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Individual members of the Toll-like receptor (TLR) family have different subcellular locations but most of these receptors activate a common signalling pathway through the adaptor protein myeloid differentia- tion primary response protein 88 (MYD88). It has been unclear how the myddosome (a protein complex containing MYD88 and other signalling molecules) can be activated from multiple organelles. Now, Kagan and colleagues show that the sorting adaptor TIRAP (TIR domain-containing adaptor protein; also known as MAL), through the promiscuity of its lipid-binding domain, triggers the assembly of the myddosome at both the plasma membrane and endosomes. Previous studies have shown that TIRAP functions as a sorting adap- tor that links TLRs on the plasma membrane, such as TLR4, and MYD88 through its interaction with phosphatidylinositol-4,5 bisphos- phate (PtdIns(4,5)P 2 ) in the plasma membrane. But, TIRAP was previ- ously excluded as a sorting adaptor for endosomal TLRs, such as TLR9. However, the requirement for TIRAP at the plasma membrane can be bypassed by high concentrations of ligands. Primary macrophages are highly phagocytic and syn- thetic nucleic acid ligands, such as unmethylated CpG-containing DNA (CpG DNA), are nuclease resistant, which led the authors to speculate that a role for TIRAP in endosomal TLR signalling could have been masked by the accumulation of these ligands at high concentrations in endosomes. To determine whether TIRAP has a role in signalling from endosomes, wild-type and TIRAP-deficient bone marrow-derived macrophages were stimulated with CpG DNA or herpes simplex virus (HSV) substrains that only activate endosomal TLR9. The authors found that HSV-induced, but not CpG DNA-induced, cytokine pro- duction was defective in the absence of TIRAP. Immortalized macrophages are less phagocytic than primary mac- rophages, and, therefore, CpG DNA may not build up to high concentra- tions in these cells. Indeed, TIRAP- deficient immortalized macrophages were completely unresponsive to CpG DNA stimulation. Furthermore, interferon-α (IFNα) production, but not interleukin-12p40 (IL-12p40), was dependent on TIRAP in response to HSV or influenza virus infection of plasmacytoid dendritic cells (pDCs), which exclusively use endosomal TLRs. Of note, cytokine and IFN production occurs from distinct endosomes, with IFN production occurring from endosomes rich in 3ʹ phosphoinositides, such as PtdIns3P. These data indicate that TIRAP is required for signalling by TLRs in 3ʹ phosphoinositide-rich endosomes. Further studies showed that TIRAP promotes the assembly and is a component of myddosomes at both the plasma membrane and endosomes. In addition, confocal microscopy showed that TIRAP can localize to both PtdIns(4,5)P 2 -rich areas of the plasma membrane and to endosomes independently of PtdIns(4,5)P 2 . To investigate this further, the authors gener- ated immortalized macrophage cell lines in which a modified TIRAP bound either exclusively to PtdIns(4,5)P 2 on the plasma membrane or to PtdIns3P on endo- somal membranes. Macrophages with PtdIns(4,5)P 2 -localized TIRAP were responsive to the TLR4 ligand lipopolysaccharide (LPS) but not the TLR9 ligand CpG DNA, whereas macrophages with PtdIns3P-localized TIRAP responded robustly to CpG DNA but not to LPS. Together, these data show that promiscuous lipid binding by TIRAP allows this sorting adaptor to promote TLR signalling from PtdIns(4,5)P 2 -rich areas of the plasma membrane and from PtdIns3P-rich endosomes. However, endosomal MYD88-dependent IL-12p40 produc- tion seems to occur independently of TIRAP, suggesting the existence of additional sorting adaptors that can also mediate compartment-specific TLR responses. Olive Leavy INNATE IMMUNE SIGNALLING TIRAP diversifies the sites of TLR signalling ORIGINAL RESEARCH PAPER Bonham, K. S. et al. A promiscuous lipid-binding protein diversifies the subcellular sites of Toll-like receptor signal transduction. Cell 156, 705–716 (2014) TIRAP is required for signalling by TLRs in 3ʹ phospho- inositide-rich endosomes NPG RESEARCH HIGHLIGHTS NATURE REVIEWS | IMMUNOLOGY VOLUME 14 | APRIL 2014 Nature Reviews Immunology | AOP, published online 7 March 2014; doi:10.1038/nri3645 © 2014 Macmillan Publishers Limited. All rights reserved
