Specialised pro-resolving mediators of inflammation in inflammatory arthritis

Prostaglandins, Leukotrienes and Essential Fatty Acids 107 (2016) 24–29

Contents lists available at ScienceDirect

Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa

Specialised pro-resolving mediators of inflammation in inflammatory arthritis Anne E. Barden a,n, Mahin Moghaddami b,c, Emilie Mas a, Michael Phillips d, Leslie G. Cleland b,c,1, Trevor A. Mori a,1 a

School of Medicine and Pharmacology, Royal Perth Hospital Unit, University of Western Australia, GPO Box X2213, Perth, WA 6847, Australia Rheumatology Unit, Royal Adelaide Hospital, North Terrace, Adelaide, SA, Australia c Discipline of Medicine, Adelaide University, Australia d Harry Perkins Institute for Medical Research, Perth, WA, Australia b

art ic l e i nf o

a b s t r a c t

Article history: Received 26 November 2015 Received in revised form 12 February 2016 Accepted 2 March 2016

Introduction: Specialised pro-resolving mediators (SPM) are derived from n-3 long chain polyunsaturated fatty acids (n-3FA). They promote resolution of inflammation and may contribute to the beneficial effects of n-3FA in patients with arthritis. This study compared SPM in knee effusions and plasma of patients with arthritis taking n-3FA, and plasma of healthy volunteers taking n-3FA. Methods: Thirty six patients taking n-3FA undergoing arthrocentesis for an inflammatory knee effusion and 36 healthy volunteers who had taken n-3FA (2.4 g/day) for 4 weeks were studied. SPM in synovial fluid and plasma were measured by liquid chromatography-tandem mass spectrometry included 18hydroxyeicosapentaenoic acid (18-HEPE), the precursor of the E-series SPM (RvE1, RvE2, RvE3, 18R-RvE3), and 17-hydroxydocosahexaenoic acid (17-HDHA), the precursor of the D-series SPM (RvD1, 17R-RvD1, RvD2). Other SPM included protectin D1 (PD1), 10S,17S-dihydroxydocosahexaenoic acid (10,17S-DHDHA), maresin-1 (MaR-1) and 14-hydroxydocosahexaenoic acid (14-HDHA) derived from docosahexaenoic acid (DHA). Results: E- and D-series SPM and the precursors 18-HEPE and 17-HDHA were present in synovial fluid and plasma of the patients with inflammatory arthritis. Plasma SPM were negatively related to erythrocyte sedimentation rate in arthritis patients (Po0.01) and synovial fluid RvE2 was negatively associated with pain score (P¼0.02). Conversion from 18-HEPE and 17-HDHA to E- and D-series SPM was greater in synovial fluid (Po0.01). Most plasma SPM in arthritis patients were elevated (Po0.05) compared with healthy volunteers, and conversion to E- and D-series SPM was greater (Po0.01). Conclusions: SPM are present in chronic knee effusions and although the levels are lower than in plasma, the association between synovial fluid RvE2 and reduced pain scores suggests that synthesis of SPM at the site of inflammation is a relevant mechanism by which n-3FA alleviate the symptoms of arthritis. & 2016 Elsevier Ltd. All rights reserved.

Keywords: Arthritis Synovial fluid Specialised pro-resolving mediators Resolvins Pain Fish oil

1. Introduction Specialised pro-resolving mediators (SPM) are a family of oxylipids that include resolvins, protectins, maresins and lipoxins.

Abbreviations: SPM, specialised pro-resolving mediators; n-3FA, n-3 long chain polyunsaturated fatty acids; 18-HEPE, 18-hydroxyeicosapentaenoic acid; 17-HDHA, 17-hydroxydocosahexaenoic acid; 10S, 17S-DHDHA, 10S, 17Sdihydroxydocosahexaenoic acid; MaR-1, maresin-1; 14-HDHA, 14-hydroxydocosahexaenoic acid; AA, arachidonic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; RvE1, resolvin E1; RvE2, resolvin E2; RvE3, resolvin E3; 18R-RvE3, 18R-resolvin E3; RvD1, resolvin D1; 17R-RvD1, 17R resolvin D1; RvD2, resolvin D2; LC–MS/MS, liquid chromatography–tandem mass spectrometry; CCC, concordance correlation co-efficient n Corresponding author. Tel.: þ 61 8 9224 0272; fax: þ 61 8 9224 0246. E-mail address: [email protected] (A.E. Barden). 1 Equal senior authorship. http://dx.doi.org/10.1016/j.plefa.2016.03.004 0952-3278/& 2016 Elsevier Ltd. All rights reserved.

SPM contribute actively to the resolution of inflammation through engagement, at nanomolar concentrations, of cognate Gprotein coupled receptors [1]. SPM arise from n-3 long chain polyunsaturated fatty acids (n-3FA) through the action of lipoxygenase enzymes and other remodelling steps [2]. The lipoxins are formed from arachidonic acid (AA, 20:4 n-6) [1,3] but there is particular interest in SPM derived from eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA; 22:6 n-3). The E-series resolvins, (RvE1-E3) are formed from EPA via a hydroxyl intermediate 18-hydroxyeicosapentaenoic acid (18-HEPE) and initially require acetylated COX-2 or cytochrome P450. 18-HEPE is then converted by 5-lipoxygenase to RvE1, RvE2 or by 15lipoxygenase to RvE3 [4,5] (Fig. 1). DHA can be metabolised by acetylated COX-2 or 15-lipoxygenase, to the unstable intermediate 17-hydroperoxydocosahexaenoic acid (17-HpDHA) that

A.E. Barden et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 107 (2016) 24–29

25

form includes questions on type, dose and mode of ingestion of fish oil taken (fish oil on juice or as capsules). Answers to these questions allowed calculation of the dose of n-3FA (18% EPAþ12% DHA w/w, 1 mL fish oil weighs 0.92 g) as g/d. A small minority of patients took a concentrate of fish oil n-3FA as natural triglycerides, in which case the daily dose was computed as the equivalent dose, with regard to EPA and DHA content, of standard fish oil in g/day. Pain score was estimated from the VALI form as previously described [19].

Fig. 1. Synthesis of E-series SPM from EPA and D-series SPM and maresins from DHA. The SPM measured in this study are identified in bold letters.

can form protectin D1 (PD1), 10S,17S-dihydroxydocosahexaenoic acid (10S,17S-DHDHA) and 17-hydroxydocosahexaenoic acid (17HDHA). The D-series resolvins (RvD1–RvD6) form as a result of metabolism of 17-HDHA by 5-lipoxygenase [6] (Fig. 1). In humans, the maresins are formed by metabolism of DHA by macrophage 12-lipoxygenase giving rise to 14hydroxydocosahexaenoic acid (14-HDHA) and maresin-1 (MaR1) [7] [8] (Fig. 1). Fish oils are a rich source of EPA and DHA, that have been shown to enhance management of rheumatoid arthritis (RA) [9] and systemic lupus [10,11] and to reduce reoccurrence rates in Crohn's disease in patients who are at high risk for relapse [12]. While a number of potential anti-inflammatory actions of n-3FA have been identified [13], it is conceivable that conversion of EPA and DHA to SPM could be a significant contributor to the disease mitigating effects of fish oil in inflammatory diseases. However, in spite of animal studies, which have demonstrated the presence of SPM in experimentally-induced inflammation [14,15] and the demonstration of SPM in healthy subjects taking fish oil [3], reports regarding SPM in human arthropathies are confined to a limited exploratory analysis of a small number of synovial fluid samples [16]. Based on favourable results of randomised controlled trials [17], at the Royal Adelaide Hospital patients with inflammatory arthropathies are routinely given advice to take fish oil supplements in addition to disease-modifying anti-inflammatory drugs. This study was undertaken to determine whether SPM are present in chronic inflammatory knee effusions of patients with arthritis of various aetiologies, to examine how well plasma SPM concentrations reflected those of synovial fluid from the inflamed joint, and to compare plasma SPM concentrations in patients with arthritis with those of healthy controls taking n-3FA.

2. Patients and methods 2.1. Recruitment of patients and healthy volunteers 2.1.1. Arthritic patients All patients gave informed written consent and the study protocol was approved by the Human Research Ethics Committee, Royal Adelaide Hospital. All procedures were performed in accordance with the Declaration of Helsinki. Synovial fluid and peripheral blood samples were obtained contemporaneously from 36 patients undergoing arthrocentesis of an inflammatory knee effusion. Most patients had been told previously to take n-3FA as fish oil 10–15 mL daily as a complement to therapy with anti-rheumatic drugs. Subjects completed a Vital Activities and Lifestyle Index (VALI) form [18] and erythrocyte sedimentation rate (ESR) and CRP were measured as a routine component of clinical assessment. Our modification of the VALI

2.1.2. Healthy volunteers Thirty six healthy volunteers were selected as a comparison group on the basis of age and gender. They were recruited from the general population in Perth, Western Australia and were nonsmokers; with no history of chronic disease, not taking antihypertensive or lipid lowering agents; aspirin or non-steroidal anti-inflammatory drugs; and not consuming fish meals or n-3FA supplements prior to the study. The volunteers gave informed written consent and the study protocol was approved by the Human Research Ethics Committee, at the University of Western Australia. All procedures were performed in accordance with the Declaration of Helsinki. Plasma SPM were measured after 4 weeks of supplementation with daily dose of 4 capsules of Omega Daily (Blackmores Australia), each 1 g capsule provides 360 mg of EPA and 240 mg of DHA as natural marine triglycerides In terms of EPA þDHA content, the dose given this equates to  8 g/d of standard fish oil given to the arthritis patients. 2.2. Measurement of plasma and synovial fluid SPM SPM in plasma and synovial fluid were measured by liquid chromatography-tandem mass spectrometry (LC–MS/MS) as previously described [20]. Briefly, internal standard (leukotriene B4-d4, 0.5 ng) was added to 1 mL of plasma or synovial fluid that was acidified to pH 3, and applied to a Bond Elut C18 cartridge (500 mg, Agilent Technologies, Lake Forrest, CA, USA). After washing with water and hexane, the SPMs were eluted with ethyl acetate (2 mL), dried under nitrogen and reconstituted in 5 mM ammonium acetate (pH¼8.9) and methanol (1/1; v/v) for analysis by LC–MS/MS, using a Thermo Scientific TSQ Quantum Ultra triple quadrupole LC– MS system equipped with an electrospray ionisation source operated in the negative ion mode. LC was performed on a Zorbax Eclipse XDB C18 column under conditions previously described [20]. Plasma and synovial fluid SPM were identified on the basis of the retention time, mass spectrum, and the parent and product ions of authentic standards as previously reported [20]. The standards 18-HEPE, 17-HDHA, resolvin D1 (RvD1) 17R-resolvin D1 (17R-RvD1), resolvin D2 (RvD2), 10S,17S-DHDHA, MaR-1 and leukotriene B4-d4 (LTB4-d4) were purchased from Cayman Chemicals (Ann Arbor, MI, USA). PD1 standard was provided by Professor Charles N Serhan (Harvard Medical School, Boston, Massachusetts, USA). Resolvin E1 (RvE1) and 14-HDHA were made available by Cayman Chemicals. Resolvin E2 (RvE2), resolvin E3 (RvE3) and 18R-resolvin E3 (18RRvE3) standards were provided by Professor Makoto Arita (Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Japan). 2.3. Assessment of E series and D-series SPM in relation to their precursors The ability to form E-series SPM and D-series SPM from their respective precursor substrates 18-HEPE and 17-HDHA was inferred from the ratios (RvE1 þRvE2 þRvE3 þ 18R-RvE3)/18-HEPE and (RvD1þ 17R-RvD1 þRvD2)/17-HDHA, respectively. MaR-1, 14HDHA, PD1 and 10S,17S-DHDHA that derive from DHA were excluded from this equation as the pathway for their synthesis does not involve 17-HDHA.

26

A.E. Barden et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 107 (2016) 24–29

2.4. Fatty acid analysis Aliquots of synovial fluid and plasma were treated with chloroform/isopropanol to extract lipids. The phospholipid fraction was separated by thin layer chromatography and subjected to methanolysis. The fatty acid methyl esters were quantified by gasliquid chromatography as previously described [21,22].

Table 1 Characteristics of the arthritis patients. Diagnosis

Psoratic arthritis (n¼6)

Gout (n¼ 4)

Pauci arthritis (n¼ 2) B27 spondyloarthritis (n ¼2)

3. Results The characteristics of the patients are shown in Table 1. Thirty three of the patients were taking n-3 FA as fish oil supplements in stated doses ranging from 4 to 28 g daily; (mean intake 11 g, median 11 g, IQR 6.4 g, 14 g). The duration of fish oil intake varied from 1 month to 12 years (mean 4 yrs, median 4 yrs, IQR 3 months, 6 yrs). The synovial fluid levels of EPA and DHA were 3.570.5% and 5.470.3% of total phospholipid fatty acids, respectively, and corresponding plasma levels were EPA 4.570.6% and DHA 5.770.3%. Duration of arthritis ranged from 1 to 41 years with the average duration 16 (95% CI 9, 20) years. The distribution of arthropathies has been previously reported [19] and was rheumatoid arthritis (n¼15, 45.5%); psoriatic arthritis (n¼ 6, 16.7%); gout (n¼4, 11%); monoarthritis (n¼ 2); pauci arthritis (n¼2); B27þspondyloarthritis (n¼2), and 1 patient each diagnosed with Crohn's arthritis, calcium pyrophosphate crystal deposition, mixed connective tissue disease, anterior cruciate ligament insufficiency and ulcerative colitis. The healthy volunteers were well matched in terms of age (57.372.4 yrs compared with patients 58.671.1 yrs) and gender distribution, (males/female¼15/21 compared with patients males/ female¼ 11/25). Plasma EPA and DHA increased from 1.570.1% and 4.770.2% respectively, to 5.770.1% and 7.570.1% respectively, after 4weeks of n-3FA supplements, indicating excellent compliance. 3.1. Comparison of SPMs in synovial fluid with plasma of arthritis patients EPA derived 18-HEPE, RvE1, RvE2, RvE3 and 18R-RvE3 and DHA derived 17-HDHA, RvD1, 17R-RvD1, RvD2, PD1, 10S,17S-HDHA, MaR-1, and 14-HDHA were quantified in synovial fluid and plasma of patients with arthritis (Table 2). The concentrations of all SPM were elevated in plasma compared with synovial fluid. We found no significant relationship between the dose of n-3 FA and any SPM in synovial fluid or plasma (Supplementary Table 1). The duration of treatment was not related to 18-HEPE or 17-HDHA but was significantly related to several of E- and D-series resolvins (Supplementary Table 1).

Length of n-3 Dose n-3 FA intake (yrs) FA (g/day)

F F F F F F F F F F F F F M M M M F M F F M M M M F F M F F F F F

83 82 48 61 73 57 60 62 82 52 66 68 63 63 84 19 56 58 60 58 55 64 56 33 54 33 35 62 53 34 62 46 67

8 6 5 9 10 7 9 0.1 11 4 1 0.3 11 9 5 0.7 5 5 6 6 0.8 0.8 4 0 0 2 2 0.1 12 0 0.2 1 0.1

2.7 4.1 4.1 1.5 1.8 2.8 3.0 4.1 2.8 3.0 2.8 4.1 8.3 1.5 1.8 5.5 3.6 1.2 4.1 3.6 4.1 8.3 8.3 0 0 2.8 4.1 4.1 1.4 0 4.1 3.0 2.8

F

55

3.0

2.7

M

50

0

0

F

50

0.1

1.9

Rheumatoid arthritis (n ¼15)

2.5. Statistical analysis Statistical analysis was carried out using STATA (Statacorp Texas USA). The agreement between concentrations of SPM in synovial fluid and plasma of the patients was examined using the method of Bland Altman [23]. Lin's concordance correlation coefficient (CCC) [24] was used to assess concordance between SPM in synovial fluid and plasma. Plasma SPM in the arthritis group was compared with the healthy control group after n-3 LC-PUFA supplementation using a linear regression model with bootstrapping to obtain a more accurate estimate of the variance. P-values were adjusted for multiple testing using the method of Holm [25]. The effect of duration and dose of n3FA on SPM in patients and controls was examined using these terms as covariates in the model. The respective SPM ratios for E- and Dseries SPM for synovial fluid and plasma of the patients were compared using a Wilcoxin signed-rank test. The respective SPM ratios for plasma in the patients and healthy controls were compared using a Mann–Whitney U test. Spearman correlation co-efficients were used to explore relationships between SPM, pain score, CRP and ESR.

Sex Age (yrs)

Monoarthritis (n¼2) Crohn's Arthritis (n¼ 1) Calcium pyrophosphate crystal deposition (n ¼1) Mixed connective tissue disease (n¼ 1) Anterior cruciate ligament insufficiency (n¼ 1) Ulcerative colitis with arthropathy (n¼1)

The relationship between SPM in synovial fluid and plasma was further examined using the method of Bland Altman, which evaluates bias in findings by comparison of differences between the means of corresponding data sets from the mean for the sets combined, and Lin's concordance coefficient, which evaluates the degree to which plots of pairs of observations approach the 45° line through the origin. Although the concentrations of all plasma SPM were significantly higher than in synovial fluid (Table 3) there was excellent concordance between synovial fluid and plasma 18-HEPE (CCC¼ 0.60, CI 0.45,0.75) and synovial fluid and plasma 17-HDHA (CCC¼0.71, CI 0.59,0.83). 3.2. Relationships between SPM markers of inflammation and pain in arthritis patients The relationship between plasma and synovial fluid SPM with the inflammation markers ESR and CRP was assessed. All plasma SPM were significantly negatively related to ESR (18-HEPE, ρ ¼  0.54, P¼ 0.002; 17-HDHA, ρ ¼  0.70, Po0.0001; 14-HDHA, ρ ¼  0.51, Po0.005; RvE2, ρ ¼  0.68, Po0.0001; RvD1, ρ ¼  0.66, P¼0.0001; 17R-RvD1, ρ ¼  0.70, Po0.0001; PD1, ρ ¼  0.62, Po0.0004 and MaR1 ρ ¼  0.56, P¼0.0015). In synovial fluid ESR was only significantly related to 17-HDHA (ρ ¼  0.74, Po0.0001); 14-HDHA (ρ ¼  0.70, Po0.0001) and RvD1 (ρ ¼  0.41, P¼ 0.03). SPM in synovial fluid and plasma were not significantly correlated with CRP. There were no significant correlations between any of the

A.E. Barden et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 107 (2016) 24–29

27

Table 2 SPM in synovial fluid and plasma in patients with arthritis and plasma SPM in healthy volunteers, taking n-3FA. SPM (pg/mL)

18-HEPE RvE1 RvE2 RvE3 18R-RvE3 17-HDHA RvD1 17R-RvD1 RvD2 PD1 10S,17S, DHDHA 14-HDHA MaR1

Synovial fluid

Plasma

Arthritis (n¼36)

Arthritis (n¼ 36)

242.8 24.6 68.8 17.9 13.9 227.3 72.2 104.5 48.9 65.6 38.5 223.3 54.8

3067.1 96.1 774.2 99.3 95.7 3949.3 122.7 153.1 120.0 86.7 93.7 6076 71.4

(162.0,363.9) (18.5,32.7) (49.7,95.2) (13.2,24.2) (9.1,21.1) (132.6,389.4) (61.4,84.9) (89.6,121.9) (40.6,58.8) (56.2,76.7) (32.4,45.9) (138.6,359.7) (47.4,63.5)

(954.1,5180.2) (8.4,183.8) (201.9,1346) (25.9,172.6) (43.8,147.7) (1099.2, 6799.3) (83.8,161.5) (122.3,183.9) (71.1-168.9) (71.7,101.7) (44.7,142.6) (3930,8222) (59.2,83.6)

P value

Adjusted P-value

Healthy controls (n¼ 36)

Arthritis v's control

Arthritis v's control

543.2 17.0 26.3 17.8 31.3 923.7 45.5 65.6 29.2 41.1 23.4 18700 34.6

0.02 0.08 0.01 0.03 0.018 0.038 o0.0001 o0.0001 o0.0001 o0.0001 0.005 0.001 o0.0001

NS NS Po 0.05 NS NS NS Po 0.05 Po 0.05 Po 0.05 Po 0.05 Po 0.05 Po 0.05 Po 0.05

(458.6,627.7) (11.6,22.2) (24.3,28.3) (15.6,20.0) (22.9,39.7) (678.0,1169.3) (42.5,48.5) (62.9,68.3) (27.1,31.2) (39.3,42.8) (22.5,24.3) (12242,25159) (33.2,36.1)

Values are mean and 95% CI (P-value for differences between plasma SPM in arthritic patients and healthy controls from the regression analysis before and after adjustment for multiple comparisons). Table 3 Comparison of SPM in synovial fluid with SPM in plasma of patients with arthritis, using Bland Altman average difference and Lin's concordance correlation coefficient.

Ln 18-HEPE Ln RvE1 Ln RvE2 Ln RvE3 Ln 18R-RvE3 Ln 17-HDHA Ln RvD1 Ln 17R-RvD1 Ln RvD2 Ln PD1 Ln 10S,17S, DHDHA Ln MaR-1 Ln 14-HDHA

Δ, pg/mL (95% LOA)

CCC (95% CI)

 1.17  0.163  0.702  0.479  0.814  1.258  0.275  0.229  0.392  0.148  0.419

0.600 (0.453,0.746) 0.153 (0.144,0.450) 0.399 (0.193,0.604) 0.486 (0.273,0.698) 0.437 (0.200,0.674) 0.707 (0.585,0.829) 0.412 (0.174,0.649) 0.214 (  0.072,0.501) 0.373 (0.154,0.591) 0.305 (0.014,0.595) 0.305(0.078,0.533)

(  3.018,0.678) Po 0.0001 (  3.043, 2.717) P o0.028 (  3.622, 2.219) P o 0.0001 (  2.668,1.709) P o0.0008 (  3.630,2.002) Po 0.0009 (  2.809,0.293) Po0.0001 (  1.449,0.899) P¼ 0.0038 (  1.411,0.953) P ¼0.067 (  2.007,1.224) P o 0.0001 (  1.225,0.928) P ¼0.267 (  1.992,1.154) P ¼0.0001

 0.148 (  1.221,0.926) P¼ 0.267 0.244 (  0.056,0.543)  2.625 (  4.407, 0.843) Po 0.0001 0.262 (0.147,0.376)

Values are average difference (Δ ¼ synovial fluid SPM  plasma SPM, pg/mL) and 95% limits of agreement (95% LOA). P values refer to the probability of lack of bias in levels between synovial fluid and plasma. The negative Δ values indicate bias toward higher levels in plasma. Lin's Concordance correlation coefficient (CCC) and 95% confidence intervals (95% CI). Values are log transformed to approximate normality.

plasma SPM and pain score but synovial fluid RvE2 was significantly negatively related to pain score (ρ ¼ 0.411, P¼0.02). 3.3. Comparison of plasma SPM in patients and healthy controls taking n-3 LC-PUFA Table 2 compares plasma SPM in the patients with those of healthy volunteers supplemented with n-3FA. After correction for multiple comparisons, patients with arthritis had significantly higher plasma concentrations of RvE2, the measured D-series resolvins, PD1 MaR-1 but 14-HDHA, was significantly lower than the healthy controls. The difference in plasma levels of SPM between arthritis patients and controls was not affected by the dose or duration of n-3 FA intake. 3.4. Relationships between SPM and fatty acid (EPA and DHA) levels In patients with arthritis, the relationship between individual SPM in synovial fluid and plasma and their fatty acid substrate (EPA or DHA) was examined using Spearman correlation coefficients (Supplementary Table 1). EPA was significantly correlated with

synovial fluid 18-HEPE but not any other measured E-series SPM in synovial fluid. Plasma EPA was not significantly related to 18-HEPE or E-series SPMs. DHA was not significantly correlated with any SPM in synovial fluid or plasma. In synovial fluid and plasma of arthritic patients, most of the E-series and D-series SPM were significantly correlated with their precursor's 18-HEPE and 17-HDHA, respectively (Supplementary Table 1). In healthy controls, plasma EPA was significantly associated with plasma 18-HEPE and RvE2 but DHA was not significantly correlated with 17-HDHA, 14-HDHA, the D series SPM or MaR-1. Plasma 18-HEPE and 17-HDHA were not significantly related to their respective downstream SPM. 3.5. Assessment of E-series and D-series SPM in relation to their precursors Although plasma levels of 18-HEPE and E-series SPM were higher than synovial fluid in patients with arthritis (Table 2), the ratio E-series SPM to 18-HEPE was significantly higher in synovial fluid suggesting increased conversion, (P o0.01, Fig. 2a). Similarly, conversion of 17-HDHA to D-series SPM was elevated in synovial fluid relative to corresponding plasma samples (P o0.01, Fig. 2b). Conversion of plasma 18-HEPE to E-series SPM, and 17-HDHA to D-series SPM, was significantly elevated in patients, compared with healthy controls (P o0.01, Fig. 2a and b).

4. Discussion This is the first study to comprehensively measure a range of SPM in synovial fluid and plasma of arthritis patients taking n-3FA and healthy volunteers of similar age and gender. We have shown that a broad spectrum of SPM derived from both EPA and DHA are present in synovial fluid and plasma of patients taking n-3FA for arthritis. A large number of SPM in plasma and synovial fluid were negatively correlated with ESR a marker of inflammation that associates with severity of arthritis. Synovial fluid RvE2 was negatively related to patientdetermined visual analogue pain scores. The concentrations of SPM were higher in plasma but assessment of E- and D-series SPM in relation to their precursors suggested that conversion of 18-HEPE and 17-HDHA to E- and D-series SPM, respectively, was greater in synovial fluid than plasma. Most plasma SPM in these patients were significantly higher than those of healthy volunteers supplemented with n-3FA and conversion to E- and D-series SPM was greater. The concentrations of SPM in synovial fluid were of the order of 0.1–0.2 nM, a concentration range that has been shown to affect neutrophil function in vitro [26]. The findings indicate that, at least in

28

A.E. Barden et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 107 (2016) 24–29

Fig. 2. (a) E-series SPM/18-HEPE in synovial fluid and plasma of patients with arthritis and healthy controls (†Po 0.01, Wilcoxin signed-rank test, comparing synovial fluid with plasma in patients) (†P o 0.01, a Mann–Whitney U test comparing plasma of patients with healthy controls). (b) D-series SPM/17-HDHA in synovial fluid and plasma of patients and healthy controls (†P o 0.01, Wilcoxin signed-rank test, comparing synovial fluid with plasma in patients) (†Po 0.01, Mann–Whitney U test comparing plasma of patients with healthy controls).

patients well-supplied with dietary EPA and DHA, n-3 derived SPM are a usual accompaniment of chronic joint inflammation. While these observations raise the question as to whether SPM influence chronic inflammation, it must be kept in mind that chronic inflammatory joint effusions typically involve a state of altered homoeostasis in which the intensity of inflammation is far less than that of an acute inflammatory episode. The negative relationship between patient perceived pain score and synovial fluid RvE2 suggest SPM may have a modulating effect in chronic inflammatory situations. In animal models RvE1, RvD1 and MaR1 have all been shown to attenuate pain [27,28]. The analgesic effects are mediated by specific G-protein-coupled receptors that are known to bind RvE2 [29] Although plasma SPM were generally higher than SPM of synovial fluid, the concordance between 18-HEPE and 17-HDHA, the precursors to the E- and D-series resolvins, measured in plasma and synovial fluid was good. These results suggest plasma measurements of SPM are a good reflection of SPM levels in synovial fluid. Importantly, the concentration of E- and D-series SPM relative to their respective precursor's 18-HEPE and 17-HDHA, was significantly elevated in synovial fluid compared with plasma suggesting enhanced conversion of Eand D-series SPMs at the site of inflammation. Plasma EPA and DHA levels were similar between the patients and healthy controls supplemented with similar amounts of n-3FA for 4 weeks, but plasma SPM concentrations in patients were substantially higher than those of healthy volunteers, of similar age and

gender. The findings suggest an increase in circulating bioactive SPM may be a feature of patients with arthritis who are taking n-3FA. Ours is the first study to measure a range of SPM in synovial fluid of patients with arthritis. Giera et al. [16], have previously reported untargeted LC–MS/MS screening of lipids in synovial fluid samples coupled with targeted analysis of a selection of SPM (MaR-1, lipoxin A4 and RvD5). The targeted analysis also included the dual lipoxygenase product 5S,12S-diHETE, which had been identified in the untargeted screening. The targeted analytes were present in each of five synovial fluid samples obtained from knees of patients with rheumatoid arthritis undergoing arthroscopy [16]. While it is difficult to generalise from such small numbers, their findings allowed the possibility that SPM may be a usual feature of rheumatoid joint effusions. Our study extends these preliminary findings to show that a broad range of n-3FA-derived SPM is a usual finding in synovial fluid in rheumatoid arthritis and other inflammatory arthropathies. The distribution of SPM in synovial fluid was similar to that of plasma. The presence of mono-hydroxyprecursors and their enhanced conversion to E-series and D-series SPM is consistent with local synthesis of SPM, for which cells and enzymes known to be present in arthritic synovium provide relevant machinery. For example synovial macrophages, neutrophils and mast cells express 5-lipoxygenase and 15-lipoxygenase is expressed by macrophages fibroblasts and endothelial cells [30]. The neutrophils in rheumatoid synovial fluid also express 5-lipoxygenase (albeit at lower levels than their peripheral blood counterparts) [31]. We have reported previously that stated intakes of fish oil taken for arthritis in a routine clinic setting correlate poorly with EPA and DHA in plasma and synovial fluid [19]. Most of the patients were taking fish oil and mean levels of EPA and DHA were substantially higher than those in healthy volunteers [32] or patients with rheumatoid arthritis not taking n-3FA [33]. In this study, we showed that EPA and DHA were poorly correlated with SPM with significant correlations found only for EPA and 18-HEPE in synovial fluid and plasma of controls. The lack of correlation between SPM and their fatty acid substrate may be due to different rates of synthesis and metabolic breakdown of individual SPM. Our findings should be viewed as an addition to knowledge regarding inhibitory effects of medicinal doses of n-3FA supplements on pro-inflammatory mediator production [34]. In patients with arthritis these effects include inhibition of leukotriene B4 synthesis [17], prostaglandin E2 [33,35], interleukin-1 and tumour necrosis factor-alpha [35–37]. In addition to these effects on humoral factors, cellular immunity may be inhibited. For example, diminished T cell responsiveness has been observed in rats fed diets supplemented with EPA- or DHA-ethyl esters [38] and the dendritic cells of rats fed fish oil diets display reduced levels of surface expression of MHC class II and adhesion molecules, as well as reduced antigen presentation activity [39]. SPM are present in chronic knee effusions and although the levels are lower than in plasma, the association between synovial fluid RvE2 and reduced patient perceived pain suggests that synthesis of SPM at the site of inflammation is a relevant mechanism by which n-3FA alleviate the symptoms of arthritis.

Authors’ contributions AEB was responsible for recruitment and collection of samples from healthy controls, writing the manuscript and assisting with statistical analysis. MM was involved in design of the study, collection and preparation of samples from patients with inflammatory arthritis, collection and interpretation of initial data and revising the manuscript.

A.E. Barden et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 107 (2016) 24–29

EM was responsible for assaying the specialised proresolving mediators of inflammation in the study interpreting data and revising the manuscript. MP was responsible for the statistical analysis of the data and contributed to revising the manuscript. LGC planned and initiated the arthritis component of the study, recruited and enroled patients, undertook arthrocenteses and statistical analyses and contributed substantially to manuscript preparation. TAM was responsible for recruitment and collection of samples from healthy controls and drafting the manuscript. All authors read and approved the final manuscript.

Conflict of interest None of the authors has any competing interests to declare.

Acknowledgements This work was supported by a grant-in-aid from Arthritis Australia and a project grant from the National Heart Foundation of Australia.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.plefa.2016.03.004.

References [1] C.N. Serhan, S. Krishnamoorthy, A. Recchiuti, N. Chiang, Novel anti-inflammatory—pro-resolving mediators and their receptors, Curr. Top. Med. Chem. 11 (2011) 629–647. [2] C.N. Serhan, N. Chiang, T.E. Van Dyke, Resolving inflammation: dual antiinflammatory and pro-resolution lipid mediators, Nat. Rev. Immunol. 8 (2008) 349–361. [3] E. Mas, K.D. Croft, P. Zahra, A. Barden, T.A. Mori, Resolvins D1, D2, and other mediators of self-limited resolution of inflammation in human blood following n-3 fatty acid supplementation, Clin. Chem. 58 (2012) 1476–1484. [4] M. Spite, J. Claria, C.N. Serhan, Resolvins, specialized proresolving lipid mediators, and their potential roles in metabolic diseases, Cell. Metab. 19 (2014) 21–36. [5] Y. Isobe, M. Arita, S. Matsueda, R. Iwamoto, T. Fujihara, H. Nakanishi, R. Taguchi, K. Masuda, K. Sasaki, D. Urabe, M. Inoue, H. Arai, Identification and structure determination of novel anti-inflammatory mediator resolvin E3, 17,18-dihydroxyeicosapentaenoic acid, J. Biol. Chem. 287 (2012) 10525–10534. [6] C.N. Serhan, N.A. Petasis, Resolvins and protectins in inflammation resolution, Chem. Rev. 111 (2011) 5922–5943. [7] B. Deng, C.-W. Wang, H.H. Arnardottir, Y. Li, C.-Y.C. Cheng, J. Dalli, C.N. Serhan, Maresin Biosynthesis and identification of maresin 2, a new anti-inflammatory and pro-resolving mediator from human macrophages, PLoS One 9 (2014). [8] C.N. Serhan, R. Yang, K. Martinod, K. Kasuga, P.S. Pillai, T.F. Porter, S.F. Oh, M. Spite, Maresins: novel macrophage mediators with potent antiinflammatory and proresolving actions, J. Exp. Med. 206 (2009) 15–23. [9] S.M. Proudman, M.J. James, L.D. Spargo, R.G. Metcalf, T.R. Sullivan, M. Rischmueller, K. Flabouris, M.D. Wechalekar, A.T. Lee, L.G. Cleland, Fish oil in recent onset rheumatoid arthritis: a randomised, double-blind controlled trial within algorithm-based drug use, Ann. Rheum. Dis. 74 (2015) 89–95. [10] A.J.E. Walton, M.L. Snaith, M. Locniskar, A.G. Cumberland, W.J.W. Morrow, D. A. Isenberg, Dietary fish oil and the severity of symptoms in patients with systemic lupus erythematosus, Ann. Rheum. Dis. 50 (1991) 463–466. [11] E.M. Duffy, G.K. Meenagh, S.A. McMillan, J.J. Strain, B.M. Hannigan, A.L. Bell, The clinical effect of dietary supplementation with omega-3 fish oils and/or copper in systemic lupus erythematosis, J. Rheumatol. 31 (2004) 1551–1556. [12] A. Belluzzi, C. Brignola, M. Campieri, A. Pera, S. Boschi, M. Miglioli, Effect of an Enteric-coated Fish-oil Preparation on Relapses in Crohn's Disease, N. Engl. J. Med. 334 (1996) 1557–1560. [13] L.G. Cleland, M.J. James, S.M. Proudman, Fish oil: what the prescriber needs to know, Arthritis Res. Ther. 8 (2006) 202–211.

29

[14] C.N. Serhan, C.B. Clish, J. Brannon, S.P. Colgan, N. Chiang, K. Gronert, Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2–nonsteroidal antiinflammatory drugs and transcellular processing, J. Exp. Med. 192 (2000) 1197–1204. [15] C.N. Serhan, S. Hong, K. Gronert, S.P. Colgan, P.R. Devchand, G. Mirick, R. L. Moussignac, Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals, J. Exp. Med. 196 (2002) 1025–1037. [16] M. Giera, A. Ioan-Facsinay, R. Toes, F. Gao, J. Dalli, A.M. Deelder, C.N. Serhan, O. A. Mayboroda, Lipid and lipid mediator profiling of human synovial fluid in rheumatoid arthritis patients by means of LC–MS/MS, Biochim. Biophys. Acta 2012 (1821) 1415–1424. [17] L.G. Cleland, J.K. French, W.H. Betts, G.A. Murphy, M.J. Elliott, Clinical and biochemical effects of dietary fish supplements in rheumatoid arthritis, J. Rheumatol. 15 (1988) 1471–1475. [18] T. Pincus, J.A. Summey, S.A.J. Soraci, K.A. Wallston, N.P. Hummon, Assessment of patient satisfaction in activities of daily living using a modified Stanford Health Assessment Questionnaire, Arthritis Rheum. 26 (1983) 1346–1353. [19] M. Moghaddami, M. James, S. Proudman, L.G. Cleland, Synovial fluid and plasma n3 long chain polyunsaturated fatty acids in patients with inflammatory arthritis, Prostag. Leukotr. Essent. Fat. Acids 97 (2015) 7–12. [20] A. Barden, E. Mas, K.D. Croft, M. Phillips, T.A. Mori, Short-term n-3 fatty acid supplementation but not aspirin increases plasma proresolving mediators of inflammation, J. Lipid Res. 55 (2014) 2401–2407. [21] R.G. Metcalf, M.J. James, E. Mantzioris, L.G. Cleland, A practical approach to increasing intakes of n-3 polyunsaturated fatty acids: use of novel foods enriched with n-3 fats, Eur. J. Clin. Nutr. 57 (2003) 1605–1612. [22] T.A. Mori, G.F. Watts, V. Burke, E. Hilme, I.B. Puddey, L.J. Beilin, Differential effects of eicosapentaenoic acid and docosahexaenoic acid on vascular reactivity of the forearm microcirculation in hyperlipidemic, overweight men, Circulation 102 (2000) 1264–1269. [23] J.M. Bland, D.G. Altman, Statistical methods for assessing agreement between two methods of clinical measurement, Lancet 1 (1986) 307–310. [24] L.I. Lin, A concordance correlation coefficient to evaluate reproducibility, Biometrics 45 (1989) 255–268. [25] S. Holm, A simple sequentially rejective multiple test procedure, Scand. J. Stat. 6 (1979) 65–70. [26] L.V. Norling, J. Dalli, R.J. Flower, C.N. Serhan, M. Perretti, Resolvin D1 limits polymorphonuclear leukocyte recruitment to inflammatory loci receptordependent actions, Arterioscler. Thromb. Vasc. Biol. 32 (2012) 1970–1978. [27] Z.-Z. Xu, L. Zhang, T. Liu, J.Y. Park, T. Berta, R. Yang, C.N. Serhan, R.-R. Ji, Resolvins RvE1 and RvD1 attenuate inflammatory pain via central and peripheral actions, Nat. Med. 16 (2010) 592-U129. [28] C.N. Serhan, J. Dalli, S. Karamnov, A. Choi, C.-K. Park, Z.-Z. Xu, R.-R. Ji, M. Zhu, N.A. Petasis, Macrophage proresolving mediator maresin 1 stimulates tissue regeneration and controls pain, FASEB J. 26 (2012) 1755–1765. [29] S.F. Oh, M. Dona, G. Fredman, S. Krishnamoorthy, D. Irimia, C.N. Serhan, Resolvin E2 formation and impact in inflammation resolution, J. Immunol. 188 (2012) 4527–4534. [30] K.R. Gheorghe, M. Korotkova, A.I. Catrina, L. Backman, E.A.F. Klint, H.E. Claesson, O. Radmark, P.-J. Jakobsson, Expression of 5-lipoxygenase and 15lipoxygenase in rheumatoid arthritis synovium and effects of intraarticular glucocorticoids, Arthritis Res. Ther. 11 (2009). [31] C. Jobin, C. Kreis, J. Gauthier, J. Letarte, A.D. Beaulieu, Differential synthesis of 5-lipoxygenase in peripheral-blood and synovial-fluid neutrophils in rheumatoid-arthritis, J. Immunol. 146 (1991) 2701–2707. [32] E. Mantzioris, L.G. Cleland, R.A. Gibson, M.A. Neumann, M. Demasi, M.J. James, Biochemical effects of a diet containing foods enriched with n-3 fatty acids, Am. J. Clin. Nutr. 72 (2000) 42–48. [33] G.E. Caughey, M.J. James, S.M. Proudman, L.G. Cleland, Fish oil supplementation increases the cyclooxygenase inhibitory activity of paracetamol in rheumatoid arthritis patients, Complement. Ther. Med. 18 (2010) 171–174. [34] T.A. Mori, L.J. Beilin, Omega-3 fatty acids and inflammation, Curr. Atheroscler. Rep. 6 (2004) 461–467. [35] G.E. Caughey, E. Mantzioris, R.A. Gibson, L.G. Cleland, M.J. James, The effect on human tumor necrosis factor-alpha and interleukin 1-beta production of diets enriched in n-3 fatty acids from vegetable oil or fish oi1, Am. J. Clin. Nutr. 63 (1996) 116–122. [36] J.M. Kremer, D.A. Lawrence, W. Jubiz, R. DiGiacomo, R. Rynes, L. E. Bartholomew, M. Sherman, Dietary fish oil and olive oil supplementation in patients with rheumatoid arthritis, Arthritis Rheum. 33 (1990) 810–820. [37] M.J. James, R.A. Gibson, L.G. Cleland, Dietary polyunsaturated fatty acids and inflammatory mediator production, Am. J. Clin. Nutr. 71 (Suppl.) (2000) 343S–348S. [38] C.A. Jolly, Y.-H. Jiang, R.S. Chapkin, D.N. McMurray, Dietary (n-3) polyunsaturated fatty acids suppress murine lymphoproliferation, interleukin-2 secretion, and the formation of diacylglycerol and ceramide, J. Nutr. 127 (1997) 37–43. [39] P. Sanderson, G.G. MacPherson, C.H. Jenkins, C. P.C., Dietary fish oil diminishes the antigen presentation activity of rat dendritic cells, J. Leukoc. Biol. 62 (1997) 771–777.