Award details

Discovery of innate immune signaling molecules that trigger wound-healing and tissue fibrosis.

Reference	BB/G016445/1
Principal Investigator / Supervisor	Prof. Derek Mann
Co-Investigators / Co-Supervisors	Professor Stuart Farrow, Professor Andrew Fisher
Institution	Newcastle University
Department	Institute of Cellular Medicine
Funding type	Skills
Value (£)	74,410
Status	Completed
Type	Training Grants
Start date	02/11/2009
End date	01/11/2013
Duration	48 months

Abstract

unavailable

Summary

1. Background: Wound-healing is a critical physiological response that promotes the repair and regeneration of damaged tissue. However, as animals age their capacity for effective wound-healing is diminished and as a result damaged tissues either fail to repair or are replaced by scar tissue (fibrosis). The goal of the studentship is to discover molecules released by damaged epithelia that trigger wound-healing signaling as these may be exploited to promote more effective tissue repair. Epithelial damage is a key trigger for wound-healing but is mechanistically ill-defined. Microbial components (e.g. LPS) induce inflammatory signaling via activation of cell surface Toll-like- and IL-1-receptors (TIRs). TIRs also trigger activation of fibrogenic cells under sterile conditions in response to cell damage. Damaged cells release intracellular molecules (so-called 'alarmins' including IL-1) recognised by specific TIRs. (Myo)fibroblasts are the major fibrogenic cell and express a variety of TIRs including IL-1R. Fibrogenic activities of fibroblasts are stimulated by TIR activation but the identity of the alarmins that trigger TIRs is unknown. The student will identify epithelial-derived alarmins that drive fibrogenesis. The lung will be used as a model experimental system to probe generic fibrogenic mechanisms. Applicants Mann and Fisher are part of a large group of fibrosis researchers in Newcastle that span multiple organ systems. The student will repeat key experiments in cellular models from at least one other organ to identify generic fibrogenic alarmins. 2. Hypothesis to be tested: Alarmins released by damaged human lung epithelia provoke the activation of fibrogenic cells. 3. Aims of the studentship: (i) Investigate IL-1 as a mediator of inflammation and fibrosis resulting from damage of lung epithelia. (ii) Generate and functionally characterise a panel of hybridomas recognising alarmins released by damaged human lung epithelia. (iii) Determine the expression of IL-1 and antigens recognised by antibodies raised in aim (ii) in human idiopathic pulmonary fibrosis (IPF) and other fibrotic organs. 4. Brief experimental plan: Aim (i). Standardised wounds will be created by scraping a line of cells from the centre of a confluent monolayer of human lung fibroblasts. Wound closure will be monitored in the presence of protein supernatants from damaged human epithelial cells. Production of fibrogenic proteins (collagen I and III, MMP-2/9, TIMP-1 and TGFb) by fibroblasts will be monitored by established assays. Effects of antibody neutralisation of IL-1a, IL-1b and blockade of IL-1R (Anakinra) on fibroblast responses will determine a role for the IL-1 system. Inhibitors of IKK, ERK1/2, p38 MAPK and JNK will confirm a role for the TIR signaling cascade. Aim (ii). Pilot experiments will determine effects of oxidant stress, radiation and simple freeze/thaw on the release of alarmins by damaged human lung epithelia. Protein supernatants from these cells will be added to cultures of human lung fibroblasts and expression of fibrogenic molecules (TGFb, Collagen I, TIMP-1) quantified by qRT-PCR. The protocol that generates the most potent fibrogenic effect will be employed to generate supernatants for immunisation of Armenian hamsters for production of hybridomas expressing antibodies against alarmins. Hybridomas will be screened for ability to neutralise induction of fibrogenic molecules by human lung fibroblasts responding to supernatants from damaged lung epithelia. Subsequent work would involve functional characterisation of antibodies in wounding assays and identification of antigens. Aim (iii). Archives of paraffin-embedded and frozen tissue from IPF and cirrhotic patients removed during transplantation will be subjected to immunohistochemistry using anti-IL1a/b and antibodies raised in aim (ii). Cell location of alarmins will be determined by confocal microcoscopy and stains for cell-specific markers.

Committee	Not funded via Committee
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	Training Grant - Industrial Case

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search