P201 Anti-inflammatory and immunologic actions of pinolenic acid in rheumatoid arthritis
Authors: Takala Rabaa , Ramji Dipak , Choy Ernest 
Published: April 2020

Abstract

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.