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Tribbles (Trib) protein was first described in Drosophila as a regulator of proliferation, later being implicated as a G2/M modulator. In mammalian systems, three Trib gene family members have been identified, which share a conserved motif similar to the catalytic domain of serine/threonine kinases. However, they lack several conserved residues in the ATP-binding pocket and the core motif of the catalytic domain necessary for catalytic function. Tribbles 1 (Trib1) is involved in inflammation through its ability to regulate MAPK, NF-κB and the CCAAT Enhancer Binding Protein (C/EBP). Moreover, Trib1 is associated with human disease, such as atherosclerosis and acute myeloid leukaemia. In this thesis, I investigated the functional role of Trib1 in Toll-like Receptor (TLR)-induced inflammatory responses together with pro- or anti-inflammatory cytokines. The RAW264.7 myeloid cell line was stimulated with TLR2/9 ligands in the presence or absence of IFN-γ or IL-10. I observed a high level of Trib1 expression in the presence of IFN-γ and TLR2 ligands, but weak Trib1 expression following treatment with IL-10 and TLR9 ligands. In gene knock-down experiments using small interfering RNAs (siRNA) to reduce Trib1 expression, C/EBPβ was up-regulated in both stimulated (by IFN-γ and TLR2 ligands) and resting macrophage populations. TNF-α production was increased following Trib1 knockdown after treatment with IFN-γ and/or TLR2 ligands but IL-6 secretion remained unchanged. Furthermore, ERK1/2 expression was reduced in Trib1 siRNA-treated cells and failed to induce chemokinesis in macrophages. Finally, Trib1 was demonstrated to act as a modulator of cell cycle (G2/M) transition and displays a delayed apoptotic phenotype. The work in this thesis demonstrates that mammalian Trib1 contributes to the pro-inflammatory response and functions as a regulator of the ERK1/2 and C/EBPβ pathways following TLR ligand-mediated activation. Its novel functions include acting as a modulator of G2/M arrest and suppressing macrophage migration.

We used Experimental Autoimmune Uveoretinitis (EAU) as a model system to investigate the involvement of CCR2 and CX₃CR1 in regulating the trafficking and function of monocytes and microglia in an autoimmune context. METHODS: W.T. or CX₃CR1^GFP/GFPmonocytes were adoptively transferred into mice with EAU.  At 48 hours post transfer their phenotype was examined by flow cytometry and monocytes trafficking to the retina was imaged using Scanning Laser Ophthalmoscopy. An anti-CCR2 antibody (MC21) or antagonist (JE(9-76)) was used to examine the effect of CCR2 blockade on W.T. monocytes trafficking.  Infiltration of monocytes into the inflamed retina and activation of retinal microglia were examined by confocal microscopy on retinal flatmounts from W.T. and CX₃CR1^GFP/GFP mice and immunohistochemistry on cryosections from eyes. RESULTS: CCR2 increased on W.T. monocytes at 48 hours post transfer and at 24 hours on CX₃CR1^GFP/GFP monocytes.  However, blocking CCR2 by either method did not reduce the number of W.T. monocytes rolling along retinal vessels and infiltrating the retina.  Lack of CX₃CR1 did not alter microglial activation but infiltrating monocytes lacking the receptor could not migrate through the retina and clustered around vessels. CONCLSIONS: CCR2 may  not always be needed for recruitment into an inflammatory site.  CX₃CR1 has a role in neuroprotection in the retina by enhancing the migratory ability and distribution of infiltrating monocytes within inflamed tissue.  This work stresses the importance for careful dissection of the chemokine receptors’ mechanism of action before therapeutic possibilities are explored.