Plasma cell but not CD20-mediated B-cell depletion protects from bleomycin-induced lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease associated with chronic inflammation and tissue remodelling leading to fibrosis, reduced pulmonary function, respiratory failure and death. Bleomycin (Blm)-induced lung fibrosis in mice replicates several clinical features of human IPF, including prominent lymphoid aggregates of predominantly B-cells that accumulate in the lung adjacent to areas of active fibrosis. We have shown previously a requirement for B-cells in the development of Blm-induced lung fibrosis in mice. To determine the therapeutic potential of inhibiting B-cell function in pulmonary fibrosis, we examined the effects of anti-CD20 B-cell ablation therapy to selectively remove mature B-cells from the immune system and inhibit Blm-induced lung fibrosis. Anti-CD20 B-cell ablation did not reduce fibrosis in this model; however, immune phenotyping of peripheral blood and lung resident cells revealed that anti-CD20-treated mice retained a high frequency of CD19+ CD138+ plasma cells. Interestingly, high levels of CD138+ cells were also identified in the lung tissue of patients with IPF, consistent with the mouse model. Treatment of mice with bortezomib, which depletes plasma cells, reduced the level of Blm-induced lung fibrosis, implicating plasma cells as important effector cells in the development and progression of pulmonary fibrosis.

Reduced SOCS1 Expression in Lung Fibroblasts from Patients with IPF Is Not Mediated by Promoter Methylation or Mir155.

The interleukin (IL)-6 family of cytokines and exaggerated signal transducer and activator of transcription (STAT)3 signaling is implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis, but the mechanisms regulating STAT3 expression and function are unknown. Suppressor of cytokine signaling (SOCS)1 and SOCS3 block STAT3, and low SOCS1 levels have been reported in IPF fibroblasts and shown to facilitate collagen production. Fibroblasts and lung tissue from IPF patients and controls were used to examine the mechanisms underlying SOCS1 down-regulation in IPF. A significant reduction in basal SOCS1 mRNA in IPF fibroblasts was confirmed. However, there was no difference in the kinetics of activation, and methylation of SOCS1 in control and IPF lung fibroblasts was low and unaffected by 5'-aza-2'-deoxycytidine' treatment. SOCS1 is a target of microRNA-155 and although microRNA-155 levels were increased in IPF tissue, they were reduced in IPF fibroblasts. Therefore, SOCS1 is not regulated by SOCS1 gene methylation or microRNA155 in these cells. In conclusion, we confirmed that IPF fibroblasts had lower levels of SOCS1 mRNA compared with control fibroblasts, but we were unable to determine the mechanism. Furthermore, although SOCS1 may be important in the fibrotic process, we were unable to find a significant role for SOCS1 in regulating fibroblast function.

Inhibition of collagen production delays malignant mesothelioma tumor growth in a murine model.

Malignant mesothelioma is an aggressive fibrous tumor, predominantly of the pleura, with a very poor prognosis. Cell-matrix interactions are recognized important determinants of tumor growth and invasiveness but the role of the extracellular matrix in mesothelioma is unknown. Mesothelioma cells synthesize collagen as well as transforming growth factor-beta (TGF-ß), a key regulator of collagen production. This study examined the effect of inhibiting collagen production on mesothelioma cell proliferation in vitro and tumor growth in vivo. Collagen production by mesothelioma cells was inhibited by incubating cells in vitro with the proline analogue thiaproline (thiazolidine-4-carboxylic acid) or by oral administration of thiaproline in a murine tumor model. Cell cytotoxicity was measured using neutral red uptake and lactate dehydrogenase assays. Proliferation was measured by tritiated thymidine incorporation, and inflammatory cell influx, proliferation, apoptosis and angiogenesis in tumors examined by immunohistochemical labelling. Tumor size was determined by tumor weight and collagen production was measured by HPLC. Thiaproline at non-toxic doses significantly reduced basal and TGF-ß-induced collagen production by over 50% and cell proliferation by over 65%. In vivo thiaproline administration inhibited tumor growth at 10 days, decreasing the median tumor weight by 80%. The mean concentration of collagen was 50% lower in the thiaproline-treated tumors compared with the controls. There were no significant differences in vasculature or inflammatory cell infiltration but apoptosis was increased in thiaproline treated tumors at day 10. In conclusion, these observations strongly support a role for collagen in mesothelioma growth and establish the potential for inhibitors of collagen synthesis in mesothelioma treatment.

InForm software: a semi-automated research tool to identify presumptive human hepatic progenitor cells, and other histological features of pathological significance.

Hepatic progenitor cells (HPCs) play an important regenerative role in acute and chronic liver pathologies. Liver disease research often necessitates the grading of disease severity, and pathologists' reports are the current gold-standard for assessment. However, it is often impractical to recruit pathologists in large cohort studies. In this study we utilise PerkinElmer's "InForm" software package to semi-automate the scoring of patient liver biopsies, and compare outputs to a pathologist's assessment. We examined a cohort of eleven acute hepatitis samples and three non-alcoholic fatty liver disease (NAFLD) samples, stained with HPC markers (GCTM-5 and Pan Cytokeratin), an inflammatory marker (CD45), Sirius Red to detect collagen and haematoxylin/eosin for general histology. InForm was configured to identify presumptive HPCs, CD45+ve inflammatory cells, areas of necrosis, fat and collagen deposition (p < 0.0001). Hepatitis samples were then evaluated both by a pathologist using the Ishak-Knodell scoring system, and by InForm through customised algorithms. Necroinflammation as evaluated by a pathologist, correlated with InForm outputs (r2 = 0.8192, p < 0.05). This study demonstrates that the InForm software package provides a useful tool for liver disease research, allowing rapid, and objective quantification of the presumptive HPCs and identifies histological features that assist with assessing liver disease severity, and potentially can facilitate diagnosis.

BARD1 serum autoantibodies for the detection of lung cancer.

PURPOSE: Currently the screening for lung cancer for risk groups is based on Computed Tomography (CT) or low dose CT (LDCT); however, the lung cancer death rate has not decreased significantly with people undergoing LDCT. We aimed to develop a simple reliable blood test for early detection of all types of lung cancer based on the immunogenicity of aberrant forms of BARD1 that are specifically upregulated in lung cancer. METHODS: ELISA assays were performed with a panel of BARD1 epitopes to detect serum levels of antibodies against BARD1 epitopes. We tested 194 blood samples from healthy donors and lung cancer patients with a panel of 40 BARD1 antigens. Using fitted Lasso logistic regression we determined the optimal combination of BARD1 antigens to be used in ELISA for discriminating lung cancer from healthy controls. Random selection of samples for training sets or validations sets was applied to validate the accuracy of our test. RESULTS: Fitted Lasso logistic regression models predict high accuracy of the BARD1 autoimmune antibody test with an AUC = 0.96. Validation in independent samples provided and AUC = 0.86 and identical AUCs were obtained for combined stages 1-3 and late stage 4 lung cancers. The BARD1 antibody test is highly specific for lung cancer and not breast or ovarian cancer. CONCLUSION: The BARD1 lung cancer test shows higher sensitivity and specificity than previously published blood tests for lung cancer detection and/or diagnosis or CT scans, and it could detect all types and all stages of lung cancer. This BARD1 lung cancer test could therefore be further developed as i) screening test for early detection of lung cancers in high-risk groups, and ii) diagnostic aid in complementing CT scan.

An Official American Thoracic Society Workshop Report: Use of Animal Models for the Preclinical Assessment of Potential Therapies for Pulmonary Fibrosis.

Numerous compounds have shown efficacy in limiting development of pulmonary fibrosis using animal models, yet few of these compounds have replicated these beneficial effects in clinical trials. Given the challenges associated with performing clinical trials in patients with idiopathic pulmonary fibrosis (IPF), it is imperative that preclinical data packages be robust in their analyses and interpretations to have the best chance of selecting promising drug candidates to advance to clinical trials. The American Thoracic Society has convened a group of experts in lung fibrosis to discuss and formalize recommendations for preclinical assessment of antifibrotic compounds. The panel considered three major themes (choice of animal, practical considerations of fibrosis modeling, and fibrotic endpoints for evaluation). Recognizing the need for practical considerations, we have taken a pragmatic approach. The consensus view is that use of the murine intratracheal bleomycin model in animals of both genders, using hydroxyproline measurements for collagen accumulation along with histologic assessments, is the best-characterized animal model available for preclinical testing. Testing of antifibrotic compounds in this model is recommended to occur after the acute inflammatory phase has subsided (generally after Day 7). Robust analyses may also include confirmatory studies in human IPF specimens and validation of results in a second system using in vivo or in vitro approaches. The panel also strongly encourages the publication of negative results to inform the lung fibrosis community. These recommendations are for preclinical therapeutic evaluation only and are not intended to dissuade development of emerging technologies to better understand IPF pathogenesis.

Secretory leukocyte protease inhibitor gene deletion alters bleomycin-induced lung injury, but not development of pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a progressive, fatal disease with limited treatment options. Protease-mediated transforming growth factor-ß (TGF-ß) activation has been proposed as a pathogenic mechanism of lung fibrosis. Protease activity in the lung is tightly regulated by protease inhibitors, particularly secretory leukocyte protease inhibitor (SLPI). The bleomycin model of lung fibrosis was used to determine the effect of increased protease activity in the lungs of Slpi(-/-) mice following injury. Slpi(-/-), and wild-type, mice received oropharyngeal administration of bleomycin (30 IU) and the development of pulmonary fibrosis was assessed. Pro and active forms of matrix metalloproteinase (MMP)-2 and MMP-9 were measured. Lung fibrosis was determined by collagen subtype-specific gene expression, hydroxyproline concentration, and histological assessment. Alveolar TGF-ß activation was measured using bronchoalveolar lavage cell pSmad2 levels and global TGF-ß activity was assessed by pSmad2 immunohistochemistry. The active-MMP-9 to pro-MMP-9 ratio was significantly increased in Slpi(-/-) animals compared with wild-type animals, demonstrating enhanced metalloproteinase activity. Wild-type animals showed an increase in TGF-ß activation following bleomycin, with a progressive and sustained increase in collagen type I, alpha 1 (Col1α1), III, alpha 1(Col3α1), IV, alpha 1(Col4α1) mRNA expression, and a significant increase in total lung collagen 28 days post bleomycin. In contrast Slpi(-/-) mice showed no significant increase of alveolar TGF-ß activity following bleomycin, above their already elevated levels, although global TGF-ß activity did increase. Slpi(-/-) mice had impaired collagen gene expression but animals demonstrated minimal reduction in lung fibrosis compared with wild-type animals. These data suggest that enhanced proteolysis does not further enhance TGF-ß activation, and inhibits sustained Col1α1, Col3α1, and Col4α1 gene expression following lung injury. However, these changes do not prevent the development of lung fibrosis. Overall, these data suggest that the absence of Slpi does not markedly modify the development of lung fibrosis following bleomycin-induced lung injury.

Epigenetic regulation of cyclooxygenase-2 by methylation of c8orf4 in pulmonary fibrosis.

Fibroblasts derived from the lungs of patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) produce low levels of prostaglandin (PG) E2, due to a limited capacity to up-regulate cyclooxygenase-2 (COX-2). This deficiency contributes functionally to the fibroproliferative state, however the mechanisms responsible are incompletely understood. In the present study, we examined whether the reduced level of COX-2 mRNA expression observed in fibrotic lung fibroblasts is regulated epigenetically. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5AZA) restored COX-2 mRNA expression by fibrotic lung fibroblasts dose dependently. Functionally, this resulted in normalization of fibroblast phenotype in terms of PGE2 production, collagen mRNA expression and sensitivity to apoptosis. COX-2 methylation assessed by bisulfite sequencing and methylation microarrays was not different in fibrotic fibroblasts compared with controls. However, further analysis of the methylation array data identified a transcriptional regulator, chromosome 8 open reading frame 4 (thyroid cancer protein 1, TC-1) (c8orf4), which is hypermethylated and down-regulated in fibrotic fibroblasts compared with controls. siRNA knockdown of c8orf4 in control fibroblasts down-regulated COX-2 and PGE2 production generating a phenotype similar to that observed in fibrotic lung fibroblasts. Chromatin immunoprecipitation demonstrated that c8orf4 regulates COX-2 expression in lung fibroblasts through binding of the proximal promoter. We conclude that the decreased capacity of fibrotic lung fibroblasts to up-regulate COX-2 expression and COX-2-derived PGE2 synthesis is due to an indirect epigenetic mechanism involving hypermethylation of the transcriptional regulator, c8orf4.

BARD1 mediates TGF-ß signaling in pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a rapid progressive fibro-proliferative disorder with poor prognosis similar to lung cancer. The pathogenesis of IPF is uncertain, but loss of epithelial cells and fibroblast proliferation are thought to be central processes. Previous reports have shown that BARD1 expression is upregulated in response to hypoxia and associated with TGF-ß signaling, both recognized factors driving lung fibrosis. Differentially spliced BARD1 isoforms, in particular BARD1ß, are oncogenic drivers of proliferation in cancers of various origins. We therefore hypothesized that BARD1 and/or its isoforms might play a role in lung fibrosis. METHODS: We investigated BARD1 expression as a function of TGF-ß in cultured cells, in mice with experimentally induced lung fibrosis, and in lung biopsies from pulmonary fibrosis patients. RESULTS: FL BARD1 and BARD1ß were upregulated in response to TGF-ß in epithelial cells and fibroblasts in vitro and in vivo. Protein and mRNA expression studies showed very low expression in healthy lung tissues, but upregulated expression of full length (FL) BARD1 and BARD1ß in fibrotic tissues. CONCLUSION: Our data suggest that FL BARD1 and BARD1ß might be mediators of pleiotropic effects of TGF-ß. In particular BARD1ß might be a driver of proliferation and of pulmonary fibrosis pathogenesis and progression and represent a target for treatment.

Role of a functional polymorphism in the F2R gene promoter in sarcoidosis.

Sarcoidosis is a multisystem granulomatous disease of unknown aetiology characterized by increased inflammation, and results from gene-environment interactions. Proteinase-activated receptor-1 mediates the interplay between coagulation and inflammation. The rs2227744G > A promoter single nucleotide polymorphism has been linked to inflammation, cardiovascular disease and chronic obstructive pulmonary disease exacerbations. Using a case-control study (184 cases with sarcoidosis and 368 controls), we show that the rs2227744A allele significantly associates with protection from sarcoidosis (P = 0.003, OR = 0.68 (0.52-0.88)).

Harnessing the potential of lung stem cells for regenerative medicine.

In response to recurrent exposure to environmental insults such as allergens, pollution, irritants, smoke and viral/bacterial infection, the epithelium of the lung is continually damaged. Homeostasis of the lung requires a balance between immune regulation and promotion of tissue regeneration, which requires the co-ordinated proliferation and differentiation of stem and progenitor cells. In this review we reflect on the current understanding of lung epithelial stem and progenitor cells and advocate a model hierarchy in which self-renewing multipotent lung epithelial stem cells give rise to lineage restricted progenitor cells that repopulate airway and alveolar epithelial cell lineages during homeostasis and repair. We also discuss the role of mesenchymal progenitor cells in maintaining the structural integrity of the lung and propose a model in which mesenchymal cells act as the quintessential architects of lung regeneration by providing molecular signals, such as FGF-10, to regulate the fate and specificity of epithelial stem and progenitor cells. Moreover, we discuss the current status and future prospects for translating lung stem cell therapies to the clinic to replace, repair, or regenerate diseased lung tissue. This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation.

Impact of a functional polymorphism in the PAR-1 gene promoter in COPD and COPD exacerbations.

Proteinase-activated receptor-1 (PAR-1) plays a key role in mediating the interplay between coagulation and inflammation in response to injury. The aim of this study was to investigate the role of the promoter single-nucleotide polymorphism (SNP) rs2227744G>A in modulating PAR-1/F2R gene expression in the context of chronic obstructive pulmonary disease (COPD) and COPD exacerbations. The function of the rs2227744G>A SNP was investigated by using reporter gene assays. The frequency of the polymorphism in the UK population was assessed by genotyping 8,579 healthy individuals from the Whitehall II and English Longitudinal Study of Ageing cohorts. The rs2227744G>A SNP was genotyped in a carefully phenotyped cohort of 203 COPD cases and matched controls. The results were further replicated in two different COPD cohorts. The minor allele of the rs2227744G>A polymorphism was found to increase F2R expression by 2.6-fold (P < 0.001). The rs2227744G>A SNP was not significantly associated with COPD, or with lung function, in all cohorts. The minor allele of the SNP was found to be associated with protection from frequent exacerbations (P = 0.04) in the cohort of COPD patients for which exacerbation frequency was available. Considering exacerbations as a continuous variable, the presence of the minor allele was associated with a significantly lower COPD exacerbation rate (3.03 vs. 1.98 exacerbations/year, Mann-Whitney U-test P = 0.04). Taken together, these data do not support a role for the rs2227744G>A F2R polymorphism in the development of COPD but suggest a protective role for this polymorphism from frequent exacerbations. Studies in separate cohorts to replicate these findings are warranted.

Mapping the future for pulmonary fibrosis: report from the 17th International Colloquium on Lung and Airway Fibrosis.

Pulmonary fibrosis is the ultimate outcome of various interstitial lung diseases, many of which have a dismal prognosis. Pulmonary fibrosis, therefore, represents a critical unmet medical need. Progress in research over the past 30 years has been encouraging. This work, which has been funded by governments, charitable trusts, industries and patient groups, has resulted in clinical trials testing novel drugs, giving hope to patients. In late September 2012, representatives from academia, industry and funding agencies met at the 17th International Colloquium on Airway and Lung Fibrosis to discuss state-of-the-art knowledge of pulmonary fibrosis. This manuscript summarises the outcomes of the meeting, highlighting the most relevant results and discoveries. It also attempts to provide a roadmap for future studies. It is hoped that such a roadmap may help interested parties to generate new research, which will be vital to continued progress. We are encouraged by the commitment expressed by all participants at this meeting and the shared vision of promoting future progress through international collaboration, the pooling of valuable resources, and the involvement of a new generation of physicians and scientists.

Airway deposition of nebulized gene delivery nanocomplexes monitored by radioimaging agents.

Receptor-targeted nanocomplexes are nonviral vectors developed for gene delivery to the airway epithelium for the treatment of pulmonary disease associated with cystic fibrosis. The present study aimed to optimize the delivery of the nanocomplex by nebulization, and to monitor the in vivo deposition of radiolabeled vector in the airways of a large animal model by γ-camera scintigraphy. Large White weaner pigs were nebulized with nanocomplexes mixed with technetium-99m radiopharmaceuticals. The aerosol deposition scans suggested that the nebulized radiovectors were deposited mainly in the trachea-main bronchi and in the midregion of the lungs. The plasmid biodistribution, assessed by real-time PCR, correlated with the scintigraphy images. The highest plasmid copy numbers were found in the bronchial areas and in the tissues proximal to the main bronchi bifurcation. Immunohistochemistry detected transgene expression in the tracheal and bronchial ciliated epithelium. Histological analysis of lung tissue showed no evidence of inflammation, and no increase in inflammatory cytokines or inflammatory cells was detected in the bronchoalveolar lavage. The deposition of nebulized nanocomplexes coassociated with technetium-99m can be monitored by nuclear medicine techniques. The use of a noninvasive strategy to follow the delivery of the vector could improve the clinical management of patients undergoing cystic fibrosis gene therapy.