Award details

Amphiregulin -- a novel regulatory t cell effector molecule?

Reference	BB/H016376/1
Principal Investigator / Supervisor	Dr Oliver Garden
Co-Investigators / Co-Supervisors	Ms Karen Affleck
Institution	Royal Veterinary College
Department	Comparative Biomedical Sciences CBS
Funding type	Skills
Value (£)	83,281
Status	Completed
Type	Training Grants
Start date	01/10/2010
End date	30/09/2014
Duration	48 months

Abstract

unavailable

Summary

CD4+CD25+FoxP3+ regulatory T cells (Tregs) play a crucial role in the maintenance of peripheral tolerance, underlined by the plethora of autoimmune, inflammatory and allergic diseases associated with Treg dysfunction. While a number of Treg effector mechanisms have been elucidated, there are still large deficits in our molecular understanding of Tregs. Amphiregulin (Areg) is a member of the epidermal growth factor (EGF) family, existing as a transmembrane precursor that is cleaved into a soluble form by the sheddase enzyme 'a disintegrin and metalloproteinase' (ADAM)-17. The soluble form of Areg binds to the EGF receptor (EGFR) and mediates autocrine or paracrine functions, while the uncleaved form mediates juxtacrine interactions. Though T cells are known to express Areg, the function of this exciting EGFR ligand in the adaptive immune system remains unknown. We have recently shown that murine CD4+CD25+FoxP3+ Tregs express high levels of Areg when compared to conventional CD4+CD25-CD45RBlow T cells (Tconv), assessed both at the level of mRNA (Treg / Tconv by microarray = 3.15, quantitative (q) PCR = 6.99; p<0.001) and protein product by flow cytometry. Moreover, the Areg-/- mouse has prominent submucosal inflammatory lesions of the gastric fundus, accompanied by increased mucosal mRNA and serum concentrations of pro-inflammatory cytokines, suggesting abnormalities of peripheral tolerance. We speculate that Treg function in this model is defective and our AIM is to test the HYPOTHESIS that Areg is a novel Treg effector molecule, facilitating interactions of Tregs with both Tconv and intestinal epithelial cells. We have put together a project that combines excellent science with an outstanding training programme in cellular and molecular immunology, delivering against the BBSRC 'Healthy Organism' strategic area and a number of research and policy priorities (outlined in Project Details). OBJECTIVE 1: To determine the expression kinetics of Areg, EGFR and ADAM-17 in Tregs, Tconv and an intestinal epithelial cell line in vitro over 24 hours, examining transcript abundance by qPCR, protein product by flow cytometry and/or Western blots, and the concentration of soluble Areg by ELISA. T cells will be stimulated with anti-CD3/CD28 Dynabeads in the presence of a cAMP agonist or antagonist, respectively anticipated to increase or decrease Areg expression; enterocytes will be similarly cultured in epithelial medium. Cells derived from both wild-type (WT) and Areg-/- mice will be examined, the latter as a negative control for Areg. OBJECTIVE 2: To determine involvement of the ERK/MAPK signalling pathway in the stimulation of Tregs, Tconv and intestinal epithelial cells by recombinant Areg, examining Western blots of cell lysates for phospho-EGFR, ERK1/2 and MAPK, with and without pre-treatment of the cells with an EGFR tyrosine kinase inhibitor. OBJECTIVE 3: To determine the role of Areg in Treg suppressive function Syngeneic and 'cross-over' co-cultures of Treg with Tconv cells from Areg-/- or WT mice will be established in vitro, examining proliferation by CFSE dilution and 3H-TdR incorporation as a read-out. If Areg plays a cell-intrinsic role in suppression, Areg-/- Tregs will not be able to suppress WT Tconv; the addition of a neutralizing antibody will abolish any residual Areg, and juxtacrine versus autocrine/paracrine mechanisms will be explored by pre-treatment of cells with an ADAM-17 inhibitor. Conclusions derived in vitro will be verified using the adoptive transfer model of colitis, examining the ability of Areg-/- Tregs to ameliorate the colitogenic potential of WT CD45RBhigh T cells in Rag-/- mice. OBJECTIVE 4: To model putative paracrine interactions of Tregs with intestinal epithelium in vitro, applying Treg culture supernatants to enterocytes in the presence or absence of anti-Areg or EGRF tyrosine kinase inhibitor, examining as a read-out proliferation and upregulation of phospho-EGFR, ERK1/2 and

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

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