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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-β (TGFβ) 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 TGFβ 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 TGFβ2 deposition, as well as kidney abnormalities. Tamoxifen-induced mesenchymal Gαq/11 gene deletion in adult mice resulted in emphysema associated with reduced TGFβ2 and elastin deposition. Cyclical mechanical stretch-induced TGFβ activation required Gαq/11 signalling and serine protease activity, but was independent of integrins, suggesting an isoform-specific role for TGFβ2 in this model. These data highlight a previously undescribed mechanism of cyclical stretch-induced Gαq/11-dependent TGFβ2 signalling in mesenchymal cells, which is imperative for normal alveologenesis and maintenance of lung homeostasis.

The αvβ6 integrin plays a key role in the activation of transforming growth factor-β (TGFβ), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvβ6 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 αvβ6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFβ signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvβ6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvβ6, induces prolonged inhibition of TGFβ 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., The αvβ6 integrin is key in activating the pro-fibrotic cytokine TGFβ in idiopathic pulmonary fibrosis. Here, the authors show an inhaled small molecule αvβ6 inhibitor GSK3008348 induces prolonged inhibition of TGFβ signaling pathways in human and murine models of lung fibrosis via αvβ6 degradation.

BackgroundAirway 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-β (TGF-β) activation by ASM cells is influenced by ECM in asthma and sought to investigate the mechanisms involved.MethodsThis study combines in vitro and in vivo approaches: human ASM cells were used in vitro to investigate basal TGF-β 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.ResultsWe found that asthmatic ASM cells activated more TGF-β basally than nonasthmatic controls and that diseased cell-derived ECM influences levels of TGF-β 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-β activation in vitro, and can reduce subepithelial collagen deposition and ASM thickness, two features of airway remodelling, in an OVA mouse model of asthma.ConclusionThese 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.

Airway smooth muscle cells (ASM) are fundamental to asthma pathogenesis, influencing bronchoconstriction, airway hyper-responsiveness, and airway remodelling. Extracellular matrix (ECM) can influence tissue remodelling pathways, however, to date no study has investigated the effect of ASM ECM stiffness and crosslinking on the development of asthmatic airway remodelling. We hypothesised that TGFβ activation by ASM is influenced by ECM in asthma and sought to investigate the mechanisms involved.This study combines in vitro and in vivo approaches: human ASM cells were used in vitro to investigate basal TGFβ activation and expression of ECM crosslinking enzymes. Human bronchial biopsies from asthmatic and non-asthmatic donors were used to confirm LOXL2 expression ASM. A chronic ovalbumin model of asthma was used to study the effect of LOXL2 inhibition on airway remodelling.We found that ASM cells from asthmatics activated more TGFβ basally than non-asthmatic controls and that diseased cell-derived ECM influences levels of TGFβ activated. Our data demonstrate that the ECM crosslinking enzyme LOXL2 is increased in asthmatic ASM cells and in bronchial biopsies. Crucially, we show that LOXL2 inhibition reduces ECM stiffness and TGFβ activation in vitro, and can reduce subepithelial collagen deposition and ASM thickness, two features of airway remodelling, in an ovalbumin mouse model of asthma.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.

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-β (TGFβ) 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 TGFβ 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 TGFβ2 deposition, as well as kidney abnormalities. Tamoxifen-induced mesenchymal Gαq/11 gene deletion in adult mice resulted in emphysema associated with reduced TGFβ2 and elastin deposition. Cyclical mechanical stretch-induced TGFβ activation required Gαq/11 signalling and serine protease activity, but was independent of integrins, suggesting an isoform-specific role for TGFβ2. These data highlight a previously undescribed mechanism of cyclical stretch-induced Gαq/11-dependent TGFβ2 signalling in mesenchymal cells, which is imperative for normal alveologenesis and maintenance of lung homeostasis.Summary statementMesenchymal cell Gαq/11 signalling regulates myofibroblast function and stretch-mediated TGFβ2 signalling, which are important for alveologenesis and organ homeostasis. These mechanisms are relevant to both developmental and adult lung disease.

In December 2019, SARS-CoV-2 emerged causing the COVID-19 pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilizes ACE2 and 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 localization of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analyzed 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, 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. 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 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 alternate receptors for SARS-CoV-2 exist to facilitate initial host cell infection.

Idiopathic Pulmonary Fibrosis (IPF) is a progressive, fatal disease with limited treatment options. Protease mediated transforming growth factor-β (TGF-β) 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-2 (MMP-2) and MMP-9 were measured. Lung fibrosis was determined by collagen subtype specific gene expression, hydroxyproline concentration, and histological assessment. Alveolar TGF-β activation was measured using bronchoalveolar lavage cell pSmad2 levels and global TGF-β 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-β 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-β activity following bleomycin, above their already elevated levels, although global TGF-β 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-β 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 dramatically modify the development of lung fibrosis following bleomycin-induced lung injury.

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.

ETS domain-containing protein-1 (ELK1) is a transcriptional repressor important in regulating αvβ6 integrin expression. αvβ6 integrins activate the profibrotic cytokine Transforming Growth Factor β1 (TGFβ1) 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 avb6 mediated TGFb 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 genes expression in the four organs potentially explaining the phenotypic differences in these organs. To understand the potential causes of reduced ELK1in human disease Finally we exposed human cells and murine lung slices to cigarette smoke extract which lead to reduced ELK1 expression which may explain the loss of ELK1 in human disease. These data support a fundament 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.

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.

Introduction Idiopathic pulmonary fibrosis (IPF) is a debilitating interstitial lung disease with a poor prognosis and limited treatment options. It is a chronic, progressive, condition characterised by the excessive deposition of extracellular matrix by fibroblasts. Transforming growth factor-β (TGF- β) is central to this process and is regarded as a key pro-fibrotic mediator in IPF. Recently, emerging evidence suggests that the cytokine interleukin-33 (IL-33) may also be important in the development of IPF. However, the cellular and molecular mechanisms by which IL-33 promotes fibrosis are unknown. In particular, whether TGF-β and IL-33 have independent pro-fibrotic effects on fibroblasts remains unclear. Methods Primary human lung fibroblasts (HLFs) from non-IPF and IPF patients were treated with either 2 ng/ml TGF-β or 10 ng/ml IL-33. The levels of IL6 and IL8 mRNA were used as readouts of IL-33-mediated NF-κβ activation whilst ACTA2 and COL1A1 mRNA were used as readouts of fibrosis. mRNA levels were measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Finally, the levels of IL-33, and its receptor ST2, were measured by qRT-PCR to assess mRNA expression and western blotting to measure protein. Results Stimulation with TGF-β for 8 hours induced statistically significant increases in IL33 gene expression by both non-IPF and IPF HLFs. Moreover, TGF-β stimulation of HLFs from patients with and without IPF induced IL-33 protein as assessed by immunoblotting. To assess the functional consequence of TGF-b induced IL-33 from fibroblasts, the effect of recombinant IL-33 on HLFs was determined. Increasing concentrations of IL-33 failed to stimulate IL6, IL8, ACTA2 and COL1A1 gene expression by either IPF or non-IPF fibroblasts. To understand the lack of IL-33 responsiveness in HLFs, ST2 levels were measured and neither ST2 mRNA or protein were detectable in IPF, or non-IPF, HLFs. Conclusion TGF-β increases IL-33 production by HLFs, however fibroblast-derived IL-33 does not act directly on fibrotic fibroblasts as they fail to express the receptor required for IL-33 responsiveness. Therefore, the pro-fibrotic effects of IL-33 are likely mediated via alternative cell types.

Rationale Idiopathic pulmonary fibrosis is a chronic fibroproliferative disease with a median survival of approximately 3 years. αvβ6 integrins are upregulated in lung fibrosis and are associated with increased activation of the profibrotic cytokine TGF-β. The transcription factor Elk1 can repress gene expression of β6. We therefore hypothesise that animals lacking functional Elk1 (Elk1-/0) will develop age related pulmonary fibrosis. Methods Elk1 knock-out (Elk1-/0) and wild-type (Elk1+/0) mice were allowed to age for 365 days. At 365 days old mice were sacrificed and their lungs harvested for evaluation of collagen gene expression, lung hydroxyproline concentration and histological assessment. Results Lungs were extracted and lung wet weights were measured in Elk1-/0 mice and wild-type (Elk1+/0) controls and no significant difference between the two genotypes was shown (175.7 mg, 161.8 mg respectively n=6–8). However, assessment of total lung hydroxyproline established that there was significantly more hydroxyproline in Elk1-/0 mice compared with Elk1+/0 controls (852.3, 758.4 µg/lung set, respectively, n=5–8, p=0.0346). Assessment of Masson’s trichrome stained Elk1-/0 lung tissue sections found a small number of fibrotic lesions were present. Furthermore, there was a trend towards increased alveolar wall median thickness in Elk1-/0 mice compared with Elk1+/0 animals (5.94 vs 5.56 µm respectively). In a small number of 12 week old mice we identified a trend towards increased α-smooth muscle actin (αSMA) mRNA expression in the lungs of Elk1-/0 mice compared with Elk1+/0 controls (9.40, 2.24 median relative expression, respectively n=3). We therefore performed immunohistochemical staining for αSMA in the lungs of mice aged to 1 year and demonstrated visible increases in expression of αSMA in the alveolar epithelium of Elk1-/0 mice but not in Elk1+/0 controls. Conclusion These data suggest that Elk1 gene deletion result in age-related early fibrotic changes associated with the development of pulmonary fibrosis.

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.

Transforming growth factor b activation by the avb6 integrin is central to the pathogenesis of idiopathic pulmonary fibrosis. Expression of the avb6 integrin is increased in fibrotic lung tissue and is a promising therapeutic target for treatment of the disease. Currently, measurement of avb6 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 avb6 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 111In-labeled avb6-specific (diethylenetriamine pentaacetate-tetra [DTPA]-A20FMDV2) or control (DTPAA20FMDVran) peptide by nanoSPECT/CT imaging. Development of fibrosis was assessed by lung hydroxyproline content, and avb6 protein and itgb6 messenger RNA were measured in the lungs. Results: Maximal binding of 111In-labeled A20FMDV2 peptide to avb6 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 avb6-blocking antibodies. Levels of 111 In-labeled A20FMDV2 peptide correlated positively with hydroxyproline, avb6 protein, and itgb6 messenger RNA levels. Conclusion: We have developed a highly sensitive, quantifiable, and noninvasive technique for measuring avb6 integrin levels within the lung. Measurement of avb6 integrins by SPECT/CT scanning has the potential for use in stratifying therapy for patients with pulmonary fibrosis.

Heterotrimeric guanine nucleotide–binding protein (G protein) signaling is a ubiquitous signaling system that links hundreds of G protein–coupled receptors (GPCRs) with four G protein signaling pathways. Two of these pathways, one mediated by Gq and G11 and the other by G12 and G13, are implicated in the force-dependent activation of transforming growth factor–β (TGFβ) in lung epithelial cells. Reduced TGFβ activation in alveolar cells leads to emphysema, whereas enhanced TGFβ activation promotes acute lung injury, and idiopathic pulmonary fibrosis, therefore precise control of alveolar TGFβ activation is essential for alveolar homeostasis. Here, we investigated whether the Gq/G11 or G12/G13 pathways in epithelial cells are required to generate TGFβ and suppress alveolar inflammation. Mice deficient in both Gαq and Gα11 developed inflammation primarily due to alternatively activated (M2-polarized) macrophages, enhanced production of matrix metalloprotease 12 (MMP12), and age-related alveolar airspace enlargement consistent with emphysema. We found that mice with impaired Gq/G11 signaling had reduced stretch-mediated generation of TGFβ by epithelial cells and elevated macrophage MMP12 synthesis, but were protected from the effects of ventilator-induced lung injury. Furthermore, synthesis of the pleiotropic 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 TGFβ. Our results suggest that alveolar Gq/G11 signaling maintains alveolar homeostasis and is likely to independently upregulate mechanotransduced epithelial TGFβ activation and downregulate epithelial IL-33 synthesis. Together, these findings suggest that disruption of Gq/G11 signaling promotes inflammatory emphysema, but protects against mechanostransduced lung injury.

Fibrosis is the formation of scar tissue due to injury or long-term inflammation and is a leading cause of morbidity and mortality in disorders that include IPF. The αvβ6 integrin has been identified as playing a key role in the activation of TGFβ that is hypothesized to be pivotal in …

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.

Severe asthma is associated with airway remodeling, characterized by structural changes including increased smooth muscle mass and matrix deposition in the airway, leading to deteriorating lung function. TGF-β is a pleiotropic cytokine leading to increased synthesis of matrix molecules by human airway smooth muscle (HASM) cells and is implicated in asthmatic airway remodeling. TGF-β is synthesized as a latent complex, sequestered in the extracellular matrix, and requires activation for functionality. Activation of latent TGF-β is the rate-limiting step in its bioavailability. This study investigated the effect of the contraction agonists LPA and methacholine on TGF-β activation by HASM cells and its role in the development of asthmatic airway remodeling. The data presented show that LPA and methacholine induced TGF-β activation by HASM cells via the integrin αvβ5. Our findings highlight the importance of the β5 cytoplasmic domain because a polymorphism in the β5 subunit rendered the integrin unable to activate TGF-β. To our knowledge, this is the first description of a biologically relevant integrin that is unable to activate TGF-β. These data demonstrate that murine airway smooth muscle cells express αvβ5 integrins and activate TGF-β. Finally, these data show that inhibition, or genetic loss, of αvβ5 reduces allergen-induced increases in airway smooth muscle thickness in two models of asthma. These data highlight a mechanism of TGF-β activation in asthma and support the hypothesis that bronchoconstriction promotes airway remodeling via integrin mediated TGF-β activation.

CXCL8 is a neutrophil and mast cell chemoattractant that is involved in regulating inflammatory cell influx in asthma. Here, we investigated the transcriptional mechanism involved in CXCL8 induction by TNF-α in cultured human airway smooth muscle (HASM) cells and compared these in cells from nonasthmatic and asthmatic individuals. Transfection studies with mutated CXCL8 promoter constructs identified NF-κB, activating protein-1, and CAAT/enhancer binding protein (C/EBP)β as key transcription factors, and binding of these three transcription factors to the CXCL8 promoter after TNF-α stimulation was confirmed by chromatin immunoprecipitation analysis. Cells derived from asthmatic individuals produced significantly higher levels of CXCL8 than nonasthmatic cells both basally and following 24 h of stimulation with TNF-α (p < 0.001). Furthermore, chromatin immunoprecipitation studies detected increased binding of NF-κB p65 and RNA polymerase II to the CXCL8 promoter of asthmatic HASM cells both in the presence and absence of TNF-α stimulation. This was not due to either an increased activation or phosphorylation of NF-κB per se or to an increase in its translocation to the nucleus. Increased binding of C/EBPβ to the CXCL8 promoter of unstimulated cells was also detected in the asthmatic HASM cells. Collectively these studies show that HASM cells from asthmatic individuals have increased CXCL8 production due to the presence of a transcription complex on the CXCL8 promoter, which contains NF-κB, C/EBPβ, and RNA polymerase II. This is the first description of an abnormality in transcription factor binding altering chemokine expression in airway structural cells in asthma.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with high mortality. Active TGFβ1 is considered central to the pathogenesis of IPF. A major mechanism of TGFβ1 activation in the lung involves the epithelially restricted αvβ6 integrin. Expression of the αvβ6 integrin is dramatically increased in IPF. How αvβ6 integrin expression is regulated in the pulmonary epithelium is unknown. Here we identify a region in the β6 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 αvβ6 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 αvβ6 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.

CCL28 is a recently identified chemokine ligand for CCR10 and CCR3 that has been identified in mucosal epithelial surfaces in diverse tissues. CCL28-mediated eosinophil chemotaxis and peroxidase release were inhibited by preincubation of cells with anti-CCR3. CCL28 was constitutively expressed in lung tissue collected from nonsensitized control mice but increased levels were found in mice sensitized and rechallenged with cockroach antigen (CRA). CCL28 levels peaked in the lungs 24 hours after intratracheal challenge with CRA, whereas eotaxin expression peaked at 8 hours. Increased expression of CCR3 but not CCR10 could be detected during the induction of the CRA-induced pulmonary inflammation. To investigate the role of CCL28 in allergic airway responses, mice were treated with CCL28 antiserum 1 hour before receiving the final CRA challenge. The level of airway hyperresponsiveness in mice treated with anti-CCL28 was significantly reduced at 24 hours, but not 8 hours, compared to mice receiving control serum. This reduction was not related to decreased Th2 cytokine, chemokine, or leukotriene levels at 24 hours although peribronchial eosinophilia was significantly reduced. Thus, CCL28 appears to play a role in regulating eosinophil recruitment to peribronchial regions of the lung possibly by coordinated temporal production with eotaxin.

Severe respiratory syncytial virus (RSV) infection has a significant impact on airway function and may induce or exacerbate the response to a subsequent allergic challenge. In a murine model combining early RSV infection with later cockroach allergen (CRA) challenge, we examined the role of RSV-induced CCL5/RANTES production on allergic airway responses. RSV infection increased CCL5 mRNA and protein levels, peaking at days 8 and 12, respectively. Administration of CCL5 antiserum during days 0-14 of the RSV infection did not significantly alter viral protein expression when compared to mice treated with control serum. In mice receiving the combined RSV-allergen challenge, lungs collected on day 22 exhibited significantly increased numbers of CD4- and CD8-positive T cells. This increase in T cell numbers was not observed in mice receiving alpha-CCL5. On day 43, peribronchial eosinophilia and leukotriene levels were increased in RSV-allergen mice. Pretreatment with CCL5 antiserum resulted in decreased recruitment of inflammatory cells to bronchoalveolar and peribronchial regions of the lungs and these reductions were associated with a reduction in both T cell recruitment into the bronchoalveolar space, leukotriene release and chemokine generation. Thus, CCL5 released during RSV infection has a significant effect on the inflammatory response to subsequent allergic airway challenges.

Peribronchial inflammation contributes to the pathophysiology of allergic asthma. In many vascular beds, adhesive interactions between leukocytes and the endothelial surface initiate the recruitment of circulating cells. Previous studies using OVA-induced airway hyperreactivity indicated that P-selectin, a member of the selectin family expressed by activated platelets and endothelium, contributed to both inflammation and bronchoconstriction. The current study used cockroach allergen (CRA), an allergen that induces asthmatic responses in both humans and mice, to further investigate the role of selectins in the development of peribronchial inflammation and airway hyperreactivity. P- and E-selectin mRNAs were detected in extracts of CRA-sensitized animals beginning shortly after intratracheal challenge with CRA. The P-selectin mRNA was transiently induced at early time points while up-regulation of the E-selectin mRNA was more prolonged. Mice with targeted deletions in E-selectin (E−), P-selectin (P−), and both genes (E−/P−) showed 70–85% reductions in airway hyperreactivity, peribronchial inflammation, and eosinophil accumulation. The P− and E−/P− groups showed the most profound reductions. The transfer of splenic lymphocytes from CRA-primed E−/P− into naive wild-type (WT) mice produced the same level of airway hyperreactivity as transfers from CRA-primed WT into naive WT hosts, indicating that peripheral immunization was similar. The observed changes in the selectin-deficient animals were not related to inadequate sensitization, because CRA priming and challenge increased serum IgE levels. Furthermore, pulmonary Th2-type cytokines and chemokines in the E-selectin−/− and WT animals were similar. The findings indicate that both P- and E-selectin contribute to CRA-induced peribronchial inflammation and airway hyperreactivity.

The severity of allergic asthma is dependent, in part, on the intensity of peribronchial inflammation. P-selectin is known to play a role in the development of allergen-induced peribronchial inflammation and airway hyperreactivity. Selective inhibitors of P-selectin-mediated leukocyte endothelial-cell interactions may therefore attenuate the inflammatory processes associated with allergic airway disease. Novel P-selectin inhibitors were created using a polyvalent polymer nanoparticle capable of displaying multiple synthetic, low molecular weight ligands. By assembling a particle that presents an array of groups, which as monomers interact with only low affinity, we created a construct that binds extremely efficiently to P-selectin. The ligands acted as mimetics of the key binding elements responsible for the high-avidity adhesion of P-selectin to the physiologic ligand, PSGL-1. The inhibitors were initially evaluated using an in vitro shear assay system in which interactions between circulating cells and P-selectin-coated capillary tubes were measured. The nanoparticles were shown to preferentially bind to selectins expressed on activated endothelial cells. We subsequently demonstrated that nanoparticles displaying P-selectin blocking arrays were functionally active in vivo, significantly reducing allergen-induced airway hyperreactivity and peribronchial eosinophilic inflammation in a murine model of asthma.

Vascular endothelial growth factor (VEGF), a key angiogenic molecule, is aberrantly expressed in several diseases including asthma where it contributes to bronchial vascular remodeling and chronic inflammation. Asthmatic human airway smooth muscle cells hypersecrete VEGF, but the mechanism is unclear. In this study, we defined the mechanism in human airway smooth muscle cells from nonasthmatic and asthmatic patients. We found that asthmatic cells lacked a repression complex at the VEGF promoter, which was present in nonasthmatic cells. Recruitment of G9A, trimethylation of histone H3 at lysine 9 (H3K9me3), and a resultant decrease in RNA polymerase II at the VEGF promoter was critical to repression of VEGF secretion in nonasthmatic cells. At the asthmatic promoter, H3K9me3 was absent because of failed recruitment of G9a; RNA polymerase II binding, in association with TATA-binding protein-associated factor 1, was increased; H3K4me3 was present; and Sp1 binding was exaggerated and sustained. In contrast, DNA methylation and histone acetylation were similar in asthmatic and nonasthmatic cells. This is the first study, to our knowledge, to show that airway cells in asthma have altered epigenetic regulation of remodeling gene(s). Histone methylation at genes such as VEGF may be an important new therapeutic target.

Regulation of phenotypic plasticity in smooth muscle requires an understanding of the mechanisms regulating phenotype-specific genes and the processes dysregulated during pathogenesis. Decades of study in airway smooth muscle has provided extensive knowledge of the gene expression patterns and signaling pathways necessary to maintain and alter smooth muscle cell phenotype. With this solid foundation, the importance and complexity of inheritable epigenetic modifications and mechanisms silencing gene expression have now emerged as fundamental components regulating aspects of inflammation, proliferation and remodeling.

AIMS: Fractalkine (CX3CL1) is a membrane-bound chemokine that signals through the G protein-coupled receptor CX3CR1 that is implicated in the development of atherosclerosis. We have previously reported that CX3CR1 is expressed by primary human coronary artery smooth muscle cells (CASMC), where it mediates chemotaxis towards CX3CL1. We sought to determine the effect of CX3CL1 on CASMC survival and proliferation and elucidate the signalling mechanisms involved. METHODS AND RESULTS: CX3CL1 significantly reduces staurosporine-induced apoptosis of CASMC, as quantified by caspase 3 immunostaining and Annexin-V flow cytometry. Furthermore, CX3CL1 is a potent mitogen for primary CASMC and induces phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, measured by western blotting. Inhibition of either ERK or phosphoinositide 3-kinase (PI3K) signalling abrogates proliferation, while only PI3K signalling is involved in the anti-apoptotic effects of CX3CL1. We describe a novel and specific small molecule antagonist of CX3CR1 (AZ12201182) which abrogates the mitogenic and anti-apoptotic effects of CX3CL1 on CASMC. Pharmacological inhibition of the epidermal growth factor receptor (EGFR) blocks CASMC survival and DNA synthesis, indicating a previously undocumented role for EGFR signalling in response to CX3CL1 involving release of a soluble EGFR ligand. Specifically, CX3CL1 induces shedding of epiregulin and increases epiregulin mRNA expression 20-fold within 2 h. Finally, antibody neutralization of epiregulin abrogates the mitogenic effect of CX3CL1. CONCLUSION: We have demonstrated two novel and important functions of CX3CL1 on primary human SMCs: anti-apoptosis and proliferation, both mediated via epiregulin-induced EGFR signalling. Our data have important implications in vascular pathologies including atherosclerosis, restenosis, and transplant accelerated arteriosclerosis, where the balance of SMC proliferation and apoptosis critically determines both plaque stability and vessel stenosis.

The Duffy antigen/receptor for chemokines (DARC) is an unusual chemokine receptor that binds a large number of inflammatory chemokines of both the CC and CXC families with nanomolar affinity, yet it lacks the ability to signal upon ligand binding. Using bioluminescent resonant energy transfer, we have demonstrated for the first time that DARC exists as a constitutive homo-oligomer in living cells and furthermore that DARC hetero-oligomerizes with the CC chemokine receptor CCR5. DARC-CCR5 interaction impairs chemotaxis and calcium flux through CCR5, whereas internalization of CCR5 in response to ligand binding remains unchanged. These results suggest a novel mechanism by which DARC could modulate inflammatory responses to chemokines in vivo.

Atherosclerosis is the leading cause of morbidity and mortality in Westernized societies and is the underlying basis of myocardial infarction, stroke, and peripheral arterial disease (Libby, 2002). Atherosclerosis is a multifactorial disease associated with a number of genetic and environmental risk factors. However, it has become increasingly evident that inflammation plays a prominent role in both the initiation and the progression of atherosclerotic lesion development as well as in the complications related to plaque destabilization and rupture (Ross, 1999). Early atherosclerotic lesions consist primarily of monocyte-derived macrophages and T-cells (Stary et al., 1994), but these cells persist even in advanced lesions. Given the complexity of the inflammatory processes involved in disease pathogenesis, enormous amounts of research have been directed towards identifying the key mediators of this inflammation so that new targets for therapeutic intervention can be discovered. This brings us to chemokines. Chemokines play essential roles in inflammation and disease (D'Ambrosio et al., 2003; John and Lukacs, 2003; Proudfoot et al., 2000; Sheikine and Hansson, 2004). This family of inflammatory mediators has diverse roles in both immune and inflammatory responses, including regulation of cellular recruitment, activation, and differentiation (Gerard and Rollins, 2001). Given the importance of chemokines in the regulation of inflammatory processes, numerous strategies have been devised to modulate chemokine-receptor interactions in an attempt to identify novel therapeutic candidates for intervention in inflammatory disease. © 2005 Elsevier Inc. All rights reserved.

Airway remodelling is a common feature of severe asthma. Transforming growth factor-s (TGF-s) is a pro-fibrotic, pleiotropic cytokine implicated in airway remodelling. TGF-s is sequestered in the extracellular matrix as a latent complex and requires activation to function. We have previously shown that contraction agonists cause aVs5-mediated TGF-s activation by human airway smooth muscle cells. The study aims were to investigate the role of the aVs5integrin in airway remodelling in vivo using two distinct mouse models of asthma. A blocking antibody directed against the aVs5 was used in the ovalbumin (OVA) model of asthma. Mice were sensitised with OVA/Alum on days 0 and 12, then challenged byoropharyngeal administration of OVA 10 times over 2 weeks. The anti-aVs5antibody or an isotype matched control antibody was administered for the duration of the OVA challenges. The second invivo model utilised itgb5 −/− mice. Aspergillous fumigatus antigen preparation was administered intra-nasally (10 μg/mice) to itgb5 −/− and wild type controls 9 times over a 21-day period. a-Smooth muscle actin (a-SMA) was quantified in lung sections from both studies by immunofluorescence. Murine airway smooth muscle cells express aVs5 integrin and can activate TGF-s in vivo in response to allergen challenge as measured by αvβ5 and phospho-Smad2 immunostaining. Treatment with both OVA and Asp.f resulted in an increase in a-SMA staining around the smaller airways. The aVs5blocking antibody significantly reduced a-SMA staining compared with the isotype control antibody. Furthermore, itgb5 −/− mice had significantly less a-SMA around their airways than wild type control mice in response to Asp.f treatment. However, both itgb5 −/− andanti-aVs5 treated mice had significantly more airway inflammation and more inflammatory cells present in the bronchoalveolar lavage compared with their matched controls. These data provide evidence that airway smooth muscle cells can activate TGF-s in vivo. Inhibition of, or genetic loss of, the aVs5 integrin significantly reduces allergen-induced increases in airway smooth muscle mass, however, peribronchial inflammation is increased consistent with the known effects of TGFβ. Targeted inhibition of the aVs5 integrin may reduce airway remodelling, but global inhibition is unlikely to be useful due to the enhanced inflammatory response.

Introduction The bleomycin mouse mode can be used as a model of pulmonary fibrosis. The Influenza A virus can infects epithelial cells leading to cell death and injury. Acute exacerbations of Idiopathic Pulmonary Fibrosis (IPF) are characterised by epithelial cell apoptosis with unknown cause. The role of infection in acute exacerbations of IPF is unclear. The aim of this study is to investigate the effect of influenza infection on bleomycin-induced pulmonary fibrosis. Materials and Methods 60 U of bleomycin was instilled into lungs of 6–8 week old male C57Bl/6 mice. After 28 days mice were exposed intranasally with 10, 20 Units of influenza virus ‘x31’ or PBS, and lungs harvested 5 or 21 days later. Lung tissue harvested for mRNA analysis, histology and hydroxyproline levels. Animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals. Results Influenza infection increased in lung collagen levels: COL1 mRNA but not COL3 was increased. There was also an increase in matrix deposition on Masson’s trichrome staining. There were increased hydroxyproline levels in influenza infected mice with fibrotic lungs due to bleomycin administration, compared with mice exposed only to bleomycin. Non-fibrotic, influenza- infected mice showed apoptosis on histological TUNEL staining. CCNA2 mRNA in influenza infected mice with fibrotic lungs was increased compared to fibrotic mice alone indicating an increase in epithelial apoptosis. Conclusion These data suggest that influenza infection may enhance the fibrotic response in the lung by promoting epithelial apoptosis and fibrogenesis.

Introduction Transforming Growth Factor-beta (TGFβ) promotes anti-proliferative and pro-apoptotic pathways in lung epithelial cells, both of which have been implicated in the pathogenesis of IPF. TGFβ must be activated before it can mediate these events. Acute exacerbations of IPF are characterised by widespread epithelial cell apoptosis. The precise cause of these exacerbations is not known. The Influenza A virus is a single-stranded segmented RNA virus that infects epithelial cells leading to cell death and injury, and can also activate TGFβ. The role of infection in acute exacerbations of IPF is unclear. The aim of this study is to investigate the effect of influenza infection on bleomycin-induced pulmonary fibrosis and TGFβ activation in vivo. Materials and Methods 60 U of bleomycin was instilled into the lungs of 6–8 week old male C57Bl/6 mice. After 28 days mice were exposed intranasally with 10, 20 Units of influenza virus ‘x31’ or PBS, and the lungs harvested 5 days later. Bronchoalveolar lavage (BAL) was performed and lung tissue harvested for mRNA analysis, histology and hydroxyproline levels. All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals. Results Mice exposed to bleomycin and infected with influenza lost less weight compared with saline-exposed influenza-infected animals. However, the lungs from bleomycin-exposed, influenza-infected mice showed increased lung damage with more matrix deposition on trichrome staining than saline-exposed, influenza-infected mice. Saline-exposed, influenza-infected mice demonstrated the anticipated dose dependent increase in BAL lymphocytosis as well as apoptosis staining in histological TUNEL assessment. However, in bleomycin-exposed mice, influenza infection did not promote enhanced BAL lymphocytosis or apoptosis. However, influenza appeared to enhance the fibrotic response demonstrated by an increase in matrix deposition on masson’s trichrome and increased lung hydroxyproline levels in influenza infected bleomycin exposed mice, as early as 5 days post infection. Conclusions These data suggest that influenza infection may exacerbate lung fibrosis by promoting epithelial apoptosis.

Introduction TGFβ activation by αvβ6 integrins is central to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Furthermore, αvβ6 expression is increased in both human, and murine, fibrotic lung tissue. Current assessment of αvβ6 integrin levels in the lung requires immunohistochemical analysis of biopsy samples and repeated measurement of αvβ6. Although αvβ6 may be useful as a biomarker in IPF, currrent methods of detection make this approach clinically impractical. We have developed a non-invasive radioimaging CT/SPECT strategy for measuring αvβ6 integrin levels in the lungs facilitating the monitoring of disease progression and therapeutic response in IPF. Methods C57Bl/6 mice received intratracheal bleomycin or saline, 28 days prior to intravenous injection with Indium111-labelled A20FMDV2, an αvβ6 binding peptide derived from the VP1 coat protein of foot-and-mouth virus. A scrambled sequence peptide was used as control. Mice were injected with 5µg (10–24MBQ) of peptide and imaged by CT/SPECT scanning one, and three, hours later. Results Maximal binding of Indium111-labelled A20FMDV2 peptide was detected in skin, and lungs, of all mice at one hour. No significant binding was detected in mice injected with control peptide. Binding to αvβ6 integrin was significantly higher in the lungs of bleomycin, compared with saline, exposed mice (0.12±0.01MBQ vs 0.06±0.009 p

Activation of latent TGFs by the epithelially-restricted αvs6 integrin is critical in the pathogenesis of lung injury and fibrosis, and disruption of this pathway promotes emphysema development. We have previously shown that Gαq and RhoA signalling pathways are central to avb6 integrin induced TGFs activation in vitro. To assess the role of the Gq/11 signalling pathway in the lungs, we generated mice with deletion of the Gq and G11 α-subunits in Surfactant protein C (SftpC)-positive epithelial cells (Gq/11DKO). SftpC-Cre mice were crossed with constitutive Gα11-deficient animals ( Gna11 −/− ; G11KO) carrying floxed alleles of the Gaq gene ( Gnaq fl/fl ) and then backcrossed onto appropriate null mice. Lungs were perfused, inflated and fixed prior to processing for histological and immunohistochemical analysis at 2, 4, 6 and 8 weeks. Bronchoalveolar lavage (BAL) cells were collected at 6 weeks for mRNA, nuclear protein extraction or histological analysis. Focal inflammatory infiltrates were visible in the Gq/11DKO lungs as early as 2 weeks, but became larger and more widespread at later timepoints. Gq/11DKO mice also exhibited significant age-related airspace enlargement compared with G11KO mice from 4 weeks onwards. From 6 weeks, inflammation was closely associated with localised disruption of the alveolar architecture and the appearance of enlarged and vacuolated macrophages within the airspaces. BAL fluid from Gq/11DKO mice contained significantly higher cells numbers (12.5±2.5×10 5 ) than G11KO mice (0.96±0.2×10 5 ) with increases in the percentage of neutrophils, lymphocytes and enlarged and vacuolated alveolar macrophages. mRNA analysis of Gq/11DKO BAL cells showed significantly increased MMP12, RELMα and Arginase 1 suggesting an increase in the number of alternatively activated macrophages. To assess levels of active TGFs in the lungs, phosphorylated SMAD2 (pSMAD2), a component of the TGFs signalling pathway, was measured by ELISA of nuclear extracts from BAL cells. Gq/11DKO BAL cells contained significantly lower levels of pSMAD2 than those from G11KO mice, suggesting decreased levels of active TGFs in the lungs of Gq/11DKO mice. These data suggest that the Gαq/11 signalling pathway in SftpC-positive epithelial cells regulates TGFs activation in the lungs and that deficiency in this pathway results in pulmonary inflammation and disruption of the alveolar architecture of the lung.

Airway remodelling is a common feature of severe asthma. Transforming growth factor-β (TGF-β) is a pro-fibrotic, pleiotropic cytokine implicated in airway remodelling. TGF-β is sequestered in the extracellular matrix as a latent complex and requires activation to function. Lysophosphatidic acid (LPA) causes TGF-β activation in airway epithelial cells. The study aims were to investigate the effect of LPA on TGF-β activation by ASM cells in asthma. TGF-β activation was assessed by a reporter cell co-culture assay, by determining expression of the TGF-β-inducible gene plasminogen activator inhibitor-1 (PAI1) and by detecting the nuclear translocation of Smad proteins. The effect of LPA on TGF-β activation in asthma was investigated by comparing the responses of ASM cells from non-asthmatic (n=3) and asthmatic (n=3) donors. TGFb activation was also assessed using a chronic ovalbumin model of airway remodelling in mice. LPA induced a time, and concentration, dependent increase in TGF-β activation by ASM cells that was abrogated by an integrin αVβ5 antibody. An inhibitor of cytoskeletal reorganisation inhibited the effects of LPA. Furthermore, the β2-agonist formoterol inhibited LPA-induced PAI1 expression. Primary asthmatic ASM cells activated more TGF-β via αVβ5 in response to LPA than control cells, however they did not express more αVβ5 on the cell surface. Phosphorylation of Smad2 and expression ofpai1 in the lungs was increased in a chronic ovalbumin model of asthmatic airway remodelling in mice. Furthermore, αVβ5 integrin staining and α-smooth muscle actin staining in the ASM layer around the airways is increased in this model. Collectively, these data show that ASM cells can activate TGF-β via the αVβ5 integrin and highlight a novel pathway of TGF-β activation in ASM cells, which may be important in development of asthmatic airway remodelling.