Pathogenesis Of Obliterative Bronchiolitis Research Articles

Identification of Hub Genes in the Pathogenesis of Bronchiolitis Obliterans via Bioinformatic Analysis and Experimental Verification.

Bronchiolitis obliterans (BO) is a chronic disease that can arise as a complication of severe childhood pneumonia and can also impact the long-term survival of patients after lung transplantation. However, the precise molecular mechanism underlying BO remains unclear. We aimed to identify BO-associated hub genes and their molecular mechanisms. BO-associated transcriptome datasets (GSE52761, GSE137169, and GSE94557) were downloaded from the Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs). Additional bioinformatics analyses, such as Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Protein-Protein Interaction (PPI) analyses, were performed to determine functional roles and DEG-associated regulatory networks. Prediction of hub genes using the 12 algorithms available in the Cytohubba plugin of Cytoscape software was also performed. Verification was performed using the BO mouse model. Our results revealed 57 DEGs associated with BO, of which 18 were down-regulated and 39 were up-regulated. The Cytohubba plugin data further narrowed down the 57 DEGs into 9 prominent hub genes (CCR2, CD1D, GM2A, TFEC, MPEG1, CTSS, GPNMB, BIRC2, and CTSZ). Genes such as CCR2, TFEC, MPEG1, CTSS, and CTSZ were dysregulated in 2,3-butanedione-induced BO mice, whereas TFEC, CTSS, and CTSZ were dysregulated in nitric acid-induced BO mouse models. Our study identified and validated four novel BO biomarkers, which may allow further investigation into the development of distinct BO diagnostic markers and novel therapeutic avenues.

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.

Bronchiolitis obliterans murine model induced by nitric acid aerosol inhalation: An economical and reproducible model

ABSTRACTPurpose: Bronchiolitis obliterans (BO) is a highly debilitative and fatal syndrome associated with a series of severe lower airway disorders. The pathogenesis of BO is complicated and not entirely understood. An appropriate animal model of BO may aid research into its pathogenesis. Here, we establish a mouse model of BO to provide insight into this disease. Materials and Methods: 6–8 week old BABL/c mice were exposed to 5% nitric acid (NA) aerosol through a nebulizer for 3 hours, and controls were exposed to distilled water instead. Symptoms, airway resistance and pathological process were observed dynamically. The levels of matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), 8-isoprostane and myeloperoxidase (MPO) in lung tissue and bronchoalveolar lavage fluids (BLAF) were determined by ELISA on day 3, 7, 14, 28 and 56 after the aerosol nebulization. Results: Typical BO lesions were observed in NA nebulized mice characterized histologically by initial necrotizing bronchiolitis and final airway fibrosis at day 28 after the aerosol nebulization. NA nebulized mice also exhibited labored breathing and significantly increased airway resistance. Expression of MMP-2, MMP-9, TIMP-1, 8-isoprostane and MPO were significantly elevated in NA nebulized mice in different time frame. Conclusion: A murine BO model was established by NA aerosol inhalation. It provides an easy, economic, and reproducible mice model for BO research.

IL-17 contributes to the pathogenesis of obliterative bronchiolitis via regulation of M1 macrophages polarization in murine heterotopic trachea transplantation models

Acute allograft rejection is a principal conundrum in lung obliterative bronchiolitis (OB). Monocytes/macrophages infiltration has been proved to be the main reason for acute rejection. IL-17 contributes to the recruitment and function of macrophages. However, the mechanism of IL-17 underlying OB progression remains elusive. In the present study, we showed that the deficiency of IL-17 attenuated the pathology of murine heterotopic trachea allografts. Compared to WT recipients, IL-17−/− mice displayed higher frequency of CD206+ cells and lower ratio of CD86+ cells among F4/80+ macrophages in allografts and spleens on day 7 post heterotopic trachea transplantation. Moreover, mRNA levels of pro-inflammatory cytokines including IL-6, TNF-α, and IL-1β decreased in allografts of IL-17−/− recipients, but these of MRC1 and Arg-1 increased in comparison with WT. IL-17 deficiency can inhibit LPS induced M1 while promote IL-4 induced M2 polarization of bone marrow-derived macrophages. Further data demonstrated that the deficiency of IL-17 suppressed the lipopolysaccharide-induced M1 polarization and function through prevention of phosphorylation of both STAT3 and STAT5. Therefore, IL-17 contributes to OB pathogenesis through regulating macrophages function, thereby it may unravel part of the complexity of IL-17 in OB and enhance future therapeutic development.

Airway injury in an in vitro human epithelium-fibroblast model of diacetyl vapor exposure: diacetyl-induced basal/suprabasal spongiosis

Inhalation exposure to diacetyl (DA) is associated with obliterative bronchiolitis (OB) in workers and induces OB-like fibrotic airway lesions in rats. The pathogenesis of OB is poorly understood in part due to complex interactions between airway epithelial, mesenchymal and blood-derived inflammatory cells. DA-induced airway toxicity in the absence of recruited-inflammatory/immune cells was characterized using an air-liquid interface (ALI) model consisting of human airway epithelium with (Epi/FT) and without (Epi) a mesenchymal component. ALI cultures were exposed to 25 mM DA-derived vapors (using vapor cups) for 1 h on day 0, 2 and 4. In some experiments, the tissues were exposed to 2,3-hexanedione (Hex) which is structurally-similar, but much less fibrogenic than DA. Lactate dehydrogenase activity and day 6 histopathologic changes associated with epithelial injury, including basal/suprabasal spongiosis, were increased following exposure of Epi/FT tissues to DA but not control or Hex vapors. IL-1a, IL-6, IL-8, sIL-1Ra, TGFa, MCP-3 and TNFa proteins were increased following DA exposure of Epi/FT tissues; only IL-1a, IL-8, sIL-1Ra and TGFa were increased following exposure of Epi tissues. MMP-1, MMP-3 and TIMP-1 proteins were increased following DA exposure of Epi/FT tissues; whereas MMP-2, MMP-7 and TIMP-2 were decreased, and production was largely dependent upon the presence of sub-epithelial stromal matrix/fibroblasts. Hex-induced protein changes were minimal. This in vitro study demonstrated that exposure of human airways to DA vapors induced epithelial injury (with the histopathologic feature of basal/suprabasal spongiosis) and increased release of pro-inflammatory and pro-fibrotic cytokines/chemokines as well as MMPs/TIMPs in the absence of recruited-inflammatory cells.

STAT6 and T-bet double deficient mouse serving as allograft recipient is a new viable animal model to study lung transplantation

Abstract The long-term success of lung transplantation is mainly limited by the development of obliterative bronchiolitis (OB) in which IL-17 plays a critical role. However, murine orthotopic allogeneic lung transplant model in wild type recipients develops IFNγ-dominant acute allograft rejection. Our previous research demonstrated that additional STAT6 deficiency to T-bet deficiency resulted in Th17-dominant immune responses, and also restored CD4 T cells to produce IFNγ which is essential to immunosuppression and immunological tolerance. In this study, murine orthotopic lung transplants were performed in C57BL/6 wild type or T-bet/STAT6 double knout-out (DKO) mice using BALB/c donors. Vigorous allograft rejection was observed in T-bet/STAT6 DKO recipients with much higher levels of IL-17 but comparable levels of IFNγ expression to that in wild type recipients. Analysis of the graft-infiltrating cells displayed a significantly decreased CD4:CD8 ratio in T-bet/STAT6 DKO recipients compared to wild type recipients, but not as extremely as that observed in T-bet KO recipients, demonstrating more balanced CD4 and CD8 responses in DKO recipients. Our results indicate T-bet and STAT6 double deficient recipients of lung allografts develop obliterative airway inflammation that is mediated by both CD4 and CD8 responses, more closely resembling OB pathogenesis, and provide a new viable model to study lung transplant rejection.

Pathology of diacetyl and 2,3-pentanedione airway lesions in a rat model of obliterative bronchiolitis.

Inhalation of diacetyl vapors by workers has been associated with obliterative bronchiolitis (OB), a poorly understood fibroproliferative disease of the small airways. Significant insights into the pathogenesis of OB have been obtained through the use of a rat model. Inhalation exposure of rats to diacetyl or 2,3-pentanedione, a related flavoring agent, can cause severe injury to the airway epithelium and underlying basement membrane. Repeated exposure to diacetyl or 2,3-pentanedione leads to aberrant repair, fibroproliferation and partial to complete occlusion of the airway lumen. Fibroproliferative lesions in rat airways were found to include both intraluminal polyps and circumferential intramural lesions. Intraluminal polyps have been observed to form secondary attachments spanning the airway lumen causing increasing obstruction. These airway lesions in rats are accompanied by inflammation in the form of peribronchial and perivascular infiltrates of lymphocytes, eosinophils and neutrophils. Diacetyl-induced OB lesions in the rat are similar to OB lesions in humans and provide a good model for studying the pathogenesis of this disease.

Th17 and Th1 play distinct roles in obliterative bronchiolitis pathogenesis after transplantation.

Abstract Development of obliterative bronchiolitis (OB) is the biggest obstacle that limits long-term allograft survival and clinical application of lung transplantation. Mouse orthotopic trachea or lung transplantations were utilized to examine roles of Th17 and Th1 during OB pathogenesis after transplantation. Allografts of wild type recipients displayed elevated levels of Th1 and Th17 cytokines IFNγ, IL-6 and IL-17 expression. Recipients deficient in Th1 key transcriptional factor T-bet resulted in decreased cell infiltration and improved airway integrity in orthotopic transplanted trachea grafts; Lack of Th2 transcriptional factor Stat6 had little effect; While absence of Th17 master transcriptional factor RORγt had striking effects in abolishing airway epithelial injury, a leading factor of OB pathogenesis. Administering anti-IL-6 or anti-IL-17 but not anti-IFNγ mAbs restored normal airway epithelial morphology. Furthermore, we found that anti-CD40L treatment resulted in increased FoxP3 and regulatory T cells, and decreased IFNγ expression; However it had very limited effect on IL-17 suppression and airway epithelial protection. While CTLA4-Ig treatment significantly inhibit IL-17 expression and reduced epithelial injury, and the combination of CTLA4-Ig and anti-CD40L markedly restored airway morphology of grafts. Together, our findings provide new insights for understanding Th17 and Th1 responses during OB pathogenesis and demonstrate the critical importance of suppressing Th17 for OB development after transplantation.

The role of soluble and insoluble gastric fluid components in the pathogenesis of obliterative bronchiolitis in rat lung allografts.

Repetitive gastric fluid aspirations have been shown to lead to obliterans bronchiolitis (OB), but the component or components of gastric fluid that are responsible are unknown. This study investigates the role of particulates and, separately, soluble material in gastric fluid during the development of OB. Whole gastric fluid (WGF) was collected from male Fischer 344 (F344) rats and separated by centrifugation into particle reduced gastric fluid (PRGF) and particulate components resuspended in normal saline (PNS). Orthotopic left lung transplants from male Wistar-Kyoto rats into F344 rats were performed using a modification of the nonsuture external cuff technique with prolonged cold ischemia. Rats were subjected to weekly aspiration of 0.5 ml/kg of WGF (n = 9), PRGF (n = 10), PNS (n = 9), or normal saline (control, NS; n = 9) for 8 weeks following transplantation. Lung allografts treated with WGF, PRGF, or PNS developed a significantly greater percentage of OB-like lesions compared with the control. No statistical difference was observed when comparing the fibrosis grades or the percentage of OB lesions of WGF, PRGF, and PNS groups, suggesting that both soluble and insoluble components of gastric fluid can promote the development of aspiration-induced OB and fibrosis in lung allografts.

The Role of Lysophosphatidic Acid on Airway Epithelial Cell Denudation in a Murine Heterotopic Tracheal Transplant Model.

Chronic rejection is the major leading cause of morbidity and mortality after lung transplantation. Obliterative bronchiolitis (OB), a fibroproliferative disorder of the small airways, is the main manifestation of chronic lung allograft rejection. However, there is currently no treatment for the disease. We hypothesized that lysophosphatidic acid (LPA) participates in the progression of OB. The aim of this study was to reveal the involvement of LPA on the lesion of OB. Ki16198, an antagonist specifically for LPA1 and LPA3, was daily administered into the heterotopic tracheal transplant model mice at the day of transplantation. At days 10 and 28, the allografts were isolated and evaluated histologically. The messenger RNA levels of LPAR in microdissected mouse airway regions were assessed to reveal localization of lysophosphatidic acid receptors. The human airway epithelial cell was used to evaluate the mechanism of LPA-induced suppression of cell adhesion to the extracellular matrix (ECM). The administration of Ki16198 attenuated airway epithelial cell loss in the allograft at day 10. Messenger RNAs of LPA1 and LPA3 were detected in the airway epithelial cells of the mice. Lysophosphatidic acid inhibited the attachment of human airway epithelial cells to the ECM and induced cell detachment from the ECM, which was mediated by LPA1 and Rho-kinase pathway. However, Ki16198 did not prevent obliteration of allograft at day 28. The LPA signaling is involved in the status of epithelial cells by distinct contribution in 2 different phases of the OB lesion. This finding suggests a role of LPA in the pathogenesis of OB.

IL-17 induces type V collagen overexpression and EMT via TGF-β-dependent pathways in obliterative bronchiolitis.

Obliterative bronchiolitis (OB), a fibrotic airway lesion, is the leading cause of death after lung transplantation. Type V collagen [col(V)] overexpression and IL-17-mediated anti-col(V) immunity are key contributors to OB pathogenesis. Here, we report a previously undefined role of IL-17 in inducing col(V) overexpression, leading to epithelial mesenchymal transition (EMT) and subsequent OB. We observed IL-17-mediated induction of col(V) α1 chains [α1 (V)] in normal airway epithelial cells in vitro and detected α1 (V)-specific antibodies in bronchoalveolar lavage fluid of lung transplant patients. Overexpression of IL-17 and col(V) was detected in OB lesions in patient lung biopsies and in a murine OB model. IL-17 is shown to induce EMT, TGF-β mRNA expression, and SMAD3 activation, whereas downregulating SMAD7 expression in vitro. Pharmacological inhibition of TGF-βRI tyrosine kinase, p38 MAPK, or focal adhesion kinase prevented col(V) overexpression and EMT. In murine orthotopic lung transplants, neutralizing IL-17 significantly decreased TGF-β mRNA and protein expression and prevented epithelial repair/OB. Our findings highlight a feed-forward loop between IL-17 and TGF-β, leading to induction of col(V) and associated epithelial repair, thus providing one possible link between autoimmunity and OB after lung transplantation.

Epithelial to Mesenchymal Transition in Murine Tracheal Allotransplantation: An Immunohistochemical Observation

Background: Aberrant epithelial repair is a crucial event in the airway remodeling which characterizes obliterative bronchiolitis (OB) in the transplanted lungs. Epithelial to mesenchymal transition (EMT) has been implicated in the pathogenesis of OB after lung transplantation. During EMT, cells lose epithelial properties and gain properties of mesenchymal cells. Recent data from epithelial cell lines and human airway tissue from recipients of lung transplantations suggest that EMT may have important role in OB. However, few studies have proved the occurrence of EMT in animal models. The aim of this study was to clarify in animal transplant model whether EMT was involved in airway remodeling. Methods: We performed orthotopic tracheal transplantation from BALB/c to C57BL/6 mice. As a control group, syngeneic tracheas were implanted into BALB/c mice. After transplantation, five allogeneic and two syngeneic recipients were sacrificed at predetermined days from day 2 to 12, day 14, and 21. Histology was evaluated on hematoxylin-eosin (H&E) staining. Expression of the epithelial marker (E-cadherin), the mesenchymal marker (α-SMA and S100A4), and EMT-related transcription factor (ZEB1) were studied by immunohistochemistry. Results: In allogeneic grafts, following remarkable pseudostratified respiratory epithelium and subepithelial inflammatory cell infiltration (by day 8), denuded and flattened epithelium and subepithelial fibrosis (from day 8) was observed on serial H&E stains. Epithelial changes were dynamic and earlier than subepithelial fibrosis. At day 14, epithelium was flattened. At day 21, subepithelial spindle-shaped cells like fibroblasts were numerous (Figure 1–). On serial immunohistochemical evaluation of these rejecting tracheas, α-SMA positive epithelial cells emerged by day 6 and are most prominent at day 7 (Figure 1–B). α-SMA positive epithelial cells were frequently lining just above basement membrane. After day 9 few intraepithelial α-SMA positive cells were observed correspond with epithelial denuded stage. S100A4 was also expressed in epithelial cells. The expression of E-Cadherin attenuated in α-SMA positive epithelial cells (Figure 1–C). Before a few days prior to intraepithelial expression of α-SMA, transcription factor ZEB1 emerged in the nucleus of epithelial cells. The expression was greatest from day 2 to 4 (Figure 1–D).[Figure 1]Conclusions: After alloimmune injury, remarkable phenotypic change was observed in the epithelial cells in the allografts: remarkable expression of EMT-related transcription factor ZEB1, subsequent emergence of mesenchymal marker α-SMA and S100A4, and attenuation of E-cadherin expression in α-SMA positive epithelial cells. These findings were observed several days prior to subepithealial prominent fibrous formation, and may suggest that epithelial cells play an important role in airway remodeling through epithelial to mesenchymal transition.

Aspiration of gastric fluid in pulmonary allografts: Effect of pH

BackgroundChronic aspiration of gastric fluid potentially plays a central role in the pathogenesis of obliterative bronchiolitis, which is often associated with chronic pulmonary allograft failure. It remains unknown whether pharmaceutical-induced increases in gastric pH might effectively prevent any putative pulmonary injury associated with chronic aspiration. Materials and methodsTo test the hypothesis that neutralization of gastric fluid would affect the development of aspiration-associated obliterative bronchiolitis, an established rat lung transplant model (WKY-to-F344) was utilized. Pulmonary allograft recipients were subjected to eight weekly aspirations of gastric fluid at pH 2.5 (low-pH), gastric fluid at pH 7.4 (neutralized-pH), or saline as a control. ResultsHistologic analysis revealed that the fraction of airways affected with lesions consistent with obliterative bronchiolitis was 0.55 ± 0.08 (mean ± SEM) in rats receiving aspiration with low-pH gastric fluid, 0.49 ± 0.07 in animals receiving neutralized-pH gastric fluid, and 0.07 ± 0.05 in rats receiving normal saline only. The difference between groups receiving gastric fluid, regardless of pH, was significantly different from the saline control (P < 0.0001), whereas the difference between the groups receiving low-pH gastric fluid and neutralized-pH gastric fluid was not significant (P = 0.75). ConclusionsEffective management of gastroesophageal reflux disease in lung transplant recipients should probably include more than neutralization of gastric fluid.

62 T-bet Deficiency in Mouse Orthotopic Lung Transplant

T-bet is critical in the development of Th1 responses and negatively regulates type 17 responses which are implicated in the pathogenesis of obliterative bronchiolitis. We characterized the rejection pathology and allospecific responses that develop in mouse orthotopic lung transplant in the absence of T-bet and investigated the effects of anti-CD154 in this setting.

Lung transplantation in the fischer 344–wistar kyoto strain combination is a relevant experimental model to study the development of bronchiolitis obliterans in the rat

ABSTRACTChronic allograft rejection and bronchiolitis obliterans (BO) limited successful long-term outcome after lung transplantation (LTX). Reliable animal models are needed to study the pathogenesis of BO and to develop effective therapeutic strategies. The relevance of an available experimental LTX model without immunosuppression—the Fischer (F344)–Wistar Kyoto (WKY) rat strain combination—was analyzed. The kinetics of acute and chronic rejection and respective graft histopathology were described in the F344-to-WKY rat strain combination after allogeneic LTX. A modified classification of chronic lung allograft rejection was introduced to describe obliterative changes in the airways after rat LTX. Animals from Harlan Winkelmann (HW) and Charles River (CR) were examined. Within 14 days after LTX, allografts showed moderate to severe acute vascular and bronchiolar inflammation. In contrast to rats from CR, animals from HW showed a delayed acute rejection process. Mid-term phase after LTX (days 20–40) presented a “borderline form” consisting of both acute and first signs of chronic airway rejection. On postoperative day (POD) 60, first signs of airway remodelling and distinct BO were diagnosed in 80% of animals from HW. At the same time, the allografts with BO-like lesions increased up to 100% in rats from CR. The F344-to-WKY rat LTX model allows detailed assessment of all features of acute and chronic pulmonary rejection representing a clinically relevant model. However, due to breeding differences resulting in various sublines of the same rat strain, the source and husbandary history of the animals is important for analysis of immuno-mediated processes.

Green Tea Epigallo-Catechin-Galleate Ameliorates the Development of Obliterative Airway Disease

ABSTRACTLung transplantation has the worst outcome compared to all solid organ transplants due to chronic rejection known as obliterative bronchiolitis (OB). Pathogenesis of OB is a complex interplay of alloimmune-dependent and -independent factors, which leads to the development of inflammation, fibrosis, and airway obliteration that have been resistant to therapy. The alloimmune-independent inflammatory pathway has been the recent focus in the pathogenesis of rejection, suggesting that targeting this may offer therapeutic benefits. As a potent anti-inflammatory agent, epigallo-catechin-galleate (EGCG), a green tea catechin, has been very effective in ameliorating inflammation in a variety of diseases, providing the rationale for its use in this study in a murine heterotopic tracheal allograft model of OB. Mice treated with EGCG had reduced inflammation, with significantly less neutrophil and macrophage infiltration and significantly reduced fibrosis. On further investigation into the mechanisms, inflammatory cytokines keratinocyte (KC), interleukin-17 (IL-17), and tumor necrosis factor-α (TNF-α), involved in neutrophil recruitment, were reduced in the EGCG-treated mice. In addition, monocyte chemokine monocyte chemoattractant protein-1 (MCP-1) was significantly reduced by EGCG treatment. Antifibrotic cytokine interferon-γ–inducible protein-10 (IP-10) was increased and profibrotic cytokine transforming growth factor-β (TGF-β) was reduced, further characterizing the antifibrotic effects of EGCG. These findings suggest that EGCG has great potential in ameliorating the development of obliterative airway disease.

Chronic lung allograft rejection and airway microvasculature: Is HIF-1 the missing link?

Chronic lung allograft rejection, known as obliterative bronchiolitis (OB), is the leading cause of death in lung transplant patients. Although OB pathogenesis is not fully understood, in this issue of the JCI, Jiang and colleagues report that tissue hypoxia resulting in dysfunctional airway microvasculature precedes the airway fibrosis characteristic of OB. In addition, a relative deficiency of allograft endothelial cell-derived HIF-1α contributes to this process. Data showing that overexpressing HIF-1α restores the microvascular airway normoxia and prevents airway fibrosis highlight a novel role for vascular biology in OB pathogenesis.

Immunosuppression-induced bronchial epithelial–mesenchymal transition: A potential contributor to obliterative bronchiolitis

Obliterative bronchiolitis is the predominant histopathologic finding in patients with chronic rejection after lung transplant. This fibroproliferative transformation within small airways of lung allograft is poorly understood; however, studies suggest epithelial-mesenchymal transition plays a role. Transplant immunosuppressive therapy has been shown to cause epithelial-mesenchymal transition in renal tubular epithelial cells, with subsequent fibrosis. This study explored whether immunosuppressive therapy contributes to epithelial-mesenchymal transition in airway epithelial cells. Bronchial epithelial cell line RL-65 was treated 3 to 5 days with several immunosuppressive agents, including cyclosporine (INN ciclosporin), tacrolimus, azathioprine, mycophenolic acid, sirolimus, prednisone, and transforming growth factor β1 as control. We then analyzed cells for presence of mesenchymal morphology and protein markers. Treatment with cyclosporine, azathioprine, mycophenolate, and sirolimus resulted in elongated and irregular cell shape, and all but azathioprine showed loss of cell-cell adhesions relative to vehicle-treated cells. Expressions of extracellular matrix proteins, fibronectin and collagen, along with mesenchymal marker, vimentin, were significantly upregulated. Immunofluorescence showed loss of E-cadherin at cell membranes and cytoskeletal rearrangements typical of epithelial-mesenchymal transition. These immunosuppressive agents also increased transforming growth factor produced by cells; however, tacrolimus- and prednisone-treated cells maintained epithelial morphology, baseline levels of matrix protein expression, and transforming growth factor production levels. Overall, we found that certain immunosuppressive agents may contribute to partial epithelial-mesenchymal transition in bronchial epithelial cells, specifically increasing production of excessive extracellular matrix proteins. This may provide novel insights into the pathogenesis of obliterative bronchiolitis after lung transplant.

Chronic rejection of a lung transplant is characterized by a profile of specific autoantibodies

Obliterative bronchiolitis (OB) continues to be the major limitation to long-term survival after lung transplantation. The specific aetiology and pathogenesis of OB are not well understood. To explore the role of autoreactivity in OB, we spotted 751 different self molecules onto glass slides, and used these antigen microarrays to profile 48 human serum samples for immunoglobulin G (IgG) and IgM autoantibodies; 27 patients showed no or mild bronchiolitis obliterans syndrome (BOS; a clinical correlate of OB) and 15 patients showed medium to severe BOS. We now report that these BOS grades could be differentiated by a profile of autoantibodies binding to 28 proteins or their peptides. The informative autoantibody profile included down-regulation as well as up-regulation of both IgM and IgG specific reactivities. This profile was evaluated for robustness using a panel of six independent test patients. Analysis of the functions of the 28 informative self antigens showed that eight of them are connected in an interaction network involved in apoptosis and protein metabolism. Thus, a profile of autoantibodies may reflect pathological processes in the lung allograft, suggesting a role for autoimmunity in chronic rejection leading to OB.

Inflammation and Epithelial to Mesenchymal Transition in Lung Transplant Recipients: Role in Dysregulated Epithelial Wound Repair

Epithelial to mesenchymal transition (EMT) has been implicated in the pathogenesis of obliterative bronchiolitis (OB) after lung transplant. Although TNF-alpha accentuates TGF-beta1 driven EMT in primary human bronchial epithelial cells (PBECs), we hypothesized that other acute pro-inflammatory cytokines elevated in the airways of patients with OB may also accentuate EMT and contribute to dysregulated epithelial wound repair. PBECs from lung transplant recipients were stimulated with TGF-beta1+/-IL-1beta, IL-8, TNF-alpha or activated macrophages in co-culture and EMT assessed. The quality and rate of wound closure in a standardized model of lung epithelial injury was assessed in response to above stimuli. Co-treatment with TGF-beta1+TNF-alpha or IL-1beta significantly accentuates phenotypic and some functional features of EMT compared to TGF-beta1 alone. Co-treatment with TGF-beta1+TNF-alpha or IL-1beta accelerates epithelial wound closure however the quality of repair is highly dysregulated. Co-treatment with TGF-beta1+IL-8 has no significant effect on EMT or the speed or quality of wound healing. Activated macrophages dramatically accentuate TGF-beta1-driven EMT and cause dysregulated wound repair. Crosstalk between macrophage-derived acute inflammation in the airway and elevated TGF-beta1 may favor dysregulated airway epithelial repair and fibrosis in the lung allograft via EMT.