Characterization of Tie2 signalling during acute inflammation

The inflammatory response is essential for the eradication of lipopolysaccharide (LPS) presenting microbial invaders. However, resolution is also important to prevent detrimental vascular inflammation to the host. Endothelial cells play active roles in inflammation by detecting LPS using Toll-Like Receptor 4 (TLR4), leading to the activation of Nf-κB, but also participate in the resolution of inflammation, in part, through the actions of the receptor tyrosine kinase, Tie2. The process by which Tie2 can attenuate LPS-TLR4 driven inflammation is poorly understood. The work herein centers upon the characterization of Tie2 signalling during acute inflammation. The effects of Tie2 activation using the Tie2 agonist, Vasculotide (VT), was monitored in an animal model of endotoxin induced inflammation. Activation of Tie2 resulted in reductions in vascular permeability, levels of circulating inflammatory cytokines, and the number of leukocytes at the site of inflammation following LPS stimulation, confirming the anti-inflammatory effect of Tie2 signalling following LPS-TLR4 driven acute inflammation. To investigate the crosstalk between Tie2 and TLR4, Nf-κB activation was monitored in cells expressing Tie2 mutants harboring tyrosine (Y) to phenylalanine mutations in the carboxy-terminal tyrosine residues essential for downstream Tie2 signalling. Tie2 signalling reduced LPS induced Nf-κB activation in a Y1100 and Erk1/2 dependent manner. Tie2 signalling decreased the expression of the TLR4 signalling proteins, TRAF6 and IRAK1 and preserved the expression of the Nf-κB inhibitor, IκBα, though Y1100 initiated Erk1/2 signalling. The ability of Tie2 to increase the expression of miRNA-146b-5p was investigated and found to be dependent on functioning of all three carboxy-terminal tyrosine residues. Our results show that Y1100 initiated Erk1/2 signalling is essential for the anti-inflammatory effect of Tie2 on TLR4 driven Nf-κB activation. Characterization of the role of Tie2 signalling during an inflammatory cascade will further our understanding of the biology of Tie2 and ultimately contribute to our understanding of the regulation of vascular inflammation.Ph.D.2019-07-11 00:00:0