DHEA Inhibits Leukocyte Recruitment through Regulation of the Integrin Antagonist DEL-1 This information is current as Athanasios Ziogas, Tomoki Maekawa, Johannes R. of September 27, 2021. Wiessner, Thi Trang Le, David Sprott, Maria Troullinaki, Ales Neuwirth, Vasiliki Anastasopoulou, Sylvia Grossklaus, Kyoung-Jin Chung, Markus Sperandio, Triantafyllos Chavakis, George Hajishengallis and Vasileia Ismini Alexaki Downloaded from J Immunol published online 24 January 2020 http://www.jimmunol.org/content/early/2020/01/24/jimmun ol.1900746 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published January 24, 2020, doi:10.4049/jimmunol.1900746 The Journal of Immunology DHEA Inhibits Leukocyte Recruitment through Regulation of the Integrin Antagonist DEL-1 Athanasios Ziogas,*,1 Tomoki Maekawa,†,‡,1 Johannes R. Wiessner,x Thi Trang Le,* David Sprott,* Maria Troullinaki,* Ales Neuwirth,* Vasiliki Anastasopoulou,* Sylvia Grossklaus,* Kyoung-Jin Chung,* Markus Sperandio,x Triantafyllos Chavakis,*,{,2 George Hajishengallis,†,2 and Vasileia Ismini Alexaki*,2 Leukocytes are rapidly recruited to sites of inflammation via interactions with the vascular endothelium. The steroid hormone dehydroepiandrosterone (DHEA) exerts anti-inflammatory properties; however, the underlying mechanisms are poorly understood. In this study, we show that an anti-inflammatory mechanism of DHEA involves the regulation of developmental endothelial locus 1 b (DEL-1) expression. DEL-1 is a secreted homeostatic factor that inhibits 2-integrin–dependent leukocyte adhesion, and the subse- Downloaded from quent leukocyte recruitment and its expression is downregulated upon inflammation. Similarly, DHEA inhibited leukocyte adhesion to the endothelium in venules of the inflamed mouse cremaster muscle. Importantly, in a model of lung inflammation, DHEA limited neutrophil recruitment in a DEL-1–dependent manner. Mechanistically, DHEA counteracted the inhibitory effect of inflammation on DEL-1 expression. Indeed, whereas TNF reduced DEL-1 expression and secretion in endothelial cells by diminishing C/EBPb binding to the DEL-1 gene promoter, DHEA counteracted the inhibitory effect of TNF via activation of tropomyosin receptor kinase A (TRKA) b and downstream PI3K/AKT signaling that restored C/EBP binding to the DEL-1 promoter. In conclusion, DHEA restrains neutrophil http://www.jimmunol.org/ recruitment by reversing inflammation-induced downregulation of DEL-1 expression. Therefore, the anti-inflammatory DHEA/DEL-1 axis could be harnessed therapeutically in the context of inflammatory diseases. The Journal of Immunology, 2020, 204: 000–000. ctivation of the endothelium is integral to leukocyte DEL-1 causes elevated leukocyte infiltration under different in- recruitment into inflamed tissues (1, 2). Upon activation flammatory conditions in mice (8, 9, 12, 15, 17–20). Inflammatory A by proinflammatory cytokines, such as TNF, endothelial cytokines, such as IL-17 and TNF, inhibit endothelial DEL-1 ex- cells orchestrate inflammation and leukocyte recruitment, which pression, thereby facilitating leukocyte recruitment and inflamma- is mediated by a cascade of leukocyte–endothelial adhesive in- tion (9, 17, 21). The IL-17–dependent downregulation of DEL-1 teractions (2–4). This cascade is initiated by selectin-mediated expression is reversed by D-series resolvins (RvDs) (21). However, by guest on September 27, 2021 rolling and deceleration of leukocytes on the endothelial surface. little is known about other factors regulating DEL-1 expression. Rolling triggers integrin activation, and activated integrins Dehydroepiandrosterone (DHEA; 5-androsten-3b-hydroxy-17- (primarily of the b2 family) promote firm adhesion of leukocytes one) and its sulfate ester are abundant circulating steroid hor- to the activated endothelium, a prerequisite step for the subse- mones in human adults, whereas their concentration declines with quent leukocyte extravasation (5, 6). age and in inflammatory diseases, such as arthritis and systemic Developmental endothelial locus 1 (DEL-1; also designated lupus erythematosus (22–26). In humans, DHEA is produced in EGF-like repeats and discoidin domains 3 [EDIL3]) is a glycoprotein the adrenal cortex, the gonads, and the CNS (27–30). In tissues, secreted by endothelial and other cells and has anti-inflammatory DHEA displays anti-inflammatory properties, including inhibition properties (7–16). DEL-1 interferes with b2-integrin–dependent of leukocyte recruitment (31, 32). DHEA can bind to nuclear re- adhesion of leukocytes to endothelial ICAM-1, thereby restraining ceptors, such as estrogen receptor a and b (33, 34). Moreover, it leukocyte recruitment (8, 9). Consistently, genetic deletion of was shown to bind to G protein–coupled receptors in endothelial *Institute of Clinical Chemistry and Laboratory Medicine, University Clinic Grant Agreement (765704 to V.I.A.), the European Research Council (DEMETINL Carl Gustav Carus, Technische Universita¨t Dresden, 01307 Dresden, Germany; to T.C.), and by National Institutes of Health grants (DE015254, DE024153, and †Department of Microbiology, Penn Dental Medicine, University of Pennsylvania, DE024716 to G.H. and DE026152 to G.H. and T.C.). Philadelphia, PA 19104; ‡Research Center for Advanced Oral Science, Graduate Address correspondence and reprint requests to Dr. Athanasios Ziogas and School of Medical and Dental Sciences, Niigata University, 951-8514 Niigata, Japan; x Dr. Vasileia Ismini Alexaki, Institute for Clinical Chemistry and Laboratory Walter Brendel Centre of Experimental Medicine and Institute of Cardiovascular Medicine, Technische Universita¨t Dresden, Fetscherstrasse 74, 01307 Dresden, Physiology and Pathophysiology, BioMedical Centre, Ludwig Maximilians Univer- { Germany. E-mail addresses: [email protected] (A.Z.) sity of Munich, 81377 Planegg-Martinsried, Germany; and Centre for Cardiovascu- and [email protected] (V.I.A.) lar Science, Queen’s Medical Research Institute, University of Edinburgh, EH16 4TJ Edinburgh, United Kingdom Abbreviations used in this article: BAL, bronchoalveolar lavage; ChIP, chroma- tin immunoprecipitation; DEL-1, developmental endothelial locus 1; DHEA, 1Equally contributing first authors. dehydroepiandrosterone; EAE, experimental autoimmune encephalomyelitis; 2Equally contributing senior authors. hDEL-1-promoter-Luc, human DEL-1 promoter/luciferase reporter plasmid; NGF, nerve growth factor; qPCR, quantitative real-time PCR; siRNA, small interfering ORCIDs: 0000-0002-4597-1316 (T.M.); 0000-0002-0471-1462 (J.R.W.); 0000-0002- RNA; TRKA, tropomyosin-related kinase A; WT, wild-type. 4060-1797 (T.T.L.); 0000-0003-4668-4316 (A.N.); 0000-0002-7689-3613 (M.S.). This article is distributed under The American Association of Immunologists, Inc., Received for publication July 2, 2019. Accepted for publication December 27, 2019. Reuse Terms and Conditions for Author Choice articles. This work was supported by grants from the Deutsche Forschungsgemeinschaft (AL1686/2-2 and AL1686/3-1 to V.I.A., CH279/6-2 to T.C., SFB/TRR 205 to V.I. Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 A. and T.C., and SP621/5-1 and SFB914 TP B01 to M.S.), the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900746 2 THE DHEA/DEL-1 ANTI-INFLAMMATORY AXIS and neuronal cells (35, 36). Additionally, it binds and activates the analysis using Ly-6G– allophycocyanin (1A8; BD Pharmingen, Heidel- nerve growth factor (NGF) receptor, tropomyosin-related kinase A berg, Germany) and CD11b–Alexa Fluor 488 (M1/7; BD Pharmingen) (TRKA), in neuronal and microglial cells, thereby triggering Abs. Experiments were approved by the Landesdirektion Sachsen, Dresden, Germany. downstream AKT signaling (30, 37, 38). However, its exact mech- anisms of action, especially in the context of recruitment regulation, Cell lines, primary cells, and cell treatments remain largely unknown (39, 40). HEK-293T cells (American Type Culture Collection, Manassas, VA) were In the current study, we demonstrate that DHEA mitigates cultured in DMEM (Life Technologies) supplemented with 10% FBS, 100 U/ml leukocyte adhesion efficiency in the LPS-induced cremaster muscle penicillin, and 100 mg/ml streptomycin at 37˚C and 5% CO2.HUVECs inflammation model and reduces neutrophil recruitment in the LPS- (Lonza, Basel, Switzerland) were cultured on 0.2% gelatin-coated plates in induced lung inflammation model. Mechanistic studies revealed Endothelial Cell Growth Medium-2