Lisa Jolly

Integrin αvβ5-Mediated TGF-β Activation by Airway Smooth Muscle Cells in Asthma

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.

Influenza Promotes Collagen Deposition via αvβ6-integrin Mediated Transforming Growth Factor β Activation

Background: The mechanism of influenza mediated TGFβ activation, and its role in pathogenesis is unclear. Results: H1N1 infection induced αvβ6-dependent TGFβ activity in iHBECs and increased epithelial cell death and collagen deposition in vivo. Conclusion: αvβ6 integrin-mediated TGFβ activation is involved in cell death and fibrogenesis following virus-induced epithelial injury. Significance: Viral infection may promote acute exacerbations of fibrotic lung disease. Influenza infection exacerbates chronic pulmonary diseases, including idiopathic pulmonary fibrosis. A central pathway in the pathogenesis of idiopathic pulmonary fibrosis is epithelial injury leading to activation of transforming growth factor β (TGFβ). The mechanism and functional consequences of influenza-induced activation of epithelial TGFβ are unclear. Influenza stimulates toll-like receptor 3 (TLR3), which can increase RhoA activity, a key event prior to activation of TGFβ by the αvβ6 integrin. We hypothesized that influenza would stimulate TLR3 leading to activation of latent TGFβ via αvβ6 integrin in epithelial cells. Using H1152 (IC50 6.1 μm) to inhibit Rho kinase and 6.3G9 to inhibit αvβ6 integrins, we demonstrate their involvement in influenza (A/PR/8/34 H1N1) and poly(I:C)-induced TGFβ activation. We confirm the involvement of TLR3 in this process using chloroquine (IC50 11.9 μm) and a dominant negative TLR3 construct (pZERO-hTLR3). Examination of lungs from influenza-infected mice revealed augmented levels of collagen deposition, phosphorylated Smad2/3, αvβ6 integrin, and apoptotic cells. Finally, we demonstrate that αvβ6 integrin-mediated TGFβ activity following influenza infection promotes epithelial cell death in vitro and enhanced collagen deposition in vivo and that this response is diminished in Smad3 knock-out mice. These data show that H1N1 and poly(I:C) can induce αvβ6 integrin-dependent TGFβ activity in epithelial cells via stimulation of TLR3 and suggest a novel mechanism by which influenza infection may promote collagen deposition in fibrotic lung disease.

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

Signaling by Gq/11 is required for optimal TGFβ activation in the lung to prevent inflammation. Gq/11 signaling maintains healthy lungs Loss of signaling by the cytokine transforming growth factor–β (TGFβ) in mice results in emphysema-like symptoms, whereas excessive TGFβ signaling results in pulmonary fibrosis and ventilator-associated lung injury. G proteins of the Gq/11 and G12/13 families mediate the integrin-dependent activation and release of latent TGFβ from the epithelial cells. John et al. found that mice deficient in Gq/11, but not those deficient in G12/13, in lung epithelial cells had defective TGFβ activation and emphysema-like symptoms. In addition, the Gq/11-deficient mice had lung inflammation associated with increased amounts of the cytokine IL-33. However, the mice were protected from ventilator-induced injury. Together, these data suggest that Gq/11 signaling is required for optimal TGFβ activation in the lung and the prevention of inflammation. Heterotrimeric guanine nucleotide–binding protein (G protein) signaling links hundreds of G protein–coupled receptors with four G protein signaling pathways. Two of these, one mediated by Gq and G11 (Gq/11) and the other by G12 and G13 (G12/13), are implicated in the force-dependent activation of transforming growth factor–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. We investigated the involvement of the Gq/11 and G12/13 pathways in epithelial cells in generating active TGFβ and regulating alveolar inflammation. Mice deficient in both Gαq and Gα11 developed inflammation that was primarily caused by alternatively activated (M2-polarized) macrophages, enhanced matrix metalloproteinase 12 (MMP12) production, and age-related alveolar airspace enlargement consistent with emphysema. Mice with impaired Gq/11 signaling had reduced stretch-mediated generation of TGFβ by epithelial cells and enhanced macrophage MMP12 synthesis but were protected from the effects of ventilator-induced lung injury. Furthermore, synthesis of the cytokine interleukin-33 (IL-33) was increased in these alveolar epithelial cells, resulting in the M2-type polarization of alveolar macrophages independently of the effect on TGFβ. Our results suggest that alveolar Gq/11 signaling maintains alveolar homeostasis and likely independently increases TGFβ activation in response to the mechanical stress of the epithelium and decreases epithelial IL-33 synthesis. Together, these findings suggest that disruption of Gq/11 signaling promotes inflammatory emphysema but protects against mechanically induced lung injury.

An Epithelial Integrin Regulates the Amplitude of Protective Lung Interferon Responses against Multiple Respiratory Pathogens.

The healthy lung maintains a steady state of immune readiness to rapidly respond to injury from invaders. Integrins are important for setting the parameters of this resting state, particularly the epithelial-restricted αVβ6 integrin, which is upregulated during injury. Once expressed, αVβ6 moderates acute lung injury (ALI) through as yet undefined molecular mechanisms. We show that the upregulation of β6 during influenza infection is involved in disease pathogenesis. β6-deficient mice (β6 KO) have increased survival during influenza infection likely due to the limited viral spread into the alveolar spaces leading to reduced ALI. Although the β6 KO have morphologically normal lungs, they harbor constitutively activated lung CD11b+ alveolar macrophages (AM) and elevated type I IFN signaling activity, which we traced to the loss of β6-activated transforming growth factor-β (TGF-β). Administration of exogenous TGF-β to β6 KO mice leads to reduced numbers of CD11b+ AMs, decreased type I IFN signaling activity and loss of the protective phenotype during influenza infection. Protection extended to other respiratory pathogens such as Sendai virus and bacterial pneumonia. Our studies demonstrate that the loss of one epithelial protein, αVβ6 integrin, can alter the lung microenvironment during both homeostasis and respiratory infection leading to reduced lung injury and improved survival.

P144 Influenza infection affects the degree of fibrosis and apoptosis in the bleomycin mouse model

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

S127 Influenza A and Poly(I:C) Induce α Vβ6-Integrin-Mediated TGFβ Activity in Human Epithelial Cells Via Stimulation of TLR3

People with chronic lung disease are more susceptible to influenza infection which may lead to exacerbation of pre-existing conditions such as fibrosis. Transforming growth factor-β (TGFβ) is a profibrotic cytokine, but its role during influenza infection remains unclear. Toll-like-receptor 3 is located on the endosomal membrane and binds dsRNA, an intermediate product from replicating ssRNAviruses such as influenza. TLR3 activation has been shown to increase RhoA activity, and we have previously shown that RhoA is a key intermediary inactivation of TGFβ by the αVβ6-integrin. Therefore, we hypothesised that influenza infection could stimulate TLR3 leading to activation of latent TGFβ via this integrin in epithelial cells. Immortalised human bronchial epithelial cells (iHBECs) were used in all experiments. To determine whether influenza virus (A/PR/8/34 H1N1), or poly (I:C) (20µg/ml) were able to activate TGFβ the following TGFβ activation assays were used; detection of phospho-smad2/3 in nuclear extracts of cell lysates by ELISA; analysis of TGFβ activity in cells transiently transfected with a TGFβ-sensitive reporterconstruct; and a co-culture of iHBECS with a TGFβ reporter cell line (TMLCs). To confirm the involvement of TLR3, cells were dual transfected with a TGFβ-sensitive reporter and a dominant negative TLR3 construct designed to prevent TLR3 signalling. The role of the RhoA-ROCK pathway, and αVβ6-integrin were investigated using the ROCK inhibitor H1152, and the αVβ6-integrin blocking antibody 6.3G9, respectively. H1N1 infection and poly(I:C) caused an increase in luciferase in iHBECs transiently transfected with a TGFβ reporter construct. Similarly, both H1N1 and poly(I:C) caused an increase in nuclear phospho-smad2/3 which could be blocked by 6.3G9 peaking at 4h. Both agents caused an increase in TGFβ as measured by a co-culture assay and this could be blocked by H1152 and 6.3G9 suggesting the involvement of ROCK, αVβ6-integrin and the requirement for cell-to-cell contact. Finally, arole for TLR3 in this process was confirmed in cells transfected with a dnTLR3 construct which lost the ability to activate TGFβ in response to poly(I:C) orH1N1. In conclusion, these data show that both influenza A and poly (I:C) lead to increased TGFβ activity in iHBECs. This supports the hypothesis that influenza A infection activates TGFβ via TLR3 and the αVβ6 integrin. These data suggest anovel mechanism by which influenza infection of epithelial cells may promoteairway and lung fibrosis.

S113 The influenza virus activates TGFß via an αVß6-integrin mediated pathway

Introduction and Objectives Idiopathic pulmonary fibrosis is a chronic progressive lung disease of unknown cause. Its pathogenesis is poorly understood but activation of latent TGFß on lung epithelium is an important factor. TGFß must be activated, as it is secreted in a latent complex with its propeptide, the latency associated peptide, and the avß6 integrin is a key activator in the lung. The Influenza A virus is a single-stranded segmented RNA virus that infects epithelial cells leading to cell death and injury. Toll-like receptors (TLRs) detect pathogens, such as influenza. TLR3 activation has been found to increase RhoA activity. We previously showed that RhoA is a key intermediary in avß6 integrin-mediated TGFß activation. The aim of this study is to investigate whether influenza can activate TGFß and stimulate TLR3 leading to activation of TGFß through the avß6 integrin in epithelial cells. Materials and Methods Immortalised human bronchial epithelial cells (iHBECs) were infected with influenza A (H2N3) virus at a multiplicity of infection 1 with, or without, the avß6 blocking antibody 6.3G9. iHBECs were also stimulated with the synthetic TLR3 ligand poly(I:C). TGFß activity was determined by: (1) immunoblotting for phosphorylated (phospho-) Smad2, and (2) Transformed mink lung cells (TMLC)-iHBEC cocultures. Infection efficiency was measured by Interferon ß mRNA levels by real-time qPCR. Results Infection with H2N3 and stimulation with poly(I:C) led to increase in phospho-smad2 and luciferase activity in coculture indicating increase in TGFß activation levels in a dose- and time-dependent manner. In both cases this was blocked with the addition of 6.3G9. qPCR data following infection showed increased IFNß1 and PAI-1, indicating the ability of the virus to infect the cells and activate TGFß. Conclusions Influenza infection and poly(I:C) activates TGFß in iHBECs in an avß6 integrin dependent manner. The data suggests a novel mechanism by which influenza infection of epithelial cells may promote lung fibrosis.

S122 Sputum cytokine profiles in asthma and the impact of smoking-a factor analysis

Introduction Cigarette smokers with asthma have a distinct clinical phenotype from non-smokers with asthma. This may reflect altered airway inflammation although how cigarette smoking directs this is unclear. We employed exploratory factor analysis to examine the impact of smoking on airway inflammation. Methods 22 smokers (sm), 10 ex-smokers (ex-sm) and 21 never smokers (ns) with asthma performed spirometry, induced sputum and completed asthma control questionnaires pre and post an oral corticosteroid trial. Sputum fluid cytokines were quantified using a 25-plex bead system (Invitrogen, Paisley). Factor analysis was performed (SPSS V.17) using principal component analysis and Varimax rotation. Factors were identified according to; visual inspection of the scree plot, eigenvalues >1.1, minimum of three cytokines loading >0.4. Sequential removal of cytokines was performed in stages according to; moderate to strong (>0.4) loadings, followed by requirement for ‘strong’ loading (>0.6) to only one factor then removal of cytokines that reduced the reliability of the data set. In a final step cytokines with the lowest loadings were removed if a factor had >3. Results The subjects were well matched except for higher asthma control questionnaires scores and inhaled corticosteroid dose in sm. Sm failed to demonstrate a lung function response to oral corticosteroids in contrast to nsm. No sputum cell differential differences were evident between smokers and non-smokers with asthma. A number of pre-steroid sputum cytokines were elevated in sm compared to nsm. The greatest difference present for interleukin 6 (sm 34.4 pg/ml (IQR 14.1, 72.4), nsm 8.1 pg/ml (4.4, 11.1), p<0.001). Factor analysis of the pre-steroid cytokines demonstrated that three factors explained 90% of the variance in the data. Sequential processing revealed three cytokines per factor (Abstract S122 table 1).Abstract S122 Table 1 Factor loadings Rotated component matrix Factor 1 2 3 IFN-? 0.991 IL-4 0.986 IL-5 0.986 IL-6 0.864 CXCL9 0.964 CXCL10 0.908 CCL2 0.813 CCL3 0.889 CCL4 0.920 Discussion Sputum cytokine profiling of subjects with asthma with differing smoking histories reveals distinct groupings when examined by exploratory factor analysis providing insight into airway inflammation in asthma and the impact of smoking. Larger cohorts of patients with asthma should be examined to confirm these preliminary findings.

S46 Activation of TGF-β by airway smooth muscle cells via the αVβ5 integrin in asthmatic airway remodelling

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.

S123 Mononuclear inflammation and disruption of normal alveolar structure following deletion of Gαq/11, but not Gα12/13, in type II alveolar epithelial cells

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.