Identification of Novel Chondroprotective Mediators in Resolving Inflammatory Exudates This information is current as Magdalena K. Kaneva, Karin V. Greco, Sarah E. Headland, of September 27, 2021. Trinidad Montero-Melendez, Prashant Mori, Kevin Greenslade, Costantino Pitzalis, Adrian Moore and Mauro Perretti J Immunol 2017; 198:2876-2885; Prepublished online 27 February 2017; Downloaded from doi: 10.4049/jimmunol.1601111 http://www.jimmunol.org/content/198/7/2876 Supplementary http://www.jimmunol.org/content/suppl/2017/02/24/jimmunol.160111 http://www.jimmunol.org/ Material 1.DCSupplemental References This article cites 60 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/198/7/2876.full#ref-list-1 Why The JI? Submit online. by guest on September 27, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Identification of Novel Chondroprotective Mediators in Resolving Inflammatory Exudates Magdalena K. Kaneva,* Karin V. Greco,* Sarah E. Headland,* Trinidad Montero-Melendez,* Prashant Mori,† Kevin Greenslade,† Costantino Pitzalis,* Adrian Moore,† and Mauro Perretti* We hypothesized that exudates collected at the beginning of the resolution phase of inflammation might be enriched for tissue protective molecules; thus an integrated cellular and molecular approach was applied to identify novel chondroprotective bioac- tions. Exudates were collected 6 h (inflammatory) and 24 h (resolving) following carrageenan-induced pleurisy in rats. The resolving exudate was subjected to gel filtration chromatography followed by proteomics, identifying 61 proteins. Fractions were added to C28/I2 chondrocytes, grown in micromasses, ions with or without IL-1b or osteoarthritic synovial fluids for 48 h. Three proteins were selected from the proteomic analysis, a1-antitrypsin (AAT), hemopexin (HX), and gelsolin (GSN), and tested against Downloaded from catabolic stimulation for their effects on glycosaminoglycan deposition as assessed by Alcian blue staining, and gene expression of key anabolic proteins by real-time PCR. In an in vivo model of inflammatory arthritis, cartilage integrity was determined histologically 48 h after intra-articular injection of AAT or GSN. The resolving exudate displayed protective activities on chondrocytes, using multiple readouts: these effects were retained in low m.w. fractions of the exudate (46.7% increase in glycosaminoglycan deposition; ∼20% upregulation of COL2A1 and aggrecan mRNA expression), which reversed the effect of IL-1b. Exogenous administration of HX, GSN, or AAT abrogated the effects of IL-1b and osteoarthritic synovial fluids on http://www.jimmunol.org/ anabolic gene expression and increased glycosaminoglycan deposition. Intra-articular injection of AATor GSN protected cartilage integrity in mice with inflammatory arthritis. In summary, the strategy for identification of novel chondroprotective activities in resolving exudates identified HX, GSN and AAT as potential leads for new drug discovery programs. The Journal of Immunology, 2017, 198: 2876–2885. here is an emerging appreciation of the complex, inte- Persistent inflammation of the joint structures and progressive grated cellular and tissue processes that regulate the end alteration/destruction of articular elements with absent or inade- T phase of the acute inflammatory response (1). Timely quate tissue repair are typical of rheumatoid arthritis and osteo- by guest on September 27, 2021 resolution of a localized inflammatory response requires engage- arthritis (OA) (5). Although the primary risk factor is age, OA ment of specific pathways and mediators to remove immune cells frequently results from traumatic joint injuries, such as those and reprogram tissue resident macrophages, yet ultimately to en- resulting from a sports injury or traffic accidents (5, 6). Surgical act tissue reparative processes (2). This latter aspect of the reso- restoration of joint stability following such injuries seldom lution program is the most important in avoiding a maladaptation prevents future development of OA (6), which often leads to of the tissue (3): efficient repair in the absence of scarring and/or depression and social isolation of the affected persons, thus fibrosis prevents chronicity of the local inflammatory reaction and presenting a significant social and healthcare burden. Currently favors the regain of tissue functionality and homeostasis (4). available treatment options rely heavily on conservative pain management strategies, through the use of analgesics and non- steroidal anti-inflammatory drugs. These methods offer only *William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, United Kingdom; temporary, modest pain relief, cause numerous side effects and and †UCB Celltech, Slough SL1 3WE, United Kingdom comorbidities, and fail to address the underlying causes of ar- ORCIDs: 0000-0003-4819-7132 (S.E.H.); 0000-0003-1696-0068 (P.M.); 0000-0002- ticular tissue inflammation and degeneration. Given the near 6782-6611 (K.G.); 0000-0003-1326-5051 (C.P.); 0000-0001-5560-300X (A.M.); complete lack of reparative therapies for cartilage defects in 0000-0003-2068-3331 (M.P.). routine clinical practice, innovative approaches to drug discovery Received for publication June 29, 2016. Accepted for publication January 29, 2017. are essential to advance this field. This work was supported by the William Harvey Research Foundation through an Recent studies have indicated that endogenous peptides, such as unrestricted grant from UCB Celltech, Slough, U.K. the melanocortins, could limit chondrocyte inflammation and car- Address correspondence and reprint requests to Dr. Magdalena K. Kaneva and Dr. Mauro Perretti, William Harvey Research Institute, Barts and The London School of tilage degradation, an effect linked to prevention of cell death (7). Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, Furthermore, the delivery of platelet-rich plasma into the joint London EC1M 6BQ, U.K. E-mail addresses: [email protected] (M.K.K.) and seems to ameliorate cartilage defects; however, the molecules re- [email protected] (M.P.) sponsible for these chondroprotective effects remain elusive (8–10). The online version of this article contains supplemental material. Interrogation of the inflammatory environment has led to the Abbreviations used in this article: AAT, a1-antitrypsin; AB, Alcian blue; ACAN, aggrecan; Ct, cycle threshold; 3D, three-dimensional; GSN, gelsolin; HMW, high discovery of endogenous proresolving molecules, such as resolvin m.w.; HX, hemopexin; i.a., intra-articular; LMW, low m.w.; MM, micromass; MMP, E1 and protectin D1 (11). Similarly, we hypothesized that tissue- matrix metalloproteinase; MS, mass spectrometry; OA, osteoarthritis; OASF, OA protective mediators might be present in resolving pleural exu- synovial fluid; sGAG, sulfated glycosaminoglycan. dates. We used acute pleurisy (12) as a source of exudates and a Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 combination of in vitro and proteomic analyses, to identify molecules www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601111 The Journal of Immunology 2877 in resolving exudates that afford chondroprotection. Delivery of these 3000 RSLCnano; Thermo Fisher Scientific, Hertfordshire, U.K.) and eluted mediators during ongoing murine experimental arthritis in vivo with a 40 min gradient (2–30% B in 35 min, 30–40% B in 5 min, 99% B in limited cartilage destruction in the knee joint. Altogether, this 10 min, and 2% B in 20 min, where A = 2% acetonitrile, 0.1% formic acid in HPLC-grade H O and B = 80% acetonitrile, 0.1% formic acid in HPLC- study identifies novel protein activities that promote chondrocyte 2 grade H2O). The column was coupled to a PicoView nanospray source (New anabolism providing novel cues to cartilage repair in joint disease. Objective). Spectra were collected from an ion trap mass analyzer (LTQ Orbitrap XL; Thermo Fisher Scientific) using full mass spectrometry (MS) over the mass-to-charge range 400–1600. MS/MS was performed on the top Materials and Methods six ions in each full MS scan using the data-dependent acquisition mode with dynamic exclusion enabled. MS/MS peak lists were generated by Models of disease extract_msn.exe software and matched to a human database (UniProtKB/ Animals were maintained on a standard chow pellet diet with free access to Swiss-Prot_2013_08) using Mascot 2.3.01 (MatrixScience, London, U.K.). water, and a 12 h light-dark cycle. All animal experiments were approved by Carboxyamidomethylation of cysteine was chosen as fixed modification and the local Animal Use and Care Committee in accordance with the U.K.