DHEA Inhibits Leukocyte Recruitment Through Regulation of the Integrin Antagonist DEL-1

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/

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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 © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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, chromatin 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 mechanisms 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 (Lonza) at 37˚C and 5% CO2. For in vitro stimulations, HUVEC were prestarved in Endothelial Cell that DHEA counteracts the inhibitory effect of TNF on endothelial Growth Basal Medium-2 (EBM-2; Lonza) containing 1% FCS (Invitrogen, DEL-1 expression, suggesting that DEL-1 might mediate the Carlsbad, CA) for up to 24 h, depending on the assay. DHEA was freshly antirecruitment effect of DHEA. Consistent with this notion, dissolved in ethanol at 10 mM prior to each experiment. Cells were pre- the anti-inflammatory effect of DHEA in the lung inflammation treated for 30 min with DHEA (100 nM or as indicated in the figure legends) or vehicle control (ethanol), followed by addition of LPS (100 ng/ml, model is lost in DEL-1–deficient animals. Furthermore, we show E. coli 0111:B4; InvivoGen) or human rTNF (10 ng/ml; R&D Systems, that DHEA restores the TNF-mediated reduction in DEL-1 ex- Wiesbaden-Nordenstadt, Germany), as described in the figure legends. In pression in endothelial cells by a mechanism that involves the certain experiments, the cells were pretreated with NGF (100 ng/ml; Merck TRKA receptor and PI3K/AKT signaling. These findings sup- Millipore, Darmstadt, Germany) in lieu of DHEA. In experiments investi- gating the receptor involved or downstream signaling molecules, prior to port an anti-inflammatory role of DHEA through restoration of Downloaded from addition of DHEA, the cells were earlier treated with one of the following endothelial DEL-1 expression under inflammatory conditions. reagents; TRKA inhibitor 648450 (Merck Millipore), AKT inhibitor MK2206 (Cayman Chemical, Ann Arbor, MI), PI3K inhibitor LY294002 (Sigma-Aldrich) or its inactive analogue LY303511 (Sigma-Aldrich), or Materials and Methods DMSO (as control), as described in the figure legends. In all experiments, Intravital microscopy of the cremaster muscle sequential treatments were performed without intermediate washing steps; for instance, treatment with TNF was performed in the presence of afore-

Eight to twelve-week-old male C57BL/6 mice (purchased from Janvier mentioned pretreatments. All treatments were performed in 1% FCS EBM-2. http://www.jimmunol.org/ Labs, Le Genest-Saint-Isle, France) were injected i.p. with 2 mg DHEA (Sigma-Aldrich, Munich, Germany) diluted in PBS containing 4.5% eth- Quantitative real-time PCR anol and 1% BSA or the same amount of control vehicle diluent (4.5% ethanol, 1% BSA in PBS), as previously described (37). Thirty minutes Total RNA extraction, cDNA synthesis and quantitative real-time PCR after DHEA injection, 50 ng of LPS (Escherichia coli O111:B4; Sigma- (qPCR) were performed as previously described (21, 46–48). Total RNA Aldrich) were injected intrascrotally. Intravital microscopy was performed was extracted using the NucleoSpin RNA isolation kit (Macherey-Nagel, 3.5 h later. The cremaster muscle preparation was performed as previously Duren, Germany) according to the manufacturer’s instructions. RNA was described (41). Briefly, the scrotum of the mouse was incised, the cre- reverse transcribed with the iScript cDNA Synthesis kit (Bio-Rad Labo- master muscle was exteriorized, additional tissue was removed, and the ratories, Munich, Germany). qPCR analysis was performed using the muscle was then opened through a longitudinal incision and mounted onto SsoFast Eva Green Supermix (Bio-Rad Laboratories), a CFX384 real-time a self-customized stage. During intravital microscopy, the cremaster System C1000 Thermal Cycler (Bio-Rad Laboratories) and the Bio-Rad by guest on September 27, 2021 muscle was constantly superfused with warm superfusion buffer (41). In- CFX Manager 3.1 software. In other experiments, total RNA was extracted travital microscopy was conducted on a BX51WI microscope (Olympus, using the PerfectPure RNA cell kit (5 Prime; Fisher, Gaithersburg, MD). Center Valley, PA) equipped with a 403 saline immersion objective RNA was reverse transcribed using the High-Capacity cDNA Archive kit (MplanFI/RI, 0.8 numerical aperture; Olympus) and a charge-coupled (Applied Biosystems, Foster City, CA) and real-time PCR with cDNA was device camera (Kappa CF8 HS). VirtualDub (version 1.9.11) was used performed using the ABI 7500 Fast System, according to the manufac- for recording of postcapillary venules. Leukocyte rolling was assessed as a turers protocol (Applied Biosystems). The relative amounts of mRNA were percentage of rolling leukocytes relative to the number of leukocytes quantified with the comparative DDCt method (49). The internal control passing the vessel (rolling flux fraction), and leukocyte adhesion efficiency for normalization was GAPDH mRNA. TaqMan probes were purchased as was assessed as the number of adherent leukocytes per square from Life Technologies (Carlsbad, CA) and primers for detection and millimeter relative to the WBC count per microliter. Adherent leuko- quantification of genes were from Invitrogen or Life Technologies. cytes were defined as nonmoving cells or cells with a displacement smaller than one cell diameter during 1 min of observation (42). Leukocyte Gene silencing by RNA interference rolling velocities were measured as averages over a 1-s time window using HUVEC were transfected with small interfering RNA (siRNA) (20 nM; the Fiji software (43). Vessel diameters were measured using a digital image Dharmacon, Lafayette, CO) in combination with Lipofectamine RNAi- processing system (44). Centerline RBC velocity in cremaster muscle MAX reagent (Life Technologies) according to the manufacturer’s in- microvessels was obtained using a dual photodiode and a digital on-line structions. For DEL-1 silencing, HUVEC were transfected with On Target cross-correlation program (CircuSoft Instrumentation, Hockessin, plus SMART pool siRNA (Dharmacon) against EDIL3 (the gene encoding Germany). Wall shear rates were assessed as 4.9 (8 v/d), where v is the DEL-1; hereafter referred to as DEL-1) and control-treated cells were mean blood flow velocity and d is the diameter of the vessel (45). Blood transfected with nontargeting control siRNA pool (Dharmacon). C/EBPb cell numbers were determined through IDEXX ProCyte Dx Hematology silencing was described elsewhere (21). Analyzer (IDEXX Europe B.V.). Experiments were approved by the Regierung of Oberbayern, Germany. Static cell adhesion assay LPS-induced lung inflammation Assays were performed according to a previously described protocol (50). Nunc-Immuno MicroWell 96-well plates (Sigma-Aldrich) were coated LPS-induced lung inflammation was performed in DEL-1+/+ wild-type with 10 mg/ml ICAM-1 (R&D systems) in PBS overnight at 4˚C, washed (WT) and DEL-12/2 mice. DEL-12/2 mice were previously described twice with PBS and blocked with 3% BSA (Sigma-Aldrich) in PBS for 1 h (8). Eight to twelve-week-old male mice were injected i.p. with DHEA in RT. Human neutrophils were isolated from peripheral heparinized blood (80 mg/kg) diluted in PBS containing 4.5% ethanol and 1% BSA or the of healthy volunteers (the procedure was approved by the ethics committee same amount of control vehicle solution (4.5% ethanol, 1% BSA in PBS), of the Technische Universita¨t Dresden), using double gradient separation followed by a second i.p. injection of the same dose of DHEA or vehicle (Histopaque 1119 and 1077; Sigma-Aldrich) (11). Erythrocytes were lysed solution after 24 h. One and a half hours later, anesthetized mice received with RBC lysis buffer (eBioscience, San Diego, CA). Neutrophils were LPS intranasally (0.2 mg/kg E. coli 0111:B4; InvivoGen, San Diego, CA). stained with 4 mM BCECF (Life Technologies) for 15 min and washed After 24 h, mice were anesthetized prior to bronchoalveolar lavage (BAL) with PBS. Cells were then pretreated with 100 nM DHEA or equivalent collection and euthanasia. The total cell number in the BAL fluid was amount of ethanol (control vehicle) in RPMI 1640 0.1% BSA medium for determined in a blinded manner by using a hemocytometer, and the 30 min at 37˚C and plated onto precoated wells (5 3 104 cells/well) in percentage of recruited neutrophils was determined by flow cytometry the presence of 50 ng/ml PMA (Sigma-Aldrich) or equivalent amount The Journal of Immunology 3

FIGURE 1. DHEA reduces leukocyte adhesion and increases leukocyte rolling in the cremaster muscle model. C57BL/6 WT mice were injected i.p. with DHEA or vehicle solution (ctrl); after 30 min, LPS was injected intrascrotally. Intravital microscopy was conducted in cremaster muscle venules 3.5 h later. (A) Adhesion efﬁciency is shown as the number of adherent leukocytes per square millimeter of vessel surface area relative to WBC count per microliter of blood. Data are presented as mean 6 SEM (n = 21–23 analyzed vessels pooled from six mice per group). (B) Leukocyte rolling ﬂux fraction is shown as percentage of rolling leukocytes relative to the number of leukocytes entering the vessel. Data are presented as mean 6 SEM (n = 21–23 analyzed vessels pooled from six mice per group). (C) Leukocyte rolling velocity. Data are presented as mean 6 SEM (n =189 leukocytes from ethanol plus LPS–treated mice [ctrl], Downloaded from and n = 234 leukocytes from DHEA plus LPS–treated mice [DHEA]); leukocytes pooled from six mice per group. (D) Cumulative histogram representing the rolling velocity of 189 leukocytes from ethanol plus LPS–treated mice (ctrl) and 234 leukocytes from DHEA plus LPS–treated mice (DHEA); leukocytes , pooled from six mice per group. *p 0.05. http://www.jimmunol.org/

of DMSO (as control) in 0.1% BSA RPMI 1640 medium without any (Thermo Fisher Scientific, Schwerte, Germany) and diluted in PBS intermediate washing steps. Following an incubation period of 30 min, supplemented with 2% FCS. Samples were analyzed by flow cytometry the wells were washed with PBS. Fluorescence was measured before (FACSCanto II flow cytometer; FACSDiva 6.1.3 software). (cell input) and after washing (adherent cells) using a SynergyTM HT multidetection microplate reader (Biotek Instruments, Winooski, VT). The ELISA percentage of adhesion is defined as the fluorescence of adherent cells by guest on September 27, 2021 divided by the fluorescence of cell input (51). Detection of human DEL-1 protein in culture supernatants was performed by a sandwich ELISA, as described (21), using serial dilutions of recombinant Flow cytometry human DEL-1 (R&D systems) for standard curve generation. Human neutrophils were isolated from peripheral blood of healthy vol- Western blot analysis unteers as described in the static adhesion assay, treated with RBC lysis buffer and washed with HBSS (Invitrogen). Neutrophils were thereafter Western blot was performed as previously described (37). HUVEC were incubated with 100 nM DHEA or equivalent amount of ethanol (control lysed in RIPA lysis buffer supplemented with 1 mM sodium orthovanadate, vehicle) in HBSS containing 1 mM Ca2+, 1 mM Mg2+ and 10 mM HEPES 2 mM PMSF and protein inhibitor mixture (Santa Cruz Biotechnology, for 20 min at 37˚C. Next, they were incubated additionally (without an Heidelberg, Germany), centrifuged and the total protein concentration was intermediate washing step) with 10 nM fMLF (Sigma-Aldrich) or equiv- determined with BCA Protein Assay Kit (Thermo Fisher Scientific). Pro- alent amount of DMSO (control vehicle) for 10 min at 37˚C in the pres- tein lysates were diluted with NuPAGE LDS sample buffer (Thermo Fisher ence of Alexa Fluor 488-conjugated mAb24 Ab (BioLegend, San Diego, Scientific) and NuPAGE sample reducing agent (Thermo Fisher Scientific) CA), which binds the open high-affinity conformation of the human aLb2 and boiled at 95˚C for 3 min. Equal amounts of cellular protein were integrin (52, 53). Cells were washed with cold HBSS containing 1 mM loaded in NuPAGE 4–12% polyacrylamide gradient Bis-Tris Protein Gels Ca2+, 1 mM Mg2+ and 10 mM HEPES, centrifuged and mAb24 binding (Invitrogen) and transferred onto nitrocellulose membranes (GE Health- was analyzed by flow cytometry with a FACSCanto II flow cytometer (BD care, Freiburg, Germany). Membranes were washed in TBS-T buffer, Biosciences, San Jose, CA) and the FACSDiva 6.1.3 software. blocked in TBS-T 5% nonfat milk (BD Biosciences) and immunoblotted HUVEC were treated with DHEA (100 nM) or vehicle control (equal with anti–phospho-AKT (Ser473) (#4060; Cell Signaling, Danvers, MA) amount of ethanol) followed by addition of TNF (10 ng/ml) or LPS (10 ng/ml) and anti-vinculin (#4650; Cell Signaling) in TBS-T 5% BSA, followed by (i.e., TNF or LPS treatment was done in the presence of DHEA [or vehicle incubation with HRP-conjugated secondary Ab (R&D Systems). In other control]). HUVEC were then fixed with 0.5% formaldehyde solution for experiments, membranes were immunoblotted with anti–phospho-TRKA 3 min at 4˚C, washed with PBS and incubated for 30 min with anti-ICAM- (Tyr490) (ab1445; Abcam, Cambridge, MA) in TBS-T 5% BSA, followed 1-PE (HA58; BD Biosciences), anti-VCAM-1- allophycocyanin (51-10C9; by incubation with HRP-conjugated secondary Ab. After extensive BD Biosciences) or anti-E-selectin- allophycocyanin (68-5H11; BD washing, membranes were reblotted with anti-vinculin (#4650; Cell Sig- Biosciences) diluted in PBS containing 0.1% BSA under shaking con- naling) followed by incubation with secondary Ab. Signal detection was ditions on ice. Next, cells were washed with PBS, detached with accutase performed using SuperSignalTM West Pico or Femto Chemiluminescent

Table I. Hemodynamic parameters

Vessel Diameter (mm) Centerline Velocity (mm/s) Shear Rate (s21) WBC (Cells/ml) Control 33.6 6 1.1 2019.0 6 132.8 1506.3 6 115.6 5423.3 6 464.1 DHEA 32.4 6 0.8 1747.8 6 104.6 1325.1 6 74.7 6233.3 6 569.1 Hemodynamic parameters observed in cremaster muscle postcapillary venules of WT mice stimulated with intrascrotal injection of LPS. WBC indicates systemic WBC count (n = 6 mice per group). 4 THE DHEA/DEL-1 ANTI-INFLAMMATORY AXIS

FIGURE 2. DHEA does not directly affect leukocyte b2-integrin–dependent adhesion or endothelial adhesion molecule expression. (A) Neutrophils were pretreated for 30 min with DHEA (100 nM) or ethanol (ctrl), followed by stimulation with vehicle control (2) or PMA for 30 min, and static adhesion to immobilized ICAM-1 was examined. Adhesion is presented as percentage of adherent cells. Data are presented as mean 6 SEM (n = 3). (B)

Neutrophils were pretreated with DHEA (100 nM) or Downloaded from ethanol (ctrl) for 20 min, followed by addition of vehicle control (2) or fMLF (10 nM) for 10 min in the presence of Alexa Fluor 488–conjugated mAb24 Ab. mAb24 binding, which detects an activation-dependent epitope of b2-intergrin, was analyzed by ﬂow cytometry. Data are expressed as relative mean ﬂuorescence intensity (MFI). MFI in ethanol

(ctrl) and vehicle control–treated (2) cells was set as http://www.jimmunol.org/ 100%. Data are presented as mean 6 SEM (n = 4). (C–E) HUVEC were pretreated for 30 min with DHEA or ethanol (ctrl) followed by stimulation with or without LPS or TNF for 6 h. Surface expression of ICAM-1, VCAM-1, and E-Selectin was determined by FACS analysis. Data are expressed as relative MFI and are shown as percentage of control. MFI of control vehicle–treated unstimulated cells (in the absence of LPS or TNF) was set as 100%. Data are presented as mean 6 SEM (n = 5 wells of HUVEC by guest on September 27, 2021 cultures from one experiment, representative of two).

Substrates (Thermo Fisher Scientific) and detected with the Fusion FX7 (21). HEK-293T cells or HUVEC were seeded on 96-well plates at a Multimaging System image analyzer (Peqlab, Erlangen, Germany). density of 5 3 103 cells per well and cotransfected with hDEL-1-promoter- Luc and pGL3 firefly luciferase reporter plasmid (Promega, Madison, WI) Chromatin immunoprecipitation assay followed by qPCR as an internal transfection control, using 4D-Nucleofector V4XC (Lonza) for HEK293T cells or FuGENE HD transfection reagent (Promega) for b Chromatin immunoprecipitation (ChIP) analysis of C/EBP binding to the HUVEC. In experiments in which cells were treated with DHEA or NGF, DEL-1 promoter in HUVEC was performed using the SimpleChIP Plus HEK-293T cells were additionally transiently cotransfected with pCMV6- Enzymatic Chromatin IP Kit with magnetic beads (Cell Signaling), as Entry Tagged Cloning control vector (Origene, Rockville, MD) or with previously reported (21). Cross-linked chromatin was immunoprecipitated NTRK1 cDNA expression plasmid (RC213091; Origene), using TurboFectin b TRKA with nonimmune rabbit IgG or rabbit IgG Ab to C/EBP (Santa Cruz 8.0 transfection reagent (Origene), to overexpress TRKA (HEK-293T ). Biotechnology). Nonimmunoprecipitated cell extracts were used as input Six hours after transfection, the cells were pretreated either with DHEA samples. For qPCR, customized TaqMan-specific primers flanking a C/ (or ethanol; vehicle control) or with NGF (or medium-only control). b 2 2 EBP binding site in the DEL-1 promoter ( 328 to 589 bp) were used These pretreatments were followed without an intermediate washing step by b (Thermo Fisher Scientific). ChIP-qPCR data obtained with the C/EBP stimulation with TNF, as described in the figure legends. HEK-293T cells Ab or nonimmune IgG were normalized using the percentage input express TNFR1 and low levels of TNFR2 (56, 57). Luciferase assay was method that normalizes to chromatin input based on the following performed using the Dual-Glo Luciferase Assay System and GloMax-Multi 3 (Ct[adjusted input] 2 Ct[IP]) equation: % Input = 100 2 (54, 55). Detection System (Promega) according to the manufacturer’s instructions. Luciferase reporter assay Statistical analysis The luciferase reporter assay and the construct of human DEL-1 promoter/ A Mann–Whitney U test or Student t test was used for the comparison of two luciferase reporter plasmid (hDEL-1-promoter-Luc) are described elsewhere groups. One-way ANOVA followed by Dunnett or Tukey multiple-comparison The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. DHEA restores TNF-mediated inhibition of DEL-1 expression in HUVEC. (A and B) HEK-293T cells (A) or HUVEC (B) were transfected with ahDEL-1-promoter-Luc reporter plasmid, followed by stimulation with or without TNF (10 ng/ml) for 16 h and analyzed for luciferase activity. Luciferase activity of untreated cells (2) was set as 1. Data are presented as mean 6 SEM (n = 5 wells of HEK-293T or HUVEC cultures from one experiment representative of three). (C) HUVEC were stimulated with or without TNF (10 ng/ml) for 24 h, and DEL-1 mRNA expression was examined by qPCR. Data were normalized to GAPDH mRNA, and relative gene expression of untreated cells (2) was set in each experiment as 1. Data are presented as mean 6 SEM (n =4 independent experiments). (D) HUVEC were stimulated with or without TNF (10 ng/ml) for 6 h, and DEL-1 protein concentrations were determined in cell culture supernatants by ELISA. Data are presented as mean 6 SEM (n = 4 independent experiments). (E) DEL-1 protein concentrations in cell culture supernatants of HUVEC transfected with siRNA against DEL-1 or nontargeting (ctrl) siRNA were measured by ELISA. Data are presented as mean 6 SEM (n = 5 independent experiments). (F) HUVEC were transfected with a hDEL-1-promoter-Luc reporter plasmid, followed by pretreatment with DHEA (100 nM) or ethanol (ctrl) for 30 min and subsequent treatment with or without TNF (10 ng/ml) for 16 h. Luciferase activity of control vehicle–treated cells (ctrl) was set as 1. Data are presented as mean 6 SEM (n = 5 wells of HUVEC cultures from one experiment representative of three). (G) HUVEC were pretreated for 30 min with increasing concentrations of DHEA or vehicle control (ethanol) at a concentration equal to that present in the highest DHEA dose (vehicle control groups are indicated as DHEA 0 nM), followed by stimulation with or without TNF (10 ng/ml) for 6 h. DEL-1 protein concentrations were determined in the cell culture supernatants by ELISA. Data are presented as mean 6 SEM (n = 5 wells of HUVEC cultures from one experiment representative of three). (H) HUVEC were treated for 24 h with the indicated concentrations of DHEA or vehicle control (ethanol) at a concentration equal to that present in the highest DHEA dose (vehicle control group is indicated as DHEA 0 nM). DEL-1 mRNA expression was analyzed by qPCR. Data were normalized to GAPDH mRNA and relative gene expression of control vehicle–treated cells was set in each experiment as 1. Data are presented as mean 6 SEM; n =3.*p , 0.05. test was used for multiple group comparisons. A p value ,0.05 was endothelium were monitored. Intravital microscopy revealed that considered to be statistically significant. Statistical analysis was performed leukocyte adhesion efficiency, assessed as the number of adherent using GraphPad Prism 6 (GraphPad Software, La Jolla, CA). leukocytes per square millimeter vessel surface area relative to WBC count per microliter of blood, was reduced in DHEA-treated Results mice, as compared with control vehicle–treated animals (Fig. 1A). DHEA inhibits leukocyte adhesion Consistent with its antiadhesive action, DHEA treatment increased We initially examined whether DHEA affects leukocyte–endothelial the leukocyte rolling flux fraction (Fig. 1B) and leukocyte interactions in vivo by performing real-time intravital microscopy rolling velocity (Fig. 1C, 1D). Circulating WBC numbers and in inflamed mouse cremaster muscle venules. To this end, mice hemodynamic parameters, such as shear rates, vessel diameter, were injected i.p. with DHEA or control vehicle solution followed and centerline velocity, did not significantly change upon DHEA by intrascrotal injection of LPS. After the endotoxin challenge, treatment (Table I). These data suggest that DHEA inhibits the cremaster muscle was exposed, and leukocytes adherent to the leukocyte adhesion to the endothelium in vivo. 6 THE DHEA/DEL-1 ANTI-INFLAMMATORY AXIS

FIGURE 4. TNF inhibits DEL-1 expression in a C/EBPb– dependent manner. (A) HUVEC were transfected with a siRNA pool against C/EBPb or a nontargeting siRNA pool, and C/EBPb expression was examined by qPCR. Data were normalized to GAPDH mRNA, and relative gene expression of control siRNA-treated cells was set as 1. Data are presented as mean 6 SEM (n = 5 wells of HUVEC cultures from one experiment representative of three). (B and C) HUVEC were transfected with a siRNA pool against C/EBPb or a nontargeting siRNA pool and stimulated with or without TNF (10 ng/ml) for 6 h. (B) DEL-1 mRNA expression was analyzed by qPCR, using GAPDH mRNA for normalization. Relative gene expression of control siRNA-treated cells was set as 1. Data are presented as mean 6 SEM (n = 5 wells of HUVEC cultures from one experiment representative of three). (C) Downloaded from DEL-1 protein concentrations in cell culture supernatants were determined by ELISA. Data are presented as mean 6 SEM (n = 5 wells of HUVEC cultures from one experiment representative of three). *p , 0.05. http://www.jimmunol.org/

DHEA does not directly affect b2-integrin activation or reduced luciferase activity in HEK-293T cells compared with endothelial adhesion molecule expression untreated cells (Fig. 3A), thus confirming the validity of this ex- Adhesive interactions between b2-integrins on leukocytes and perimental model for mechanistic studies. Similarly, TNF reduced their endothelial counterreceptors are key steps in leukocyte the DEL-1 promoter activity in HUVEC transfected with a hDEL- adhesion. To this end, we first examined whether the inhibitory 1-promoter-Luc plasmid (Fig. 3B). Consistently, TNF reduced by guest on September 27, 2021 effect of DHEA on leukocyte adhesion is associated with al- DEL-1 mRNA expression in HUVEC (Fig. 3C), as we observed tered leukocyte b2-integrin activation. DHEA did not affect before (17). Moreover, TNF diminished the levels of secreted PMA-induced neutrophil adhesion to ICAM-1 (Fig. 2A). Consis- DEL-1 protein in HUVEC, as assessed by ELISA of culture su- tently, DHEA did not affect b2-integrin activation. Specifically, pernatants (Fig. 3D). The validity of the DEL-1 ELISA was DHEA failed to influence b2-integrin activation in fMLF-activated confirmed by the reduced DEL-1 concentrations in supernatants neutrophils, as assessed by flow cytometry using mAb24 (Fig. 2A), of HUVEC pretreated with siRNA against DEL-1 (Fig. 3E). Im- an Ab that recognizes an activation-dependent epitope on portantly, DHEA pretreatment reversed the inhibitory effect of b2-integrins (58). TNF on DEL-1 promoter activity (Fig. 3F) and restored DEL-1 Next, we asked whether DHEA could alter endothelial cell secreted protein levels in a dose-dependent manner in HUVEC adhesion molecule expression. However, surface expression of (Fig. 3G). In fact, the endothelial DEL-1 protein expression in E-selectin, ICAM-1, or VCAM-1 in LPS- or TNF-treated HUVEC TNF-stimulated HUVEC was fully restored in the presence of was not affected by DHEA (Fig. 2C–E). These data collectively a DHEA concentration of 100 nM (Fig. 3G). On the contrary, suggest that the inhibitory effect of DHEA on leukocyte adhe- DHEA did not affect DEL-1 mRNA expression in the absence sion is neither because of regulation of b2-integrin activation in of TNF (Fig. 3H), suggesting that DHEA regulates DEL-1 ex- leukocytes nor because of reduction of adhesion molecule expression only under inflammatory conditions. Moreover, DHEA pression in endothelial cells. posttreatment did not reverse the inhibitory effect of TNF on DEL-1 expression, suggesting that DHEA acts in a preventive DHEA reverses the inhibitory effect of TNF on manner (data not shown). DEL-1 expression In the absence of direct mechanisms that could explain the in- TNF inhibits DEL-1 expression via a b hibitory effect of DHEA on leukocyte adhesion, we interrogated C/EBP –dependent mechanism alternative indirect mechanisms of DHEA. Because DEL-1 is an To understand the mechanism, by which DHEA regulates DEL-1, it inhibitor of leukocyte–endothelial adhesion (8), we sought to in- was imperative to first investigate how TNF downregulates DEL-1 vestigate whether DHEA acts upstream of DEL-1. Specifically, expression. Because we have previously shown that the tran- we determined whether DHEA could regulate endothelial DEL-1 scription factor C/EBPb is recruited to the DEL-1 promoter and expression under inflammatory conditions. Given the importance maintains constitutive expression of DEL-1 (21), we examined of TNF on endothelial cell activation in inflammatory conditions whether TNF could regulate DEL-1 expression via C/EBPb. In- (59), we first examined the effect of TNF on DEL-1 production. To deed, siRNA-mediated C/EBPb silencing in HUVEC (Fig. 4A) this end, HEK-293T cells were transfected with a hDEL-1-promoter- reduced constitutive DEL-1 mRNA expression and secretion Luc plasmid and then treated or not with TNF. TNF significantly (Fig. 4B, 4C). TNF did not further decrease DEL-1 mRNA or The Journal of Immunology 7 protein levels in C/EBPb-deficient HUVEC (Fig. 4B, 4C), revealing that the C/EBPb transcription factor is a critical target of the inhibitory effect of TNF on DEL-1 expression. DHEA reverses TNF-mediated downregulation of DEL-1 via the PI3K–AKT–C/EBPb pathway We next sought to elucidate the mechanisms mediating the promotional effect of DHEA on DEL-1 expression in TNF-stimulated endothelial cells. Given that DHEA activates and exerts anti- inflammatory actions through the PI3K/AKT pathway (36, 37), we initially identified that DHEA induces AKT phosphorylation in HUVEC (Fig. 5A). Next, we asked whether the PI3K/AKT pathway mediates the effect of DHEA on DEL-1 expression. Because C/EBPb is implicated in the regulation of DEL-1 expression by TNF, we examined the effect of DHEA on C/EBPb binding on the DEL-1 promoter in TNF-treated HUVEC pretreated with the PI3K inhibitor LY294002 or its inactive control (LY303511) by ChIP. TNF significantly diminished

C/EBPb binding to the DEL-1 promoter, although this ef- Downloaded from fect was reversed by DHEA. PI3K inhibition with LY294002 abrogated the reversal effect of DHEA (Fig. 5B). Moreover, PI3K inhibition with LY294002 or AKT inhibition with MK2206 abolished the effect of DHEA on restoring DEL-1 mRNA expression in TNF-treated HUVEC (Fig. 5C), thus revealing a crit-

ical role of the DHEA–PI3K/AKT signaling pathway in the http://www.jimmunol.org/ restoration of the endothelial DEL-1 expression under inflammatory conditions. DHEA restores DEL-1 expression in endothelial cells through TRKA We next set out to identify the receptor mediating the promotional effect of DHEA on DEL-1 expression in TNF-treated HUVEC. In this regard, the neurotrophin receptor TRKA is expressed in endothelial cells and can activate the PI3K/AKT signaling pathway by guest on September 27, 2021 in response to DHEA in other cell types (37, 38, 60, 61). We thus reasoned that TRKA may mediate the effect of DHEA on DEL-1 expression in TNF-stimulated endothelial cells. To this end, we first showed that DHEA induced TRKA phosphorylation in HUVEC (Fig. 6A). Next, we performed a luciferase assay in HEK-293T cells cotransfected with the hDEL-1-promoter-Luc construct and a plasmid overexpressing human TRKA (HEK- TRKA 293T ). DHEA reversed the TNF-mediated decrease in DEL-1 FIGURE 5. DHEA restores DEL-1 expression in TNF-treated HUVEC TRKA promoter activity in HEK-293T but failed to do so in HEK- via the PI3K-AKT–C/EBPb pathway. (A) HUVEC were treated with 293T cells (Fig. 6B), which do not express TRKA (38, 62). DHEA for indicated time points, and the phosphorylation status of AKT Moreover, a TRKA inhibitor abolished the effect of DHEA on was examined by Western blot using vinculin as a loading control. One DEL-1 mRNA expression in TNF-treated HUVEC (Fig. 6C). representative out of at least three experiments is shown. (B) HUVEC were Consistent with these findings, the prototype ligand of TRKA, preincubated for 1 h with the PI3K inhibitor LY294002 (20 mM) or its m NGF, also restored DEL-1 mRNA expression in TNF-treated inactive analogue LY303511 (20 M) or DMSO as control, followed by HUVEC and DEL-1 promoter activity in HEK-293TTRKA cells additional pretreatment with DHEA (100 nM) or ethanol as control for 30 min and subsequent stimulation with or without TNF (10 ng/ml) for transfected with hDEL-1-promoter-Luc plasmid (Fig. 6D, 6E). 4 h. Chromatin was immunoprecipitated with nonimmune IgG or anti– These data collectively suggest that DHEA restores DEL-1 C/EBPb IgGandsubjectedtoqPCRtoamplifythe2328- to 2589-bp expression in TNF-activated endothelial cells via the TRKA region of the DEL-1 promoter. Nonimmunoprecipitated cell extracts receptor. were used as input samples. Data are expressed as percentage of input. Means 6 SEM are shown (n = 3 independent experiments). (C)HUVEC DHEA inhibits neutrophil recruitment in WT but not were preincubated for 1 h with the AKT inhibitor MK2206 (20 mM), DEL-1–deficient mice PI3K inhibitor LY294002 (20 mM), or its inactive analogue LY303511 We next determined the biological significance of the ability of (20 mM) or DMSO as vehicle control, followed by additional pre- DHEA to rescue endothelial DEL-1 expression under inflammatory treatment with DHEA (100 nM) or ethanol as vehicle control for 30 min conditions. Specifically, we tested whether DHEA could inhibit and subsequent stimulation with or without TNF (10 ng/ml) for 4 h. DEL-1 mRNA expression was analyzed by qPCR. Data were normalized neutrophil recruitment in an in vivo model of inflammation. We to GAPDH mRNA, and relative gene expression of control vehicle– engaged the model of LPS-induced lung inflammation, in which treated cells was set as 1. Data are presented as mean 6 SEM (n =5 neutrophil recruitment is regulated by DEL-1 (8). DEL-1–sufficient wells of HUVEC cultures from one experiment, representative of three). +/+ 2/2 (DEL-1 or WT) and –deficient (DEL-1 ) mice received i.p. *p , 0.05. injections of DHEA or vehicle control, followed by intranasal 8 THE DHEA/DEL-1 ANTI-INFLAMMATORY AXIS Downloaded from http://www.jimmunol.org/

FIGURE 6. DHEA restores TNF-reduced DEL-1 expression via TRKA. (A) HUVEC were treated with DHEA for 5 min and phospho-TRKA levels were examined by Western blot using vinculin as a loading control. One representative out of at least three experiments is shown. (B) HEK-293T cells were cotransfected with a hDEL-1-promoter-Luc reporter plasmid and either a control vector (left panel) or TRKA cDNA plasmid (right panel). Cells were pretreated with ethanol (ctrl) or DHEA (100 nM) for 30 min followed by stimulation with or without TNF (10 ng/ml) for 16 h. Luciferase activity of control vehicle–treated cells was set as 1. Data are presented as mean 6 SEM (n = 5 wells of HEK-293T or HEK-293TTRKA cultures from one experiment representative of three). (C) HUVEC were pretreated for 30 min with TRKA inhibitor (1 mM) or DMSO as vehicle control, followed by additional pretreatment with DHEA or ethanol as vehicle control for 30 min and subsequent stimulation with or without TNF (10 ng/ml) for 24 h, and DEL-1 mRNA expression was analyzed by qPCR. Data were normalized to GAPDH mRNA, and relative gene expression of vehicle control–treated cells was set in each experiment as 1. Data are presented as mean 6 SEM (n = 8 independent experiments). (D) HUVEC were pretreated with or without NGF (100 ng/ml) for by guest on September 27, 2021 30 min, followed by stimulation with or without TNF (10 ng/ml) for 24 h. DEL-1 mRNA expression was analyzed by qPCR. Data were normalized to GAPDH mRNA, and relative gene expression of cells that were left untreated (ctrl) was set in each experiment as 1. Data are presented as mean 6 SEM (n = 5 experiments). (E) HEK-293T cells were cotransfected with hDEL-1-promoter-Luc reporter plasmid and TRKA cDNA plasmid (HEK-293TTRKA). Cells were pretreated with or without NGF (100 ng/ml) for 30 min, followed by stimulation or not with TNF (10 ng/ml) for 16 h and analyzed for luciferase activity. Relative luciferase activity in untreated cells was set as 1. Data are presented as mean 6 SEM (n = 5 wells of HEK-293TTRKA cultures from one experiment representative of three). *p , 0.05. administration of LPS, and the number of infiltrated neutrophils effect of DHEA on neutrophil recruitment requires the presence was determined in the BAL fluid. WT mice pretreated with of DEL-1 (Fig. 7B). DHEA displayed reduced numbers of recruited neutrophils in the alveolar space as compared with vehicle-treated animals Discussion (Fig.7A).Incontrast,theanti-inflammatory effect of DHEA Endothelial cell–derived DEL-1 can regulate the initiation of in- was abrogated in DEL-12/2 mice, suggesting that the inhibitory flammation by controlling leukocyte–endothelial adhesion and,

FIGURE 7. DHEA reduces neutrophil recruitment in an acute lung inflammation model in WT but not DEL-1–deficient mice. (A)WTand(B) DEL-12/2 mice were injected i.p. with DHEA or vehicle control solution (ctrl), followed by a second i.p. injection of DHEA or vehicle control solution after 24 h, as described in Materials and Methods. One and one-half hours later, mice received LPS intranasally, and after a further 24 h, neutrophil numbers were counted in the BAL fluid. Absolute neutrophil numbers are shown. Data are presented as mean 6 SEM (n = 13 mice per group). *p , 0.05. The Journal of Immunology 9 thereby, leukocyte recruitment (8, 9). Reduced DEL-1 expression linked and may represent an “inflamm-aging” signature. Fur- is observed in several inflammatory conditions, such as human thermore, it is possible that the aging-related decline in DEL-1 and murine periodontitis, multiple sclerosis, and its murine expression might, in part, be secondary to the reduced levels counterpart, experimental autoimmune encephalomyelitis (EAE), of DHEA, given our current findings that the latter positively inflammation-related adrenal gland dysfunction, and in human and regulate the former. murine inflammatory lung pathologies (7, 14, 15, 17–19, 63). We have recently shown that the spatial cellular distribution of Whereas reduced DEL-1 expression leading to increased inflam- DEL-1 determines distinct functions of DEL-1. Whereas endothelial matory cell recruitment may be beneficial in acute infections, cell–derived DEL-1 regulates leukocyte recruitment, macrophage- decreased DEL-1 levels in a chronic setting may exacerbate in- derived DEL-1 promotes resolution of inflammation by mediating flammatory pathologic conditions as seen in naturally occurring phagocytosis of apoptotic neutrophils (efferocytosis) by macro- periodontitis in mice (9). Thus, identification of factors restoring phages, leading to the reprogramming of the latter toward a pro- DEL-1 expression is of particular importance as an anti-inflammatory resolving phenotype (11). Given our current findings that DEL-1 therapeutic approach. acts downstream of DHEA, it can be reasoned that DHEA may have In this study, we demonstrate that the steroid hormone DHEA a proresolving action. Thus, future studies could address whether can restore the inflammation-mediated decrease in endothelial DHEA is present at sufficient levels during the resolution phase DEL-1 expression, thereby inhibiting leukocyte recruitment and, and promotes the expression of DEL-1 in macrophages, thereby hence, inflammation. Although DHEA diminished leukocyte adhe- indirectly contributing to successful inflammation resolution. sion efficiency, resulting in increased leukocyte rolling flux fraction In conclusion, our findings introduce the DHEA–DEL-1 axis as and rolling velocity, these effects of DHEA were not mediated by a novel immune-endocrine homeostatic mechanism that regulates Downloaded from direct interference with leukocyte–endothelial cell interactions. neutrophil recruitment and inflammation, suggesting that DHEA Indeed, DHEA did not regulate b2-integrin–dependent adhesion could provide a novel therapeutic approach in diseases associated or the expression of endothelial cell adhesion molecules but, with excessive neutrophil recruitment or related with reduced rather, the expression of the b2-integrin antagonist DEL-1. The DEL-1 expression. requirement of DEL-1 for the anti-inflammatory effects of DHEA was conclusively demonstrated in LPS-induced lung inflamma- Disclosures http://www.jimmunol.org/ tion, in which DHEA inhibited acute neutrophil recruitment in The authors have no financial conflicts of interest. DEL-1–sufficient but not –deficient mice. Endothelial DEL-1 expression is diminished by acute inflammation (8, 9, 21), in which TNF plays a major role as an acti- References vator of the endothelium (64). In this study, we show that the 1. Nourshargh, S., and R. Alon. 2014. Leukocyte migration into inflamed tissues. Immunity 41: 694–707. TNF-mediated downregulation of endothelial DEL-1 expression 2. Pober, J. S., and W. C. Sessa. 2007. Evolving functions of endothelial cells in (8, 17) occurs at the transcriptional level through blockage of inflammation. Nat. Rev. Immunol. 7: 803–815. C/EBPb bindingtotheDEL-1promoter.DHEAcanreversethe 3. Chavakis, E., E. Y. Choi, and T. Chavakis. 2009. Novel aspects in the regulation of the leukocyte adhesion cascade. Thromb. Haemost. 102: 191–197. by guest on September 27, 2021 inhibitory effect of TNF on endothelial DEL-1 expression through 4. Kourtzelis, I., I. Mitroulis, J. von Renesse, G. Hajishengallis, and T. Chavakis. a pathway that involves the TRKA receptor and downstream 2017. From leukocyte recruitment to resolution of inflammation: the cardinal PI3K/AKT signaling pathway, thereby restoring C/EBPb binding role of integrins. J. Leukoc. Biol. 102: 677–683. 5. Ley, K., C. Laudanna, M. I. Cybulsky, and S. Nourshargh. 2007. Getting to to the DEL-1 promoter. Our findings thus provide a mechanistic the site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. understanding of the anti-inflammatory action of DHEA. Con- Immunol. 7: 678–689. 6. Mitroulis, I., V. I. Alexaki, I. Kourtzelis, A. Ziogas, G. Hajishengallis, and sistently, NGF, the prototype ligand of TRKA, displayed a similar T. Chavakis. 2015. Leukocyte integrins: role in leukocyte recruitment and as regulatory effect on DEL-1 expression. Additionally, it would be therapeutic targets in inflammatory disease. Pharmacol. Ther. 147: 123–135. of interest to know if other steroids with known anti-inflammatory 7. Hajishengallis, G., and T. Chavakis. 2019. DEL-1-regulated immune plasticity and inflammatory disorders. Trends Mol. Med. 25: 444–459. actions may share the regulatory effect of DHEA on DEL-1 ex- 8. Choi, E. Y., E. Chavakis, M. A. Czabanka, H. F. Langer, L. Fraemohs, pression under inflammatory conditions (65). M. Economopoulou, R. K. Kundu, A. Orlandi, Y. Y. Zheng, D. A. Prieto, et al. Steroid hormones may regulate inflammation, although their 2008. Del-1, an endogenous leukocyte-endothelial adhesion inhibitor, limits inflammatory cell recruitment. Science 322: 1101–1104. synthesis is strongly affected by inflammation (66–68), thus sug- 9. Eskan, M. A., R. Jotwani, T. Abe, J. Chmelar, J. H. Lim, S. Liang, P. A. 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DEL-1 promotes inflammation in rodent models of LPS-induced brain inflamma- macrophage efferocytosis and clearance of inflammation. Nat. Immunol. 20: 40–49. tion or EAE (32, 37, 73, 74). Similarly, DEL-1 levels are de- 12. Klotzsche-von Ameln, A., S. Cremer, J. Hoffmann, P. Schuster, S. Khedr, creased in the CNS of multiple sclerosis patients and in the course I. Korovina, M. Troullinaki, A. Neuwirth, D. Sprott, A. Chatzigeorgiou, et al. 2017. Endogenous developmental endothelial locus-1 limits ischaemia-related of EAE (19). Interestingly, high expression of DEL-1 is observed angiogenesis by blocking inflammation. [Published erratum appears in 2019 in immunoprivileged organs, such as the brain, which also ex- Thromb. Haemost.] Thromb. Haemost. 117: 1150–1163. hibit relatively high concentrations of NGF and DHEA (8, 19, 29, 13. Hajishengallis, G., and T. Chavakis. 2013. Endogenous modulators of inflammatory cell recruitment. Trends Immunol. 34: 1–6. 75–77). Intriguingly, aging in humans is associated with increased 14. Yan, S., L. Chen, Q. Zhao, Y. N. Liu, R. Hou, J. Yu, and H. Zhang. 2018. De- circulating levels of TNF and decreased levels of DHEA, although velopmental endothelial locus-1 (Del-1) antagonizes interleukin-17-mediated an age-related decline of DEL-1 expression is described in mice allergic asthma. Immunol. Cell Biol. 96: 526–535. 15. Kang, Y. Y., D. Y. Kim, S. H. Lee, and E. Y. Choi. 2014. Deficiency of devel- and humans as well (9, 26, 63, 78). It is thus tempting to speculate opmental endothelial locus-1 (Del-1) aggravates bleomycin-induced pulmonary that the inverse associations between inflammatory cytokines, such fibrosis in mice. Biochem. Biophys. Res. Commun. 445: 369–374. 16. Baban, B., J. Y. Liu, R. Abdelsayed, and M. S. Mozaffari. 2013. Reciprocal as TNF, and the anti-inflammatory factors DHEA and DEL-1 in the relation between GADD153 and Del-1 in regulation of salivary gland inflam- context of inflammatory diseases and aging may be mechanistically mation in Sjo¨gren syndrome. Exp. Mol. Pathol. 95: 288–297. 10 THE DHEA/DEL-1 ANTI-INFLAMMATORY AXIS

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