BackgroundAnti-Citrullinated Protein Antibodies (ACPAs) are specific for Rheumatoid Arthritis (RA) and have been implicated in disease pathogenesis. The fragment antigen-binding domain of ACPA was recently shown to be extensively glycosylated. It is known that glycans play a key role in controlling innate and adaptive immunity, however to date there is limited understanding on the mode of action of glycans in RA. We hypothesise that the glycans on ACPA interact with glycan binding receptors and thus modulate immune responses in RA. Therefore, our aim is to elucidate the glycan effect of ACPA and other glycans on immune cells of RA patients to increase our understanding of RA pathogenesis.Materials and methodsA whole blood flow assay is used to study glycan interactions with leukocytes. Leukocytes were isolated from blood using Ficoll density centrifugation and lysis of erythrocytes. Cells were incubated for 4 hours with 4 µg/ml glycan at 4°C. Used biotinylated glycans include: sialic acid, Lewis-x, mannose, lactosamine, galactosamine and as a negative control glucitol. Glycan binding and identification of immune cell subsets was assessed with flow cytometry using a whole blood flow antibody panel.ResultsA whole blood flow assay of four healthy donors showed consistent high binding of mannose to B-cells. Interestingly, no binding of mannose was observed to other immune cells indicating that mannose binds specifically to B cells. At this concentration there was no binding of other glycans to leukocytes. When there is a difference in glycan binding between healthy and RA patients CyTOF3-Helios mass cytometer will be used to have a more in-depth analyse of the interacting leukocyte cell subsets.ConclusionThis study examines the glycan-binding capacity of leukocytes in healthy donors and RA patients via the whole blood flow assay. Our preliminary data indicates specific binding of mannose to B cells. This is an important finding because B cells play a key role in the pathogenesis of RA, as they produce ACPA and are very efficient in antigen presentation. Further studies on glycan binding to other key immune cells in RA may aid in elucidating their role in the pathogenesis of RA.
Methods Arthritis was induced by collagen-induced arthritis (CIA) and passive collagen antibody induced arthritis (CAIA) in respectively C57BL/6 and RAG2-/-(T-and B-cell deficient) mice. Animals were subjected to different regimens of mechanical strain. Increased strain occurred in voluntary running mice whereas tail suspension (unloading) abolished mechanical strain; both were compared to control housing conditions. The impact of different loading conditions was measured on clinical disease score, histology, micro-CT images and erosion quantification, gene induction in tendon and synovial tissue, immune cell recruitment in situ, development of anti-collagen antibodies and their pattern of siaylation and galactosylation. Results Voluntary running of CIA in C57BL/6 mice markedly induced an early onset and increased progression whereas no disease onset could be observed in the hind paws from animals in unloaded conditions. CAIA in running RAG2-/-mice also induced early arthritic symptoms and severe progression. Intriguingly, running conditions were sufficient to induce arthritis without the need of LPS as an inflammatory trigger. Mechanical strain did not alter however IgG autoantibody levels nor their levels of galactosylation and sialylation. Furthermore, we demonstrate that mechanical strain on stromal cells results in recruitment of classical monocytes into specialised mechano-sensitive regions characterised by a unique microanatomy. This promotes local inflammation and differentiation into local osteoclasts which induce regional erosions. A striking similarity was observed in the pattern of joint erosions in human patients with RA and SpA which were also confined to these mechanosensitive regions. Conclusions This study provides the first evidence that mechanical strain controls the transition from systemic autoimmunity into site-specific joint inflammation. Homing of inflammation and development of erosions was confined to mechano-sensitive regions, characterised by a high number of attachment-and contact points for tendons.This represents a novel paradigm and explains why arthritis in mice and humans is characterised by a regional and patchy distribution. Curiously, this pathway does not rely on adaptive immunity but rather on stromal cells. Mechanostimulation of mesenchymal cells induced CXCL1 and CCL2 permitting recruitment of classical monocytes which can differentiate into bone-resorbing osteoclasts. Thus, mechanical strain controls the site-specific direction of inflammation and tissue damage in arthritis.
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