centers with distinct transcriptomes.

Despite advances in the field, chronic graft-versus-host-disease (cGVHD) remains a leading cause of morbidity and mortality following allogenic hematopoietic stem cell transplant. Because treatment options remain limited, we tested efficacy of anticancer, chromatin-modifying enzyme inhibitors in a clinically relevant murine model of cGVHD with bronchiolitis obliterans (BO). We observed that the novel enhancer of zeste homolog 2 (EZH2) inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 each improved pulmonary function; impaired the germinal center (GC) reaction, a prerequisite in cGVHD/BO pathogenesis; and JQ5 reduced EZH2-mediated H3K27me3 in donor T cells. Using conditional EZH2 knockout donor cells, we demonstrated that EZH2 is obligatory for the initiation of cGVHD/BO. In a sclerodermatous cGVHD model, JQ5 reduced the severity of cutaneous lesions. To determine how the 2 drugs could lead to the same physiological improvements while targeting unique epigenetic processes, we analyzed the transcriptomes of splenic GCB cells (GCBs) from transplanted mice treated with either drug. Multiple inflammatory and signaling pathways enriched in cGVHD/BO GCBs were reduced by each drug. GCBs from JQ5- but not JQ1-treated mice were enriched for proproliferative pathways also seen in GCBs from bone marrow-only transplanted mice, likely reflecting their underlying biology in the unperturbed state. In conjunction with in vivo data, these insights led us to conclude that epigenetic targeting of the GC is a viable clinical approach for the treatment of cGVHD, and that the EZH2 inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 demonstrated clinical potential for EZH2i and BETi in patients with cGVHD/BO.

Inhibition of Vascular Endothelial Growth Factor Receptors 1 and 2 Attenuates Natural Killer Cell and Innate Immune Responses in an Experimental Model for Obliterative Bronchiolitis.

Obliterative bronchiolitis (OB) after lung transplantation is a nonreversible, life-threatening complication. Herein, the role of vascular endothelial growth factor receptor (Vegfr)-1 and -2 was investigated in the development of obliterative airway disease (OAD), an experimental model for OB. The nonimmunosuppressed recipients underwent transplantation with fully major histocompatibility complex mismatched heterotopic tracheal allografts and received Vegfr1 and -2-specific monoclonal antibodies either alone or in combination, or rat IgG as a control. The treatment with Vegfr1- or -2-blocking antibody significantly decreased intragraft mRNA expression of natural killer cell activation markers early after transplantation. This was followed by reduced infiltration of Cd11b+ cells and Cd4+ T cells as well as down-regulated mRNA expression of proinflammatory chemokines and profibrotic growth factors. However, blocking of both Vegfr1 and -2 was necessary to reduce luminal occlusion. Furthermore, concomitant inhibition of the calcineurin activation pathway almost totally abolished the development of OAD. This study proposes that blocking of Vegf receptors blunted natural killer cell and innate immune responses early after transplantation and attenuated the development of OAD. The results of this study suggest that further studies on the role of Vegfr1 and -2 blocking in development of obliterative airway lesions might be rewarding.

Caspase-1 and interleukin-18 in children with post infectious bronchiolitis obliterans: a case-control study.

The exact immunological mechanisms of post infectious bronchiolitis obliterans (PIBO) in childhood are not fully known. It has been shown that the inflammasome and IL-18 pathway play important roles in the pathogenesis of lung fibrosis. We aimed to investigate the role of caspase-1, IL-18, and IL-18 components in PIBO. From January to May 2020, children with PIBO, children with history of influenza infection without PIBO, and healthy children were asked to participate in the study in three pediatric pulmonology centers. Serum caspase-1, IL-18, IL-18BP, IL-18R, and INF-γ levels were measured by ELISA and compared between the 3 groups. There were 21 children in the PIBO group, 16 children in the influenza group, and 39 children in the healthy control group. No differences in terms of age and gender between the 3 groups were found. IL-18 and IL-18BP levels were higher in the healthy control group (p = 0.018, p = 0.005, respectively). IL-18R was higher in the PIBO group (p = 0.001) and caspase-1 was higher in the PIBO and influenza group than the healthy control group (p = 0.002). IFN-γ levels did not differ between the 3 groups. IL-18BP/IL-18 was higher in the influenza group than the PIBO group and the healthy control group (p = 0.003). CONCLUSIONS: Caspase-1 level was increased in patients with PIBO which suggests that inflammasome activation may have a role in fibrosis; however, IL-18 level was found to be low. Mediators other than IL-18 may be involved in the inflammatory pathway in PIBO. Further immunological studies investigating inflammasome pathway are needed for PIBO with chronic inflammation. WHAT IS KNOWN: • Post infectious bronchiolitis obliterans (PIBO) is a rare, severe chronic lung disease during childhood which is associated with inflammation and fibrosis which lead to partial or complete luminal obstruction especially in small airways. • The exact immunological mechanisms of PIBO in childhood are not fully known. WHAT IS NEW: • Inflammasome activation persists even years after acute infection and may play a role in fibrosis in PIBO. • Mediators other than IL-18 may be involved in these inflammatory pathway.

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.

MicroRNA-17-92 is required for T-cell and B-cell pathogenicity in chronic graft-versus-host disease in mice.

Chronic graft-versus-host disease (cGVHD) is characterized as autoimmune-like fibrosis and antibody production mediated by pathogenic T cells and B cells. MicroRNA-17-92 (miR-17-92) influences the survival, differentiation, and function of lymphocytes in cancer, infections, and autoimmunity. To determine whether miR-17-92 regulates T- and B-cell responses in cGVHD, we generated mice conditionally deficient for miR-17-92 in T cells, B cells, or both. Using murine models of allogeneic bone marrow transplantation, we demonstrate that expression of miR-17-92 in donor T and B cells is essential for the induction of both scleroderma and bronchiolitis obliterans in cGVHD. Mechanistically, miR-17-92 expressed in T cells not only enhances the differentiation of pathogenic T helper 1 (Th1) and Th17 cells, but also promotes the generation of follicular Th cells, germinal center (GC) B cells, and plasma cells. In B cells, miR-17-92 expression is required for autoantibody production and immunoglobulin G deposition in the skin. Furthermore, we evaluated a translational approach using antagomirs specific for either miR-17 or miR-19, key members in miR-17-92 cluster. In a lupus-like cGVHD model, systemic administration of anti-miR-17, but not anti-miR-19, alleviates clinical manifestations and proteinuria incidence in recipients through inhibiting donor lymphocyte expansion, B-cell activation, and GC responses. Blockade of miR-17 also ameliorates skin damage by reducing Th17 differentiation in a scleroderma-cGVHD model. Taken together, our work reveals that miR-17-92 is required for T-cell and B-cell differentiation and function, and thus for the development of cGVHD. Furthermore, pharmacological inhibition of miR-17 represents a potential therapeutic strategy for the prevention of cGVHD.

Pirfenidone ameliorates murine chronic GVHD through inhibition of macrophage infiltration and TGF-ß production.

Allogeneic hematopoietic stem cell transplantation is hampered by chronic graft-versus-host disease (cGVHD), resulting in multiorgan fibrosis and diminished function. Fibrosis in lung and skin leads to progressive bronchiolitis obliterans (BO) and scleroderma, respectively, for which new treatments are needed. We evaluated pirfenidone, a Food and Drug Administration (FDA)-approved drug for idiopathic pulmonary fibrosis, for its therapeutic effect in cGVHD mouse models with distinct pathophysiology. In a full major histocompatibility complex (MHC)-mismatched, multiorgan system model with BO, donor T-cell responses that support pathogenic antibody production are required for cGVHD development. Pirfenidone treatment beginning one month post-transplant restored pulmonary function and reversed lung fibrosis, which was associated with reduced macrophage infiltration and transforming growth factor-ß production. Pirfenidone dampened splenic germinal center B-cell and T-follicular helper cell frequencies that collaborate to produce antibody. In both a minor histocompatibility antigen-mismatched as well as a MHC-haploidentical model of sclerodermatous cGVHD, pirfenidone significantly reduced macrophages in the skin, although clinical improvement of scleroderma was only seen in one model. In vitro chemotaxis assays demonstrated that pirfenidone impaired macrophage migration to monocyte chemoattractant protein-1 (MCP-1) as well as IL-17A, which has been linked to cGVHD generation. Taken together, our data suggest that pirfenidone is a potential therapeutic agent to ameliorate fibrosis in cGVHD.

Precision medicine: integration of genetics and functional genomics in prediction of bronchiolitis obliterans after lung transplantation.

PURPOSE OF REVIEW: Lung transplantation (LTx) can be a life saving treatment in end-stage pulmonary diseases, but survival after transplantation is still limited. Posttransplant development of chronic lung allograft dysfunction with bronchiolits obliterans syndrome (BOS) as the major subphenotype, is the main cause of morbidity and mortality. Early identification of high-risk patients for BOS is a large unmet clinical need. In this review, we discuss gene polymorphisms and gene expression related to the development of BOS. RECENT FINDINGS: Candidate gene studies showed that donor and recipient gene polymorphisms affect transplant outcome and BOS-free survival after LTx. Both selective and nonselective gene expression studies revealed differentially expressed fibrosis and apoptosis-related genes in BOS compared with non-BOS patients. Significantly, recent microarray expression analysis of blood and broncho-alveolar lavage suggest a role for B-cell and T-cell responses prior to the development of BOS. Furthermore, 6 months prior to the development of BOS differentially expressed genes were identified in peripheral blood cells. SUMMARY: Genetic polymorphisms and gene expression changes are associated with the development of BOS. Future genome wide studies are needed to identify easily accessible biomarkers for prediction of BOS toward precision medicine.

SOCS3 overexpression in T cells ameliorates chronic airway obstruction in a murine heterotopic tracheal transplantation model.

PURPOSE: Suppressor of cytokine signaling-3 (SOCS3) is a negative feedback inhibitor of cytokine signaling with T-cell-mediated immunosuppressive effects on obliterative bronchiolitis (OB). In this study, we aimed to investigate the impact of T-cell-specific overexpression of SOCS3 using a murine heterotopic tracheal transplantation (HTT) model. METHODS: Tracheal allografts from BALB/c mice were subcutaneously transplanted into wild-type C57BL/6J (B6; WT) mice and SOCS3 transgenic B6 (SOCS3TG) mice. Tracheal allografts were analyzed by immunohistochemistry and quantitative polymerase chain reaction assays at days 7 and 21. RESULTS: At day 21, allografts in SOCS3TG mice showed significant amelioration of airway obstruction and epithelial loss compared with allografts in WT mice. The intragraft expression of IFN-γ and CXCL10 was suppressed, while that of IL-4 was enhanced in SOCS3TG mice at day 7. The T-bet levels were lower in SOCS3TG allografts than in WT allografts at day 7. CONCLUSION: We revealed that the overexpression of SOCS3 in T cells effectively ameliorates OB development in a murine HTT model by inhibiting the Th1 phenotype in the early phase. Our results suggest that the regulation of the T-cell response, through the modulation of SOCS expression, has potential as a new therapeutic strategy for chronic lung allograft dysfunction.

Bronchiolitis obliterans syndrome is associated with increased p-glycoprotein expression and loss of glucocorticoid receptor from steroid-resistant proinflammatory CD8+ T cells.

Immunosuppressive therapy fails to suppress the production of proinflammatory cytokines, particularly by CD8+ T cells, in stable lung transplant recipients and those undergoing chronic rejection, suggesting that some patients may become relatively resistant to immunosuppressants such as glucocorticoids (GC). We have shown loss of GC receptor (GCR) from the CD8+ cells, and we hypothesized that the drug membrane efflux pump, p-glycoprotein-1 (Pgp), may also be involved in lymphocyte steroid resistance following lung transplant. Pgp/GCR expression and interferon (IFN)-γ/tumour necrosis factor (TNF)-α proinflammatory cytokine production was measured in blood lymphocytes from 15 stable lung transplant patients, 10 patients with bronchiolitis obliterans syndrome (BOS) and 10 healthy aged-matched controls (± prednisolone ± Pgp inhibitor, cyclosporin A ± GCR activator, Compound A) using flow cytometry. Both Pgp+ and Pgp- lymphocyte subsets from all subjects produced IFN-γ/TNF-α proinflammatory cytokines. Pgp expression was increased in CD8+ Pgp+ T cells and correlated with IFN-γ/TNF-α expression and BOS grade. Reduced GCR was observed in CD8+ Pgp- T, natural killer (NK) T-like and NK cells from stable patients compared with controls, and reduced further in CD8+ Pgp- T cells in BOS. The addition of 2·5 ng/ml cyclosporin A and 1 µM prednisolone inhibit IFN-γ/TNF-α production significantly by CD8+ Pgp+ T cells from BOS patients. The addition of 10 µM Compound A and 1 µM prednisolone inhibit IFN-γ/TNF-α production significantly by CD8+ Pgp- T cells from BOS patients. BOS is associated with increased Pgp expression and loss of GCR from steroid-resistant proinflammatory CD8+ T cells. Treatments that inhibit Pgp and up-regulate GCR in CD8+ T cells may improve graft survival.

1H NMR To Evaluate the Metabolome of Bronchoalveolar Lavage Fluid (BALf) in Bronchiolitis Obliterans Syndrome (BOS): Toward the Development of a New Approach for Biomarker Identification.

This report describes the application of NMR spectroscopy to the profiling of metabolites in bronchoalveolar lavage fluid (BALf) of lung transplant recipients without bronchiolitis obliterans syndrome (BOS) (stable, S, n = 10), and with BOS at different degrees of severity (BOS 0p, n = 10; BOS I, n = 10). Through the fine-tuning of a number of parameters concerning both sample preparation/processing and variations of spectra acquisition modes, an efficient and reproducible protocol was designed for the screening of metabolites in a pulmonary fluid that should reflect the status of airway inflammation/injury. Exploiting the combination of mono- and bidimensional NMR experiments, 38 polar metabolites, including amino acids, Krebs cycle intermediates, mono- and disaccharides, nucleotides, and phospholipid precursors, were unequivocally identified. To determine which signature could be correlated with the onset of BOS, the metabolites' content of the above recipients was analyzed by multivariate (PCA and OPLS-DA) statistical methods. PCA analysis (almost) totally differentiated S from BOS I, and this discrimination was significantly improved by the application of OPLS-DA, whose model was characterized by excellent fit and prediction values (R2 = 0.99 and Q2 = 0.88). The analysis of S vs BOS 0p and of BOS 0p vs BOS I samples showed a clear discrimination of considered cohorts, although with a poorer efficiency compared to those measured for S vs BOS I patients. The data shown in this work assess the suitability of the NMR approach in monitoring different pathological lung conditions.

Molecular Profiling in Lung Biopsies of Human Pulmonary Allografts to Predict Chronic Lung Allograft Dysfunction.

Chronic lung allograft dysfunction (CLAD) is the main reason for poor long-term outcome of lung transplantation, with bronchiolitis obliterans (BO) representing the predominant pathological feature. BO is defined as a progressive fibrous obliteration of the small airways, thought to be triggered by a combination of nonimmune bronchial injury and alloimmune and autoimmune mechanisms. Because biopsy samples are too insensitive to reliably detect BO and a decline in lung function test results, which is clinically used to define CLAD, does not detect early stages, there is need for alternative biomarkers for early diagnosis. Herein, we analyzed the cellular composition and differential expression of 45 tissue remodeling-associated genes in transbronchial lung biopsy specimens from two cohorts with 18 patients each: patients who did not develop CLAD within 3 years after transplantation (48 biopsy specimens) and patients rapidly developing CLAD within the first 3 postoperative years (57 biopsy specimens). Integrating the mRNA expression levels of the five most significantly dysregulated genes from the transforming growth factor-ß axis (BMP4, IL6, MMP1, SMAD1, and THBS1) into a score, patient groups could be confidently separated and the outcome predicted (P < 0.001). We conclude that overexpression of fibrosis-associated genes may be valuable as a tissue-based molecular biomarker to more accurately diagnose or predict the development of CLAD.

Increased T follicular helper cells and germinal center B cells are required for cGVHD and bronchiolitis obliterans.

Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Having shown that germinal center (GC) formation and immunoglobulin deposition are required for multiorgan system cGVHD and associated bronchiolitis obliterans syndrome (BOS) in a murine model, we hypothesized that T follicular helper (Tfh) cells are necessary for cGVHD by supporting GC formation and maintenance. We show that increased frequency of Tfh cells correlated with increased GC B cells, cGVHD, and BOS. Although administering a highly depletionary anti-CD20 monoclonal antibody (mAb) to mice with established cGVHD resulted in peripheral B-cell depletion, B cells remained in the lung, and BOS was not reversed. BOS could be treated by eliminating production of interleukin-21 (IL-21) by donor T cells or IL-21 receptor (IL-21R) signaling of donor B cells. Development of BOS was dependent upon T cells expressing the chemokine receptor CXCR5 to facilitate T-cell trafficking to secondary lymphoid organ follicles. Blocking mAbs for IL-21/IL-21R, inducible T-cell costimulator (ICOS)/ICOS ligand, and CD40L/CD40 hindered GC formation and cGVHD. These data provide novel insights into cGVHD pathogenesis, indicate a role for Tfh cells in these processes, and suggest a new line of therapy using mAbs targeting Tfh cells to reverse cGVHD.

Hepatocyte growth factor enhances the inflammation-alleviating effect of umbilical cord-derived mesenchymal stromal cells in a bronchiolitis obliterans model.

BACKGROUND AIMS: Specific and effective therapy for prevention or reversal of bronchiolitis obliterans (BO) is lacking. In this study, we evaluated the therapeutic effect of hepatocyte growth factor (HGF) gene modified mesenchymal stromal cells (MSCs) on BO. METHODS: A mouse model of experimental BO was established by subcutaneously transplanting the tracheas from C57BL/6 mice into Balb/C recipients, which were then administered saline, Ad-HGF-modified human umbilical cord-MSCs (MSCs-HGF) or Ad-Null-modified MSCs (MSCs-Null). The therapeutic effects of MSCs-Null and MSCs-HGF were evaluated by using fluorescence-activated cell sorting (FACS) for lymphocyte immunophenotype of spleen, enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (rt-PCR) for cytokine expression, and histopathological analysis for the transplanted trachea. RESULTS: The histopathologic recovery of allograft tracheas was improved significantly after MSCs-Null and MSCs-HGF treatment and the beneficial effects were particularly observed in MSCs-HGF-treated mice. Furthermore, the allo-transplantation-induced immunophenotype disorders of the spleen, including regulatory T (Treg), T helper (Th)1, Th2 and Th17, were attenuated in both cell-treated groups. MSCs-HGF treatment reduced expression and secretion of inflammation cytokines interferon-gamma (IFN-γ), and increased expression of anti-inflammatory cytokine interleukin (IL)-4 and IL-10. It also decreased the expression level of the profibrosis factor transforming growth factor (TGF)-ß. CONCLUSION: Treatment of BO with HGF gene modified MSCs results in reduction of local inflammation and promotion in recovery of allograft trachea histopathology. These findings might provide an effective therapeutic strategy for BO.

MicroRNA screening and functional study of obliterative bronchiolitis in a rat model simulating lung transplantation.

The aim of this study was to provide the experimental basis for effective prevention and treatment of obliterative bronchiolitis (OB) by studying the changes on the microRNA (miRNA) expression profile after an orthotopic tracheal transplantation (OTT) simulating lung transplantation (LT). The OTT was performed on inbred rats to establish an OB animal model simulating LT, which was confirmed successful through pathological examination after 4 weeks. A miRNA microarray was used to screen for the most significantly differentially expressed miRNA in the OB tissues of donor transplanted trachea and real-time quantitative PCR was then used to validate the reliability of the microarray results. The microarray detection obtained 29 OB-related miRNAs, composed of 15 and 14 significantly up- and down-regulated miRNAs, respectively, among which miR-146a, miR-155, and miR-451, whose function is involved in the immune and inflammatory reactions, were subjected to relative quantitation research. The LT-simulated OTT-induced OB showed significantly differential expressions of multiple miRNAs, among which miR-146a and miR-155 were highly expressed, while miR-451 was lowly expressed, suggesting that these miRNAs may play an important regulatory role in the OB pathological process after LT.

Diacetyl induces amphiregulin shedding in pulmonary epithelial cells and in experimental bronchiolitis obliterans.

Diacetyl (DA), a component of artificial butter flavoring, has been linked to the development of bronchiolitis obliterans (BO), a disease of airway epithelial injury and airway fibrosis. The epidermal growth factor receptor ligand, amphiregulin (AREG), has been implicated in other types of epithelial injury and lung fibrosis. We investigated the effects of DA directly on the pulmonary epithelium, and we hypothesized that DA exposure would result in epithelial cell shedding of AREG. Consistent with this hypothesis, we demonstrate that DA increases AREG by the pulmonary epithelial cell line NCI-H292 and by multiple independent primary human airway epithelial donors grown under physiologically relevant conditions at the air-liquid interface. Furthermore, we demonstrate that AREG shedding occurs through a TNF-α-converting enzyme (TACE)-dependent mechanism via inhibition of TACE activity in epithelial cells using the small molecule inhibitor, TNF-α protease inhibitor-1, as well as TACE-specific small inhibitor RNA. Finally, we demonstrate supportive in vivo results showing increased AREG transcript and protein levels in the lungs of rodents with DA-induced BO. In summary, our novel in vitro and in vivo observations suggest that further study of AREG is warranted in the pathogenesis of DA-induced BO.

[Aberrant reparative tissue remodeling: histopathology and molecular pathology]. / Aberrierender reparativer Gewebeumbau : Histo- und Molekularpathologie.

Not only tumorous infiltrations can lead to destruction of parenchymal organs but also the aberrant proliferation and matrix production of mesenchymal cells and vessels during a dysregulated repair attempt. This fibrogenesis is the result of a complex pathogenesis, which can be investigated in animal models but also in situ to harvest new biomarkers. This article deals particularly with the second method and summarizes molecular pathological findings in various model diseases for aberrant reparative tissue reconstruction. These model diseases include plexiform vasculopathy in pulmonary arterial hypertension (PAH), Quilty lesions in heart transplantation, bronchiolitis obliterans (BO), inflammatory airway remodeling and Epstein-Barr virus (EBV) induced smooth muscle proliferation (posttransplantation smooth muscle tumor, PTSMT).Using in situ molecular pathology, we were able to dismiss an assumed involvement of myofibroblastic cells in vessel reconstruction of the lung in PAH. We could also for the first time perform a comprehensive molecular characterization of the vascular remodeling and prove that plexiform vasculopathy represents a complex-regulated epiphenomenon of excessive pulmonary hypertension. This method also allowed us to describe for the first time the miRNA expression in PAH in a compartment-specific manner and to draw conclusions regarding the damaged overriding regulatory mechanisms. In the same way, we were also able to describe the chimeric character of the complex neoangiogenesis in the donor organ after heart transplantation.After lung transplantation, we identified for the first time a group of tissue-based molecular markers, which can predict later occurrence of BO even in morphologically normal transbronchial biopsies. In addition, we have documented for the first time the molecular characteristics of the morphologically analogous airway reconstruction in lung-transplanted and non-transplanted patients. We could further elucidate the role of matrix metalloproteinases (MMP) and their antagonists in inflammatory airway reconstruction and deduce from this the resulting therapeutic implications. Accordingly, we were able to further clarify the origin, pathogenesis and the malignant potential of EBV-induced PTSMT and for the first time provide an evidence-based therapy recommendation and risk assessment.In summary, this article documents that in situ diagnostics can meet the requirements of the challenging parameters and issues of life sciences. It is to be expected that the technical possibilities will develop analogously to the increasing demands and the in situ method will move further into the focus of molecular pathology.

Obliterative airway remodeling: molecular evidence for shared pathways in transplanted and native lungs.

Obliteration of the small airways is a largely unresolved challenge in pulmonary medicine. It represents either the irreversible cause of functional impairment or a morphologic disorder of limited importance in a multitude of diseases. Bronchiolitis obliterans is a key complication of lung transplantation. No predictive markers for the onset of obliterative remodeling are currently available. To further elucidate the molecular mechanisms of airway remodeling, compartment-specific expression patterns were analyzed in patients. For this purpose, remodeled and nonremodeled bronchioli were isolated from transplanted and nontransplanted lung explants using laser-assisted microdissection (n = 24). mRNA expression of 45 fibrosis-associated genes was measured using quantitative real-time RT-PCR. For 20 genes, protein expression was also analyzed by immunohistochemistry. Infiltrating cells were characterized at conventional histology and immunohistochemistry. Obliterative remodeling of the small airways in transplanted and nontransplanted lungs shared similar grades of chronic inflammation and pivotal fibrotic pathways such as transforming growth factor ß signaling and increased collagen expression. Bone morphogenetic protein and thrombospondin signaling, and also matrix metalloproteinases and tissue inhibitor of metalloproteinases, were primarily up-regulated in obliterative airway remodeling in nontransplanted lungs. In transplanted lungs, clinical remodeled bone morphogenetic protein but nonremodeled bronchioli were characterized by a concordant up-regulation of matrix metalloproteinase-9, RANTES, and tissue inhibitor of metalloproteinase-1. These distinct expression patterns warrant further investigation as potential markers of impending airway remodeling, especially for prospective longitudinal molecular profiling.

Single-cell transcriptome mapping identifies a local, innate B cell population driving chronic rejection after lung transplantation.

Bronchiolitis obliterans syndrome (BOS) is the main reason for poor outcomes after lung transplantation (LTx). We and others have recently identified B cells as major contributors to BOS after LTx. The extent of B cell heterogeneity and the relative contributions of B cell subpopulations to BOS, however, remain unclear. Here, we provide a comprehensive analysis of cell population changes and their gene expression patterns during chronic rejection after orthotopic LTx in mice. Of 11 major cell types, Mzb1-expressing plasma cells (PCs) were the most prominently increased population in BOS lungs. These findings were validated in 2 different cohorts of human BOS after LTx. A Bhlhe41, Cxcr3, and Itgb1 triple-positive B cell subset, also expressing classical markers of the innate-like B-1 B cell population, served as the progenitor pool for Mzb1+ PCs. This subset accounted for the increase in IgG2c production within BOS lung grafts. A genetic lack of Igs decreased BOS severity after LTx. In summary, we provide a detailed analysis of cell population changes during BOS. IgG+ PCs and their progenitors - an innate B cell subpopulation - are the major source of local Ab production and a significant contributor to BOS after LTx.

Ferret Lung Transplantation Models Differential Lymphoid Aggregate Morphology Between Restrictive and Obstructive Forms of Chronic Lung Allograft Dysfunction.

BACKGROUND: Long-term survival after lung transplantation remains limited by chronic lung allograft dysfunction (CLAD). CLAD has 2 histologic phenotypes, namely obliterative bronchiolitis (OB) and restrictive alveolar fibroelastosis (AFE), which have distinct clinical presentations, pathologies, and outcomes. Understanding of OB versus AFE pathogenesis would improve with better animal models. METHODS: We utilized a ferret orthotopic single-lung transplantation model to characterize allograft fibrosis as a histologic measure of CLAD. Native lobes and "No CLAD" allografts lacking aberrant histology were used as controls. We used morphometric analysis to evaluate the size and abundance of B-cell aggregates and tertiary lymphoid organs (TLOs) and their cell composition. Quantitative RNA expression of 47 target genes was performed simultaneously using a custom QuantiGene Plex Assay. RESULTS: Ferret lung allografts develop the full spectrum of human CLAD histology including OB and AFE subtypes. While both OB and AFE allografts developed TLOs, TLO size and number were greater with AFE histology. More activated germinal center cells marked by B-cell lymphoma 6 Transcription Repressor, (B-cell lymphoma 6) expression and fewer cells expressing forkhead box P3 correlated with AFE, congruent with greater diffuse immunoglobulin, plasma cell abundance, and complement 4d staining. Furthermore, forkhead box P3 RNA induction was significant in OB allografts specifically. RNA expression changes were seen in native lobes of animals with AFE but not OB when compared with No CLAD native lobes. CONCLUSIONS: The orthotopic ferret single-lung transplant model provides unique opportunities to better understand factors that dispose allografts to OB versus AFE. This will help develop potential immunomodulatory therapies and antifibrotic approaches for lung transplant patients.

Survival difference between high-risk and low-risk CFTR genotypes after lung transplant.

BACKGROUND: While cystic fibrosis transmembrane conductance regulator (CFTR) genotypes are associated with clinical outcomes in cystic fibrosis patients, it is unknown if genotype impacts lung transplant outcomes. We sought to compare lung transplant survival and time to bronchiolitis obliterans syndrome (BOS) between high-risk, low-risk, and not yet classified CFTR genotypes. METHODS: We used merged data from the Organ Procurement and Transplantation Network (2005-2017) and United States Cystic Fibrosis Foundation Patient Registry (2005-2016). Cox Proportional Hazards models compared graft failure after lung transplant and time to BOS among high-risk, low-risk, and not yet classified risk CFTR genotype classes. RESULTS: Among 1,830 cystic fibrosis lung transplant recipients, median survival for those with low-risk, high-risk, and not yet classified genotype was 9.83, 6.25, and 5.75 years, respectively. Adjusted Cox models showed recipients with a low-risk genotype had 39% lower risk of death or re-transplant compared to those with high-risk genotype (adjusted HR 0.61, 95% CI = 0.40, 0.91). A subset of 1,585 lung transplant recipients were included in the BOS subgroup analysis. Adjusted analyses showed no significant difference of developing BOS among high-risk, low-risk, or not yet classified genotypes. CONCLUSIONS: Lung transplant recipients with low-risk CFTR genotype have better survival after transplant compared to recipients with high-risk or not yet classified genotypes. Given these differences, future studies evaluating the mechanism by which CFTR genotype affects post-transplant survival could identify potential targets for intervention.