This PhD project will take a molecular approach to study the modulation of innate immune responses by ß-glucans and focus on pattern-recognition receptors (PRRs) on myeloid cells that respond to pathogen-associated molecular patterns (PAMPs) such as ß-glucans that compose fungal cell walls. PRRs that are predicted to be central to this process are TOLL-like receptors (TLRs).
Danilo Pietretti started his PhD in March 2009. The PhD project is part of an EU-funded Initial Training Network (ITN) on “Protective immune modulation in warm water fish by feeding glucans’ (short name NEMO, website http://www.nemo-network.eu/Home/Intro_page.html ). The scientific aim of NEMO is to establish optimal protocols for the use of ß-glucans (components of fungal cell walls) in the strategic improvement of animal health. The training aim of NEMO is to form a nucleus of 16 young scientists by training-through-research and by provision of complementary skills. I started my great interest in fish during my MSc thesis in Italy, where I worked on the immune system of sea bass (Dicentrarchus Labrax), now during my PhD, I am still working on the immune system of fish, in this case, common carp.
The focus of this project is on TLR4 and TLR20 expressed on carp myeloid cells, on the signaling pathways of the TLRs and on their possible functions as activating receptors involved in innate immune responses to, for example, stimulation with ß-glucans or infections with parasites (Trypanoplasma borreli or Trypanosoma Carassii). Once TLRs sequences are obtained, they will allow for the design of specific primers for real-time quantitative PCR analysis of gene expression induced by ß-glucan and parasites or different ligands. Furthermore, a microarray approach will be taken to identify in an unbiased manner transcripts in ß-glucan-stimulated macrophages for further study. The sequences will also allow for the design of transfection experiments aimed at studying the degree of evolutionary conservation of TLRs activation mechanisms in both human cell lines and in carp myeloid cells. Recombinant protein of carp TLRs produced by bacterial expression will be used for immunization of chickens in order to obtain polyclonal antibodies.