Abstract
Abstract Toll-like receptor 9 (TLR9) is responsible for proinflammatory cytokine production via recognizing pathogenic unmethylated CpG DNA. In the process, CpG oligodeoxynucleotides (CpG ODN) uptake into endosomes and TLR9 trafficking from endoplasmic reticulum (ER) to endosomes are two critical steps in eliciting innate immune responses. ADP-ribosylation factors (ARFs) are members of the Ras superfamily of guanine nucleotide-binding proteins, which are involved in mediating a wide variety of cellular events including endocytosis and vesicle trafficking. Here, we found that inhibition of ARF3 or ARF6 by dominant mutants and siRNA impaired CpG ODN-mediated responses, whereas constitutive active ARF3 or ARF6 mutant enhanced these responses. Additional studies showed that CpG ODN uptake was increased in ARF6-activated cells but impaired in ARF6-defective cells. Cells pre-treated with CpG ODN had increased CpG ODN uptake due to CpG ODN-induced ARF6 activity. Further studies with ARF6-defective and ARF6-activated cells demonstrated that class III phosphatidylinositol 3-kinases (PI3K) were required for downstream ARF6 regulation of CpG ODN uptake. These results propose that a novel class III PI3K-ARF6 axis pathway mediates TLR9 signaling by regulating the cellular uptake of CpG ODN. In contrast to the involvement of ARF6 in CpG ODN/TLR9-mediated responses, ARF3 activity is essential for TLR9 trafficking into endolysosomes without affecting CpG ODN uptake, thereby regulating the formation of functional cleaved TLR9. In addition, experiment with immnuoprecipitation indicated that activation of ARF3 interacted with the TLR9 and the carrier protein, UNC93B1. Thus, the results demonstrate that ARF3 mediates CpG ODN-driven responses by regulating TLR9 trafficking from ER into endolysosomes. Collectively, class I ARF3 and class III ARF6 play distinct roles in CpG ODN/TLR9-mediated responses. ARF6 directly participates in CpG ODN internalization, while ARF3 drives TLR9 trafficking to activate CpG ODN/TLR9-induced immune responses.