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

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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 © 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 . Fractions were added to C28/I2 , 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), (HX), and gelsolin (GSN), and tested against Downloaded from catabolic stimulation for their effects on deposition as assessed by Alcian blue staining, and expression of key anabolic proteins by real-time PCR. In an in vivo model of inflammatory , 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 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 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 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 ; MS, mass spectrometry; OA, ; 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 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 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. oxidation of methionine as variable modification. The mass tolerance was Animals (Scientific Procedures) Act, 1986. set at 10 ppm for the precursor ions and at 0.8 Da for fragment ions. Two missed cleavages were allowed. Scaffold (v4.0.6; Proteome Software, Carrageenan-induced pleurisy Portland, OR) was used to calculate the spectral counts and to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted Male Wistar rats (n = 6; 120–150 g; Charles River, U.K.) were anesthetized if they could be established at .95% probability as specified by the Peptide l with isoflurane and 1% (w/v) -Carrageenan (Sigma-Aldrich, Poole, U.K.) Prophet algorithm. Protein identifications were accepted if established solution was injected into the pleural cavity (12, 13). Pleural exudates were at .99% probability with at least two independent peptides. harvested at 6 h (inflammatory exudate) and 24 h (resolving exudate) Real-time PCR. Total RNA was purified in quadruplicate from C28/I2 postinjection by washing each cavity with 1 ml of 100 U/ml Heparin in PBS Downloaded from MMs (0.5 3 106 chondrocytes per MM) using RNeasy Plus Mini Kit as an anticoagulant. Exudates contaminated with blood were discarded. Rat (Qiagen, Manchester, U.K.). cDNA was synthesized using SuperScript III exudates were centrifuged at 1500 rpm for 10 min and the resulting cell-free First-Strand Synthesis System (Invitrogen). OligodT primers were used to supernatants were pooled and filtered using 0.22 mm filters (Amicon synthesize the first strand cDNA (Promega, Southampton, U.K.). Following Ultrafree-MC; Millipore) for 7 h at 4˚C to remove any residual carrageenan. spectrophotometric quantification, RNA was normalized to 1 mg total RNA K/BxN serum arthritis per cDNA reaction. cDNA, diluted 1:5, was used as template to determine relative amounts of ∼

Male C57BL/6 mice (n = 4–6; 30 g body weight) were purchased from mRNA by real-time PCR (ABI Prism 7900 Sequence Detection System; http://www.jimmunol.org/ Charles River and administered 100 ml K/BxN serum on day 0 and day 2 Applied Biosystems) using specific primers (QuantiTect Primer Assay; of arthritis. On day 3, a1-antitrypsin (AAT) (100 ng in 5 ml per mouse) or Qiagen) and 23 Power SYBR Green Mastermix (Applied Biosystems, gelosin (GSN) (30 ng in 5 ml per mouse) was administered via intra- Thermo Fisher Scientific). The expression of COL2A1, ACAN, SOX9, articular (i.a.) injection into the knee joint. The contralateral knees re- matrix metalloproteinase (MMP)-13, and IL6 was interrogated. Cycle ceived saline (5 ml). On day 5, joints were collected in formalin for 48 h threshold (Ct) values were measured and calculated by Sequence Detector before decalcification in formic acid (10% w/v). Following processing and Software v2.4. Relative amounts of mRNA were normalized to endogenous paraffin embedding, samples were prepared for histology. Coronal sections control (GAPDH) and to vehicle. Relative mRNA contents were calculated m 2DDCt of the knee joint were taken (6 m thickness) and stained with toluidine as x =2 , where DDCt = DE 2 DC and DE = Ctsample 2 CtGAPDH and blue for the analysis of cartilage integrity. Images were captured using DC = Ctcontrol 2 CtGAPDH (20). EVOS XL Core Imaging System (Thermo Fisher Scientific, Paisley, U.K.); Alcian blue staining of sulfated in vitro. Quantitation percentage area toluidine blue positive was measured via ImageJ (National of cartilage-specific sulfated glycosaminoglycans (sGAGs) deposition was by guest on September 27, 2021 Institutes of Health) by splitting each image into its RGB channels and performed as previously described (15). Washed MMs were fixed with 4% quantifying the positive area after applying a threshold. glutaraldehyde solution (v/v in double distilled H2O), and submerged in , Chondrocyte cultures Alcian blue (AB) 8GS dye (48 h at 1% w/v) in 0.1 N HCl (pH 1; Carl Roth, Karlsruhe, Germany) at room temperature. AB dye was extracted in The immortalized chondrocytes C-28/I2 were kindly provided by Dr. guanidine-HCl (Sigma-Aldrich) for 48 h at RT. A630 of the extracted dye M. Goldring (14). Cells were cultured in complete medium: DMEM/Ham’s was measured and concentration was quantified by interpolation with AB F12 (1:1; Life Technologies-Invitrogen, Paisley, U.K.), supplemented with standard curve and normalized to DNA content (nanogram per micro- 10% nonheat-inactivated FCS (Life Technologies-Invitrogen) and maintained gram). DNA content was measured in the extracted dye solution by at 5% CO2. High-density three-dimensional (3D) micromass (MM) cultures fluorescence (485/535 nm). were generated as previously described (15). The rat chondrocyte-restricted Quantification of cytokine, , and ACAN release. Levels of IL-6, IL-8, RCJ3.1C5.18 cell line (16–18) was maintained in DMEM supplemented and MMP13 in cell-free supernatants were measured using a FlowCytomix 2 with 10% NI-FCS, 1% dexamethasone (1 310 7 M), 100 U/ml penicillin, multiple analyte detection system (eBioscience, Hatfield, U.K.). ACAN 100 mg/ml streptomycin (Omega Scientific, Tarzana, CA), and 5% CO2. release by the chondrocyte MMs was measured by DuoSet ELISA Devel- For experiments, the RCJ3.1C5.18 cells were plated in 24-well plates at opment Systems (R&D Systems, Minneapolis, MN). 5 3 104 cells per cm2 and maintained for 24 h in complete medium without dexamethasone. Statistical analysis All chondrocyte cultures were serum starved for 24 h in phenol red-free All PCR data are reported as mean 6 SEM unless otherwise indicated in DMEM/Ham’s F12 (1:1) supplemented with 1% insulin-transferrin- individual figure legends. Significant differences in PCR experiments were selenium G supplement (ITS; Invitrogen) to allow for collagen type 2 determined with the nonparametric Kruskal–Wallis ANOVA test, followed and aggrecan (ACAN) production. Chondrocytes were then stimulated as by Dunn multiple comparison post hoc test. AB-staining experiments were indicated in the individual figure legends. analyzed using one-way ANOVA, followed by Dunnett multiple comparison Molecular and biochemical analyses post hoc test; histology-staining experiments were analyzed via two-way ANOVA, followed by Bonferroni post hoc correction for multiple compari- Exudate fractionation. The filtered resolving exudate was subjected to size- sons. All statistical analyses were performed using GraphPad Prism 5.0, exclusion chromatography using a pre-equilibrated HiLoad Superdex-200 (GraphPad Software, CA). Values were considered significant for p , 0.05. 16/60 column with a flow rate of 1 ml/min. A total of two column volumes (equivalent to 240 ml) of buffer were used for isocratic elution and 1 ml fractions collected. Fractions corresponding to specifically chosen peak Results regions were pooled together into nine reconstituted fractions (see Fig. 2A) and later used for differential stimulation of chondrocytes. Discrete alteration of chondrocyte metabolism by pleural Mass spectrometry analysis of rat pleural exudate fractions. The resulting exudates pooled rat pleural exudate fractions were reduced in Laemmli sample buffer Acute and resolving pleurisy was produced by injection of car- as previously described (19). After electrophoresis and silver staining, bands were subjected to in-gel digestion with trypsin using an Investigator rageenan into the pleural cavity of rats. Thus, as expected, immune ProGest (DIGILab) robotic digestion system. Tryptic peptides from the di- cell migration and exudate formation peaked at 6 h (12.6 6 2.5 3 gests were separated on a reverse-phase nanoflow HPLC system (UltiMate 106 cells; n = 6), whereas by 24 h the inflammation had subsided 2878 TISSUE REPARATIVE ACTIVITIES IN RESOLUTION with reduced cell numbers (4.8 6 1.3 3 106 cells; p , 0.05), a trough with very low protein content. Fractionated bioactivities of alongside published data (12, 13). The 6 h exudate (onset of in- the resolving exudate were tested on human C-28/I2 chondrocytes flammation) hereafter referred to as the inflammatory exudate, and grown in high-density 3D MMs. Initially samples were pooled into the 24 h exudate, coincident with the initiation of resolution, high m.w. (HMW) and low m.w. (LMW) fractions (Fig. 2A). In- hereafter referred to as the resolving exudate, were used. cubation of chondrocytes with HMW sample (1:30 dilution) re- Serial dilutions of cell-free exudates were tested on RCJ3.1C5.18 duced deposition (.60%; p , 0.05). This effect was cells to monitor COL2A1, ACAN,andMMP13 gene expression paralleled by downregulation of COL2A1 and ACAN gene prod- (Fig. 1). The inflammatory exudates displayed typical catabolic ac- ucts (p , 0.05). Conversely, treatment with the LMW sample tivity: at its highest concentration (dilution 1:10), the exudate pro- resulted in a significant increase in proteoglycan deposition (∼50%, voked ∼20-fold inhibition of COL2A1 and ∼8-fold inhibition of p , 0.05) and modest anabolic effects on gene expression (Fig. 2B). ACAN gene products, an effect paralleled by a significant increase in The pooled LMW fraction was tested against IL-1b (20 ng/ml): in MMP13 gene transcription (Fig. 1; p , 0.05). The resolving exudate these settings, the anabolic properties of the LMW fractions were was mostly inactive on its own, except for a modest modulation of magnified with augmented COL2A1 (∼6-fold) and ACAN (∼5-fold) ACAN expression (p , 0.05; Fig. 1). Gene modulation was time- mRNA levels compared with IL-1b (p , 0.01; Fig. 2C). dependent with 48 h being optimal (Fig. 1B); this incubation period The individual fractions (1:30 dilution) were tested against the was selected for subsequent experiments. Stimulation of RCJ3.1C5.18 catabolic stimulus IL-1b observing that the first three fractions rat chondrocytes with both the inflammatory (at 1:100 fixed dilution) were mostly inactive (Fig. 2D). HMW fractions 5 and 6 poten- and resolving exudate significantly inhibited MMP13 transcrip- tiated the cellular response to IL-1b with significant upregulation tion by 20%, alongside augmented expression of COL2A1 and of catabolic IL6 (p , 0.001) and MMP13 (p , 0.05, Downloaded from ACAN mRNAs (p , 0.05; Fig. 1). Thus, the resolving exudate compared with IL-1b stimulation alone). HMW fractions 4 and 5 significantly reverted the catabolic effect of the inflammatory had a moderate effect on COL2A1 transcription (p , 0.05; exudate. Fig. 2D), whereas fractions 8 and 9 counteracted IL-1b with a significant stimulation of the anabolic response, as evident from Definition of exudate chondromodulatory activities increased COL2A1 (p , 0.01) and ACAN gene products. These

Size-exclusion chromatography of the resolving exudate yielded effects prompted identification of the protein content of these http://www.jimmunol.org/ eight fractions (Fig. 2A). A ninth fraction was included, representing chondromodulatory fractions.

FIGURE 1. Pleural exudates promote discrete alterations in rat chondrocyte metabolism. Rats by guest on September 27, 2021 were injected with 1.5 mg carrageenan in 150 mlat time 0. Exudates were harvested at either 6 h (in- flammatory) or 24 h (resolving). (A) RCJ3.1C5.18 chondrocytes were incubated for 48 h with a range of dilutions of either the inflammatory or resolving exudate. In a further group, chondrocytes were treated for 48 h with the inflammatory exudate (1:100 dilution) and the indicated dilution of re- solving exudate simultaneously. (B) RCJ3.1C5.18 chondrocytes were stimulated with inflammatory exudate (fixed dilution of 1:100) for 0–48 h. IL-1b (20 ng/ml) was used as a reference catabolic stimulus (dashed line). Total RNA extracted from each sample was reverse transcribed into cDNA and expression of COL2A1, ACAN, and MMP13 was quantified by PCR. The GAPDH gene was used as internal control. Results are expressed as arbitrary units based on calculation of 22DDCt method; relative amount of target genes were nor- malized to GAPDH and to untreated controls, with expression set to 1.0. Data are mean 6 SEM of four experiments performed in duplicate. Kruskal– Wallis test followed by Dunn multiple comparison post hoc test were used to compare the effect of resolving exudate dilution on vehicle/inflammatory exudate (dashed line). *p , 0.05, **p , 0.01 [against inflammatory exudate alone; value 1 in (B)]. #p , 0.05, ##p , 0.01, ###p , 0.001 [against vehicle, value 1 in (A)]. The Journal of Immunology 2879

FIGURE 2. Anabolic and catabolic activities are contained in distinct fractions of resolving pleural exudates. (A) Gel-filtration chromatogram. Pleural exudates collected at 6 and 24 h postcarrageenan- injection were pooled together and filtered using 0.22 mM filter at 7500 rpm for 7 h at 4˚C. The exudate material was subjected to gel filtration on HiPrep S200 16/60 column. The labeled peaks (1–9) were later reconstituted, as shown. (B) Human C-28/I2 MMs were stimulated for 48 h with a pool of HMW fractions 4–6, or LMW fractions 7–9. Spectrophoto- metric quantification of guanidine-HCL–extracted AB dye is normalized to DNA content (microgram per nanogram). Data are mean 6 SEM of n = 2 experi- ments performed in quadruplicate. Statistical analyses were conducted using one-way ANOVA, followed by Downloaded from Dunnett multiple comparison post hoc test (versus vehicle). C-28/I2 MMs were stimulated for 48 h with: (C)IL-b and pooled LMW fractions as indicated, or (D) simultaneously stimulated with IL-1b (20 ng/ml) and single fractions (1–9). The expression of COL2A1, ACAN, IL6,andMMP13 mRNA was quantified by PCR. GAPDH was used as internal control. Results http://www.jimmunol.org/ are expressed as arbitrary units based on calculation of 22DDCt method; relative amount of target genes were normalized to GAPDH and vehicle, with expression set to 1.0. Data are presented as mean 6 SEM of n =3 experiments performed in duplicate. Statistical analy- ses were conducted using Kruskal–Wallis test followed by Dunn multiple comparison post hoc test (versus IL-1b, dashed line). *p , 0.05, **p , 0.01. by guest on September 27, 2021

Identification of candidates for chondroprotection catabolic response by upregulating the anabolic genes (p , 0.05; Tandem gel-LC-MS-MS proteomic analysis was performed on all Fig. 4A–C), although concomitantly inhibiting MMP13 transcrip- , fractions. The proteomic analysis identified a total of 61 proteins tion in a concentration-dependent manner (p 0.01; Fig. 4D). b across all fractions, which are listed in Supplemental Table I IL-1 –induced IL-6 secretion was also inhibited by GSN (maxi- m , alongside the number of proteolytic peptides sequenced for each mum effect at 0.1 g/ml; p 0.05; Supplemental Table II). hit. Based on their relative abundance, and detailed literature re- Varied results were obtained for HX, which on its own produced view, we selected and tested .7 candidate proteins (data not shown) a more marked yet balanced response, with increased expression of that had indications for functions relevant to chondroprotection. Of anabolic and catabolic gene products (Fig. 3). In the presence of b those, AAT, GSN, and hemopexin (HX) were brought forward (see IL-1 , HX significantly reverted the catabolic response on all , Discussion) for more in-depth functional analysis. anabolic genes tested (p 0.05; Fig. 4A–C), although only being b Addition of these three proteins to unstimulated chondrocyte effective counteracting IL-1 -induced MMP13 transcription at the , MMs displayed minimal changes in the cellular response: as an lowest concentration tested (p 0.05; Fig. 4D). HX significantly b example, AAT triggered 2-fold upregulation of COL2A1 (p , 0.05), reduced IL-1 –induced IL-6 production (Supplemental Table II). and a concentration-dependent downregulation of MMP13, optimal at 30 mg/ml (p , 0.001; Fig. 3A–D). However, bioactions were more AAT, GSN, and HX modulated sGAG deposition in catabolic acute in the presence of IL-1b; in these settings, addition of AAT settings improved COL2A1 and ACAN gene expression by ∼4-fold and Functional data on sGAG deposition were obtained to complement SOX9 transcription was enhanced by ∼3-fold, compared with IL-1b gene expression studies. MMs were incubated for 48 h with a stimulation alone (p , 0.01; Fig. 4A, 4B). Simultaneously, incuba- concentration range of AAT, GSN, and HX (0.1–30 mg/ml) in the tion with this reduced (∼70%) 1) IL-1b–induced presence of IL-1b. Fig. 5A and 5B shows that AAT counteracted MMP13 gene expression and 2) secretion of IL-6 and IL-8 the action of IL-1 b by bringing sGAG deposition levels back to (Supplemental Table II). those of vehicle-treated MMs. For GSN (0.1–10 mg/ml) and HX On its own, GSN afforded minimal modulation on COL2A1 and (0.1–30 mg/ml) a bell-shaped concentration-response curve was ACAN expression, with a modest effect on SOX9 (p , 0.01; produced, with optimal effects at 0.3 and 1.0 mg/ml, respectively Fig. 3A–C). Again, in the presence of IL-1b, GSN prevented the (.3-fold increase; p , 0.001; Fig. 5B). 2880 TISSUE REPARATIVE ACTIVITIES IN RESOLUTION

FIGURE 3. Effect of AAT, GSN, and HX on chondrocyte gene expression profiles. C-28/I2 chondrocyte MM were stimulated for 48 h with AAT, GSN, and HX (0–30 mg/ml). Total RNA was extracted from each MM and reverse tran- scribed into cDNA. The expression of COL2A1 (A), ACAN (B), SOX9 (C), and MMP13 (D) was determined by PCR. GAPDH was used as internal control. Results are expressed Downloaded from as arbitrary units based on calculation of 22DDCt method; relative amount of target genes were normalized to GAPDH and vehicle, with expression set to 1.0. Data are presented as mean 6 SEM of three experiments performed in duplicate. Statistical analyses were conducted using Kruskal–Wallis test followed by Dunn multiple comparison post hoc test (versus vehicle, V). *p , 0.05, **p , 0.01, ***p , 0.001. http://www.jimmunol.org/ by guest on September 27, 2021

Next, we assessed the functions of the polypeptides in more cartilage was eroded by ∼60% (sGAG content; p , 0.05 compared complex settings like those promoted by chondrocyte stimulation with naive mice). Importantly, therapeutic i.a. injection of either with OA synovial fluid (OASF), rich in variety of proinflammatory AAT (100 ng) or GSN (30 ng) recovered cartilage integrity by 52 cytokines and cartilage degrading enzymes (21). AB staining and 34%, respectively, compared with vehicle-injected contralateral revealed that OASF-stimulated chondrocyte MMs (1:100 dilution joints (p , 0.05; Fig. 6). as selected from preliminary analyses, data not shown) markedly inhibited sGAG deposition (p , 0.01, Fig. 5C, 5D). Treatment of Discussion MMs with AAT, GSN, or HX abrogated the catabolic effect of We applied an unbiased approach to identify proteins with chondro- the fluids. Similar counteracting properties were evident when protective activity in resolving exudates. Using a combination of we quantified OASF-induced release of IL-6, IL-8, and MMP13 in vitro and in vivo techniques, complemented by analyses with (Fig. 5E). human synovial fluids, we describe the presence of the anabolic factors AAT, GSN, and HX in resolving exudates and characterize In vivo efficacy of AAT and GSN on knee joint arthritis their chondroprotective properties. This approach could become To complete these in vitro discoveries we selected an animal model paradigmatic for identifying novel therapeutic leads. of inflammatory arthritis, where immune cell and exudation are Acute inflammation is tightly regulated in space and time, with also present but are additionally coupled to significant cartilage an onset phase followed by a resolution phase that paves the way to destruction, complementing the initial studies with the acute in- restoring the homeostatic balance. This second phase of inflam- flammatory exudates. The serum-transfer model of inflammatory mation has gained interest in the last decade, with the character- arthritis has been applied to unveil chondroprotective mechanisms ization of specific proresolving mediators (1) and receptors (25) as evoked by extracellular vesicles (22) or calcitonin nanomedicines and definition of fundamental proresolving processes, which until (23, 24). GSN or AAT were administered by i.a. injection and cu- recently have been predominantly studied in the context of im- mulative data for both GSN and AAT are reported in Fig. 6. To- mune cell reactivity and behavior (4, 26). However, the end point luidine blue staining of multiple focal sections of each joint showed of resolution is the repair of the affected tissue with recovery of widespread loss of staining in sham arthritic mice, where articular normal physiological functions. As reasoned in the Introduction, The Journal of Immunology 2881

FIGURE 4. AAT, GLS and HX counteract the cata- bolic effect of IL-1b on chondrocyte gene expression profiles. C-28/I2 chondrocyte MM were stimulated for 48 h with AAT, GSN, and HX in the presence of IL-1b (20 ng/ml). Total RNA was extracted from each MM and reverse transcribed into cDNA. The expression of Downloaded from COL2A1 (A), ACAN (B), SOX9 (C), and MMP13 (D) was determined by PCR. GAPDH used as internal control. Results are expressed as arbitrary units based on calculation of 22DDCt method; relative amount of target genes were normalized to GAPDH and vehicle, with expression set to 1.0. Data are presented as mean 6 http://www.jimmunol.org/ SEM of three experiments performed in duplicate. Sta- tistical analyses were conducted using Kruskal–Wallis test followed by Dunn multiple comparison post hoc test (versus IL-1b). *p , 0.05, **p , 0.01, ***p , 0.001. by guest on September 27, 2021

few studies have addressed the pharmacological modulation of with 3D cultures of human cells. Relevant gene expression patterns skin repair, applying models of wound healing (27, 28). However, were measured together with deposition of , cul- several other tissues would undergo reparative processes at the end minating with proteomic analysis coupled with mass spectrometry—a of inflammatory episodes and, equally, could be amenable to powerful tool for the exploration and discovery of novel biomarkers, therapeutic exploitation using approaches that emerge from the and an approach well known in the identification of OA-driving science of resolution. This overarching hypothesis guided the players (32). A comprehensive review of the proteomics hits was current investigation by employing a model of resolving inflam- conducted, taking into account their relative abundance in the various mation that has previously allowed identification of novel tissue- fractions in parallel with a comprehensive literature review of the protective pathways, including those centered on hemoxygenase-1 identified proteins, which was cross-referenced against any possible (13), cyclo-oxygenase type 2 (12), and more recently, lipidomics anabolic or anti-inflammatory properties that they exhibit in various approaches, which have led to the identification of novel lipid tissue systems. The candidate proteins fitting the criteria were taken mediators in biological fluids (29), e.g., resolvin D1, endowed with forward for further testing in our chondrocyte assays, based on the cartilage-protective actions during ongoing inflammatory arthritis hypothesis that such effects might be translatable to chondrocytes (30). This approach is especially pertinent for the attempted repair and/or cartilage and/or experimental arthritis. of cartilage, as cartilage erosions and sport injuries often lead to Using this approach, we identified several candidate mediators osteoarthritis with no treatable parameters. and selected three of them, namely AAT, GSN, and HX, for which Distinct biological activities were detected in pleural exudates we define new properties in the context of chondrocyte biology. All harvested at onset and peak of inflammation, the latter coinciding three factors were detected by Western blotting in both the re- with the onset of resolution (31). We applied a streamlined ex- solving and inflammatory exudates, with levels more pronounced perimental strategy, where rat chondrocytes were rapidly substituted in the resolving exudate (data not shown). Herein, we progressed 2882 TISSUE REPARATIVE ACTIVITIES IN RESOLUTION Downloaded from http://www.jimmunol.org/

FIGURE 5. AAT, GLS and HX rescue sGAG deposition by chondrocytes under catabolic status. C-28/I2 chondrocytes were stimulated for 48 h with 20 ng/ml IL-1b (A and B) or OASF (1:100; C and D) in the presence or absence of AAT, GSN, or HX (0–30 mg/ml). (A and C) Representative images of MMs stained with AB for the detection of sGAGs. (B and D) Spectrophotometric quantification of guanidine-HCL–extracted AB dye normalized to DNA content (microgram per nanogram). Data are mean 6 SEM of six experiments. Statistical analyses were conducted with one-way ANOVA, followed by Dunnett multiple comparison post hoc test, (versus vehicle or IL-1b). Vehicle effects are indicated by dotted line and OASF-stimulated controls by dashed line. (E) AAT and GSN modulate OASF-induced inflammatory mediator release. Human C-28/I2 chondrocytes were stimulated for 48 h with OASF (1:100) in the presence or absence of AAT (10 mg/ml) or GSN (0.3 mg/ml). Cell-free supernatants were collected and analyzed for IL-6, IL-8, and MMP13 levels by Flowcytomix CBA Assay. Data are mean 6 SEM of three independent experiments. Statistical analyses were conducted using one-way ANOVA, followed by guest on September 27, 2021 by Bonferroni multiple comparison post hoc test. *p , 0.05, **p , 0.01, ***p , 0.001. our investigation by utilizing a dual translational remit: 1) testing murine acute gouty arthritis with reduced release of IL-1b and the identified factors against human arthritic synovial fluids, which induction of IL-1 receptor antagonist (43). Equally pertinent is the allowed for assessment of their effects against the numerous cat- observation that AAT treatment reduces autoimmunity and delays abolic and proinflammatory mediators, contained in the complex arthritis development in a mouse model of collagen-induced ar- arthritic synovial fluids, which drive cartilage destruction in ar- thritis (44). Our data add to the current wealth of AAT biology thritis (21), and 2) in vivo administration of those proteins in a showing that at low nanomolar concentrations (1–10 mg/ml model of inflammatory arthritis. Of interest, and in support of our equivalent to 19–192 nM), this peptide partially blocked the cat- approach, recent proteomic analysis of human arthritic synovial abolic response evoked by IL-1b in chondrocytes both for ana- fluids identified these proteins though without postulating relevant bolic genes and MMP13. In the AB assay, where IL-1b was used bioactions (33). as a stimulus, AAT was active at concentrations as low as 0.1 mg/ml AAT, GSN, and HX are acute phase proteins released by the liver (1.9 nM), whereas full protection against the catabolic status induced in settings of systemic stress. AAT is a 52-kDa glycoprotein by complex OASF was evident at 10 mg/ml (192 nM), probably due functioning as the major natural inhibitor of serine (34) to the presence of multiple catabolic and proinflammatory mediators with potent effects on neutrophil elastase (35, 36). It is produced in the inflamed synovial fluid (21). mainly by hepatocytes, yielding circulating steady-state levels of GSN and HX displayed similar, yet not overlapping, profiles to 1–3 mg/ml; these levels rise during infection and inflammation AAT. GSN, an 82-kDa Ca2+-regulated actin filament severing, (33) and remain elevated for 7–10 d (37). Local inflammation- capping, and nucleating protein, seems to be involved in the driven production of AAT by endothelial and myeloid cells is also control of biological processes beyond filament remodeling. There reported (37). The property of AAT to inactivate protease activity are two isoforms of GSN: plasma or pGSN, and cytosolic or cGSN allows both direct and indirect reduction of inflammation as some (45). Circulating levels of pGSN in the blood of healthy individ- of these proteases activate protease-activated receptors, thus af- uals are ∼200 6 50 mg/l and may be a key component of an fecting the release of proinflammatory cytokines (38). In addition, extracellular actin scavenging system operating during tissue AAT possesses a broad spectrum of anti-inflammatory (37, 39) damage (46). pGSN decreases during acute injury and inflam- and immunomodulatory properties, unrelated to protease inhibi- mation, and rGSN treatment of animals improves outcomes fol- tion (37, 40), including elevation of cAMP in target cells (41) and lowing major trauma (47, 48), sepsis (49, 50), and burn injuries inhibition of the ADAMTS-4 (42). A recent study (51). Interestingly, circulating levels of pGSN are decreased in the reported the antiarthritic properties of an ATT-Fc fusion protein in plasma of patients suffering from rheumatoid arthritis (52) and The Journal of Immunology 2883

FIGURE 6. Intra-articular injection of AAT and GSN rescue cartilage degradation in inflammatory arthritis. Arthritis was induced via i.p. injection of K/BxN serum at day 0 and day 2 (100 ml each). At disease onset (day 3) AAT (100 ng) or GSN (30 ng) was injected in the right knee, whereas the left knee received saline (5 ml; see scheme in D). (A) Forty-eight hours later (day 5), knees were harvested and multiple focal sections (6 mm) were paraffin-embedded, stained with toluidine blue and analyzed. Representative im- ages of knee joint microstructure with evident signs of erosion are shown. CE, cartilage erosion. (B and C) Cumulative data for both naive and arthritic mice upon treatment with GSN or AAT. Data are presented as Downloaded from mean 6 SEM of n = 4 (GSN), n = 8 (AAT), or n =4 (naive) mice. Matched two-way ANOVA followed by Bonferroni post hoc test were used to compare the effect of the two drugs on cartilage degradation, compared with vehicle-injected contralateral joint. *p , 0.05. http://www.jimmunol.org/

analyses of the synovial fluids of those patients show consumption it was important to establish efficacy against the human synovial fluids, of pGSN into the inflamed joints (52). The modulatory functions thus indicating a translational potential for the chondromodulatory by guest on September 27, 2021 of GSN we identified on chondrocytes are further supported by the proteins identified in the resolving exudates, because both contain a findings that GSN2/2 mice experience exacerbated arthritis (53). variety of stimuli, rather than a single cytokine (e.g., IL-1b). Additionally, GSN is expressed by both normal and hypertrophic Equally important was to expand these in vitro analyses and chondrocytes (54) yet its functions have been scarcely investigated establish proof-of-concept activity by monitoring efficacy of AAT in the context of the biology of this cell type. In analogy to ATT, and GSN on knee cartilage during ongoing arthritis. Local ad- abundant GSN has been identified in human inflammatory fluids ministration of a low dose of either AAT or GSN, calculated to fall (33, 52, 55). within the range that afforded in vitro chondroprotection, prevented HX (60 kDa) is primarily produced by hepatocytes, with the loss of cartilage integrity. A therapeutic approach was used, moderate amounts also synthesized by the nervous system, skeletal where the peptides were injected after initial signs of arthritis were muscle, and kidney (56). HX binds heme with high affinity con- evident: it was important to establish pharmacological efficacy trolling heme-iron availability in tissues and cells. In the context against internal controls, hence the vehicle-injected contralateral of inflammation, HX can inhibit granulocyte recruitment and—in knees. The exact mechanism of action of both GSN and AAT is systemic sepsis—the production of proinflammatory cytokines currently being further investigated. We can postulate that for GSN (57). A negative modulatory function on Th17 T-cell differentia- we might have replenished the protein, which would be naturally tion has been reported (58), with clear potential for antiarthritic lacking in the synovial fluids of arthritic mice, thus restoring benefits (59). chondroprotection. For AAT, it is plausible that along with IL-1b In our experimental settings, (p)GSN promoted anabolic gene reduction and IL-1RA augmentation, this protein can reduce modulation with significant changes in vehicle (e.g., SOX9) and neutrophil infiltration and modify the activity of neutrophil elas- cytokine-stimulated (COL2A1, ACAN, and MMP13) conditions. tase in the inflamed joints. In both cases, the in vivo mechanisms HX was quite potent on IL-1b–stimulated MMs, with significant are currently being investigated in follow-up studies. effects at 3–10 mg/ml, (50–167 nM). Both GSN and HX—in In conclusion, we propose that innovation in the identification analogy to AAT—promoted proteoglycan deposition against and/or development of novel antiarthritic strategies may benefit IL-1b and OASF stimulation with optimal concentrations of 0.3 from the exploitation of endogenous pathways of the resolution of and 10 mg/ml, respectively. inflammation. Reasoning that the end point of resolution is tissue A common thread is emerging here, with the notion that acute repair, healing, and regaining of physiological functions, we have phase proteins—previously categorized passive regulators of analyzed experimental inflammatory exudates and focused our inflammation—are endowed with specific and relevant properties at analyses on proteins identified in human synovial fluids. The different cellular levels, including those relevant to joint diseases, properties of these proteins were then characterized on human from skeletal muscle to T cells and, from our data, to chondrocytes. In chondrocytes, detailing partial over-lapping but also distinct biology our rationale of identification of novel effectors of chondroprotection, for AAT, GSN, and HX on basal or activated cells. Importantly, an 2884 TISSUE REPARATIVE ACTIVITIES IN RESOLUTION in vivo proof-of-concept study held true on the in vitro data produced 22. Headland, S. E., H. R. Jones, L. V. Norling, A. Kim, P. R. Souza, E. Corsiero, C. D. Gil, A. Nerviani, F. Dell’Accio, C. Pitzalis, et al. 2015. Neutrophil-derived for AAT and GSN. Collectively, the data presented here add AAT, microvesicles enter cartilage and protect the joint in inflammatory arthritis. Sci. HX, and GSN to the list of arthritic disease modifiers with a potential Transl. Med. 7: 315ra190. therapeutic value. 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