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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-β (TGFβ) via the alpha-V beta-6 (αvβ6) 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 αvβ6 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 αvβ6 with high affinity and demonstrated fast binding kinetics. In primary human lung epithelial cells, GSK3335103 induced concentration- and time-dependent internalisation of αvβ6 with a rapid return of integrin to the cell surface observed after washout. Following sustained engagement of the αvβ6 integrin in vitro, lysosomal degradation was induced by GSK3335103. GSK3335103 was shown to engage with the αvβ6 integrin and inhibit the activation of TGFβ in both ex vivo IPF tissue and in a murine model of bleomycin-induced lung fibrosis, as measured by αvβ6 engagement, TGFβ signalling and collagen deposition, with a prolonged duration of action observed in vivo. In summary, GSK3335103 is a potent αvβ6 inhibitor that attenuates TGFβ 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.

The novel coronavirus SARS-CoV-2 utilizes Angiotensin Converting Enzyme-2 (ACE2) receptors to internalize cells, which are expressed in the nasal and ocular mucosa, and at low levels in the pulmonary epithelium Despite significant sequence similarities there are substantial differences in transmission dynamics and clinical phenotype between SARS-CoV-2 and SARS-CoV-1 The SARS-CoV-2 spike protein (S1), which is used to internalize cells, contains RGD integrin binding domains which are not present within SARS-CoV-1 S1 We investigated whether SARS-CoV-2 S1 binds integrins while exploring mechanisms that might upregulate ACE2 expression to help explain SARS-CoV-2 viral entry and associated respiratory disease Lung cell line ACE2 expression was determined using QPCR and western blotting, and in primary lung cells using single cell RNAseq data from publicly available datasets The effect of IL6 and TGFb on ACE2 expression levels in lung epithelial cells and precision cut lung slices (PCLS) was explored Solid phase binding assays were used to investigate S1 binding to ACE2 or av containing integrins Immunohistochemistry was used to stain sections of COVID-19 infected lung tissue for ACE2 and av containing integrins Single Cell RNA-seq showed that normal lung expresses low levels of ACE2 and only a small proportion of Alveolar type 2 epithelial cells are ACE2 positive (1 5%) Supporting this we found low level ACE2 mRNA and protein expression in small airway epithelial cells, immortalized human bronchial epithelial cells (iHBECs) and A549 cells IL6 had no effect on ACE2 mRNA or protein expression in the above cells, nor did it affect ACE2 protein in PCLS TGFb increased ACE2 mRNA in iHBECs and increased ACE2 protein in PCLS Binding assays demonstrated that SARS-CoV-2 S1 binds avb3 and avb6 integrins in an RGD dependent manner, albeit with a lower affinity than to ACE2 Crucially avb3 integrins are upregulated in COVID-19 infected lung tissue, whereas ACE2 levels remain low even in patients with high viral RNA and protein expression in alveolar tissue Our data suggests SARS-CoV-2 is able to bind integrins, and may utlise this mechanism to facilitate internalization into lung epithelial cells, which may help explain severe pathology despite low ACE2 expression levels in the lung

Introduction Bronchopulmonary dysplasia (BPD) is characterised by arrested alveolarisation and lifelong lung function impairment. There are no treatments that can reactivate normal alveologenesis. Alveolarisation involves tightly regulated signalling between several cell types. Pericytes are mesenchymal cells that drive many developmental processes, particularly by differentiating into myofibroblasts. Transforming growth factor-β (TGFβ) regulates cellular differentiation and is important in alveologenesis, but must be activated to exert any effects. Signalling by the G protein α subunit Gαq/11 activates TGFβ in epithelial cells. However, the role of mesenchymal cell Gαq/11 in TGFβ signalling and alveolarisation is unknown. Aim Understand how mesenchymal Gαq/11 influences TGFβ signalling in alveologenesis. Methods Pdgfrb-Cre± and Gnaqfl/fl;Gna11-/- mice were crossed, producing Pdgfrb-Cre±;Gnaqfl/fl;Gna11-/- offspring which lack Gαq/11 in pericytes, fibroblasts, and myofibroblasts. Lungs were collected for histology. Breathing-related cyclical mechanical stretch (CMS) was applied to wild-type (WT) and Gnaq-/-;Gna11-/-murine embryonic fibroblasts (MEFs), and human lung fibroblasts (HLFs) using Flexcell® apparatus. Integrin (αv and β1), ROCK (Y27632) and serine protease (AEBSF) inhibitors were used. TGFβ signalling was assessed by Smad2 phosphorylation. Gene expression was assessed using qPCR. Results Pdgfrb-Cre±;Gnaqfl/fl;Gna11-/- lungs had a BPD-like appearance, with enlarged airspaces, thickened alveolar walls, and reduced cellular proliferation and secondary septation compared with controls (p Pdgfrb-Cre±;Gnaqfl/fl;Gna11-/- lungs contained less α-smooth muscle actin (αSMA), elastin and collagen than controls. Furthermore, Gnaq-/-;Gna11-/- MEFs expressed less Acta2, Col1a1, Col3a1, and Eln mRNA compared with WT MEFs (p CMS-induced TGFβ signalling was reduced in MEFs and HLFs lacking Gαq/11 (p Conclusion These data demonstrate a novel mechanism of breathing-related CMS-induced TGFβ2 activation. This Gαq/11- and serine protease-dependent pathway controls essential processes in alveolarisation, including pericyte-to-myofibroblast differentiation, myofibroblast function, and pericyte migration. This is the first study to identify an isoform-specific role for TGFβ2 in alveologenesis. Further study of this pathway may identify novel therapeutic targets for BPD.

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.

Rationale We recently identified a polymorphism associated with the AKAP13 gene resulted in higher levels of AKAP13 gene expression and Idiopathic pulmonary fibrosis (IPF) susceptibility. Furthermore, higher expression of AKAP13 were associated with diseased epithelium.1 AKAP13 is a Rho-GEF for RhoA, a key intermediate signal in the activation of the TGF-β activating integrin, αvβ6, in lung epithelial cells. However, the role for AKAP13 in the lung is still not understood. Aim To investigate the role of AKAP13 in lung epithelial cells, including Rho-A and TGFβ. Method Localisation of protein expression of AKAP13 and αvβ6 was assessed in IPF human lung tissue via immunohistochemistry, using serial sections. AKAP13 gene expression was assessed via qPCR in primary human lung fibroblasts (normal, n=3; IPF, n=4) and primary epithelial cell lines (small airway SAEC, n=9; human bronchial HBEC, n=4). Immortalised human bronchial epithelial cells (iHBECs) were treated with AKAP13 siRNA to knockdown expression of AKAP13 and assessed for changes to mRNA after 48 hrs. iHBECs were treated with 10uM of A13, an inhibitor for AKAP13-RhoA interaction, to assess for functional changes to Rho-A activation in response to LPA. Results Assessment of serial lung sections from IPF patients (n=106) show that positive staining for AKAP13 and αvβ6 is observed in lung epithelial cells, within the same regions of lung. AKAP13 gene expression was found to be 19-fold higher in epithelial cells, compared to fibroblasts, which had very low expression for AKAP13 (both normal and IPF), confirming our previous and current immunohistochemistry findings.1 Knockdown of the AKAP13 gene in iHBECS also resulted in a significant decrease in ITGB6 expression, the gene for αvβ6 (n=4, p=0.03). In addition, treatment of iHBECS with 10uM of A13 was able to supress RhoA activation in response to LPA. Conclusion AKAP13 expression is found predominantly in epithelial cells in the lung. AKAP13 appears to regulate RhoA activation in iHBECs and influence αvβ6 expression. This suggests that it is involved in the RhoA- αvβ6 pathway that drives TGF-β activation in epithelial cells Reference Allen RJ, et al. Genetic variants associated with susceptibility to idiopathic „pulmonary fibrosis in people of European ancestry: a genome-wide association study. The Lancet Respiratory Medicine 2017;5(11):869–880.

Rationale: We recently identified a polymorphism associated with the AKAP13 gene resulted in higher levels of AKAP13 expression in the lung, particularly epithelium, and increased IPF susceptibility. AKAP13 is a Rho-GEF for RhoA, a key intermediate signal in the activation of the TGF-β activating integrin, αvβ6, in lung epithelial cells. However, the role for AKAP13 in the lung is still not understood. Aim: to investigate the role of AKAP13 in lung epithelial cells Method: AKAP13 gene expression was assessed via qPCR in primary human lung fibroblasts (normal, n=3; IPF, n=4) and primary epithelial cell lines (small airway SAEC, n=9; human bronchial HBEC, n=4). Immortalised human bronchial epithelial cells (iHBECs) were treated with AKAP13 siRNA to knockdown expression of AKAP13 and assessed for changes to mRNA after 48hrs. iHBECs were treated with 10μM of A13, an inhibitor for AKAP13-RhoA interaction, to assess for functional changes to RhoA activation in response to LPA. Results: AKAP13 gene expression was found to be 19-fold higher in epithelial cells, compared to fibroblasts, which had very low expression for AKAP13 (both normal and IPF), confirming our previous immunohistochemistry findings. Knockdown of the AKAP13 gene in iHBECS also resulted in a significant decrease in ITGB6 expression, the gene for αvβ6 (n=4, p=0.03). In addition, treatment of iHBECS with 10μM of A13 was able to supress RhoA activation in response to LPA. Conclusion: AKAP13 expression is found predominantly in epithelial cells in the lung. AKAP13 appears to regulate RhoA activation in iHBECs and influence αvβ6 expression. This suggests that it is involved in the RhoA-αvβ6 pathway that drives TGF-β activation in epithelial cells.

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.

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.

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.

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.

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.

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.

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.

Activation of latent TGFβ by the epithelially restricted αvβ6 integrin is induced by activators of the RhoA signalling pathway and is critical in the pathogenesis of lung injury and fibrosis. The G-proteins, Gα12 and Gα13 are known to activate RhoA and we have previously shown that the αvβ6 integrin can mediate TGFβ activation via Gαq and RhoA. To establish the role of these G-proteins in both normal lung development and following lung injury, we generated mice with a targeted deletion of Gαq/11 or Gα12/13 in SpC-positive Type II alveolar epithelial cells. SpC-Cre mice were crossed with either Gαq(flox-flox)/11(-/-) or Gα12(-/-)/13(flox-flox) mice and the lungs analysed histologically at 6 and 8 weeks after birth. At 6 weeks, lungs from mice with a homozygous deficiency in SpC-Gαq/11 contained focal inflammatory infiltrates consisting primarily of mononuclear leukocytes. Inflammation was associated with the localised disruption of normal alveolar architecture and the appearance of abnormal Type I and Type II alveolar epithelial cells, identified by SpC and T1α immunohistochemistry, within in the alveolar airspaces. Furthermore, immunohistochemical analysis of phosho-Smad2 levels in these lungs detected increased staining in the inflammatory foci within the homozygous SpC-Gαq/11 knockout lungs. At 8 weeks, the inflammatory foci were more numerous and lung architecture was severely disrupted with multiple abnormally large alveolar airspaces detected. In contrast, mice with at least one floxed Gαq or null Gα11 allele showed no abnormalities at either 6 or 8 weeks. We also detected no abnormal lung phenotype in 6- and 8-week old mice with a homozygous or heterozygous deficiency in SpC-Gα12/13. These data suggest that Gα11/q signalling is required to prevent pulmonary inflammation and our findings would be consistent with impaired epithelial TGFβ activation in the lungs of these mice. Further studies are required to determine the origin of the cells activating TGFβ in these lungs.

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.