Since the X-ray structure of the zebra fish P2X4 receptor in the closed state was published in 2009 homology modeling has been used to generate structural models for P2X receptors. In this chapter, we outline how to use the MODELLER software to generate such structural models for P2X receptors whose structures have not been solved yet.

P2X receptors are trimeric ATP-gated ion channels. In response to ATP binding, conformational changes lead to opening of the channel and ion flow. Current flow can decline during continued ATP binding in a process called desensitisation. The rate and extent of desensitisation is affected by multiple factors, for instance the T18A mutation in P2X2 makes the ion channel fast desensitising. We have used this mutation to investigate whether the gate restricting ion flow is different in the desensitised and the closed state, by combining molecular modelling and cysteine modification using MTSET (2-(Trimethylammonium)ethyl methanethiosulfonate). Homology modelling of the P2X2 receptor and negative space imaging of the channel suggested a movement of the restriction gate with residue T335 being solvent accessible in the desensitised, but not the closed state. This was confirmed experimentally by probing the accessibility of T335C in the P2X2 T18A/T335C (fast desensitisation) and T335C (slow desensitisation) mutants with MTSET which demonstrates that the barrier to ion flow is different in the closed and the desensitised states. To investigate the T18A induced switch in desensitisation we compared molecular dynamics simulations of the wild type and T18A P2X2 receptor which suggest that the differences in time course of desensitisation are due to structural destabilization of a hydrogen bond network of conserved residues in the proximity of T18.

The human P2X1 receptor (hP2X1R) is a trimeric ligand-gated ion channel opened by extracellular ATP. The intracellular amino and carboxyl termini play significant roles in determining the time-course and regulation of channel gating—for example, the C terminus regulates recovery from the desensitized state following agonist washout. This suggests that the intracellular regions of the channel have distinct structural features. Studies on the hP2X3R have shown that the intracellular regions associate to form a cytoplasmic cap in the open state of the channel. However, intracellular features could not be resolved in the agonist-free apo and ATP-bound desensitized structures. Here we investigate the organization of the intracellular regions of hP2X1R in the apo and ATP-bound desensitized states following expression in HEK293 cells. We couple cysteine scanning mutagenesis of residues R25-G30 and H355-R360 with the use of bi-functional cysteine reactive cross-linking compounds of different lengths (MTS-2-MTS, BMB, and BM(PEG)2), which we use as molecular calipers. If two cysteine residues come into close proximity, we predict they will be cross-linked and result in ∼66% of the receptor subunits running on a Western blot as dimers. In the control construct (C349A) that removed the free cysteine C349, and some cysteine-containing mutants, cross-linker treatment does not result in dimerization. However, we detect efficient dimerization for R25C, G30C, P358C, K359C, and R360C. This selective pattern indicates that there is structural organization to these regions in the apo and desensitized states in a native membrane environment. The existence of such precap (apo) and postcap (desensitized) organization of the intracellular domains would facilitate efficient gating of the channel.

Schmallenberg virus (SBV) caused a large scale epidemic in Europe from 2011 to 2013, infecting ruminants and causing foetal deformities after infection of pregnant animals. The main impact of the virus was financial loss due to restrictions on trade of animals, meat and semen. Although effective vaccines were produced, their uptake was never high. Along with the subsequent decline in new SBV infections and natural replacement of previously exposed livestock, this has resulted in a decrease in the number of protected animals. Recent surveillance has shown that a large population of naïve animals is currently present in Europe and that the virus is circulating at a low level. These changes in animal status, in combination with favourable conditions for insect vectors, may open the door to the re-emergence of SBV and another large scale outbreak in Europe. This review details the potential and preparedness for SBV re-emergence in Europe, discusses possible co-ordinated sentinel monitoring programmes for ruminant seroconversion and the presence of SBV in the insect vectors, and provides an overview of the economic impact associated with diagnosis, control and the effects of non-vaccination.

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.

Signaling by G q/11 is required for optimal TGFβ activation in the lung to prevent inflammation.

Activated TGFβ promotes pro-apoptotic pathways in lung epithelial cells, which has been implicated in the pathogenesis of IPF. The Influenza-A virus infects epithelial cells leading to cell death and injury, and can also activate TGFβ. The rhinovirus causes common cold. Pulmonary fibrosis is characterised by deposition of collagen within the lung. The role of viral respiratory tract infections in IPF is unclear. The aim of this study is to investigate the role of viral infections in collagen deposition within the lung. C57Bl6 mice were infected with 20HAU influenza ‘x319 for 5 days or rhinovirus for 3,7 days or PBS. Lungs were harvested and a hydroxyproline (HYP) assay was performed. 60U of bleomycin were instilled into the lungs of C57Bl/6 mice to induce pulmonary fibrosis. After 28 days mice were exposed intranasally to 10, 20HAU influenza virus or PBS. Lungs were harvested 5 or 21 days later for mRNA analysis, histology and HYP levels. Mice with non-fibrotic lungs infected with influenza showed increased lung collagen deposition as early as 5 days post-infection. Mice infected with rhinovirus showed no increase in lung collagen levels 3 days or 7 days post-infection. Mice that were given bleomycin for 28 days showed an enhanced fibrotic response after influenza infection: there were increased lung HYP levels and an increase in matrix deposition on Masson9s trichrome. There also was an increase in CCNA2 mRNA in influenza-infected, bleomycin-exposed mice, indicating an increase in epithelial apoptosis. These data suggest that influenza infection promotes exacerbations of lung collagen deposition perhaps via epithelial cell apoptosis and TGFβ activation.

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.

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.

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.

Introduction Transforming Growth Factor-beta (TGFβ) promotes anti-proliferative and pro-apoptotic pathways in lung epithelial cells, both of which have been implicated in the pathogenesis of IPF. TGFβ must be activated before it can mediate these events. Acute exacerbations of IPF are characterised by widespread epithelial cell apoptosis. The precise cause of these exacerbations is not known. The Influenza A virus is a single-stranded segmented RNA virus that infects epithelial cells leading to cell death and injury, and can also activate TGFβ. 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 and TGFβ activation in vivo. Materials and Methods 60 U of bleomycin was instilled into the 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 the lungs harvested 5 days later. Bronchoalveolar lavage (BAL) was performed and lung tissue harvested for mRNA analysis, histology and hydroxyproline levels. All 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 Mice exposed to bleomycin and infected with influenza lost less weight compared with saline-exposed influenza-infected animals. However, the lungs from bleomycin-exposed, influenza-infected mice showed increased lung damage with more matrix deposition on trichrome staining than saline-exposed, influenza-infected mice. Saline-exposed, influenza-infected mice demonstrated the anticipated dose dependent increase in BAL lymphocytosis as well as apoptosis staining in histological TUNEL assessment. However, in bleomycin-exposed mice, influenza infection did not promote enhanced BAL lymphocytosis or apoptosis. However, influenza appeared to enhance the fibrotic response demonstrated by an increase in matrix deposition on masson’s trichrome and increased lung hydroxyproline levels in influenza infected bleomycin exposed mice, as early as 5 days post infection. Conclusions These data suggest that influenza infection may exacerbate lung fibrosis by promoting epithelial apoptosis.

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.