TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-beta

Abstract

Toll-like receptor 4 (TLR4) induces two distinct signaling pathways controlled by the TIRAP-MyD88 and TRAM-TRIF pairs of adaptor proteins, which elicit the production of proinflammatory cytokines and type I interferons, respectively. How TLR4 coordinates the activation of these two pathways is unknown. Here we show that TLR4 activated these two signaling pathways sequentially in a process organized around endocytosis of the TLR4 complex. We propose that TLR4 first induces TIRAP-MyD88 signaling at the plasma membrane and is then endocytosed and activates TRAM-TRIF signaling from early endosomes. Our data emphasize a unifying theme in innate immune recognition whereby all type I interferon-inducing receptors signal from an intracellular location.

Figure 1

Inhibition of TLR4 endocytosis selectively disrupts TRAM-TRIF signaling. (a) Fluorescence microscopy of macrophages transfected with plasmid encoding a TLR4 construct with the ectodomain of the receptor replaced with the ectodomain of CD4 (CD4-TLR4) or Rab5 tagged with red fluorescent protein (RFP-Rab5). Outlined areas are enlarged in bottom left corners. Scale bars, 5 μm. Data are representative of at least three independent experiments with over 500 cells per condition, in which over 95% of the cells had similar staining patterns. (b) Flow cytometry of TLR4 surface staining on wild-type or TLR4-deficient (TLR4-KO) macrophages left untreated (left) or treated with dynasore (right) and left unstimulated (Untreated) or stimulated with LPS (100 ng/ml), assessed with the TLR4-specific antibody Sa15-21. PE, phycoerythrin. Data are representative of two independent experiments. (c) Immunoblot analysis of macrophages treated for 0, 20 or 40 min with LPS (100 ng/ml) and either dimethyl sulfoxide (DMSO) or 80 μM dynasore and probed for phosphorylated (p-) or total IRF3 or p38. Data are representative of two independent experiments. (d,e) RT-PCR analysis (d) and ELISA (e) of LPS-induced gene expression in macrophages left untreated or treated with dynasore. Data are representative of three independent experiments.

Figure 2

Distinct subcellular distribution of TRAM and TIRAP. (a,b) Fluorescence microscopy of macrophages transfected with plasmids encoding GFP-TRAM (a) or GFP-TIRAP (b) and RFP-Rab5. (c,d) Fluorescence microscopy of macrophages transfected with plasmid encoding GFP-TRAM (c) or CD4-TLR4 (d) and treated for 30 min with dextran labeled with Texas red. Outlined areas are enlarged in bottom left corners. Scale bars, 5 μm. Data are representative of at least three independent experiments with over 500 cells per condition, in which over 95% of the cells had similar staining patterns.

Figure 3

A bipartite localization motif regulates the localization of TRAM. (a–d) Fluorescence microscopy of macrophages transfected with plasmids encoding chimeric proteins consisting of full-length TRAM (a) or various amino acids (in parentheses) of TRAM (b–d) fused in-frame to GFP. Outlined areas are enlarged in bottom left corners. Scale bars, 5 μm. Data are representative of at least three independent experiments with over 500 cells per condition, in which over 95% of the cells had similar staining patterns (unless stated otherwise elsewhere). (e) Isoelectric points of sequential 20–amino acid segments of TRAM. Data are representative of experiments done two times. (f) Primary structure of mouse TRAM. Below, bipartite localization motif (amino acids 1–20), with the myristoylation motif (red letters) followed by the polybasic motif (substituted amino acids are underlined). (g) Amino acid alignment of human proteins identified as containing a bipartite localization motif, with putative myristoylation motifs (underlined residues) followed by the polybasic motif, and with basic and aromatic residues indicated by red lettering.

Figure 4

TLR4 can induce the production of type I interferon from early endosomes. (a,b) Fluorescence microscopy of macrophages transfected with plasmids encoding a GFP-tagged TRAM construct with substitutions in the polybasic domain (TRAM3X) and RFP-Rab5 (a) or with a myristoylation-deficient TRAM mutant (TRAMG2A; b). Outlined areas are enlarged in the bottom left and top right corners (a). Scale bars, 5 μm. Data are representative of at least three independent experiments with over 500 cells per condition, in which over 95% of the cells had similar staining patterns. (c) ‘PIP strips’ of various lipids (identified at right) overlaid with GST-tagged wild-type TRAM (left) or TRAM with substitutions in the polybasic domain (middle); lipid binding was identified by standard protein-protein immunoblot techniques. LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine 1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine. Data are representative of two independent experiments. (d) ELISA of IL-6 and RANTES in TRAM-deficient macrophages transduced with retroviral vectors encoding GFP-tagged TRAM mutants (horizontal axes) and challenged with 1, 10 or 100 ng/ml (key) of LPS. The transduction efficiency of each resulting macrophage population was determined to be 30–40% by flow cytometry and fluorescence microscopy. Data are representative of three independent experiments.

Figure 5

The subcellular localization of TRAF3 dictates the ability of TLRs to produce type I interferon. (a,b) Fluorescence microscopy of macrophages transfected with plasmids encoding hemagglutinin-tagged constructs of wild-type TRAF3 (a) or a chimera of the pleckstrin homology domain of PLC-δ1 and TRAF3 (PLC-TRAF3; b). Scale bars, 5 μm. Data are representative of at least three independent experiments with over 500 cells per condition, in which over 95% of the cells had similar staining patterns. (c,d) Luciferase production by 293T cells 24 h after transfection with expression vectors encoding GFP only, wild-type TRAF3 or the chimera of PLC-δ1 and TRAF3, plus a luciferase reporter dependent on ISRE (c) or NF-κB (d). Values are relative to those of the GFP vector, set as 1. Data are representative of three independent experiments. (e) RT-PCR analysis of the expression of genes encoding IFN-β, IL-1β or HPRT by RAW267 macrophage-like cells stably expressing either wild-type TRAF3 (left) or the chimera of PLC-δ1 and TRAF3 (right), left untreated (None) or treated for 4 h with the TLR4 agonist LPS (100 ng/ml; TLR4) or the TLR2 agonist PAM₃CSK (1 μg/ml; TLR2). Data are representative of three independent experiments done in duplicate.