Pivotal Role of ADP-ribosylation Factor 6 in Toll-like Receptor 9-mediated Immune Signaling

Jing-Yiing Wu,Cheng-Chin Kuo

doi:10.1074/jbc.m111.295113

Abstract

CpG oligodeoxynucleotide (CpG ODN) cellular uptake into endosomes, the rate-limiting step of Toll-like receptor 9 (TLR9) signaling, is critical in eliciting innate immune responses. ADP-ribosylation factor 6 (ARF6) is a member of the Ras superfamily, which is critical to a wide variety of cellular events including endocytosis. Here, we found that inhibition of ARF6 by dominant mutants and siRNA impaired CpG ODN-mediated responses, whereas cells expressing the constitutively active ARF6 mutant enhanced CpG ODN-induced cytokine production. Inhibition of ARF6 impaired TLR9 trafficking into endolysosomes, thereby inhibiting proceed functional cleavage of TLR9. Additional studies showed that CpG ODN uptake was increased in ARF6-activated cells but impaired in ARF6-defective cells. Furthermore, cells pretreated with CpG ODN but not GpC 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) was required for downstream ARF6 regulation of CpG ODN uptake. Together, our findings demonstrate that a novel class III PI3K-ARF6 axis pathway mediates TLR9 signaling by regulating the cellular uptake of CpG ODN.

Highlights

ADP-ribosylation factor 6 is critical to a wide variety of cellular events, including vesicular trafficking and endocytosis
Our findings demonstrate that a novel class III phosphatidylinositol 3-kinases (PI3K)-ADPribosylation factor 6 (ARF6) axis pathway mediates Toll-like receptor 9 (TLR9) signaling by regulating the cellular uptake of CpG oligodeoxynucleotide (CpG ODN)
ARF6 Is Associated with CpG ODN/TLR9 Signaling—To evaluate the influence of ARF6 on CpG ODN/TLR9-mediated responses, we first examined whether ARF6 is involved in CpG ODN-stimulated NF-␬B activation

Summary

Background

ADP-ribosylation factor 6 is critical to a wide variety of cellular events, including vesicular trafficking and endocytosis. Conclusion: A novel class III PI3K-ARF6 axis pathway mediates TLR9 signaling by regulating the cellular uptake of CpG ODN. Our findings demonstrate that a novel class III PI3K-ARF6 axis pathway mediates TLR9 signaling by regulating the cellular uptake of CpG ODN. ARFs have important functions in cellular processes, studies demonstrating the precise role of each ARF in cellular biological responses have been limited because of a lack of specific inhibitors to individual ARFs. Recent reports have shown that the ARF-inhibitor brefeldin A impaired CpG ODN-induced NF-␬B activation by blocking TLR9 trafficking through Golgi but not by inhibiting cellular CpG ODN uptake [33], suggesting that brefeldin A-sensitive ARFs plays a critical role in CpG ODNmediated responses. Our findings indicate that ARF6 is involved in CpG ODN/TLR9mediated responses by regulating cellular CpG ODN uptake as well as the proteolytic processing of TLR9

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION