Career situation of first and presenting authorStudent for a master or a PhDIntroductionIn rheumatoid arthritis (RA) activated neutrophils produce neutrophil extracellular traps (NETs), which provide a source of autoantigens that drives the autoimmune process.1 To identify novel immune processes that dampened neutrophil activity, we investigated a family of inhibitory glycan-binding receptors (Siglecs) that bind a specific type of glycan; called sialic acids.2 We hypothesize that sialic acid-mediated triggering of siglecs on neutrophils, which express siglec-5, –9 and −14, will reduce their activation. In this study we focused on dampening the activity of neutrophils and thereby NET formation.MethodsPolymorphonuclear cells (PMNs) were isolated from healthy donors. Neutrophil binding of sialic acid-containing glycoconjugates was assessed by flow cytometry. Neutralizing antibodies for siglec-5/14 and −9 were used to block the interaction with the sialic acid glycoconjugates. For functional assays a branched synthetic molecule containing sialic acids (sialic acid dendrimer) was used. PMNs were rested for 1 hour at 37°C followed by stimulation of sialic acids dendrimers for 30 min. Subsequently, IgA coated beads were added for 30 min to activate the neutrophils. NETosis was quantified via Sytoxgreen and visualised via microscopy, and phagocytosis was measured by flow cytometry.ResultsBinding of sialic acid glycoconjugates was observed on neutrophils. Neutralizing siglec-5/14 and −9 receptor almost completely abolished sialic acid glycoconjugate binding to neutrophils. Neutrophils activated with IgA beads released NETs, as confirmed via microscopy. Triggering neutrophils with sialic acid dendrimer reduced this process of NETosis. The capacity to engulf IgA beads was not affected by sialic acid dendrimer stimulation.ConclusionsNeutrophils stimulated with sialic acid dendrimers show reduced activation. Patients with RA might benefit from treatment with sialic acid to dampen neutrophil-mediated autoimmune response.ReferencesWright HL, Moots RJ, Edwards SW. The multifactorial role of neutrophils in rheumatoid arthritis. Nat Rev Rheumatol 2014;10(10):593–601.Macauley MS, Crocker PR, Paulson JC. Siglec-mediated regulation of immune cell function in disease. Nat Rev Immunol 2014;14(10):653–66.Disclosure of InterestNone declared.
Career situation of first and presenting authorStudent for a master or a PhD.IntroductionIn rheumatoid arthritis the imbalance between osteoblasts and osteoclasts leads to an increased bone resorption, resulting in extensive bone destruction. Osteoclasts are multinucleated cells formed after fusion of macrophages. Macrophages are known to acquire an anti-inflammatory phenotype upon engagement of the carbohydrate sialic acid1, however whether triggering by sialic acids affects osteoclastogenesis is unknown. Therefore, we investigated the effect of sialic acids on osteoclastogenesis and their expression of the sialic acid binding receptors, called Siglecs.MethodsHuman monocytes were plated on either plastic, glass or bone slices and stimulated with M-CSF and RANK-L to generate osteoclasts. We designed a novel sorting methodology to separate the osteoclasts and the residual macrophages in our osteoclast-derived cultures. Purified osteoclasts were reanalyzed by microscopy and assessed for gene expression of osteoclast-specific genes by RT-PCR. Flow cytometry was used to obtain a full expression profile of the different siglec receptors in the osteoclasts, macrophages and monocytes precursor cells. Stimulation of pre-osteoclasts with sialic acids was done at day 7 and 10 of the culture. Supernatants were assessed for IL-6 and IL-10 production.ResultsOur novel osteoclast purification method resulted in pure osteoclasts expressing the classical genetic osteoclast markers such as cathepsin K and TRAP, while being negative for CD14. During the transition to osteoclasts, expression of siglec-1, −5/14 and −7 was lost, while siglec-6, –9 and −15 expression was maintained or gained. Osteoclasts are known for their effectively bone resorption, however after adding sialic acids to the pre-osteoclasts, both osteoclast formation and bone resorption were reduced, while an increase of IL-6 and IL-10 cytokines was observed.ConclusionsPre-osteoclasts stimulated with sialic acids display reduced osteoclast formation and bone resorption. Based on the siglec profiling we hypothesize that sialic acids engage siglec-9 on osteoclasts. Macrophages stimulated via siglec-9 produce IL-10 cytokine,1 similarly as we observed in our stimulation experiments. Currently, we are addressing the role of siglec-9 in osteoclastogenesis.ReferenceAndo M, et al. Siglec-9 enhances IL-10 production in macrophages via tyrosine-based motifs. Biochem Biophys Res Commun 2008;369(3):878–83.Disclosure of InterestNone declared.
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