S100A11 (calgizzarin), a member of S100 family, is associated with several autoimmune diseases, including rheumatoid arthritis (RA). Neutrophil extracellular traps (NETs) are implicated in the pathogenesis of RA and in the externalization of some S100 family members. Therefore, we aimed to determine the association between S100A11 and NETs in RA. For this purpose, the levels of S100A11 and NETosis markers were detected in the RA synovial fluid by immunoassays. The expression of S100A11 by neutrophils in the RA synovial tissue was assessed. Neutrophils isolated from peripheral blood were exposed to S100A11 or stimulated to release NETs. The levels of NETosis- and inflammation-associated proteins were analysed by immunoassays. NETs were visualized by immunofluorescence. We showed that S100A11 was expressed by the neutrophils in the RA synovial tissue. Moreover, S100A11 in the RA synovial fluid correlated with several NETosis markers. In vitro, S100A11 was abundantly released by neutrophils undergoing NETosis compared to untreated cells (p < 0.001). Extracellular S100A11 increased the secretion of IL-6 (p < 0.05) and TNF (p < 0.05) by neutrophils but did not induce NETosis. This study demonstrates, for the first time, that the release of S100A11 is dependent on NETosis and that extracellular S100A11 augments the inflammatory response by inducing pro-inflammatory cytokines in neutrophils.

BackgroundInterleukin 40 (IL-40) is a newly identified B cell-associated cytokine implicated in humoral immune responses and B cell homeostasis. As B cells play a pivotal role in autoimmunity, we investigated the function of IL-40 in rheumatoid arthritis (RA).MethodsIL-40 expression was determined in the synovial tissue from RA and osteoarthritis (OA) patients. IL-40 was analysed in the serum/synovial fluid of patients with RA (n=50), systemic lupus erythematosus (SLE, n=69), OA (n=44), and healthy controls (HC, n=50). We assessed the changes of IL-40 levels in RA patients following the B cell depletion by rituximab (n=29) or after the TNF inhibition by adalimumab (n=25). We examined the relationship between IL-40, disease activity, autoantibodies, cytokines, and NETosis markers. Effect of IL-40 on synovial fibroblasts was determined.ResultsIL-40 was overexpressed in RA synovial tissue, particularly by synovial lining and infiltrating immune cells. The levels of IL-40 were up-regulated in the synovial fluid of RA versus OA patients (p&lt;0.0001). Similarly, IL-40 was increased in the serum of RA patients compared to HC, OA, or SLE (p&lt;0.0001 for all) and decreased after 16 and 24 weeks (p&lt;0.01 and p&lt;0.01) following rituximab treatment. No significant effect of adalimumab on IL-40 was observed. IL-40 levels in RA patients correlated with rheumatoid factor-IgM and anti-cyclic citrullinated peptides (anti-CCP) in the serum (p&lt;0.0001 and p&lt;0.01), as well as in the synovial fluid (p&lt;0.0001 and p&lt;0.001). Synovial fluid IL-40 was also associated with disease activity score DAS28 (p&lt;0.05), synovial fluid leukocyte count (p&lt;0.01), neutrophil attractants IL-8 (p&lt;0.01), MIP-1α (p&lt;0.01), and markers of neutrophil extracellular traps externalization (NETosis) such as proteinase 3 (p&lt;0.0001) and neutrophil elastase (p&lt;0.0001). Synovial fibroblasts exposed to IL-40 increased the secretion of IL-8 (p&lt;0.01), MCP-1 (p&lt;0.05), and MMP-13 (p&lt;0.01) compared to the unstimulated cells.ConclusionsWe show the up-regulation of IL-40 in RA and its decrease following B cell depleting therapy. The association of IL-40 with autoantibodies, chemokines, and markers of NETosis may imply its potential involvement in RA development. Moreover, IL-40 up-regulates the secretion of chemokines and MMP-13 in synovial fibroblasts, indicating its role in the regulation of inflammation and tissue destruction in RA.

Our previous study demonstrated that heat shock protein 90 (Hsp90) is overexpressed in the involved skin of patients with systemic sclerosis (SSc) and in experimental dermal fibrosis. Pharmacological inhibition of Hsp90 prevented the stimulatory effects of transforming growth factor-beta on collagen synthesis and the development of dermal fibrosis in three preclinical models of SSc. In the next step of the preclinical analysis, herein, we aimed to evaluate the efficacy of an Hsp90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), in the treatment of established experimental dermal fibrosis induced by bleomycin. Treatment with 17-DMAG demonstrated potent antifibrotic and anti-inflammatory properties: it decreased dermal thickening, collagen content, myofibroblast count, expression of transforming growth factor beta receptors, and pSmad3-positive cell counts, as well as leukocyte infiltration and systemic levels of crucial cytokines/chemokines involved in the pathogenesis of SSc, compared to vehicle-treated mice. 17-DMAG effectively prevented further progression and may induce regression of established bleomycin-induced dermal fibrosis to an extent comparable to nintedanib. These findings provide further evidence of the vital role of Hsp90 in the pathophysiology of SSc and characterize it as a potential target for the treatment of fibrosis with translational implications due to the availability of several Hsp90 inhibitors in clinical trials for other indications.

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

Background:S100A11 protein, a member of S100 family, has been associated with several autoimmune inflammatory conditions such as rheumatoid arthritis (RA). Although the pathogenesis of autoimmune diseases is not fully understood, the formation of neutrophil extracellular traps (NETs) seems to play a certain role. Recent data indicate that S100A8/A9 is released via NETosis and can further augment inflammatory responses.Objectives:The aim of our study was to examine the association of S100A11 with NETs in RA.Methods:To assess the expression of S100A11 by neutrophils of RA synovial tissue (n=8), immunofluorescence staining of S100A11 and myeloperoxidase (MPO) was performed. The levels of S100A11 and MPO in RA synovial fluid (n=23) were measured by ELISAs (RayBiotech and Abcam), and the activity of peptidyl arginine deiminases (PADs) was measured by an in-house immunoassay. NETosis was induced by adding phorbol 12-myristate 13-acetate (PMA) to neutrophils from RA patients (n=7). Release of NETs was visualised by immunocytochemistry (n=7) and the presence of S100A11 in supernatants was analysed by ELISA (RayBiotech). Neutrophils purified from healthy donors (n=5) were stimulated by S100A11 and the release of cytokines TNF and IL-6 was measured by ELISA (RayBiotech).Results:S100A11 was expressed by synovial tissue neutrophils of the RA patients (n=8). The levels of S100A11 in the synovial fluid of RA patients (n=23) correlated with the levels of a NETosis marker MPO (r=0.562, p=0.005) and with PADs activity (r=0.690, p<0.001), which affects NETs immunogenicity. Neutrophils treated with LPS (n=7) did not up-regulate the secretion of S100A11 compared to unstimulated controls (0.23±0.05 vs. 0.29±0.07 ng/ml; p=ns). However, the release of S100A11 was markedly up-regulated in PMA-stimulated neutrophils undergoing NETosis compared to untreated controls (1.16±0.17 vs. 0.29±0.07 ng/ml; p<0.001). Moreover, diphenyleneiodoinum treatment abolished PMA-induced S100A11 secretion. By immunofluorescence staining (n=8) we demonstrated that neutrophils activated by PMA release NETs containing S100A11 protein. In addition, extracellular S100A11 augmented the inflammatory response of neutrophils from healthy donors (n=5) via IL-6 and TNF in comparison with unstimulated cells (0.39±0.11 vs. 0.05±0.01 pg/ml; p<0.05 and 0.31±0.06 vs. 0.09±0.03 pg/ml; p<0.05).Conclusion:Here we show for the first time that release of S100A11 by neutrophils is dependent on NETosis. Moreover, extracellular S100A11 augments the inflammatory response by inducing TNF and IL-6 secretion in neutrophils.Acknowledgments:Supported by MHCR 023728Disclosure of Interests:Adela Navratilova: None declared, Viktor Becvar: None declared, Jiří Baloun: None declared, Dres Damgaard: None declared, Claus Henrik Nielsen: None declared, Karel Pavelka Consultant of: Abbvie, MSD, BMS, Egis, Roche, UCB, Medac, Pfizer, Biogen, Speakers bureau: Abbvie, MSD, BMS, Egis, Roche, UCB, Medac, Pfizer, Biogen, Jiří Vencovský: None declared, Ladislav Šenolt: None declared, Lucie Andres Cerezo: None declared, David Veigl: None declared