Abstract
The retention of intracellular Toll-like receptors (TLRs) in the endoplasmic reticulum prevents their activation under basal conditions. TLR9 is activated by sensing ligands in specific endosomal-lysosomal compartments. Here we identified IRAP+ endosomes as major cellular compartments for the early steps of TLR9 activation in dendritic cells (DCs). Both TLR9 and its ligand, the dinucleotide CpG, were present as cargo in IRAP+ endosomes. In the absence of the aminopeptidase IRAP, the trafficking of CpG and TLR9 to lysosomes and signaling via TLR9 were enhanced in DCs and in mice following bacterial infection. IRAP stabilized CpG-containing endosomes by interacting with the actin-nucleation factor FHOD4, which slowed the trafficking of TLR9 toward lysosomes. Thus, endosomal retention of TLR9 via the interaction of IRAP with the actin cytoskeleton is a mechanism that prevents hyper-activation of TLR9 in DCs.
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References
Lee, B.L. & Barton, G.M. Trafficking of endosomal Toll-like receptors. Trends Cell Biol. 24, 360–369 (2014).
Latz, E. et al. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat. Immunol. 5, 190–198 (2004).
Leifer, C.A. et al. TLR9 is localized in the endoplasmic reticulum prior to stimulation. J. Immunol. 173, 1179–1183 (2004).
Fukui, R. et al. Unc93B1 restricts systemic lethal inflammation by orchestrating Toll-like receptor 7 and 9 trafficking. Immunity 35, 69–81 (2011).
Garcia-Cattaneo, A. et al. Cleavage of Toll-like receptor 3 by cathepsins B and H is essential for signaling. Proc. Natl. Acad. Sci. USA 109, 9053–9058 (2012).
Lee, B.L. et al. UNC93B1 mediates differential trafficking of endosomal TLRs. eLife 2, e00291 (2013).
Asagiri, M. et al. Cathepsin K-dependent toll-like receptor 9 signaling revealed in experimental arthritis. Science 319, 624–627 (2008).
Ewald, S.E. et al. Nucleic acid recognition by Toll-like receptors is coupled to stepwise processing by cathepsins and asparagine endopeptidase. J. Exp. Med. 208, 643–651 (2011).
Hipp, M.M. et al. Processing of human toll-like receptor 7 by furin-like proprotein convertases is required for its accumulation and activity in endosomes. Immunity 39, 711–721 (2013).
Maschalidi, S. et al. Asparagine endopeptidase controls anti-influenza virus immune responses through TLR7 activation. PLoS Pathog. 8, e1002841 (2012).
Park, B. et al. Proteolytic cleavage in an endolysosomal compartment is required for activation of Toll-like receptor 9. Nat. Immunol. 9, 1407–1414 (2008).
Sepulveda, F.E. et al. Critical role for asparagine endopeptidase in endocytic Toll-like receptor signaling in dendritic cells. Immunity 31, 737–748 (2009).
Hayashi, K., Sasai, M. & Iwasaki, A. Toll-like receptor 9 trafficking and signaling for type I interferons requires PIKfyve activity. Int. Immunol. 27, 435–445 (2015).
Hazeki, K., Uehara, M., Nigorikawa, K. & Hazeki, O. PIKfyve regulates the endosomal localization of CpG oligodeoxynucleotides to elicit TLR9-dependent cellular responses. PLoS One 8, e73894 (2013).
Sasai, M., Linehan, M.M. & Iwasaki, A. Bifurcation of Toll-like receptor 9 signaling by adaptor protein 3. Science 329, 1530–1534 (2010).
Saveanu, L. et al. IRAP identifies an endosomal compartment required for MHC class I cross-presentation. Science 325, 213–217 (2009).
Weimershaus, M. et al. Conventional dendritic cells require IRAP-Rab14 endosomes for efficient cross-presentation. J. Immunol. 188, 1840–1846 (2012).
Hosaka, T. et al. p115 Interacts with the GLUT4 vesicle protein, IRAP, and plays a critical role in insulin-stimulated GLUT4 translocation. Mol. Biol. Cell 16, 2882–2890 (2005).
Hirata, Y. et al. Vimentin binds IRAP and is involved in GLUT4 vesicle trafficking. Biochem. Biophys. Res. Commun. 405, 96–101 (2011).
Tojo, H. et al. The Formin family protein, formin homolog overexpressed in spleen, interacts with the insulin-responsive aminopeptidase and profilin IIa. Mol. Endocrinol. 17, 1216–1229 (2003).
Cheng, H. et al. Identification of a missense variant in LNPEP that confers psoriasis risk. J. Invest. Dermatol. 134, 359–365 (2014).
Stow, J.L., Low, P.C., Offenhauser, C. & Sangermani, D. Cytokine secretion in macrophages and other cells: pathways and mediators. Immunobiology 214, 601–612 (2009).
Leuchowius, K.J., Weibrecht, I. & Soderberg, O. In situ proximity ligation assay for microscopy and flow cytometry. In Current Protocols in Cytometry (ed. Robinson, J.P.) Ch 9, Unit 9 36 (John Wiley and Sons, 2011).
Greene, C.M. et al. TLR-induced inflammation in cystic fibrosis and non-cystic fibrosis airway epithelial cells. J. Immunol. 174, 1638–1646 (2005).
Benmohamed, F. et al. Toll-Like receptor 9 deficiency protects mice against Pseudomonas aeruginosa lung infection. PLoS One 9, e90466 (2014).
Saveanu, L. & van Endert, P. The role of insulin-regulated aminopeptidase in MHC class I antigen presentation. Front. Immunol. 3, 57 (2012).
Onji, M. et al. An essential role for the N-terminal fragment of Toll-like receptor 9 in DNA sensing. Nat. Commun. 4, 1949 (2013).
Fratti, R.A., Backer, J.M., Gruenberg, J., Corvera, S. & Deretic, V. Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest. J. Cell Biol. 154, 631–644 (2001).
Lakadamyali, M., Rust, M.J. & Zhuang, X. Ligands for clathrin-mediated endocytosis are differentially sorted into distinct populations of early endosomes. Cell 124, 997–1009 (2006).
Kim, S. & Coulombe, P.A. Intermediate filament scaffolds fulfill mechanical, organizational, and signaling functions in the cytoplasm. Genes Dev. 21, 1581–1597 (2007).
Goode, B.L. & Eck, M.J. Mechanism and function of formins in the control of actin assembly. Annu. Rev. Biochem. 76, 593–627 (2007).
Heng, T.S. & Painter, M.W. The Immunological Genome Project: networks of gene expression in immune cells. Nat. Immunol. 9, 1091–1094 (2008).
Rifkin, I.R., Leadbetter, E.A., Busconi, L., Viglianti, G. & Marshak-Rothstein, A. Toll-like receptors, endogenous ligands, and systemic autoimmune disease. Immunol. Rev. 204, 27–42 (2005).
Brinkmann, M.M. et al. The interaction between the ER membrane protein UNC93B and TLR3, 7, and 9 is crucial for TLR signaling. J. Cell Biol. 177, 265–275 (2007).
Kuhn, S. & Geyer, M. Formins as effector proteins of Rho GTPases. Small GTPases 5, e29513 (2014).
Fernandez-Borja, M., Janssen, L., Verwoerd, D., Hordijk, P. & Neefjes, J. RhoB regulates endosome transport by promoting actin assembly on endosomal membranes through Dia1. J. Cell Sci. 118, 2661–2670 (2005).
Prete, F. et al. Wiskott-Aldrich syndrome protein-mediated actin dynamics control type-I interferon production in plasmacytoid dendritic cells. J. Exp. Med. 210, 355–374 (2013).
Seth, A., Otomo, C. & Rosen, M.K. Autoinhibition regulates cellular localization and actin assembly activity of the diaphanous-related formins FRLα and mDia1. J. Cell Biol. 174, 701–713 (2006).
Wimmer, N. et al. Lymphotoxin beta receptor activation on macrophages induces cross-tolerance to TLR4 and TLR9 ligands. J. Immunol. 188, 3426–3433 (2012).
Acharya, M. et al. alphav Integrins combine with LC3 and atg5 to regulate Toll-like receptor signalling in B cells. Nat. Commun. 7, 10917 (2016).
Han, C. et al. Integrin CD11b negatively regulates TLR-triggered inflammatory responses by activating Syk and promoting degradation of MyD88 and TRIF via Cbl-b. Nat. Immunol. 11, 734–742 (2010).
Etienne-Manneville, S. Cdc42–the centre of polarity. J. Cell Sci. 117, 1291–1300 (2004).
Goncalves de Moraes, V.L., Singer, M., Vargaftig, B.B. & Chignard, M. Effects of rolipram on cyclic AMP levels in alveolar macrophages and lipopolysaccharide-induced inflammation in mouse lung. Br. J. Pharmacol. 123, 631–636 (1998).
Descamps, D. et al. Toll-like receptor 5 (TLR5), IL-1beta secretion, and asparagine endopeptidase are critical factors for alveolar macrophage phagocytosis and bacterial killing. Proc. Natl. Acad. Sci. USA 109, 1619–1624 (2012).
Vander Kooi, C.W. Megaprimer method for mutagenesis of DNA. Methods Enzymol. 529, 259–269 (2013).
Saveanu, L. et al. Concerted peptide trimming by human ERAP1 and ERAP2 aminopeptidase complexes in the endoplasmic reticulum. Nat. Immunol. 6, 689–697 (2005).
Tiscornia, G., Singer, O. & Verma, I.M. Production and purification of lentiviral vectors. Nat. Protoc. 1, 241–245 (2006).
Acknowledgements
We thank S. Lory (Harvard Medical School) for P. aeruginosa strain PAK; S. Keller (University of Virginia) for IRAP-deficient mice and rabbit anti-IRAP ; D. Billadeau (Mayo Clinic College of Medicine) for rabbit polyclonal anti-FHOD4 and anti-FHOD1; F. Benvenutti (International Centre for Genetic Engineering and Biotechnology, Trieste) for the VAMP3-GFP plasmid; F. Perez (Curie Institut, Paris) for the pmCherry plasmid; S. Blystone (SUNY Upstate Medical University) for the FHOD4-GFP plasmid; N. Goudin and S. Benadda for advice on analysis and quantification of confocal microscopy images; and 'ARC pour la Recherche sur le cancer' for acquisition of the Leica SP8 confocal microscope. Supported by the 'Agence Nationale de Recherche' (ANR IRAPDC; ANR-15-CE15-0005 to L.S.; ANR 2010 MIDI 008 01 to B.M.; and post-doctoral support for D.D.), Fondation pour la Recherche Médicale (A.C.A.), Institut Curie (M.T.), the Initiative and Networking Fund of the Helmholtz Association (VH-NG-637 to M.M.B.) and 'ARC pour la Recherche sur le cancer' (J.B. and S.M.).
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J.B., D.D., A.C.A., M.T., S.M., I.E., L.R.V., M.D.L., F.-X.M., B.M. and L.S. designed and did the experiments and analyzed the data; M.G.-T. contributed to the acquisition and analysis of confocal images; M.M.B. provided the mice with transgenic expression of TLR9-GFP; M.C. supervised the in vivo models of mice infection; and B.M. and L.S. wrote the paper, supervised the project and edited the paper.
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Integrated supplementary information
Supplementary Figure 1 IRAP-deficient BMDCs have an enhanced TLR7 response and normal trafficking of proinflammatory cytokines.
(a-b) WT and KO BMDCs or splenic pDCs were stimulated with TLR7 ligand (imiquimod) and the secretion of IL-6, IL-12(p40) and TNF was measured by ELISA. (n=3 experiments, mean ± SEM, *p<0.031, **p<0.007,***p<0.0012).
(c-d) Immunofluorescence microscopy of BMDCs from WT or KO mice stimulated or not (NS) with CpG-B for 6 hours and stained for IRAP and IL-6 or IL-12(p40) (a), and IRAP and GM-130 (b) using specific antibodies. Quantification of colocalization showed that 70% (+/-5%) and 80% (+/- 7) of IL-6 is found in Golgi stacks in WT and KO cells, respectively (n=10 cells, mean+/-SEM).
Supplementary Figure 2 The innate inflammatory infiltrate of lungs infected with Pseudomonas aeruginosa is not altered by the absence of IRAP.
(a) The presence of macrophages/monocytes (CD11b+/GR-1Int) and neutrophils (CD11b+/GR-1high) was analyzed by flow cytometry of single cell suspensions in BAL fluids from WT and KO mice 24 h post infection (n=8 animals; mean ± SEM). (b) Myeloperoxidase (MPO) activity (lower panel) was measured in BAL fluids from WT and KO mice 24 h post infection (n=8 animals; mean ± SEM).
Supplementary Figure 3 Stx6 and VAMP3 are components of IRAP+ endosomes.
WT BMDCs were transfected with VAMP3-GFP by nucleofection. Two days later, the cells were stained with antibodies specific for IRAP (a), STX6 (b) or Rab14 (c) and analyzed by confocal microscopy. (d) Quantification of colocalization between the two markers (n=10 cells, mean ± SEM).
Supplementary Figure 4 The trafficking of TLR3 is not affected by deletion of IRAP.
(a-b) WT BMDCs were transfected with TLR3-HA by nucleofection. Two days later the cells were stimulated or not (NS) with polyIC for the indicated time points, fixed and stained with antibodies against IRAP (a) or LAMP1 (b). (c) Quantification of TLR3-HA and LAMP1 co-localization in the experiment shown in (b) using Image J Software (n=10 cells from 2 independent experiments, mean ± SEM).
Supplementary Figure 5 The interaction of IRAP with FHOD4 can be reconstituted in wild-type fibroblasts.
(a) WT fibroblasts were transfected by electroporation with a plasmid expressing FHOD4-GFP. Thirty-six hours later IRAP and FHOD4 were immunoprecipitated with anti-IRAP and anti-GFP respectively and the precipitates were split in two and analyzed by immunoblot as indicated. (b) WT and KO fibroblasts were transfected as in (a) and a proximity ligation assay for detection of IRAP/FHOD4 interaction was performed with antibodies against IRAP and GFP.
Supplementary Figure 6 Inactivation of FHOD4, similar to IRAP deficiency, increases the activation of TLR9.
(a) WT and KO BMDCs were transduced with shNT (non-targeting) and shFHOD4 (17) lentiviruses and stimulated with different TLR ligands for 6 h. The secretion of IL-12p40 and IL-6 in supernatants was measured by ELISA (n=2 experiments, mean ± SEM, **p<0.009, *p<0.018). (b) BMDCs from TLR9-GFP transgenic mice were transduced with lentiviruses coding for 5 different shRNA against FHOD1 (shFHOD1 42, 91, 47, 49, 02) or a non-targeting shRNA (shNT) and the knock-down efficiency was analyzed by immunoblotting with antibodies specific for FHOD1. WT fibroblasts transfected with a plasmid coding for FHOD1 were used as positive control for anti-FHOD1 antibodies. (c-d) BMDCs from TLR9-GFP transgenic mice were transduced with lentiviruses expressing shNT or shFHOD1 (42) and used to analyze TLR9-GFP localization in steady state conditions by confocal microscopy using an anti-LAMP1 antibody (c) or to measure the secretion of IL-6 and IL-12(p40) after TLR4 and TLR9 activation (d) (n=2 experiments, mean ± SEM).
Supplementary Figure 7 Integration of data in the literature and our results for a model of the trafficking of TLR9.
Under basal conditions, TLR9 is retained in the ER. Upon cell stimulation, the TLR9-Unc93b complex traffics to the cell surface and is internalized via AP2 and clathrin mediated endocytosis7. Once intracellular, TLR9 reaches IRAP+ endosomes that contain CpG. IRAP vesicles are Rab14 and Stx6 positive and overlap with both EEA1+8,9 and VAMP3+ vesicles. In VAMP3+ compartment, TLR9 recruits MyD88 and induces the NF-κB response. The sorting of TLR9 from VAMP3+ vesicles is dependent on the clathrin adaptor AP310 and allows TLR9 transport, probably via microtubules11, to lysosomes, from where it can signal via both, MyD88 and IRFs. Actin polymerization around early endosomal compartments delays CpG and TLR9 transport to lysosomes12. IRAP interaction with the actin nucleator FHOD4 anchors the endosomes containing CpG and TLR9 to the actin network, blocking their transport towards lysosomes and limiting TLR9 activation.
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Babdor, J., Descamps, D., Adiko, A. et al. IRAP+ endosomes restrict TLR9 activation and signaling. Nat Immunol 18, 509–518 (2017). https://doi.org/10.1038/ni.3711
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DOI: https://doi.org/10.1038/ni.3711
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