IRAP+ endosomes restrict TLR9 activation and signaling

Abstract

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 describe the identification of insulin responsive aminopeptidase (IRAP) endosomes as major cellular compartments for the early steps of TLR9 activation in dendritic cells (DCs). Both TLR9 and its ligand CpG were found as cargo in IRAP endosomes. In the absence of IRAP, CpG and TLR9 trafficking to lysosomes and TLR9 signaling were enhanced in DCs and in mice following bacterial infection. IRAP stabilized CpG-containing endosomes by interacting with the actin nucleation factor FHOD4, slowing down TLR9 trafficking towards lysosomes. Thus, endosome retention of TLR9 via IRAP interaction with the actin cytoskeleton is a mechanism that prevents TLR9hyper-activation in DCs. discriminate between different classes of microbial products and initiate specific signaling cascades. While microbial products with no equivalent in mammalian cells, such as the components of the bacterial wall, are recognized by surface TLRs (1, 2, 4, 5 and 6), pathogen derived nucleic acids are sensed by intracellular TLRs (3,7, 8 and 9). Recognition of nucleic acids by intracellular TLRs has the potential to trigger autoimmune diseases through interaction with self nucleic acids 1 . To avoid inappropriate activation of endosomal TLRs, their trafficking is tightly controlled. Thus, in basal conditions the receptors are located in the endoplasmic reticulum (ER) and translocate to endocytic vesicles only after cell stimulation by TLR ligands. Although all intracellular TLRs reside in the ER 2,3 , the trafficking pathways that move the receptors into the endocytic pathway show considerable variation among intracellular TLRs 4-6 . For example, TLR7 traffics from Golgi stacks directly to endosomes using the clathrin adaptor AP4, whereas the TLR9 is directed to the cell surface and reaches the endosomes via AP2-mediated clathrin-dependent endocytosis 6 .In addition to the transfer into the endocytic pathway, a second step that controls the activation of endosomal TLRs is their partial proteolysis by an array of different proteases, specific for each TLR 5,7-12 .Although less often mentioned, the intracellular trafficking of its ligand also controls the activation of TLR9. TLR9 ligands (CpG) are internalized via clathrin-mediated endocytosis in early endosomes and translocate to late LAMP + compartments 2 . TLR9 activation depends on CpG localization, since the abrogation of CpG translocation to LAMP + compartments by specific inhibitors decreased TLR9 signaling 13,14 . Thus, the intracellular trafficking of both, the ligand and the receptor are essential for the control of TLR9 activation. RESULTS IRAP deletion increases TLR9 responseTo address the role of IRAP in TLRs signaling, wild-type and IRAP-deficient bone marrow derived dendritic cells (BMDCs) were stimulated with specific TLR ligands: polyIC for TLR3, Imiquimod fo...

STIM1 promotes migration, phagosomal maturation and antigen cross-presentation in dendritic cells

“…Cathepsin/AEP protease activity assays were performed as previously described 66 using 1 µg of protein in lysis buffer (50 mM Tris/150 mM NaCl/1% NP40) on a Mithras LB 940 microplate reader by measuring the release of fluorescent N-Acetyl-Methyl-Coumarin substrate (100 µM) in citrate buffer (pH 5.5) at 37 °C, detected using 380/440 nm excitation/emission. Leucyl aminopeptidase activity was measured as previously described 67 in 50 mM Tris buffer, pH 7.5/1 mM DTT and 100 µM substrate except that activity was measured on 3 µg of whole-cell or purified phagosome lysates without anti-IRAP immunoprecipitation. Specific substrates for AEP (Z-Ala-Ala-Asn- NHMec), CatB/L (Z-Phe-Arg-NHMec), CatS (Z-Val-Val-Arg-NHMec) and IRAP substrate (Leu-AMC) were purchased from Bachem.…”

Is There an Interplay Between the Functional Domains of IRAP?

“…The N-terminus of IRAP is also believed to be important for tethering specialized endosomes to the cytoskeleton via binding of interacting proteins including tankyrase ( Chi and Lodish, 2000 ), acyl-coenzyme A dehydrogenase ( Katagiri et al, 2002 ), formin homology domain (FHOD) ( Babdor et al, 2017 ), p115 ( Hosaka et al, 2005 ), Vps35 ( Pan et al, 2019 ), and Akt substrate of 160 kDa (AS160) ( Figure 2 ; Peck et al, 2006 ). This has been clearly demonstrated in dendritic cells, in which the role of the N-terminal domain of IRAP has only recently been implicated in immune function.…”

“…This has been clearly demonstrated in dendritic cells, in which the role of the N-terminal domain of IRAP has only recently been implicated in immune function. A comprehensive study by Babdor et al (2017) revealed that anchorage of endosomes containing toll-like receptor 9 (TLR9) to the actin cytoskeleton by the N-terminus of IRAP via the interacting protein, FHOD4, prevents TLR9 activation. Thus, in the absence of IRAP, the trafficking of TLR9 to lysosomes and subsequent activation was enhanced in cultured dendritic cells and in mice following bacterial infection, leading to an increased secretion of pro-inflammatory cytokines ( Babdor et al, 2017 ).…”

“…A comprehensive study by Babdor et al (2017) revealed that anchorage of endosomes containing toll-like receptor 9 (TLR9) to the actin cytoskeleton by the N-terminus of IRAP via the interacting protein, FHOD4, prevents TLR9 activation. Thus, in the absence of IRAP, the trafficking of TLR9 to lysosomes and subsequent activation was enhanced in cultured dendritic cells and in mice following bacterial infection, leading to an increased secretion of pro-inflammatory cytokines ( Babdor et al, 2017 ). This study is crucial as it not only suggests another intracellular trafficking mechanism regulated by IRAP, but also has potential implications for understanding the mechanisms underlying certain autoimmune disorders such as arthritis, where there is inappropriate activation of endosomal TLRs ( Rifkin et al, 2005 ).…”

Reciprocal Expression Patterns of Placental Leucine Aminopeptidase/Insulin-Regulated Aminopeptidase and Vasopressin in the Murine Brain

“…These results indicate that P-LAP/IRAP plays important roles in the modulation of several brain functions. This enzyme is also an important player in host defense systems, such as antigen cross-presentation, and Toll-like receptor 9 signaling (Saveanu et al, 2009;Babdor et al, 2017). P-LAP/IRAP trims antigenic peptides presented by MHC class I molecules, and also modulates Toll-like receptor 9 trafficking to lysosomes via interaction with FHOD4 (Saveanu et al, 2009;Babdor et al, 2017).…”