Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10

Abstract

Toll-like receptor (TLR)-10 remains an orphan receptor without well-characterized ligands or functions. Here, we reveal that TLR10 is predominantly localized to endosomes and binds dsRNA in vitro at endosomal pH, suggesting that dsRNA is a ligand of TLR10. Recognition of dsRNA by TLR10 activates recruitment of myeloid differentiation primary response gene 88 for signal transduction and suppression of interferon regulatory factor-7 dependent type I IFN production. We also demonstrate crosstalk between TLR10 and TLR3, as they compete with each other for dsRNA binding. Our results suggest for the first time that dsRNA is a ligand for TLR10 and propose novel dual functions of TLR10 in regulating IFN signaling: first, recognition of dsRNA as a nucleotide-sensing receptor and second, sequestration of dsRNA from TLR3 to inhibit TLR3 signaling in response to dsRNA stimulation.

Keywords: IFN; TLR10; dsRNA; interferon regulatory factor; ligand sequestration; myeloid differentiation primary response gene 88; nucleotide-sensing receptor; toll-like receptor.

Figure 1

Sub-cellular localization of toll-like receptor (TLR)-10 in THP-1 cells. (A) Confocal micrograph of resting wild-type THP-1 cells stained for TLR10 (red), organelle markers (green), and nuclei (blue) stained with DNA-binding dye 4′,6-Diamidin-2-phenylindol (DAPI). Co-localization of TLR10 and respective organelle marker (yellow). Scale bars, 5 µm. (B) Relative expression of TLR10 in different organelles. The expression level of TLR10 in different organelles was compared with those expressed in the Golgi apparatus (GIANTIN⁺). Signals of more than 30 randomly picked cells from three independent experiments were computed using ImageJ with the co-localization plug-in. Data are presented as mean with SEM.

Figure 2

Toll-like receptor (TLR)-10 regulates dsRNA-mediated type I IFN expression. (A) Basal expression of TLR10 in unstimulated wild-type (WT), TLR10 overexpressed (OE), and knockdown (KD) THP-1 cells. (B) Expression of IFNβ in WT, TLR10 OE, and TLR10 KD THP-1 cells upon challenge by 10 µg/ml poly(I:C) at 4 h post-stimulation. Intracellular: poly(I:C) transfected by cationic lipid delivery; surface: poly(I:C) added to cell culture medium directly. (C,D) Expression of IFNβ in WT, TLR10 OE, and KD THP-1 cells at different time points (C) and concentrations (D) upon poly(I:C) stimulation. (E) Basal expression of TLR3 and RIG-I compared with TLR10 in WT, TLR10 OE, and KD THP-1 cells. (F) Expression of IFNβ in WT, TLR10 OE, and KD THP-1 cells upon stimulation by 2′3 ′-cGAMP, 5′pppdsRNA synthesized in vitro (dsRNA WT) or its variant (dsRNA M5). Data are mean with SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant.

Figure 3

Toll-like receptor (TLR)-10 binds dsRNA in vitro. (A,B) Cell lysates of THP-1 cells were incubated with biotin-poly(I:C) (5 ng/ml) at (A) pH 5.5 or (B) pH 7.4, with or without addition of competitive unlabeled poly(I:C) (50 ng/ml) for 1 h. Complexes were pulled-down using streptavidin beads and analyzed by Western blotting using anti-TLR10 antibody. Data shown are representative of at least three independent experiments.

Figure 4

Interaction between toll-like receptor (TLR)-10 and poly(I:C) was observed by fluorescent resonance energy transfer (FRET) after acceptor photo bleaching. (A) Confocal micrograph of THP-1 stained TLR10 (red), organelle marker (green), and transfected fluorophore-conjugated poly(I:C) (cyan). Arrows indicate the co-localization of TLR10 and poly(I:C) in corresponding endosomal compartments (white). (B) Fluorophore-conjugated poly(I:C) were transfected to THP-1 cells. Channels corresponding to TLR10 (red) and poly(I:C) (green). Gradual photo-bleaching of the acceptor by 561 nm laser followed by signal capture from both channels starts after the fifth frame. Merged images depicting co-localization of TLR10 and poly(I:C) with the region of interest for acceptor and donor images before and after bleaching circled. Quantification of FRET for the circled region is displayed graphically as fluorescence intensity over frame. Scale bars, 5 µm. Data are mean with SEM of eight individual samples.

Figure 5

Myeloid differentiation primary response gene 88 (MyD88) is the adaptor protein for toll-like receptor (TLR)-10 signaling following stimulation by dsRNA. (A) Alignment of toll/interleukin-1 receptor domain sequences of human TLRs. Sequence logo (top) represents the conserved motif identified by MEME. Sequence in black box is the BB-loop sequence in TLR10. The alanine/proline residues highlighted in green determine the adaptor protein bound by TLRs. All human TLRs, except TLR3, have proline in the BB-loop. (B) Confocal micrograph of THP-1 cells stimulated with 10 µg/ml poly(I:C) stained at different time points for TLR10 (red), MyD88 (green), with nuclei stained with DNA-binding dye 4′,6-Diamidin-2-phenylindol (DAPI) (blue). Unstimulated (US) cells were included as a control. Arrows indicate the co-localization of TLR10 and MyD88 (yellow). Inset (at 10 min post-challenge) is an enlargement of the white square box. Scale bars, 5 µm. (C) TLR10 interacts with MyD88 upon poly(I:C) stimulation. Cell lysates of THP-1 cells stimulated with 10 µg/ml poly(I:C) at different time points were immunoprecipitated using anti-TLR10 antibody and then analyzed by Western blotting using anti-MyD88 or anti-TRIF antibodies. β-ACTIN was the input control. Data shown are representative of at least two independent experiments.

Figure 6

Toll-like receptor (TLR)-10 stimulated by dsRNA regulates type I IFN responses through phosphorylation of interferon regulatory factor (IRF)-7. (A,B) Phosphorylation level of (A) IRF7 and (B) IRF3 in wild-type (WT) and TLR10 overexpressed (OE) THP-1 cells upon stimulation by 10 µg/ml poly(I:C) at different time points post-challenge was analyzed by Western blotting with anti-phospho-IRF7 (Ser477) and anti-phospho-IRF3 (Ser396) antibodies, respectively. A representative blot (left) and mean (with SEM, right) from three independent experiments are shown. (C) Augmented type I IFN signaling in TLR10 knockdown THP-1 cells through an IRF-inducible luciferase reporter. Luciferase activity measured in THP-1 reporter cells upon transfection with 10 µg/ml poly(I:C) in TLR10 small interfering RNA (siRNA) (si-TLR10) or a non-targeting control siRNA (NC) treated THP-1 cells. Data are mean with SEM from at least three independent experiments. *p < 0.05, **p < 0.01.

Figure 7

Crosstalk between toll-like receptor (TLR)-10 and TLR3. (A) THP-1 cells were challenged with fluorophore-conjugated poly(I:C) (cyan) and stained for TLR10 (red) and TLR3 (green). Arrow indicates co-localization of TLR10, TLR3, and poly(I:C) (white). Scale bars, 5 µm. (B,C) The ectodomains (ECD) of TLR10 or TLR3 recombinant proteins were incubated with biotin-conjugated poly(I:C) alone or together for 1 h at pH 5.5. The biotin-poly(I:C) bound complexes were pulled-down by streptavidin beads and analyzed by immunoblotting using anti-TLR10 (B) or anti-TLR3 (C) antibodies. (D) Expression of IFNβ in wild-type (WT), TLR3 knockdown (KD), TLR10 KD, and TLR3/10 double KD THP-1 cells upon poly(I:C) challenge. (E,F) Expression of TLR3 (E) and sterile alpha and TIR motif-containing protein 1 (F) in WT and TLR10 overexpressed (OE) cells in response to poly(I:C) challenge (10 µg/ml, 6 h post-stimulation). The mRNA expression was quantitated using RT-qPCR and denoted as fold change compared with corresponding unstimulated cells. Data are mean with SEM from three independent experiments. *p < 0.05, ***p < 0.001.

Figure 8

Proposed model for the novel dual functions of toll-like receptor (TLR)-10 in regulating IFN signaling. (A) Sensing of dsRNA by TLR10 in endosomes. If TLR10 forms homodimer/heterodimer or co-factors needed for signaling is not clear. (B) Activation of TLR10 recruits myeloid differentiation primary response gene 88 (MyD88), subsequently leading to decrease in interferon regulatory factor (IRF)-7 phosphorylation and suppress IFNβ expression. (C) Ligand sequestration: TLR10 competes with TLR3 for dsRNA, attenuates TLR3 mediated IFNβ expression. (D) Signaling of TLR10 negatively regulates TLR3 expression and promotes expression of negative regulator of the signaling, sterile alpha and TIR motif-containing protein 1 (SARM1) to further suppress TLR3 signaling and the subsequent IFNβ expression.