Background: Axial spondylarthritis (axSpA) is a chronic inflammatory condition driven by complex molecular pathways involving an aberrant innate and adaptive immune system response. Key players in the pathogenesis of axSpA include T cells, which drive pro-inflammatory cytokine production, and neutrophils, which contribute to tissue damage through the release of reactive oxygen species, proteases and neutrophil extracellular traps (NETs). Current clinical approaches comprise non-steroidal anti-inflammatory drugs and biological treatments. Although valuable therapeutic response is usually met, a significant proportion of patients still fail to respond. This highlights the urgent need for innovative therapeutic strategies to mitigate inflammation and address associated comorbidities. Phenolic compounds derived from olive leaves exhibit potent anti-inflammatory and antioxidant properties capable of modulating immune system activity. Thus, the supplementation of regular therapies with these compounds could become a new therapeutic strategy in the management of patients with axSpA.

Objectives: 1 ) To evaluate the in vitro effect of polyphenolic compounds derived from olive leaves (tyrosol, hydroxytyrosol and hydroxytyrosol acetate) on the inflammatory proteome of immune cells from patients with axSpA and 2 ) to analyze the impact of these compounds on the on the reduction of NETs in neutrophils isolated from axSpA patients.

Methods: Twenty-one patients with axSpA [median age 46.09 ± 12.15 years; 15 male/6 female] were enrolled in the study. CD4 ⁺ T cells were isolated from peripheral blood mononuclear cells (PBMCs) using positive inmuno-isolation and cultured in 24-well plates. Cells were treated with olive leaf-derived polyphenols at concentrations of 12.5 μM and 25 μM, with or without phytohemagglutinin (PHA, 10 μg/mL) as a positive control. After 72 hours, supernatants were collected for proteomic analysis using Olink Proteomics technology (inflammatory panel). Enrichment analysis was conducted using EnrichR-KG to identify the biological pathways and functional processes associated with the significantly altered protein profiles for each compound. Neutrophils were isolated from the same patients using negative inmuno-selection and seeded into 96-well and 24-well plates then treated with olive leaf-derived polyphenols at the same concentrations, with or without phorbol myristate acetate (PMA, 250 nM) serving as a positive control to induce NET formation. NET formation was assessed after 6 hours using Sytox Green dye for fluorescence quantification and by immunofluorescence imaging to detect extracellular free DNA-MPO complexes. Images were acquired using a THUNDER IMAGER 3D ASSAY fluorescence microscope (Leica Microsystems, Advanced Optical Microscopy Unit, IMIBIC). Fluorescence images were analyzed using NIH FIJI/ImageJ, measuring the percentage of myeloperoxidase overlapping with NETs structures in each sample.

Results: Proteomic analysis of CD4+ T-cell supernatants revealed that hydroxytyrosol and hydroxytyrosol acetate significantly downregulated several inflammation-related proteins in a dose-dependent manner, whereas tyrosol showed no significant effects. Hydroxytyrosol at concentrations of 12.5 µM and 25 µM significantly reduced the expression of IL-10RA, IL-10RB, IL-10, OPG, IL-2, IL-4, TNFSF14, and OSM. Hydroxytyrosol acetate exhibited a more pronounced impact, downregulating 16 proteins at 12.5 µM and 33 proteins at 25 µM. The top 10 modulated proteins at 12.5 µM included IL-10, TGFB-1, TRAIL, OPG, CD8A, TNFSF14, SLAMF-1, IL-18R1, IL-10RB, and OSM, whereas at 25 µM, the most modulated proteins were OPG, IL-10, IL-4, TRAIL, TGFB-1, CD8A, SLAMF-1, TWEAK, LIF, and CCL19. These proteins are primarily associated with cytokine-mediated signaling pathways and positive regulation of cytokine production, supporting the potential anti-inflammatory effects of olive leaf-derived polyphenolic compounds in CD4+ T cells. In neutrophils, hydroxytyrosol acetate significantly reduced free DNA levels during spontaneous NETosis (6 hours after isolation) at both 12.5 µM and 25 µM. Additionally, hydroxytyrosol acetate at 25 µM (p < 0.1) also suppressed PMA-induced NETosis (3 hours post-stimulation). Similar results were obtained when analysing the area of NETs and the co-localization of MPO by microscopy.

Conclusion: 1 ) Hydroxytyrosol and hydroxytyrosol acetate demonstrate significant potential as complementary therapies for axSpA due to their pronounced anti-inflammatory and immunomodulatory effect in vitro ; 2 ) The findings highlight a dose-dependent modulation of inflammatory proteins, with hydroxytyrosol acetate exhibiting the most robust effects, particularly at higher concentrations, suggesting its potential as a lead candidate for further clinical investigation; 3 ) The ability of hydroxytyrosol acetate to reduce spontaneous and PMA-induced NETosis in neutrophils underscores its potential to mitigate neutrophil-driven inflammatory pathways in axSpA, which may contribute to reducing tissue damage and disease progression.

REFERENCES: NIL.

Acknowledgements: Projects “PID2023-152503OB-I00” and “DIN2022-012766” funded by the Minister of Science, Innovation and Universities co-financed by the European Union.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.