The Clathrin-Mediated Endocytic Pathway Participates in dsRNA-Induced IFN-β Production

Abstract

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.

Defined TLR3-specific adjuvant that induces NK and CTL activation without significant cytokine production in vivo

“…What we experienced in developing an RNA adjuvant was that: very little in vitro transcribed dsRNAs entered the human cells 22 , whereas poly(I:C) as well as CpG or control GpC phosphorothioate oligodeoxynucleotides (sODNs) reached the endosome in human myeloid DCs and epithelial cells. Poly(I:C) and sODNs appeared to share the uptake receptor 23 . To deliver dsRNA to endosome TLR3, we have connected sODN to 5 0 sense RNA and annealed it with antisense RNA (Fig.…”

Toll-like receptor 3 recognizes incomplete stem structures in single-stranded viral RNA

“…The features of the TLR3-recognizing PV-RNAs are consistent with our previous results that raftlin mediates poly(I:C) cellular uptake through interaction with the clathrin-AP-2 complex in human myeloid DCs and epithelial cells 35 . In addition, uptake of B/C-type CpG ODNs that share their uptake receptor with poly(I:C) was also mediated by raftlin 33,35 . Given that PV5-induced TLR3 activation in HEK293 cells was inhibited by pre-treatment with the B-type CpG ODN ( Supplementary Fig.…”

“…TLR3 activation by poly(I:C) requires clathrin-mediated internalization of extracellular poly(I:C) 33 . In human myeloid DCs and epithelial cells such as HeLa cells, the cytoplasmic raft protein, raftlin, induces poly(I:C) uptake through interaction with clathrin-AP-2 complex 34,35 .…”

Double-Stranded DNA Activates Glomerular Endothelial Cells and Enhances Albumin Permeability via a Toll-Like Receptor-Independent Cytosolic DNA Recognition Pathway

“…9 -11,24 Consistently, rhodaminelabeled dsDNA was not detectable in the intracellular cytosol of GEnCs, unless complexed with CL, as assessed by confocal microscopy ( Figure 2A). To identify the possible intracellular uptake mechanism, we stimulated GEnCs with B-DNA/CL in the presence or absence of chlorpromazine, known to specifically block clathrin-dependent endocytosis 25,26 ; methyl-␤ cyclodextrin, blocking caveolae-dependent endocytosis 27 ; and cytochalasin D, blocking phagocytosis. 26 The B-DNA-induced production of CXCL10/IP-10 was inhibited by chlorpromazine, but not by any of the other compounds ( Figure 2B), suggesting a role for clathrin in the endocytosis of complexed B-DNA.…”