NF‐κB is activated from endosomal compartments in antiphospholipid antibodies‐treated human monocytes

Abstract

To cite this article: Brandt KJ, Fickentscher C, Boehlen F, Kruithof EKO, de Moerloose P. NF-jB is activated from endosomal compartments in antiphospholipid antibodies-treated human monocytes. J Thromb Haemost 2014; 12: 779-91.See also Groot PG, Urbanus RT. Cellular signaling by antiphospholipid antibodies. This issue, pp 773-5 and Gris JC. The hidden activism of the misnamed. This issue, pp 776-8.Summary. Background: The antiphospholipid antibody syndrome (APS) is an autoimmune disease associated with arterial or venous thrombosis and/or recurrent fetal loss and is caused by pathogenic antiphospholipid antibodies (aPLA). We recently demonstrated that Toll-like receptor 2 (TLR2) and CD14 contribute to monocyte activation of aPLA. Objective: To study the mechanisms of cell activation by aPLA, leading to pro-coagulant and pro-inflammatory responses. Methods and Results: For this study, we used purified antibodies from the plasmas of 10 different patients with APS and healthy donors. We demonstrate that aPLA, but not control IgG, co-localizes with TLR2 and TLR1 or TLR6 on human monocytes. Blocking antibodies to TLR2, TLR1 or TLR6, but not to TLR4, decreased TNF and tissue factor (TF) responses to aPLA. Pharmacological and siRNA approaches revealed the importance of the clathrin/dynamin-dependent endocytic pathway in cell activation by aPLA. In addition, soluble aPLA induced NF-jB activation, while bead-immobilized aPLA beads, which cannot be internalized, were unable to activate NF-jB. Internalization of aPLA in monocytes and NF-jB activation were dependent on the presence of CD14. Conclusion: We show that TLR2 and its co-receptors, TLR1 and TLR6, contribute to the pathogenicity of aPLA, that aPLA are internalized via clathrin-and CD14-dependent endocytosis and that endocytosis is required for NF-jB activation. Our results contribute to a better understanding of the APS and provide a possible therapeutic approach.

Update on the pathogenesis and treatment of the antiphospholipid syndrome

“…The role of TLR2 and TLR4 has been confirmed in a recent ex-vivo study [12]: an increased mRNA expression of these innate immunity receptors and a markedly raised phosphorylation level of interleukin-1 receptor-associated kinase 1 (IRAK-1) -a major mediator in TLR transduction pathwaywere observed in peripheral blood mononuclear cells (PBMCs) from APS patients. Consistently, TLR1, TLR2 and TLR6 have been shown to co-localize with aPL IgG; in addition, antibodies blocking TLR1, TLR2 and TLR6 decreased aPL-mediated upregulation of tumour necrosis factor (TNF) and tissue factor (TF) in human monocytes [13]. The lack of evidence in support of the TLR4 role emerged in this study might be ascribable to a cell-specific orchestra of receptors, co-receptors and accessory molecules deputed to aPL binding.…”

“…The cellular steps leading to NFkB translocation in monocytes have been recently clarified: aPLs engaged NF-kB via a clathrin-dependent endocytic pathway, a mechanism that requires CD14 (TLR4 co-receptor) and AnnexinA2 [13]. NF-kB might also provide a potential future pharmacological target: in BALB/c mice, treatment with a specific NF-kB inhibitor, Dehydroxymethylepoxyquinomicin, prevented aPL-mediated thrombus formation [16].…”

Cellular signaling by antiphospholipid antibodies

“…It is unlikely that every cell type has its own receptor for the autoantibodies because most of the proposed receptors are available on all cell types. Experiments performed by Brandt et al [7] and published in this issue of the Journal support an important role of TLR2 in the activation of monocytes by the autoantibodies. The significance of this publication is not that the authors have added another article with additional experiments showing the importance of their favorite receptor: TLR2.…”

“…Diffusion of second messengers is sufficient for information transfer over a short distance from the plasma membrane, whereas long-distance signaling needs protection against inactivation by cytosolic phosphatases [10]. Monocyte and endothelial cell activation by antiphospholipid antibodies results, among others, in the induction of tissue factor via the transcription factor NF-jB [7,11]. In this respect, transport of the antibody-receptor complex to the nucleus via endosomes could be the essential step in the induction of a prothrombotic cellular response.…”

The hidden activism of the misnamed

“…This issue of the Journal of Thrombosis and Haemostasis contains a very elegant and accurate article by Brandt et al from the P. de Moerloose laboratory, which provides ex vivo experimental results demonstrating that nuclear factor-jB (NF-jB) activation in human monocytes triggered by human aPLAbs is regulated via clathrin/dynamin-dependent and CD14-dependent endocytosis, and that TLR2 and its coreceptors, TLR1 and TLR6, contribute to the TNF-a and tissue factor cellular response to aPLAbs [11]. These findings reinforce the growing evidence for a crucial role for internalization in the pathogenicity of aPLAbs [5][6][7][8][9][10], and point to the importance of endosome signaling.…”