Amanda Goodwin

Role of integrin-mediated TGFβ activation in the pathogenesis of pulmonary fibrosis

IPF (idiopathic pulmonary fibrosis) is a chronic progressive disease of unknown aetiology without effective treatment. IPF is characterized by excessive collagen deposition within the lung. Recent evidence suggests that the lung epithelium plays a key role in driving the fibrotic response. The current paradigm suggests that, after epithelial injury, there is impaired epithelial proliferation and enhanced epithelial apoptosis. This in turn promotes lung fibrosis through impaired basement membrane repair and increased epithelial-mesenchymal transition. Furthermore, fibroblasts are recruited to the wounded area and adopt a myofibroblast phenotype, with the up-regulation of matrix-synthesizing genes and down-regulation of matrix-degradation genes. There is compelling evidence that the cytokine TGFbeta (transforming growth factor beta) plays a central role in this process. In normal lung, TGFbeta is maintained in an inactive state that is tightly regulated temporally and spatially. One of the major TGFbeta-activation pathways involves integrins, and the role of the (alpha)vbeta6 integrin has been particularly well described in the pathogenesis of IPF. Owing to the pleiotropic nature of TGFbeta, strategies that inhibit activation of TGFbeta in a cell- or disease-specific manner are attractive for the treatment of chronic fibrotic lung conditions. Therefore the molecular pathways that lead to integrin-mediated TGFbeta activation must be precisely defined to identify and fully exploit novel therapeutic targets that might ultimately improve the prognosis for patients with IPF.

Caffeine inhibits TGFβ activation in epithelial cells, interrupts fibroblast responses to TGFβ, 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 TGFβ. 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 TGFβ activation by lung epithelial cells in a concentration-dependent manner but had no effect on TGFβ activation in fibroblasts. Importantly, however, caffeine abrogated profibrotic responses to TGFβ in lung fibroblasts. It inhibited basal expression of the α-smooth muscle actin gene and reduced TGFβ-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.

Amplification of TGFβ Induced ITGB6 Gene Transcription May Promote Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating, progressive disease with poor survival rates and limited treatment options. Upregulation of αvβ6 integrins within the alveolar epithelial cells is a characteristic feature of IPF and correlates with poor patient survival. The pro-fibrotic cytokine TGFβ1 can upregulate αvβ6 integrin expression but the molecular mechanisms driving this effect have not previously been elucidated. We confirm that stimulation with exogenous TGFβ1 increases expression of the integrin β6 subunit gene (ITGB6) and αvβ6 integrin cell surface expression in a time- and concentration-dependent manner. TGFβ1-induced ITGB6 expression occurs via transcriptional activation of the ITGB6 gene, but does not result from effects on ITGB6 mRNA stability. Basal expression of ITGB6 in, and αvβ6 integrins on, lung epithelial cells occurs via homeostatic αvβ6-mediated TGFβ1 activation in the absence of exogenous stimulation, and can be amplified by TGFβ1 activation. Fundamentally, we show for the first time that TGFβ1-induced ITGB6 expression occurs via canonical Smad signalling since dominant negative constructs directed against Smad3 and 4 inhibit ITGB6 transcriptional activity. Furthermore, disruption of a Smad binding site at -798 in the ITGB6 promoter abolishes TGFβ1-induced ITGB6 transcriptional activity. Using chromatin immunoprecipitation we demonstrate that TGFβ1 stimulation of lung epithelial cells results in direct binding of Smad3, and Smad4, to the ITGB6 gene promoter within this region. Finally, using an adenoviral TGFβ1 over-expression model of pulmonary fibrosis we demonstrate that Smad3 is crucial for TGFβ1-induced αvβ6 integrin expression within the alveolar epithelium in vivo. Together, these data confirm that a homeostatic, autocrine loop of αvβ6 integrin activated TGFβ1-induced ITGB6 gene expression regulates epithelial basal αvβ6 integrin expression, and demonstrates that this occurs via Smad-dependent transcriptional regulation at a single Smad binding site in the promoter of the β6 subunit gene. Active TGFβ1 amplifies this pathway both in vitro and in vivo, which may promote fibrosis.

Novel approaches to pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating condition with a poor prognosis and few treatment options. However, recent research into this condition has led to considerable insights into the pathophysiology of the disease, resulting in the identification of potential biomarkers to aid diagnosis and stratification of patients and the development of novel therapies. In this review we will discuss the recent developments in this field and review how this knowledge has been translated into clinical trials and a paradigm shift in our approach to patients with IPF.

S69 Transcriptional Mechanisms Regulating Expression of the Avb6 Integrin in IPF

TGF-β is fibrogenic cytokine implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). The integrin αVβ6 can activate TGF-β, and dysregulation of this pathway is thought to play a role in the pathogenesis of pulmonary fibrosis. TGF-β induces αVβ6 integrin expression and aVb6 integrins are upregulated in fibrotic regions of lungs from patients with IPF. This raises the possibility that dysregulation of a self-regulating feedback loop may promote IPF. This study aims to investigate the mechanisms involved TGF-β-induced β6 expression and how this may be dysregulated in IPF. Using QPCR and flow cytometry we assessed expression of the integrin β6 (ITGB6) in lung epithelial cells. An ITGB6 promoter-luciferase construct and truncated mutants were used to identify the regulatory region of the promoter. Dominant negative (dn) constructs were used to assess the role of Smad proteins. Binding of transcription factors to the promoter was assessed by chromatin immunoprecipitation (ChIP). TGF-β caused concentration- and time-dependent increases in αVβ6 and ITGB6 mRNA, and increased activity of the ITGB6 promoter. Truncated mutants of the promoter showed that loss of 2 Smad binding sites resulted in loss of promoter activity. Co-transfection of dnSmad3 with the ITGB6 promoter reporter inhibited basal and TGF-β-induced promoter activity whereas dnSmad2 had little effect. dnSmad3 reduced TGF-β-induced αvβ6 cell surface expression. ChIP demonstrated binding of Smad3 and Smad4 to the promoter in response to TGF-β. Binding of Smad3 to the ITGB6 promoter was higher in lung tissue derived from IPF patients compared with controls. Finally, we identified a region of the ITGB6 promoter responsible for repressing transcription of the gene and demonstrate that siRNA targeting the transcription factor ELK1 increases αvβ6 expression. In conclusion, TGF-β increases expression of αVβ6 by transcriptional regulation involving Smad3. Furthermore, enhanced binding of Smad3 to the ITGB6 promoter in patients with IPF suggests dysregulated synthesis of αvβ6 integrins may promote IPF. Finally, we identified ELK1 as a potentially important negative regulator of αvβ6 expression. This highlights a positive feedback loop which could be dysregulated through hyperstimulation and impaired repression leading to amplification of αvβ6 mediated TGF-β activation that could be fundamental to IPF pathogenesis.

Breathlessness in an ex-miner: an unusual consideration

A 73-year-old retired miner, with 36 years ‘at the coal face’, presented with a 5-year history of exertional dyspnoea on climbing steep inclines. His medical history was limited to quiescent chronic lymphocytic lymphoma, for which he required no chemotherapy or radiotherapy and had annual haematology follow-up. He was a smoker, with a 15 pack-year history, and took no regular medications. The patient had a widely split second heart sound, with accentuation of the pulmonary component, and bilateral intercostal bruits. Echocardiography demonstrated normal left sided chambers, moderate mitral and tricuspid regurgitation, and a raised estimated systolic pulmonary artery pressure of 80 mm Hg. Pulmonary function tests showed an obstructive pattern (FEV1 1.91 L (70% predicted), FVC 3.62 L (101%), FEV1/FVC ratio 53%), with a mildly reduced diffusion capacity (transfer coefficient (KCO) 0.86 mmol/min/KPa/L, 70% predicted). On the incremental shuttle walking test, his oxygen saturations dropped from …

S66 Caffeine Inhibits TGFβ Activation by Epithelial Cells, Interrupts Fibroblast Responses to TGFβ, and Reduces Pulmonary Fibrosis in Ex Vivo Precision-cut Lung Slices

Caffeine (1, 3, 7-tri-methylxanthine) is a common food additive found naturally in many products. It is a non-selective competitive antagonist of G-protein coupled adenosine receptors and can inhibit phosphodiesterases. Caffeine has anti-fibrotic effects in the liver and increased caffeine consumption has been associated with reduced liver fibrosis in patients with chronic hepatitis C infection. The effect of caffeine on pulmonary fibrosis has not been investigated, however, it has been shown to inhibit TGFβ-induced Smad signalling in epithelial cells. This study aimed to investigate the anti-fibrotic effects of caffeine in the lung using lung epithelial cells, fibroblasts and an ex vivo precision-cut lung slice (PCLS) model of fibrosis. Immortalised human bronchial epithelial cells (iHBECs) and primary human lung fibroblasts from were used. TGFβ activation was assessed using an in vitro TGFβ reporter cell assay and assessment of phosphorylated Smad2. Expression of pro-fibrotic genes was assessed by quantitative polymerase chain reaction. Proliferation of fibroblasts was assessed by brdU incorporation assay. Finally, the effect of caffeine on established lung fibrosis was investigated ex vivo using PCLS. Mice were instilled with saline or 60 IU bleomycin and PCLS obtained after 28 days. PCLS were treated with increasing concentrations of caffeine for five days prior to measurement of collagen by high-performance liquid chromatography. Viability of the PCLS following caffeine treatment was assessed by MTT assay. Caffeine induced a concentration-dependent decrease in TGFβ activation in iHBECs but had no effect on TGFβ activation in lung fibroblasts. Furthermore, caffeine reduced expression of the TGFβ-inducible genes PAI1 and Col1A and reduced TGFB1 transcript in epithelial cells. Additionally, caffeine reduced TGFβ-induced proliferation of lung fibroblasts and reduced expression of pro-fibrotic genes including COL1A and ACTA2. Crucially, ex vivo treatment of fibrotic PCLS from bleomycin treated animals with caffeine caused a dose-dependent reduction in collagen deposition after five days. Caffeine had no effect on collagen deposition in PCLS isolated from saline treated animals nor did caffeine affect tissue viability in PCLS from either saline or bleomycin treated animals. In conclusion, caffeine has anti-fibrotic effects in the lung via concomitant inhibition of epithelial TGF activation and fibroblast responses to TGFb.