Multi-platform studies of a pro-fibrotic network in IPF

Aims:
To characterise key pro-fibrotic interactions relevant to pathogenesis of idiopathic pulmonary fibrosis (IPF) functionally and structurally and so define novel targets for therapeutic development.

Background:
A cell surface interactome (the ‘gal-3-fibrosome’) mediates fibrotic responses to extracellular insults in multiple organs including the lung in models of the interstitial lung disease (ILD) idiopathic pulmonary fibrosis (IPF). IPF has a poor prognosis (median survival of 3 years from diagnosis) and is responsible for 1% of UK deaths each year. Galectin-3 is found at greatly increased levels in IPF and circulates as a lung fibrosis biomarker (Mackinnon et al, 2012). Galectin-3 can self-associate and simultaneously bind to modified glycans to cluster and/or stabilise pro-fibrotic factors. These include CD98 in complex with β1-integrin (that provides mechanotransductive cross-talk between the extracellular matrix and cytoskeleton) and the TGF-β receptor II (TGFβRII). We have recently characterised the major molecular pathway mediating IL-6/-8 responses to experimental conditions that mimic bacterial and mechanical drivers of IPF exacerbation and deterioration, in vitro and in vivo. Our data demonstrate a gal-3-fibrosome protein is key to this process, supporting its potential as an important mechanism coupling inflammatory exacerbations to progression of fibrosis in IPF.

Approaches:
1. The student will characterise the co-localisation of previously described identified gal-3-fibrosome components and our recently defined exacerbation pathway at the cell surface in human IPF and healthy lung tissue and cells. We will use ex vivo tissue samples (BRC-Resp), primary fibroblasts/myofibroblasts derived ex vivo and cell lines modelling AEC behaviour. Levels of each protein and their co-localisation will be correlated in tissue with collagen deposition.
2. Cell phenotypes of interest to the Gooptu group/collaborators will be correlated with cell surface levels of the different proteins/ colocalisation using methods already in successful use in our labs. The relationship between phenotype and protein will then be probed by knock-down studies. Specifically the student will assess: (i) Alveolar epithelial models – IL-6, IL-8 and TNF-α secretion (ELISA), epithelial mesenchymal transition (EMT) in vitro; (ii) Myofibroblasts – collagen-I and fibronectin secretion and α-SMA expression; (iii) All models: TGF- β1 canonical/non-canonical signalling pathways, bioenergetics and cell cycle behaviour.
3. The spatial organisation of elements that show robust co-localisation in tissue and cells will be assessed at higher resolution using biophysical techniques including FRET/FLIM (in cells), and proximity ligation assays (cells and tissue).
4. Negative stain and cryo-electron microscopy studies of the gal-3-fibrosome, working with the new Leicester cryo-EM facility established at the Leicester Institute of Structural and Chemical Biology (LISCB).