-Derived B, a Potent Cysteine Activator of Plasma Chemerin

This information is current as Paulina Kulig, Brian A. Zabel, Grzegorz Dubin, Samantha J. of October 2, 2021. Allen, Takao Ohyama, Jan Potempa, Tracy M. Handel, Eugene C. Butcher and Joanna Cichy J Immunol 2007; 178:3713-3720; ; doi: 10.4049/jimmunol.178.6.3713 http://www.jimmunol.org/content/178/6/3713 Downloaded from

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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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Staphylococcus aureus-Derived Staphopain B, a Potent Activator of Plasma Chemerin1

Paulina Kulig,2* Brian A. Zabel,2‡§ Grzegorz Dubin,† Samantha J. Allen,¶ Takao Ohyama,‡§ Jan Potempa,†ʈ Tracy M. Handel,¶ Eugene C. Butcher,‡§ and Joanna Cichy3*

Chemerin is an attractant for cells that express the serpentine receptor CMKLR1, which include immature plasmacytoid dendritic cells (pDC) and . Chemerin circulates in the blood where it exhibits low biological activity, but upon proteolytic cleavage of its C terminus, it is converted to a potent chemoattractant. that contribute to this conversion include host serine of the , fibrinolytic, and inflammatory cascades, and it has been postulated that recruitment of pDC and macrophages by chemerin may serve to balance local tissue immune and inflammatory responses. In this work, we describe a potent, pathogen-derived proteolytic activity capable of chemerin activation. This activity is mediated by staphopain B (SspB), a cysteine protease secreted by Staphylococcus aureus. Chemerin activation is triggered by growth medium of clinical isolates of Downloaded from SspB-positive S. aureus, but not by that of a SspBnull mutant. C-terminal processing by SspB generates a chemerin isoform identical with the active endogenous attractant isolated from human ascites fluid. Interestingly, SspB is a potent trigger of chemerin even in the presence of plasma inhibitors. SspB may help direct the recruitment of specialized host cells, including immunoregulatory pDC and/or macrophages, contributing to the ability of S. aureus to elicit and maintain a chronic inflammatory state. The Journal of Immunology, 2007, 178: 3713–3720.

hemoattractants target host-defense effector cells to local nate receptors, but no longer promote , acting instead as http://www.jimmunol.org/ sites of infection and guide developing immune cells to antagonists capable of dampening inflammation (4). C microenvironments suitable for their maturation. The Protease activity can also modulate the function of the chemokine presence of a chemoattractant in a tissue, and the presence of its CX3CL1 that is tethered to the cell surface through a mucin-like cognate receptor on a leukocyte, help define migration specificities stalk. Although it is attached to the cell surface, CX3CL1 acts as (1). It is clear that transcriptional control is important for regulat- a cell adhesion molecule, but after proteolytic cleavage, it func- ing the expression of numerous chemoattractants (2). However, tions as a soluble chemokine (5, 6). Finally, recent advances reveal recent literature shows that posttranslational proteolytic processing the potential for proteases to activate chemoattractants (7, 8).

also provides an important mechanism for the regulation of che- Chemerin, also known as tazarotene-induced 2, has been by guest on October 2, 2021 moattractant function (3). For example, metalloprotease activity recently characterized as a ligand for the seven transmembrane, G leads to the inactivation of several , including the -coupled receptor CMKLR1 (also known as ChemR23 in monocyte chemotactic CCL2, 7, 8, and 13 (MCP1–4), humans and DEZ in mice). Chemerin mRNA is present in most and the general leukocyte attractant CXCL12 (SDF1) (4). In cer- tissues, including liver, pancreas, and skin (7, 8). The liver, how- tain cases, proteolytically truncated chemokines bind to their cog- ever, appears to be a primary source of chemerin, and is likely to be responsible for the high levels of this protein in plasma (8, 9). Chemerin circulates as an inactive precursor (prochemerin) in *Department of Immunology, †Department of Microbiology, Faculty of Biochemis- blood and requires proteolytic processing for activation. Recently, try, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; ‡Stan- ford University Medical School, Stanford, CA 94305; §Veterans Affairs Palo Alto we and others have demonstrated that active chemerin is generated Health Care System, Palo Alto, CA 94304; ¶Skaggs School of Pharmacy and Phar- by serine proteases of the inflammatory, coagulation and fibrino- maceutical Sciences, University of California at San Diego, La Jolla, CA 92093; and lytic cascades (9, 10). These include neutrophil-derived elastase ʈDepartment of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602 and G, mast cell tryptase, as well as factors VIIa, XIIa, Received for publication July 28, 2006. Accepted for publication January 2, 2007. tissue plasminogen activator, -type plasminogen activa- tor, and plasmin. Proteolytically processed chemerin selectively The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance attracts specific subsets of immunoregulatory APCs, such as im- with 18 U.S.C. Section 1734 solely to indicate this fact. mature plasmacytoid dendritic cells (pDCs)4 and macrophages that 1 This work was supported in part by grants from the Polish Ministry of Scientific express CMKLR1 (7–11) providing a potential mechanism for Research 2P04A07629 (to J.C.), 158/E-338/SPB/5.PR UE/DZ 19/2003 (to J.P.), and controlling immune responses at sites of inflammation or tissue 2P04A01129 (to J.P. and G.D.), by a Fogarty International Research Collaborative Award R03TW007174-01 (to E.C.B. and J.C.), a grant from the Commission of the injury. European Communities, Programme QLRT-2001-01250, the European Social Fund With the growing prevalence of bacteria-mediated chronic inflam- and National Budget in the Frame of The Integrated Regional Operational Programme (to G.D.). G.D. is a recipient of the Polish Science Foundation Scholarship for young matory diseases (12–15), there is a need to define the mechanisms scientists. B.A.Z., T.O., and E.C.B. were supported by National Institutes of Health underlying recruitment of Ag presenting and immune regulatory cells Grants AI-59635 and GM-37734. S.J.A. was supported by a postdoctoral fellowship from the Cancer Research Institute, New York. T.M.H. was supported by Grant AI37113-09 from the National Institutes of Health. 4 2 Abbreviations used in this paper: pDC, plasmacytoid dendritic cell; SspB, sta- P.K. and B.A.Z. contributed equally to this work. phopain B; Spl, serine protease-like protein; ScpA, staphopain A; Aur, ; 3 Address correspondence and reprint requests to Dr. Joanna Cichy, Faculty of Bio- TFA, trifluoroacetic acid; MS, mass spectrometry. chemistry, Biophysics and Biotechnology, Ulica. Gronostajowa 7, 30-387 Krakow, Poland. E-mail address: [email protected] Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org 3714 CHEMERIN ACTIVATION BY CYSTEINE PROTEASE to sites of bacteria infection. Staphylococcus aureus is among the plasma for 10 min at 37°C and then tested in an in vitro chemotaxis assay most successful of human pathogens. It is a major cause of skin using 5-␮m pore Transwell inserts (Costar). In each case, enzymatic di- and soft tissue infections and has been implicated in sepsis as well gestion was stopped by placing samples on ice and diluting them 1/15 into chemotaxis medium (RPMI 1640 containing 10% FBS). A total of 100 ␮l as in several chronic inflammatory disorders, including atopic der- of cells (2.5 ϫ 105 cells/well) were added to the top well and tested sam- matitis and psoriasis (15). The success of S. aureus as a pathogen ples were added to the bottom well in a 600 ␮l volume. Migration was may reflect an ability to regulate the recruitment and modulate the assayed for2hat37°C. The inserts were then removed and cells that had function of specialized host cells. migrated through the filter to the lower chamber were collected and counted by flow cytometry (FACSCalibur; BD Biosciences). The results In this work, we demonstrate that the secreted cysteine protease are presented as percent input migration. staphopain B (SspB), a potential virulence factor that is likely to contribute to the chronicity of S. aureus infections, is a potent Human blood pDC transendothelial migration activator of chemerin. Processing of chemerin by SspB may there- The Institutional Review Board (Stanford University) approved all human fore contribute to the pathologic inflammatory response to staph- subject protocols and informed consent was obtained for all donations. ylococcal invasion through recruitment of immunomodulatory Human blood was collected and PBMC were harvested following His- pDC and/or macrophages to sites of infection. topaque 1077 gradient separation. Total PBMC were preincubated1hin chemotaxis medium (RPMI 1640 plus 10% FCS, supplemented with L- glutamine, penicillin/streptomycin, sodium pyruvate, and nonessential Materials and Methods amino acids) at 107 cells/ml to allow for recovery of receptor expression. Bacterial strains and growth conditions Transwell inserts (5-␮m pure; Costar) were coated with 2% gelatin and seeded with 105 HUVECs (passage Ͻ5), and incubated for 24–48 h. Con- Wild-type S. aureus strains 8325-4 and COL (16, 17), and isogenic knock- fluent HUVEC monolayers were confirmed by DiffQuick staining (IMEB). out mutants of individual protease in the 8325-4 genetic background ⌬ ⌬ Monolayers were rinsed with chemotaxis medium before use. A total of Downloaded from were used in this study. Staphopain A ( ScpA), SspB ( SspB), SspB and 6 ␮ ⌬ ⌬ ⌬ 10 PBMC (100 l) were added to the top wells, and various concentra- V8 protease ( SspB V8), and aureolysin ( Aur) deficient strains (18) tions of chemoattractants were added to the bottom wells in a 600-␮l vol- were obtained from Dr. L. Shaw (Department of Biochemistry and Molecular ⌬ ume. Migration was assayed for2hat37°C, then the inserts were removed, Biology, University of Georgia, Athens, GA) while the Spl proteases ( Spl) and the cells that had migrated through the filter to the lower chamber were mutant (19) was a gift from Dr. K. Bayles (Department of Microbiology, harvested, stained (Lin FITC (CD3, CD14, CD16, CD19, CD20, CD56), Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID). Col- CD11c PE, CD123 CyChrome, HLA-DR allophycocyanin; mAbs from BD lection of clinical isolates of S. aureus sampled from joint, blood, and le- Pharmingen and eBioscience), and analyzed by flow cytometry. An equiv- sional skin of patients suffering from bone infection, sepsis, or atopic der- alent number of beads were added to each tube to allow the cell count to http://www.jimmunol.org/ matitis were donated by Dr. S. Eick (Institute of Medical Microbiology, be normalized. University Hospital, Jena, Germany) and Dr. M. Kapinska-Mrowiecka (Zeromski General Hospital, Krakow, Poland). The S. aureus strains were Intracellular calcium mobilization grown in tryptic soy broth (Sigma-Aldrich) at 37°C with shaking at 200 rpm for 14 h. The bacterial density was measured spectrophotometrically Chemoattractant-stimulated Ca2ϩ mobilization was performed following and the cell number was calculated by using previously determined stan- Alliance for Cell Signaling protocol ID PP00000210. CMKLR1/L1.2 ϫ 6 ␮ dard curves. Typically, the bacterial OD (OD600) was within 8–10. No transfectants (5 10 /ml) were loaded with 4 M Fluo4-AM, 0.16% plu- major growth differences were observed among S. aureus strains as con- ronic acid F-127 (Invitrogen Life Technologies) in modified Iscove’s me- firmed by serial dilution and CFU counting on tryptic soy agar. The bac- dium (Invitrogen Life Technologies) plus 1% FBS for 30 min at 37°C. The teria were centrifuged at 5000 ϫ g for 15 min and conditioned medium was samples were mixed every 10 min during loading, washed once, resus- ϭ ϫ 6 by guest on October 2, 2021 collected, diluted with tryptic soy broth to OD600 7, and used in che- pended at 2 10 /ml in the same buffer, and allowed to rest at room motaxis assays and zymography. temperature in the dark for 20 min. Changes in Fluo4 fluorescence in the labeled cells following treatment with chemoattractants were measured Recombinant prochemerin over real-time with a FACScan flow cytometer and CellQuest software (BD Biosciences) at room temperature under stirring conditions (500 rpm). Prochemerin with a C-terminal HIS tag was cloned into pACGP67 (BD 6 Fluorescent data were acquired continuously up to 512 s at 1-s intervals. Biosciences) and transfected into Sf-9 cells. The expressed protein has the The samples were analyzed for 45 s to establish their basal state, then sequence NH2-ADPELTEA. . .LPRSPHHHHHH-COOH, where the un- removed from the nozzle for addition of stimuli, then returned to the nozzle derlined residues are nonnative. After viral amplification, prochemerin was for continued data acquisition. Mean channel fluorescence over time was expressed by adding high titer virus to shaker flasks containing Hi-5 insect analyzed with FlowJo software (Tree Star). cells in Ex-cell 405 medium (JRH Biosciences). After incubation for 2–3 days at 27.5°C, the supernatant was harvested by centrifugation, filtered to SDS-PAGE 0.22 ␮m, and concentrated at 4°C using a tangential flow concentrator (Filtron) with a 3-kDa cutoff filter. After a Ͼ100-fold buffer exchange Recombinant prochemerin was incubated with pure SspB at a 2:1 molar into 50 mM HEPES, 0.3 M NaCl (pH 8.0), prochemerin was purified by ratio at 37°C for 10 min in a buffer containing 50 mM Tris-HCl (pH 7.6), running the solution over nickel-nitrilotriacetic acid (Amersham Bio- 2.5 mM EDTA, and 1 mM DTT. The enzymatic digestion was stopped by sciences) and C-18 reverse-phase HPLC columns (Vydac). The protein addition of the SDS gel loading buffer. Samples were resolved by SDS- was lyophilized and checked for purity using electrospray mass spec- PAGE (13%) under nonreducing conditions. Bands were then visualized trometry (9). using the Coomassie G250 stain (Bio-Rad). Protease purification Gelatin zymography S. aureus V8 protease (glutamylendopeptidase), Aur, and SspB were pu- Bacteria growth medium were resolved by SDS-PAGE (8%) containing rified as previously described (20–35). Proteolytically inactive SspB (gen- 0.1% gelatin (type I from porcine skin; Sigma-Aldrich). After electro- erated by replacing Cys24 with Ala (23)) was a gift from Dr. R. Filipek phoresis, the gel was washed in 2.5% Triton X-100 for 30 min at room (International Institute of Molecular and Cell Biology, Warsaw, Poland). temperature, followed by 40-h incubation at 37°C in buffer containing 50 mM Tris (pH 7.7), 5 mM CaCl and 0.02% NaN . The gel was then stained Cell culture 2 3 with Coomassie to allow the bands of proteolytic activity to be detected as Murine pre-B lymphoma cell line L1.2 and L.1.2 cells stably transfected clear bands of lysis against a stained background. with human recombinant CMKLR1 (CMKLR1/L1.2) (8) were cultured in RPMI 1640 supplemented with 50 ␮g/ml gentamicin and 10% FBS. The Purification of chemerin fragments by reverse-phase HPLC medium for CMKLR1/L1.2 transfectants was also supplemented with 1 For analytical applications, recombinant prochemerin was incubated with mg/ml geneticin. pure SspB at 2:1 molar ratio at 37°C for 10 min in a buffer containing 50 Chemotaxis assay mM Tris-HCl (pH 7.6), 2.5 mM EDTA, and 1 mM DTT. The reaction was stopped by addition of trifluoroacetic acid (TFA) to a final concentration of Growth medium derived from S. aureus, or purified staphylococcal pro- 0.5%. Chemerin cleavage products were isolated using a Waters 501 HPLC teases, were incubated with recombinant human prochemerin or human System on a ␮Bondapak C18 column (3.9 ϫ 300 mm) also obtained from The Journal of Immunology 3715 Downloaded from

FIGURE 1. Activation of recombinant prochemerin by S. aureus-con- ditioned medium. A, A total of 160 nM recombinant prochemerin was FIGURE 2. Among S. aureus-derived proteases only SspB is directly http://www.jimmunol.org/ incubated for 10 min at 37°C with S. aureus growth medium (S. aureus involved in triggering chemerin activity. A total of 160 nM recombinant GM). Samples were diluted 15 times and CMKLR1/L1.2 migration was prochemerin was incubated for 10 min at 37°C with the indicated concentra- assessed by in vitro transwell chemotaxis. Cell migration to growth me- tions of purified Aur, V8, SspB, and enzymatically inactive SspB (inSspB) (A) dium only was used as a negative control. B, A total of 160 nM recombi- or with growth medium from wild-type S. aureus strain 8325-4 (WT), nant prochemerin was incubated for 10 min at 37°C with S. aureus growth growth medium from SspB-deficient mutant (⌬SspB), or growth medium medium from either wild-type S. aureus strain 8325-4 (WT) or various from the SspB-deficient mutant supplemented with 100 nM purified SspB ‚ protease-deficient ( ) strains: ScpA; SspB; V8; Aur; Spl. After the samples (B). Samples were diluted 15 times and CMKLR1/L1.2 migration was were diluted 15 times, CMKLR1/L1.2 migration was assessed by in vitro assessed by in vitro transwell chemotaxis. The mean from duplicate wells transwell chemotaxis. Cell migration to prochemerin alone was used as a of two experiments Ϯ range is shown. control. The mean from duplicate wells of three or four experiments Ϯ SD by guest on October 2, 2021 (p Ͻ 0.001 by Student’s t test comparing (A ,ءء ;p Ͻ 0.05 ,ء .is shown prochemerin alone vs prochemerin ϩ S. aureus GM or (B) comparing various protease-deficient mutants with WT. 2880 ϫ g for 15 min to separate the plasma from leukocytes and platelets. Serum was obtained by allowing the blood to clot for1hat37°C before centrifugation twice at 2880 ϫ g for 15 min. Waters. The column was run for 40 min with a linear gradient from 0 to 80% acetonitrile in water containing 0.1% TFA at a flow rate of 1 ml/min. Absorbance at 220 nm was monitored to detect the eluting peptides. Frac- Results tions containing peptides were collected, flash-frozen in liquid nitrogen, Recombinant prochemerin is activated by a cysteine protease and lyophilized. secreted by S. aureus For preparative applications, prochemerin was resuspended and mixed with SspB at a 100:1 prochemerin:active SspB molar ratio at 37°C. The To determine whether proteolytic enzymes produced by the hu- final buffer conditions for cleavage were 50 mM HEPES, 100 mM NaCl man pathogen S. aureus generate active chemerin, bacteria (pH 7.2), and prochemerin samples were cleaved at a concentration of 1 growth medium was incubated with recombinant full-length mg/ml. Samples containing 2 mg of prochemerin were removed at 150 min (proform) chemerin. As demonstrated in Fig. 1A, CMKLR1- and the components present were separated using a semipreparative C18 Reverse-Phase HPLC column (Grace Vydac), using a gradient of 25–90% transfected L1.2 cells migrated significantly in response to acetonitrile in 0.1% TFA. Fractions from the HPLC purification were ly- prochemerin treated with S. aureus growth medium. No cell ophilized and identified using electrospray mass spectrometry. migration was detected when the growth medium was tested in the absence of prochemerin, or when prochemerin was tested MALDI-TOF mass spectrometry alone, indicating that prochemerin is activated by factors se- The HPLC-purified chemerin fragments were suspended in 10 ␮l of 0.1% creted by S. aureus. TFA in water and mixed 1:1 (v/v) with saturated solutions of either A number of different proteases are secreted by S. aureus. These ␣-cyano-4-hydroxycinnaminic acid or 3,5-dimetoksy-4-hydroxycin- naminic acid, both in water- acetonitrile (2:1; v/v). Samples were then include a metalloprotease (Aur), a serine glutamyl applied to a stainless steel target plate, allowed to dry, and analyzed on a (V8 protease), and serine protease-like proteins (Spl), as well as MALDI-TOF Reflex IV mass spectrometer (Bru¨ker Daltonics) operated in two cysteine proteases known as ScpA and SspB. The involvement a positive-ion mode. of each protease in S. aureus-mediated prochemerin processing Serum and plasma collection was examined using growth medium of isogenic mutants deficient in selected proteases. As shown in Fig. 1B, prochemerin activation The Institutional Review Board at Jagiellonian University approved all was completely abrogated in the SspB mutant (⌬SspB) and in human subject protocols. Plasma and serum were obtained from whole ⌬ ⌬ blood of healthy individuals. To prevent clotting when purifying plasma, the double-deficient SspB V8 mutant. Generation of active blood was collected in the presence of sodium citrate. The blood was then chemerin was also significantly diminished in the absence of Aur centrifuged once at 400 ϫ g for 10 min followed by two centrifugations at (⌬Aur). In contrast, deficiency of the Spl proteases (⌬Spl), or 3716 CHEMERIN ACTIVATION BY CYSTEINE PROTEASE

FIGURE 3. Analysis of SspB-mediated chemerin cleavage products. Recombinant prochemerin was incu- bated with SspB protease at a 2:1 molar ratio for 10 min at 37°C in a buffer containing 50 mM Tris-HCl (pH 7.6), 2.5 mM EDTA, and 1 mM DTT. The reaction was terminated either by addition of TFA to a final concen- tration of 0.5% (for HPLC) or sample buffer (for SDS- PAGE) and the cleavage products were analyzed by HPLC followed by MALDI-TOF or SDS-PAGE. A, HPLC chromatogram of chemerin (20 ␮g) cleavage products, undigested prochemerin, and SspB serve as negative controls. Insets, MALDI-TOF traces of frac- Downloaded from tions I, II, and III. Chemerin fragments are labeled with their mass values. B, SDS-PAGE analysis of chemerin cleavage products. Undigested prochemerin and SspB serve as negative controls. C, The amino acid sequence of recombinant prochemerin used in the study. Arrows point to identified SspB cleavage sites. Underlined cys- teines form the three S-S bonds (3). D, Summary of http://www.jimmunol.org/ chemerin cleavage products. The experimental mass data, the theoretical mass value (calculated mass) of the predicted peptide/protein, and their mass differences are shown for the indicated chemerin fragments collected in HPLC fractions I, II, and III. by guest on October 2, 2021

ScpA (⌬ScpA), did not have a significant effect on chemerin ac- Identification of the SspB cleavage site tivation. These data indicate that SspB and to a lesser extent Aur To determine the SspB-mediated cleavage site(s), recombinant are responsible for S. aureus-mediated chemerin activation. prochemerin was incubated with purified SspB and then subjected Because Aur contributes to the activation of the V8 protease, to a separation by HPLC (Fig. 3A) or SDS-PAGE (Fig. 3B). As which in turn activates SspB (26–28), the lack of Aur or V8 pro- expected, coincubation of prochemerin with SspB resulted in lim- tease may indirectly affect chemerin activation by reducing the ited proteolysis of the chemoattractant (Fig. 3B). HPLC fractions levels of functional SspB produced by these mutants. To determine (labeled I-III) were analyzed by MALDI-TOF mass spectrometry whether Aur and the V8 protease are capable of activating (Fig. 3A, inset). Fraction I (1590.8 Da) corresponds to the C-ter- chemerin directly, we incubated recombinant prochemerin with purified Aur, the V8 protease, and SspB. As demonstrated in Fig. minal fragment of chemerin encompassing residues from 141 to 2A, while SspB was able to activate prochemerin, neither Aur nor 146, plus the proline linker and C-terminal HIS6 tag (Fig. 3, C and the V8 protease were able to activate the chemoattractant. Thus, D). The larger portion of chemerin (16,155.4 Da) corresponding to among enzymes secreted by S. aureus, SspB is an exclusive residues 1–140, was collected in fractions II and III. Identification prochemerin-activating agent. Furthermore, purified SspB restored of two matching chemerin fragments corresponding to the C- and the ability of SspB-deficient medium to trigger the chemerin ac- N-terminal portion of the molecule (1,590.8 and 16,155.4 Da, re- tivity (Fig. 2B). This finding supports the notion that prochemerin spectively) revealed the SspB-mediated chemerin cleavage site to 2 activation by S. aureus growth medium results from a direct effect be NH2..AFS KAL..COOH (Fig. 3, C and D). Fraction II also mediated by SspB and not from clonal or growth differences contained an additional peptide of molecular mass 14,963.1 Da, among staphylococcal strains. Moreover, purified, enzymatically corresponding to residues 1–130, indicating an additional cleavage inactive SspB (inSspB) protein, (the catalytic Cys to Ala mutant) event. In this case, however, the matching C-terminal peptide or its (23) was unable to trigger prochemerin activation, confirming that further degradation products could not be recovered. The 16,155.4 proteolytic activity of SspB is required for S. aureus-mediated che- Da cleavage product of SspB, further referred to as “chem16”, was moattractant activation (Fig. 2A). the primary product observed under conditions when prochemerin The Journal of Immunology 3717 was treated with SspB at 100:1 molar ratio for 150 min, in a buffer containing 50 mM HEPES, 100 mM NaCl (pH 7.2), or at 1000:1 molar ratio for 90 min. in a buffer containing 50 mM Tris-HCl (pH 7.6), 2.5 mM EDTA, and 1 mM DTT (data not shown). Longer incubation resulted in further truncation of prochemerin and the generation of the 14,963.1 Da product, as described above and referred to as “chem15”. Similar SspB cleavage fragments were detected when recombinant prochemerin lacking five of the six histidines in the C-terminal HIS6 tag was used, indicating that SspB-mediated prochemerin cleavage is not affected by the pres- ence of the HIS6 tag (data not shown). To confirm the C-term processing site for the primary chemerin product generated by SspB, we performed MALDI-TOF MS (mass spectrometry) of chem16 and identified a mass value correspond- ing to the nontryptic C-terminal peptide predicted by the MS anal- ysis presented in Fig. 3. Microsequencing of this peptide by col- lision induced dissociation (tandem MS/MS) confirmed the sequence, thus identifying the primary SspB processing site (data not shown). Downloaded from SspB-activated chemerin triggers a functional response in CMKLR1-positive cells We characterized the bioactivity of the two SspB-generated chemerin isoforms using in vitro chemotaxis and calcium mobili- zation assays. Chem16 is a potent chemoattractant, eliciting a ro- bust, dose-dependent chemotactic response in CMKLR1/L1.2 http://www.jimmunol.org/ transfectants. Just 4 pM of chem16 was sufficient to induce a de- tectable migratory response, with 1 nM eliciting the maximal am- plitude of migration (Fig. 4A). Chem16 also triggers intracellular calcium mobilization (Fig. 4B) in a dose-dependent fashion. In contrast, the bioactivity profile of chem15 was similar to the rel- atively inactive prochemerin in both chemotaxis and calcium flux assays (Fig. 4). Chem15 is a poor chemoattractant, requiring con- centrations over 6000 times higher than chem16 to elicit the same by guest on October 2, 2021 magnitude of response (Fig. 4A). Taken together, these data sug- gest that SspB-mediated prochemerin cleavage results in genera- FIGURE 4. Comparison of cell-activating potential of chemerin cleav- tion of the biologically active chem16 form, which is then further age products. Recombinant prochemerin was incubated with SspB at a truncated to an inactive chem15 form. To characterize any inhib- 100:1 molar ratio for 150 min at 37°C in a buffer containing 50 mM itory effect chem15 may have on chem16, CMKLR1/L1.2 trans- HEPES and 100 mM NaCl (pH 7.2). Two cleavage products of SspB, fectants were simultaneously exposed to both isoforms. Chem15 representing chemerin 14963.4 Da (chem15) and chemerin 16155.4 Da did not inhibit the chemotactic response to chem16, even at a mo- (chem16) (see also Fig. 3.) were purified by HPLC and used for cell mi- gration (A and C) and calcium mobilization (B) experiments. A, CMKLR1/ lar ratio of 500:1, respectively (Fig. 4C). We conclude that chem16 L1.2 migration was assessed by in vitro transwell chemotaxis assays using exerts biological effects on CMKLR1-expressing cells despite the indicated doses of SspB-cleaved chemerin (15 and 16 kDa forms). Un- presence of the truncated 15 kDa form. cleaved prochemerin is shown as a control. Representative data for three We next tested to see whether SspB-activated chemerin could experiments are shown. B, CMKLR1/L1.2 were loaded with Fluo4-AM; act as a chemoattractant for human blood plasmacytoid dendritic intracellular calcium mobilization was examined using continuous-acqui- cells, a circulating leukocyte population that expresses CMKLR1 sition flow cytometry. Indicated doses of chem15 and chem16 SspB cleav- and is chemerin responsive (8). In in vitro transendothelial migra- age products and prochemerin were added at the time designated by the tion assays, pDC migrated significantly to picomolar concentra- arrow. Representative data for three experiments are shown. C, CMKLR1/ tions of SspB-activated chemerin (mean 9.5 Ϯ 3.5% SEM input L1.2 migration to chem16 alone (4 pM), chem15 alone (4, 40, 400, and migration to 16–64 pM chem16 vs 0.2 Ϯ 0.1% background mi- 2000 pM), and a mixture of chem16 (4 pM) with indicated concentrations of chem15 was assessed by in vitro transwell chemotaxis. Cell migration to gration) (Fig. 5). pDC also migrated well to nanomolar concen- chemotaxis medium (no chem.) was used as a negative control. The mean trations of the general leukocyte attractant CXCL12 (SDF1) from duplicate wells of four experiments Ϯ SD is shown. (19.6 Ϯ 7.2%). SspB-activated chemerin may therefore contribute to pDC influx to sites of S. aureus infection. medium (Fig. 6A). The effect of clinical isolates on the generation S. aureus strains isolated from clinical patients activate of active chemerin was compared with the effects of the widely chemerin used laboratory strains 8325-4 and COL. As demonstrated in Fig. To determine whether SspB can serve as the pathophysiologic trig- 6B, chemerin activity was triggered by all three clinical isolates ger of chemerin activity, we evaluated sets of clinical S. aureus tested, as well as by strain 8325-4, but not by the laboratory strain isolates for their ability to activate prochemerin. These included COL. Strain 8325-4 was the most potent in inducing prochemerin isolates from patients suffering from sepsis, septic arthritis and activation, which is consistent with its high-level of proteolytic atopic dermatitis. The selected bacterial strains displayed proteo- activity (18). Likewise, the lack of prochemerin activation by the lytic activity as evidenced by zymographic analysis of the growth COL strain can be explained by the negligible SspB proteolytic 3718 CHEMERIN ACTIVATION BY CYSTEINE PROTEASE

FIGURE 5. SspB-activated chemerin is a potent chemoattractant for hu- man blood pDC. Transendothelial migration was investigated using Trans- well inserts coated with HUVEC monolayers. Total PBMC were tested and the migrated cells were collected and stained. pDC were identified as Lin- HLA-DRϩCD123ϩCD11cϪ. A total of 20 nM CXCL12 was used as a positive control. The optimal concentration of chem16 for eliciting pDC chemotaxis was 16–64 pM. The percent input migration of pDC is dis- FIGURE 7. played (mean Ϯ SEM), n ϭ 7 experiments with three different donors; SspB activates human plasma chemerin in the presence of p Ͻ 0.05 in pairwise comparisons with background migration “(Ϫ)no endogenous plasma protease inhibitors. Human plasma was collected in the ,ء ϫ Ϫ7 chem” by Mann-Whitney rank sum test. presence of sodium citrate and incubated (1:1 v/v) with 1 10 Mpu- rified SspB at 37°C for 10 min. Matched serum isolated from the same donor was also collected. Plasma treated with SspB, and an equivalent Downloaded from volume of plasma or serum, was diluted 1/15 into chemotaxis medium, and activity (Fig. 6A). These data indicate that SspB may be involved then tested in a chemotaxis assay with CMKLR1/L1.2 and L1.2 cells. The in generating a chemotactic gradient of chemerin at sites of S. mean from duplicate wells of two experiments Ϯ range is shown. aureus infection in humans.

SspB activates endogenous human plasma chemerin effect of SspB-treated plasma on CMKLR1-expressing cells was http://www.jimmunol.org/ Plasma represents a significant reservoir for proform chemerin in comparable to the effect of serum (Fig. 7), which contains high vivo, where it circulates at low nanomolar concentrations (3). Be- levels of active chemerin that results from processing by serine cause plasma contains a high concentration of protease inhibitors proteases activated during clotting/fibrinolysis (9). (10% of total protein content), to evaluate the pathophysiologic relevance of chemerin activation by SspB it was important to de- Discussion termine whether or not SspB can process prochemerin in the pres- In this work, we show that SspB, an S. aureus-derived cysteine ence of plasma inhibitors. As shown in Fig. 7, incubation of pu- protease, is a potent chemerin activating . The predicted rified SspB with human plasma resulted in prochemerin activation, three-dimensional structure of chemerin suggests that it is related suggesting that the chemerin-activating potential of SspB is not by guest on October 2, 2021 to the cystatin family (3, 7). The physiologic role of cystatins as blocked by plasma protease inhibitors. Moreover, the chemotactic inhibitors of cysteine proteases is exerted through tight complex formation with target substrates (29). Thus, the homology shared between cystatin and prochemerin is predictive of interactions be- tween prochemerin and cysteine proteases. Initially, we tested to see whether prochemerin could function as a cystatin, but found that it did not display significant inhibitory activity toward human cysteine proteases () (data not shown). We did, however, discover that a specific bacterial-derived cysteine protease inter- acts directly with prochemerin, as described in this report. This interaction does not result in inhibition of SspB proteolytic activity (data not shown) but, instead, results in activation of chemerin. It will be interesting to see whether host cysteine proteases, or other pathogen-derived cysteine proteases, are similarly able to activate chemerin. Several lines of evidence from our present work indicate that SspB may have a physiologically relevant role in chemerin acti- vation. First, endogenous SspB released into S. aureus growth me- dium activates chemerin, suggesting that SspB triggers the FIGURE 6. Clinical isolates of S. aureus activate chemerin. Growth chemerin chemotactic activity in the context of other S. aureus medium was harvested from cultures of S. aureus derived from the indi- secreted enzymes. This is important, because high levels of pro- cated anatomical sites from patients with sepsis, bone infection, or atopic teolytic activity often result in protein cleavage at multiple sites, dermatitis, or from the standard laboratory strains 8325 and COL. The leading to nonspecific protein degradation. Second, SspB gener- growth medium was then tested by gelatin zymography (A) or incubated ates active chemerin with a six amino acid C-terminal truncation. with 160 nM recombinant chemerin for 10 min at 37°C (B). A, Protease This particular isoform is identical with an endogenous active hu- activity of indicated strains is visualized using a gelatin-containing gel. The position of SspB is indicated. Two bands represent different maturation man chemerin isoform isolated from ascites fluid (7). Third, the products of SspB (18). B, CMKLR1/L1.2 migration was assessed by in concentration of host proteases known to activate chemerin, in- vitro transwell chemotaxis, after diluting the samples 1/15 with chemotaxis cluding factor XII and VII, as well as plasmin, are in the range of Ϫ7 Ϫ5 medium. The mean from duplicate wells of two experiments Ϯ range is 10 –10 M (9). Therefore, the effect of SspB appears to be shown. pathophysiologically significant because at the concentration of The Journal of Immunology 3719

1 ϫ 10Ϫ7 M, SspB is a very effective trigger of chemerin activa- by active chemerin are capable of complex regulation of immune tion in plasma, yielding chemotactic activity comparable to that responses. One possibility therefore is that S. aureus recruits these generated by all combined host proteases released during blood cells and manipulates their immunoregulatory potential to its own coagulation and fibrinolysis. Fourth, active chemerin accumulates survival advantage. This would be consistent with the ability of S. in SspB-treated plasma despite an abundance of various inhibitors aureus to both initiate and maintain a state of chronic pathologic that keep host-derived proteases under the tight control. Impor- inflammation. tantly, host proteases implicated thus far as chemerin activators, such as neutrophil elastase, cathepsin G, plasmin, urokinase-type plasminogen activator, and tissue plasminogen activator (9, 10) Acknowledgments would be expected to be opposed by plasma inhibitors, including We are grateful to Drs. L. Shaw, K. Bayles, S. Eick, R. Filipek, and M. ␣1-protease inhibitor, ␣1-antichymotrypsin, ␣2-antiplasmin, and Kapinska-Mrowiecka for the reagents and Dr. David King, Berkeley Uni- versity, for help with mass spectrometry. plasminogen activator inhibitor (30–32). At early stages of acute inflammation, the protease-antiprotease balance likely shifts in fa- vor of proteolysis. However, as the inflammatory response contin- Disclosures ues, the plasma concentration of protease inhibitors increases (30, The authors have no financial conflict of interest. 31). Therefore, at later stages of the inflammatory response or during ongoing inflammation, host proteases are neutralized by plasma inhibitors. In contrast to host proteases, SspB remains ac- References tive in plasma, suggesting that it can serve as a chemerin activator 1. Butcher, E. C., and L. J. Picker. 1996. Lymphocyte homing and homeostasis.

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