The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor

Abstract

Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells. The ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Notably, although both the full-length and cleaved forms of TLR9 are capable of binding ligand, only the processed form recruits MyD88 on activation, indicating that this truncated receptor, rather than the full-length form, is functional. Furthermore, conditions that prevent receptor proteolysis, including forced TLR9 surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acids.

Figure 1. A truncated version of TLR9 is exclusively present in phagosomes

(a) Lysates or phagosome preparations from RAW-TLR9 cells fed latex beads were separated by SDS-PAGE and probed with antibodies specific for the indicated proteins. Full-length TLR9 (FL TLR9) and the truncated protein (cleaved TLR9) are indicated. (b) Analysis of phagosomes from RAW-TLR9 cells fed unconjugated latex beads or beads conjugated to stimulatory CpG oligos. Lysates or purified phagosomes from the cells were separated by SDS-PAGE and probed with antibodies against the indicated proteins. (c) The truncated form of TLR9 is a product of the full-length protein. Pulse/chase analysis of RAW-TLR9 cells or control cells (RAW). Asterisk (*) indicates the high molecular weight full-length TLR9 band. ”NS” indicates a non-specific band present in both RAW-TLR9 and control RAW cells. (d) Modelled structure of the truncated ectodomain (residues 477−790) of TLR9. Leucine rich repeats (LRR) 15 − 26 are labelled. (e) The TLR9 cleavage product is present in multiple cell types. Hematopoetic stem cells were transduced with a retrovirus encoding TLR9-HA and then differentiated into macrophages with M-CSF (MØ), dendritic cells with GM-CSF (cDC), or plasmacytoid dendritic cells with Flt3L (pDC). (f) Lysates from RAW cells expressing TLR4-HA or TLR7-HA were probed with anti-HA antibody. Note that TLR4 is not cleaved. The two bands of TLR4 are due to glycosylation. Note: unless indicated otherwise, the data presented in each panel are representative of at least 3 independent experiments.

Figure 2. Prior to cleavage, TLR9 exits the ER and traffics through the Golgi

(a) Cleaved TLR9 is EndoH-resistant. Proteins from lysates or purified phagosomes from RAW-TLR9 cells were immunoprecipitated with anti-HA resin and treated with EndoH (E), PNGaseF (P), or left untreated (-). (b) A longer exposure of TLR9 immunoprecipated from RAW-TLR9 cells and subjected to analysis as described in (a). An asterisk (*) indicates the high molecular weight full-length TLR9 band that is EndoH-resistant. (c) TLR9 cleavage requires passage through the Golgi. Pulse/chase analysis was performed, at the indicated chase time points, on RAW-TLR9 cells treated with brefeldinA (B) or vehicle (—). Note: the data presented in each panel are representative of at least 3 experiments.

Figure 3. Unc93b1 is required for TLR9 ER exit and cleavage

(a) Expression of Unc93b1 transcript was measured by quantitative RT-PCR in RAW cells transduced with a control retrovirus or a retrovirus encoding an shRNA specific for Unc93b1. (b) Unc93b1-shRNA RAW cells respond poorly to TLR7 and TLR9 ligands but not TLR4 ligands. RAW cells or Unc93b1-shRNA RAW cells were stimulated with the indicated ligands and TNFα production was measured by ELISA (representative of two experiments). (c) Unc93b1 is required for TLR9 cleavage. Lysates from control RAW-TLR9 cells (vector) or RAW-TLR9 cells transduced with the Unc93b1 shRNA retrovirus (Unc. shRNA) were probed with anti-HA antibodies or anti-tubulin antibodies. (d) Lysates or purified phagosomes from the same cells described in (c) were analyzed for the presence of TLR9 by anti-HA immunoblot. (e) Overexpression of Unc93b1 in MEFs enhances TLR9 cleavage. TLR9 in lysates of MEFs stably expressing TLR9-HA or TLR9-HA with Unc93b1 was detected by anti-HA immunoblot. RAW-TLR9 cells were included as a control for TLR9 cleavage product. (f) Cells described in (e) were stimulated with 5μM CpG or 25ng/mL recombinant mouse IL-1β. KC production was measured by ELISA (representative of 2 experiments). Unless noted otherwise, all data presented are representative of at least 3 experiments. For indicated statistical comparisons, * indicates p<0.05; ** indicates p<0.01.

Figure 4. Processing of TLR9 is necessary to generate a functional receptor

(a) TLR9 cleavage requires the activity of acid-dependent proteases. Pulse/chase analysis was performed on RAW-TLR9 cells treated with 50nM bafilomycinA1 (Baf) or DMSO (-) vehicle control. (b) In vitro proteolysis assay performed on TLR9-HA immunoprecipitated from 293T cells. TLR9-HA was incubated with recombinant CtsK, CtsS or no cathepsin (—) and detected by anti-HA immunoblot. (c) The cleaved form of TLR9 can bind ligand. (Left) Whole cell lysates or lysates of purified phagosomes were immunoprecipitated with anti-HA antibody, separated by SDS-PAGE and immunoblotted with anti-HA antibody. (Right) Lysates of purified phagosomes were incubated with (+) or without (—) 0.2 μM Biotin-CpG followed by steptavidin precipitation. TLR9-HA was detected by anti-HA immunoblot. Data are representative of two experiments. (d) Cell lysates were incubated with (+) or without (-) Biotin-CpG and precipitated with streptavidin as described in (c). Where indicated, unconjugated CpG was also added, prior to precipitation. Unmanipulated lysate (No IP) is shown as a control. (e) Cleaved TLR9 recruits MyD88. RAW cells expressing TLR9-HA and FLAG-MyD88 were stimulated with CpG oligos followed by lysis at the indicated time points. After lysis, MyD88 was immunoprecipitated and TLR9 or MyD88 were detected by anti-HA or anti-FLAG immounoblot, respectively. (f) TLR9 does not signal from the cell surface in Unc93b1-Ist2 cells. 293T cells were transiently transfected with an NF-κB luciferase reporter together with the indicated expression plasmids. Luciferase production was assayed 12h after stimulation with 3μM CpG, with or without pre-treatment with 50μM chloroquine (CHQ). (g) TLR9, TLR9-Ist2, and TLR9-Ist2δ18 expressed in RAW cells were analyzed for sensitivity to EndoH or PNGaseF as described in Fig. 2a. (h) 293T cells were transiently transfected with the indicated expression plasmids and NF-κB activation was measured as described in (f). (i) Schematic of compartmentalized proteolysis and regulation of TLR9 and TLR7. Note: for indicated statistical comparisons, * indicates p<0.05; ** indicates p<0.1. Unless noted otherwise, all data presented are representative of at least 3 independent experiments.