Wiskott-Aldrich syndrome protein restricts cGAS/STING activation by dsDNA immune complexes
Giulia Maria Piperno, Asma Naseem, Giulia Silvestrelli, Roberto Amadio, Nicoletta Caronni, Karla Evelia Cervantes-Luevano, Nalan Liv, Judith Klumperman, Andrea Colliva, Hashim Ali, Francesca Graziano, Philippe Benaroch, Hans Haecker, Richard N. Hanna, Federica Benvenuti
Giulia Maria Piperno, Asma Naseem, Giulia Silvestrelli, Roberto Amadio, Nicoletta Caronni, Karla Evelia Cervantes-Luevano, Nalan Liv, Judith Klumperman, Andrea Colliva, Hashim Ali, Francesca Graziano, Philippe Benaroch, Hans Haecker, Richard N. Hanna, Federica Benvenuti
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Research Article Cell biology Immunology

Wiskott-Aldrich syndrome protein restricts cGAS/STING activation by dsDNA immune complexes

Abstract

Dysregulated sensing of self–nucleic acid is a leading cause of autoimmunity in multifactorial and monogenic diseases. Mutations in Wiskott-Aldrich syndrome protein (WASp), a key regulator of cytoskeletal dynamics in immune cells, cause autoimmune manifestations and increased production of type I IFNs by innate cells. Here we show that immune complexes of self-DNA and autoantibodies (DNA-ICs) contribute to elevated IFN levels via activation of the cGAS/STING pathway of cytosolic sensing. Mechanistically, lack of endosomal F-actin nucleation by WASp caused a delay in endolysosomal maturation and prolonged the transit time of ingested DNA-ICs. Stalling in maturation-defective organelles facilitated leakage of DNA-ICs into the cytosol, promoting activation of the TBK1/STING pathway. Genetic deletion of STING and STING and cGAS chemical inhibitors abolished IFN production and rescued systemic activation of IFN-stimulated genes in vivo. These data unveil the contribution of cytosolic self–nucleic acid sensing in WAS and underscore the importance of WASp-mediated endosomal actin remodeling in preventing innate activation.

Authors

Giulia Maria Piperno, Asma Naseem, Giulia Silvestrelli, Roberto Amadio, Nicoletta Caronni, Karla Evelia Cervantes-Luevano, Nalan Liv, Judith Klumperman, Andrea Colliva, Hashim Ali, Francesca Graziano, Philippe Benaroch, Hans Haecker, Richard N. Hanna, Federica Benvenuti

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Figure 6

The cGAS/STING pathway controls responses to DNA-ICs in WASp-null cells.

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The cGAS/STING pathway controls responses to DNA-ICs in WASp-null cells....
(A) WKO DCs were treated with C-178 (0.25 μM) or vehicle (DMSO) and stimulated with DMXAA (5 μg/mL), cyclic di-GMP (cGAMP, 3 μg/mL), LPS (1 μg/mL), and DNA-ICs (IC, 90 μg/mL). Type I IFN protein (for DMXAA, cGAMP, and LPS) or transcripts (for DNA-ICs) were evaluated by B16-Blue reporter assay or RT-PCR, respectively. Data are expressed as ratio between inhibitor-treated and DMSO-treated cells. Significance was determined by. Mean ± SEM of 3–4 biological replicates. ****P ≤ 0.0001, ***P ≤ 0.001, 2-way ANOVA for protein and by unpaired t test for transcript. (B) Transcription level of Isg15 in resting or stimulated (DNA-IC, 90 μg/mL) WKO DCs that had been pretreated with C-178 (0.25 μM). Mean ± SEM of 4 biological replicates. *P ≤ 0.5, **P ≤ 0.001, 2-way ANOVA. (C) WKO and WKO STING-KO DCs were stimulated with DMXAA (5 μg/mL), LPS (1 μg/mL), and DNA-ICs (90 μg/mL). Type I IFN protein (DMXAA and LPS) or transcripts (DNA-ICs) are expressed as ratio between values in WKO STING-KO and values in WKO. Significance was determined by. Mean ± SEM of 3–6 biological replicates. *P ≤ 0.5, ***P ≤ 0.001, ****P ≤ 0.0001 2-way ANOVA for protein and unpaired t test for transcript. (D) WKO DCs were treated with RU.521 (50 μM) or vehicle (DMSO) and stimulated with dsDNA (2.5 μg), DMXAA (5 μg/mL), and DNA-ICs (90 μg/mL). Values were measured and expressed as in A. (E) WKO mice were treated for 11 consecutive days with 500 mM C-176 or DMSO. Total splenocytes were harvested after 12 days, and the levels of Isg15 mRNA were evaluated by RT-PCR. Data are from n = 5 C-176 and n = 4 DMSO animals. Significance was evaluated by t test