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

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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/

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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 © 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 considered 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 hydrazide) mimicked the effects of hLF1-11 on the inflammatory response by monocytes and on monocyte–macrophage differentiation. 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 manufacturer’s protocol. The resulting suspension comprised 96% labeled streptavidin (Invitrogen) for 30 min. After rinsing, the cells were monocytes with a viability exceeding 98% as determined by annexin V and incubated with rhodamine-labeled wheat germ agglutinin (Invitrogen) for propidium iodide staining 2 h after isolation. Monocytes were resuspended 20 min to stain the membranes. Thereafter, cells were washed and mounted in RPMI-1640 (Invitrogen, Breda, The Netherlands) supplemented with 2 with hard Vectashield containing DAPI (Vector Laboratories, Amsterdam, mM L-glutamine (Invitrogen), 100 U/ml penicillin (PAA, Pasching, Ger- The Netherlands). High-quality, three-dimensional stacks of optical sec- many), 100 mM streptomycin (PAA), and 10% inactivated FBS (Invi- tions were obtained at room temperature with a Marianas digital imaging trogen), further referred to as standard medium. Monocytes were cultured microscopy workstation (Zeiss 200M; Zeiss, Go¨ttingen, Germany) using Downloaded from 3 6 at a concentration of 1 10 cells/ml of standard medium at 37˚C and 5% a 363 oil-immersion objective (EC-plan neofluar, NA 1.25), a CCD CO2. Monocytes were exposed to the different peptides or the myeloper- camera (Sensicam; PCO, Kelheim, Germany) and a Z-step size of 0.25 oxidase (MPO) inhibitor aminobenzoic acid hydrazide (ABAH; Sigma- mm. Finally, three-dimensional image acquisition, deconvolution, and Aldrich Chemie BV, Zwijndrecht, The Netherlands) immediately at the automated image analysis were performed under full software control start of the culture, and 1 h thereafter these cells were stimulated with 100 (SlideBook version 5.0; Intelligent Imaging Innovations, Denver, CO). ng/ml LPS (Sigma-Aldrich). About 18–20 h thereafter, the supernatants were collected for assessment of cytokine levels using cytoset ELISA kits Isolation of the cellular target(s) of hLF1-11 in human

(Invitrogen). To investigate the involvement of MPO in the effects of monocytes http://www.jimmunol.org/ hLF1-11 on GM-CSF–driven monocyte–macrophage differentiation, monocytes were incubated right after isolation in standard medium supple- Monocytes (3 3 107/ml) were lysed by keeping them on ice for 30 min mented with GM-CSF (5 ng/ml) in the presence of either hLF1-11, control in TNE buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 2 mM peptide, the MPO inhibitor ABAH, or solvent control. MPO oxidizes EDTA) containing a protease inhibitor mixture (Pierce Biotechnology, ABAH to a radical that reduces MPO to its ferrous intermediate. Ferrous Rockford, MA) and 1% Nonidet P-40. Lysates were centrifuged for 10 min MPO reacts either with oxygen to allow enzyme turnover or with hydrogen at 10,000 3 g, and supernatants were stored at 280˚C. Next, hLF1-11 peroxide to give irreversible inactivation (20). After 6 d of culture, the cells target proteins were isolated from these supernatants using the biotinylated were stimulated with LPS (100 ng/ml) for 20 h; thereafter, supernatants protein interaction pull-down kit from Pierce Biotechnology according to were collected for assessment of the production of IL-10 using cytoset the manufacturer’s protocol. In short, hLF1-11–biotin or control peptide– ELISA kits. biotin was immobilized on a streptavidin column. Next, these columns by guest on September 25, 2021

FIGURE 1. Binding to and penetration of human monocytes by hLF1-11. Human monocytes were incubated with biotinylated hLF1-11 for several intervals at 37˚C. Thereafter, the cells were washed, incubated with streptavidin–PE for 15 min, washed again, and then the MFI of the monocytes was assessed by flow cytometry. Results are expressed as boxes and whiskers; boxes represent medians and second and third interquartiles, and whiskers represent range within four independent experiments (A). To determine whether hLF1-11 enters human monocytes or detaches from the cells, monocytes were incubated with biotinylated hLF1-11 for 60 min at 37˚C (B) and 4˚C (C), fixed with 4% paraformaldehyde, permeabilized with 90% methanol, and washed with PBS. Thereafter, the interaction of this peptide with the cells was visualized with Alexa 647-labeled streptavidin (left). The cells were stained with rhodamine-conjugated wheat germ agglutinin (a membrane marker; second image from left) and DAPI (to stain the nucleus; second image from right). Thereafter, cells were analyzed on a Marianas digital imaging microscope using a 633 oil-immersion objective and a Z-step size of 0.25 mm. An overlay of the three images is displayed in the right-most panel. 5014 hLF1-11 INHIBITS MYELOPEROXIDASE ACTIVITY were incubated for 2 h with monocyte lysate supernatant, and after several 0.1% trifluoroacetic acid was used to extract peptides from the gel plugs, washings, proteins were eluted with the manufacturer’s elution buffer. and this extract was pooled with extract 1. Eluted samples were dried in a centrifugal vacuum concentrator (Eppen- MALDI-TOF/TOF analyses were performed on an Ultraflex II mass dorf, Hamburg, Germany) and taken up in a small volume of elution buffer. spectrometer (Bruker Daltonics, Bremen, Germany) using dihydrox- Next, 20 ml of this sample was mixed with 5 mlof53 concentrated ybenzoic acid (5 mg/ml of 50% ACN/0.1% trifluoroacetic acid) as a matrix. nonreducing loading buffer (100 mM Tris-HCl, 4% w/v SDS, 50% glyc- The mass spectrometer was used in the positive ion reflectron mode. Spectra erol, and 0.05% bromophenol blue) and then subjected to SDS-PAGE on were imported in Flexanalysis 3.0 (Bruker) for smoothing, baseline sub- a 10% SDS-polyacrylamide gel. After electrophoresis, the gel was fixed traction, and peak picking. Peak lists were searched against the human IPI overnight in 50% methanol, 12% acetic acid, 0.05% formaldehyde, and database (date of release August 23, 2010, 89,486 sequences) using the then washed three times for 20 min in 35% ethanol. Next, the gel was Mascot search algorithm (Mascot 2.2; Matrix Science, London, U.K.). sensitized for 2 min in 0.02% Na2S2O3 and washed three times for 5 min in Trypsin was selected as the enzyme, and one missed cleavage was allowed. water, followed by staining of the proteins for 20 min in 0.2% (w/v) silver Carbamidomethylcysteine was selected as a fixed modification and oxi- nitrate and 0.076% formaldehyde. After washing, the gel was developed dation of methionine as a variable modification. The mass tolerance was set using 6% (w/v) Na2CO3, 0.05% formaldehyde, and 0.0004% (w/v) to 50 ppm. Na2S2O3 for a maximum of 5 min, after which the reaction was stopped with 50% methanol and 12% acetic acid. Assays for MPO enzymatic activity In-gel tryptic digestion and mass spectrometry The effect of hLF1-11 and the other peptides on MPO enzymatic activities was assessed using an MPO inhibitor screening assay kit of Cayman Europe Protein bands were excised from the gel, cut into small pieces, and washed (Tallinn, Estonia). The assay for OCl2 (hypochlorite) and other strong with 25 mM NH4HCO3 followed by dehydration with 100% acetonitrile oxidants uses the nonfluorescent 2-[6-(4-aminophenoxy)-3-oxo-3H- (ACN) for 10 min. For reduction and alkylation, dried gel particles were xanthen-9-yl]-benzoic acid (APF) to yield the highly fluorescent com- first incubated with 10 mM DTT for 30 min at 56˚C. After dehydration pound fluorescein. The peroxidation assay uses the peroxidase component with ACN, gel plugs were subsequently incubated in 55 mM iodoaceta- of MPO. The reaction between hydrogen peroxide and 10-acetyl-3,7- Downloaded from mide for 20 min at room temperature. After two rounds of washing with 25 dihydroxyphenoxazine (ADHP) produces the highly fluorescent com- mM NH4HCO3 and dehydration with 100% ACN, the gel particles were pound resorufin. Both enzymatic activities of MPO were assessed fol- completely dried in a centrifugal vacuum concentrator (Eppendorf). The lowing the manufacturer’s instructions. In short, formation of hypochlorite dried gel particles were reswollen for 15 min on ice after the addition of 15 (and other strong oxidants) was assayed by mixing several concentrations ml of a trypsin solution (Sequencing Grade Modified Trypsin; Promega, of hLF1-11 or control peptide (range 0.5–250 mg/ml) with MPO (final Madison, WI; 5 ng/mlin25mMNH4HCO3). Subsequently, 20 mlof25 concentration of 114 ng/ml) in the presence of a reaction mixture con- mM NH4HCO3 was added, and the samples were kept on ice for an ad- taining H2O2 (40 mM) and APF (final concentration of 40 mM). For the http://www.jimmunol.org/ ditional 30 min. Tryptic digestion was subsequently performed overnight peroxidation assay, similar concentrations of hLF1-11 or control peptide at 37˚C. The overlaying digestion solution containing the tryptic peptides and MPO were mixed with H2O2 (final concentration of 20 mM) and was collected (extract 1). One additional round of extraction with 20 mlof ADHP (final concentration of 100 mM). MPO alone was used as a positive by guest on September 25, 2021

FIGURE 2. Identiﬁcation of human MPO as the principal binding partner of hLF1-11 in monocytes. To investigate the intracellular binding partner of hLF1-11, biotinylated hLF1-11–coupled and control peptide (CP)-coupled streptavidin columns were used in pull-down assays with monocytic cell lysates as input. After washing, bound proteins were eluted and subjected to SDS-PAGE under nonreducing conditions. hLF1-11 speciﬁcally bound to a protein with an apparent molecular mass of 110 kDa (inset). Tryptic digestion followed by MALDI-TOF MS analysis and database searches using the Mascot search algorithm showed that this protein corresponds to human MPO (IPI00236554, Mascot score 241). Fragments indicated with an asterisk correspondto matched tryptic peptides of MPO (total sequence coverage 35%). The Journal of Immunology 5015

FIGURE 3. MPO activity in the presence of hLF1-11 and control peptide. The peroxidation activity (A) and the formation of hypochlorite (and other strong oxidants) (B) by human MPO were assessed in the presence of several concentrations of hLF1-11 (dark gray) or control peptide (light gray). The results of a represen- tative experiment (out of three independent experiments) are displayed.

control and assay buffer as a negative control. The MFIs of both enzymatic Results activities were measured at an excitation wavelength of 485 nm and an Binding and internalization of hLF1-11 by human monocytes emission wavelength of 535 nm. To investigate if hLF1-11 binds to and penetrates monocytes, we Measurement of intracellular reactive oxygen species in human incubated human monocytes for various time intervals with biotin– monocytes Downloaded from hLF1-11 and assessed fluorescence of these cells by flow cytom- Intracellular reactive oxygen species (ROS) production by human mono- etry. Results revealed that hLF1-11 binds rapidly to monocytes cytes was assayed using the fluorescent probe 29,79-dichlorofluorescein- (Fig. 1A), after which the peptide either detaches, degrades, or diacetate (DCFH-DA; Invitrogen). In short, monocytes were incubated with hLF1-11, control peptide, no peptide, or the MPO inhibitor ABAH for enters the cells as the fluorescence decreased over time. To dis- 1 h at 37˚C. Next, 10 mM DCFH-DA was added to the culture and incu- criminate between these possibilities, monocytes were incubated bated for 20 min at 37˚C in the dark. Thereafter, monocytes were stimu- with biotin–hLF1-11 for 15 or 60 min at 37˚C or 4˚C and analyzed lated with 100 ng/ml LPS for various intervals. ROS production was using fluorescence microscopy. Results showed that biotin–hLF1- http://www.jimmunol.org/ measured on a FACSCalibur and analyzed with BD CellQuest software. 11 mainly associated with the cell membrane of monocytes after Medians of MFI are expressed as boxes and whiskers. 15-min incubation (data not shown). After 60 min of incubation, In silico docking of MPO–hLF1-11 biotin–hLF1-11 was also localized intracellularly (Fig. 1B). Simi- The crystal structures of the human MPO (Protein Data Bank ID: 3F9P) (21) lar results were obtained after incubation of monocytes with la- and of human lactoferricin (Protein Data Bank ID: 1Z6V) (22) were beled peptide at 4˚C (Fig. 1C), suggesting that hLF1-11 passively downloaded from the Research Collaboratory for Structural Bioinformatics enters the cells. Protein Data Bank (http://www.rcsb.org). To generate the hLF1-11 peptide, only the first 11 aa of 1Z6V were selected. Automatic docking using the Identification of the intracellular target of hLF1-11 ClusPro server (23) was applied to model the MPO–hLF1-11 complex. Next, we sought to isolate the intracellular binding partner(s) of by guest on September 25, 2021 The highest ranked model was selected and shown. Docking models were visualized using the Pymol program. Hydrogen bonds and polar contacts hLF1-11 by using a pull-down assay with monocyte lysates and an were defined by the Pymol command “dist name” using default parameters hLF1-11–coated column followed by SDS-PAGE of bound pro- that control the identification. teins. Results revealed that the hLF1-11 column bound a single ∼ Statistical analyses protein with an apparent molecular mass of 110 kDa, whereas no protein was retained by the control peptide-coated column Friedman followed by Dunn’s multiple comparisons post hoc test or, where (Fig. 2, inset). After elution from the column, the hLF1-11 indicated, Wilcoxon’s test was used to determine the differences between the results for hLF1-11–treated, ABAH, and control (peptide-treated) cells. binding protein was subjected to in-gel tryptic digestion and Data are expressed as median and range. Two-sided p values are reported, identified as human MPO using MALDI-TOF mass spectrometry and the level of significance was set at p , 0.05. followed by database searching (Fig. 2). MPO was identified as

FIGURE 4. Intracellular ROS production by hLF1-11–incubated, ABAH-incubated, or control peptide-incubated monocytes in response to LPS. Monocytes were cultured in the presence of hLF1-11 (100 mg/ml, dark gray), control peptide (100 mg/ml, light gray), or no peptide (open) for 1 h and then labeled with DCFH-DA. Next, the labeled monocytes were stimulated with LPS (100 ng/ml), and the MFI was assessed directly by flow cytometry and after 30, 60, and 90 min as a measure of ROS production. Data are expressed as boxes and whiskers; boxes represent medians and second and third interquartiles, and whiskers represent range within experiments with eight different donors (A). In addition, monocytes were cultured in the presence of ABAH (100 mM) or hLF1-11 (72 mM) and then labeled with DCFH-DA. Next, the labeled monocytes were stimulated with LPS for 60 min. Thereafter, intracellular ROS production was assessed by flow cytometry. Data are expressed as boxes and whiskers; boxes represent medians and second and third interquartiles, and whiskers represent range within experiments with seven different donors (B). *p , 0.05 (compared with control monocytes and control peptide-incubated monocytes). 5016 hLF1-11 INHIBITS MYELOPEROXIDASE ACTIVITY the single binding partner of hLF1-11 in three out of three independent experiments. Effects of hLF1-11 on the enzymatic activities of human MPO To investigate whether hLF1-11 affects the enzymatic activities of MPO, we determined both the formation of hypochlorite (and other strong oxidants) and the peroxidation activity of MPO in the presence of hLF1-11 or a control peptide. hLF1-11 inhibited both activities of MPO in a dose-dependent fashion (Fig. 3A, 3B). Surprisingly, the control peptide did not inhibit the peroxidation activity of MPO, but inhibited the formation of hypochlorite (and other strong oxidants), although the control peptide was ∼25 times less efficient than hLF1-11.

Effect of hLF1-11 on the production of ROS by monocytes in response to LPS As MPO ampliﬁes the potency of ROS after initiation of the oxidative burst, we considered the possibility that binding of hLF1-11 to MPO results in reduced ROS production by human monocytes. Downloaded from Results showed that hLF1-11–incubated monocytes produced signiﬁcantly less ROS after LPS stimulation than that of control (peptide-treated) monocytes (Fig. 4A). In addition, the MPO inhibitor ABAH also reduced the LPS-stimulated ROS production by human monocytes (Fig. 4B), indicating that inhibition of MPO

by this chemical mimics the effect of hLF1-11 on the LPS-induced http://www.jimmunol.org/ ROS production.

Comparison of the effects of hLF1-11 and the MPO inhibitor ABAH on LPS-induced responses by monocytes and on monocyte–macrophage differentiation To investigate whether inhibition of MPO by hLF1-11 can explain the immunomodulatory actions of hLF1-11, we compared the effects of hLF1-11 and ABAH on IL-10 production by LPS- stimulated human monocytes (18) and on the GM-CSF–driven by guest on September 25, 2021 monocyte–macrophage differentiation (13). For this purpose, we incubated human monocytes with 100 mM ABAH, 72 mM hLF1- 11 peptide, or the combination of these two MPO inhibitors, as well as with the proper controls (diluents of these compounds) for 1 h and then stimulated the cells for 18 h with LPS. Thereafter, the IL-10 levels in the supernatants of the various monocyte cultures were quantified by ELISA. Results showed that hLF1-11–incu- FIGURE 5. Comparison of the LPS-induced IL-10 production by bated and ABAH-incubated monocytes produced significantly ABAH-incubated and hLF1-11–incubated monocytes and by macrophages higher levels of IL-10 upon stimulation with LPS than those of resulting from monocytes differentiated by GM-CSF in the presence or control monocytes (Fig. 5A). In addition, monocytes incubated absence of these agents. Monocytes were cultured in the presence of with the combination of ABAH (100 mM) and the hLF1-11 pep- ABAH (100 mM), hLF1-11 (72 mM), both, or diluent control for 1 h and tide (72 mM) did not produce significantly more IL-10 in response then stimulated for 24 h with LPS (100 ng/ml). Thereafter, supernatants A to LPS than that of monocytes exposed to either of these com- were collected and assessed for IL-10 production ( ). In addition, monocytes were cultured with recombinant human GM-CSF in the pres- pounds alone (Fig. 5A). Notably, the LPS-stimulated IL-10 pro- ence of ABAH (100 mM) or hLF1-11 (72 mM) for 7 d. Thereafter, IL-10 duction by monocytes did not differ if the cells were exposed first production by the resulting macrophages after LPS was assessed by to ABAH, washed, and then exposed to the peptide or to these ELISA. (B) Lastly, human monocytes were incubated with hLF1-11 or compounds in the reversed order before addition of LPS (data not derived peptides in which one amino acid within the hLF1-11 sequence shown). To investigate the possibility that inhibition of MPO by was replaced by an alanine (all 100 mg/ml) for 1 h; thereafter, the ABAH reduces the responsiveness of the monocytes to hLF1-11, monocytes were stimulated with LPS (100 ng/ml) for 24 h after which the we preincubated monocytes with 500 mM ABAH or its diluent supernatants were collected for assessment of the IL-10 levels (C). All data (0.5% DMSO) for 1 h, washed and reincubated the cells for an are expressed as boxes and whiskers; boxes represent medians and second additional hour with hLF1-11 or no peptide, and then LPS was and third interquartiles, and whiskers represent range within experiments , added. Eighteen hours thereafter, the culture supernatants were with 6–24 different donors. *p 0.05 (compared with control monocytes incubated with hLF1-11), **p , 0.01, ***p , 0.001 (compared with harvested and the levels of IL-10 quantitated. Results revealed that control monocytes). hLF1-11 enhanced the LPS-stimulated IL-10 production by DMSO-preincubated monocytes (p = 0.007), but not by ABAH- preincubated monocytes (p 5 0.24); (the LPS-induced IL-10 cytes; the respective values were 1543 (802–3707; n = 8) and 1020 production by DMSO-preincubated cells exposed to hLF1-11 (557–2143; n = 8) pg of IL-10/ml. These results suggest that MPO amounted to 1718 [487–3180; n = 8] pg/ml and by control cells to is a major mediator of the modulatory effects of the hLF1-11 483 [101–2817; n = 8] pg/ml) and by ABAH-pretreated mono- peptide on monocytes. The Journal of Immunology 5017

Furthermore, monocytes differentiated by GM-CSF to macro- of hypochlorite/hydroxyl radical by MPO (data not shown), in- phages in the presence of ABAH displayed significantly enhanced dicating that the cysteine is indeed able to scavenge radicals levels of IL-10 in response to LPS similar to macrophages that produced by MPO. Moreover, experiments with cysteine-con- differentiated from monocytes in the presence of GM-CSF and taining peptides without any homology to hLF1-11 showed con- hLF1-11 (Fig. 5B). To determine which amino acid(s) in hLF1-11 siderable inhibition of the MPO activity, as has been observed is (are) most essential for this immunomodulatory activity, we previously (25). Together, these data indicate that the presence of compared the effect of a set of peptides with a single amino acid cysteines within the assay mixture was sufficient to inhibit the substitution to alanine on IL-10 production by LPS-stimulated hypochlorite (and other strong oxidants) formation by MPO. monocytes. Notably, results revealed that the cysteine at position As the docking model (Fig. 6A, 6B) suggested that also the 10 was pivotal for the LPS-induced IL-10 production by mono- arginines were of importance for the interaction of hLF1-11 with cytes (Fig. 5C). MPO, we considered the possibility that the arginines interact with human MPO but are not involved in the inhibition of its enzymatic Structural model of MPO with hLF1-11 and identification of activities. To investigate this possibility, we immobilized human amino acids within hLF1-11 primarily responsible for its MPO (purified from extracts of human neutrophils, a kind gift MPO-inhibiting activity of Mrs. N. Klar-Mohamad, Department of Nephrology, Leiden As both the crystal structures of human MPO and hLF1-11 (as part University Medical Center) on the surface of a 96-well plate, of human lactoferricin) have been solved (21, 22), we could preincubated this with hLF1-11 or other peptides, washed away generate a binding model of the two molecules by submitting the the unbound peptides (thereby removing the unspecific scavenging respective structures as receptor and ligand to the ClusPro pro- activity), and then determined the hypochlorite (and other strong Downloaded from tein–protein docking server (see Materials and Methods). This oxidants) formation. Results revealed that peptides in which two predicted that hLF1-11 docks in a crevice containing the heme or more arginines had been replaced by alanines were much less pocket (Fig. 6A). This crevice has previously been identified as effective than the hLF1-11 peptide in inhibiting this enzymatic the catalytic site of MPO (24). The docking model furthermore activity. Substitution of all four arginines with lysine did not predicts a binding mode in which several electrostatic interactions affect the inhibitory activity of the peptide. Furthermore, po-

between hLF1-11 peptide and MPO are involved (Fig. 6B). In sitioning of the cysteine immediately after the arginine stretch http://www.jimmunol.org/ summary, the predicted docking site of hLF1-11 on MPO may did not affect the inhibitory activity of the hLF1-11 peptide. occlude the active site and block the catalytic activity of MPO. Substitution of cysteine with cysteic acid rendered the hLF1-11 These ﬁndings thus provide a molecular model to explain the peptide virtually ineffective in inhibiting the hypochlorite (and observation that hLF1-11 is able to inhibit the enzymatic activities other strong oxidants) formation by MPO (Fig. 6C). Together, of MPO. these results suggest that the arginines in the hLF1-11 peptide Next, we wanted to study in more detail which amino acids are are necessary to bind to the crevice of the active site and that important for the MPO-inhibitory effect of hLF1-11 using an assay the cysteine subsequently interferes with the ROS within the for the formation of hypochlorite (and other strong oxidants) by active site.

MPO. In agreement with the results observed with the cellular by guest on September 25, 2021 assay (Fig. 5C), the cysteine was also essential for the inhibitory Discussion activity of hLF1-11 on this enzymatic activity of MPO (data not We previously reported that the hLF1-11 peptide modulates the shown). As a cysteine can be oxidized and can thereby act as LPS-induced inﬂammatory responses of monocytes and drives a radical scavenger, we wanted to discriminate whether hLF1-11 the GM-CSF–driven monocyte differentiation toward macrophages acted mainly by scavenging or that the peptide inhibited the en- displaying enhanced antimicrobial activities (13, 18). In addition, zymatic activity of MPO differently. Substitution of the cysteine a 60-min exposure of monocytes to hLF1-11 before addition of with cysteic acid (the oxidized form of cysteine) rendered the GM-CSF was sufﬁcient to direct the differentiation toward mac- hLF1-11 peptide virtually ineffective in affecting the production rophages with enhanced effector functions compared with control

FIGURE 6. Model of human MPO with the hLF1-11 peptide docked in the active site and effects of peptides on the hypochlorite (and other strong oxidants) formation by immobilized human MPO. This model shows the molecular surfaces for human MPO. Atoms of hLF1-11 residues are shown in a red-stick representation. Residues surrounding hLF11, making up the peroxidase substrate pocket, are colored blue for better visualization (A). Hydrogen bonds and polar contacts between side chains of hLF1-11 (red) and human MPO (blue) are shown (dotted line). Numbering of residues according to PDB 3F9P. The interaction model was generated by the fully automatic ClusPro protein–protein docking server and visualized using Pymol. (B) To investigate the predicted role of the arginines and cysteine, wells were coated overnight with 100 mlof19mg/ml human MPO at 4˚C. The next day, the nonadherent MPO was removed by washes, the peptide was added, and 1 h thereafter, unbound hLF1-11 was removed by washes, and the amount of hypochlorite (and other strong oxidants) formed was assessed using the reaction mixture containing hydrogen peroxide and substrate. Results are expressed as IC50%, i.e., the concentration of peptide inhibiting 50% of the chlorination activity (C). Data are expressed as means and SEM of three to four independent experiments. U, Cysteic acid. 5018 hLF1-11 INHIBITS MYELOPEROXIDASE ACTIVITY

GM-CSF macrophages (18). Together, a short exposure of mono- Alongside its roles in the host defense, MPO also promotes cytes to hLF1-11 is sufficient to exert its immunomodulatory ef- oxidative damage of host tissues at sites of inflammation and has fects. Because this peptide also inhibits the enzymatic activities of been implicated in diseases such as coronary artery disease (32), MPO, we suggest that these effects may be linked. This suggestion Alzheimer’s disease (33), and kidney disease (34). Accordingly, is supported by the following observations. First, hLF1-11 binds to there is considerable interest in the development of therapeutically and subsequently penetrates monocytes. Second, hLF1-11, but not useful MPO inhibitors (35). In this context, Liu et al. (36) have a control peptide, bound to a single protein in lysates of mono- shown that rabbits pretreated with ABAH and subsequently sub- cytes. This protein was subsequently identified as MPO. Third, mitted to myocardial ischemia and reperfusion displayed signifi- hLF1-11 inhibited the peroxidation and chlorination activity of cantly reduced cardiac caspase-3 activity, suggesting that MPO is purified human MPO. Lastly, the MPO-inhibitor ABAH mimicked a significant contributor to postischemic cardiomyocyte apoptosis. the modulatory effects of hLF1-11 on LPS-induced inflammatory Consistent with this notion, MPO deficiency appeared to be as- responses of monocytes and the GM-CSF–driven monocyte– sociated with lower incidence of cardiovascular heart diseases macrophage differentiation. In addition, monocytes preincubated (37). Finally Askari et al. (38) reported a significant enhanced with a high concentration of ABAH (500 mM) were less respon- preservation of left ventricle function after myocardial infarction sive to LPS than control monocytes using IL-10 production as in MPO-deficient mice compared with that after myocardial in- readout. farction in wild-type mice. Other major findings of this study pertain to the specific amino In conclusion, immunomodulatory effects of hLF1-11 are me- acids in hLF1-11 that are pivotal to its inhibitory effects on MPO. In diated by its ability to inhibit MPO activity. As repeated i.v. our experiments with immobilized MPO in which we removed administrations of 5 mg hLF1-11 were safe for healthy human Downloaded from excess peptide by washes, we found that hLF1-11, but not peptides volunteers (39), the current findings merit further research into the with two or fewer arginines, inhibited the production of hypo- possibility of hLF1-11 as a potential therapeutic agent in diseases chlorite (and other strong oxidants) by MPO. In addition, this in which MPO plays an unfavorable role. peptide, but not peptides only lacking the cysteine residue and such peptides substituted with cysteic acid, inhibited this enzymatic Disclosures activity of MPO. In agreement, the structural docking model of http://www.jimmunol.org/ MPO–hLF1-11 indicated that the four arginine residues of this The authors have no financial conflicts of interest. peptide might bind to the edge of the cleft of the active site of MPO and that the cysteine is close to the heme group in the ac- References tivity center. As hLF1-11 contains 5 nmol (4 nmol of arginine and 1. Valencia, R., L. A. Arroyo, M. Conde, J. M. Aldana, M. J. Torres, F. Fernańdez- 1 nmol of cysteine) of a residue that can compete with APF, the Cuenca, J. Garnacho-Montero, J. M. Cisneros, C. Ortı´z, J. Pachoń, and J. Aznar. 2009. Nosocomial outbreak of infection with pan-drug-resistant Acinetobacter possibility that this peptide competes with the fluorescent sub- baumannii in a tertiary care university hospital. Infect. Control Hosp. Epidemiol. strate for reaction with hypochlorite was considered. However, in 30: 257–263. experiments in which excess peptide remained present during the 2. Pantosti, A., and M. Venditti. 2009. What is MRSA? Eur. Respir. J. 34: 1190– 1196. by guest on September 25, 2021 enzymatic reaction, a peptide with cysteine substituted by alanine 3. Migliori, G. B., K. Dheda, R. Centis, P. Mwaba, M. Bates, J. O’Grady, (still having four arginines) did not inhibit the cleavage of APF by M. Hoelscher, and A. Zumla. 2010. Review of multidrug-resistant and exten- MPO, indicating that the guanidinium groups of the arginines in sively drug-resistant TB: global perspectives with a focus on sub-Saharan Africa. Trop. Med. Int. Health. 15: 1052–1066. hLF1-11 do not effectively compete with APF for hypochlorite. 4. Hancock, R. E., and R. Lehrer. 1998. Cationic peptides: a new source of anti- On the basis of these results, we suggest that hLF1-11 comprises biotics. Trends Biotechnol. 16: 82–88. 5. Hancock, R. E., and H. G. Sahl. 2006. 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