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Department of Microbiology and Immunology, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo, Japan
TLR3 and cytoplasmic RIG-I-like receptor (RLR) recognize virus-derived dsRNA and induce type I IFN production in a distinct manner. Human TLR3 localizes to the endosomal compartments in myeloid dendritic cells (mDCs), while it localizes to both the cell surface and interior in fibroblasts and epithelial cells. TLR3 signaling arises in the intracellular compartment in both cell types and requires endosomal maturation. The mechanisms by which extracellular dsRNA is delivered to the TLR3-containing organelle remain largely unknown. Among various synthetic dsRNAs, poly(I:C) is preferentially internalized and activates TLR3 in mDCs. In vitro transcribed dsRNAs hardly induce IFN-β production in mDCs. In this study, we demonstrate that the clathrin-dependent endocytic pathway mediates cell entry of poly(I:C) to induce IFN-β gene transcription. Furthermore, poly(I:C)-induced IFN-β production is inhibited by pretreatment of cells with B- and C-type oligodeoxynucleotides (ODNs) but not with TLR7/8 ligands. The binding and internalization of B-type ODNs by mDCs was reduced in the presence of poly(I:C), suggesting that poly(I:C) shares the uptake receptor with B- and C-type ODNs. Hence, foreign dsRNA is recognized by differently categorized receptors, cytoplasmic RIG-I-like receptor, membrane-bound TLR3 and cell-surface RNA capture. The endocytic pathway is critical for dsRNA-induced TLR3-mediated cell activation.
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1 This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, and Culture, the Ministry of Health, Labor, and Welfare of Japan, and by the Uehara Memorial Foundation, the Mitsubishi Foundation, the NorthTec Foundation, and the Akiyama Foundation. Financial supports by the Sapporo Biocluster "Bio-S" the Knowledge Cluster Initiative of the MEXT, and the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases, MEXT are gratefully acknowledged.
2 K. Itoh and A. Watanabe contributed equally to this work.
3 Current address: Center for Integrated Medical Research, Keio University, Tokyo, Japan.
4 Address correspondence and reprint requests to Dr. Misako Matsumoto, Department of Microbiology and Immunology, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo, Japan. E-mail address: matumoto{at}pop.med.hokudai.ac.jp
5 Abbreviations used in this paper: DC, dendritic cell; mDC, myeloid DC; TICAM-1, Toll-IL-1 receptor-containing adaptor molecule-1; ODN, oligodeoxynucleotide; AcLDL, acetylated low density lipoprotein; CTXB, cholera toxin subunit B; MV, measles virus; CIAP, calf intestine alkaline phosphatase; iDC, immature DC; pDC, plasmacytoid DC; CpG-B, B-type CpG ODN; CpG-A, a-type CpG ODN; DPBS, Dulbecco’s PBS; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate; DAPI, 4',6-diamidino-2-phenylindole.
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