Human Lung Transplantation Research Articles

Elevated CXCL10 (IP-10) in Bronchoalveolar Lavage Fluid Is Associated With Acute Cellular Rejection After Human Lung Transplantation

CXCL10 (IP-10) is a potent chemoattractant for T cells that has been postulated to play a role in infection and acute cellular rejection (ACR) in animal models. We measured CXCL10 (IP-10) (and other cytokines previously implicated in the pathogenesis of ACR) in the bronchoalveolar lavage (BAL) of lung transplant recipients (LTRs) to determine the association between CXCL10 (IP-10) and ACR in LTRs. In a prospective study of 85 LTRs, expression of cytokines (tumor necrosis factor, interferon-γ, interleukin [IL]-6, IL-8, IL-15, IL-16, IL-17, CXCL10 [IP-10], and MCP-1 [CCL2]) in BAL samples (n=233) from patients with episodes of ACR (n=44), infection ("Infect"; n=25), concomitant "Infect+ACR" (n=10), and "No Infect and No ACR" (n=154) were analyzed. The levels of both CXCL10 (IP-10) and IL-16 were significantly increased in histologically proven ACR compared with the "No Infect and No ACR" group (CXCL10 [IP-10]: 107.0 vs. 31.9 pg/mL [P=0.001] and IL-16: 472.1 vs. 283.01 pg/mL [P=0.01]). However, in a linear mixed-effects model, significant association was found only between CXCL10 (IP-10) and ACR. A one-log increase of CXCL10 (IP-10) was associated with a 40% higher risk of ACR (odds ratio, 1.4; 95% confidence interval, 1.12-1.84). Higher values of CXCL10 (IP-10) in BAL fluid are associated with ACR in LTRs, suggesting a potential mechanistic role in the pathogenesis of ACR in LTRs. These results suggest that therapeutic strategies to inhibit CXCL10 (IP-10) and or its cognate receptor, CXCR3, warrant investigation to prevent and/or treat ACR in clinical lung transplantation.

Role of defensins in the pathogenesis of chronic lung allograft rejection

Chronic rejection predominantly manifested as bronchiolitis obliterans syndrome (BOS), still remains a major problem affecting long-term outcomes in human lung transplantation (LTx). Donor specific antibodies (DSA) and infiltration of neutrophils in the graft have been associated with the development of BOS. This study determines the role of defensins, produced by neutrophils, and its interaction with α-1-antitrypsin (AAT) towards induction of airway inflammation and fibrosis which are characteristic hallmarks of BOS. Bronchoalveolar lavage (BAL) and serum from LTx recipients, BOS+ (n=28), BOS− (n=26) and normal healthy controls (n=24) were analyzed. Our results show that BOS+ LTx recipients had higher α-defensins (HNP1–3) and β-defensin2 HBD2 concentration in BAL and serum compared to BOS-DSA-recipients and normal controls (p=0.03). BOS+ patients had significantly lower serum AAT along with higher circulating concentration of HNP–AAT complexes in BAL (p=0.05). Stimulation of primary small airway epithelial cells (SAECs) with HNPs induced expression of HBD2, adhesion molecules (ICAM and VCAM), cytokines (IL-6, IL-1β, IL-13, IL-8 and MCP-1) and growth-factor (VEGF and EGF). In contrast, anti-inflammatory cytokine, IL-10 expression decreased 2-fold (p=0.002). HNPs mediated SAEC activation was completely abrogated by AAT. In conclusion, our results demonstrates that neutrophil secretory product, α-defensins, stimulate β-defensin production by SAECs causing upregulation of pro-inflammatory and pro-fibrotic signaling molecules. Hence, chronic stimulation of airway epithelial cells by defensins can lead to inflammation and fibrosis the central events in the development of BOS following LTx.

α1-Antitrypsin inhibits ischemia reperfusion-induced lung injury by reducing inflammatory response and cell death

Pulmonary ischemia-reperfusion (IR)-induced lung injury is a severe complication that increases the likelihood of primary graft dysfunction and early death after lung transplantation. Inflammatory cytokine release and cell death play a critical role in the development of IR-induced lung injury. α1-Antitrypsin (A1AT) is a protease inhibitor clinically used for the treatment of A1AT-deficiency emphysema. On the basis of a literature review, we hypothesize that A1AT may have the potential to reduce IR-induced lung injury through its anti-inflammatory and anti-apoptotic effects. A human pulmonary cell culture model was used to simulate IR processes in lung transplantation. Effects of A1AT on cell death and cytokine production were examined. A rat pulmonary IR model, in which the left pulmonary hilum was clamped for 90 minutes, followed by reperfusion for 2 hours, was used to determine the effects of A1AT on acute lung injury, function, cell death, and inflammatory response. A1AT significantly inhibited cell death and inflammatory cytokine release dose-dependently in vitro and significantly improved lung oxygenation and lung mechanics and reduced pulmonary edema in vivo. Moreover, A1AT inhibited neutrophil infiltration in the lung and reduced cell death and significantly reduced IR-induced inflammatory mediators in plasma, including interleukin (IL)-1α, IL-4, IL-12p70, monocyte chemotactic protein 1, and tumor necrosis factor-α. Considering its current clinical use, our findings indicate that administration of A1AT may be an effective and safe therapy for the treatment of IR injury in human lung transplantation.

Promotion of airway anastomotic microvascular regeneration and alleviation of airway ischemia by deferoxamine nanoparticles

Airway tissue ischemia and hypoxia in human lung transplantation is a consequence of the sacrifice of the bronchial circulation during the surgical procedure and is a major risk factor for the development of airway anastomotic complications. Augmented expression of hypoxia-inducible factor (HIF)-1α promotes microvascular repair and alleviates allograft ischemia and hypoxia. Deferoxamine mesylate (DFO) is an FDA-approved iron chelator which has been shown to upregulate cellular HIF-1α. Here, we developed a nanoparticle formulation of DFO that can be topically applied to airway transplants at the time of surgery. In a mouse orthotopic tracheal transplant (OTT) model, the DFO nanoparticle was highly effective in enhancing airway microvascular perfusion following transplantation through the production of the angiogenic factors, placental growth factor (PLGF) and stromal cell-derived factor (SDF)-1. The endothelial cells in DFO treated airways displayed higher levels of p-eNOS and Ki67, less apoptosis, and decreased production of perivascular reactive oxygen species (ROS) compared to vehicle-treated airways. In summary, a DFO formulation topically-applied at the time of surgery successfully augmented airway anastomotic microvascular regeneration and the repair of alloimmune-injured microvasculature. This approach may be an effective topical transplant-conditioning therapy for preventing airway complications following clinical lung transplantation.

A relevant experimental model for human bronchiolitis obliterans syndrome

The long-term success of human lung transplantation is limited by the development of bronchiolitis obliterans syndrome. Acute rejection episodes and infections are important risk factors and seem to play major pathogenic roles. We established a relevant experimental model that mimics important aspects of human bronchiolitis obliterans syndrome. The Fischer 344-to-Lewis rat strain combination was used for orthotopic left lung transplantation. Isogeneic transplantations were performed in the Lewis rat. Recipients were treated with ciclosporin for 10 days. Lipopolysaccharide or vehicle was instilled into the airways 28 days after transplantation. Grafts were monitored by computed tomography, and recipients were euthanized on Days 28-90. The messenger RNA expression of selected chemokines and their receptors was measured on Days 28, 29, 33, 40 after transplantation. Graft histopathology on Day 90 was compared with lungs from patients who underwent re-transplantation due to end-stage allograft dysfunction. Lung allografts treated with ciclosporin and vehicle only sporadically displayed tissue remodeling. In contrast, lipopolysaccharide treatment induced severe inflammation. In the long-term, severe vascular remodeling, lung fibrosis, and fibroproliferative remodeling of airways were found that closely resemble the histopathologic changes in grafts from human patients with bronchiolitis obliterans syndrome. Chronic damage was virtually absent from pulmonary isografts and native right lungs. Chemokine (C-C motif) ligand 5 and chemokine (C-X-C motif) ligand 9-11, and their receptors, were over-expressed in allografts. Our experimental model mirrors key aspects of human bronchiolitis obliterans syndrome. It will be useful to elucidate its pathogenesis and to develop therapeutic approaches improving the long-term outcome of human lung transplantation.

MicroRNAs implicated in dysregulation of gene expression following human lung transplantation

BackgroundLung transplantation remains the only viable treatment option for the majority of patients with advanced lung diseases. However, 5-year post-transplant survival rates remain low primarily secondary to chronic rejection. Novel insights from global gene expression profiles may provide molecular phenotypes and therapeutic targets to improve outcomes after lung transplantation.MethodsWhole-genome gene expression profiling was performed in a cohort of patients that underwent lung transplantation as well as healthy controls using the Affymetrix Human Exon 1.0ST Array. To explore the potential roles of microRNAs (miRNAs) in regulating lung transplantation-associated gene dysregulation, miRNA expression levels were also profiled in the same samples using the Exiqon miRCURY LNA Array.ResultsIn a cohort of 18 lung transplant patients, 364 dysregulated genes were identified in Caucasian patients relative to normal individuals without pulmonary disorders. Pathway enrichment analysis of the dysregulated genes pointed to Gene Ontology biological processes such as “defense response”, “immune response” and “response to wounding”. We then compared the expression profiles of potential regulating miRNAs, suggesting that dysregulation of a number of lung transplantation-associated genes (e.g., ATR, FUT8, LRRC8B, NFKBIA) may be attributed to the dysregulation of their respective regulating miRNAs.ConclusionsFollowing human lung transplantation, a substantial proportion of genes, particularly those genes involved in certain biological processes like immune response, were dysregulated in patients relative to their healthy counterparts. This exploratory analysis of the relationships between miRNAs and their gene targets in the context of lung transplantation warrants further investigation and may serve as novel therapeutic targets in lung transplant complications.Electronic supplementary materialThe online version of this article (doi:10.1186/2213-0802-1-12) contains supplementary material, which is available to authorized users.

Antibodies to Lung Self-Antigens (Ka1 Tubulin or Collagen V) Lead to Epitope Spreading, Loss of Tolerance and Rejection of CTLA4/MR1 Induced Tolerant Murine Lung Allograft

Purpose Immune responses against lung self-antigens (SAg) [Kα1tubulin (Kα1T) and Collagen V (ColV)] are critical in the pathogenesis of chronic rejection in human lung transplantation (LT). Our goal was to determine the mechanisms by which antibodies (Abs) to SAgs lead to rejection in co-stimulatory blockade induced tolerant murine LT. Methods and Materials LT was done from Balb/c to Bl/6 and tolerance was induced with MR1 (250μg) and CTLA4-Ig (200μg) on post-LT days 1 and 2. Abs to Kα1T or ColV or both (200μg/dose, i/p) or isotype control were given on days -10, -2, 0 & weekly following LT. Lungs were analyzed by H&E, trichrome and immunostains. Kα1T and ColV specific IFN-γ, IL-10, IL-4 and IL-17 cells were enumerated by ELISpot. Abs to allo-MHC was determined by FACS. Cytokine and growth factors in lung were done by RTPCR. Results Allo-LT with co-stimulatory blockade did not show evidence of rejection for >100 days. However, administration of Abs to Kα1T or ColV or both resulted in loss of tolerance and rejection (cellular infiltration, airway remodeling) by day 45-60. Isotype controls did not demonstrate rejection pathology. Abs to SAgs in tolerant mice led to Infiltration of CD4 and CD8 T cells, B-cells and neutrophils, increase in SAg specific T-cells secreting IFNγ (p=0.002), IL-4 (p=0.047) and IL-17 (p=0.001) and decrease in IL-10 (p=0.03). Abs to either SAgs resulted in immune responses to both the SAgs and donor specific Abs to allo-MHC (MFI>2000 against Balb/c - H2kd), demonstrating epitope spreading to other SAgs and linked allo-MHC. Upregulation of IL-6 (7.1 fold), IL-1β (17 fold), IFN-γ(4.8 fold), IL-17 (10.1 fold), VEGF (2.9 fold), TGF-β (3.8 fold) was also noted in the lung tissue. Conclusions Administration of Abs to lung Sags resulted in epitope spreading of immune responses not only to Sags but also to the linked allo-MHC leading to loss of tolerance induced by co-stimulatory blockade.

Cross-Reactive Anti-Viral T Cells Increase Prior to an Episode of Viral Reactivation Post Human Lung Transplantation

Human Cytomegalovirus (CMV) reactivation continues to influence lung transplant outcomes. Cross-reactivity of anti-viral memory T cells against donor human leukocyte antigens (HLA) may be a contributing factor. We identified cross-reactive HLA-A*02:01-restricted CMV-specific cytotoxic T lymphocytes (CTL) co-recognizing the NLVPMVATV (NLV) epitope and HLA-B27. NLV-specific CD8+ T cells were expanded for 13 days from 14 HLA-A*02:01/CMV seropositive healthy donors and 11 lung transplant recipients (LTR) then assessed for the production of IFN-γ and CD107a expression in response to 19 cell lines expressing either single HLA-A or -B class I molecules. In one healthy individual, we observed functional and proliferative cross-reactivity in response to B*27:05 alloantigen, representing approximately 5% of the NLV-specific CTL population. Similar patterns were also observed in one LTR receiving a B27 allograft, revealing that the cross-reactive NLV-specific CTL gradually increased (days 13–193 post-transplant) before a CMV reactivation event (day 270) and reduced to basal levels following viral clearance (day 909). Lung function remained stable with no acute rejection episodes being reported up to 3 years post-transplant. Individualized immunological monitoring of cross-reactive anti-viral T cells will provide further insights into their effects on the allograft and an opportunity to predict sub-clinical CMV reactivation events and immunopathological complications.

Critical Role for IL-17A/F in the Immunopathogenesis of Obliterative Airway Disease Induced by Anti-MHC I Antibodies

The IL-17 axis is implicated in pathogenesis of chronic rejection after human lung transplantation. Using a murine model of obliterative airway disease (OAD), we recently demonstrated that Abs to MHC class I antigens can induce immune responses to self-antigens that contributes to immunopathogenesis of chronic rejection. Using a murine model of OAD, we determined the role of IL-17 family members in induction of autoimmunity leading to OAD after ligation of MHC class I. Anti-MHC class I or control antibodies (Abs) were administered intrabronchially to wild-type (WT) and IL-17a knock out (IL-17A-/-) C57BL/6. By day 30, anti-MHC I administered endobronchially in IL-17A-/- mice demonstrated significant reduction in cellular infiltration, a 36.8% reduction in CD4 T cells, 62.7% in CD11b macrophages, 37.5% in degree of fibrosis, 1.94 fold and 2.17 fold decrease in anti-KAT and anti-Col-V, respectively, when compared with wild-type mice. Analysis of lung infiltrating cells in anti-MHC I WT revealed increase in IL-17A (KAT:92+21,Col-V:103+19spm) and IL-17F (KAT:5.03%,Col-V:2.75%) secreting CD4+ T cells. However, administration of anti-MHC I in IL-17A-/- demonstrated increase only in IL-17F for KAT (13.70%) and Col-V (7.08%). Anti-IL-17(A-F) mAb administration after anti-MHC I abrogated OAD in both WT and IL-17A-/-. Our findings indicate that IL-17A and IL-17F secreted by CD4+Th17 cells specific to lung self-antigens are critical mediators of autoimmunity leading to the pathogenesis of OAD.

Experimental models of lung transplantation

Lung transplantation is a life saving treatment for end stage pulmonary diseases. The development and refinement of this therapy required the utilization of various animal models, without which this procedure would not have become a clinical reality. Canine models were critical in the initial breakthroughs in surgical technique and immunosuppressive regimens, which directly led to the first successful human lung transplantation. Orthotopic lung transplant models in the rat provided a platform for more detailed investigation of immune responses to pulmonary grafts. Investigation of chronic rejection of lungs has significantly been advanced through the use of mouse tracheal transplant experiments. And finally, the advent of orthotopic, vascularized lung transplantation in the mouse opened the door to the use of genetic and molecular tools that are necessary for the rigorous mechanistic study of alloimmune and non-alloimmune factors contributing to lung graft failure. Taken together, animal models will continue to be a cornerstone in the advancement of clinical success in lung transplantation.

Cross-Reactive CMV-Specific T Cells Significantly Increase in Frequency and TCR Clonality in the Setting of Viral Reactivation in Human Lung Transplantation

Introduction: Cross-reactive anti-viral memory T cells constitute a significant proportion of the alloresponse and potentially play a pivotal role in mediating adverse post-transplant outcomes in HLA mismatched allografts. In addition, chronic DNA viruses such as cytomegalovirus (CMV) commonly reactivate and continue to influence episodes of post-transplant morbidity and mortality. This study identified a novel cross-reactive HLA-A2-restricted CMV-specific CD8+ T cell recognising both the CMV epitope NLVPMVATV (NLV) and the HLA-B27 alloantigen. We examine the functionality and TCR clonal distribution dynamics of cross-reactive NLV-specific CD8+ T cells both preceding and following CMV reactivation and their role in mediating allograft dysfunction. Method: NLV-specific CD8+ T cell expansion was performed on HLA-A2/CMV healthy donors (n=14) and LTR (n=11) by stimulating peripheral blood mononuclear cells with NLV peptide for 13 days. Allo-reactivity of NLV-specific CD8+ T cells was assessed using single expressing HLA class I-specific transfected cell lines and multiparameter flow cytometry which included A2/NLV-tetramer staining (specificity), IFN-γ production (cytolytic ability), CFSE (proliferation) and CD107a expression (cytotoxicity). Additionally, paired TCR variable (V) alpha and beta chains from the complementarity-determining region (CDR3) of cross-reactive NLV-specific CD8+ T cells were characterised using novel single-cell multiplex nested RT-PCR for simultaneous detection of V alpha and V beta chains. The dynamics of cross-reactive NLV-specific CD8+ T cells were then analysed in relation to the patient's CMV viral loads, lung function and acute rejection events. Results: NLV-specific CD8+ T cells from 3 healthy individuals underwent initial screening for allo-reactivity using cell lines encompassing 19 HLA-A and -B alleles. Cross-reactivity was observed in one individual based on proliferation, cytokine and cytolytic activity in response to B*2705 alloantigen, representing approximately 5% of the NLV-specific CD8+ T cell population. Further investigation elucidated a hierarchical response in allelic variation of B27 (B*2709>B*2705>B*2703). Similar patterns were observed in one LTR receiving a B27 allograft and we show that the cross-reactive A2/NLV-specific CD8+ T cells gradually increased (days 13-193 post-transplant) prior to a clinically detectable CMV reactivation event (day 270) and these cross-reactive cells reduced to basal levels following viral clearance on day 909. The overall NLV-specific TCR repertoire was dominated by one TCR clone (81%), but upon stimulation with HLA-B27, the main NLV-specific TCR repertoire shifted to 100% clonality for a second cross-reactive clonotype. However, following viral clearance the TCR repertoire greatly diversified in both the NLV (70%) and B27-cross-reactive pools (53%). Conclusion: Individualised monitoring of cross-reactive antiviral T cells and their TCR repertoire will provide further insights into the potential interaction between sub-clinical CMV reactivation and long term allograft outcomes.

MicroRNA 144 Involved in the Modulation of TGF-b Signaling and Fibrosis Is Upregulated in Lung Biopsies from Transplant Recipients Undergoing Chronic Rejection - Bronchiolitis Obliterans Syndrome (BOS)

Introduction: Development of BOS following human lung transplantation (LTx) remains a major impediment for long term function of the transplanted lungs. BOS is a fibroproliferative process resulting in progressive decline in pulmonary function and eventual allograft failure. The immune mechanisms leading to the development of BOS remains poorly defined. MicroRNAs (miR), through the regulation of gene expression, control various biological processes. Specifically, miR 144 has been reported to upregulate TGF-b signaling leading to fibrosis and therefore may play a significant role in the pathogenesis of BOS. Objective: The objective of this study was to determine whether miR 144 is upregulated in the lung biopsies from LTx recipients diagnosed with BOS and to define the role of miR 144 in the development of BOS following human LTx. Methods: Serum concentrations of TGF-β1 and IL-6 were determined by ELISA. Levels of miR 144 in the lung biopsies of BOS+ and BOS-LTx recipients (n=15 each) were analyzed by quantitative real time PCR (qPCR). The levels of downstream targets of miR 144 TGFB-induced factor homeobox 1 (TGIF-1), SMAD, TGF-b1, IL-6, and connective tissue growth factor (CTGF) were determined by qPCR using gene specific primers. Results: Analysis of the serum levels of TGF-β1 and IL-6 demonstrated a significant increase in the levels of TGF-β1 (430 vs 110 ng/ml, p< 0.05) and IL-6 (278 vs 63 ng/ml, p< 0.05) levels in BOS+ patients. Based on this we analyzed miR 144 levels in the lung biopsies from a different cohort of LTx which showed significant increase (3.4±1.2 fold) in the levels of miR 144 in the lung biopsies of BOS+ LTx when compared to the BOS- LTx (p< 0.05). qPCR analysis for the down stream targets also demonstrated significant decrease in the expression of TGIF (3.8±0.9 fold) and marked increases in SMAD (4.3±1.2 fold), TGFβ1 (5.3 fold increase), IL-6 (2.9±0.9 fold increase) and CTGF (3.9±1.1 fold increase) in comparison to BOS- LTx recipients. Analysis of TGF-β and IL-6 in serum samples from part of this cohort of BOS+ LTx also demonstrated significant increases in the levels of TGF-β (262ng/ml) and IL-6 (62.6ng/ml). Conclusion: Our results demonstrated increased levels of miR 144 in the lung biopsies from BOS+ LTx recipients. We propose that miR144 can down regulate the expression of TGIF-1, a negative regulator of SMAD which is critical for the TGF-β signaling cascade. This will result in increased expression of TGF-β1, IL-6 and CTGF which can promote the development of fibrosis, a hallmark of the lesion for the development of BOS in LTx recipients. Therefore, agents that can target miR 144 may aid in the prevention of fibrosis development and therefore BOS following LTx.

Microarray Meta-Analysis Identifies Acute Lung Injury Biomarkers in Donor Lungs That Predict Development of Primary Graft Failure in Recipients

ObjectivesTo perform a meta-analysis of gene expression microarray data from animal studies of lung injury, and to identify an injury-specific gene expression signature capable of predicting the development of lung injury in humans.MethodsWe performed a microarray meta-analysis using 77 microarray chips across six platforms, two species and different animal lung injury models exposed to lung injury with or/and without mechanical ventilation. Individual gene chips were classified and grouped based on the strategy used to induce lung injury. Effect size (change in gene expression) was calculated between non-injurious and injurious conditions comparing two main strategies to pool chips: (1) one-hit and (2) two-hit lung injury models. A random effects model was used to integrate individual effect sizes calculated from each experiment. Classification models were built using the gene expression signatures generated by the meta-analysis to predict the development of lung injury in human lung transplant recipients.ResultsTwo injury-specific lists of differentially expressed genes generated from our meta-analysis of lung injury models were validated using external data sets and prospective data from animal models of ventilator-induced lung injury (VILI). Pathway analysis of gene sets revealed that both new and previously implicated VILI-related pathways are enriched with differentially regulated genes. Classification model based on gene expression signatures identified in animal models of lung injury predicted development of primary graft failure (PGF) in lung transplant recipients with larger than 80% accuracy based upon injury profiles from transplant donors. We also found that better classifier performance can be achieved by using meta-analysis to identify differentially-expressed genes than using single study-based differential analysis.ConclusionTaken together, our data suggests that microarray analysis of gene expression data allows for the detection of “injury" gene predictors that can classify lung injury samples and identify patients at risk for clinically relevant lung injury complications.

T Regulatory Cells Play a Significant Role in Modulating MHC Class I Antibody-Induced Obliterative Airway Disease

The molecular mechanisms leading to the development of chronic lung allograft dysfunction following de novo development of antibodies to mismatched donor MHC remain undefined. We demonstrated that intrabronchial administration of antibodies to MHC class I resulted in induction of both innate and adaptive cellular immune responses characterized by a predominance of Th17 specific to lung associated self-antigens Kα1-tubulin and Collagen-V leading to the development of obliterative airway lesions (OAD), correlate of chronic rejection following human lung transplantation. To determine the role of regulatory T cells (Treg) in the pathogenesis of OAD, we administered anti-MHC class I to mice, in which Treg were depleted by conditional ablation of FoxP3+cells. Under this condition, we observed a threefold increase in pulmonary cellular infiltration, luminal occlusion and fibrous deposition when compared anti-MHC class I Ab administered mice maintaining FoxP3. OAD lesions were accompanied with enhanced accumulation of neutrophils along with self-antigen-specific Th17 and humoral responses. However, IL-17-blockade or adoptive transfer of Treg abrogated OAD. We conclude that Treg exerts a suppressive effect on anti-MHC induced IL-8-mediated neutrophil infiltration and innate immune responses that leads to inhibition of Th17 immune responses to lung associated self-antigens which is critical for development of OAD.

A Pilot Study to Examine the Effect of Chronic Treatment with Immunosuppressive Drugs on Mucociliary Clearance in a Vagotomized Murine Model

BackgroundPreviously, we have demonstrated that mucociliary clearance (MCC) is diminished within the first months after surgery in lung transplant patients and the explanation for the reduction in MCC is unknown. We hypothesized that chronic treatment with a commonly prescribed regimen of immunosuppressive drugs significantly impairs MCC. We tested this hypothesis in a murine model of lung transplantation.MethodsFifteen C57BL/6 mice underwent vagotomy on the right side to simulate denervation associated with lung transplantation in humans. For 6 days, seven mice (controls) were intraperitoneally injected with three 100 µL doses of phosphate buffered saline and eight mice (immunosuppressed) were injected with three 100 µL injections of tacrolimus (1 mg/kg), mycophenolate mofetil (30 mg/kg), and prednisone (2 mg/kg) once daily. Then, mice inhaled the radioisotope 99mtechnetium and underwent gamma camera imaging of their lungs for 6.5 hrs. Counts in the right lung at 1–1.5 hrs and at 6–6.5 hrs were first background-corrected and then decay-corrected to time 0 counts. Decay-corrected counts were then divided by time 0 counts. Retention at each time point was subtracted from 1.00 and multiplied by 100% to obtain percent removed by mucociliary clearance.ResultsAlthough there was a slowing of MCC at 1–1.5 hrs for the immunosuppressed mice, there was no statistical difference in MCC measured at 1–1.5 hrs for the two groups of mice. At 6–6.5 hrs, MCC was significantly slower in the immunosuppressed mice, compared to controls, with 7.78±5.9% cleared versus 23.01±11.7% cleared, respectively (p = 0.006).ConclusionsThese preliminary results suggest that chronic treatment with immunosuppressive medications significantly slows MCC in vagotomized C57BL/6 mice. These findings could shed light on why MCC is reduced in lung transplant patients whose lungs are denervated during surgery and who are chronically treated with immunosuppressive drugs post surgery.

Complement activation is not required for obliterative airway disease induced by antibodies to major histocompatibility complex class I: Implications for chronic lung rejection

The role of non-complement activating antibodies (ncAbs) to mismatched donor human leukocyte antigen (HLA) in the pathogenesis of chronic lung rejection is not known. We used a murine model of obliterative airway disease (OAD) induced by Abs to major histocompatibility major histocompatibility complex (MHC) class I and serum from donor-specific Abs developed in human lung transplant (LTx) recipients to test the role of ncAbs in the development of OAD and bronchiolitis obliterans syndrome (BOS). Anti-MHC ncAbs were administered intrabronchially in B.10 mice or in C3 knockout (C3KO) mice. Lungs were analyzed by histopathology. Lymphocytes secreting interleukin (IL)-17, interferon-γ, or IL-10 to collagen V and K-α1 tubulin (Kα1T) were enumerated by enzyme-linked immunospot assay. Serum antibodies to collagen V and Kα1T were determined by enzyme-linked immunosorbent assay. Cytokine and growth factor expression in lungs was determined by real-time polymerase chain reaction. Donor-specific Abs from patients with BOS and control BOS-negative LTx recipients were analyzed by C1q assay. Administration of ncAbs in B.10 mice or C3KO resulted in OAD lesions. There were significant increases in IL-17- and interferon-γ-secreting cells to collagen V and Kα1T, along with serum Abs to these antigens. There was also augmented expression of monocyte chemotactic protein-1, IL-6, IL-1β, vascular endothelial growth factor, transforming growth factor-β, and fibroblastic growth factor in mice administered ncAbs by Day 3. Among 5 LTx recipients with BOS, only 1 had C1q binding donor-specific Abs. Complement activation by Abs to MHC class I is not required for development of OAD and human BOS. Therefore, anti-MHC binding to epithelial and endothelial cells can directly activate pro-fibrotic and pro-inflammatory cascades leading to immune response to self-antigens and chronic rejection.

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Our previous results demonstrated that 200 μg complement fixing antibodies (Abs) to MHC class I (cfAbs) or non-complement fixing Abs (ncAbs) result in obliterative airway disease (OAD) in mouse model of intrabronchial anti-MHC administration. In human lung transplantation, recipients may develop both complement fixing and non-complement fixing DSA that may augment chronic rejection. The aim of this study was to test if low levels of cfAbs and ncAbs can synergize leading to OAD. Low dose anti-MHC class I ncAbs mouse IgG1 (100 μg) or cfAbs IgG2a (25 μg) were given alone or in combination intrabronchially in F1 C57Bl/6 × B.10 mice on day 0,1,2,6 and weekly after. Isotype IgG were given in control. Lungs analyzed by histopathology. Serum tested for Abs to Kalpha1Tubulin (K α 1T) and collagenV (ColV) by ELISA. IFNγ, IL17 and IL10 cells specific to Kα1T and ColV enumerated by ELISPOT. Cytokines and growth factors in lung determined by RTPCR. Low dose of ncAbs (100 μg) or cfAbs (25 μg) alone did not lead to OAD including isotype control. Combination of ncAbs and cfAbs at same low dose resulted in OAD with cellular infiltration, fibrosis and luminal occlusion of vessels and bronchioles. Mice given both ncAbs and cfAbs developed serum Abs to Kα1T (667 ± 63 μg/mL, p = 0.065) and ColV (323 ± 41, p = 0.0034) compared to ncAbs alone, cfAbs alone or isotype mice. This was associated with increased IFNγ (p = 0.002), IL17 (p = 0.004) specific to Kα1T and ColV and suppression of IL10 compared to other groups. Further, there was upregulation of TGFβ, MCP-1, IL-6, IL-1β and VEGF. High dose ncAbs and cfAbs to MHC lead to OAD, but low dose of ncAbs or cfAbs given alone did not induce OAD. However, low levels of ncAbs and cfAbs in combination, can synergize and potentiate each other resulting in activation of inflammatory and fibrotic cascades leading to OAD. Hence, in human lung transplant recipients low titers of de novo DSA which are cfAbs and ncAbs may synergize augmenting chronic lung allograft rejection.

A Nonhuman Primate Model of Lung Regeneration: Detergent-Mediated Decellularization and Initial In Vitro Recellularization with Mesenchymal Stem Cells

Currently, patients with end-stage lung disease are limited to lung transplantation as their only treatment option. Unfortunately, the lungs available for transplantation are few. Moreover, transplant recipients require life-long immune suppression to tolerate the transplanted lung. A promising alternative therapeutic strategy is decellularization of whole lungs, which permits the isolation of an intact scaffold comprised of innate extracellular matrix (ECM) that can theoretically be recellularized with autologous stem or progenitor cells to yield a functional lung. Nonhuman primates (NHP) provide a highly relevant preclinical model with which to assess the feasibility of recellularized lung scaffolds for human lung transplantation. Our laboratory has successfully accomplished lung decellularization and initial stem cell inoculation of the resulting ECM scaffold in an NHP model. Decellularization of normal adult rhesus macaque lungs as well as the biology of the resulting acellular matrix have been extensively characterized. Acellular NHP matrices retained the anatomical and ultrastructural properties of native lungs with minimal effect on the content, organization, and appearance of ECM components, including collagen types I and IV, laminin, fibronectin, and sulfated glycosaminoglycans (GAG), due to decellularization. Proteomics analysis showed enrichment of ECM proteins in total tissue extracts due to the removal of cells and cellular proteins by decellularization. Cellular DNA was effectively removed after decellularization (∼92% reduction), and the remaining nuclear material was found to be highly disorganized, very-low-molecular-weight fragments. Both bone marrow- and adipose-derived mesenchymal stem cells (MSC) attach to the decellularized lung matrix and can be maintained within this environment in vitro, suggesting that these cells may be promising candidates and useful tools for lung regeneration. Analysis of decellularized lung slice cultures to which MSC were seeded showed that the cells attached to the decellularized matrix, elongated, and proliferated in culture. Future investigations will focus on optimizing the recellularization of NHP lung scaffolds toward the goal of regenerating pulmonary tissue. Bringing this technology to eventual human clinical application will provide patients with an alternative therapeutic strategy as well as significantly reduce the demand for transplantable organs and patient wait-list time.

Lung transplant acceptance is facilitated by early events in the graft and is associated with lymphoid neogenesis.

Early immune responses are important in shaping long-term outcomes of human lung transplants. To examine the role of early immune responses in lung rejection and acceptance, we developed a method to retransplant mouse lungs. Retransplantation into T-cell-deficient hosts showed that for lungs and hearts alloimmune responses occurring within 72 h of transplantation are reversible. In contrast to hearts, a 72-h period of immunosuppression with costimulation blockade in primary allogeneic recipients suffices to prevent rejection of lungs upon retransplantation into untreated allogeneic hosts. Long-term lung acceptance is associated with induction of bronchus-associated lymphoid tissue, where Foxp3(+) cells accumulate and recipient T cells interact with CD11c(+) dendritic cells. Acceptance of retransplanted lung allografts is abrogated by treatment of immunosuppressed primary recipients with anti-CD25 antibodies. Thus, events contributing to lung transplant acceptance are established early in the graft and induction of bronchus-associated lymphoid tissue can be associated with an immune quiescent state.

524 The Role of Lymphotoxin beta Receptor in De Novo Formation of Lymphoid Tissue in Chronic Graft Dysfunction after Lung Transplantation

Lymphoid neogenesis is de novo formation of lymph-node-like tissue observed in many chronic inflammatory disorders. In human lung transplantation, lymphoid neogenesis is found in transplanted lungs affected by chronic graft dysfunction represented by bronchiolitis obliterans syndrome. Lymphotoxin beta receptor (LTbR) is expressed by activated stromal cells forming lymphoid-like stroma involved in lymphoid neogenesis. The purpose of the study is to determine the role of LTbR pathway in the formation of lymphoid-like stroma in chronic graft dysfunction after lung transplantation.