Size-Dependent Attenuation of TLR9

Gold nanoparticles (GNPs), which are generally thought to be bio-inert and non-cytotoxic, have become one of the most ideal nano-materials for medical applications. Once engulfed by phagocytes, the immunological effects of GNPs are still of concern and require detailed investigation. Therefore, this study explored the immunological significance of GNPs on TLR-mediated innate immunity in murine macrophages. GNP causes specific inhibition of TLR9 (CpG oligodeoxynucleotides; CpG-ODNs) signal in macrophages. The impaired CpG-ODN–induced TNF-a production is GNP concentration-and size-dependent in murine Raw264.7 cells: a GNP of 4 nm in size is more potent than a GNP of 11, 19, 35, or 45 nm in size. Consistent with cytokine inhibition, the CpG-ODN– induced phosphorylation of NF-kB and JNK as well as NF-kB activation are suppressed by GNPs. GNPs accumulate in lysosomes after phagocytosis and also increase TLR9-associated lysosomal cathepsin expression and activities, but this is irrelevant to TLR9 inhibition by GNPs in our studies. In addition, GNPs affected TLR9 translocation in response to CpG-ODNs and to phagosomes. Further exploring how GNPs inhibited TLR9 function, we found that GNPs could bind to high-mobility group box-1 (which is involved in the regulation of TLR9 signaling) inside the lysosomes. The current studies demonstrate that size-dependent inhibition of TLR9 function by GNP may be attributed to its binding to high-mobility group box-1. M acrophages are professional phagocytic cells that are resident in lymphoid and nonlymphoid tissues and play an important role in the body's defenses (1). Macro-phages express a broad range of pathogen recognition receptors (PRRs) to recognize and internalize microorganisms, induce cy-tokine production, process Ag, then present Ag to T cells, and consequently induce adaptive immunity (2–4). TLRs are one of the most important PRRs to recognize distinct microbial components and directly activate APCs (2–4). Eleven TLRs have been identified in humans and 13 in mice and can be divided into two kinds: TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11, which are expressed on the cell surface to sense various foreign components from outside, such as TLR4 for LPS and TLR2 for lipoteichoic acid (LTA) (5); and TLR3, TLR7/8, TLR9, and TLR10, which are localized in endolysosomes and recognize internalized components from pathogens, including dsRNA for TLR3, single-stranded RNA for TLR7, and unmethylated CpG oligodeoxy-nucleotides (CpG-ODNs) for TLR9 (5). Upon stimulation, TLRs require additional proteins to be activated by their respective ligands such as high-mobility group box-1 (HMGB-1), the nucleic acid sensor. Recently, HMGB-1 has been …