Direct Supervisor: Daniele D’Ambrosio, MD, PhD
Location: Pre-clinical research branch
Descriptive title of the research activity: Identification of novel G protein coupled receptors (GPCRs) regulating leukocyte migration and/or adhesion in chronic inflammatory diseases
Overall goals: Our project focuses on the identification of novel G protein coupled receptors (GPCRs) involved in regulating migration and/or leukocyte adhesion, whose inappropriate, eccessive or disregulated activity is implicated in the pathogenesis of diseases such as asthma, chronic obstructive pulmonary disease (COPD), psoriasis, atopic dermatitis (AD), inflammatory bowel disease (IBD), rheumatoid arthritis (RA) and multiple sclerosis (MS).
Rationale and Significance: Since various cell surface molecules that regulate cell migration belong to different classes of GPCRs (e.g.: chemokine, leukotriene, prostaglandin and lysophospholipid receptors), we anticipate that a significant fraction of the identified targets will likely be involved directly or indirectly in the regulation of cell migration. We have extensive experience and know-how on the isolation, differentiation, manipulation and functional characterization of immune cell types. In the recent past we have deciphered the gene expression profile of Th1 and Th2 cells and have dissected the expression pattern and function of chemokine receptors expressed on different subsets of T cells. Thus far, two chemokine receptors (CCR7 and CXCR3) and the enzyme fucosyltransferase VII (FucTVII), which are involved in the migration of T cells and DCs into sites of inflammation and secondary lymphoid tissues, have been selected for the identification of small molecular weight antagonists with the purpose of developing a new class of anti-inflammatory drugs aimed at interfering with the migration of inflammatory cells in chronic inflammatory and allergic diseases.
Description of work and methodology: By utilizing a custom-made Affimetrix gene chip to monitor the expression level of >700 human GPCRs, we will analyze specific GPCR mRNA expression in different human cell types including in vitro-derived and freshly isolated T helper (Th) 1 and Th2 cells, skin-homing (cutaneous lymphocyte antigen, CLA+) and gut-homing T cells (integrin 4+7+), CD25+ T regulatory (Treg) cells, immature and mature dendritic cells (DCs), mast cells.
The different cell types obtained either by direct isolation from human blood of healthy donors or following well established in vitro culture conditioning protocols will permit isolation of RNA that will be subject to labelling and hybridization onto the GPCR gene chip. Similar analysis has been already carried out on almost 100 different normal human tissues and cell types and will allow identification of those GPCRs that exhibit the most selective expression profile on discrete immune cell types (e.g. Th1 versus Th2 cells or immature versus mature DCs) by computational analysis. Expression of selected receptors will subsequently be verified in human tissues obtained from relevant inflammatory diseases. Selected receptors will be cloned and ectopically expressed by transfection into suitable cell lines to facilitate their functional characterization. Antibodies will also be raised and siRNA technology-based deletion will be implemented to help elucidating the function of selected receptors. Site-directed mutagenesis of critical intracytoplasmic residues of GPCRs will be used to generate constitutively active forms of selected GPCRs and by transient transfection of such mutated receptors into appropriate cell lines it will be possible to screen for small molecular weight agonist/antagonist compounds using a high-throughput cell based functional assay.