Anthony Habgood

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.

Preclinical SPECT/CT Imaging of αvβ6 Integrins for Molecular Stratification of Idiopathic Pulmonary Fibrosis

Transforming growth factor β activation by the αvβ6 integrin is central to the pathogenesis of idiopathic pulmonary fibrosis. Expression of the αvβ6 integrin is increased in fibrotic lung tissue and is a promising therapeutic target for treatment of the disease. Currently, measurement of αvβ6 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 αvβ6 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 αvβ6-specific (diethylenetriamine pentaacetate-tetra [DTPA]-A20FMDV2) or control (DTPA-A20FMDVran) peptide by nanoSPECT/CT imaging. Development of fibrosis was assessed by lung hydroxyproline content, and αvβ6 protein and itgb6 messenger RNA were measured in the lungs. Results: Maximal binding of 111In-labeled A20FMDV2 peptide to αvβ6 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 αvβ6-blocking antibodies. Levels of 111In-labeled A20FMDV2 peptide correlated positively with hydroxyproline, αvβ6 protein, and itgb6 messenger RNA levels. Conclusion: We have developed a highly sensitive, quantifiable, and noninvasive technique for measuring αvβ6 integrin levels within the lung. Measurement of αvβ6 integrins by SPECT/CT scanning has the potential for use in stratifying therapy for patients with pulmonary fibrosis.

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.

Caffeine inhibits TGFβ activation in epithelial cells, interrupts fibroblast responses to TGFβ, and reduces established fibrosis in ex vivo precision-cut lung slices

Caffeine is a commonly used food additive found naturally in many products. In addition to potently stimulating the central nervous system caffeine is able to affect various systems within the body including the cardiovascular and respiratory systems. Importantly, caffeine is used clinically to treat apnoea and bronchopulmonary dysplasia in premature babies. Recently, caffeine has been shown to exhibit antifibrotic effects in the liver in part through reducing collagen expression and deposition, and reducing expression of the profibrotic cytokine TGFβ. The potential antifibrotic effects of caffeine in the lung have not previously been investigated. Using a combined in vitro and ex vivo approach we have demonstrated that caffeine can act as an antifibrotic agent in the lung by acting on two distinct cell types, namely epithelial cells and fibroblasts. Caffeine inhibited TGFβ activation by lung epithelial cells in a concentration-dependent manner but had no effect on TGFβ activation in fibroblasts. Importantly, however, caffeine abrogated profibrotic responses to TGFβ in lung fibroblasts. It inhibited basal expression of the α-smooth muscle actin gene and reduced TGFβ-induced increases in profibrotic genes. Finally, caffeine reduced established bleomycin-induced fibrosis after 5 days treatment in an ex vivo precision-cut lung slice model. Together, these findings suggest that there is merit in further investigating the potential use of caffeine, or its analogues, as antifibrotic agents in the lung.

ECM crosslinking enhances fibroblast growth and protects against matrix proteolysis in lung fibrosis

&NA; Idiopathic pulmonary fibrosis (IPF) is characterized by accumulation of extracellular matrix (ECM) proteins and fibroblast proliferation. ECM cross‐linking enzymes have been implicated in fibrotic diseases, and we hypothesized that the ECM in IPF is abnormally cross‐linked, which enhances fibroblast growth and resistance to normal ECM turnover. We used a combination of in vitro ECM preparations and in vivo assays to examine the expression of cross‐linking enzymes and the effect of their inhibitors on fibroblast growth and ECM turnover. Lysyl oxidase‐like 1 (LOXL1), LOXL2, LOXL3, and LOXL4 were expressed equally in control and IPF‐derived fibroblasts. Transglutaminase 2 was more strongly expressed in IPF fibroblasts. LOXL2‐, transglutaminase 2‐, and transglutaminase‐generated cross‐links were strongly expressed in IPF lung tissue. Fibroblasts grown on IPF ECM had higher LOXL3 protein expression and transglutaminase activity than those grown on control ECM. IPF‐derived ECM also enhanced fibroblast adhesion and proliferation compared with control ECM. Inhibition of lysyl oxidase and transglutaminase activity during ECM formation affected ECM structure as visualized by electron microscopy, and it reduced the enhanced fibroblast adhesion and proliferation of IPF ECM to control levels. Inhibition of transglutaminase, but not of lysyl oxidase, activity enhanced the turnover of ECM in vitro. In bleomycin‐treated mice, during the postinflammatory fibrotic phase, inhibition of transglutaminases was associated with a reduction in whole‐lung collagen. Our findings suggest that the ECM in IPF may enhance pathological cross‐linking, which contributes to increased fibroblast growth and resistance to normal ECM turnover to drive lung fibrosis.

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.

Secretory leukocyte protease inhibitor gene deletion alters bleomycin-induced lung injury, but not development of pulmonary fibrosis

Idiopathic pulmonary fibrosis 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 (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 markedly modify the development of lung fibrosis following bleomycin-induced lung injury.

Investigating lung responses with functional hyperpolarized xenon-129 MRI in an ex vivo rat model of asthma

Asthma is a disease of increasing worldwide importance that calls for new investigative methods. Ex vivo lung tissue is being increasingly used to study functional respiratory parameters independent of confounding systemic considerations but also to reduce animal numbers and associated research costs. In this work, a straightforward laboratory method is advanced to probe dynamic changes in gas inhalation patterns by using an ex vivo small animal ovalbumin (OVA) model of human asthma.

Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvβ6 Expression

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.

S66 Caffeine Inhibits TGFβ Activation by Epithelial Cells, Interrupts Fibroblast Responses to TGFβ, and Reduces Pulmonary Fibrosis in Ex Vivo Precision-cut Lung Slices

Caffeine (1, 3, 7-tri-methylxanthine) is a common food additive found naturally in many products. It is a non-selective competitive antagonist of G-protein coupled adenosine receptors and can inhibit phosphodiesterases. Caffeine has anti-fibrotic effects in the liver and increased caffeine consumption has been associated with reduced liver fibrosis in patients with chronic hepatitis C infection. The effect of caffeine on pulmonary fibrosis has not been investigated, however, it has been shown to inhibit TGFβ-induced Smad signalling in epithelial cells. This study aimed to investigate the anti-fibrotic effects of caffeine in the lung using lung epithelial cells, fibroblasts and an ex vivo precision-cut lung slice (PCLS) model of fibrosis. Immortalised human bronchial epithelial cells (iHBECs) and primary human lung fibroblasts from were used. TGFβ activation was assessed using an in vitro TGFβ reporter cell assay and assessment of phosphorylated Smad2. Expression of pro-fibrotic genes was assessed by quantitative polymerase chain reaction. Proliferation of fibroblasts was assessed by brdU incorporation assay. Finally, the effect of caffeine on established lung fibrosis was investigated ex vivo using PCLS. Mice were instilled with saline or 60 IU bleomycin and PCLS obtained after 28 days. PCLS were treated with increasing concentrations of caffeine for five days prior to measurement of collagen by high-performance liquid chromatography. Viability of the PCLS following caffeine treatment was assessed by MTT assay. Caffeine induced a concentration-dependent decrease in TGFβ activation in iHBECs but had no effect on TGFβ activation in lung fibroblasts. Furthermore, caffeine reduced expression of the TGFβ-inducible genes PAI1 and Col1A and reduced TGFB1 transcript in epithelial cells. Additionally, caffeine reduced TGFβ-induced proliferation of lung fibroblasts and reduced expression of pro-fibrotic genes including COL1A and ACTA2. Crucially, ex vivo treatment of fibrotic PCLS from bleomycin treated animals with caffeine caused a dose-dependent reduction in collagen deposition after five days. Caffeine had no effect on collagen deposition in PCLS isolated from saline treated animals nor did caffeine affect tissue viability in PCLS from either saline or bleomycin treated animals. In conclusion, caffeine has anti-fibrotic effects in the lung via concomitant inhibition of epithelial TGF activation and fibroblast responses to TGFb.