A study of the modulation of Toll-like receptor signalling in macrophages by Annexin-1
Annexin-A1 (AnxA1) is an endogenous anti-inflammatory protein that has been shown to exert a protective role against the lethal effects induced by the Toll-like receptor (TLR) 4 agonist lipopolysaccharide (LPS). The aim of this PhD studentship was to expand these observations and investigate the possible cross-talk between AnxA1 and other TLR signalling pathways in macrophages. To this aim, we compared the response in vitro of AnxA1-/- to AnxA1+/+ bone marrow-derived macrophages (BMDMs) after stimulation with TLR2, 3, 5, 7 and 9 ligands. AnxA1-/- BMDMs exhibited higher expression of the activation markers MHC II and co-stimulatory molecules CD40, CD80, CD86 at the basal level, but a similar upregulation after stimulation with different TLR agonists. Stimulation of AnxA1-/- BMDMs with MyD88-dependent TLR agonists caused an increased production of TNF- and IL-6 compared to AnxA1+/+. Conversely, stimulation with the TRIF-dependent ligand poly (I:C) caused a decreased production of IL-6, but not TNF-, by these cells. Interestingly, comparison of MyD88 and TRIF-dependent downstream signalling pathways in AnxA1+/+ and AnxA1-/- BMDMs showed different kinetics of NF-B DNA-binding activity, IB- degradation and ERK1/2 phosphorylation. Consistent with this, measurement of MyD88-dependent or TRIF-regulated genes in AnxA1+/+ and AnxA1-/- BMDMs indicated a different time course of expression following stimulation with TLR3 (TRIF-dependent), TLR9 (MyD88-dependent) and TLR4 (TRIF and MyD88-dependent) ligands. Finally, AnxA1-/- mice showed an increased survival after challenge with poly (I:C), in contrast to increased lethality after injection of LPS, compared to AnxA1+/+ mice. These results suggest that endogenous AnxA1 influences mainly the MyD88-dependent pathway and to a lesser extent the TRIF-dependent pathway both in vitro and in vivo. In addition, this study provides future venues for the investigation of molecular mechanisms by which endogenous AnxA1 preferentially interferes with specific TLR signalling pathways.
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