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Activity by Specific Inhibition of Myeloperoxidase Hlf1-11 Exerts Immunomodulatory Effects the Human Lactoferrin-Derived Peptide

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Activity by Specific Inhibition of Myeloperoxidase Hlf1-11 Exerts Immunomodulatory Effects the Human Lactoferrin-Derived Peptide The Human Lactoferrin-Derived Peptide hLF1-11 Exerts Immunomodulatory Effects by Specific Inhibition of Myeloperoxidase Activity This information is current as of September 25, 2021. Anne M. van der Does, Paul J. Hensbergen, Sylvia J. Bogaards, Medine Cansoy, André M. Deelder, Hans C. van Leeuwen, Jan W. Drijfhout, Jaap T. van Dissel and Peter H. Nibbering J Immunol 2012; 188:5012-5019; Prepublished online 20 Downloaded from April 2012; doi: 10.4049/jimmunol.1102777 http://www.jimmunol.org/content/188/10/5012 http://www.jimmunol.org/ References This article cites 39 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/188/10/5012.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average 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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The Human Lactoferrin-Derived Peptide hLF1-11 Exerts Immunomodulatory Effects by Specific Inhibition of Myeloperoxidase Activity Anne M. van der Does,* Paul J. Hensbergen,† Sylvia J. Bogaards,‡ Medine Cansoy,* Andre´ M. Deelder,† Hans C. van Leeuwen,x Jan W. Drijfhout,{ Jaap T. van Dissel,* and Peter H. Nibbering* Because of their ability to eliminate pathogens and to modulate various host immune responses, antimicrobial peptides are consid- ered as candidate agents to fight infections by (antibiotic-resistant) pathogens. We recently reported that hLF1-11 (GRRRRSVQWCA), an antimicrobial peptide derived from the N terminus of human lactoferrin, displays diverse modulatory activities on monocytes, thereby enhancing their actions in innate immune responses. The aim of this study was to identify the cellular target of hLF1-11 Downloaded from that mediates these effects. Results revealed that hLF1-11 binds and subsequently penetrates human monocytes, after which it inhibits the enzymatic activities of myeloperoxidase (MPO). Moreover, a chemical inhibitor of MPO (aminobenzoic acid hy- drazide) mimicked the effects of hLF1-11 on the inflammatory response by monocytes and on monocyte–macrophage differ- entiation. Computer-assisted molecular modeling predicted that hLF1-11 can bind to the edge of and within the crevice of the active site of MPO. Experiments with a set of hLF1-11 peptides with amino acid substitutions identified the stretch of arginines and the cysteine at position 10 as pivotal in these immunomodulatory properties of hLF1-11. We conclude that hLF1-11 may http://www.jimmunol.org/ exert its modulatory effects on human monocytes by specific inhibition of MPO activity. The Journal of Immunology, 2012, 188: 5012–5019. ver the past 60 years, the excessive use of antibiotics in peptides that are active against a variety of microorganisms, in- humans and in animals has resulted in the emergence of cluding multidrug-resistant pathogens. In addition to their direct O multidrug resistance of a variety of microorganisms (1– antimicrobial activities, many antimicrobial peptides also display 3). Today’s acknowledgment of antibiotic resistance as a global immunomodulatory properties. For example, they have been re- threat underscores the need for better antibiotic stewardship and ported to mediate chemotaxis of neutrophils and monocytes (7) by guest on September 25, 2021 novel antibiotics with a mode of action different from that of and induce the production of cytokines/chemokines by immune current anti-infectives. In the past decades, the development of cells (8, 9). They can also stimulate angiogenesis and wound antimicrobial agents has mainly focused on ways to eliminate the healing (10–12) and even modulate monocyte–macrophage dif- pathogen, either by a direct microbicidal activity or by affecting ferentiation (13) and monocyte–dendritic cell differentiation (14). bacterial multiplication, allowing the host’s phagocytic cells to Obviously, identification of the intracellular target(s) of antimi- ingest and kill the invaders. Recently, research has shifted toward crobial peptides in immune cells could provide the basis for fur- exploring the possibility for an alternative way of coping with ther development of agents that modulate the (innate) immune infections through modulation of the host’s immune system, response. thereby enhancing its ability to cope with pathogens. In addition to its antimicrobial effects (15–17), the lactoferrin- Promising candidates in this respect may be found in the class derived peptide hLF1-11—a synthetic peptide comprising the first of antimicrobial peptides (4–6); that is, cationic, relatively short 11 N-terminal residues of human lactoferrin—enhances cytokine and chemokine production by murine and human monocytes in response to microbial stimuli (18). Moreover, this peptide directs *Department of Infectious Diseases, Leiden University Medical Center, Leiden, 2333 ZA, The Netherlands; †Biomolecular Mass Spectrometry Unit, Department of Para- the GM-CSF–driven monocyte–macrophage differentiation to- sitology, Leiden University Medical Center, Leiden, 2333 ZA, The Netherlands; ward an IL-10–producing macrophage subset that shows increased ‡Department of Physiology, VU University Medical Center, Amsterdam, 1081 BT, The Netherlands; xDepartment of Medical Microbiology, Leiden University Medical responsiveness toward microbial stimuli and enhanced phagocy- Center, Leiden, 2333 ZA, The Netherlands; and {Department of Immunohematology tosis and intracellular killing of pathogens (13). The aim of this and Blood Transfusion, Leiden University Medical Center, Leiden, 2333 ZA, The study was to identify the cellular target(s) of hLF1-11 that medi- Netherlands ate(s) its immunomodulatory effects. Received for publication September 26, 2011. Accepted for publication March 16, 2012. Address correspondence and reprint requests to Dr. Peter H. Nibbering, Department Materials and Methods of Infectious Diseases, Leiden University Medical Center, C5-P, Albinusdreef 2, Peptides Leiden, 2333 ZA, The Netherlands. E-mail address: [email protected] The synthetic peptide comprising the first 11 aa of human lactoferrin Abbreviations used in this article: ABAH, aminobenzoic acid hydrazide; ACN, ace- (further referred to as hLF1-11; GRRRRSVQWCA) was purchased from tonitrile; ADHP, 10-acetyl-3,7-dihydroxyphenoxazine; APF, 2-[6-(4-aminophenoxy)- 3-oxo-3H-xanthen-9-yl]-benzoic acid; DCFH-DA, 29,79-dichlorofluorescein-diace- Peptisyntha (Torrance, CA). The control peptide GAARRAVQWAA, N- tate; MFI, mean fluorescence intensity; MPO, myeloperoxidase; ROS, reactive oxy- terminal biotinylated hLF1-11 (biotin–hLF1-11), N-terminal biotinylated gen species. control peptide (biotin–control peptide), and a set of alanine-substituted peptides were from Isogen (De Meern, The Netherlands). The purity of the Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 peptides was determined by reverse-phase HPLC and exceeded 97%. An www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102777 The Journal of Immunology 5013 additional set of peptides (i.e., GAAAASVQWCA, GAARRSVQWCA, Interaction of hLF1-11 with human monocytes GARRASVQWCA, GKKKKSVQWCA, GRRRRSVQWCA, GRRRRSV- m QWUA, GRRRRCSVQWA, GRRRRUSVQWA, and GAARRAVQWAA) Monocytes were incubated with 10 g hLF1-11–biotin/ml at 37˚C and 4˚C was synthesized and purified as described before (19); the purity of these for several intervals up to 70 min, then washed with ice-cold PBS and peptides exceeded 88%. U represents cysteic acid. The peptides were incubated with PE-labeled streptavidin (Invitrogen) for 15 min on ice. tested for endotoxin contamination by incubation with human monocytes Afterward, the monocytes were washed, and the mean fluorescence in- for 24 h. No IL-6, IL-10, IL-12p40, or TNF-a were detected in the tensity (MFI) of the cells was assessed by flow cytometry on a FACSCa- supernatants of these cultures, indicating that these peptides were endo- libur and analyzed using BD CellQuest software (BD Biosciences, toxin free. Heidelberg, Germany). To find out whether the hLF1-11 peptide enters cells, we incubated monocytes with 100 mg hLF1-11–biotin/ml or saline Isolation and culture of human monocytes for 15 or 60 min at 37˚C or for 60 min at 4˚C. Thereafter, the cells were fixed with 4% paraformaldehyde for 10 min, washed three times with PBS, Human monocytes were isolated from buffycoats from healthy donors by and adhered on Labtek II cc2 slides (Nunc, Rochester, NY) by a 15-min Ficoll amidotrizoate density centrifugation. Monocytes were further puri- incubation. Next, the cells were carefully washed with PBS supplemented fied by CD14-positive selection using anti-CD14–conjugated magnetic with 0.05% Tween 20 and permeabilized with 90% methanol for 10 min. microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to Finally, these cells were washed with PBS and incubated with Alexa 647- ∼ the
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