title: Investigating NF-κB signaling in lung fibroblasts in 2D and 3D culture systems.
abstract: BACKGROUND
  Inflammatory respiratory diseases are amongst major global health challenges. Lung fibroblasts have been shown to play a key role in lung inflammatory responses. However, their exact role in initiation and maintenance of lung diseases has remained elusive partly due to the limited availability of physiologically relevant in vitro models. Therefore, developing new tools that enable investigating the molecular pathways (e.g. nuclear factor-kappa B (NF-κB) activation) that underpin inflammatory responses in fibroblasts could be a valuable resource for scientists working in this area of research.
  
  
  RESULTS
  In order to investigate NF-κB activation in response to pro-inflammatory stimuli in real-time, we first developed two detection systems based on nuclear localization of NF-κB by immunostaining and luciferase reporter assay system. Furthermore using electrospun porous scaffolds, with similar geometry to human lung extracellular matrix, we developed 3D cultures of lung fibroblasts allowing comparing NF-κB activation in response to pro-inflammatory stimuli (i.e. TNF-α) in 2D and 3D. Our data clearly show that the magnitude of NF-κB activation in 2D cultures is substantially higher than 3D cultures. However, unlike 2D cultures, cells in the 3D model remained responsive to TNF-α at higher concentrations. The more subdued and wider dynamic range of NF-κB responses in 3D culture system was associated with a different expression pattern for TNF receptor I in 3D versus 2D cultures collectively reflecting a more in vivo like TNF receptor I expression and NF-κB activation pattern in the 3D system.
  
  
  CONCLUSIONS
  Our data suggest that lung fibroblasts are actively involved in the pathogenesis of lung inflammation by activation of NF-κB signaling pathway. The 3D culture detection system provides a sensitive and biologically relevant tool for investigating different pro-inflammatory events involving lung fibroblasts.
authors.affiliation: Cellular Immunology and Allergy Research Group, Division of Immunology, School of Life Science, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham, UK. mrxsshtw1@nottingham.ac.uk.
authors.forename: Su Su
authors.initials: SS
authors.lastname: Htwe
authors.affiliation: Cellular Immunology and Allergy Research Group, Division of Immunology, School of Life Science, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham, UK. Helen.Harrington@nottingham.ac.uk.
authors.forename: Helen
authors.initials: H
authors.lastname: Harrington
authors.affiliation: Division of Respiratory Medicine, University of Nottingham, City Hospital, Nottingham, UK. alan.knox@nottingham.ac.uk.
authors.forename: Alan
authors.initials: A
authors.lastname: Knox
authors.affiliation: Division of Drug Delivery and Tissue Engineering, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham, UK. felicity.rose@nottingham.ac.uk.
authors.forename: Felicity
authors.initials: F
authors.lastname: Rose
authors.affiliation: Laboratory of Biophysics and Surface Analysis, School of Pharmacy, University of Nottingham, Nottingham, UK. jon.aylott@nottingham.ac.uk.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Aylott
authors.affiliation: Department of Materials Science and Engineering, University of Sheffield, Sheffield, UK. j.w.haycock@sheffield.ac.uk.
authors.forename: John W
authors.initials: JW
authors.lastname: Haycock
authors.affiliation: Cellular Immunology and Allergy Research Group, Division of Immunology, School of Life Science, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham, UK. amg@nottingham.ac.uk.
authors.forename: Amir M
authors.initials: AM
authors.lastname: Ghaemmaghami
mesh_terms: D018929:Cell Culture Techniques; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D017930:Genes, Reporter; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism*; D008168:Lung / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D011014:Pneumonia / Q000378:metabolism* / Q000473:pathology; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D018836:Inflammation Mediators; C498143:RELA protein, human; D047888:Receptors, Tumor Necrosis Factor, Type I; C506235:TNFRSF1A protein, human; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha