{
  "abstract": "Background IPF is characterised by an increase in aberrant basaloid epithelial cells which have high levels of Keratin 17 (KRT17) and low levels of Keratin 5 (KRT5) although their source is still unclear. One potential source is airway basal cells which express high levels of both KRT17 and KRT5. Cell extrusion is a process whereby individual epithelial cells are expelled from contiguous epithelium, whilst barrier integrity and tissue architecture is maintained. I hypothesise that extrusion of epithelial cells from small airways in lung leads to loss of KRT5 and is a critical first step in fibrogenesis and generation of aberrant basaloid cells.Methods KRT5 and KRT17 gene expression was evaluated in ihBECs (immortalised human bronchial epithelial cells) using real time qPCR in monolayer, extruded and replated extruded cells. ihBECs were seeded at high density to induce overcrowding to promote cell extrusion. Once 95% confluent, extruded cells were collected from supernatant and replated for sub-culture. The monolayer cells remained in culture for another three days until collected along with extruded cells for RNA extraction. KRT5 and KRT17 protein in monolayer and extruded ihBECs was evaluated by flow cytometry (FACS Canto BD) and flow imaging cytometry (Imagestream MKII) using fluorescent antibodies ((Anti-Cytokeratin 5 antibody [EP1601Y] and Anti-Cytokeratin 17 antibody [EP1623]); 2ug/mL).Results There was a five-fold decrease in KRT5 and two-fold increase in KRT17 mRNA in extruded ihBEC (eihBEC) cells compared ihBEC in monolayer (mihBEC). KRT5 protein was downregulated by 50% in eihBECs compared with mihBEC in both flow cytometry and flow imaging cytometry analyses. Flow cytometry of KRT17 in eihBEC compared with miHBEC showed upregulation by 35%. The gene expression pattern in extruded cells showing loss of KRT5 and increase of KRT17 was maintained upon sub-culture of replated eihBEC cells compared with mihBEC.Conclusion Extrusion of ihBECs leads to loss of KRT5 and increased KRT17 consistent with the phenotype of aberrant basaloid cells seen in IPF. We propose that extrusion of small airway epithelial cells may be a critical first step in development of aberrant basaloid cells.",
  "authors": [
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "K Bhatti"
    },
    {
      "affiliations": [
        "Kings College London, London, UK"
      ],
      "name": "I Uwagboe"
    },
    {
      "affiliations": [
        "Kings College London, London, UK"
      ],
      "name": "J Rosenblatt"
    },
    {
      "affiliations": [
        "Kings College London, London, UK"
      ],
      "name": "M Parsons"
    },
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "G Jenkins"
    },
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "A John"
    },
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "I Stewart"
    },
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "C Dean"
    }
  ],
  "title": "P242 Epithelial cell extrusion promotes the loss of KRT5 with implications for pulmonary fibrosis",
  "uid": "f9b66ac0-7398-5a78-ae74-b1e95e2c2d90"
}