Defining the mechanism of galectin-3-mediated TGF-ß1 activation and its role in lung fibrosis.

Integrin-mediated activation of the profibrotic mediator transforming growth factor-ß1 (TGF-ß1), plays a critical role in idiopathic pulmonary fibrosis (IPF) pathogenesis. Galectin-3 is believed to contribute to the pathological wound healing seen in IPF, although its mechanism of action is not precisely defined. We hypothesized that galectin-3 potentiates TGF-ß1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-ß1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L-α-lysophosphatidic acid sodium salt-induced integrin-mediated TGF-ß1 activation. Surface plasmon resonance analysis confirmed that galectin-3 binds to αv integrins, αvß1, αvß5, and αvß6, and to the TGFßRII subunit in a glycosylation-dependent manner. This binding is heterogeneous and not a 1:1 binding stoichiometry. Binding interactions were blocked by small molecule inhibitors of galectin-3, which target the carbohydrate recognition domain. Galectin-3 binding to ß1 integrin was validated in vitro by coimmunoprecipitation in HLFs. Proximity ligation assays indicated that galectin-3 and ß1 integrin colocalize closely (≤40 nm) on the cell surface and that colocalization is increased by TGF-ß1 treatment and blocked by galectin-3 inhibitors. In the absence of TGF-ß1 stimulation, colocalization was detectable only in HLFs from IPF patients, suggesting the proteins are inherently more closely associated in the disease state. Galectin-3 inhibitor treatment of precision cut lung slices from IPF patients' reduced Col1a1, TIMP1, and hyaluronan secretion to a similar degree as TGF-ß type I receptor inhibitor. These data suggest that galectin-3 promotes TGF-ß1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-ß1 signaling cascade.

Stretch regulates alveologenesis and homeostasis via mesenchymal Gαq/11-mediated TGFß2 activation.

Alveolar development and repair require tight spatiotemporal regulation of numerous signalling pathways that are influenced by chemical and mechanical stimuli. Mesenchymal cells play key roles in numerous developmental processes. Transforming growth factor-ß (TGFß) is essential for alveologenesis and lung repair, and the G protein α subunits Gαq and Gα11 (Gαq/11) transmit mechanical and chemical signals to activate TGFß in epithelial cells. To understand the role of mesenchymal Gαq/11 in lung development, we generated constitutive (Pdgfrb-Cre+/-;Gnaqfl/fl;Gna11-/-) and inducible (Pdgfrb-Cre/ERT2+/-;Gnaqfl/fl;Gna11-/-) mesenchymal Gαq/11 deleted mice. Mice with constitutive Gαq/11 gene deletion exhibited abnormal alveolar development, with suppressed myofibroblast differentiation, altered mesenchymal cell synthetic function, and reduced lung TGFß2 deposition, as well as kidney abnormalities. Tamoxifen-induced mesenchymal Gαq/11 gene deletion in adult mice resulted in emphysema associated with reduced TGFß2 and elastin deposition. Cyclical mechanical stretch-induced TGFß activation required Gαq/11 signalling and serine protease activity, but was independent of integrins, suggesting an isoform-specific role for TGFß2 in this model. These data highlight a previously undescribed mechanism of cyclical stretch-induced Gαq/11-dependent TGFß2 signalling in mesenchymal cells, which is imperative for normal alveologenesis and maintenance of lung homeostasis.

The IL-33:ST2 axis is unlikely to play a central fibrogenic role in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease (ILD) with limited treatment options. Interleukin-33 (IL-33) is proposed to play a role in the development of IPF however the exclusive use of prophylactic dosing regimens means that the therapeutic benefit of targeting this cytokine in IPF is unclear. METHODS: IL-33 expression was assessed in ILD lung sections and human lung fibroblasts (HLFs) by immunohistochemistry and gene/protein expression and responses of HLFs to IL-33 stimulation measured by qPCR. In vivo, the fibrotic potential of IL-33:ST2 signalling was assessed using a murine model of bleomycin (BLM)-induced pulmonary fibrosis and therapeutic dosing with an ST2-Fc fusion protein. Lung and bronchoalveolar lavage fluid were collected for measurement of inflammatory and fibrotic endpoints. Human precision-cut lung slices (PCLS) were stimulated with transforming growth factor-ß (TGFß) or IL-33 and fibrotic readouts assessed. RESULTS: IL-33 was expressed by fibrotic fibroblasts in situ and was increased by TGFß treatment in vitro. IL-33 treatment of HLFs did not induce IL6, CXCL8, ACTA2 and COL1A1 mRNA expression with these cells found to lack the IL-33 receptor ST2. Similarly, IL-33 stimulation had no effect on ACTA2, COL1A1, FN1 and fibronectin expression by PCLS. Despite having effects on inflammation suggestive of target engagement, therapeutic dosing with the ST2-Fc fusion protein failed to reduce BLM-induced fibrosis measured by hydroxyproline content or Ashcroft score. CONCLUSIONS: Together these findings suggest the IL-33:ST2 axis does not play a central fibrogenic role in the lungs with therapeutic blockade of this pathway unlikely to surpass the current standard of care for IPF.

Differential remodeling in small and large murine airways revealed by novel whole lung airway analysis.

Airway remodeling occurs in chronic asthma leading to increased airway smooth muscle (ASM) mass and extracellular matrix (ECM) deposition. Although extensively studied in murine airways, studies report only selected larger airways at one time-point meaning the spatial distribution and resolution of remodeling are poorly understood. Here we use a new method allowing comprehensive assessment of the spatial and temporal changes in ASM, ECM, and epithelium in large numbers of murine airways after allergen challenge. Using image processing to analyze 20-50 airways per mouse from a whole lung section revealed increases in ASM and ECM after allergen challenge were greater in small and large rather than intermediate airways. ASM predominantly accumulated adjacent to the basement membrane, whereas ECM was distributed across the airway wall. Epithelial hyperplasia was most marked in small and intermediate airways. After challenge, ASM changes resolved over 7 days, whereas ECM and epithelial changes persisted. The new method suggests large and small airways remodel differently, and the long-term consequences of airway inflammation may depend more on ECM and epithelial changes than ASM. The improved quantity and quality of unbiased data provided by the method reveals important spatial differences in remodeling and could set new analysis standards for murine asthma models.

Lysyl oxidase like 2 is increased in asthma and contributes to asthmatic airway remodelling.

BACKGROUND: Airway smooth muscle (ASM) cells are fundamental to asthma pathogenesis, influencing bronchoconstriction, airway hyperresponsiveness and airway remodelling. The extracellular matrix (ECM) can influence tissue remodelling pathways; however, to date no study has investigated the effect of ASM ECM stiffness and cross-linking on the development of asthmatic airway remodelling. We hypothesised that transforming growth factor-ß (TGF-ß) activation by ASM cells is influenced by ECM in asthma and sought to investigate the mechanisms involved. METHODS: This study combines in vitro and in vivo approaches: human ASM cells were used in vitro to investigate basal TGF-ß activation and expression of ECM cross-linking enzymes. Human bronchial biopsies from asthmatic and nonasthmatic donors were used to confirm lysyl oxidase like 2 (LOXL2) expression in ASM. A chronic ovalbumin (OVA) model of asthma was used to study the effect of LOXL2 inhibition on airway remodelling. RESULTS: We found that asthmatic ASM cells activated more TGF-ß basally than nonasthmatic controls and that diseased cell-derived ECM influences levels of TGF-ß activated. Our data demonstrate that the ECM cross-linking enzyme LOXL2 is increased in asthmatic ASM cells and in bronchial biopsies. Crucially, we show that LOXL2 inhibition reduces ECM stiffness and TGF-ß activation in vitro, and can reduce subepithelial collagen deposition and ASM thickness, two features of airway remodelling, in an OVA mouse model of asthma. CONCLUSION: These data are the first to highlight a role for LOXL2 in the development of asthmatic airway remodelling and suggest that LOXL2 inhibition warrants further investigation as a potential therapy to reduce remodelling of the airways in severe asthma.

Pharmacological characterisation of GSK3335103, an oral αvß6 integrin small molecule RGD-mimetic inhibitor for the treatment of fibrotic disease.

Fibrosis is the formation of scar tissue due to injury or long-term inflammation and is a leading cause of morbidity and mortality. Activation of the pro-fibrotic cytokine transforming growth factor-ß (TGFß) via the alpha-V beta-6 (αvß6) integrin has been identified as playing a key role in the development of fibrosis. Therefore, a drug discovery programme to identify an orally bioavailable small molecule αvß6 arginyl-glycinyl-aspartic acid (RGD)-mimetic was initiated. As part of a medicinal chemistry programme GSK3335103 was identified and profiled in a range of pre-clinical in vitro and in vivo systems. GSK3335103 was shown to bind to the αvß6 with high affinity and demonstrated fast binding kinetics. In primary human lung epithelial cells, GSK3335103-induced concentration- and time-dependent internalisation of αvß6 with a rapid return of integrin to the cell surface observed after washout. Following sustained engagement of the αvß6 integrin in vitro, lysosomal degradation was induced by GSK3335103. GSK3335103 was shown to engage with the αvß6 integrin and inhibit the activation of TGFß in both ex vivo IPF tissue and in a murine model of bleomycin-induced lung fibrosis, as measured by αvß6 engagement, TGFß signalling and collagen deposition, with a prolonged duration of action observed in vivo. In summary, GSK3335103 is a potent αvß6 inhibitor that attenuates TGFß signalling in vitro and in vivo with a well-defined pharmacokinetic/pharmacodynamic relationship. This translates to a significant reduction of collagen deposition in vivo and therefore GSK3335103 represents a potential novel oral therapy for fibrotic disorders.

COVID-19 and pulmonary fibrosis: A potential role for lung epithelial cells and fibroblasts.

The COVID-19 pandemic rapidly spread around the world following the first reports in Wuhan City, China in late 2019. The disease, caused by the novel SARS-CoV-2 virus, is primarily a respiratory condition that can affect numerous other bodily systems including the cardiovascular and gastrointestinal systems. The disease ranges in severity from asymptomatic through to severe acute respiratory distress requiring intensive care treatment and mechanical ventilation, which can lead to respiratory failure and death. It has rapidly become evident that COVID-19 patients can develop features of interstitial pulmonary fibrosis, which in many cases persist for as long as we have thus far been able to follow the patients. Many questions remain about how such fibrotic changes occur within the lung of COVID-19 patients, whether the changes will persist long term or are capable of resolving, and whether post-COVID-19 pulmonary fibrosis has the potential to become progressive, as in other fibrotic lung diseases. This review brings together our existing knowledge on both COVID-19 and pulmonary fibrosis, with a particular focus on lung epithelial cells and fibroblasts, in order to discuss common pathways and processes that may be implicated as we try to answer these important questions in the months and years to come.

Translational pharmacology of an inhaled small molecule αvß6 integrin inhibitor for idiopathic pulmonary fibrosis.

The αvß6 integrin plays a key role in the activation of transforming growth factor-ß (TGFß), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvß6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and disease-related end points. Here, we report, GSK3008348 binds to αvß6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFß signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvß6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvß6, induces prolonged inhibition of TGFß signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy.

Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.

In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, causing the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilises angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147 and GRP78 may also function as receptors for SARS-CoV-2.To determine the expression and in situ localisation of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analysed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, proteomic datasets, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples.We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung, confirmed with proteomics. We present confirmatory evidence for the presence of TMPRSS2, CD147 and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 and GRP78 in the respiratory mucosa.Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternative receptors for SARS-CoV-2 exist to facilitate initial host cell infection.

Loss of ELK1 has differential effects on age-dependent organ fibrosis.

ETS domain-containing protein-1 (ELK1) is a transcription factor important in regulating αvß6 integrin expression. αvß6 integrins activate the profibrotic cytokine Transforming Growth Factor ß1 (TGFß1) and are increased in the alveolar epithelium in idiopathic pulmonary fibrosis (IPF). IPF is a disease associated with aging and therefore we hypothesised that aged animals lacking Elk1 globally would develop spontaneous fibrosis in organs where αvß6 mediated TGFß activation has been implicated. Here we identify that Elk1-knockout (Elk1-/0) mice aged to one year developed spontaneous fibrosis in the absence of injury in both the lung and the liver but not in the heart or kidneys. The lungs of Elk1-/0 aged mice demonstrated increased collagen deposition, in particular collagen 3α1, located in small fibrotic foci and thickened alveolar walls. Despite the liver having relatively low global levels of ELK1 expression, Elk1-/0 animals developed hepatosteatosis and fibrosis. The loss of Elk1 also had differential effects on Itgb1, Itgb5 and Itgb6 expression in the four organs potentially explaining the phenotypic differences in these organs. To understand the potential causes of reduced ELK1 in human disease we exposed human lung epithelial cells and murine lung slices to cigarette smoke extract, which lead to reduced ELK1 expression andmay explain the loss of ELK1 in human disease. These data support a fundamental role for ELK1 in protecting against the development of progressive fibrosis via transcriptional regulation of beta integrin subunit genes, and demonstrate that loss of ELK1 can be caused by cigarette smoke.

Extracellular Matrix Cross-Linking Enhances Fibroblast Growth and Protects against Matrix Proteolysis in Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by accumulation of extracellular matrix (ECM) proteins and fibroblast proliferation. ECM cross-linking enzymes have been implicated in fibrotic diseases, and we hypothesized that the ECM in IPF is abnormally cross-linked, which enhances fibroblast growth and resistance to normal ECM turnover. We used a combination of in vitro ECM preparations and in vivo assays to examine the expression of cross-linking enzymes and the effect of their inhibitors on fibroblast growth and ECM turnover. Lysyl oxidase-like 1 (LOXL1), LOXL2, LOXL3, and LOXL4 were expressed equally in control and IPF-derived fibroblasts. Transglutaminase 2 was more strongly expressed in IPF fibroblasts. LOXL2-, transglutaminase 2-, and transglutaminase-generated cross-links were strongly expressed in IPF lung tissue. Fibroblasts grown on IPF ECM had higher LOXL3 protein expression and transglutaminase activity than those grown on control ECM. IPF-derived ECM also enhanced fibroblast adhesion and proliferation compared with control ECM. Inhibition of lysyl oxidase and transglutaminase activity during ECM formation affected ECM structure as visualized by electron microscopy, and it reduced the enhanced fibroblast adhesion and proliferation of IPF ECM to control levels. Inhibition of transglutaminase, but not of lysyl oxidase, activity enhanced the turnover of ECM in vitro. In bleomycin-treated mice, during the postinflammatory fibrotic phase, inhibition of transglutaminases was associated with a reduction in whole-lung collagen. Our findings suggest that the ECM in IPF may enhance pathological cross-linking, which contributes to increased fibroblast growth and resistance to normal ECM turnover to drive lung fibrosis.

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.

Methods for the Assessment of Active Transforming Growth Factor-ß in Cells and Tissues.

The potent and pluripotent cytokine TGFß has important roles in normal homeostasis and disease pathogenesis. Once released from cells, TGFß exists in both latent and functionally active forms. Large amounts of latent TGFß are secreted from cells and sequestered in extracellular matrix, only a small proportion of which is activated at any given time. Accurate assessment of TGFß activity levels is an important measurement in biological research and requires methods distinct from measuring total levels of TGFß expression as small changes in TGFß activity levels could be masked by the large amounts of latent TGFß available to be measured. In this chapter, we describe detailed experimental methods for assessing levels of active TGFß in cells and tissues. This chapter includes methods for the assessment of TGFß activity in cells in vitro, in ex vivo precision cut tissue, and in vivo.

Loss of epithelial Gq and G11 signaling inhibits TGFß production but promotes IL-33-mediated macrophage polarization and emphysema.

Heterotrimeric guanine nucleotide-binding protein (G protein) signaling links hundreds of G protein-coupled receptors with four G protein signaling pathways. Two of these, one mediated by Gq and G11 (Gq/11) and the other by G12 and G13 (G12/13), are implicated in the force-dependent activation of transforming growth factor-ß (TGFß) in lung epithelial cells. Reduced TGFß activation in alveolar cells leads to emphysema, whereas enhanced TGFß activation promotes acute lung injury and idiopathic pulmonary fibrosis. Therefore, precise control of alveolar TGFß activation is essential for alveolar homeostasis. We investigated the involvement of the Gq/11 and G12/13 pathways in epithelial cells in generating active TGFß and regulating alveolar inflammation. Mice deficient in both Gαq and Gα11 developed inflammation that was primarily caused by alternatively activated (M2-polarized) macrophages, enhanced matrix metalloproteinase 12 (MMP12) production, and age-related alveolar airspace enlargement consistent with emphysema. Mice with impaired Gq/11 signaling had reduced stretch-mediated generation of TGFß by epithelial cells and enhanced macrophage MMP12 synthesis but were protected from the effects of ventilator-induced lung injury. Furthermore, synthesis of the cytokine interleukin-33 (IL-33) was increased in these alveolar epithelial cells, resulting in the M2-type polarization of alveolar macrophages independently of the effect on TGFß. Our results suggest that alveolar Gq/11 signaling maintains alveolar homeostasis and likely independently increases TGFß activation in response to the mechanical stress of the epithelium and decreases epithelial IL-33 synthesis. Together, these findings suggest that disruption of Gq/11 signaling promotes inflammatory emphysema but protects against mechanically induced lung injury.

Amplification of TGFß 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 αvß6 integrins within the alveolar epithelial cells is a characteristic feature of IPF and correlates with poor patient survival. The pro-fibrotic cytokine TGFß1 can upregulate αvß6 integrin expression but the molecular mechanisms driving this effect have not previously been elucidated. We confirm that stimulation with exogenous TGFß1 increases expression of the integrin ß6 subunit gene (ITGB6) and αvß6 integrin cell surface expression in a time- and concentration-dependent manner. TGFß1-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 αvß6 integrins on, lung epithelial cells occurs via homeostatic αvß6-mediated TGFß1 activation in the absence of exogenous stimulation, and can be amplified by TGFß1 activation. Fundamentally, we show for the first time that TGFß1-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 TGFß1-induced ITGB6 transcriptional activity. Using chromatin immunoprecipitation we demonstrate that TGFß1 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 TGFß1 over-expression model of pulmonary fibrosis we demonstrate that Smad3 is crucial for TGFß1-induced αvß6 integrin expression within the alveolar epithelium in vivo. Together, these data confirm that a homeostatic, autocrine loop of αvß6 integrin activated TGFß1-induced ITGB6 gene expression regulates epithelial basal αvß6 integrin expression, and demonstrates that this occurs via Smad-dependent transcriptional regulation at a single Smad binding site in the promoter of the ß6 subunit gene. Active TGFß1 amplifies this pathway both in vitro and in vivo, which may promote fibrosis.

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.

Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvß6 Expression.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with high mortality. Active TGFß1 is considered central to the pathogenesis of IPF. A major mechanism of TGFß1 activation in the lung involves the epithelially restricted αvß6 integrin. Expression of the αvß6 integrin is dramatically increased in IPF. How αvß6 integrin expression is regulated in the pulmonary epithelium is unknown. Here we identify a region in the ß6 subunit gene (ITGB6) promoter acting to markedly repress basal gene transcription, which responds to both the Ets domain-containing protein Elk1 (Elk1) and the glucocorticoid receptor (GR). Both Elk1 and GR can regulate αvß6 integrin expression in vitro We demonstrate Elk1 binding to the ITGB6 promoter basally and that manipulation of Elk1 or Elk1 binding alters ITGB6 promoter activity, gene transcription, and αvß6 integrin expression. Crucially, we find that loss of Elk1 causes enhanced Itgb6 expression and exaggerated lung fibrosis in an in vivo model of fibrosis, whereas the GR agonist dexamethasone inhibits Itgb6 expression. Moreover, Elk1 dysregulation is present in epithelium from patients with IPF. These data reveal a novel role for Elk1 regulating ITGB6 expression and highlight how dysregulation of Elk1 can contribute to human disease.

CXCL8 histone H3 acetylation is dysfunctional in airway smooth muscle in asthma: regulation by BET.

Asthma is characterized by airway inflammation and remodeling and CXCL8 is a CXC chemokine that drives steroid-resistant neutrophilic airway inflammation. We have shown that airway smooth muscle (ASM) cells isolated from asthmatic individuals secrete more CXCL8 than cells from nonasthmatic individuals. Here we investigated chromatin modifications at the CXCL8 promoter in ASM cells from nonasthmatic and asthmatic donors to further understand how CXCL8 is dysregulated in asthma. ASM cells from asthmatic donors had increased histone H3 acetylation, specifically histone H3K18 acetylation, and increased binding of histone acetyltransferase p300 compared with nonasthmatic donors but no differences in CXCL8 DNA methylation. The acetylation reader proteins Brd3 and Brd4 were bound to the CXCL8 promoter and Brd inhibitors inhibited CXCL8 secretion from ASM cells by disrupting Brd4 and RNA polymerase II binding to the CXCL8 promoter. Our results show a novel dysregulation of CXCL8 transcriptional regulation in asthma characterized by a promoter complex that is abnormal in ASM cells isolated from asthmatic donors and can be modulated by Brd inhibitors. Brd inhibitors may provide a new therapeutic strategy for steroid-resistant inflammation.

Preclinical SPECT/CT imaging of αvß6 integrins for molecular stratification of idiopathic pulmonary fibrosis.

UNLABELLED: Transforming growth factor ß activation by the αvß6 integrin is central to the pathogenesis of idiopathic pulmonary fibrosis. Expression of the αvß6 integrin is increased in fibrotic lung tissue and is a promising therapeutic target for treatment of the disease. Currently, measurement of αvß6 integrin levels in the lung requires immunohistochemical analysis of biopsy samples. This procedure is clinically impractical for many patients with pulmonary fibrosis, and a noninvasive strategy for measuring αvß6 integrin levels in the lungs is urgently required to facilitate monitoring of disease progression and therapeutic responses. METHODS: Using a murine model of bleomycin-induced lung injury, we assessed the binding of intravenously administered (111)In-labeled αvß6-specific (diethylenetriamine pentaacetate-tetra [DTPA]-A20FMDV2) or control (DTPA-A20FMDVran) peptide by nanoSPECT/CT imaging. Development of fibrosis was assessed by lung hydroxyproline content, and αvß6 protein and itgb6 messenger RNA were measured in the lungs. RESULTS: Maximal binding of (111)In-labeled A20FMDV2 peptide to αvß6 integrins was detected in the lungs 1 h after intravenous administration. No significant binding was detected in mice injected with control peptide. Integrin binding was increased in the lungs of bleomycin-, compared with saline-, exposed mice and was attenuated by pretreatment with αvß6-blocking antibodies. Levels of (111)In-labeled A20FMDV2 peptide correlated positively with hydroxyproline, αvß6 protein, and itgb6 messenger RNA levels. CONCLUSION: We have developed a highly sensitive, quantifiable, and noninvasive technique for measuring αvß6 integrin levels within the lung. Measurement of αvß6 integrins by SPECT/CT scanning has the potential for use in stratifying therapy for patients with pulmonary fibrosis.

Epilepsy surgery in Neurofibromatosis Type 1.

Epilepsy is relatively uncommon in patients with Neurofibromatosis Type 1 (NF1) and seizures are usually well controlled with antiepileptic treatment. However, pharmacoresistance has been reported in patients with NF1 and MRI evidence of malformations of cortical development or glioneuronal tumours. Available information on epilepsy surgery in NF1 is limited to a few patients with gliomas and glioneuronal tumours who underwent lesionectomies. We conducted a survey amongst 25 European epilepsy surgery centres to collect patients with NF1 who had undergone surgery for drug-resistant seizures and identified 12 patients from eight centres. MRI abnormalities were present in all patients but one. They were unilateral temporal in eight, bilateral temporal in one and multilobar or hemispheric in two. Seizures originated from the temporal lobe in ten patients, from the temporo-parieto-occipital region in one, and were bitemporal in one. One year after surgery eight patients were seizure free, one had worthwhile improvement and the remaining three had experienced no benefit. Postoperative outcome, available at 2 years in ten patients and at 5 years in three, remained stable in all but one whose seizures reappeared. Histology revealed dysembryoplastic neuroepithelial tumour (DNET) in five patients, hippocampal sclerosis in four, mixed pathology in one and polymicrogyria in one. No histological abnormality was observed in the remaining patient. Epilepsy surgery can be performed effectively in patients with NF1 provided a single and well-delimited epileptogenic zone is recognized. The high prevalence of DNETs in this series might suggest a non-fortuitous association with NF1.