Obliterative Airway Disease Research Articles

Murine Intrapulmonary Tracheal Transplantation: A Model for Investigating Obliterative Airway Disease After Lung Transplantation.

Murine intrapulmonary tracheal transplantation (IPTT) is used as a model of obliterative airway disease (OAD) following lung transplantation. Initially reported by our team, this model has gained use in the study of OAD due to its high technical reproducibility and suitability for investigating immunological behaviors and therapeutic interventions. In the IPTT model, a rodent tracheal graft is directly inserted into the recipient's lung through the pleura. This model is distinct from the heterotopic tracheal transplantation (HTT) model, wherein grafts are transplanted into subcutaneous or omental sites, and from the orthotopic tracheal transplantation (OTT) model in which the donor trachea replaces the recipient's trachea. Successful implementation of the IPTT model requires advanced anesthetic and surgical skills. Anesthetic skills include endotracheal intubation of the recipient, setting appropriate ventilatory parameters, and appropriately timed extubation after recovery from anesthesia. Surgical skills are essential for precise graft placement within the lung and for ensuring effective sealing of the visceral pleura to prevent air leakage and bleeding. In general, the learning process takes approximately 2 months. In contrast to the HTT and OTT models, in the IPTT model, the allograft airway develops airway obliteration in the relevant lung microenvironment. This allows investigators to study lung-specific immunological and angiogenic processes involved in airway obliteration after lung transplantation. Furthermore, this model is also unique in that it exhibits tertiary lymphoid organs (TLOs), which are also seen in human lung allografts. TLOs are comprised of T and B cell populations and characterized by the presence of high endothelial venules that direct immune cell recruitment; therefore, they are likely to play a crucial role in graft acceptance and rejection. We conclude that the IPTT model is a useful tool for studying intrapulmonary immune and profibrotic pathways involved in the development of airway obliteration in the lung transplant allograft.

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.

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.

Hydrogen water alleviates obliterative airway disease in mice.

Bronchiolitis obliterans syndrome arising from chronic airway inflammation is a leading cause of death following lung transplantation. Several studies have suggested that inhaled hydrogen can protect lung grafts from ischemia-reperfusion injury via anti-inflammatory and -oxidative mechanisms. We investigated whether molecular hydrogen-saturated water can preserve lung allograft function in a heterotopic tracheal allograft mouse model of obliterative airway disease METHODS: Obliterative airway disease was induced by heterotopically transplanting tracheal allografts from BALB/c donor mice into C57BL/6 recipient mice, which were subsequently administered hydrogen water (10ppm) or tap water (control group) (n = 6 each) daily without any immunosuppressive treatment. Histological and immunohistochemical analyses were performed on days 7, 14, and 21. Hydrogen water decreased airway occlusion on day 14. No significant histological differences were observed on days 7 or 21. The cluster of differentiation 4/cluster of differentiation 3 ratio in tracheal allografts on day 14 was higher in the hydrogen water group than in control mice. Enzyme-linked immunosorbent assay performed on day 7 revealed that hydrogen water reduced the level of the pro-inflammatory cytokine interleukin-6 and increased that of forkhead box P3 transcription factor, suggesting an enhancement of regulatory T cell activity. Hydrogen water suppressed the development of mid-term obliterative airway disease in a mouse tracheal allograft model via anti-oxidant and -inflammatory mechanisms and through the activation of Tregs. Thus, hydrogen water is a potential treatment strategy for BOS that can improve the outcome of lung transplant patients.

MiR-155 Plays an Essential Role in Anti-MHC-Induced Obliterative Airway Disease

Purpose Previous studies in our lab demonstrated that development of Abs to HLA often precedes development of Abs to self-Antigens (SAgs), Collagen V (Col-V) and K-alpha1 Tubulin (Kα1T), increasing the risk of bronchiolitis obliterans syndrome as a significant risk factor for acute and chronic rejection (CR). Circulating exosomes expressing donor HLA, SAgs have been proposed to stimulate immune responses leading to lung allograft rejection. miR-155 was among the first miRNAs linked to inflammation, potent upregulation of Toll like receptors in multiple cell lineages leading to production of inflammatory cytokines including IL17 which has been proposed to play an important role in the development of CR following lung transplantation. In our present study, we elucidated the role of miR-155 in anti-MHC-induced obliterative airway disease (OAD) model and their role in inducing circulating exosomes with lung SAgs and contribution to OAD. Methods Wild-type C57BL/6 miR-155 knock-out (KO) B6.Cg-Mir155tm1Rsky/J mice (8-12 wks) were procured. Anti-H-2Kb (AF6-88.5.5.3, BioXcell,) Abs was administered intrabronchially. Mouse Abs to lung-SAgs, Col-V or Kα1T, was detected by ELISA. Circulating exosomes were isolated using total exosome isolation kit and analyzed for lung SAgs, CIITA and 20S proteosome by western blots. Results Anti-MHC class I Ab administration into the wild type mice developed Abs against lung SAgs, Col-V days 8, 16, 30, and K-α1T day 8. Anti-MHC class I Abs administrated to wild-type mice released exosomes with increased levels of SAgs, Col-V or K-α1T, CIITA and 20S proteasome on day 2; which increased by day 16. In contrast, miR-155KO mice demonstrated neither Abs to SAgs nor exosomes containing SAgs. Histopathological studies revealed that lungs from wild-type animals administered with anti-MHC class I Ab demonstrated significant inflammatory cells around the vessels and bronchiole with increase in fibrosis. miR-155KO mice did not show any significant lesions. Conclusion Our results demonstrate that Abs to lung SAgs and induction of circulating exosomes with lung SAgs occurs prior to development of OAD following anti-MHC administration into native lungs. Further, miR-155 plays an obligatory role in the pathogenesis of anti-MHC induced OAD since miR-55KO mice did not develop Abs to lung SAgs, induce circulating exosomes with lung SAgs, nor result in OAD.

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.

Azithromycin Fails to Prevent Accelerated Airway Obliteration in T-bet-/- Mouse Lung Allograft Recipients

BackgroundCellular and molecular mechanisms of acute and chronic lung allograft rejection have yet to be clearly defined, and obliterative bronchiolitis (OB) remains the primary limitation to survival in lung transplant recipients (LTRs). We have previously shown that T-bet–deficient recipients of full major histocompatibility complex (MHC)-mismatched, orthotopic left lung transplants develop accelerated obliterative airway disease (OAD) in the setting of acute cellular rejection characterized by robust alloimmune CD8+ interleukin (IL)-17 and interferon (IFN)-γ responses that are attenuated with neutralization of IL-17. Azithromycin has been shown to be beneficial in some LTRs with bronchiolitis obliterans syndrome/OB. Here, we evaluated the effects of azithromycin on rejection pathology and T-cell effector responses in T-bet-/- recipients of lung transplants. MethodsOrthotopic left lung transplantation was performed in BALB/c → B6 wild type or BALB/c → B6 T-bet-/- strain combinations as previously described. Mice treated with azithromycin received 10 mg/kg or 50 mg/kg subcutaneously daily. Lung allograft histopathology was analyzed at day 10 or day 21 post-transplantation, and neutrophil staining for quantification was performed using anti-myeloperoxidase. Allograft mononuclear cells were isolated at day 10 for T-cell effector cytokine response assessment using flow cytometry. ResultsWe show that while azithromycin significantly decreases lung allograft neutrophilia and CXCL1 levels and attenuates allospecific CD8+ IL-17 responses early post-transplantation, OAD persists in T-bet–deficient mice. ConclusionsOur results indicate that lung allograft neutrophilia is not essential for the development of OAD in this model and suggest allospecific T-cell responses that remain despite marked attenuation of CD8+ IL-17 are sufficient for obliterative airway inflammation and fibrosis.

(300) - Extracorporeal Photopheresis Inhibits the Development of Obliterative Airway Disease and Promotes the Expansion of Intragraft Retinoic Acid-Producing CD103+ Dendritic Cells and Tregs

(300) - Extracorporeal Photopheresis Inhibits the Development of Obliterative Airway Disease and Promotes the Expansion of Intragraft Retinoic Acid-Producing CD103+ Dendritic Cells and Tregs

Long-Term Persistence of Donor Alveolar Macrophages in Human Lung Transplant Recipients That Influences Donor-Specific Immune Responses.

Steady-state alveolar macrophages (AMs) are long-lived lung-resident macrophages with sentinel function. Evidence suggests that AM precursors originate during embryogenesis and populate lungs without replenishment by circulating leukocytes. However, their presence and persistence are unclear following human lung transplantation (LTx). Our goal was to examine donor AM longevity and evaluate whether AMs of recipient origin seed the transplanted lungs. Origin of AMs was accessed using donor-recipient HLA mismatches. We demonstrate that 94-100% of AMs present in bronchoalveolar lavage (BAL) were donor derived and, importantly, AMs of recipient origin were not detected. Further, analysis of BAL cells up to 3.5years post-LTx revealed that the majority of AMs (>87%) was donor derived. Elicitation of de novo donor-specific antibody (DSA) is a major post-LTx complication and a risk factor for development of chronic rejection. The donor AMs responded to anti-HLA framework antibody (Ab) with secretion of inflammatory cytokines. Further, in an experimental murine model, we demonstrate that adoptive transfer of allogeneic AMs stimulated humoral and cellular immune responses to alloantigen and lung-associated self-antigens and led to bronchiolar obstruction. Therefore, donor-derived AMs play an essential role in the DSA-induced inflammatory cascade leading to obliterative airway disease of the transplanted lungs.

The Selective JAK1/3-Inhibitor R507 Mitigates Obliterative Airway Disease Both With Systemic Administration and Aerosol Inhalation.

The efficacy of selective Janus kinase 1/3 inhibitor R507 to prevent obliterative airway disease was analyzed in preclinical airway transplantation models. Orthotopic trachea transplantations were performed between Lewis donors and Brown Norway rat recipients. Oral everolimus (4 mg/kg once per day) or oral respective inhaled R507 (60 mg/kg twice per day, each) was used for immunosuppression. Grafts were retrieved after 6 or 60 days. Toxicity and anti-inflammatory effects of R507 were analyzed on human airway epithelial cells. In 6-day animals, oral and inhaled R507 more potently diminished mononuclear graft infiltration than everolimus and preserved ciliated pseudostratified columnar respiratory epithelium. Everolimus and R507 similarly suppressed systemic cellular and humoral immune activation. In untreated rats, marked obliterative airway disease developed over 60 days. Oral and inhaled R507 was significantly more effective in reducing airway obliteration and preserved the morphology of the airway epithelium. Luciferase-positive donors revealed that a substantial amount of smooth muscle cells within the obliterative tissue was of donor origin. Only everolimus but not R507, adversely altered kidney function and lipid profiles. The R507 aerosol did not show airway toxicity in vitro but effectively suppressed activation of inflammatory signaling pathways induced by IL-1β. The Janus kinase 1/3 inhibitor R507 is a very well-tolerated immunosuppressant that similarly diminished obliterative airway disease with systemic or inhaled administration.

(500) - CD8+ IL-17 Responses and Obliterative Airway Disease Persist in T-bet-/- Mouse Recipients of Lung Transplant Treated with Low Dose Azithromycin Therapy

(500) - CD8+ IL-17 Responses and Obliterative Airway Disease Persist in T-bet-/- Mouse Recipients of Lung Transplant Treated with Low Dose Azithromycin Therapy

B Cell-Activating Transcription Factor Plays a Critical Role in the Pathogenesis of Anti-Major Histocompatibility Complex-Induced Obliterative Airway Disease.

Antibodies (Abs) against major histocompatibility complex (MHC) results in T helper-17 (Th17)-mediated immunity against lung self-antigens (SAgs), K-α1 tubulin and collagen V and obliterative airway disease (OAD). Because B cell-activating transcription factor (BATF) controls Th17 and autoimmunity, we proposed that BATF may play a critical role in OAD. Anti-H2K(b) was administered intrabronchially into Batf (-/-) and C57BL/6 mice. Histopathology of the lungs on days 30 and 45 after Ab administration to Batf (-/-) mice resulted in decreased cellular infiltration, epithelial metaplasia, fibrosis, and obstruction. There was lack of Abs to SAgs, reduction of Sag-specific interleukin (IL)-17 T cells, IL-6, IL-23, IL-17, IL-1β, fibroblast growth factor-6, and CXCL12 and decreased Janus kinase 2, signal transducer and activator of transcription 3 (STAT3), and retinoid-related orphan receptor γT. Further, micro-RNA (miR)-301a, a regulator of Th17, was reduced in Batf (-/-) mice in contrast to upregulation of miR-301a and downregulation of protein inhibitor of activated STAT3 (PIAS3) in anti-MHC-induced OAD animals. We also demonstrate an increase in miR-301a in the bronchoalveolar lavage cells from lung transplant recipients with Abs to human leukocyte antigen. This was accompanied by reduction in PIAS3 mRNA. Therefore, we conclude that BATF plays a critical role in the immune responses to SAgs and pathogenesis of anti-MHC-induced rejection. Targeting BATF should be considered for preventing chronic rejection after human lung transplantation.

Increased myeloid cell hypoxia-inducible factor-1 delays obliterative airway disease in the mouse

Obliterative bronchiolitis after lung transplantation is characterized by chronic airway inflammation leading to the obliteration of small airways. Hypoxia-inducible factor-1 (HIF-1) is a master regulator of cellular responses to hypoxia and inflammation. The Von Hippel-Lindau protein (pVHL) drives the degradation of oxygen-sensitive subunit HIF-1α that controls the activity of HIF-1. We investigated the effect of myeloid cell-targeted gene deletion of HIF-1α or its negative regulator pVHL on the development of obliterative airway disease (OAD) in the recipients of tracheal allografts, a mouse model for obliterative bronchiolitis after lung transplantation. Tracheal allografts were heterotopically transplanted from BALB/c donor mice to fully major histocompatibility complex-mismatched recipient mice with HIF-1α or VHL gene deletion in myeloid cells. The recipients were left non-immunosuppressed or received tacrolimus daily. Histologic, immunohistochemical, and real-time reverse transcription polymerase chain reaction analyses were performed at 3, 10, and 30 days. In the absence of immunosuppression, myeloid cell-specific VHL deficiency of the recipient mice improved epithelial recovery, decreased inflammatory cell infiltration and expression of pro-inflammatory cytokines, increased regulatory forkhead box P3 messenger RNA expression, and reduced OAD development in tracheal allografts. In the presence of tacrolimus immunosuppression, loss of HIF-1α activity in myeloid cells of the recipient by HIF-1α gene deletion accelerated OAD development in mouse tracheal allografts. Activity of the HIF-pathway affects the development of allograft rejection, and our results suggest that myeloid cell-specific VHL-deficiency that potentially increases HIF-activity decreases allograft inflammation and the subsequent development of OAD in mouse tracheal allografts.

De novo development of dsa and autoantibodies to lung associated self-antigens is associated with upregulation of transcription factor Zbtb7a: Role in chronic rejection following human lung transplantation

Aim Administration of anti-MHC class I (H-2Kb) leads to induction of transcription factor Zinc finger and BTB domain containing 7A (Zbtb7a) leading to inflammatory cascade and chronic rejection in a murine obliterative airway disease model. Development of DSA and Abs to lung-associated self-antigens (K-alpla-1 Tubulin (K α 1T) and Collagen V (Col-V) are significant risk factors in development of bronchiolitis obliterans syndrome (BOS) following human lung transplantation (LTx). The current study focused on the role of Zbtb7a in development of BOS following human LTx. Methods We analyzed 25 LTx patients that developed BOS years after LTx as well as 10 stable LTx performed at Barnes-Jewish Washington University. Kinetics of Zbtb7a expression was studied in bronchoalveolar lavage (BAL) cells. Quantitative PCR analysis was performed to evaluate mRNA levels and data was compared with the DSA, autoantibody and immunosuppression. Results Increases in the Zbtb7a expression was observed in BAL cells collected at visits immediately prior to clinical BOS diagnosis compared with earlier visits. Zbtb7a expression was significantly (p 0.01) increased (150%) in pre-BOS BAL cells. On the contrary, stable patients did not demonstrate any change in Zbtb7a levels. All of the BOS+ patients had developed DSA and Abs to K α 1T and Col-V. Zbtb7a expression in 13 patients who received Ab directed therapy following DSA demonstrated significant reduction along with decrease in DSA and Abs to lung self-antigens. Therefore, Zbtb7a activation is an important inducer of the DSA and autoantibody. Conclusions Up regulation of Zbtb7a was positively associated with de novo DSA and Abs to lung-associated self-antigens. Ab directed therapy significantly reduced Zbtb7a expression. We propose that Zbtb7a may be used as a prognostic marker in evaluating Ab-induced inflammation in the lung as well as efficacy of Ab directed therapy following development of DSA.

OR7 Antibodies to MHC class I induce transcription factor Zbtb7a and regulate development of autoimmunity leading to chronic rejection

Aim Chronic rejection, bronchiolitis obliterans syndrome (BOS), is a major hurdle following human lung transplantation. Antibodies (Ab) to HLA (DSA) and lung-associated antigens (Collagen V (Col-V) and K-alpha-1 Tubulin (K α 1T) have been associated with development of BOS. Our goal was to demonstrate early events (genes and their role in inflammation) associated with administration of anti-MHC class I (H-2 Kb) into murine (C57BL/6) lungs that precede cellular and humoral autoimmunity and chronic rejection. Methods We analyzed molecular signatures arising from anti-MHC administration by genechip microarray. Zbtb7a was selected for functional analysis. siRNA-Lentivirus was used to knockdown Zbtb7a in lungs to study its role in Ab induced chronic rejection. Kinetics of Ab development and T cell responses was tested by ELISA and ELISPOT. Changes in leukocyte profile were analyzed by flow cytometry and gene expression levels by real-time PCR. Results In genechip assay, 12 genes including Zbtb7a were significantly ( p 1.5-fold) at 4 h post Ab administration. Use of siRNA-Lentivirus knocked down Zbtb7a expression. Following anti-MHC I, Zbtb7a knockdown demonstrated significant ( p α 1T and anti-Col-V, and remained free from inflammation and fibrosis. Further, anti-MHC elicited lower K α 1T and Col-V specific Th17 and Th1 cells. Moreover, less infiltration of neutrophil and B cell were seen in lungs and decreased levels of B cell (CXCL13) and neutrophil (CXCL15) chemoattractants were observed. Conclusions We demonstrate that DSA activates unique molecular signature involved in lung autoimmunity. Zbtb7a, as a transcription factor induced by DSA, is a “master regulator” of B and T cell development and has novel inflammatory functions leading to chronic rejection. By targeted knockdown, we established that Zbtb7a has an obligatory role in the amplification of inflammatory circuits where its loss rendered protection from Ab induced obliterative airway disease.

De novo-developed antibodies to donor MHC antigens lead to dysregulation of microRNAs and induction of MHC class II.

Immune responses to HLA and development of anti-donor HLA (DSA) were shown to play a role in chronic rejection following transplantation. We hypothesized that Abs to MHC change microRNAs (miRNAs), leading to chronic lung allograft rejection. Microarray analysis was performed in a murine model of anti-MHC-induced obliterative airway disease (OAD), a correlate of obliterative bronchiolitis. A unique profile of dysregulated miRNAs was detected in OAD mice on days 7 and 15 after Ab administration compared with control. Sixty-seven miRNAs were increased and 42 miRNAs were decreased in OAD mice on day 7. In addition, 15 miRNAs were overexpressed and 16 miRNAs were underexpressed in OAD mice on day 15. The expression of miR-16 and miR-195 was significantly decreased in lungs of OAD mice, as assessed by quantitative RT-PCR and in situ hybridization, with increases in H-2 Aa and H-2 Dma mRNA levels. Significant reductions in miR-16 and miR-195 levels were also noted in lung transplant (LTx) patients with DSA compared with LTx patients without DSA. Bioinformatic TargetScan and reporter assays identified the binding of miR-16 and miR-195 to the 3'-untranslated region of regulatory factor X 5. Quantitative PCR and immunohistochemistry indicated posttranscriptional increases in regulatory factor X 5 mRNA and protein expression in OAD mice, as well as in LTx recipients with DSA, which was associated with increased expression of HLA-DPA1, HLA-DQA1, and HLA-DRA mRNA. Therefore, our results demonstrated that miRNAs induced by alloimmunity may play important roles in chronic rejection after LTx.

A Novel MHC Class I Transgenic Mouse Model With Lung Specific Expression Demonstrates That Antibodies to MHC Class I Induces Epithelial Injury, Autoimmunity and Obliterative Airway Disease (OAD).

Aim: Antibodies (Abs) to mismatched Donor HLA and lung associated self-antigens (SAgs) have been shown to play an important role in immunopathogenesis of chronic rejection (Bronchiolitis Obliterans Syndrome, BOS) following lung transplantation. Antibody mediated epithelial injury has been implicated in development of BOS. Clara cells in the lung epithelium secrete anti-inflammatory Clara Cell Secretory Protein (CCSP) to protect the bronchiolar epithelium. This study is aimed at developing a novel MHC class I transgenic mouse model to elucidate the role of clara cells in inducing epithelial injury following administration of antibodies to MHC Class I. Methods: We developed a transgenic mouse model where lung specific expression of MHC class I is obtained by cloning H2KD gene under CCSP promoter. Anti-MHC or isotype control were administered intravenously. The lungs were harvested on day 15 and 30 for histopathological analysis. Morphometric analysis of cellular infiltration, epithelial abnormalities and fibrosis was determined by H&E and Trichome staining. The alloimmune responses to MHC and the induction of autoimmune responses to lung SAgs (Kα1 Tubulin and Col V) were determined by ELISA. Using ELISpot the frequency of SAg specific IFN-γ, IL-10 and IL-17 secreting T cells were enumerated. Results: The transgenic mouse model demonstrated lung specific expression of H2KD. Intravenous administration of anti-MHC developed cellular infiltration around vessels and bronchiole by day 15 and further increased on day 30, followed by epithelial injury, fibrosis, and occlusion of the distal airways similar to BOS following lung transplantation. This was accompanied by decreased expression of CCSP in the lungs. Mice receiving the anti-H2KD showed increased development of Abs to SAgs. Administration of anti-H2KD also showed increased frequency of IFN-γ, IL-4 and IL-17 secreting T cells against SAgs in the lungs. Conclusion: Using a novel transgenic mouse model with lung specific expression of MHC we demonstrate that anti-MHC induces damage to Clara cells and decrease in CCSP expression resulting in the development of autoimmunity and OAD. This model of OAD will enable us to define the role of Clara cells in antibody mediated cellular injury leading to chronic rejection.

Activation of Eosinophil CCR3 Signaling and Eotaxin Using a Bioinformatics Analysis of a Mouse Model of Obliterative Airway Disease

The role of eosinophils in the development and progression of chronic allograft rejection is recognized in multiple organ transplantation settings. The CCR3 signaling pathway is one of the key regulatory pathways in eosinophil migration to the engrafted tissue. Eotaxin is a ligand for CCR3 and reflects eosinophilic inflammation, which can lead to fibrosis. We hypothesized that the CCR3 pathway would be upregulated in obliterative airway disease (OAD) in an established model of chronic airway allograft rejection. The mouse gene microarray data from a heterotopic mouse model of OAD in the NIH Gene Expression Omnibus (GEO) repository were analyzed for differentially expressed eosinophil pathways, using the Partek Suite and Ingenuity Pathway Analysis. A P value of <0.005 was defined as significant for differential expression, and P value of <0.05 for pathways. Day 25 allografts were defined as chronic allograft rejection and day 4 as acute allograft rejection. The isografts and allografts at day 25 showed significant upregulation of the eosinophil CCR3 pathway (P=0.04), based on the analysis of 1,299 uniquely expressed genes. The isografts at day 4 were compared with those at day 25 based on the identification of 1,859 unique genes, and there was a trend toward the CCR3 pathway upregulation over time (P=0.06). CCR3 pathways were not upregulated during the progression of alloimmune rejection in the allografts at day 4 versus day 25 in comparison, based on the analysis of 1,603 genes. Eotaxin was upregulated in chronic allograft rejection by 2.5-fold. The eosinophil signaling pathway CCR3 and eotaxin were significantly expressed in chronic allograft rejection and our results imply a role in controlling early alloimmune damage in controls.

(250) - Ligation of Transgenic MHC Class I Molecule Expressed Only in the Lungs By Its Specific Antibodies Induces Epithelial Injury, Autoimmunity and Obliterative Airway Disease (OAD): A Novel Transgenic Mouse Model of OAD

(250) - Ligation of Transgenic MHC Class I Molecule Expressed Only in the Lungs By Its Specific Antibodies Induces Epithelial Injury, Autoimmunity and Obliterative Airway Disease (OAD): A Novel Transgenic Mouse Model of OAD