The discovery of mammalian TLRs and their critical role in detecting pathogen associated molecular patterns has greatly expanded our understanding of the innate immune response to infection. By contrast, the molecular mechanisms underlying sterile inflammatory conditions remain less well understood. Atherosclerosis and Alzheimer’s disease are characterized by a protracted sterile inflammatory response in which cells of the monocyte lineage accumulate prominently in plaques that underlie the pathology of these diseases14,17
. A common characteristic of these age-related diseases is the anomalous deposition of modified self-components in affected tissues: oxidized LDL and β-amyloid peptide accumulate in plaques of the artery wall and brain respectively, and are believed to fuel the recruitment and activation of mononuclear cells. Our work identifies a previously undescribed TLR heterodimer composed of TLR4 and TLR6 that senses these endogenous ligands and initiates innate immune activation of macrophages and microglia. Notably, this pathway is triggered through binding of atherogenic lipids and β-amyloid to a shared receptor, CD36, that provides the proximal signalling event required for TLR4-TLR6 activation. Together, this heterotrimeric signalling complex regulates the expression of proinflammatory mediators directly implicated in the deleterious effects of these modified self-components in vivo
, including chemokines and reactive oxygen and nitrogen species. Additionally, CD36-TLR4-TLR6 signaling in macrophages and microglia results in pro-IL1β transcription, priming these cells for inflammasome activation and IL-1β secretion. This work thus provides the molecular mechanism of how these endogenous ligands can act as TLR mimetics and identifies a common means for chronic mononuclear cell activation in two important age-related chronic inflammatory diseases.
Targeted deletion of the TLR adaptor MyD88 in a mouse model of atherosclerosis causes a dramatic reduction in atherosclerotic lesion size, attributable in part to decreased chemokine expression in the aorta15
. Upstream activation of TLR4 or TLR2 has been implicated in this MyD88-dependent inflammation of the artery wall16,23,24
, however the mechanism by which these microbial sensing pathways are triggered and the ligands responsible are not known. Notably, we find that oxLDL induced CD36-TLR4-TLR6 signaling in macrophages triggers a MyD88-dependent chemokine signature similar to that observed in vivo
, and additionally elicits expression of the TRIF-dependent chemokine RANTES. Consistent with these findings, expression of these chemokines is reduced in the aortae of hyperlipidemic Apoe−/−
mice lacking CD36, emphasizing the important role of this co-receptor in facilitating TLR4-TLR6 signaling. In addition to oxLDL-induced chemokine expression, MyD88-dependent signaling initiated by CD36-TLR4-TLR6 activates the macrophage respiratory burst and production of reactive oxygen species. This innate defense function of macrophages amplifies oxidative stress in the artery wall, thereby promoting the generation of oxidized lipoprotein ligands and chronic macrophage activation. Our identification of a CD36-TLR4-TLR6 complex that regulates these inflammatory responses to oxidized LDL thus provides mechanistic insight into how MyD88-dependent signaling is triggered during atherogenesis.
The inflammatory cytokine IL-1β is found prominently in atherosclerotic and Alzheimer’s disease plaques14,17
, and targeted inhibition of this cytokine reduces markers of disease18,25,26
. The secretion of IL-1β is a two step process requiring both transcriptional upregulation of pro-IL-1β via NF-κB and activation of a cytosolic multiprotein complex called the inflammasome that processes pro-IL-1β protein to its mature form27
. The recent identification of β-amyloid peptide18
and cholesterol crystals (E. Latz, personal communication) as activators of the NALP-3 inflammasome has emphasized the importance in understanding the regulation of pro-IL-1β expression in atherosclerosis and Alzheimer’s disease. Monocytes deficient in CD36 show impaired IL-1β expression in response to oxLDL and β-amyloid10,11
. Our work now identifies TLR4-TLR6 as critical components of the signaling pathway that primes macrophages and microglia for pro-IL-1β expression. These data identify signaling via this novel CD36-TLR4-TLR6 complex as the “first hit” in the induction of this highly regulated cytokine and we show that disruption of TLR4-TLR6 signaling in microglia abrogates IL-1β, nitric oxide, and reactive oxygen production and protects surrounding neurons from β-amyloid-induced death. TLR4 expression in the brain is increased in a mouse model of Alzheimer’s disease28
, and consistent with our model, targeted deletion of TLR4 blocks the upregulation of cerebral IL-1β amounts and microglial production of nitric oxide28,29
. Although the role of IL-1β and neuroinflammation in Alzheimer’s disease remains controversial30
, it is notable that a TLR4 polymorphism that attenuates receptor signalling (Asp299Gly) is associated with decreased risk of atherosclerosis, acute coronary events and Alzheimer’s disease31-33
Although we focus on two endogenous ligands involved in sterile inflammatory processes, this TLR heterodimer may also recognize as yet unidentified pathogen associated molecular patterns as several microbial ligands for CD36 have been identified4,5
. Moreover, this work has also revealed some generally applicable insights into TLR recognition. First, our work identifies a novel TLR heterodimer composed of TLR4-TLR6. We find that atherogenic lipids and β-amyloid initiate signalling that requires co-expression of both TLR4 and TLR6, and is independent of known components of the TLR4 homodimeric complex, such as MD-2 and CD14. This signaling leads to the expression of both MyD88- and TRIF-dependent genes, indicating that TLR4-TLR6 engages both of these adaptor pathways. Second, our data suggest a new model of TLR heterodimerization in which dimer assembly and activation is regulated by signaling initiated from a cell surface co-receptor, CD36. Co-immunoprecipitation assays showed a ligand-dependent physical association of TLR4 and TLR6, requiring expression of functional CD36. The early description of TLR2-TLR1 and TLR2-TLR6 heterodimers led to the speculation that heterodimerization might further diversify the TLR repertoire3
. However, it is notable that no other heterodimers with ascribed ligands have since been described. Our identification of a TLR4-TLR6 heterodimer was only made possible because of coexpression with the relevant coreceptor, CD36, which was required for its activation. While several TLR co-receptors, such as CD14 and the vitronectin receptor, have been suggested to concentrate microbial molecules and deliver them to pattern recognition complexes2,6
, our data indicate a more active role for CD36 in modulating TLR4-TLR6 signal transduction. The signals that induce TLR4-TLR6 heterodimerization occur from the C-terminus of CD36 within the cell, leading us to compare this to the “inside-out” signaling described for integrins34
. Our data are consistent with TLR4-TLR6 signaling being triggered by a proximal membrane event initiated by CD36 interaction with Lyn kinase. This model is supported by the following observations: first, mutation of tyrosine 463 of CD36 blocks its ability to induce TLR4-TLR6 heterodimerization and NF-κB activation; second, tyrosine 463 of CD36 regulates its interaction with Lyn kinase; third, chemical inhibitors of Lyn kinase block CD36-ligand induced TLR4-TLR6 association and activation of NF-κB and fourth, Lyn-deficient macrophages show impaired responses to β-amyloid, including the production of ROS12
. Because tyrosine phosphorylation of the TIR domain of TLR4 is required for LPS-signaling22
, we suggest that through recruitment of Lyn kinase, CD36 may similarly promote TLR4 and/or TLR6 phosphorylation and activation. This model highlights the importance of co-receptor function in triggering TLR4-TLR6 signaling and suggests that other TLR heterodimers may be identified if, as we demonstrate herein, the TLRs are co-expressed with their relevant co-receptors.