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Background: Chronic lung allograft dysfunction (CLAD) and its obstructive form, the obliterative bronchiolitis (OB), are the main long-term complications related to high mortality rate postlung transplantation. CLAD treatment lacks a significant success in survival. Here, we investigated a new strategy through inhibition of the proinflammatory mitogen- and stress-activated kinase 1 (MSK1) kinase., Methods: MSK1 expression was assessed in a mouse OB model after heterotopic tracheal allotransplantation. Pharmacological inhibition of MSK1 (H89, fasudil, PHA767491) was evaluated in the murine model and in a translational model using human lung primary fibroblasts in proinflammatory conditions. MSK1 expression was graded over time in biopsies from a cohort of CLAD patients., Results: MSK1 mRNA progressively increased during OB (6.4-fold at D21 posttransplantation). Inhibition of MSK1 allowed to counteract the damage to the epithelium (56% restoration for H89), and abolished the recruitment of MHCII+ (94%) and T cells (100%) at the early inflammatory phase of OB. In addition, it markedly decreased the late fibroproliferative obstruction in allografts (48%). MSK1 inhibitors decreased production of IL-6 (whose transcription is under the control of MSK1) released from human lung fibroblasts (96%). Finally, we confirmed occurrence of a 2.9-fold increased MSK1 mRNA expression in lung biopsies in patients at 6 months before CLAD diagnosis as compared to recipients with stable lung function., Conclusions: These findings suggest the overall interest of the MSK1 kinase either as a marker or as a potential therapeutic target in lung dysfunction posttransplantation., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Bronchiolitis obliterans syndrome (BOS), the major cause of death on lung transplantation, is characterized by bronchiolar inflammation and tissue remodeling. Matrix metalloproteinases (MMPs) have been implicated in these processes, although it is still unclear whether MMP activity and binding to their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), is abnormal in BOS., Methods: We studied total MMP-1,-2,-3,-7,-8,-9,-12,-13 levels, their activity state using activity-based extraction and their binding to TIMP-1, -2, -3, and -4 in bronchoalveolar lavage (BAL) of lung transplant recipients with good outcome and BOS using a multiplex immunoassay., Results: The BAL levels of TIMP-1 and -2 and MMP-2, -3, -7, -8, and -9 were significantly increased in BOS compared to good outcome recipients. Interestingly, activity of MMP-7, but none of the other MMPs, was detected in good outcome recipients, whereas no active MMPs were observed in BOS recipients. However, BAL levels of TIMP-bound MMP-8 and -9 were higher in BOS than in good outcome recipients, suggesting activity of these MMPs in an earlier stage., Conclusions: We demonstrate that development of BOS is associated with increased levels of TIMP-1 and -2 and total MMP-2, -3, -7, -8, and -9. Although active MMP-7 was only observed in good outcome recipients, levels of TIMP-bound MMP-8 and -9 were higher in BOS. By enabling profiling of active and TIMP-bound MMPs, our novel method may open opportunities for the screening of early predictors for BOS.

Background: Long-term survival after lung transplantation is hindered by the development of bronchiolitis obliterans syndrome (BOS), and recent evidence suggests that dysregulated epithelial repair may underlie its development. Because matrix metalloproteinase (MMP) -2 and MMP-9 secretion is integral to repair, we hypothesized that airway epithelial cells from patients with BOS would over-express these matrix-degrading enzymes., Methods: Cells obtained from bronchial and bronchiolar brushings from patients with and without BOS (without acute rejection or infection) were analyzed via quantitative polymerase chain reaction and immunocytochemistry for MMP-2, and MMP-9 gene and protein expression. The expression of tissue inhibitor of metalloproteinase (TIMP)2 and TIMP1 was also assessed. MMP activity in bronchoalveolar lavage was determined via gelatin zymography., Results: MMP-2 and MMP-9 production was significantly higher in bronchoalveolar lavage (3.85- and 11.59-fold, p < 0.001) and airway epithelium (MMP-2 bronchial: 6.33-fold, bronchiolar: 3.57-fold, both p < 0.001; MMP-9 bronchial: 32.55-fold, p < 0.001; bronchiolar: 8.60-fold, p = 0.01) in patients with BOS, but expression in patients without BOS was not different from healthy controls. TIMP expression was similar in patients with and without BOS. Immunostaining confirmed that the airway epithelium was a direct source of MMP-2 and MMP-9 expression in patients with BOS., Conclusion: In patients with BOS, the airway epithelium over-expresses MMPs, even in the absence of acute rejection or infection. Dysregulated epithelial repair may be a key feature of BOS., (Copyright © 2011 International Society for Heart and Lung Transplantation. All rights reserved.)

Obliterative bronchiolitis after lung transplantation is a chronic inflammatory and fibrotic condition of small airways. The fibrosis associated with obliterative bronchiolitis might be reversible. Matrix metalloproteinases (MMPs) participate in inflammation and tissue remodeling. MMP-2 localized to myofibroblasts in post-transplant human obliterative bronchiolitis lesions and to allograft fibrosis in a rat intrapulmonary tracheal transplant model. Small numbers of infiltrating T cells were also observed within the fibrosis. To modulate inflammation and tissue remodeling, the broad-spectrum MMP inhibitor SC080 was administered after the allograft was obliterated, starting at post-transplant day 21. The allograft lumen remained obliterated after treatment. Only low-dose (2.5 mg/kg per day) SC080 significantly reduced collagen deposition, reduced the number of myofibroblasts and the infiltration of T cells in association with increased collagenolytic activity, increased MMP-2 gene expression, and decreased MMP-8, MMP-9, and MMP-13 gene expression. In in vitro experiments using cultured myofibroblasts, a relatively low concentration of SC080 increased MMP-2 activity and degradation of type I collagen. Moreover, coculture with T cells facilitated persistence of myofibroblasts, suggesting a role for T-cell infiltration in myofibroblast persistence in fibrosis. By combining low-dose SC080 with cyclosporine in vivo at post-transplant day 28, partial reversal of obliterative fibrosis was observed at day 42. Thus, modulating MMP activity might reverse established allograft airway fibrosis by regulating inflammation and tissue remodeling., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Obliteration of the small airways is a largely unresolved challenge in pulmonary medicine. It represents either the irreversible cause of functional impairment or a morphologic disorder of limited importance in a multitude of diseases. Bronchiolitis obliterans is a key complication of lung transplantation. No predictive markers for the onset of obliterative remodeling are currently available. To further elucidate the molecular mechanisms of airway remodeling, compartment-specific expression patterns were analyzed in patients. For this purpose, remodeled and nonremodeled bronchioli were isolated from transplanted and nontransplanted lung explants using laser-assisted microdissection (n = 24). mRNA expression of 45 fibrosis-associated genes was measured using quantitative real-time RT-PCR. For 20 genes, protein expression was also analyzed by immunohistochemistry. Infiltrating cells were characterized at conventional histology and immunohistochemistry. Obliterative remodeling of the small airways in transplanted and nontransplanted lungs shared similar grades of chronic inflammation and pivotal fibrotic pathways such as transforming growth factor β signaling and increased collagen expression. Bone morphogenetic protein and thrombospondin signaling, and also matrix metalloproteinases and tissue inhibitor of metalloproteinases, were primarily up-regulated in obliterative airway remodeling in nontransplanted lungs. In transplanted lungs, clinical remodeled bone morphogenetic protein but nonremodeled bronchioli were characterized by a concordant up-regulation of matrix metalloproteinase-9, RANTES, and tissue inhibitor of metalloproteinase-1. These distinct expression patterns warrant further investigation as potential markers of impending airway remodeling, especially for prospective longitudinal molecular profiling., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Bronchiolitis obliterans syndrome (BOS) is characterized by persistent alloreactive, infective and non-specific epithelial injury, loss of epithelial integrity and dysregulated repair. We have reported increased apoptosis of epithelial cells collected from the large airway in lung transplant recipients. As part of the alloreactive response, T cells induce apoptosis of target epithelial cells by secreting granzyme b. We hypothesized that granzyme b would be increased in lung transplant patients with acute rejection and BOS and that commonly used immunosuppressive agents would fail to suppress this serine protease adequately. We investigated intracellular T cell granzyme b in blood, bronchoalveolar lavage (BAL) and large airway brushing (23 controls, 29 stable transplant, 23 BOS, 28 acute rejection, 31 infection) using flow cytometry and assessed the effect of clinically relevant concentrations of cyclosporin A, tacrolimus, methylprednisolone and a protease inhibitor, gabexate mesilate, on in vitro granzyme b production. Granzyme b was increased significantly in all compartments of all transplant groups compared to controls. Surprisingly, granzyme b was even higher in patients with BOS than in patients with acute rejection. In longitudinal analysis in three patients, blood granzyme b increased prior to or at the onset of BOS. In vitro, methylprednisolone and gabexate mesilate had no effect and cyclosporin A and tacrolimus only a moderate effect on production of granzyme b by CD8(+) T cells. Increased T cell granzyme b production may contribute to BOS pathogenesis and is not curtailed by current immunosuppressants. Longitudinal investigation of granzyme b in blood may provide an adjunctive non-invasive method for predicting BOS/OB.

Background: Indoleamine 2,3-dioxygenase (IDO), an enzyme involved in the degradation of tryptophan (Try) to kynurenine (Kyn), is thought to suppress T-cell activity. Although a few experimental studies have suggested a role for IDO in graft acceptance, human data are scarce and inconclusive. We sought to establish whether, in lung transplant recipients (LTRs), plasma IDO activity mirrors the level of graft acceptance., Methods: We measured the plasma Kyn/Try ratio, reflecting IDO activity, by high-performance liquid chromatography (HPLC) in 90 LTRs, including 26 patients who were still functionally/clinically stable for >36 post-transplant months (stable LTRs) and 64 LTRs with bronchiolitis obliterans syndrome (BOS, Grades 0-p to 3). Twenty-four normal healthy controls (NHCs) were also included., Results: The Kyn/Try ratio in stable LTRs resembled that observed in NHCs, whereas, unexpectedly, patients with BOS, who had lower counts of peripheral CD4(+) T-regulatory cells and tolerogenic plasmacytoid dendritic cells than stable LTRs, showed an increased plasma Kyn/Try ratio compared with both NHCs and stable LTRs. IDO expression by in vitro-stimulated peripheral blood mononuclear cells (PBMC) did not vary between BOS and stable LTRs. Furthermore, BOS patients displayed signs of chronic systemic inflammation (increased plasma levels of interleukin-8 and tumor necrosis factor-alpha) and higher T-cell activation (increased frequency of peripheral interferon-gamma-producing clones)., Conclusions: Our results suggest that, in vivo, in lung transplantation, plasma IDO activity does not reflect the degree of lung graft acceptance, but instead is correlated with the degree of chronic inflammation.

Parenchymal disease in the allograft lung is associated with interstitial remodeling believed to be mediated by matrix metalloproteinases (MMPs). Recent studies suggest high levels of MMP-9 are associated with bronchiolitis obliterans syndrome (BOS) in lung transplant recipients. Since BOS occurs late in the posttransplant period and may be preceded by episodes of acute rejection or infection, which are associated with interstitial remodeling, we examined MMP profiles in allograft bronchoalveolar lavage (BAL) fluid in the early posttransplant period (preceding BOS). Gelatin zymography, protein array analysis and specific ELISA on BAL fluids from transplanted lungs indicated that MMP-8, MMP-9 and TIMP-1 were strongly expressed in allograft BAL fluid from stable patients, or those with infection or rejection compared to BAL fluid from normal volunteers. Elevated expression of MMP-8, MMP-9 and TIMP-1 occurred early, and was sustained for the 3.2 years covered in this study. Elevations of MMP-8, MMP-9 and TIMP-1 in the first 2 years posttransplant appear to be associated with lung transplantation itself, and not infection or rejection. These data suggest that ongoing and clinically silent MMP activity could perpetuate progressive disease in the allograft lung.

Obliterative bronchiolitis (OB) develops insidiously in nearly half of all lung transplant recipients. Although typically preceded by a CD8(+) T cell-rich lymphocytic bronchitis, it remains unresponsive to conventional immunosuppression. Using an airflow permissive model to study the role of gases flowing over the transplanted airway, it is shown that prolonged inhalation of sublethal doses of carbon monoxide (CO), but not nitric oxide (NO), obliterate the appearance of the obstructive airway lesion. Induction of the enzyme responsible for the synthesis of CO, heme oxygenase (Hmox) 1, increased carboxyhemoglobin levels and suppressed lymphocytic bronchitis and airway luminal occlusion after transplantation. In contrast, zinc protoporphyrin IX, a competitive inhibitor of Hmox, increased airway luminal occlusion. Compared with wild-type allografts, expression of inducible NO synthase (iNOS), which promotes the influx of cytoeffector leukocytes and airway graft rejection, was strikingly reduced by either enhanced expression of Hmox-1 or exogenous CO. Hmox-1/CO decreased nuclear factor (NF)-kappaB binding activity to the iNOS promoter region and iNOS expression. Inhibition of soluble guanylate cyclase did not interfere with the ability of CO to suppress OB, implicating a cyclic guanosine 3',5'-monophosphate-independent mechanism through which CO suppresses NF-kappaB, iNOS transcription, and OB. Prolonged CO inhalation represents a new immunosuppresive strategy to prevent OB.

Bronchiolitis obliterans (BO) is a survival-limiting factor in lung transplantation. There are no common BO markers in use. Since BO is associated with extracellular matrix remodeling, we asked whether matrix metalloproteases (MMPs) and their tissue inhibitors (TIMPs) could serve as BO markers. In 72 lung transplant patients (34 BO syndrome (BOS) 0, 15 BOS 0-p, and 23 BOS 1) serum and broncho-alveolar lavage (BAL) MMP and TIMP levels were examined by ELISA. The BAL cell counts were additionally analyzed. The serum MMP-2, MMP-8, MMP-9 and TIMP-2 levels were not different in all groups. In contrast, the BAL MMP-8, -9 and TIMP-1 levels were significantly elevated in BOS 0-p (p = 0.003; p = 0.007; p = 0.0003, respectively) and BOS 1 (p = 0.003; p = 0.001; p = 0.0004, respectively) as compared to BOS 0 patients. The BAL MMP-8, -9 and TIMP-1 levels were significant predictors of BOS 0-p (p = 0.01; p = 0.01; p = 0.01, respectively) and BOS-1 (p = 0.007; p = 0.01; p = 0.006, respectively) in receiver operating characteristic analysis. Except for BAL macrophages that were significantly decreased in BOS 0-p versus BOS 0 patients; other cell counts were not different between the groups. BAL MMP-8, -9 and TIMP-1 might be useful markers to detect BO in lung transplant patients.

Background: Epithelial cell injury, inflammation, fibrosis, and airway obliteration are associated in post-transplant obliterative bronchiolitis. Fibrosis is a consequence of fibroblastic activity and of collagen deposition after disturbances in the balance of protein formation and degradation. Proteolytic enzymes such as the matrix metalloproteinases mediate degradation. To assess matrix metalloproteinases during obliterative bronchiolitis development, we studied porcine, heterotopic bronchial allografts., Methods: A total of 119 allografts or autografts were harvested serially at 3 to 60 days after transplantation and processed for histology and in situ hybridization for matrix metalloproteinases 2 and 9. Immunocytochemistry for vimentin and alpha-smooth-muscle-cell actin was performed with specific antibodies., Results: Implants had initial ischemic injury to airway epithelium and to the bronchial wall. Recovery was rapid in autografts and in immunosuppressed allografts. In matrix metalloproteinase-2 mRNA activity in fibroblasts, correlation with endothelial expression and expression in macrophages occurred during intense fibroproliferation. We observed intense matrix metalloproteinase-9 positivity during onset of inflammation and fibroproliferation in endothelial cells (p < 0.01), fibroblasts (p < 0.05), macrophages (p < 0.05), and lymphocytes (p < 0.05). Matrix metalloproteinase-9 mRNA activity in fibroblasts correlated with that in endothelial and inflammatory cells and also proved predictive of early obliteration., Conclusions: Matrix metalloproteinase-2, and especially matrix metalloproteinase-9, gene activity was associated with onset of inflammation and fibroblastic proliferation in allografts, predicting early obliteration. Although this may be the case in the model described, its role in human-allograft post-transplant obliterative bronchiolitis requires further supportive data.

Bronchiolitis obliterans syndrome (BOS) is a severe complication after lung transplantation (LTX). In a retrospective cohort study 12 stable healthy recipients (non-BOS) and eight patients with BOS were enrolled after LTX and matrix metalloproteinases (MMP)-9, TIMP-1 and cell characteristics in bronchoalveolar lavage (BAL) samples (n = 145) were analysed. BALs from patients with BOS were further divided according to whether they were obtained before (pre-BOS) or after manifestation of BOS (BOS group). The MMP-9/TIMP-1 ratio was significantly increased in the BOS group compared with non-BOS or pre-BOS; furthermore, the ratio was negatively correlated with forced expiratory volume in one second. In zymography, the active form of MMP-9 was detected predominantly in the BOS group. In addition, zymography showed the banding pattern of neutrophil-derived MMP-9, indicating that polymorphonuclear neutrophils (PMNs) were the main source of MMP-9. According to that, MMP-9 was significantly correlated with the number of PMN. In immunocytochemistry, MMP-9 was also associated predominantly with PMN. This is the first study to evaluate the expression of matrix metalloproteinase-9 and tissue inhibitors of metalloproteinases-1 over time during manifestation of a fibroproliferative lung disease in patients. It demonstrates development of bronchiolitis obliterans syndrome after lung transplantation is associated with an imbalance of matrix metalloproteinases-9/tissue inhibitors of metalloproteinase-1 ratio.

Heme oxygenase-1 (HO-1) has been found to be a cytoprotective protein, and has recently been identified as a graft survival gene. This study demonstrates that HO-1 expression is increased in human lung allografts with acute cellular rejection and obliterative bronchiolitis. HO-1 expression was correlated with increased tissue iron and/or ferritin expression and increased inflammatory/oxidant load as measured by myeloperoxidase expression. Although the trigger for increased HO-1 expression in this setting is unknown, it may be related to hemorrhage and/or oxidative stress associated with rejection.

The main cause of mortality following lung transplantation is chronic rejection, manifesting morphologically as obliterative bronchiolitis (OB). It has been suggested that damage to the respiratory epithelium initiates proliferation of mesenchymal cells, leading to dense collagenous scarring in small airways. Inducible nitric oxide synthase (iNOS) is strongly expressed in the damaged epithelium in human OB, along with high levels of peroxynitrite, suggesting that endogenous NO mediates the epithelial destruction. To examine further the role of iNOS in this process, heterotopic airway implants were studied in rats, an acknowledged disease model. Specimens of iso- or allografted trachea, collected 3-60 days after implantation, were processed for histology and immunocytochemistry for iNOS and, as a marker of peroxynitrite formation, nitrotyrosine. In both iso- and allografts at the earliest stage (day 3), ischaemia was associated with severe epithelial damage or loss. These changes progressed until day 7 and were accompanied by strong expression of iNOS and nitrotyrosine in epithelial cells. In isografts, epithelial recovery was seen, with abundant iNOS immunoreactivity but little nitrotyrosine. In contrast, the epithelium in allografts did not regenerate and progressive inflammation and fibroproliferation occurred until complete obliteration of the tracheal lumen at day 60. The fibroproliferation was associated with changes in morphology of fibroblasts that were accompanied by alterations in their iNOS expression. iNOS immunoreactivity was dense in the plump fibroblasts of early lesions, in some cases as early as post-operative day 5, but very weak in elongated fibroblasts in totally occluded grafts. The intensity of immunoreactivity for nitrotyrosine corresponded to that of iNOS. These results indicate a dual role for NO in the airway obliteration that follows transplantation, through destruction of epithelium and stimulation of fibroblast activity., (Copyright 2000 John Wiley & Sons, Ltd.)

Large numbers of neutrophils with unopposed neutrophil elastase (NE) proteolytic activity are found in lower respiratory tract secretions from most patients with advanced cystic fibrosis (CF). To determine whether antielastase defenses may be overwhelmed in epithelial lining fluid after lung transplantation, we measured NE activity (cleavage of the specific substrate, MeO-Suc-Ala-Ala-Pro-Val-pNA) in bronchoalveolar lavage fluids (BALF) obtained for surveillance or diagnostic purposes at various intervals (1 mo to 7 yr after transplantation) from 52 recipients who had undergone double or bilateral lung transplantation for end-stage CF. Unopposed NE activity was found in BALF from 14 recipients, most of whom also had >= 10(5) colony forming units (cfu) of Pseudomonas aeruginosa in BALF. Ten of the 14 recipients with unopposed NE in bronchoalveolar lavage (BAL) had developed obliterative bronchiolitis (OB), but only 8 of the 38 subjects without unopposed NE activity had OB (p = 0. 002; Fisher exact test). We conclude that antiprotease defenses in lower respiratory tract secretions of CF patients receiving lung allografts are sufficient in the majority of patients to prevent unopposed NE activity. However, the presence of unopposed NE activity in BAL from lung allografts of patients with CF is associated with progressive, irreversible OB and graft failure.