Targeting PI3Kδ function for amelioration of murine chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality following allotransplant. Activated donor effector T cells can differentiate into pathogenic T helper (Th)-17 cells and germinal center (GC)-promoting T follicular helper (Tfh) cells, resulting in cGVHD. Phosphoinositide-3-kinase-δ (PI3Kδ), a lipid kinase, is critical for activated T cell survival, proliferation, differentiation, and metabolism. We demonstrate PI3Kδ activity in donor T cells that become Tfh cells is required for cGVHD in a nonsclerodermatous multiorgan system disease model that includes bronchiolitis obliterans (BO), dependent upon GC B cells, Tfhs, and counterbalanced by T follicular regulatory cells, each requiring PI3Kδ signaling for function and survival. Although B cells rely on PI3Kδ pathway signaling and GC formation is disrupted resulting in a substantial decrease in Ig production, PI3Kδ kinase-dead mutant donor bone marrow-derived GC B cells still supported BO cGVHD generation. A PI3Kδ-specific inhibitor, compound GS-649443, that has superior potency to idelalisib while maintaining selectivity, reduced cGVHD in mice with active disease. In a Th1-dependent and Th17-associated scleroderma model, GS-649443 effectively treated mice with active cGVHD. These data provide a foundation for clinical trials of US Food and Drug Administration (FDA)-approved PI3Kδ inhibitors for cGVHD therapy in patients.

Regression of allograft airway fibrosis: the role of MMP-dependent tissue remodeling in obliterative bronchiolitis after lung transplantation.

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.

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

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

Analysis of Risk Factors of Bronchiolitis Obliterans in Children with Mycoplasma pneumoniae Bronchiolitis.

OBJECTIVE: To investigate the related risk factors for bronchiolitis obliterans (BO) in children with mycoplasma pneumonia (MP) bronchiolitis. METHOD: The clinical data of 227 children with MP bronchiolitis who were admitted to the II Department of Respiratory of Children's Hospital of Hebei Province from January 2018 to June 2020 were retrospectively analyzed. According to the sequelae of BO, they were divided into 32 cases in the BO group and 195 cases in the non-BO group. The univariate analysis was performed on the clinical and laboratory parameters of the two groups, and the multifactor logistic regression was performed further to determine the independent risk factors for the occurrence of BO in MP bronchiolitis, and then, the cut-off value with the maximum diagnostic value of indicators was found through the ROC curve analysis. RESULTS: The results of univariate and multivariate logistic regression analysis showed that the independent risk factors for the occurrence of BO in MP bronchioles were longer duration of moist rales (OR = 1.203, P = 0.003), higher levels of serum lactate dehydrogenase (LDH) (OR = 1.005, P = 0.036), hypoxemia (OR = 7.442, P = 0.035), and pleural effusion (OR = 4.437, P = 0.004). The area under the ROC curve was 78.2%, 72.0%, 68.2%, and 71.0%, respectively (P < 0.001). The cut-off value of duration of moist rales and levels of serum LDH are 7.5 d and 330 U/L, respectively. CONCLUSION: Children with MP bronchiolitis with high serum LDH level (≥330 U/L), combined with hypoxemia, pleural effusion, and lung wet rale duration (≥7.5 d), may be more prone to BO, in which lung wet rale duration prediction value is the largest. Among them, duration of pulmonary moist rales has the highest predictive value.

Reciprocal regulation of airway rejection by the inducible gas-forming enzymes heme oxygenase and nitric oxide synthase.

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.

Overexpression of the MSK1 Kinase in Patients With Chronic Lung Allograft Dysfunction and Its Confirmed Role in a Murine Model.

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.

Increased levels of T cell granzyme b in bronchiolitis obliterans syndrome are not suppressed adequately by current immunosuppressive regimens.

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.

Tissue inhibitor of metalloproteinase-1 moderates airway re-epithelialization by regulating matrilysin activity.

Obliterative bronchiolitis (OB) is the histopathological finding in chronic lung allograft rejection. Mounting evidence suggests that epithelial damage drives the development of airway fibrosis in OB. Tissue inhibitor of metalloproteinase (TIMP)-1 expression increases in lung allografts and is associated with the onset of allograft rejection. Furthermore, in a mouse model of OB, airway obliteration is reduced in TIMP-1-deficient mice. Matrilysin (matrix metallproteinase-7) is essential for airway epithelial repair and is required for the re-epithelialization of airway wounds by facilitating cell migration; therefore, the goal of this study was to determine whether TIMP-1 inhibits re-epithelialization through matrilysin. We found that TIMP-1 and matrilysin co-localized in the epithelium of human lungs with OB and both co-localized and co-immunoprecipitated in wounded primary airway epithelial cultures. TIMP-1-deficient cultures migrated faster, and epithelial cells spread to a greater extent compared with wild-type cultures. TIMP-1 also inhibited matrilysin-mediated cell migration and spreading in vitro. In vivo, TIMP-1 deficiency enhanced airway re-epithelialization after naphthalene injury. Furthermore, TIMP-1 and matrilysin co-localized in airway epithelial cells adjacent to the wound edge. Our data demonstrate that TIMP-1 interacts with matrix metalloproteinases and regulates matrilysin activity during airway epithelial repair. Furthermore, we speculate that TIMP-1 overexpression restricts airway re-epithelialization by inhibiting matrilysin activity, contributing to a stereotypic injury response that promotes airway fibrosis via bronchiole airway epithelial damage and obliteration.

Allograft airway fibrosis in the pulmonary milieu: a disorder of tissue remodeling.

Obliterative bronchiolitis (OB) is thought to be a form of chronic allograft rejection. However, immunosuppressive therapy is not effective once fibrosis has developed. We hypothesize that disordered tissue remodeling is a mechanism for the pathogenesis of OB. We examined allograft airway fibrosis in an intrapulmonary tracheal transplant model of OB. Allograft airways were completely obliterated at day 21 by fibrotic tissue; however, tissue remodeling continued thereafter, as demonstrated by the change of collagen deposition density, shift from type I to type III collagen, shift from fibroblasts to myofibroblasts and shift of expression profiles and activities of matrix metalloproteinases (MMPs). We then used a broad-spectrum MMP inhibitor, SC080, to attempt to manipulate tissue remodeling. Administration of the MMP inhibitor from day 0 to day 28 reduced airway obliteration, without inhibiting T-cell activation. MMP inhibition from day 14 to day 28 showed similar effects on airway obliteration. MMP inhibition from day 21 to day 35 did not reverse the airway obliteration, but significantly reduced the collagen deposition, type III collagen and myofibroblasts in the lumen. We conclude that tissue remodeling plays a critical role in the development and maintenance of fibrosis after transplantation.

Matrix metalloproteinase induction in post-transplant obliterative bronchiolitis.

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.

Metalloproteinase Profiling in Lung Transplant Recipients With Good Outcome and Bronchiolitis Obliterans Syndrome.

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.

Effects of azithromycin and tanomastat on experimental bronchiolitis obliterans.

OBJECTIVE: Azithromycin has become a standard of care in therapy of bronchiolitis obliterans following lung transplantation. Matrix metalloprotease-9 broncho-alveolar lavage levels increase in airway neutrophilia and bronchiolitis obliterans. Interleukin-17 may play a role in lung allograft rejection, and interleukin-12 is downregulated in bronchiolitis obliterans. Whether these mechanisms can be targeted by azithromycin remains unclear. METHODS: Bronchiolitis obliterans was induced by transplantation of Fischer F344 rat left lungs to Wistar Kyoto rats. Allografts with azithromycin therapy from day 1 to 28 or 56 and mono- or combination therapy with the broad-spectrum matrix metalloprotease inhibitor tanomastat from day 1 to 56 were compared to control allografts and isografts. Graft histology was assessed, and tissue cytokine expression studied using Western blotting and immunofluorescence. RESULTS: The chronic airway rejection score in the azithromycin group did not change between 4 and 8 weeks after transplantation, whereas it significantly worsened in control allografts (P = .041). Azithromycin+tanomastat prevented complete allograft fibrosis, which occurred in 40% of control allografts. Azithromycin reduced interleukin-17 expression (P = .049) and the number of IL-17(+)/CD8(+) lymphocytes at 4 weeks, and active matrix metalloprotease-9 at 8 weeks (P = .017), and increased interleukin-12 expression (P = .025) at 8 weeks following transplantation versus control allografts. CONCLUSIONS: The expression of interleukin-17 and matrix metalloprotease-9 in bronchiolitis obliterans may be attenuated by azithromycin, and the decrease in interleukin-12 expression was prevented by azithromycin. Combination of azithromycin with a matrix metalloprotease inhibitor is worth studying further because it prevented complete allograft fibrosis in this study.

Increased expression of heme oxygenase-1 in human lung transplantation.

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.

Constrictive bronchiolitis obliterans. Characterisation of fibrogenesis and lysyl oxidase expression patterns.

The process leading to irreversible fibrotic constriction of the bronchioles was studied in two cases of bronchiolitis obliterans (BO) after bone marrow transplantation. Because lysyl oxidase (LOX) is the main collagen cross-linking enzyme that might account for irreversible fibrosis, its expression was studied together with expression of extracellular matrix (ECM) proteins. Characteristic types of lesions could be distinguished on the basis of histological and immunohistological criteria. An inflammatory stage was characterised by infiltration restricted to the bronchioles by lymphocytes and dendritic cells. A fibro-inflammatory stage was characterised by the coexistence of a persistent immune cellular lesion pattern with further focal modelling of a sub-epithelial neo-synthesised connective matrix. LOX expression was observed at the tips of intra-luminal fibrotic protrusions, together with tenascin and cellular fibronectin. A fibrotic stage was characterised by dense ECM deposits spreading throughout the peri-bronchiolar connective tissue, resulting in bronchiole obliteration and final disappearance. In contrast to reversible cases of fibrosis, persistence of long-term LOX expression reflecting continuing fibrosing activity might account for the irreversible status of BO. Our two cases illustrated that, at inflammatory and fibro-inflammatory stages, BO may be stabilised by immunosuppressive treatment, while the persistence of LOX expression in the fibrotic stage might correspond to a disease that becomes irreversible and fatal.

Experimental bronchiolitis obliterans induced by in vivo HVJ-liposome-mediated endothelin-1 gene transfer.

BACKGROUND: Bronchiolitis obliterans (OB) is a lesion that results when injury to small conducting airways is repaired by a proliferation of fibrous granulation tissue. Bronchiolitis obliterans has emerged as a main cause of morbidity and mortality in the setting of lung and heart-lung transplantation. Endothelin-1 (ET-1), initially discovered as a vasoconstrictive peptide, has a mitogenic activity on vascular smooth cells and airway epithelial cells. Overproduction of endothelin has been reported in patients with OB or chronic rejection after lung transplantation. It is still undetermined whether locally overexpressed ET-1 has a potential impact in the pathogenesis of OB. METHODS: We locally overexpressed ET-1 using ultraviolet irradiation-inactivated hemagglutinating virus of Japan (HVJ)-liposome-mediated in vivo gene transfer. Plasmid DNA of prepro-ET-1 and high mobility group 1 protein were coencapsulated in liposomes, and were introduced into airway epithelial cells by HVJ-mediated membrane fusion. Control animals received instillation of HVJ-liposome with an empty expression cassette. To confirm the efficiency of transfection, HVJ liposome with beta-galactosidase gene was introduced. The expression of ET-1 and beta-galactosidase was assessed by immunohistochemistry. RESULTS: Bronchial epithelium alveolar cells and alveolar macrophage were stained blue (X-Gal) 1 week after in vivo gene transfer of beta-galactosidase gene, indicating beta-gal activity. In animals 1 to 2 weeks after in vivo transfection of prepro-ET-1 gene, hyperplastic connective tissue plaque was seen in the alveolar duct and small conducting airway, indicating histologically distinctive bronchiolitis obliterans. Strong ET-1-like immunoactivities were seen in the airway epithelial, hyperplastic connective tissue, and alveolar cells. No histopathologic changes were seen in the control animals. CONCLUSIONS: These results suggested that ET-1 may play an important role in the pathogenesis of OB. The effective pharmacologic antagonist or inhibitor may possibly control the progression of disease in patients of OB.

The airway epithelium is a direct source of matrix degrading enzymes in bronchiolitis obliterans syndrome.

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.

MMP-8 promotes polymorphonuclear cell migration through collagen barriers in obliterative bronchiolitis.

Increased levels of MMP-8 (neutrophil collagenase) have been reported in OB, but the biological role of MMP-8 in OB is not known. MMP-8 is an interstitial collagenase highly expressed by polymorphonuclear leukocytes, which are prominent in early OB. Here, we show that MMP-8 promotes migration of PMNs through the collagen-rich matrix in a mouse heterotopic airway transplant model of OB. Overall, MMP-8(-/-) mice had significantly fewer PMNs in the airway lumen 2 and 14 days post-transplantation, and the percentage of PMNs traversing the matrix to the lumen was decreased markedly in the MMP-8(-/-) compared with WT mice at 14 days. There were significantly more PMNs outside of the lumen in the ECM in the MMP-8(-/-) mice compared with WT mice. In vitro, significantly fewer MMP-8(-/-) PMNs migrated through 3D cross-linked collagen gels than WT PMNs. MMP inhibitor GM6001 was also able to impede migration of WT PMNs through collagen gels. The decreased migration was likely a result of pericollagenase activity of MMP-8, as WT PMNs expressing MMP-8 were not able to migrate effectively through collagen that was resistant to the collagenase. Protection from OB was seen in the MMP-8(-/-) mice, as the airway lumen had significantly less obliteration and collagen deposition, suggesting that MMP-8 plays an important role in the pathogenesis of OB.

Indoleamine 2,3-dioxygenase in lung allograft tolerance.

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.