BackgroundIL-40 is a newly described cytokine associated with immune system function and malignant transformation. We have recently shown that IL-40 is up-regulated in rheumatoid arthritis (RA) and associates with disease activity, autoantibodies and NETosis1.ObjectivesAs autoantibodies and neutrophil activation are factors thought to drive the pathological processes at early phase of RA development, we aimed to investigate IL-40 in relation to neutrophils and early stages of RA (ERA).MethodsThe levels of serum IL-40 were determined in a cohort of treatment naïve patients with ERA at baseline (n=60) and three months after initiation of conventional treatment (n=60). Serum IL-40 was also determined in sex- and age- matched healthy controls (n=60). Levels of IL-40, cytokines and NETosis markers (proteinase 3, PR3 and neutrophil elastase, NE) were measured by commercially available ELISA kits. The levels of autoantibodies were analysed by routine laboratory techniques. In vitro experiments were performed on peripheral blood neutrophils from patients with ERA (n=15).ResultsLevels of IL-40 were elevated in ERA patients at baseline compared to healthy controls (p<0.0001) and normalised after three months of the treatment (p<0.0001). Baseline serum IL-40 was associated with the levels of autoantibodies RF (IgM) (p<0.01) and anti-CCP (p<0.01) and markers of NETosis PR3 and NE) (both p<0.0001). Moreover, significant decreases in the serum IL-40 following the therapy correlated with the decrease of NETosis markers PR3 (p<0.01) and NE (p<0.05). In vitro, neutrophils from patients with ERA significantly enhanced the release of IL-40 following NETosis induction (p<0.05) or after exposure to pro-inflammatory cytokines such as IL-1β, IL-8 (p<0.05), TNF (p<0.01) or to LPS (p<0.05). Lastly, recombinant IL-40 induced the secretion of IL-1β (p<0.05) and TNF (p<0.05) by ERA neutrophils.ConclusionWe demonstrated for the first time that IL-40 is upregulated in ERA and decreases after three months of conventional therapy. Moreover, we showed that neutrophils are an important source of IL-40 in RA and its release is potentiated by pro-inflammatory cytokines and NETosis. Our results suggest that IL-40 may play an important role in early stages of RA.References[1]Navrátilová A, Andrés Cerezo L, Hulejová H, Bečvář V, Tomčík M, Komarc M, et al. IL-40: A New B Cell-Associated Cytokine Up-Regulated in Rheumatoid Arthritis Decreases Following the Rituximab Therapy and Correlates With Disease Activity, Autoantibodies, and NETosis. Front Immunol. 2021 Oct 21;12:745523. doi: 10.3389/fimmu.2021.745523.AcknowledgementsSupported by AZV-NU21-05-00276, MHCR 023728, SVV 260 523 and BBMRI-CZ LM2018125Disclosure of InterestsNone declared