Association study of human leukocyte antigen variants and idiopathic pulmonary fibrosis.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial pneumonia marked by progressive lung fibrosis and a poor prognosis. Recent studies have highlighted the potential role of infection in the pathogenesis of IPF, and a prior association of the HLA-DQB1 gene with idiopathic fibrotic interstitial pneumonia (including IPF) has been reported. Owing to the important role that the human leukocyte antigen (HLA) region plays in the immune response, here we evaluated if HLA genetic variation was associated specifically with IPF risk. Methods: We performed a meta-analysis of associations of the HLA region with IPF risk in individuals of European ancestry from seven independent case-control studies of IPF (comprising 5159 cases and 27 459 controls, including a prior study of fibrotic interstitial pneumonia). Single nucleotide polymorphisms, classical HLA alleles and amino acids were analysed and signals meeting a region-wide association threshold of p<4.5×10-4 and a posterior probability of replication >90% were considered significant. We sought to replicate the previously reported HLA-DQB1 association in the subset of studies independent of the original report. Results: The meta-analysis of all seven studies identified four significant independent single nucleotide polymorphisms associated with IPF risk. However, none met the posterior probability for replication criterion. The HLA-DQB1 association was not replicated in the independent IPF studies. Conclusion: Variation in the HLA region was not consistently associated with risk in studies of IPF. However, this does not preclude the possibility that other genomic regions linked to the immune response may be involved in the aetiology of IPF.

Genome-wide SNP-sex interaction analysis of susceptibility to idiopathic pulmonary fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic lung condition that is more prevalent in males than females. The reasons for this are not fully understood, with differing environmental exposures due to historically sex-biased occupations, or diagnostic bias, being possible explanations. To date, over 20 independent genetic variants have been identified to be associated with IPF susceptibility, but these have been discovered when combining males and females. Our aim was to test for the presence of sex-specific associations with IPF susceptibility and assess whether there is a need to consider sex-specific effects when evaluating genetic risk in clinical prediction models for IPF. Methods: We performed genome-wide single nucleotide polymorphism (SNP)-by-sex interaction studies of IPF risk in six independent IPF case-control studies and combined them using inverse-variance weighted fixed effect meta-analysis. In total, 4,561 cases (1,280 females and 2,281 males) and 23,500 controls (8,360 females and 14,528 males) of European genetic ancestry were analysed. We used polygenic risk scores (PRS) to assess differences in genetic risk prediction between males and females. Findings: Three independent genetic association signals were identified. All showed a consistent direction of effect across all individual IPF studies and an opposite direction of effect in IPF susceptibility between females and males. None had been previously identified in IPF susceptibility genome-wide association studies (GWAS). The predictive accuracy of the PRSs were similar between males and females, regardless of whether using combined or sex-specific GWAS results. Interpretation: We prioritised three genetic variants whose effect on IPF risk may be modified by sex, however these require further study. We found no evidence that the predictive accuracy of common SNP-based PRSs varies significantly between males and females.

Association study of human leukocyte antigen (HLA) variants and idiopathic pulmonary fibrosis.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial pneumonia marked by progressive lung fibrosis and a poor prognosis. Recent studies have highlighted the potential role of infection in the pathogenesis of IPF and a prior association of the HLA-DQB1 gene with idiopathic fibrotic interstitial pneumonia (including IPF) has been reported. Due to the important role that the Human Leukocyte Antigen (HLA) region plays in the immune response, here we evaluated if HLA genetic variation was associated specifically with IPF risk. Methods: We performed a meta-analysis of associations of the HLA region with IPF risk in individuals of European ancestry from seven independent case-control studies of IPF (comprising a total of 5,159 cases and 27,459 controls, including the prior study of fibrotic interstitial pneumonia). Single nucleotide polymorphisms, classical HLA alleles and amino acids were analysed and signals meeting a region-wide association threshold p<4.5×10-4 and a posterior probability of replication >90% were considered significant. We sought to replicate the previously reported HLA-DQB1 association in the subset of studies independent of the original report. Results: The meta-analysis of all seven studies identified four significant independent single nucleotide polymorphisms associated with IPF risk. However, none met the posterior probability for replication criterion. The HLA-DQB1 association was not replicated in the independent IPF studies. Conclusion: Variation in the HLA region was not consistently associated with risk in studies of IPF. However, this does not preclude the possibility that other genomic regions linked to the immune response may be involved in the aetiology of IPF.

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.

Transcriptome analysis of IPF fibroblastic foci identifies key pathways involved in fibrogenesis.

INTRODUCTION: Fibroblastic foci represent the cardinal pathogenic lesion in idiopathic pulmonary fibrosis (IPF) and comprise activated fibroblasts and myofibroblasts, the key effector cells responsible for dysregulated extracellular matrix deposition in multiple fibrotic conditions. The aim of this study was to define the major transcriptional programmes involved in fibrogenesis in IPF by profiling unmanipulated myofibroblasts within fibrotic foci in situ by laser capture microdissection. METHODS: The challenges associated with deriving gene calls from low amounts of RNA and the absence of a meaningful comparator cell type were overcome by adopting novel data mining strategies and by using weighted gene co-expression network analysis (WGCNA), as well as an eigengene-based approach to identify transcriptional signatures, which correlate with fibrillar collagen gene expression. RESULTS: WGCNA identified prominent clusters of genes associated with cell cycle, inflammation/differentiation, translation and cytoskeleton/cell adhesion. Collagen eigengene analysis revealed that transforming growth factor ß1 (TGF-ß1), RhoA kinase and the TSC2/RHEB axis formed major signalling clusters associated with collagen gene expression. Functional studies using CRISPR-Cas9 gene-edited cells demonstrated a key role for the TSC2/RHEB axis in regulating TGF-ß1-induced mechanistic target of rapamycin complex 1 activation and collagen I deposition in mesenchymal cells reflecting IPF and other disease settings, including cancer-associated fibroblasts. CONCLUSION: These data provide strong support for the human tissue-based and bioinformatics approaches adopted to identify critical transcriptional nodes associated with the key pathogenic cell responsible for fibrogenesis in situ and further identify the TSC2/RHEB axis as a potential novel target for interfering with excessive matrix deposition in IPF and other fibrotic conditions.

Genome-Wide Association Study of Susceptibility to Idiopathic Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a complex lung disease characterized by scarring of the lung that is believed to result from an atypical response to injury of the epithelium. Genome-wide association studies have reported signals of association implicating multiple pathways including host defense, telomere maintenance, signaling, and cell-cell adhesion.Objectives: To improve our understanding of factors that increase IPF susceptibility by identifying previously unreported genetic associations.Methods: We conducted genome-wide analyses across three independent studies and meta-analyzed these results to generate the largest genome-wide association study of IPF to date (2,668 IPF cases and 8,591 controls). We performed replication in two independent studies (1,456 IPF cases and 11,874 controls) and functional analyses (including statistical fine-mapping, investigations into gene expression, and testing for enrichment of IPF susceptibility signals in regulatory regions) to determine putatively causal genes. Polygenic risk scores were used to assess the collective effect of variants not reported as associated with IPF.Measurements and Main Results: We identified and replicated three new genome-wide significant (P < 5 × 10-8) signals of association with IPF susceptibility (associated with altered gene expression of KIF15, MAD1L1, and DEPTOR) and confirmed associations at 11 previously reported loci. Polygenic risk score analyses showed that the combined effect of many thousands of as yet unreported IPF susceptibility variants contribute to IPF susceptibility.Conclusions: The observation that decreased DEPTOR expression associates with increased susceptibility to IPF supports recent studies demonstrating the importance of mTOR signaling in lung fibrosis. New signals of association implicating KIF15 and MAD1L1 suggest a possible role of mitotic spindle-assembly genes in IPF susceptibility.

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.

Prostanoid EP2 Receptors Are Up-Regulated in Human Pulmonary Arterial Hypertension: A Key Anti-Proliferative Target for Treprostinil in Smooth Muscle Cells.

Prostacyclins are extensively used to treat pulmonary arterial hypertension (PAH), a life-threatening disease involving the progressive thickening of small pulmonary arteries. Although these agents are considered to act therapeutically via the prostanoid IP receptor, treprostinil is the only prostacyclin mimetic that potently binds to the prostanoid EP2 receptor, the role of which is unknown in PAH. We hypothesised that EP2 receptors contribute to the anti-proliferative effects of treprostinil in human pulmonary arterial smooth muscle cells (PASMCs), contrasting with selexipag, a non-prostanoid selective IP agonist. Human PASMCs from PAH patients were used to assess prostanoid receptor expression, cell proliferation, and cyclic adenosine monophosphate (cAMP) levels following the addition of agonists, antagonists or EP2 receptor small interfering RNAs (siRNAs). Immunohistochemical staining was performed in lung sections from control and PAH patients. We demonstrate using selective IP (RO1138452) and EP2 (PF-04418948) antagonists that the anti-proliferative actions of treprostinil depend largely on EP2 receptors rather than IP receptors, unlike MRE-269 (selexipag-active metabolite). Likewise, EP2 receptor knockdown selectively reduced the functional responses to treprostinil but not MRE-269. Furthermore, EP2 receptor levels were enhanced in human PASMCs and in lung sections from PAH patients compared to controls. Thus, EP2 receptors represent a novel therapeutic target for treprostinil, highlighting key pharmacological differences between prostacyclin mimetics used in PAH.

Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung.

INTRODUCTION: Loss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport. METHODS: We investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air-liquid interface (ALI).Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (Vt), short circuit current (Isc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections. RESULTS: Nanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and ßENaC mRNA by 30%. Transfections reduced Vt, the amiloride-sensitive Isc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%. CONCLUSION: Nanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo.

Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis.

Pulmonary fibrosis is a progressive scarring disorder of the lung with dismal prognosis and no curative therapy. Clusterin, an extracellular chaperone and regulator of cell functions, is reduced in bronchoalveolar lavage fluid of patients with pulmonary fibrosis. However, its distribution and role in normal and fibrotic human lung are incompletely characterized. Immunohistochemical localization of clusterin revealed strong staining associated with fibroblasts in control lung and morphologically normal areas of fibrotic lung but weak or undetectable staining in fibrotic regions and particularly fibroblastic foci. Clusterin also co-localized with elastin in vessel walls and additionally with amorphous elastin deposits in fibrotic lung. Analysis of primary lung fibroblast isolates in vitro confirmed the down-regulation of clusterin expression in fibrotic compared with control lung fibroblasts and further demonstrated that TGF-ß1 is capable of down-regulating fibroblast clusterin expression. shRNA-mediated down-regulation of clusterin did not affect TGF-ß1-induced fibroblast-myofibroblast differentiation but inhibited fibroblast proliferative responses and sensitized to apoptosis. Down-regulation of clusterin in fibrotic lung fibroblasts at least partly due to increased TGF-ß1 may therefore represent an appropriate but insufficient response to limit fibroproliferation. Reduced expression of clusterin in the lung may also limit its extracellular chaperoning activity contributing to dysregulated deposition of extracellular matrix proteins.

Genetic variants associated with susceptibility to idiopathic pulmonary fibrosis in people of European ancestry: a genome-wide association study.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease with high mortality, uncertain cause, and few treatment options. Studies have identified a significant genetic risk associated with the development of IPF; however, mechanisms by which genetic risk factors promote IPF remain unclear. We aimed to identify genetic variants associated with IPF susceptibility and provide mechanistic insight using gene and protein expression analyses. METHODS: We used a two-stage approach: a genome-wide association study in patients with IPF of European ancestry recruited from nine different centres in the UK and controls selected from UK Biobank (stage 1) matched for age, sex, and smoking status; and a follow-up of associated genetic variants in independent datasets of patients with IPF and controls from two independent US samples from the Chicago consortium and the Colorado consortium (stage 2). We investigated the effect of novel signals on gene expression in large transcriptomic and genomic data resources, and examined expression using lung tissue samples from patients with IPF and controls. FINDINGS: 602 patients with IPF and 3366 controls were selected for stage 1. For stage 2, 2158 patients with IPF and 5195 controls were selected. We identified a novel genome-wide significant signal of association with IPF susceptibility near A-kinase anchoring protein 13 (AKAP13; rs62025270, odds ratio [OR] 1·27 [95% CI 1·18-1·37], p=1·32 × 10-9) and confirmed previously reported signals, including in mucin 5B (MUC5B; rs35705950, OR 2·89 [2·56-3·26], p=1·12 × 10-66) and desmoplakin (DSP; rs2076295, OR 1·44 [1·35-1·54], p=7·81 × 10-28). For rs62025270, the allele A associated with increased susceptibility to IPF was also associated with increased expression of AKAP13 mRNA in lung tissue from patients who had lung resection procedures (n=1111). We showed that AKAP13 is expressed in the alveolar epithelium and lymphoid follicles from patients with IPF, and AKAP13 mRNA expression was 1·42-times higher in lung tissue from patients with IPF (n=46) than that in lung tissue from controls (n=51). INTERPRETATION: AKAP13 is a Rho guanine nucleotide exchange factor regulating activation of RhoA, which is known to be involved in profibrotic signalling pathways. The identification of AKAP13 as a susceptibility gene for IPF increases the prospect of successfully targeting RhoA pathway inhibitors in patients with IPF. FUNDING: UK Medical Research Council, National Heart, Lung, and Blood Institute of the US National Institutes of Health, Agencia Canaria de Investigación, Innovación y Sociedad de la Información, Spain, UK National Institute for Health Research, and the British Lung Foundation.

Delivery of ENaC siRNA to epithelial cells mediated by a targeted nanocomplex: a therapeutic strategy for cystic fibrosis.

The inhibition of ENaC may have therapeutic potential in CF airways by reducing sodium hyperabsorption, restoring lung epithelial surface fluid levels, airway hydration and mucociliary function. The challenge has been to deliver siRNA to the lung with sufficient efficacy for a sustained therapeutic effect. We have developed a self-assembling nanocomplex formulation for siRNA delivery to the airways that consists of a liposome (DOTMA/DOPE; L), an epithelial targeting peptide (P) and siRNA (R). LPR formulations were assessed for their ability to silence expression of the transcript of the gene encoding the α-subunit of the sodium channel ENaC in cell lines and primary epithelial cells, in submerged cultures or grown in air-liquid interface conditions. LPRs, containing 50 nM or 100 nM siRNA, showed high levels of silencing, particularly in primary airway epithelial cells. When nebulised these nanocomplexes still retained their biophysical properties and transfection efficiencies. The silencing ability was determined at protein level by confocal microscopy and western blotting. In vivo data demonstrated that these nanoparticles had the ability to silence expression of the α-ENaC subunit gene. In conclusion, these findings show that LPRs can modulate the activity of ENaC and this approach might be promising as co-adjuvant therapy for cystic fibrosis.

Exploration of a potent PI3 kinase/mTOR inhibitor as a novel anti-fibrotic agent in IPF.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is the most rapidly progressive and fatal of all fibrotic conditions with no curative therapies. Common pathomechanisms between IPF and cancer are increasingly recognised, including dysfunctional pan-PI3 kinase (PI3K) signalling as a driver of aberrant proliferative responses. GSK2126458 is a novel, potent, PI3K/mammalian target of rapamycin (mTOR) inhibitor which has recently completed phase I trials in the oncology setting. Our aim was to establish a scientific and dosing framework for PI3K inhibition with this agent in IPF at a clinically developable dose. METHODS: We explored evidence for pathway signalling in IPF lung tissue and examined the potency of GSK2126458 in fibroblast functional assays and precision-cut IPF lung tissue. We further explored the potential of IPF patient-derived bronchoalveolar lavage (BAL) cells to serve as pharmacodynamic biosensors to monitor GSK2126458 target engagement within the lung. RESULTS: We provide evidence for PI3K pathway activation in fibrotic foci, the cardinal lesions in IPF. GSK2126458 inhibited PI3K signalling and functional responses in IPF-derived lung fibroblasts, inhibiting Akt phosphorylation in IPF lung tissue and BAL derived cells with comparable potency. Integration of these data with GSK2126458 pharmacokinetic data from clinical trials in cancer enabled modelling of an optimal dosing regimen for patients with IPF. CONCLUSIONS: Our data define PI3K as a promising therapeutic target in IPF and provide a scientific and dosing framework for progressing GSK2126458 to clinical testing in this disease setting. A proof-of-mechanism trial of this agent is currently underway. TRIAL REGISTRATION NUMBER: NCT01725139, pre-clinical.

Minicircle DNA Provides Enhanced and Prolonged Transgene Expression Following Airway Gene Transfer.

Gene therapy for cystic fibrosis using non-viral, plasmid-based formulations has been the subject of intensive research for over two decades but a clinically viable product has yet to materialise in large part due to inefficient transgene expression. Minicircle DNA give enhanced and more persistent transgene expression compared to plasmid DNA in a number of organ systems but has not been assessed in the lung. In this study we compared minicircle DNA with plasmid DNA in transfections of airway epithelial cells. In vitro, luciferase gene expression from minicircles was 5-10-fold higher than with plasmid DNA. In eGFP transfections in vitro both the mean fluorescence intensity and percentage of cells transfected was 2-4-fold higher with minicircle DNA. Administration of equimolar amounts of DNA to mouse lungs resulted in a reduced inflammatory response and more persistent transgene expression, with luciferase activity persisting for 2 weeks from minicircle DNA compared to plasmid formulations. Transfection of equal mass amounts of DNA in mouse lungs resulted in a 6-fold increase in transgene expression in addition to more persistent transgene expression. Our findings have clear implications for gene therapy of airway disorders where plasmid DNA transfections have so far proven inefficient in clinical trials.

Caffeine inhibits TGFß activation in epithelial cells, interrupts fibroblast responses to TGFß, and reduces established fibrosis in ex vivo precision-cut lung slices.

Caffeine is a commonly used food additive found naturally in many products. In addition to potently stimulating the central nervous system caffeine is able to affect various systems within the body including the cardiovascular and respiratory systems. Importantly, caffeine is used clinically to treat apnoea and bronchopulmonary dysplasia in premature babies. Recently, caffeine has been shown to exhibit antifibrotic effects in the liver in part through reducing collagen expression and deposition, and reducing expression of the profibrotic cytokine TGFß. The potential antifibrotic effects of caffeine in the lung have not previously been investigated. Using a combined in vitro and ex vivo approach we have demonstrated that caffeine can act as an antifibrotic agent in the lung by acting on two distinct cell types, namely epithelial cells and fibroblasts. Caffeine inhibited TGFß activation by lung epithelial cells in a concentration-dependent manner but had no effect on TGFß activation in fibroblasts. Importantly, however, caffeine abrogated profibrotic responses to TGFß in lung fibroblasts. It inhibited basal expression of the α-smooth muscle actin gene and reduced TGFß-induced increases in profibrotic genes. Finally, caffeine reduced established bleomycin-induced fibrosis after 5 days treatment in an ex vivo precision-cut lung slice model. Together, these findings suggest that there is merit in further investigating the potential use of caffeine, or its analogues, as antifibrotic agents in the lung.

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.

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.

Genetic partitioning of interleukin-6 signalling in mice dissociates Stat3 from Smad3-mediated lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease that is unresponsive to current therapies and characterized by excessive collagen deposition and subsequent fibrosis. While inflammatory cytokines, including interleukin (IL)-6, are elevated in IPF, the molecular mechanisms that underlie this disease are incompletely understood, although the development of fibrosis is believed to depend on canonical transforming growth factor (TGF)-ß signalling. We examined bleomycin-induced inflammation and fibrosis in mice carrying a mutation in the shared IL-6 family receptor gp130. Using genetic complementation, we directly correlate the extent of IL-6-mediated, excessive Stat3 activity with inflammatory infiltrates in the lung and the severity of fibrosis in corresponding gp130(757F) mice. The extent of fibrosis was attenuated in B lymphocyte-deficient gp130(757F);µMT(-/-) compound mutant mice, but fibrosis still occurred in their Smad3(-/-) counterparts consistent with the capacity of excessive Stat3 activity to induce collagen 1α1 gene transcription independently of canonical TGF-ß/Smad3 signalling. These findings are of therapeutic relevance, since we confirmed abundant STAT3 activation in fibrotic lungs from IPF patients and showed that genetic reduction of Stat3 protected mice from bleomycin-induced lung fibrosis.

STAT3-mediated signaling dysregulates lung fibroblast-myofibroblast activation and differentiation in UIP/IPF.

STAT3 is a latent transcription factor that plays a role in regulating fibroblast function in fibrotic lung diseases. To further understand the role of STAT3 in the phenotypic divergence and function of human lung fibroblasts (LFs), we investigated the effect of basal and cytokine-induced STAT3 activity on indices of LF differentiation and activation, including expression of α-smooth muscle actin (α-SMA), collagen, and adhesion molecules Thy-1/CD90 and α(v) ß(3) and ß(5) integrins. We identified a population of fibroblasts from usual interstitial pneumonia (UIP)/idiopathic pulmonary fibrosis (IPF) lungs characterized by constitutively phosphorylated STAT3, lower proliferation rates, and diminished expression of α-SMA, Thy-1/CD90, and ß(3) integrins compared with control LFs. Staining of UIP lung biopsy specimens demonstrated that phosphorylated STAT3 was not present in α-SMA-positive fibroblastic foci but was observed in the nuclei of cells located in the areas of dense fibrosis. STAT3 activation in LFs did not significantly influence basal or transforming growth factor ß(1)-induced collagen I expression but inhibited expression of α-SMA, Thy-1/CD90, and αv ß(3) integrins. Suppression of STAT3 signaling diminished resistance of IPF LFs to staurosporine-induced apoptosis and responsiveness to transforming growth factor ß(1) but increased basal α-SMA and restored ß(3) integrin expression in LFs via an ALK-5-dependent, SMAD3/7-independent mechanism. These data suggest that STAT3 activation regulates several pathways in human LFs associated with normal wound healing, whereas aberrant STAT3 signaling plays a critical role in UIP/IPF pathogenesis.