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Regulation of Chemerin Chemoattractant and Antibacterial Activity by Human Cysteine

This information is current as Paulina Kulig, Tomasz Kantyka, Brian A. Zabel, Magdalena of September 28, 2021. Banas, Agnieszka Chyra, Anna Stefanska, Hua Tu, Samantha J. Allen, Tracy M. Handel, Andrzej Kozik, Jan Potempa, Eugene C. Butcher and Joanna Cichy J Immunol 2011; 187:1403-1410; Prepublished online 29

June 2011; Downloaded from doi: 10.4049/jimmunol.1002352 http://www.jimmunol.org/content/187/3/1403

References This article cites 31 articles, 16 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/187/3/1403.full#ref-list-1

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Regulation of Chemerin Chemoattractant and Antibacterial Activity by Human Cysteine Cathepsins

Paulina Kulig,*,1 Tomasz Kantyka,†,1 Brian A. Zabel,‡,1 Magdalena Banas´,* Agnieszka Chyra,* Anna Stefan´ska,* Hua Tu,x Samantha J. Allen,{ Tracy M. Handel,{ Andrzej Kozik,‖ Jan Potempa,†,# Eugene C. Butcher,‡,** and Joanna Cichy*

Chemerin, a ligand for the G-protein coupled receptor -like receptor 1, requires C-terminal proteolytic processing to unleash its chemoattractant activity. Proteolytically processed chemerin selectively attracts specific subsets of immunoregulatory APCs, including chemokine-like receptor 1-positive immature plasmacytoid dendritic cells (pDC). Chemerin is predicted to belong to the structural /cystatin family of proteins composed of antibacterial polypeptide and inhibitors of cys- teine proteinases (cystatins). We therefore hypothesized that chemerin may interact directly with cysteine , and that it might also function as an antibacterial agent. In this article, we show that chemerin does not inhibit human cysteine proteases, but Downloaded from rather is a new substrate for (cat) K and L. cat K- and L-cleaved chemerin triggered robust migration of human blood- derived pDC ex vivo. Furthermore, cat K- and L-truncated chemerin also displayed antibacterial activity against Enterobacter- iaceae. Cathepsins may therefore contribute to host defense by activating chemerin to directly inhibit bacterial growth and to recruit pDC to sites of infection. The Journal of Immunology, 2011, 187: 1403–1410. http://www.jimmunol.org/ hemerin is a recently characterized chemoattractant pro- tease-mediated, C-terminal truncation to acquire chemotactic tein that serves as a ligand for the seven-pass G-protein activity. Truncated, bioactive chemerin lacking 6 (chemS157), 8 C coupled receptor chemokine-like receptor 1 (CMKLR1) (chemA155), or 9 (chemF154) amino acids in the C terminus have (1, 2). Two additional heptahelical receptors, GPR1 and CCRL2, been isolated from several biofluids, including ascites, serum, and have been reported to bind chemerin, although they do not appear hemofiltrate, respectively (reviewed in Ref. 5). Serine proteases of to directly support chemotaxis (3, 4). Chemerin circulates as an the coagulation, fibrinolytic, and inflammatory cascades, includ- inactive precursor (prochemerin) in blood and undergoes pro- ing elastase and cathepsin (cat) G, generate bioactive chemerin (6, 7). In addition, chemerin can be activated in a se- quential manner by plasma carboxypeptidases after initial cleav- by guest on September 28, 2021 *Department of Immunology, Faculty of Biochemistry, Biophysics and Biotechnol- age by plasmin (8). ogy, Jagiellonian University, 30-387 Krakow, Poland; †Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30- Cells that are critical in linking the innate and adaptive immune 387 Krakow, Poland; ‡Veterans Affairs Palo Alto Health Care System, Palo Alto, CA responses, such as plasmacytoid dendritic cells (pDC), NK cells, and 94304; xLakePharma, Inc., Belmont, CA 94002; {Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093; macrophages, express CMKLR1 and respond to chemerin through ‖Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and chemotaxis (1, 2, 9–12). Although the structure of chemerin has # Biotechnology, Jagiellonian University, 30-387 Krakow, Poland; Center for Oral not yet been solved, the predicted structural homology between Health and Systemic Diseases, University of Louisville School of Dentistry, Louis- ville, KY 40202; and **Stanford University Medical School, Stanford, CA 94305 chemerin and inhibitors of cysteine proteinases (cystatins) and 1P.K., T.K., and B.A.Z. contributed equally to this work. antimicrobial cathelicidins (1, 5) suggests that chemerin may in- hibit endogenous human cysteine proteases and possibly exhibit Received for publication July 13, 2010. Accepted for publication May 29, 2011. antibacterial activity. Alternatively, host cysteine proteases may This work was supported by the Ministry of Scientific Research, Poland (Grants SPUB3088 and 0724/B/P01/2011/40 to J.C.), a Fogarty International Research Col- bind and proteolytically process chemerin. In support of the latter, laborative award (R03TW007174-01 to E.C.B. and J.C.), a grant from the European we recently reported that the cysteine staphopain B Union 6th FP project (SP6MTKD-CT-2006-042586 to J.C.), a Team award from the (SspB) secreted by the human pathogen Foundation for Polish Science (TEAM/2010-5/1 to J.C.), and the National Institutes of Health (Grant AI079320 to B.A.Z. and National Institutes of Health grants to E.C.B.). selectively cleaves and activates chemerin (13). T.H. is supported by National Institutes of Health Grant R01-AI37113. J.P. is sup- The -like cysteine proteases, including cat B, cat L, and ported by the European Community Gums & Joints project (7FP-HEALTH-2010- 261460), the Ministry of Scientific Research, Poland (Warsaw, Poland; Grant 1642/B/ cat K are well-known degradative of mammalian cells, P01/2008/35), and the Foundation for Polish Science (TEAM Project DPS/424-329/ participating primarily in intracellular proteolytic pathways (such 10). The Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian as Ag processing and presentation), but also extracellular protein University is a beneficiary of structural funds from the European Union (Grant POIG.02.01.00-12-064/08). turnover. Recent studies show that lysosomal cathepsins can exert Address correspondence and reprint requests to Dr. Joanna Cichy, Faculty of Bio- their proteolytic activity at extracellular sites (14, 15), where they chemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7 contribute to a variety of pathophysiological processes, including Street, 30-387 Krakow, Poland. E-mail address: [email protected] chronic inflammation associated with obesity (16–18). Abbreviations used in this article: cat, cathepsin; chem, chemerin truncated at in- Cathelicidins consist of two distinct domains: a highly con- dicated residue position; chemS, chemerin serum form; chem/SspB, staphopain B- truncated chemerin; CID, collision-induced dissociation; CMKLR1, chemokine-like served N-terminal cathelin-like domain with homology to the cyst- receptor 1; hCAP18, human cationic antimicrobial of 18 kDa; MHB, Muel- atins and a divergent C-terminal antimicrobial region that varies ler–Hinton broth; MS, matrix-assisted laser desorption ionization time-of-flight mass among species. Only one cathelicidin has been described in hu- spectrometry; pDC, plasmacytoid ; SspB, staphopain B. mans: human cationic antimicrobial peptide of 18 kDa (hCAP18). Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 hCAP18 is cleaved by neutrophil serine proteases such as pro- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002352 1404 CHEMERIN ACTIVATION BY HOST CYSTEINE CATHEPSINS teinase 3 to generate a 37-aa antimicrobial peptide LL-37 and a harvested after LSM1077 (PAA Laboratories) gradient separation, as de- 103-aa cathelin-like domain (19, 20). scribed by the manufacturer. pDC were enriched from PBMC using negative In this study, we found that although chemerin does not inhibit selection with biotinylated mAbs directed against CD3, CD14, CD16, CD19, CD20, CD56, and anti-biotin MACS Microbeads (Miltenyi Biotec), the proteolytic activities of cat L or K, these cysteine proteases are according to the manufacturer’s recommendations. Cells were blocked potent activators of chemerin. cat L and K initially and efficiently with 50–80% autologous plasma and then stained for flow cytometry cleave prochemerin to release a 6-aa peptide from the carboxyl analysis using mAbs against CD123 and BDCA-2 to identify pDC. Stained terminus, generating chemS157; the enzymes can also cleave cells were analyzed on an LSRII flow cytometer (Becton Dickinson). chemerin to release a 38-residue C-terminal peptide, generating Chemotaxis assay chemR125. The activated chemerin S157 is a potent attractant for + Cathepsins were incubated with recombinant human prochemerin for 10 min CMKLR1 cells, including human blood pDC. In addition, we at 37˚C and then tested in an in vitro chemotaxis assay using murine pre-B demonstrate that although the smaller chemerin fragment generated lymphoma L1.2 cells stably transfected with human recombinant CMKLR1 by both cathepsins (chemerin R125) does not support chemotaxis (CMKLR1/L1.2) or purified human blood pDC. In each case, enzymatic of CMKLR1+ cells, both chemS157 and chemR125 display com- digestion was stopped by placing samples on ice and diluting with che- motaxis medium (RPMI 1640 containing 10% FBS). Where indicated, parable antimicrobial activity against Enterobacteriaceae. recombinant chemerin isoforms, Fc-chemerin fusion proteins, or Fc alone were used. A total of 100 ml cells (2.5 3 105 cells/well) was added to the Materials and Methods top well of 5-mm pore transwell inserts (Costar), and test samples were added to the bottom well in a 600-ml volume. Migration was assayed for Materials 2 h at 37˚C. The inserts were then removed, and cells that had migrated through the filter to the lower chamber were collected and counted by flow

Recombinant cat B, L, and K, and chemS157 were purchased from R&D Downloaded from Systems. Anti-CD3, -CD14, -CD16, -CD19, -CD20, -CD56 biotin-linked cytometry (FACSCalibur; BD Biosciences). The results are presented as mAbs, as well as FITC-labeled CD123 and allophycocyanin-labeled percentage input migration. CXCL12 served as a positive control. BDCA-2 were obtained from BD Pharmingen, Miltenyi Biotec, eBio- Antimicrobial assay science, and BioLegend. Recombinant chemerin isoforms, full-length prochemerin, chemerin serum form (chemA155), and SspB-truncated (HB101, a conventional laboratory strain) and Klebsiella chemerin (chem/SspB; chemS157) were produced as previously de- pneumoniae (a clinical isolate from human bronchoalveolar lavage fluid) scribed (2, 13, 21). Recombinant Fc-chemerin proteins were produced and were used in this study. The antimicrobial activity of the indicated che- purified from Chinese hamster ovary cells via transient transfection and merin forms was estimated using a microtiter broth dilution assay (22). A http://www.jimmunol.org/ protein A purification. DNA fragments corresponding to the desired single colony of bacteria was inoculated into 20 ml Mueller–Hinton broth chemerin proteins were amplified by PCR and cloned in-frame down- (MHB; Difco) and incubated overnight at 37˚C, subcultured once at 1:100 stream of human IgG1 Fc domain, which is downstream of a secretion dilution in MHB, and then grown for 2–3 h to midlogarithmic phase. Cell signal peptide in mammalian expression vector pLEV113 (LakePharma). numbers were calculated using previously determined standard curves, and There is a 9-aa glycine-rich linker between the Fc and chemerin domains. for subsequent experiments, bacteria were used at 2–7 3 105 CFU/ml. Plasmid DNA was transfected into Chinese hamster ovary cells using Bacterial suspensions (90 ml) in MHB were mixed with 10 ml diluent Lafectine transfection reagent (LakePharma), and cell culture supernatant (10 mM HEPES, 100 mM Tris, 150 mM NaCl, and 0.45% NaOAc, or was collected 3 d posttransfection. Fc fusion proteins were purified with MHB)(control)or10ml different concentrations of chemerin, Fc- Protein A resins (Mab Select SuRe GE Healthcare), and final proteins were chemerin fusion proteins, Fc alone, or synthetic LL-37 (Emory Micro- formulated in 100 mM Tris, 150 mM NaCl, and 0.45% NaOAc. chemical Facility) and incubated at 37˚C for the indicated times. After by guest on September 28, 2021 serial dilutions with MHB, the diluted mixture was plated on MHB agar Inhibition assays plates and incubated at 37˚C overnight for enumeration of CFU. In se- lected experiments, samples of the bacteria/peptide mixtures were also Inhibitory activity against cat B, K, and L was assayed fluorometrically analyzed by flow cytometry and by spectrophotometry. These methods using a Molecular Devices Gemini XS system, with an excitation of 350 produced comparable results to the colony-forming assay (data not shown). nm and emission wavelength of 460 nm. Cathepsins were preactivated in a buffer containing 100 mM sodium acetate, pH 5.5, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, and 0.01% Tween 20 for 10 min at 25˚C. Results Cathepsins (0.2 nM) were then incubated with 10 or 100 molar excess of cat L and K activate chemerin chemoattractant activity recombinant prochemerin or the nonspecific cysteine proteinase inhibitor E64 for 30 min at 37˚C in the same buffer containing the fluorogenic We initially tested chemerin for cystatin-like activity. Substrate substrates Z-Phe-Arg-AMC (10 mM; Sigma) for cat B and L or Boc-VLK- hydrolysis by cat B, L, or K was not significantly inhibited by m AMC (10 M; International) for cat K, in a total volume of 100 prochemerin, serum-form bioactive chemerin (chemA155), or ml. The progress of the reaction was monitored by fluorescence spec- troscopy, and the data were plotted versus time. Maximum velocity values SspB-activated chemerin (chemS157), even at 10:1 and 100:1 were calculated by linear fit to the time-dependent curve. The residual molar ratios of chemerin-to-cathepsin (Fig. 1, Table I). The general cathepsin activity present in each treatment group is presented as a per- cysteine proteinase inhibitor E64 efficiently abolished the activity centage of the maximum velocity determined in the absence of inhibitor of all cathepsins examined (Fig. 1). Thus, chemerin does not ap- (cathepsin + substrate only). pear to function as a cystatin. Mass spectrometry Prochemerin was incubated with purified cat B, L, or K and Matrix-assisted laser desorption ionization time-of-flight spectrometry tested for attractant activity to test whether prochemerin is a sub- (MS) was performed by the Stanford Protein and Nucleic Acid Bio- strate for human cysteine proteases. A controlled digest of pro- technology Facility (Stanford University, Stanford, CA). After trypsin chemerin, using low concentrations of either cat L or K (1000-fold digest of the cat K and L truncated-chemerin band, MS was performed less than prochemerin) generated a single primary proteolytic and the mass values were used in a Mascot search (http://www.matrixs- product (Fig. 2A). Two apparent cleavage products were generated cience.com) of public peptide databases. PeptideCutter was used to predict the mass values of tryptic chemerin fragments (http://www.expasy.org). when cat L and K concentrations were increased 10–50 times, Mass values obtained after collision-induced dissociation (CID) of the suggesting further digestion of prochemerin. Under similar con- predicted C-terminal peptide (f) were compared with the predicted CID ditions, cat B did not cleave prochemerin (data not shown). In- mass values for the peptide corresponding to residues 141–157 of chem- terestingly, the two specific .10-kDa chemerin cleavage products erin (sequence: AGEDPHSFYFPGQFAFS), using Mascot software (Ma- trix Science). generated by cat L and K can be distinguished by PAGE only under reducing conditions (Fig. 2A). Because prochemerin con- Purification of pDC tains three disulfide bonds, these data suggest that under non- The Institutional Review Board at Jagiellonian University approved all reducing conditions, the dual-cleaved chemerin products remain human subject protocols. Human blood was collected and PBMCs were associated with the holo-molecule through S–S bond(s). The Journal of Immunology 1405 7 13 13 7, 21 References This study This study 163 155 157 157 PH 148 a 125 Downloaded from http://www.jimmunol.org/ VLGRLVHCPIETQVLR C-Terminal Sequence

FIGURE 1. Chemerin does not inhibit the activity of the cysteine 111 cathepsins. Cathepsins were incubated with 10 (103) or 100 molar excess (1003) of recombinant prochemerin (pro-chem), serum chemerin (chemA155), or chem/SspB (chemS157), or 100 molar excess of the

general cysteine proteinase inhibitor E64 for 30 min at 37˚C with fluoro- VLGRLVHCPIETQVLREAEEHQETQCLRVQRAGEDPHS genic substrates. *p , 0.05, Student t test. Data are shown as mean 6 SD of three independent measurements done in duplicate. Control contained 111 VLGRLVHCPIETQVLREAEEHQETQCLRVQRAGEDPHSFYFPGQFAFS VLGRLVHCPIETQVLREAEEHQETQCLRVQRAGEDPHSFYFPGQFAFS no inhibitor. VLGRLVHCPIETQVLREAEEHQETQCLRVQRAGEDPHSFYFPGQFA by guest on September 28, 2021 111 111 VLGRLVHCPIETQVLREAEEHQETQCLRVQRAGEDPHSFYFPGQFAFSKALPRS 111 111 CMKLR1-transfected L1.2 cells migrated significantly to cat L- or K-treated prochemerin (Fig. 2B). The chemotactic response of CMKLR1/L1.2 transfectants was dependent on cathepsin con- centration, with cat L and K eliciting maximal effects on chemerin-mediated migration at a 1:100 prochemerin/cathepsin ratio (Fig. 2B). Although chemerin treated with cat L appeared to elicit slightly higher chemotactic response compared with cat K-treated chemerin (Fig. 2B), the difference was not statistically Prochem chemS157 chemR125 chemA155 significant. Freshly isolated CMKLR1+ human blood pDC also Abbreviations migrated in response to cathepsin-cleaved chemerin, suggesting Minor form chemS148 chem/SspB; chemS157 that these enzymes may be involved in pDC recruitment (Fig. 2C). No cell migration was detected in the absence of chemerin, or when prochemerin or the cathepsins were tested alone (Fig. 2). Compared with cat L and K, cat B had negligible effects on chemerin chemoattractant activity (data not shown). Taken to- gether, these data suggest that incubation of cat L and K with prochemerin results in generation of bioactive chemerin chemo- attractant. Identification of the cathepsin chemerin cleavage sites We used MS to determine the cathepsin chemerin cleavage sites. The mass value for the larger chemerin cleavage product initially and efficiently generated by cat L and K (experimental mass Chemerin Form [M+H]+, 16,152 Da; calculated mass [M+H]+, 16,155 Da; D mass, 3 Da) corresponds to chemerin residues 18–157 (ADPELT…… GQFAFS, chem157S). The larger protein band (indicated by arrowheads in Fig. 2A) was isolated, digested with trypsin, and Residue position is italic. Nonnative amino acids are shown in bold. Cat L-treated chemerin, Cat K-treated chemerin Cat L-treated chemerin, Cat K-treated chemerin Serum form SspB-treated chemerin Prochemerin (full-length chemerin) analyzed by mass spectrometry to confirm the processing site. A a

peptide fragment with a mass value of 1903.8 Da was identified, Table I. Comparison of chemerin isoforms used in this study 1406 CHEMERIN ACTIVATION BY HOST CYSTEINE CATHEPSINS

FIGURE 2. cat L- and K-activated chemerin triggers CMKLR1/L1.2 cell and human blood pDC chemo- taxis. A, Various concentrations of cat L or K were incubated with 2.9 mM chemerin, and the resulting products were separated by SDS-PAGE under either reducing (left panel) or nonreducing (right panel) conditions. Arrowheads indicate the two predominant chemerin cleavage products (two-pointed arrowheads Downloaded from signify the larger, three-pointed arrowheads signify the smaller cleavage products). Representative of n =3 performed with similar results. B and C, cat K- or L- activated chemerin was tested in in vitro transwell chemotaxis using CMKLR1/L1.2 transfectants (B)or human blood pDC (C). The chemerin samples de- scribed in A were diluted to a final concentration of 1 http://www.jimmunol.org/ nM and tested in chemotaxis assays. CXCL12 (10 nM) was used as a positive control for pDC chemotaxis (C). The mean 6 SD from duplicate wells of three or four experiments is shown. *p , 0.05 by Student t test comparing cathepsin-treated chemerin versus proche- merin, or CXCL12 versus (2) control. by guest on September 28, 2021

corresponding to a nontryptic peptide comprising amino acids likely remains linked to the holo-molecule through a disulfide 141–157 from the C terminus of chemerin (Fig. 3A,3B). This bond, as indicated by Fig. 2A. peptide confirmed the initial and predominant cat K- and L-me- Chemerin S157, but not chemerin R125, is chemotactively diated chemerin cleavage site as NH2..AFS↓KAL..COOH. Further microsequencing of this peptide by CID (tandem MS/MS) con- active firmed the sequence (Fig. 3C). Interestingly, this cleavage site is Because separation of the smaller cleavage product by HPLC identical to a previously identified endogenous active human required reducing the S-S bonds, which would likely alter its chemerin isoform isolated from ascites fluid (1), and to a main secondary structure and possibly bioactivity, to determine the chemerin isoform generated by the S. aureus-secreted cysteine relative biological activities of the two cathepsin-generated protease SspB (chem/SspB; Table I). chemerin products, we generated recombinant Fc-chemerins, ab- The mass value for the smaller chemerin cleavage product breviated further as Fc-chemS157 (the larger cleavage product) and generated by cat L and K (experimental mass [M+H]+, 12,442 Da; Fc-chemR125 (the smaller cleavage product). The Fc-chemerins calculated mass [M+H]+, 12,437 Da; D mass, 5 Da) corresponds to have a glycine linker on the N terminus that is connected to the chemerin residues 18–125 (ADPELT……ETQVLR, chemR125). Fc domain of human IgG1; thus, the C terminus of the Fc-chemerin MS and CID analysis of the tryptic digests confirmed that the most fusion proteins remained native. Fc alone or Fc-chemR125 failed to distal tryptic or nontryptic fragment was “LVHCPIETQVLR” trigger CMKLR1+ cell migration at every concentration tested (up (fragment d in Fig. 3B, data not shown). Thus, prolonged in- to 50 nM) (Fig. 4). However, chemS157 was the most potent at- cubation (data not shown) or incubation with 10–50 times higher tractant for CMKLR1+ cells, with 1 nM eliciting a maximum 42 6 concentrations of cat K or L (Fig. 2A) cleaves chemerin at position 8% cell migration. Recombinant, commercially available chem- NH2..QVLR↓EAEE..COOH, and the released C-terminal peptide erin S157 and HPLC-purified chemerin cleavage product of SspB The Journal of Immunology 1407 Downloaded from http://www.jimmunol.org/

FIGURE 3. Identification of the chemerin cat K and L processing site. Prochemerin was incubated with cat L and K, and the resulting peptide fragments by guest on September 28, 2021 were resolved by SDS-PAGE. The larger chemerin bands (upper bands indicated by two-pointed arrowheads in Fig. 2A) were digested with trypsin and subjected to MS analysis. A, Peptide fragments corresponding to predicted tryptic chemerin mass values are labeled with their mass values (a–f). Asterisk indicates carbamidomethyl modification of cysteine. B, The peptides corresponding to the chemerin fragments identified by MS are underlined and lettered (a–f). Vertical bars indicate predicted trypsin cleavage sites. The N-terminal signal peptide is indicated by italics, as is the C-terminal peptide released on cathepsin cleavage. The carboxy terminus of truncated chemerin is nontryptic C-terminal peptide (f). C, CID mass spectra (tandem MS/MS analysis) of peptide (f) confirms the sequence identity of the nontryptic C-terminal peptide. The major intensity peaks are predicted b or y series ions.

(13) were used interchangeably as chemS157, because both gave The Fc-chemS157 fusion protein also triggered statistically similar results in chemotaxis assays (data not shown). In previous significant CMKLR1/L1.2 cell migration at concentrations of 5, 10, work, we and others have shown that chemS157 is a chemo- and 50 nM, although the response was not as robust as the un- attractant for pDC, NK cells, and monocyte-derived dendritic cells modified form. ChemA155 also triggered CMKLR1+ cell migra- and macrophages (1, 11, 13). tion, although it should be noted that this recombinant protein has a nonnative “PH” C-terminal dipeptide following the native ter- minal alanine (residue 155; Table I), which may alter its activity. Chemerin displays antibacterial activity The predicted homology between chemerin and the antimicrobial cathelicidins, coupled with the expression of chemerin in the skin and other epithelial cell surfaces continually exposed to bacterial challenge (9), led us to evaluate possible antibacterial activities of chemerin. Different chemerin isoforms were first tested for anti- bacterial activity against a laboratory strain of E. coli. Human cathelicidin LL-37 (3 mM) was used as a positive control peptide inhibitor of bacterial growth (20). As demonstrated in Fig. 5A, FIGURE 4. Chemerin S157 is the most active chemoattractant chem- full-length chemerin (prochemerin, 2 mM) significantly inhibited erin. CMKLR1/L1.2 cell migration to the indicated doses of the indicated the growth of E. coli when incubated for 24 h, leading to survival chemerin isoforms was assessed by in vitro transwell chemotaxis. Mi- 6 gration to media only or to Fc only (tested at molar concentrations of 59 13% of bacteria compared with vehicle-treated E. coli set equivalent to the Fc-chemerin fusion proteins) are shown as negative as 100%. Notably, truncated chemerin was an even more effective controls. The mean 6 SD from three experiments is shown. Statistically inhibitor of bacterial growth. The primary chemerin cleavage significant differences between media or Fc-control and the chemerin product generated by cat K and L (chemS157; Table I) was sig- isoforms are indicated by asterisks. *p , 0.05, Student t test. nificantly more effective than prochemerin in inhibiting bacterial 1408 CHEMERIN ACTIVATION BY HOST CYSTEINE CATHEPSINS

effective at inhibiting K. pneumoniae growth than prochemerin. However, it should be noted that 24-h incubation of prochemerin with either E. coli or K. pneumoniae also resulted in some trun- cation of prochemerin (data not shown), suggesting that native protein requires removal of inhibitory C-terminal sequence to display full antibacterial activity. Taken together, these data sug- gest that proteolytic cleavage increases the antimicrobial activity of chemerin. The inhibition of E. coli growth by chemerin was detectable after just 8 h (Fig. 5B). Treatment with prochemerin, chemA155, or chemS157 diminished the survival of E. coli to 65 6 7, 53 6 10, and 60 6 5% of control, respectively (Fig. 5B). The chemerin- mediated decrease in bacterial viability grew even more pro- nounced at prolonged incubation times; after 24 h, for example, the percentage of viable bacteria decreased to 53 6 9, 35 6 9, and 35 6 4%, respectively (Fig. 5B). With the exception of 4 h, when prochemerin seemed to be more effective compared with chemA155 and chemS157 (87 6 5, 95 6 3, 94 6 4), the truncated

chemerin forms demonstrated stronger antibacterial activity Downloaded from against E. coli (Fig. 5B). Interestingly, chemerin-mediated in- hibition of E. coli survival was detectable over a relatively wide range of concentrations, from 2 mM (the highest concentration tested leading to statistically significant growth inhibition) to as little as 0.125 to 0.0625 mM for chemA155 and chemS157 (Fig.

5C). This was in contrast with LL-37, which was highly effective http://www.jimmunol.org/ at inhibiting E. coli growth when used at 3 mM (Fig. 5A) but had almost no effect when tested at 1.5 mM (data not shown). The enhanced antibacterial activity of the truncated chemerin isoforms compared with prochemerin suggests an inhibitory role for the prochemerin C-terminal peptide. Addition of chemically synthe- sized C-terminal peptide KALPRS that is released from proche- merin by SspB, cat K, and cat L, however, did not reduce the antibacterial activity of the larger truncated chemerin forms (data not shown). These data suggest that after release from the core by guest on September 28, 2021 protein, the C-terminal peptide no longer plays an inhibitory role. Interestingly, both chemerin Fc fusion proteins, Fc-chemS157 and Fc-chemR125, displayed comparable antibacterial activity against E. coli (Fig. 5D). These data suggest that in contrast with che- motactic activity, the antibacterial properties of chemerin appear FIGURE 5. Chemerin exhibits antibacterial activity. The antimicrobial to be localized closer to the N terminus, because removal of 38 activity of full-length (pro-chem), chemA155, chemS157 (HPLC-purified residues from the C terminus did not abrogate its antibacterial chem/SspB; Table I), Fc-chemS157, Fc-chemR125, and Fc alone were activity. tested against E. coli (A–D) and K. pneumoniae (A) using the microtiter broth dilution assay. Chemerin isoforms were tested at 2 mM(A, B), 0.5 Discussion mM(D), or at the indicated concentrations (C). Synthetic LL-37 (3 mM) In this article, we identify a novel antimicrobial activity associated was used as a positive control (A). Bacteria were incubated with the peptides for 24 h (A, C, D) or for indicated times (B). The results are with chemerin, and show that host-derived cat L and K can cleave expressed as the mean 6 SD of nine (A, E. coli), four (A, K. pneumoniae, and activate the leukocyte attractant activity of chemerin, as well as B, C), or two (D) independent experiments done in duplicate. A, Statis- enhance its antibacterial effects. tically significant differences between prochemerin and the truncated Various serine proteases have been reported to effectively con- chemerin isoforms are indicated by asterisks and p values (Student t test). vert chemerin to a potent chemoattractant in vitro. There is also B–D, Asterisks indicate statistically significant differences comparing a single example of a , S. aureus-derived SspB the indicated protein preparations with vehicle- or Fc alone-treated E. coli. that can efficiently activate human chemerin. In addition, host- *p , 0.05, **p , 0.001, Student t test. originating and have been reported to pro- cess mouse chemerin, although in this case, the proteolysis of the growth, resulting in 33 6 15% E. coli survival (Fig. 5A). Trun- C terminus generates chemerin variants equipped primarily with cated chemA155 also significantly reduced E. coli survival (39 6 anti-inflammatory properties (13, 23). Cysteine cathepsins of the 19%) versus prochemerin (Fig. 5A). We also tested chemerin papain-like family are normally confined to the endosomal/ (2 mM) for antibacterial activity against a clinical isolate of an- lysosomal network. However, there is evidence that certain cath- other genus within the family of Enterobacteriaceae, K. pneu- epsins are also active extracellularly, either in association with the moniae. As demonstrated in Fig. 5A, incubation of K. pneumoniae cell surface or in soluble form (14). Some cells such as macro- with prochemerin resulted in 58 6 14% viable bacteria compared phages and fibroblasts constitutively secrete cysteine cathepsins as with control, whereas chemA155 and chemS157 reduced K. zymogens (14). Moreover, macrophages have been reported to pneumoniae survival to 32 6 11 and 46 6 13%, respectively. deploy enzymatically active cat B, L, and S, and exhibit an elastin- Thus, similar to E. coli, the truncated form of chemerin was more degrading phenotype, indicating that macrophages can mobilize The Journal of Immunology 1409 cysteine cathepsins to participate in the pathophysiologic remod- However, because antimicrobial properties can be sensitive to pH eling of the extracellular matrix (17). Massive amounts of extra- and ionic composition of the peptide environment (31), it will be cellular cathepsins, probably released from macrophages, are important to determine whether chemerin operates in conditions found in the bronchial tree of patients suffering from acute pul- similar to those found in the skin, bronchial tree, or both. monary inflammation (24). In addition, cat K is strongly impli- Thus, our work uncovers a novel antibacterial property of cated in maintaining the homeostasis of the extracellular matrix in chemerin and characterizes the activation of chemerin by host- the lung (25). Because chemerin mRNA is abundantly produced in derived cysteine proteases of the cathepsin family, and adds a lung (1, 2), collectively, these data suggest that either cat L or K new dimension to the ways chemerin may modulate and augment may be involved in chemerin processing in this organ. Alterna- immunity. tively, significant expression and/or activity of cat K and L in the joints of patients with rheumatoid arthritis and skin dermatoses, Acknowledgments respectively (26, 27), together with reported chemerin immuno- We are grateful to Dr. M. Bulanda and K. Palaga for help with bacteria reactivity or bioactivity, or both, in psoriasis skin and inflamed collection. synovial fluid (1, 9, 28), suggest that these cathepsins may play a role in chemerin cleavage in joints and skin. Because cathepsin- Disclosures mediated processing releases chemerin attractant activity, these The authors have no financial conflicts of interest. enzymes may have an important regulatory role in immune cell migration. Notably, the presence of pDC in lung, as well as the inflamed joints and psoriatic skin (1, 9, 28, 29), supports the no- References Downloaded from tion that cat K and L, through the generation of active chemerin, 1. Wittamer, V., J. D. Franssen, M. Vulcano, J. F. Mirjolet, E. 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