POS0724 NEUTROPHIL DYSFUNCTION IN EARLY RHEUMATOID ARTHRITIS AND ITS IMPACT ON CLINICAL PHENOTYPE AND INTERFERON SIGNALLING PATHWAYS

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Background: Neutrophils are innate immune cells and migrate early to the inflamed rheumatoid arthritis (RA) synovial compartment. They are short lived cells and upon cell death can release degranulation products. These are increased in the synovial fluid of established RA patients and the extrusion of neutrophil material into the extracellular space may provide a source for citrullinated autoantigens. Furthermore, this neutrophil derived material, such as neutrophil extracellular traps (NETs), has been implicated in interferon (IFN)-alpha generation in other autoimmune diseases. Evidence of increased IFN-alpha signalling is most marked in early drug-naïve RA (eRA) and can negatively impact initial therapeutic outcomes. However, neutrophil degranulation products, such as NETs, have not been examined in eRA and it is unknown what triggers IFN-alpha release in this population. Objectives: We sought to examine for the first time any evidence of extracellular neutrophil products in the circulation of early drug-naïve RA (eRA) with particular relevance to 1) IFN-alpha production and 2) clinical characteristics. Methods: Longitudinal serum samples were obtained from the RA-MAP consortium of DMARD- and glucocorticoid-naïve early RA patients at 1) the point of RA diagnosis and 2) 6-months post-initiation of DMARD therapy. Clinical data including demographics, inflammatory markers, anti-citrullinated protein antibody (ACPA) levels, DAS-28 and 6-month EULAR response were collated. Products related to neutrophil degranulation, including those present in NETs, were measured in patient serum using commercial kits and included citrullinated histone H3, double-stranded DNA, matrix metalloproteinase-9 (MMP-9) and lactoferrin. IFN-alpha activity in the serum was measured at a protein level using the SIMOA assay and the interferon gene signature (IGS) calculated by whole blood expression of MxA, IFI6, OAS1, ISG15 and IFI44L. We performed modelling and multivariate statistical analyses using Microsoft Excel, R database and R Studio and GraphPad Prism software (version 5.0; GraphPad Software, La Jolla, Calif). Statistical significance was defined when p<0.05 and analyses were corrected for age and sex. Results: Matched baseline and 6-month serum samples were available from 80 eRA patients. Median age was 54.5 years (range 20-84 years), female to male ratio was 7:3, 5% were current or previous smokers (n=44). MMP-9 levels and lactoferrin levels were significantly higher in the baseline serum samples compared with 6-months (Wilcoxon's signed rank test, p=0.026 and p=0.009 respectively, Figure 1). There was no significant difference seen in the dsDNA and histone H3 concentrations between time points (p>0.05). An inverse association was identified between baseline ACPA values and serum MMP-9 (p=0.003) and lactoferrin (p=0.008)) after adjusting for age, sex and smoking, with lower baseline ACPA scores correlating to higher analyte levels at baseline. There was an inverse association between patient baseline ESR values and serum MMP-9, p=0.024 and lactoferrin, p=0.038 after adjusting for age and sex, with higher analyte levels at baseline correlating to a lower baseline ESR. This association was lost at 6-months. There was no significant association between any of the neutrophil degranulation products, measured at baseline, with serum IFN-alpha protein levels, baseline CRP, DAS-28 scores (baseline and 6 month) and 6-month EULAR clinical responses. The IGS demonstrated a borderline significant inverse association with histone levels only (p=0.049, adjusted for age and sex), with no significant associations seen with other neutrophil degranulation components. Figure 1Comparison of longitudinal serum MMP-9 (matrix metalloproteinase-9) and Lactoferrin analyte levels of drug naive early RA (eRA) patients at baseline (BL) versus 6-month (6M) following treatment initiation. The first horizontal line depicts the mean, with error bars depicting the upper and lower interquartile ranges. Wilcoxen's test, significance determined by P value <0.05 = *, <0.01 = **. Conclusion: For the first time we present evidence of neutrophil derived material in early RA serum at disease onset, which fell at 6 months. This could represent a change in dominance from the innate to adaptive immune response as disease evolves. However, there is conflicting evidence if this activity contributes to disease activity or pathogenesis. Furthermore, contrary to other autoimmune diseases, the increased IFN-alpha reported in eRA was independent of circulating markers of neutrophil cell death. This probably reflects disease-specific interferonogenic triggers within rheumatic diseases, despite a similar IGS being reported. Further work is therefore needed to explore both the role of neutrophils, as well as potential triggers of IFN-alpha production, in eRA. REFERENCES: NIL . Acknowledgements: This work received funding from the JGW Patterson Foundation. The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. 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.

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