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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.

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.

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.

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.