The SYK side of TLR4: signalling mechanisms in response to LPS and minimally oxidized LDL

Abstract

Spleen tyrosine kinase (SYK) is the best known for its involvement in immune receptor signalling, mediated by binding of SYK tandem Src-homology 2 domains to tandem phosphotyrosine in immunoreceptor tyrosine-based activation motifs (ITAMs). ITAM adaptors or ITAM-containing receptor tails mediate signalling from B- and T-cell receptors, Fc receptors and many C-type lectins, including dectin-1. Recent data point to constitutive binding of SYK to the cytoplasmic domain of toll-like receptor-4 (TLR4). This SYK-TLR4 binding increases upon TLR4 dimerization and phosphorylation, and SYK plays a prominent role in TLR4 signalling in response to LPS in neutrophils and monocytes. SYK also plays an important role in TLR4-mediated macrophage responses to minimally oxidized low-density lipoprotein (mmLDL), which is a form of oxidized LDL relevant to development of human atherosclerosis. Interestingly, mmLDL-induced effects in macrophages, which occur via TLR4, are predominantly MyD88 independent. This unmasks the role of the SYK branch of TLR4 signalling, which mediates modest cytokine release via activation of AP-1 transcription and robust reactive oxygen species generation and cytoskeletal rearrangements. The latter results in extensive membrane ruffling and macropinocytosis, leading to lipoprotein uptake and foam cell formation, a hallmark of atherosclerotic lesions. Because inhibitors of SYK activity, such as fostamatinib, are in advanced clinical trials for rheumatoid arthritis and other autoimmune diseases, understanding the role of SYK in signalling via TLR4 is of immediate importance. This signalling pathway seems to be particularly important in TLR4 activation by host-derived, damage-associated molecular pattern ligands, such as mmLDL, relevant to development of atherosclerosis and other chronic inflammatory diseases.

Figure 1

Signalling mechanisms of TLR4/SYK-dependent macrophage activation. SYK is constitutively bound to the cytoplasmic domain of TLR4. Macrophage activation with mmLDL results in TLR4 dimerization and phosphorylation and increased SYK binding to TLR4 (note: SYK SH2 binding to phosphotyrosine in TLR4 is hypothetical; there are yet no specific data to support the SH2-type binding). Phosphorylated (activated) SYK activates Vav1, PLCγ and JNK. In turn, Vav1 activates a Ras-Raf-MEK1-ERK1/2 signalling cascade. ERK1/2-dependent phosphorylation of paxillin and GTP binding (activation) to Rac and Cdc42 lead to robust cytoskeletal rearrangements in macrophages, in part resulting in extensive membrane ruffling. PI3K- and Rho-assisted ruffle closing into macropinosomes captures the surrounding fluid. If this occurs in a LDL-rich medium, macrophages accumulate large quantities of lipid and become the so-called foam cells, a hallmark of atherosclerotic lesions. Downstream from PLCγ, activation of PKC, together with ERK1/2-mediated activation of Rac, contributes to Nox2-dependent generation of ROS. Phosphorylation of the transcription factors Fos and Jun by ERK1/2 and JNK, respectively, results in the assembly of an AP-1 transcription complex and stimulates expression of pro-inflammatory cytokines. Nox2-produced ROS contributes to cytokine expression.