P146 TLR2-ligands induce NF-κB activation exclusively from endosomal compartments

Abstract

Introduction Antiphospholipid antibody syndrome (APS) is an auto-immune disease associated with arterial or venous thrombosis and/or recurrent fetal loss and is caused by pathogenic antiphospholipid antibodies (aPLA). We recently demonstrated TLR2 as a crucial factor in recognizing aPLA by monocytes. While TLR4 is known to transmit signals from both the plasma membrane and endosomal compartments, less is known about the function of endosomal trafficking on TLR2 signaling. Methods Primary human monocytes and HEK-Blue2™ cells were treated with TLR2 ligands, i.e. LTA and Pam 3 CSK 4 , or with aPLA, in the presence of endocytosis inhibitors. Expression of TNF and tissue factor (TF) as well as NF- κ B activation were assessed. In order to evaluate the importance of receptor internalization, clathrin expression was reduced by siRNA and NF- κ B activation assessed after treatment with TLR2 ligands and aPLA. Endocytosis of biotinylated LTA and Pam 3 CSK 4 or aPLA was evaluated by flow cytometry. Results We demonstrate that blocking antibodies to TLR2, TLR1 or TLR6, as well as antibodies to the TLR co-receptor CD14, significantly decreased TNF and TF responses to LTA, Pam 3 CSK 4 and aPLA. TLR2 ligands internalization is observed in primary human monocytes and HEK-Blue2™ cells and we show that NF-kB-dependent cell activation is associated to ligand internalization. Pharmacological blockade and siRNA approaches revealed the importance of the clathrin/dynamin-dependent endocytic pathway in ligand-induced NF- κ B activation. In addition, LTA-, Pam 3 CSK 4 - and aPLA-beads, which could not be internalized, are unable to activate NF- κ B while soluble ligands and LPS-beads (a TLR4 ligand) are competent to trigger NF- κ B activation. Furthermore, in a similar manner as TLR4, we demonstrate that TLR2 ligands uptake is regulate by CD14. Unlike LPS, TLR2 ligands do not induce phosphorylation of IRF-3 or IFN β expression. Finally, CD14-dependent endocytosis is shown to regulate the activation of NF-kB induced by LTA, Pam 3 CSK 4 and aPLA in HEK-Blue2™ cells or primary human monocytes. Conclusion Our results reveal that TLR2-ligand complexes are internalized, via clathrin- and CD14-dependent endocytosis into primary human monocytes and HEK-Blue2™ cells; endocytosis of these complexes is required for NF- κ B activation. The evidence that TLR1 and TLR6 play a part in the pathogenicity of aPLA contributes to a better understanding of the antiphospholipid syndrome and provides insight for the development of novel therapies. In conclusion, our study reveals a new important aspect of the regulation of TLR2-dependent signaling. Although present at the cell surface, TLR2-ligands induce NF- κ B activation exclusively from endosomal compartment.