Abstract 189: The Role of Spleen Tyrosine Kinase, Syk, in CD36-Toll Like Receptor Cooperative Signaling in Atherosclerosis

Abstract

Atherosclerosis is characterized by chronic sterile inflammation of the artery wall in which cells of the monocyte lineage accumulate in response to the deposition of low density lipoprotein (LDL). We previously established that recognition of oxidized LDL (oxLDL) by CD36 triggers assembly of a novel Toll-like receptor heterodimer composed of TLR4 and TLR6. Here we set out to understand the molecular mechanisms of CD36/TLR4/TLR6 activation and establish how it triggers downstream signals that lead to the expression of the pro-inflammatory mediators that have been directly implicated in the deleterious effects of oxLDL and atherosclerosis progression. By confocal microscopy we demonstrate that oxLDL induces CD36, TLR4 and TLR6 co-localization in intracellular compartments, but not on the cell surface of macrophages. Notably, inhibition of oxLDL endocytosis (with Dynasore) or lysosomal maturation (with Bafilomycin A or NH4Cl) blocks CD36-TLR4-TLR6 complex formation and oxLDL-induced cytokine responses in macrophages. These data indicate that both ligand internalization and lysosomal acidification are required for assembly of a functional CD36/TLR4/TLR6 signaling complex. Notably, CD36 contains a hemi-ITIM motif in the C-terminus that is reported to interact with the spleen tyrosine kinase Syk through its SH2 domain. As Syk has recently been implicated in the trafficking of CD14 and TLR4 to the endosome in response to LPS, we investigated the role of this kinase in CD36/TLR4/TLR6 signaling. We find that Syk is required for CD36 internalization and TLR4/TLR6 heterodimerization. Using a pharmacological inhibitor, we show that inhibition of Syk activity blocks oxLDL-induced TLR4-TLR6 co-precipitation and abrogates macrophage expression of both MyD88- (IL-1b, CXCL1) and TRIF-dependent (CCL5) cytokines/chemokines. Together, our data are consistent with a key role for Syk in the trafficking of CD36 and oxLDL to the lysosome, where it coordinates the assembly of a functional TLR4-TLR6 heterodimer to initiate signaling. This model highlights the importance of CD36 as a co-receptor that orchestrates TLR4-TLR6 trafficking and assembly to initiate the detrimental inflammatory responses that promote the progression of atherosclerosis.