The Functional Role of TLR9 in Human Platelets

Abstract 366

Human and murine platelets (PLTs) variably express toll-like receptors (TLRs), which link the innate and adaptive immune responses during infectious inflammation and atherosclerotic vascular disease. This is perhaps best exemplified by the observation that severe thrombocytopenia is associated with sepsis. While recent phenotypic and functional studies have addressed the roles of TLRs 2 and 4 in human/murine PLTs, which are primarily extracellular receptors, very little is known about the cellular localization, expression, and physiological significance of intracellular TLR9. Here we show that TLR9 localizes to unique granular compartments along the cell periphery (underlying the plasma membrane) in human PLTs. While thought to reside exclusively in the endosomes of monocytes, macrophages, and plasmacytoid dendritic cells, TLR9 expression is significantly enhanced in PLTs following incubation with thombin, ADP, PMA, CRP and type IV collagen, suggesting activation-mediated granule release. Surprisingly, TLR9 surface expression did not coincide with CD62P and CD61 expression levels upon PLT activation (which were not increased following type IV collagen incubation), and TLR9 was shown not to co-localize with known alpha-granule (fibrinogen, CD62P, PDGF-B, VEGF, CD42a, CD42b), dense granule (serotonin), endosomal (M6P, syntaxin-13), or lysosomal (LAMP-1) proteins. Immunogold electron microscopy revealed that TLR9 was expressed instead in a novel intracellular electron-dense granule (T-granule) that is mostly distributed along the plasma membrane. Incubation of resting PLTs with synthetic unmethylated Type C CpG dinucleotides (characteristic of bacterial/viral DNA) resulted in increased TLR9 surface expression, followed by increased Type C CpG sequestration and CD62P surface expression over 20 minutes. Interestingly, when TLR9 surface expression was specifically upregulated by pre-incubating PLTs with type IV collagen, subsequent incubation with Type C CpG resulted in considerably increased Type C CpG sequestration, CD62P surface expression, and PLT aggregation within 30 seconds of addition. These results imply that PLTs must be primed to express TLR9 on their surface prior to signal transduction through TLR9, and provide a mechanism for PLT regulation of the immune response to infection in human whole blood by sequestering bacterial/viral DNA and marking themselves for clearance. Taken together, this paper; (1) tracks TLR9 to a new intracellular compartment in PLTs, (2) describes a novel mechanism of TLR9 signaling, and (3) reveals a unique role for human PLTs as mediators of innate immunity at sites of vascular damage.