CD82 controls CpG-dependent TLR9 signaling

Abstract

The tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post-ER. Moreover, CD82 is essential for TLR9-dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9-dependent NF-κB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.-Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E.-M., Becker, C. E., Dagher, Z., Kim, Y.-M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG-dependent TLR9 signaling.

Keywords: TLRs; macrophages; myddosome; tetraspanins.

Figure 1

A–D) CD82 regulates TLR9 signaling in macrophages and in vivo in response to CpG. TNF-α production in WT or CD82KO immortalized macrophages was measured by ELISA in response to increasing doses of Pam₃CsK₄ (A), LPS (B), imiquimod (C), or CpG (D) for 6 h. E) WT, CD82KO, TLR9KO, or CD82KO + CD82-mRFP1 macrophages stimulated with 1 µM CpG for 16 h and assessed for TNF-α production. F) WT and CD82KO macrophages stimulated with 1 µM CpG for 2 h or unstimulated were assessed for NF-κB translocation in nuclear lysates by immunoblot. LSD1, lysine-specific histone demethylase 1A. G) Sera of WT, CD82KO, and TLR9KO mice (n = 3 mice/group) intraperitoneally injected with 20 nmol CpG and/or 20 mg D-GalN and assessed for TNF-α production. ELISA results are presented as means of technical (A–E) or biologic (G) triplicates ± sd. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001 (unpaired Student’s t test).

Figure 2

CD82 and TLR9 associate in macrophages. A, B) Confocal imaging of RAW macrophages expressing TLR9-GFP (green) and CD82-mRFP1 (red). Macrophages incubated with PBS (A) or 1 µM Alexa Fluor 647–conjugated CpG (B) (blue) for 1 h. White arrows indicate colocalization. Scale bars, 5 µm. C, D) Immunoprecipitation (IP) of TLR9-GFP or HA-CD82 in WT macrophages stimulated with either PBS (Unst.) or 1 µM CpG. C) Western blot of lysates immunoprecipitated with anti-GFP and probed for HA-CD82 (top blot) or TLR9-GFP (second blot). Lysates probed for HA-CD82 (third blot). Western blot of lysates immunoprecipitated with anti-HA and probed for TLR9-GFP (top blot) or HA-CD82 (second blot) (D). Lysates probed for TLR9-GFP (third blot). DIC, differential interference contrast.

Figure 3

Full-length TLR9 and CD82 interact in the ER, whereas cleaved TLR9 and CD82 interact post-ER. A, B) WT macrophages expressing TLR9-GFP and HA-CD82 stimulated with 1 µM CpG for 1 h or unstimulated. A) Western blot of lysates immunoprecipitated with anti-GFP and probed for HA-CD82 (top blot). Cell lysates probed for TLR9-GFP (second blot) and HA-CD82 (third blot). B) Western blot of lysates immunoprecipitated with anti-HA and probed for TLR9-GFP (top blot). Cell lysates probed for HA-CD82 (second blot) and TLR9-GFP (third blot). FL, full length; IP, immunoprecipitation.

Figure 4

CD82 controls TLR9 trafficking to acidified CpG-containing compartments. Confocal imaging of WT (A) and CD82KO (B) macrophages expressing TLR9-GFP (green) stimulated with 1 µM Alexa Fluor 647–conjugated CpG (blue) and stained with LysoTracker (red). White arrows indicate colocalization. Scale bars, 5 µm. C–E) Grap represents mean PCC ± sd (n ∼ 100 images) for colocalization between TLR9-GFP and CpG (C), TLR9-GFP and LysoTracker (D), and CpG and LysoTracker (E). DIC, differential interference contrast. ****P ≤ 0.0001 (unpaired Student’s t test; n = 100 macrophages).

Figure 5

TLR9 and VAMP3 interaction is mediated by CD82. A) WT macrophages expressing TLR9-GFP stimulated with 1 µM CpG for 1 h or unstimulated. Western blot of lysates immunoprecipitated with anti-VAMP3 and blotted for TLR9-GFP (top blot) or VAMP3 (second blot). Lysates probed for TLR9-GFP (third blot) and VAMP3 (fourth blot). Unst., unstimulated. B) WT and UNC93B1-KO macrophages expressing TLR9-GFP stimulated with 1 µM CpG for 1 h or unstimulated. Western blot of lysates immunoprecipitated with anti-GFP and probed for VAMP3 (top blot) or TLR9-GFP (second blot). Lysates probed for VAMP3 (third blot). C) WT and CD82KO macrophages expressing TLR9-GFP or CD82KO macrophages expressing TLR9-GFP and HA-CD82 stimulated with 1 µM CpG for 1 h or unstimulated. Western blot of lysates immunoprecipitated with anti-GFP and probed for VAMP3 (top blot) or TLR9-GFP (second blot). Lysates probed for VAMP3 (third blot). D) Densitometry analysis of VAMP3 found in TLR9-GFP immunoprecipitates, using the blot from panel C. FL, full length; IP, immunoprecipitation. *P ≤ 0.05, ***P ≤ 0.01, ****P ≤ 0.0001 (2-way ANOVA Tukey’s multiple comparisons test).

Figure 6

CD82 promotes TLR9-induced myddosome assembly and is dispensable for TLR7- and TLR4-induced myddosome formation. WT, TLR9KO, CD82KO, and CD82KO + CD82-mRFP1 macrophages stimulated with 1 µM CpG (A), 20 µM imiquimod (B), or 100 ng/ml LPS (C) for 2 h. MyD88 was immunoprecipitated, resolved by SDS-PAGE, and immunoblotted for IRAK-2 and IRAK4. Cell lysates were probed for actin as a loading control. IP, immunoprecipitation.

Figure 7

Schematic representation of CD82 controls CpG-dependent TLR9 signaling. CD82 interacts with TLR9 in WT macrophages in response to CpG. CD82 controls the assembly of TLR9-dependent myddosome assembly and subsequent NF-κB translocation to the nucleus and TNF-α production (left). In the absence of CD82, TLR9-mediated myddosome formation is absent, resulting in reduced TNF-α secretion (right). Illustration by Nicole Wolf, MS, ©2017. Printed with permission.