Search

Clodronate liposomes (Clo-Lip) have been widely used to deplete mononuclear phagocytes (MoPh) to study the function of these cells in vivo. Here, we revisited the effects of Clo-Lip together with genetic models of MoPh deficiency, revealing that Clo-Lip exert their anti-inflammatory effects independent of MoPh. Notably, not only MoPh but also polymorphonuclear neutrophils (PMN) ingested Clo-Lip in vivo, which resulted in their functional arrest. Adoptive transfer of PMN, but not of MoPh, reversed the anti-inflammatory effects of Clo-Lip treatment, indicating that stunning of PMN rather than depletion of MoPh accounts for the anti-inflammatory effects of Clo-Lip in vivo. Our data highlight the need for a critical revision of the current literature on the role of MoPh in inflammation.

Gut microbial dysbiosis is associated with the development of autoimmune disease, but the mechanisms by which microbial dysbiosis affects the transition from asymptomatic autoimmunity to inflammatory disease are incompletely characterized. Here, we identify intestinal barrier integrity as an important checkpoint in translating autoimmunity to inflammation. Zonulin family peptide (zonulin), a potent regulator for intestinal tight junctions, is highly expressed in autoimmune mice and humans and can be used to predict transition from autoimmunity to inflammatory arthritis. Increased serum zonulin levels are accompanied by a leaky intestinal barrier, dysbiosis and inflammation. Restoration of the intestinal barrier in the pre-phase of arthritis using butyrate or a cannabinoid type 1 receptor agonist inhibits the development of arthritis. Moreover, treatment with the zonulin antagonist larazotide acetate, which specifically increases intestinal barrier integrity, effectively reduces arthritis onset. These data identify a preventive approach for the onset of autoimmune disease by specifically targeting impaired intestinal barrier function., Intestinal dysbiosis is associated with an ever-growing list of autoimmune diseases. Here the authors show that both mice and humans with autoimmune arthritis can have dysbiosis and barrier leakiness prior to major signs of inflammatory arthritis, and treatment of mice with a zonulin antagonist can limit collagen-induced arthritis.

Inflammatory joint diseases like rheumatoid arthritis (RA) belong to the most prevalent autoimmune disorders. RA is characterized by a massive infiltration of immune cells into synovial tissue, cartilage destruction and bone erosion. The perpetuating inflammatory and destructive milieu is associated with severe pain and culminates in complete disability of synovial joints. The events initiating RA are still not fully understood and the treatments are mainly confined to strategies that modify and inhibit the body’s immune system. Macrophages and osteoclasts (OC) are myeloid cells of the innate immune system and are considered to play a central role in the inflammatory and destructive events of arthritis by production of inflammatory cytokines and mediating pathological bone resorption. In recent years, the use of novel fate mapping strategies identifying the origin and cellular development (ontogeny) of OC and macrophages in conjunction with new genetically modified mouse models, single cell analysis and advanced imaging techniques substantially changed our understanding on the ontogenetic and functional heterogeneity of these cells.

Background Glucocorticoid (GC) therapy is frequently used to treat rheumatoid arthritis due to potent anti-inflammatory actions of GCs. Direct actions of GCs on immune cells were suggested to suppress inflammation. Objectives Define the role of the glucocorticoid receptor (GR) in stromal cells for suppression of inflammatory arthritis. Methods Bone marrow chimeric mice lacking the GR in the hematopoietic or stromal compartment, respectively, and mice with impaired GR dimerisation (GRdim) were analysed for their response to dexamethasone (DEX, 1 mg/kg) treatment in serum transfer-induced arthritis (STIA). Joint swelling, cell infiltration (histology), cytokines, cell composition (flow cytometry) and gene expression were analysed and RNASeq of wild type and GRdim primary murine fibroblast-like synoviocytes (FLS) was performed. Results GR deficiency in immune cells did not impair GC-mediated suppression of STIA. In contrast, mice with GR-deficient or GR dimerisation-impaired stromal cells were resistant to GC treatment, despite efficient suppression of cytokines. Intriguingly, in mice with impaired GR function in the stromal compartment, GCs failed to stimulate non-classical, non-activated macrophages (Ly6Cneg, MHCIIneg) and associated anti-inflammatory markers CD163, CD36, AnxA1, MerTK and Axl. Mice with GR deficiency in FLS were partially resistant to GC-induced suppression of STIA. Accordingly, RNASeq analysis of DEX-treated GRdim FLS revealed a distinct gene signature indicating enhanced activity and a failure to reduce macrophage inflammatory protein (Mip)-1α and Mip-1β. Conclusion We report a novel anti-inflammatory mechanism of GC action that involves GR dimerisation-dependent gene regulation in non-immune stromal cells, presumably FLS. FLS control non-classical, anti-inflammatory polarisation of macrophages that contributes to suppression of inflammation in arthritis., publishedVersion

Eosinophils were reported to serve as an essential component of the plasma cell niche within the bone marrow. As the potential contribution of eosinophils to humoral immunity has remained incompletely understood, we aimed to further characterize their role during antibody responses and to additionally investigate their role in autoimmune disease. Contrary to our expectations and the currently prevailing paradigm, we found that eosinophils are fully dispensable for the survival of murine bone marrow plasma cells and accordingly do not contribute to antibody production and autoantibody-mediated disease. Littermate wild type and eosinophil-deficient ΔdblGATA-1 animals showed similar numbers and frequencies of plasma cells and did not differ in steady state levels of immunoglobulins or their ability to raise antigen-specific antibody responses. Eosinophils were likewise dispensable for autoantibody production or autoantibody-induced disease in a mouse model of systemic lupus erythematosus. Our findings thus argue against a role of eosinophils during the maintenance of the plasma cell pool and challenge the hitherto postulated concept of an eosinophil-sustained bone marrow niche.

Closed circulatory systems underlie the function of vertebrate organs, but in long bones their structure is unclear although they constitute the exit route for bone marrow (BM) leukocytes. To understand neutrophil migration from BM, we studied the vascular system of murine long bones. Here, in a mouse model, we show that hundreds of capillaries originate in BM, traverse cortical bone perpendicularly along the shaft and connect to the periosteal circulation. Structures similar to these trans-cortical vessels (TCVs) also exist in human limb bones. TCVs express arterial or venous markers and transport neutrophils. Furthermore, over 80% of arterial and 59% of venous blood passes through TCVs. Genetic and drug-mediated modulation of osteoclast count and activity leads to substantial changes in TCV numbers. In a murine model of chronic arthritic bone inflammation, new TCVs develop within weeks. Our data indicate that TCVs are a central component of the closed circulatory system in long bones and may represent an important route for immune cell export from BM.

Mononuclear phagocytes, including monocytes and macrophages, are a central component of the host's innate immune system designated to protect against invading pathogens. However, these cells do not only interact with various parts of the innate and adaptive immune system, but also fulfill indispensable duties during the control of tissue homeostasis and organ function. Moreover, macrophages are crucially involved in tissue remodeling and repair in response to damage. Simultaneously, mononuclear phagocytes might also contribute to the pathogenesis of various inflammatory and autoimmune diseases. In particular, their potential role in inflammatory joint diseases such as rheumatoid arthritis (RA) has drawn increasing attention and substantially shaped our general understanding of the role of monocytes and macrophages during health and disease. This review summarizes our current knowledge about the origin and function of mononuclear phagocytes within the joint and addresses their involvement in joint inflammation.

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

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.

Mononuclear phagocytes, including monocytes and macrophages, are a central component of the host's innate immune system designated to protect against invading pathogens. However, these cells do not only interact with various parts of the innate and adaptive immune system, but also fulfill indispensable duties during the control of tissue homeostasis and organ function. Moreover, macrophages are crucially involved in tissue remodeling and repair in response to damage. Simultaneously, mononuclear phagocytes might also contribute to the pathogenesis of various inflammatory and autoimmune diseases. In particular, their potential role in inflammatory joint diseases such as rheumatoid arthritis (RA) has drawn increasing attention and substantially shaped our general understanding of the role of monocytes and macrophages during health and disease. This review summarizes our current knowledge about the origin and function of mononuclear phagocytes within the joint and addresses their involvement in joint inflammation.

Macrophages are considered to contribute to chronic inflammatory diseases such as rheumatoid arthritis1. However, both the exact origin and the role of macrophages in inflammatory joint disease remain unclear. Here we use fate-mapping approaches in conjunction with three-dimensional light-sheet fluorescence microscopy and single-cell RNA sequencing to perform a comprehensive spatiotemporal analysis of the composition, origin and differentiation of subsets of macrophages within healthy and inflamed joints, and study the roles of these macrophages during arthritis. We find that dynamic membrane-like structures, consisting of a distinct population of CX3CR1+ tissue-resident macrophages, form an internal immunological barrier at the synovial lining and physically seclude the joint. These barrier-forming macrophages display features that are otherwise typical of epithelial cells, and maintain their numbers through a pool of locally proliferating CX3CR1− mononuclear cells that are embedded into the synovial tissue. Unlike recruited monocyte-derived macrophages, which actively contribute to joint inflammation, these epithelial-like CX3CR1+ lining macrophages restrict the inflammatory reaction by providing a tight-junction-mediated shield for intra-articular structures. Our data reveal an unexpected functional diversification among synovial macrophages and have important implications for the general role of macrophages in health and disease. Analysis of macrophage subsets within joints reveals a population of CX3CR1+ tissue-resident macrophages that form a tight-junction-mediated barrier at the synovial lining, protecting the joint from the invasion of inflammatory cells.

In this study, we deploy a doxycycline-dependent suicide switch integrated in a tumor challenge model. With this experimental setup, we characterized the immunological consequences of cells dying by four distinct cell death stimuli in vivo. We observed that apoptotic cell death induced by expression of the truncated form of BH3 interacting-domain death agonist (tBid) and a constitutively active form of caspase 3 (revC3), respectively, showed higher immunogenicity than cell death induced by expression of the tuberculosis-necrotizing toxin (TNT). Our data indicate that the early release of ATP induces the silent clearance of dying cells, whereas the simultaneous presence of ‘find me' signals and danger-associated molecular patterns (DAMPs) promotes inflammatory reactions and increased immunogenicity. This proposed model is supported by findings showing that the production and release of high concentrations of IL-27 by bone-marrow-derived macrophages (BMDM) is limited to BMDM exposed to those forms of death that simultaneously released ATP and the DAMPs heat-shock protein 90 (HSP90) and high-mobility group box-1 protein (HMGB1). These results demonstrate that the tissue microenvironment generated by dying cells may determine the subsequent immune response.

Background Checkpoints and mechanisms regulating the onset of rheumatoid arthritis (RA) remain largely elusive. Apart from B cells and auto-antibodies, Th17 cells were shown to critically contribute to disease development. Mice lacking IL-23, a cytokine controlling the pathogenicity of Th17 cells, are completely protected against arthritis. Yet, the exact role of the IL-23/Th17 axis during this autoantibody-driven disease remain incompletely understood. Material and methods IL23A-/- mice and mice receiving an IL23 blocking antibody were analysed during active and passive arthritis models including collagen-induced arthritis (CIA), the K/BxN arthritis model, collagen-antibody induced arthritis (CAIA) and K/BxN-serum transfer arthritis. Both clinical, histological and immunological parameters of arthritis were assessed. IgG glycosylation was analysed using the MALDI-TOF technique. IgG activity was determined by measuring the cytokine release of immune-complex-stimulated myeloid cells. To study the crosstalk between B cells and Th17 cells, co-culture experiments were performed. Results Here we report, that the IL-23/Th17 axis did not directly contribute to auto-antibody induced inflammation within inflamed joints, but controlled the glycosylation profile and inflammatory activity of auto-antibodies during the prodromal phase of disease. Th17 cells were found to accumulate in germinal centres auf secondary lymphatic organs prior to onset of experimental arthritis, where they suppressed the expression of β-glactoside α2,6-sialyltransferase 1 (St6gal1) in differentiating plasmablasts. The consecutive change in the immunoglobulin G (IgG) glycosylation profile provoked a shift towards a pro-inflammatory autoantibody repertoire and triggered the inflammatory phase of arthritis. Plasmablasts of RA patients similarly displayed a decreased St6gal1 activity, while IgG from these individuals showed corresponding changes in its glycosylation profile as well as an increased inflammatory activity, suggesting that related pathways might contribute to onset and progression of autoantibody-mediated diseases in humans. Conclusion Our current findings identify a novel IL-23/Th17-dependent checkpoint that controls autoantibody activity, unmasks a preexisting breach in humoral tolerance, and initiates the transition from a stage of asymptomatic autoimmunity into inflammatory autoimmune disease.

The checkpoints and mechanisms that contribute to autoantibody-driven disease are as yet incompletely understood. Here we identified the axis of interleukin 23 (IL-23) and the TH17 subset of helper T cells as a decisive factor that controlled the intrinsic inflammatory activity of autoantibodies and triggered the clinical onset of autoimmune arthritis. By instructing B cells in an IL-22- and IL-21-dependent manner, TH17 cells regulated the expression of β-galactoside α2,6-sialyltransferase 1 in newly differentiating antibody-producing cells and determined the glycosylation profile and activity of immunoglobulin G (IgG) produced by the plasma cells that subsequently emerged. Asymptomatic humans with rheumatoid arthritis (RA)-specific autoantibodies showed identical changes in the activity and glycosylation of autoreactive IgG antibodies before shifting to the inflammatory phase of RA; thus, our results identify an IL-23–TH17 cell–dependent pathway that controls autoantibody activity and unmasks a preexisting breach in immunotolerance.

Since their discovery in 1948, glucocorticoids have been widely used clinically to treat inflammatory disorders like rheumatoid arthritis. However, their usefulness, especially in rheumatoid arthritis therapy, is hampered by severe side effects on bone leading to glucocorticoid-induced osteoporosis. The molecular and cellular mechanisms mediating the beneficial and adverse effects remain poorly understood. Nevertheless, advanced molecular biological analyses and in vivo approaches using conditional mutant mice have helped to unravel in part the underlying mechanisms of immunosuppression and side effects of glucocorticoid therapy in arthritis, thereby contributing to an improved understanding of these therapeutically important hormones.

Alternatively activated macrophages (AAMs) contribute to the resolution of inflammation and tissue repair. However, molecular pathways that govern their differentiation have remained incompletely understood. Here, we show that uncoupling protein-2-mediated mitochondrial reprogramming and the transcription factor GATA3 specifically controlled the differentiation of pro-resolving AAMs in response to the alarmin IL-33. In macrophages, IL-33 sequentially triggered early expression of pro-inflammatory genes and subsequent differentiation into AAMs. Global analysis of underlying signaling events revealed that IL-33 induced a rapid metabolic rewiring of macrophages that involved uncoupling of the respiratory chain and increased production of the metabolite itaconate, which subsequently triggered a GATA3-mediated AAM polarization. Conditional deletion of GATA3 in mononuclear phagocytes accordingly abrogated IL-33-induced differentiation of AAMs and tissue repair upon muscle injury. Our data thus identify an IL-4-independent and GATA3-dependent pathway in mononuclear phagocytes that results from mitochondrial rewiring and controls macrophage plasticity and the resolution of inflammation.