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Jimmunol.1701048.Full.Pdf Cyclic GMP−AMP Synthase Is the Cytosolic Sensor of Plasmodium falciparum Genomic DNA and Activates Type I IFN in Malaria This information is current as Carolina Gallego-Marin, Jacob E. Schrum, Warrison A. of September 29, 2021. Andrade, Scott A. Shaffer, Lina F. Giraldo, Alvaro M. Lasso, Evelyn A. Kurt-Jones, Katherine A. Fitzgerald and Douglas T. Golenbock J Immunol published online 6 December 2017 http://www.jimmunol.org/content/early/2017/12/20/jimmun Downloaded from ol.1701048 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 29, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 27, 2017, doi:10.4049/jimmunol.1701048 The Journal of Immunology Cyclic GMP–AMP Synthase Is the Cytosolic Sensor of Plasmodium falciparum Genomic DNA and Activates Type I IFN in Malaria Carolina Gallego-Marin,*,†,1 Jacob E. Schrum,*,1 Warrison A. Andrade,* Scott A. Shaffer,‡,x Lina F. Giraldo,† Alvaro M. Lasso,† Evelyn A. Kurt-Jones,* Katherine A. Fitzgerald,*,2 and Douglas T. Golenbock*,2 Innate immune receptors have a key role in the sensing of malaria and initiating immune responses. As a consequence of infection, systemic inflammation emerges and is directly related to signs and symptoms during acute disease. We have previously reported that plasmodial DNA is the primary driver of systemic inflammation in malaria, both within the phagolysosome and in the cytosol of effector cells. In this article, we demonstrate that Plasmodium falciparum genomic DNA delivered to the cytosol of human Downloaded from monocytes binds and activates cyclic GMP–AMP synthase (cGAS). Activated cGAS synthesizes 2939-cGAMP, which we subse- quently can detect using liquid chromatography–tandem mass spectrometry. 2939-cGAMP acts as a second messenger for STING activation and triggers TBK1/IRF3 activation, resulting in type I IFN production in human cells. This induction of type I IFN was independent of IFI16. Access of DNA to the cytosolic compartment is mediated by hemozoin, because incubation of purified malaria pigment with DNase abrogated IFN-b induction. Collectively, these observations implicate cGAS as an important cytosolic sensor of P. falciparum genomic DNA and reveal the role of the cGAS/STING pathway in the induction of type I IFN http://www.jimmunol.org/ in response to malaria parasites. The Journal of Immunology, 2018, 200: 000–000. alaria remains a major cause of morbidity and mortality disease, the problems associated with malaria eradication remain worldwide. The World Health Organization has esti- significant. These include the increasing resistance of insect vec- M mated that there were ∼212 million cases of malaria tors to insecticides and the emerging resistance of Plasmodium globally in 2016 and ∼429,000 deaths, primarily (∼70%) occurring to the most efficacious antimalarial drugs (2). Current evidence in children under age 5 y (1). Despite many gains against the indicates that drug resistance to artemisinin derivatives, the last- generation treatment for asexual blood-stage infection, has de- by guest on September 29, 2021 veloped in Southeast Asia and Africa (3–5). Despite these *Program in Innate Immunity, Division of Infectious Diseases and Immunology, setbacks, efforts continue with the objective of achieving the Department of Medicine, University of Massachusetts Medical School, Worcester, global elimination of malaria. MA 01605; †Centro Internacional de Entrenamiento e Investigaciones Medicas, Cali 760001, Colombia; ‡Proteomics and Mass Spectrometry Facility, University Our understanding of the pathogenesis of malaria is still limited (6). of Massachusetts Medical School, Shrewsbury, MA 01545; and xDepartment of Therefore, a top priority in basic research is to dissect the mecha- Biochemistry and Molecular Pharmacology, University of Massachusetts Medical nisms involved in malaria disease development and provide new School, Worcester, MA 01605 approaches for therapeutic and prophylactic interventions. An im- 1 C.G.-M. and J.E.S. contributed equally to this work. portant component of the pathogenesis of malaria is the host innate 2 K.A.F. and D.T.G. contributed equally to this work. immune response to the parasite. The activation of innate immune ORCIDs: 0000-0001-7018-830X (C.G.-M.); 0000-0002-5264-3257 (S.A.S.); 0000-0002- cells and the associated systemic inflammationleadtotheinitial 3558-7249 (L.F.G.); 0000-0002-1230-6707 (A.M.L); 0000-0003-4669-2084 (E.A.K.-J.); 0000-0002-2447-2358 (D.T.G.). signs and symptoms of disease and can influence the development of Received for publication July 20, 2017. Accepted for publication November 6, 2017. severe disease (7). Inflammatory mediators during malaria infection are produced as a result of direct recognition of plasmodial pathogen- This work was supported by National Institute of Allergy and Infectious Dis- eases Grants R21AI124171 (to E.A.K.-J., D.T.G., and C.G.-M.) and associated molecular patterns (PAMPs) by innate immune receptors, R01AI079293 (to K.A.F. and D.T.G.). A.M.L. and L.F.G. were supported by including TLRs (7), Nod-like receptors (NLRs), and nucleic acid Departamento de Ciencia y Tecnologia – Colciencias Grants 0234-2014 and 0552-2015 (to C.G.-M.) and by National Institutes of Health/Fogarty International sensors (8). Concomitant with TLR activation, expression of sensor Center Training Grant D43TW006589. proteins, including NLRs, is augmented, and NLR inflammasomes Address correspondence and reprint requests to Dr. Douglas T. Golenbock, Univer- are assembled. Proinflammatory cytokines and mediators like TNF-a, sity of Massachusetts Medical School, LRB, Room 328, 364 Plantation Street, IL-12, caspase-1, and IL-1b are then released (9). Elevated expres- Worcester, MA 01605. E-mail address: [email protected] sion of IFN-stimulated genes in innate immune cells is also charac- Abbreviations used in this article: AT-rich ODN, adenine thymine–rich oligodeoxy- teristic during Plasmodium infection (8, 10). nucleotide; BMDM, bone marrow–derived macrophage; cGAMP, cyclic GMP–AMP; cGAS, cyclic GMP–AMP synthase; gDNA, genomic DNA; Hz, hemozoin; IFI16, The recognition of microbial DNA by the immune system IFN-g–inducible protein 16; iRBC, infected RBC; IRF, IFN regulatory factor; ISD, provides a general mechanism for the detection of pathogens immunostimulatory DNA; KO, knockout; LC-MS/MS, liquid chromatography– tandem mass spectrometry; MDM, monocyte-derived macrophage; NLR, Nod-like (11, 12). Delivery of foreign or self-DNA into the cytoplasm receptor; PAMP, pathogen-associated molecular pattern; p(dA:dT), poly(deoxyadenylic- (which is largely free of self-DNA) through microbial infection deoxythymidylic) acid; SeV, Sendai virus; STING, stimulator of IFN genes; TBK1, activates innate cytosolic nucleic acid sensors (13). Plasmodial tank-binding kinase 1; uRBC, uninfected RBC; WT, wild-type. DNA represents a major trigger of innate immunity during Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 infection (7, 8, 14). The plasmodial genome contains highly www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701048 2 cGAS IS THE CYTOSOLIC SENSOR OF P. FALCIPARUM DNA stimulatory CpG motifs, which are thought to activate TLR9 when extracted with phenol/chloroform/isoamyl alcohol and centrifuged at carried into the phagolysosomal compartment by the malaria 10,000 3 g for 10 min. The parasite DNA was precipitated overnight with 2 pigment hemozoin (Hz) (15–17). However, CpG-rich motifs are NaOAc and absolute ethanol at 80˚C, washed with 70% ethanol, and suspended in nuclease- and endotoxin-free water. The DNA concentration relatively rare in Plasmodium falciparum. In contrast, AT-rich was estimated by measuring absorption at 260 nm, and P. falciparum DNA motifs are abundant and induce type I IFN via a pathway gDNA was stored at 220˚C. The purity of P. falciparum gDNA was that is independent of TLRs, DNA-dependent activator of IFN- confirmed by PCR using primers for Plasmodium 18S RNA, as described regulatory factors, RNA polymerase III, and IFN-g–inducible pro- previously (29), and human TLR7 genes. tein 16 (IFI16/p204) but dependent on stimulator of IFN genes Cell culture and stimulation (STING) (18, 19), tank-binding kinase 1 (TBK1) (20, 21), and IFN PBMCs from healthy donors were obtained, as described previously, using regulatory factor (IRF)3 and IRF7 (8, 22). + Ficoll gradient separation (10). Human primary CD14 monocytes were The enzyme cyclic GMP–AMP synthase (cGAS) has been iden- purified from PBMCs using the Pan Monocyte Isolation Kit (Miltenyi tified as a cytosolic DNA sensor whose activation results in the Biotec) and MACS, according to the manufacturer’s instructions. subsequent activation of STING (23, 24). Specifically, in the presence Monocyte-derived macrophages (MDMs) were obtained by adherence and + of cytosolic dsDNA, cGAS catalyzes the synthesis of 2939–cyclic differentiation of CD14 monocytes in RPMI 1640–10% FBS medium for 7 d. Human promonocyte THP-1 cells (wild-type [WT], cGAS+/+, GMP–AMP (cGAMP) from ATP and GTP. 2939-cGAMP then cGAS2/2, IFI16+/+, and IFI162/2) were generated as previously described functions as a second messenger that binds to and activates STING. (30). WT and knockout (KO) THP-1 cells were grown in RPMI 1640/ Activated STING leads to IRF3 phosphorylation, type I IFN pro- glutamine supplemented with 10% FBS. Primary bone marrow–derived 2/2 2/2 duction, and expression of IFN-stimulated genes (12, 25).
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