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IRAP+ endosomes restrict TLR9 activation and signaling

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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Figure 1: Deletion of IRAP enhances the TLR9 response.
Figure 2: Enhanced TLR9 signaling in IRAP-deficient DCs.
Figure 3: IRAP-deficient mice display a hyper-inflammatory phenotype driven by activation of TLR9.
Figure 4: IRAP's enzymatic activity is not involved in the activation of TLR9.
Figure 5: CpG and TLR9 are cargo of IRAP+ endosomes.
Figure 6: The absence of IRAP increases the susceptibility of TLR9 to lysosomal processing.
Figure 7: Deletion of IRAP reduces the retention of CpG and TLR9 in early endosomes.
Figure 8: IRAP interacts with FHOD4.

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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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Contributions

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.

Corresponding authors

Correspondence to Bénédicte Manoury or Loredana Saveanu.

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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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