Research ArticleInnate Immunity

The interaction between IKKα and LC3 promotes type I interferon production through the TLR9-containing LAPosome

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Science Signaling  01 May 2018:
Vol. 11, Issue 528, eaan4144
DOI: 10.1126/scisignal.aan4144
  • Fig. 1 IKKα binds to LC3 after DOTAP–CpG-A stimulation.

    (A to C) Confocal microscopy images from Ikkα knockout (KO) fetal liver macrophages that were retrovirally co-transduced with wild-type (WT) IKKα-HA, TLR9-GFP, and Cherry-LC3 plasmids and then stimulated with 3 μM DOTAP–CpG-A. Images are representative of three or more independent experiments. Scale bars, 2.5 μm. Quantified co-localization data are means ± SEM. ***P < 0.001 by t test. (D) HEK293T cells were co-transfected with WT IKKα-HA and GFP-LC3, and then cell lysates were immunoprecipitated (IP) for GFP and blotted for the indicated protein. Blots are representative of three or more independent experiments. IB, immunoblot. (E) Day 7 BMMs were stimulated with DOTAP–CpG-A for 30 min, and then the prenuclear cell lysates were immunoprecipitated for GFP-LC3 and blotted for the indicated proteins. Blots are representative of three or more independent experiments. (F) RAW264.7 cells stably expressing TLR9-HA and Cherry-LC3 were stimulated with DOTAP–CpG-A for 6 hours, and then cell lysates were immunoprecipitated for LC3 and blotted for the indicated proteins. Blots are representative of three or more independent experiments. n.s., nonspecific.

  • Fig. 2 Autophagy induction is not sufficient to trigger LC3-IKKα interaction.

    (A to C) WT hematopoietic stem cells (HSCs) were retrovirally transduced with retrovirus expressing Cherry-LC3 and then differentiated into BMMs that were either treated with DOTAP alone (A), 3 μM DOTAP–CpG-A (A), or 1 μM Tat–Beclin-1 peptide (B), or starved in serum-deprived media (C) for 6 hours. After treatment, cells were stained for the indicated proteins and analyzed by confocal microscopy. Images are representative of three or more independent experiments. Scale bars, 5 μm. (D) Percentage of IKKα-LC3 colocalization from confocal analysis BMMs expressing Cherry-LC3 (A to C). Data are means ± SEM from three or more independent experiments. *P < 0.05, **P < 0.01 by Student’s t test.

  • Fig. 3 LC3-IKKα interaction depends on LAP but not canonical autophagy.

    (A) Atg5+/− or Atg5−/− fetal liver HSCs were retrovirally transduced with WT IKKα-HA and Cherry-LC3, differentiated into macrophages, and stimulated with 1 μM DOTAP–CpG-A for 6 hours. After stimulation, cells were stained for the indicated proteins and analyzed by confocal microscopy. Images are representative of three or more independent experiments. Scale bars, 5 μm. **P < 0.01. het, heterozygous. (B) WT or Atg5−/− fetal liver macrophages were stimulated with DOTAP–CpG-A for 24 hours, and secretion of IFN-α and IL-12 was measured by enzyme-linked immunosorbent assay (ELISA). Data are means ± SEM from three to five independent experiments. *P < 0.05 by t test. (C) WT and Fip200fl/fl × LysM-Cre HSCs were retrovirally transduced with Cherry-LC3, differentiated to BMMs, and stimulated with DOTAP–CpG-A for 6 hours. After treatment, cells were stained and analyzed by confocal microscopy. Images are representative of three or more independent experiments. Scale bars, 5 μm. (D) WT and Fip200fl/fl × LysM-Cre BMDCs were stimulated with 1 μM DOTAP–CpG-A for 24 hours, and the cytokine accumulation in the supernatants was assayed by ELISA. Data are means ± SEM from three or more independent experiments.

  • Fig. 4 IKKα-LC3 complex recruits TRAF3 and IRF7.

    (A) HEK293T cells were transfected with plasmids encoding WT IKKα-HA, GFP-LC3, and TRAF3. Cell lysates were immunoprecipitated for IKKα and blotted for the indicated proteins. Blots are representative of three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) HEK293T cells were transfected with plasmids encoding WT IKKα-HA, GFP-LC3b, MyD88, TRAF3, TRAF6, and IRF7-Flag. Cell lysates were subjected to immunoprecipitated for the Flag-tag and blotted for the indicated proteins. Blots are representative of three independent experiments.

  • Fig. 5 Interaction between IKKα and LC3 depends on the LC3-interacting region in IKKα.

    (A) HEK293T cells were transfected with different LIR mutants, immunoprecipitated for LC3, and blotted for the indicated proteins. Blots are representative of three or more independent experiments. Mu1, LIR-1 mutant; Mu2, LIR-2 mutant; Mu3, LIR-3 mutant. (B) IKKα-deficient bone marrow cells were retrovirally reconstituted with IKKα-HA (WT, LIR-1, LIR-2, or LIR-3 mutant) and Cherry-LC3, differentiated into BMDCs, and stimulated with 0.5 μM DOTAP–CpG-A for 6 hours. After treatment, cells were analyzed by confocal microscopy. Images are representative of three independent experiments. Scale bars, 5 μm.

  • Fig. 6 Interaction between LC3 and IKKα is crucial for type I IFN production in granulocyte-macrophage colony-stimulating factor–differentiated BMDCs.

    Ikkα−/− bone marrow cells were retrovirally reconstituted with IKKα-HA (WT, LIR-1, LIR-2, or LIR-3 mutant), differentiated into BMDCs, and stimulated with DOTAP–CpG-A. After 24 hours, the supernatants were harvested, and cytokine production was assayed by ELISA. Data are means ± SEM from three independent experiments. **P < 0.01 by t test. n.s., not significant.

  • Fig. 7 Schematic of IKKα complex recruitment to LC3-containing LAPosomes necessary for TLR9 signaling and IFN production.

    After stimulation, ligand-bound TLR9 dimerizes and recruits signaling adaptor MyD88 and downstream signaling molecules including IRAK4 and the E3 ligase TRAF6. Autoubiquitination of TRAF6 mediates recruitment of TAK1 and its regulatory proteins TAB2/3, which phosphorylates and activates IKKβ. This induces NF-κB–mediated transcription of proinflammatory genes (NF-κB endosome). Activation of TLR9 also triggers ATG5-dependent recruitment of LC3 to the endosomal membrane through the process of LAP. IKKα is recruited to the LC3 through LIR-2 and LIR-3 domains. Adaptor protein 3 (AP-3) is required for the formation of lysosome-related organelle decorated with LAMP2. IKKα recruitment to the membrane-bound LC3 brings this kinase to the proximity of IRF7, where it phosphorylates IRF7 and activates type I IFN gene transcription. IkB, NF-κB inhibitor β.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/528/eaan4144/DC1

    Fig. S1. LC3 binds to TLR9 and IKKα after DOTAP–CpG-A stimulation.

    Fig. S2. ATG5 does not interact with LC3 or IKKα.

    Fig. S3. LIR mutants of IKKα maintain intact kinase activity.

    Fig. S4. Fms-related tyrosine kinase 3 ligand–derived Ikkα-deficient pDCs show impaired IFN-α production but intact IL-12p40 after CpG-A stimulation.

    Fig. S5. WT and LIR-1 but not LIR-2 or LIR-3 mutant IKKα induces IRF7 phosphorylation.

    Fig. S6. LIR prediction based on IKKβ structure by SWISS-MODEL.

  • Supplementary Materials for:

    The interaction between IKKα and LC3 promotes type I interferon production through the TLR9-containing LAPosome

    Kachiko Hayashi, Manabu Taura, Akiko Iwasaki*

    *Corresponding author. Email: akiko.iwasaki{at}yale.edu

    This PDF file includes:

    • Fig. S1. LC3 binds to TLR9 and IKKα after DOTAP–CpG-A stimulation.
    • Fig. S2. ATG5 does not interact with LC3 or IKKα.
    • Fig. S3. LIR mutants of IKKα maintain intact kinase activity.
    • Fig. S4. Fms-related tyrosine kinase 3 ligand–derived Ikkα-deficient pDCs show impaired IFN-α production but intact IL-12p40 after CpG-A stimulation.
    • Fig. S5. WT and LIR-1 but not LIR-2 or LIR-3 mutant IKKα induces IRF7 phosphorylation.
    • Fig. S6. LIR prediction based on IKKβ structure by SWISS-MODEL.

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    © 2018 American Association for the Advancement of Science

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