Pinolenic acid Mediated Anti-inflammatory and Immunometabolic Effects in Peripheral blood-derived Monocytes from patients with Rheumatoid Arthritis is Associated with Modulation of miRNA expression

Objectives: Pinolenic acid (PNLA), an omega-6 polyunsaturated fatty acid from pine nuts, has anti-inflammatory and anti-atherogenic effects. We aimed to investigate the actions of PNLA on activated purified monocytes from peripheral blood of patients with rheumatoid arthritis (RA).Methods: Flow cytometry was used to assess the intracellular expression of TNF-α, IL-6, IL-1β, and IL-8 in purified monocytes from patients with RA after lipopolysaccharide (LPS) stimulation with/without PNLA pre-treatment. The whole genomic transcriptomic (WGT) profile of PNLA-treated, and LPS-activated monocytes from patients with active RA was investigated by RNA-sequencing.Results: PNLA reduced percentage of monocytes expressing the cytokines TNF-α by 23% (p=0.048), IL-6 by 25% (p=0.011), IL-1β by 23% (p=0.050) and IL-8 by 19% (p=0.066). Canonical pathway analysis showed that PNLA inhibited oxidative phosphorylation (p= 9.14E-09) and mitochondrial dysfunction (p=4.18E-08), while the sirtuin (SIRTs) signalling pathway was activated (p=8.89E-06). Pathway analysis predicted upstream activation of peroxisome proliferator-activated receptors (PPARs), sirtuin3, and let7miRNA, which are anti-inflammatory and antioxidative. In contrast, DAP3, LIF and STAT3, which are involved in TNF-α, and IL-6 signal transduction, were inhibited. Many miRNAs were modulated by PNLA suggesting potential post-transcriptional regulation of metabolic and immune response that has not been described previously. Multiple miRNAs target pyruvate dehydrogenase kinase-4 (PDK4), single-immunoglobulin interleukin-1 receptor-related molecule (SIGIRR), mitochondrially encoded ATP synthase membrane subunit 6 (MT-ATP6) and acetyl-CoA acyltransferase 2 (ACAA2); genes implicated in cell metabolism, inflammation, and mitochondrial dysfunction.Conclusion: PNLA has anti-inflammatory and immune-metabolic effects on monocytes that are pathogenic in RA and atherosclerosis. Dietary PNLA supplementation may regulate key miRNAs that are involved in mitochondrial, metabolic, and inflammatory pathways.

POS0346 THE THERAPEUTIC EFFECTS OF OMEGA-6 PUFA (PINOLENIC ACID) ON THE TRANSCRIPTOMIC PROFILE OF ACTIVATED PERIPHERAL BLOOD MONONUCLEAR CELLS ISOLATED FROM HEALTHY CONTROLS AND RHEUMATOID ARTHRITIS PATIENTS

Background:Globally, Rheumatoid arthritis (RA) is the most common chronic inflammatory arthritis. EULAR recommends patient education and disease modifying anti-rheumatic drugs including advanced targeted therapies for management1 2. Many patients seek advice on a dietary intervention that may improve symptoms. Foods high in omega-3 polyunsaturated fatty acids (PUFAs) is one of the most common recommended based on their anti-inflammatory properties3 4. Previously we showed pinolenic acid (PNLA), an omega-6 (PUFAs) found in pine nuts, reduced lipid uptake, endocytosis and migration of monocyte in vitro. PNLA also reduced IL-6, TNF-α, IL-1β and PGE2 produced by lipopolysaccharide (LPS) activated PBMCs from patients with RA and healthy controls (HCs).Objectives:We hypothesis that PNLA inhibits key inflammatory processes in synovities as in diagram.1. We investigated the transcriptomic profile of PNLA treatment on LPS activated PBMCs isolated from HCs and RA patients.Methods:Adult RA patients (n=6) were recruited from the Rheumatology Department of University Hospital of Wales together with 6 HCs. Blood was collected after taking signed informed consent. PBMCs were isolated by Ficoll gradient centrifugation and pre-treated with 25 μM PNLA or vehicle and then activated with 100 ng/ml LPS. RNA was extracted followed by library construction and sequencing for whole genomic transcriptome. DEGs were analysed using DESeq2 and pathway analysis was performed using Ingenuity pathway analysis. The study was approved by the Research Ethics Committee for Wales (reference no. 12/WA/0045).Results:DEGs were selected with at least differential genes using at least 1.2* fold changes and adjusted p-value of <0.05. PNLA significantly upregulates the expression of PDK4, PAI-1 (SERPINE1), FBP1, and NDRG2. These genes have important roles in metabolic pathways. Pathway analysis predicted upstream activation of nuclear receptors peroxisome proliferator-activated receptor (PPARs) involved in the anti-inflammatory process, and inhibition of NF-κB and STAT1, the major transcription factors of the pro-inflammatory cytokines TNF-α, IL-1, IL-6 and IFN-γ.Conclusion:PNLA has significant effects on the regulation of metabolic and inflammatory pathways in PBMCs from RA patients and HCs. Based on these results we demonstrate that PNLA may have beneficial anti-inflammatory effects in patients with RAReferences:[1]Agca R, Heslinga SC, Rollefstad S, Heslinga M, McInnes. IB, Peters MJL, Kvien, TK., Dougados, M, Radner, H, Atzeni, F. and Primdahl, J. 2017. EULAR recommendations for cardiovascular disease risk management in patients with rheumatoid arthritis and other forms of inflammatory joint disorders: 2015/2016 update. Annals of the rheumatic diseases, 76(1), pp.17-28.[2]Smolen JS, Landewé RBM, Bijlsma JWJ, et al. 2019. EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: updateAnnals of the Rheumatic Diseases 2020; 79:685-699.[3]Chehade L, Jaafar ZA, El Masri D, Zmerly H, Kreidieh D, Tannir H, Itani L, and El Ghoch, M. 2019. Lifestyle Modification in Rheumatoid Arthritis: Dietary and Physical Activity Recommendations Based on Evidence. Current rheumatology reviews, 15(3), pp.209-214.[4]Marchand NE, Chiu Y-H, Yoshida K, Malspeis S, Sparks JA, Costenbader K, et al. Threshold Level for Long-term Healthy Diet Adherence to Reduce the Risk of Rheumatoid Arthritis Among Women in a Prospective Cohort Using a Marginal Structural Model Approach.:25.Diagram 1.Disclosure of Interests:None declared

P201 Anti-inflammatory and immunologic actions of pinolenic acid in rheumatoid arthritis

Background Rheumatoid arthritis (RA) is a common inflammatory arthritis. Although advanced targeted therapies have improved prognosis, many patients seek advice on dietary intervention that may improve symptoms. Pinolenic acid (PNLA) is a polyunsaturated fatty acid found in pine nuts. We investigated the anti-inflammatory effects of 25-100 μM PNLA on cell line, primary culture, and peripheral blood mononuclear cells (PBMCs) from patients with RA and healthy controls (HCs). Methods 1- Migration using modified Boyden Chambers: THP-1 monocytes incubated with vehicle or PNLA were added to the apical compartment of a modified Boyden chamber. The migration of the cells through inserts of 8 μm pore size in response to the chemokine monocyte chemoattractant protein-1 (MCP-1) added to basolateral (bottom) chamber was determined. 2- Macropinocytosis using Lucifer yellow (LY): THP-1 and primary human macrophages were pre-incubated with PNLA or vehicle control followed by LY. After incubation, cells were removed, fixed and assessed by flow cytometry. 3- Lipid uptake using Dil-oxidised low-density lipoprotein (Dil-oxLDL): THP-1 and primary macrophages were pre-incubated with PNLA or vehicle control followed by Dil-oxLDL. After incubation, cells were removed, fixed and assessed by flow cytometry. 4- Cytokines release by lipopolysaccharide (LPS) stimulated PBMCs: PBMCs were isolated from blood obtained from RA patients aged ≥18 years and HCs. Monocytes were purified and cultured with PNLA or vehicle control. Cells were stimulated with LPS. IL-6, TNF-α, IL-1β and prostaglandin E2 (PGE2) in the supernatant were assessed by ELISAs. For macrophages, monocytes were left to grow and differentiate over 10 days, the differentiated macrophages were treated with PNLA or vehicle and activated with LPS and assayed in identical conditions for monocytes. Results PNLA at all concentrations reduced THP-1 monocytes migration by average of 55% (p &lt; 0.001) when compared with vehicle controls. Macropinocytosis of THP-1 macrophages and human macrophages were reduced by almost 50% (p &lt; 0.001) and 45% (p &lt; 0.001) respectively by PNLA. Similarly, Dil-oxLDL uptake by THP-1 macrophages and primary macrophages were reduced by 40% (p &lt; 0.01) and 25% (p &lt; 0.05) respectively by 25 μM PNLA. Release of IL-6 and TNF-α by LPS stimulated monocytes in RA patients were reduced with 25 and 50 μM PNLA by 60% (p &lt; 0.001) and in HC by 50% and 35% respectively (p &lt; 0.01). PGE2 levels were inhibited by the same percentage in both HC and RA monocytes (p &lt; 0.001) by 50 μM PNLA. Similarly, effects were observed with IL-6, TNF- α, and PGE2 levels in LPS-stimulated macrophages especially in RA patients 30% (p &lt; 0.05). Conclusion Our data suggest that PNLA significantly attenuated monocyte migration, significantly reduced macropinocytosis and Dil-oxLDL uptake in macrophages. Furthermore, PNLA inhibited production of IL-6, TNF-α and PGE2 levels in LPS-stimulated monocytes and macrophages from RA patients. These data inform on the potential anti-inflammatory and analgesic effects of PNLA. Disclosures R. Takala None. D. Ramji None. E. Choy None.