Abstract 5674: Toll-like Receptor-2 and -4 Signaling Regulates Inflammation and Matrix Degradation in the Human Atherosclerotic Plaque

Abstract

Background The current consensus view of the mechanisms leading to plaque rupture has assigned a central role to inflammation and matrix degradation. Toll-like receptors (TLRs), key players in innate immunity, are upregulated in atherosclerotic lesions but their functional role in matrix degradation in human atherosclerosis is unknown. Methods We explored the effects of blocking TLR-2, -4 and the downstream signaling adaptors myeloid differentiation primary response gene 88 (MyD88, shared by most TLRs and Trif-related adaptor molecule (TRAM; used by TLR-4) in an in vitro model of human atherosclerosis. Carotid endarterectomies were obtained from patients with symptomatic carotid disease. Cells were isolated via enzymatic tissue dissociation in order to obtain a single cell suspension of mixed cell types. Cells were cultured in the presence or absence of TLR signaling blockers immediately after isolation. Phenotyping by flow cytometry showed that macrophages are the most abundant cells in the mixture, being approximately double the proportions of smooth muscle cells and lymphocytes. Results A dominant-negative form of MyD88 (MyD88DN) decreased the production of MCP-1/CCL2 (P=.015), IL-8/CXCL8 (P =.002), IL-6 (P =.001), MMP-1) (P =.016), and MMP-3 (P =.0001), as well as nuclear factor kappaB (NFkB) activation (P= .0001) in atheroma cell cultures. TLR-2 neutralizing antibodies inhibited NFkB activation (P = .025), and significantly reduced MCP-1/CCL2 (P=.0001), IL-8/CXCL8 (P=.007), IL-6 (P=.0001), and MMP-1, -2, -3, -9 production (P=.0001). In contrast, a dominant negative form of the adaptor TRAM (TRAM DN ), which is used by TLR-4, significantly reduced NF κ B activity, MMP-3 ( P =.049) and MMP-1, but had no effect on gelatinases MMP-2 and -9 or inflammatory mediators. Conclusions Our data indicates that TLR-2 signaling through MyD88 plays a predominant role in inflammation and matrix degradation in human atherosclerosis. However, we also uncovered a role for TLR-4/TRAM-dependent signaling in MMP production, suggesting that the MyD88-independent pathway may play a role in mechanisms related to matrix degradation and plaque vulnerability. TLR blockade may represent a therapeutic strategy for atherosclerosis and its complications.