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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.

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.

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

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.

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.

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.

Background Developmental lung diseases cause significant mortality and lifelong morbidity. There is no treatment to restore normal organogenesis, but research into the molecular mechanisms of lung development may identify therapeutic targets. Normal lung development requires the coordinated activity of numerous cell types, and has shared signalling pathways with neoplasia. Pericytes and myofibroblasts are essential for lung development, through their roles in vascularisation and alveolarisation, respectively. The G proteins Gαq and Gα11 (Gαq/11) are key components of physiological and pathophysiological signalling pathways, but the role of mesenchymal Gαq/11 signalling in lung development is unknown. Methods Gαq floxed, Gα11 knockout homozygous mice (Gnaqfl/fl;Gna11-/-) were crossed with Pdgfrb-Cre± mice to generate offspring lacking Gαq/11 in Pdgfrb-expressing cells (pericytes, myofibroblasts). Organs were harvested from 2 week old mice for histological and biochemical analyses. Whole lung RNA from Gαq/11-deficient and control mice was analysed using ClariomTM D Mouse microarray chips (Affymetrix) and Partek Genomics Suite 6.6 software. Differentially expressed genes were considered of interest with p≤0.05 and fold change ±1.5. Results Mesenchymal Gαq/11-knockout mouse lungs had enlarged airspaces, thickened alveolar walls, fewer secondary septae, and fewer elastin fibres on histology compared with controls (p Whole lung RNA microarray demonstrated downregulation of mt-Tk, a key mitochondrial function gene, and several miRNAs known to regulate angiogenesis, proliferation, and cellular differentiation (miR-684, miRlet7f-1, miR186) in mesenchymal Gαq/11-knockout lungs compared with controls (p 1.5). High gene expression variability in mesenchymal Gαq/11-knockout lungs may indicate variations in phenotype, and these findings require validation. Neoplastic hyperproliferation of the medullary tubular epithelium in mice lacking mesenchymal Gαq/11 was found on renal histology, suggesting a role for mesenchymal Gαq/11 in tumour suppression. Conclusion Gαq/11 signalling in Pdgfrb-positive cells is essential for normal lung development, with regulation of elastin deposition, mitochondrial function, angiogenesis, and proliferation as candidate mechanisms. Mesenchymal Gαq/11 signalling regulates renal epithelial proliferation, highlighting a shared developmental and malignant pathway. This is the first study to generate mesenchymal Gαq/11-knockout mice, further investigation of which may identify therapeutic targets for developmental lung disease.

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.

Within the lungs, fibrosis can affect both the parenchyma and the airways. Fibrosis is a hallmark pathological change in the parenchyma in patients with idiopathic pulmonary fibrosis (IPF), whilst in asthma or chronic obstructive pulmonary disease (COPD) fibrosis is a component of the remodelling of the airways. In the past decade, significant advances have been made in understanding the disease behaviour and pathogenesis of parenchymal and airway fibrosis and as a result a variety of novel therapeutic targets for slowing or preventing progression of these fibrotic changes have been identified. This review highlights a number of these targets and discusses the potential for treating parenchymal or airway fibrosis through these mediators/pathways in the future.

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.

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

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.

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.