Search

Efficient cellular fusion of mononuclear precursors is the prerequisite for the generation of fully functional multinucleated bone-resorbing osteoclasts. However, the exact molecular factors and mechanisms controlling osteoclast fusion remain incompletely understood. Here we identify RANKL-mediated activation of caspase-8 as early key event during osteoclast fusion. Single cell RNA sequencing-based analyses suggested that activation of parts of the apoptotic machinery accompanied the differentiation of osteoclast precursors into mature multinucleated osteoclasts. A subsequent characterization of osteoclast precursors confirmed that RANKL-mediated activation of caspase-8 promoted the non-apoptotic cleavage and activation of downstream effector caspases that translocated to the plasma membrane where they triggered activation of the phospholipid scramblase Xkr8. Xkr8-mediated exposure of phosphatidylserine, in turn, aided cellular fusion of osteoclast precursors and thereby allowed generation of functional multinucleated osteoclast syncytia and initiation of bone resorption. Pharmacological blockage or genetic deletion of caspase-8 accordingly interfered with fusion of osteoclasts and bone resorption resulting in increased bone mass in mice carrying a conditional deletion of caspase-8 in mononuclear osteoclast precursors. These data identify a novel pathway controlling osteoclast biology and bone turnover with the potential to serve as target for therapeutic intervention during diseases characterized by pathologic osteoclast-mediated bone loss.

International audience; Autoimmune diseases (AIDs) are caused by the loss of self-tolerance and destruction of tissues by the host's immune system. Several antigen-specific immunotherapies, focused on arresting the autoimmune attack, have been tested in clinical trials with discouraging results. Therefore, there is a need for innovative strategies to restore self-tolerance safely and definitively in AIDs. We previously demonstrated the therapeutic efficacy of phosphatidylserine (PS)-liposomes encapsulating autoantigens in experimental type 1 diabetes and multiple sclerosis. Here, we show that PS-liposomes can be adapted to other autoimmune diseases by simply replacing the encapsulated autoantigen. After administration, they are distributed to target organs, captured by phagocytes and interact with several immune cells, thus exerting a tolerogenic and immunoregulatory effect. Specific PS-liposomes demonstrate great preventive and therapeutic efficacy in rheumatoid arthritis and myasthenia gravis. Thus, this work highlights the therapeutic potential of a platform for several autoimmunity settings, which is specific, safe, and with long-term effects.

The balance between bone forming osteoblasts and bone resorbing osteoclasts can be disturbed in autoimmune diseases resulting in local and systemic bone loss. It was long time believed that autoantibodies only indirectly contribute to bone loss by fueling the overall inflammation. However, in the last decade, more and more evidence emerged that autoantibodies and immune complexes directly activate osteoclasts and pre-osteoclasts by binding to Fcγ receptors (FcγRs) on the (pre)-osteoclast cell surface. This pro-osteoclastogenic effect seems to be dependent on the absence of sialic acid in the IgG Fc glycan, which is a typical feature of many autoantibodies. Clinical studies revealed the importance of autoantibody-mediated bone loss mainly in rheumatoid arthritis, but also in other diseases, such as celiac disease. In summary, the gained knowledge about autoantibody-mediated bone loss helps to better understand bone pathologies of autoimmune diseases. However, studies are still relatively limited and more research is needed to fully understand the impact of autoantibodies on bone and to develop future therapeutic strategies. Das Gleichgewicht zwischen knochenbildenden Osteoblasten und knochenresorbierenden Osteoklasten kann bei Autoimmunerkrankungen gestort sein, wodurch es zu lokalem und systemischem Knochenverlust kommt. Es wurde lange Zeit angenommen, dass Autoantikorper nur indirekt zum Knochenverlust beitragen, indem sie die Entzundung befeuern. In den letzten zehn Jahren gab es jedoch immer mehr Hinweise darauf, dass Autoantikorper und Immunkomplexe Osteoklasten und Praosteoklasten direkt aktivieren, indem sie an Fcγ-Rezeptoren (FcγRs) auf der (Pra-) Osteoklastenoberflache binden. Diese pro-osteoklastogene Wirkung scheint von der Abwesenheit von Sialinsaure im IgG-Fc-Glykan abhangig zu sein, was ein typisches Merkmal vieler Autoantikorper ist. Klinische Studien zeigten die Bedeutung des autoantikorpervermittelten Knochenverlusts hauptsachlich bei rheumatoider Arthritis, aber auch bei anderen Krankheiten wie Zoliakie. Zusammengenommen hilft das gewonnene Wissen uber autoantikorpervermittelten Knochenverlust, die Knochenpathologien von Autoimmunerkrankungen besser zu verstehen. Bisher gibt es jedoch nur relativ wenige Studien und es sind weitere Forschungsarbeiten erforderlich, um die Auswirkungen von Autoantikorpern auf den Knochen vollstandig zu verstehen und zukunftige Therapiestrategien zu entwickeln.

Neutrophil extracellular trap (NET) formation is a powerful instrument to fight pathogens, but may induce collateral damage in the affected tissues. Besides pathogen-derived factors, immune complexes are potent inducers of NET formation. Neutrophils express IgA and IgG specific Fc receptors (FcRs) and therefore respond to complexed IgA and IgG. Especially in the context of autoimmune diseases, IgA and IgG immune complexes have been shown to trigger NET formation, a process that putatively contributes to disease severity. However, it is of question if both antibody classes stimulate neutrophils to the same extent. In this study, we compared the capability of IgA and IgG complexes formed by heat aggregation to induce NET formation. While stimulation of neutrophils with IgA complexes robustly induced NET formation, complexed IgG only marginally increased the amount of NETs compared to the unstimulated control. Mixing IgA with IgG before heat aggregation did not increase the effect of complexed IgA on neutrophils. By contrast, the presence of IgG complexes seemed to disturb neutrophil stimulation by IgA complexes. The capacity of complexed IgG to induce NET formation could not be increased by the addition of autologous serum or the removal of terminal sialic acid in the Fc glycan. Together, our data show that IgA is a much more potent inducer of NET formation than IgG. IgA may thus be the main driving force in (auto)immune complex-mediated NET formation.

ObjectiveEvents triggering disease outbreak in individuals at-risk for rheumatoid arthritis (RA at-risk) remain unclear, and the role of the various anticitrullinated protein antibody (ACPA) isotypes in this process is still to be established. We aimed to investigate the prevalence of IgA ACPA in RA at-risk individuals, their role in the transition from the RA at-risk status to RA and their dynamics during this transition.MethodsCross-sectional measurement of serum IgA1 and IgA2 ACPA levels was conducted in healthy controls, RA at-risk individuals and patients with RA and compared with the frequency of RA development in at risk individuals during a follow-up of 14 months. In addition, longitudinal measurements of serum IgA1 and IgA2 ACPA levels prior to, at and after the onset of RA were performed.ResultsApproximately two-thirds of RA at-risk individuals were positive for serum IgA1 and IgA2 ACPA in levels comparable to IgG ACPA positive patients with RA. IgA1, but not IgA2 ACPA positivity was associated with the transition from the RA at-risk state to RA within the following 14 months. Interestingly, during this transition process, IgA1 ACPA levels declined at RA onset and also thereafter during the early phase of RA. This decline was confirmed in a second, independent cohort.ConclusionBoth IgA1 and IgA2 ACPA are present in RA at-risk individuals, but only IgA1 ACPA are associated with the progression to RA. The observed decline in serum IgA1 ACPA levels before the onset of RA might indicate starting barrier leakiness prior to disease outbreak.

Autoantibodies represent a hallmark of rheumatoid arthritis (RA), with the rheumatoid factor (RF) and antibodies against citrullinated proteins (ACPA) being the most acknowledged ones. RA patients who are positive for RF and/or ACPA (“seropositive”) in general display a different etiology and disease course compared to so-called “seronegative” patients. Still, the seronegative patient population is very heterogeneous and not well characterized. Due to the identification of new autoantibodies and advancements in the diagnosis of rheumatic diseases in the last years, the group of seronegative patients is constantly shrinking. Aside from antibodies towards various post-translational modifications, recent studies describe autoantibodies targeting some native proteins, further broadening the spectrum of recognized antigens. Next to the detection of new autoantibody groups, much research has been done to answer the question if and how autoantibodies contribute to the pathogenesis of RA. Since autoantibodies can be detected years prior to RA onset, it is a matter of debate whether their presence alone is sufficient to trigger the disease. Nevertheless, there is gathering evidence of direct autoantibody effector functions, such as stimulation of osteoclastogenesis and synovial fibroblast migration in in vitro experiments. In addition, autoantibody positive patients display a worse clinical course and stronger radiographic progression. In this review, we discuss current findings regarding different autoantibody types, the underlying disease-driving mechanisms, the role of Fab and Fc glycosylation and clinical implications.

Alcohol consumption is a consistent protective factor for the development of autoimmune diseases such as rheumatoid arthritis (RA). The underlying mechanism for this tolerance-inducing effect of alcohol, however, is unknown. Here we show that alcohol and its metabolite acetate alter the functional state of T follicular helper (TFH) cells in vitro and in vivo, thereby exerting immune regulatory and tolerance-inducing properties. Alcohol-exposed mice have reduced Bcl6 and PD-1 expression as well as IL-21 production by TFH cells, preventing proper spatial organization of TFH cells to form TFH:B cell conjugates in germinal centers. This effect is associated with impaired autoantibody formation, and mitigates experimental autoimmune arthritis. By contrast, T cell independent immune responses and passive models of arthritis are not affected by alcohol exposure. These data clarify the immune regulatory and tolerance-inducing effect of alcohol consumption. Moderate consumption of alcohol is associated with protection from some autoimmune diseases. Here the authors show that ethanol and its metabolite acetate can protect mice from collagen-induced arthritis and provide evidence that the mechanism of this effect might be via inhibition of the effector function of T follicular helper cells.

Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.

Jagn1 deficiency results in alterations in the endoplasmic reticulum (ER) and Golgi apparatus of antibody-producing cells, reduced antibody secretion, and aberrant IgG N-glycosylation. The data uncover a key role for JAGN1 and ER stress in antibody glycosylation and humoral immunity in mice and humans., Jagunal homolog 1 (JAGN1) has been identified as a critical regulator of neutrophil biology in mutant mice and rare-disease patients carrying JAGN1 mutations. Here, we report that Jagn1 deficiency results in alterations in the endoplasmic reticulum (ER) of antibody-producing cells as well as decreased antibody production and secretion. Consequently, mice lacking Jagn1 in B cells exhibit reduced serum immunoglobulin (Ig) levels at steady state and fail to mount an efficient humoral immune response upon immunization with specific antigens or when challenged with viral infections. We also demonstrate that Jagn1 deficiency in B cells results in aberrant IgG N-glycosylation leading to enhanced Fc receptor binding. Jagn1 deficiency in particular affects fucosylation of IgG subtypes in mice as well as rare-disease patients with loss-of-function mutations in JAGN1. Moreover, we show that ER stress affects antibody glycosylation. Our data uncover a novel and key role for JAGN1 and ER stress in antibody glycosylation and humoral immunity in mice and humans.

Monomeric serum immunoglobulin A (IgA) can contribute to the development of various autoimmune diseases, but the regulation of serum IgA effector functions is not well defined. Here, we show that the two IgA subclasses (IgA1 and IgA2) differ in their effect on immune cells due to distinct binding and signaling properties. Whereas IgA2 acts pro-inflammatory on neutrophils and macrophages, IgA1 does not have pronounced effects. Moreover, IgA1 and IgA2 have different glycosylation profiles, with IgA1 possessing more sialic acid than IgA2. Removal of sialic acid increases the pro-inflammatory capacity of IgA1, making it comparable to IgA2. Of note, disease-specific autoantibodies in patients with rheumatoid arthritis display a shift toward the pro-inflammatory IgA2 subclass, which is associated with higher disease activity. Taken together, these data demonstrate that IgA effector functions depend on subclass and glycosylation, and that disturbances in subclass balance are associated with autoimmune disease., Immunoglobulin A (IgA) has two subclasses, IgA1 and IgA2, but differential effects on inflammation are unclear. Here the authors show that IgA2, when compared with IgA1, has stronger pro-inflammatory functions associated with changed glycosylation and higher disease scores in patients with rheumatoid arthritis.

Background:Understanding key mechanisms of flare development and sustained remission is one of the acute goals in modern rheumatology. Anti-citrullinated protein antibodies (ACPA) are the most abundant and specific autoantibodies in rheumatoid arthritis (RA) patients. However, the impact of ACPA of IgA isotype is poorly defined. IgA ACPA were previously shown to have a higher percentage of IgA2 in comparison to total IgA; and a correlation between IgA2% ACPA with the DAS28 score was observed in a previous study [1]. Of note, IgA1 and IgA2 were shown to exhibit different effector functions, with IgA2 being pro-inflammatory, which might be the background for its role in RA [1].Objectives:We aimed to investigate, whether IgA ACPA could be used as a predictive factor for flare development in RA; and to look further into the changes in IgA ACPA levels in patients remaining in stable remission versus patients developing flare.Methods:We analysed serum of 111 patients from a multicentre randomized controlled trial ‘RETRO’. The study observational period was 12 months. Patients in the trial had to be in stable remission (DAS28-ESRResults:60% of patients had IgG-ACPA. IgA ACPA levels were higher among the IgG-ACPA-positive patients (median 4.7 versus 2.24 µg/ml, pConclusion:Neither IgA1 nor IgA2 ACPA levels were predictive of flare development or associated with treatment strategies (though rituximab, JAK-inhibitors and abatacept were not amongst treatment options). However, in patients remaining in sustained remission after 1 year a decrease in IgA2 and IgA2% ACPA was observed and IgA2% ACPA was associated with DAS28 score registered after flare. This could be an indication towards ACPA of IgA2 isotype contributing to the severity of flare, alongside other factors, and its reduction being associated with a prolonged state of remission.References:[1]Steffen U, Koeleman CA, Sokolova MV, et al. IgA subclasses have different effector functions associated with distinct glycosylation profiles. Nat Commun 11, 120 (2020).[2]Haschka J, Englbrecht M, Hueber AJ, et al. Relapse rates in patients with rheumatoid arthritis in stable remission tapering or stopping antirheumatic therapy: interim results from the prospective randomised controlled RETRO study. Ann Rheum Dis. 75:45-51 (2016).Disclosure of Interests:None declared

Although the control of bone-resorbing osteoclasts through osteocyte-derived RANKL is well defined, little is known about the regulation of osteoclasts by osteocyte death. Indeed, several skeletal diseases, such as bone fracture, osteonecrosis, and inflammation are characterized by excessive osteocyte death. Herein we show that osteoclasts sense damage-associated molecular patterns (DAMPs) released by necrotic osteocytes via macrophage-inducible C-type lectin (Mincle), which induced their differentiation and triggered bone loss. Osteoclasts showed robust Mincle expression upon exposure to necrotic osteocytes in vitro and in vivo. RNA sequencing and metabolic analyses demonstrated that Mincle activation triggers osteoclastogenesis via ITAM-based calcium signaling pathways, skewing osteoclast metabolism toward oxidative phosphorylation. Deletion of Mincle in vivo effectively blocked the activation of osteoclasts after induction of osteocyte death, improved fracture repair, and attenuated inflammation-mediated bone loss. Furthermore, in patients with osteonecrosis, Mincle was highly expressed at skeletal sites of osteocyte death and correlated with strong osteoclastic activity. Taken together, these data point to what we believe is a novel DAMP-mediated process that allows osteoclast activation and bone loss in the context of osteocyte death.

Background Targeting cytokines relevant to rheumatoid arthritis (RA) has proven efficient in clinical practice, but there is still demand for therapies that rebuild joint tissues which have been subjected to deterioration. Since many cytokines involved in RA rely on the intracellular janus kinase - signal transducer and activator of transcription (JAK-STAT) signaling pathway, targeting them presents itself as option. For this approach, JAK-inhibitors such as Tofacitinib and Baricitinib, targeting JAK1/JAK3 and JAK1/JAK2 respectively, seem favorable, as they have been approved for the treatment of RA1. Moreover, preliminary data indicates an impact of JAK inhibition on local bone formation. Objectives To investigate the influence of JAK-inhibition on structural bone damage in vivo and its impact on osteoclast/osteoblast-mediated bone homeostasis in vitro. Methods In vivo analysis comprised unchallenged steady-state, the ovariectomy-induced mouse model of postmenopausal osteoporosis (OVX) and the serum-induced arthritis (SIA) mouse model. For steady-state analysis C57BL/6 (WT) mice obtained tofacitinib QD by oral gavage for 6 weeks. For OVX, WT mice received tofacitinib BID by oral gavage for 6 weeks. WT mice of the SIA model were fed tofacitinib or baricitinib BID for 14 days. Experimental readout included clinical parameters, ELISA (RANKL/OPG levels in serum), qPCR (mRNA expression in bone) and µCT. For in vitro analysis, murine osteoclasts (OC) were analyzed with TRAP staining (osteoclastogenesis) and von Kossa staining (Resorptive capacity). Murine osteoblasts (OB), derived from mesenchymal stem cells (MSC) and calvariae were assessed with qPCR (differentiation) and Alizarin red staining (mineralization capacity). Results In steady-state conditions, JAK-inhibition by tofacitinib enhanced tibial trabecular bone density and decreased RANKL/OPG fraction in blood serum. These findings, and increased trabeculae numbers, were also applicable to spinal bone of tofacitinib-treated OVX mice. In SIA, both baricitinib and tofacitinib improved clinical symptoms and halted trabecular and cortical bone loss. In vitro OC-differentiation and function were not affected by JAK-inhibition. However, tofacitinib and baricitinib amplified OCN expression in MSC-derived OBs at day 1 after osteogenic induction, together with reduced Igf1 and elevated Dkk1 levels at day 7. Moreover, as a result of JAK-inhibition RANKL expression was decreased in calvaria-derived OBs. Accordingly, both MSC- and calvaria-derived OBs showed increased mineralization when treated with JAK-inhibitors. Conclusion Our results suggest that JAK-inhibition by tofacitinib and baricitinib causes increased mineralization by osteoblasts, resulting in enhanced bone density in vivo, in both unchallenged and pathological mouse models. Reference [1] Baker KF, Isaacs JD, ARD, 2018 Acknowledgement Work funded by Pfizer, Eli Lilly. The MSC were a kind gift from Farida Djouad (IRMB, Centre Hospitalier Regional Universitaire de Montpellier). Susanne Adam is graduate member of IRTG1181 and Lifa@FAU. Disclosure of Interests Susanne Adam: None declared, Nils Simon: None declared, Ulrike Steffen (nee Harre): None declared, Fabian Andes: None declared, Dorothea Muller: None declared, Stephan Culemann: None declared, Darja Andreev: None declared, Madelaine Hahn: None declared, Carina Scholtysek: None declared, Georg Schett: None declared, Gerhard Kronke Grant/research support from: Lilly, Pfizer, Speakers bureau: Novartis, Silke Frey: None declared, Axel Hueber Grant/research support from: Novartis, Pfizer, Lilly, Consultant for: Lilly, GSK, Novartis, Janssen, Celgene, Abbvie, Roche, Speakers bureau: Lilly, Janssen, Novartis, Celgene, Biogen, Abbvie, BMS

Alcohol consumption is a consistent protective factor for the development of autoimmune diseases such as rheumatoid arthritis (RA). The underlying mechanism for this tolerance-inducing effect of alcohol, however, is unknown. Here we show that alcohol and its metabolite acetate alter the functional state of T follicular helper (TFH) cells in vitro and in vivo, thereby exerting immune regulatory and tolerance-inducing properties. Alcohol-exposed mice have reduced Bcl6 and PD-1 expression as well as IL-21 production by TFH cells, preventing proper spatial organization of TFH cells to form TFH:B cell conjugates in germinal centers. This effect is associated with impaired autoantibody formation, and mitigates experimental autoimmune arthritis. By contrast, T cell independent immune responses and passive models of arthritis are not affected by alcohol exposure. These data clarify the immune regulatory and tolerance-inducing effect of alcohol consumption., Moderate consumption of alcohol is associated with protection from some autoimmune diseases. Here the authors show that ethanol and its metabolite acetate can protect mice from collagen-induced arthritis and provide evidence that the mechanism of this effect might be via inhibition of the effector function of T follicular helper cells.

Career situation of first and presenting author Student for a master or a PhD. Introduction Many cytokines relevant to rheumatoid arthritis (RA) rely on the janus kinase – signal transducer and activator of transcription (JAK-STAT) signaling pathway. The JAK-inhibitors tofacitinib and baricitinib, targeting JAK3/JAK1 and JAK1/JAK2 respectively, have been approved for treatment of RA1. While currently available therapies reduce inflammation, erosive damage in involved joints is still irreversible. However, preliminary data suggests an influence of JAK inhibition on local bone healing. Objectives To study the role of JAK-inhibition in osteoblast and osteoclast-mediated bone homeostasis and its capacity to alleviate structural bone damage in vivo. Methods For steady state analysis C57BL/6 (WT) mice received tofacitinib QD by oral gavage for 6 weeks. WT mice of the ovariectomy-induced osteoporosis model (OVX) obtained tofacitinib BID by oral gavage for 6 weeks. For the serum-induced arthritis model (SIA) WT mice received tofacitinib or baricitinib BID by oral gavage for 14 days. Readout covered serum cytokine levels (ELISA), µCT and mRNA analysis of bone (qPCR) and clinical scoring. Murine osteoclasts (OC) were assessed for differentiation (TRAP staining) and resorption (Von Kossa staining). Murine mesenchymal stem cell (MSC)-derived and primary osteoblasts (OB) were analyzed for differentiation (qPCR) and function (Alizarin red staining). Results In unchallenged WT mice, treatment with tofacitinib increased trabecular bone density in tibia and reduced RANKL/OPG ratio in serum. These results, along with elevated trabeculae numbers, also applied to spinal bone in tofacitinib-treated OVX mice. In SIA, baricitinib and tofacitinib ameliorated disease manifestation and inhibited trabecular/cortical bone loss. In vitro JAK-inhibition exhibited no effect on differentiation and function of OCs. However, it enhanced OCN expression in MSC-derived OBs at day 1 after osteoblastic induction and led to decreased Igf1 and increased Dkk1 expression at day 7. Accordingly, primary OBs showed reduced RANKL expression during JAK-inhibition. Moreover, both MSC- and primary OBs responded to JAK-inhibition with increased mineralization. Conclusions Our findings indicate that JAK-inhibition by tofacitinib and baricitinib increases bone formation in vivo, in both steady-state and pathological conditions, presumably as a result of increased mineralization capacity by osteoblasts. Reference Baker KF, Isaacs JD. ARD 2018. Acknowledgements Work funded by Pfizer and Eli Lilly. Disclosure of Interest S. Adam Grant/research support from: Pfizer, Eli Lilly, N. Simon: None declared, U. Steffen: None declared, F. Andes: None declared, D. Muller: None declared, S. Culemann: None declared, D. Andreev: None declared, M. Hahn: None declared, C. Scholtysek: None declared, G. Schett: None declared, G. Kronke: None declared, S. Frey: None declared, A. Hueber: None declared.

Career situation of first and presenting author Student for a master or a PhD. Introduction Prominent features of rheumatoid arthritis (RA) are pain, joint swelling and erosion induced by massive infiltration of immune cells into the inflamed tissue. Among these immune cells, granulocytes and monocytes play a crucial role. Both express sialic acid-binding immunoglobulin-like lectins (Siglecs) −7 and −9 (in mice Siglec-E), trans-membrane proteins generally transmitting inhibitory signals through immunoreceptor tyrosine-based inhibitory motifs in their cytoplasmic tails. So far, there has been only limited research on the effects of Siglecs in RA. Objectives To evaluate the potential role of Siglec 7- and −9 during disease progression in RA by comparing the expression on blood leukocytes with disease activity in RA patients. Additionally we monitored Siglec-E knockout mice in an experimental arthritis model. Methods Blood samples from 45 healthy donors (HD) and 37 RA patients were analyzed by flow cytometry for Siglec-7 and −9 expressions on NK cells (CD14+/CD56+/CD3-), granulocytes (CD16+/CD19-) and monocytes (CD14+/CD16-). Expression was correlated with autoantibody positivity and disease severity. Siglec-E knockout mice and wildtype controls were used in a serum transfer induced arthritis model and disease manifestation was monitored by measurement of paw swelling. Additionally we performed functional ex vivo assays to study Siglec impact on granulocytes and monocytes. Results There was a diminished Siglec-7/9 expression on NK cells from RA patients, especially in highly CCP positive patients (>500 U/ml), while no differences were observed in neutrophils and monocytes compared to healthy controls. Interestingly we observed a correlation between a higher DAS28 score and lower Siglec-9 expression levels on granulocytes in seropositive patients. Siglec-E knockout mice displayed higher disease scores and paw swelling in serum transfer induced arthritis, especially at the peak and the resolution phase. Functional studies unraveled that granulocytes are in a more active state if Siglec-7 and −9 are missing. Conclusions Together our data support that Siglec-7/9 expression negatively correlates with pathogenesis and disease manifestation in RA patients. We showed in vivo and ex vivo, that Siglecs possess an important role concerning granulocyte and monocyte function and are capable of dampening immune response. Due to their inhibitory potential, Siglecs should be considered as potential therapeutic starting point in future studies. Acknowledgements This work was supported by DFG (HA 8163/1–1) and STAEDTLER Stiftung (WW/eh20/17). Disclosure of Interest None declared.

Rheumatoid arthritis (RA) is a chronic inflammatory disease, characterized by autoimmunity that triggers joint inflammation and tissue destruction. Traditional concepts of RA pathogenesis have strongly been focused on inflammation. However, more recent evidence suggests that autoimmunity per se modulates the disease and in particular bone destruction during the course of RA. RA-associated bone loss is caused by increased osteoclast differentiation and activity leading to rapid bone resorption. Autoimmunity in RA is based on autoantibodies such as rheumatoid factor (RF) and autoantibodies against citrullinated proteins (ACPA). These autoantibodies exert effector functions on immune cells and on bone resorbing osteoclasts, thereby facilitating bone loss. This review summarizes potential pathways involved in increased destruction of bone tissue in RA, particularly focusing on the direct and indirect actions of autoantibodies on osteoclast generation and function.

Background:Alcohol consumption has emerged as consistent protective factor for the development of autoimmune diseases such as rheumatoid arthritis (RA). The underlying mechanism for this tolerance-inducing effect of alcohol, however, is unknown.Objectives:To understand the anti-arthritogenic effect of alcoholMethods:The immune-regulatory properties of alcohol consumption in vivo were tested in the collagen-induced arthritis (CIA) and serum-induced arthritis (SIA) model as well as after immunization with T cell- dependent (NP-CGG) and independent (TNP-FICOLL) antigens. Additional experiments in vivo experiments in these models were done with acetate- the metabolite of ethanol. The models were analysed for T- cell lineage and plasma cell differentiation, germinal centre formation and IgG levels and sialylation. Molecular expression of T follicular helper cell (TFH) activation such as IL-21, Bcl-6 and PD-1, as well as TFH: B cell conjugates were also assessed. Furthermore, TFH cells were generated in vitro, exposed to ethanol or acetate and tested for IL-21 production, PD1 expression and conjugate formation with B cells.Results:Ethanol exposure significantly inhibited arthritis in the active adaptive immunity-driven model of arthritis (CIA) but not in the passive innate immunity-driven model (STA) suggesting that the immune suppressive effect of alcohol is based on interference of T- and B- cell activation. In line ethanol and even more its metabolite acetate, suppressed T cell dependent antibody formation after NP-CGG immunization, while T cell independent antibody formation after TNP-FICOLL immunization was not suppressed. Ethanol, as well as its metabolite acetate, specifically altered the functional state of T follicular helper (TFH) cells in vitro and in vivo, thereby exerting immune regulatory and tolerance-inducing properties. Alcohol-exposed mice showed reduced Bcl6 and PD-1 expression as well as interleukin (IL)-21 production by TFH cells, preventing proper spatial organization of TFH cells to form TFH: B cell conjugates in the germinal centre. This effect of alcohol on TFHcells was associated with impaired autoantibody formation, higher sialylation of autoantibodies and less arthritis. In accordance, overexpression of IL-21 in vivo completely reversed the immune regulatory effects of alcohol.Conclusion:In summary, these data provide a new mechanistic explanation for the immune regulatory and tolerance-inducing effect of alcohol consumption in arthritis.Acknowledgments:Funden by DFG-FOR2886, DFG–CRC1181, Staedtler foundation, Johannes und Frieda Marohn-Stiftung, Else Kröner-Fresenius foundation, Interdisciplinary Centre for Clinical Research, Erlangen, BMBF-MASCARA, IMI funded project RTCure.Disclosure of Interests:Vugar Azizov: None declared, Maria V Sokolova: None declared, Kerstin Sarter: None declared, Vladimir Temchura: None declared, Ulrike Steffen (née Harre): None declared, Martin Herrmann: None declared, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB, Mario Zaiss: None declared

Objectives A substantial proportion of RA patients flare upon withdrawal of DMARDs, and thus the definition of prognostic markers is crucial. ACPA positivity has been identified as a risk factor for flare. However, only the role of IgG ACPA is established in this context, while the role of IgA ACPA is poorly defined. We thus aimed to investigate the role of IgA ACPA in flaring of RA. Methods Serum levels of IgA1 and IgA2 ACPA at baseline and after 12 months were measured in 108 patients from the randomized controlled RETRO study. RA patients in stable remission for at least 6 months at study recruitment were assigned to either one of the DMARD tapering arms or to continuation of DMARDs. Results In patients remaining in remission but not in the ones who flared, IgA2 ACPA levels and proportion of IgA2 in ACPA (IgA2% ACPA) significantly declined (median of 17.5%; P Conclusion In this study, IgA ACPA was identified as a risk factor for flare in combination with IgG ACPA. IgA2 ACPA levels were associated with flare severity and declined in patients in stable remission.

OBJECTIVE: B cell depletion is an established therapeutic principle in a wide range of autoimmune diseases. However, B cells are also critical for inducing protective immunity after infection and vaccination. We undertook this study to assess humoral and cellular immune responses after infection with or vaccination against SARS-CoV-2 in patients with B cell depletion and controls who are B cell-competent. METHODS: Antibody responses (tested using enzyme-linked immunosorbent assay) and T cell responses (tested using interferon-I³ enzyme-linked immunospot assay) against the SARS-CoV-2 spike S1 and nucleocapsid proteins were assessed in a limited number of previously infected (n = 6) and vaccinated (n = 8) autoimmune disease patients with B cell depletion, as well as previously infected (n = 30) and vaccinated (n = 30) healthy controls. RESULTS: As expected, B cell and T cell responses to the nucleocapsid protein were observed only after infection, while respective responses to SARS-CoV-2 spike S1 were found after both infection and vaccination. A SARS-CoV-2 antibody response was observed in all vaccinated controls (30 of 30 [100%]) but in none of the vaccinated patients with B cell depletion (0 of 8). In contrast, after SARS-CoV-2 infection, both the patients with B cell depletion (spike S1, 5 of 6 [83%]; nucleocapsid, 3 of 6 [50%]) and healthy controls (spike S1, 28 of 30 [93%]; nucleocapsid, 28 of 30 [93%]) developed antibodies. T cell responses against the spike S1 and nucleocapsid proteins were found in both infected and vaccinated patients with B cell depletion and in the controls. CONCLUSION: These data show that B cell depletion completely blocks humoral but not T cell SARS-CoV-2 vaccination response. Furthermore, limited humoral immune responses are found after SARS-CoV-2 infection in patients with B cell depletion.

Osteoclasts are the only bone-resorbing cells in the body. Together with bone-forming osteoblasts, they are responsible for bone homeostasis and constant bone remodeling. Aberrant activation of osteoclasts leads to bone loss, as seen in postmenopausal osteoporosis or in autoimmune diseases like rheumatoid arthritis. Although much research has been performed to understand and prevent osteoclast-mediated bone loss, the mechanisms of osteoclast hyperactivation are not completely understood. This unit describes several protocols for ex vivo generation of murine and human osteoclasts, allowing study of the effects of specific cells, cytokines, or chemical substances on osteoclast formation and activity without the need for expensive and time-consuming animal experiments. In addition, we provide protocols for specific staining of osteoclasts and for analysis of resorption activity using calcium phosphate-coated surfaces or bone slices. © 2019 by John Wiley & Sons, Inc.

Regulation of osteoclast differentiation and function is a central element in bone homeostasis. While the role of soluble factors, such as cytokines, hormones and growth factors, in controlling osteoclast differentiation has been intensively characterized, the function of surface receptors is less well understood. Sialic acid-binding immunoglobulin-like lectin (Siglec)-9 and its murine homolog Siglec-E are sialic acid-recognizing inhibitory receptors from the CD33-related Siglec-family and mainly expressed on myeloid cells. We found Siglec-9 and Siglec-E to be expressed at all stages of human and murine osteoclastogenesis, respectively. Siglec-E knockout mice displayed lower bone mass despite unchanged osteoclast numbers and an increased bone formation rate. Ex vivo osteoclast assays using Siglec-E knockout cells or a blocking antibody against human Siglec-9 confirmed the suppressive effect of Siglec-9/Siglec-E on osteoclast function. Although osteoclast numbers were unchanged or even slightly decreased, the blockade/absence of Siglec-9/Siglec-E resulted in an augmented resorption activity of mature osteoclasts. This increased resorption activity was associated with enlarged actin rings. Together, our results suggest Siglec-9/Siglec-E to inhibit osteoclast activation independently from osteoclast differentiation and thereby propose a new mechanism for the control of local bone resorption.