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 2

DNA-ICs accumulate in endosomes of WASp-null cells.

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DNA-ICs accumulate in endosomes of WASp-null cells. (A) WT or WKO DCs we...
(A) WT or WKO DCs were pulsed with DNA-ICs (90 μg/mL) for 7 minutes and chased for different durations. Cells were labeled with antibodies to the Fc portion of ICs and to early endosomes (EEA1). Images show representative confocal planes and the corresponding colocalization mask for early endosomes (EEs) and ICs. (B) Bars shows relative Mandel overlap coefficient (MOC) for ICs and EEs at the different time points. Mean ± SEM of 30 cells/condition from 3 independent experiments. ***P ≤ 0.001, **** P ≤ 0.0001, 2-way ANOVA. (C) The area of intracellular IC structures was measured 15 minutes or 40 minutes after pulse. Mean ± SEM. *P ≤ 0.05, ***P ≤ 0.001, unpaired t test, on 28 cells/condition from 2 independent experiments. (D) Images show representative central confocal planes of ICs and LAMP1 distribution after 40 minutes of chase. Insets are magnifications of the regions indicated by dotted lines. Graph shows MOC of ICs and Lamp1. Mean ± SEM of 28 cells/condition from 2 independent experiments. ****P ≤ 0.0001, unpaired t test. (E) DC were preloaded with labeled WGA (WGA-AF647) to mark lysosomes and incubated with DNA-ICs (90 μg/mL). Representative Airyscan micrographs and the corresponding insets show the relative distribution of ICs with respect to the WGA signal. The graph shows MOC of ICs and WGA (mean ± SEM of 28 WT and 23 WKO cells from 2 independent experiments). ***P ≤ 0.001, unpaired t test . Scale bars: 5 μm.