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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.

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Next to proinflammatory cytokines, autoimmunity has been identified as a key trigger for osteoclast activation and bone loss. IgG-rheumatoid factor (IgG-RF) immune complexes, which are present in patients with rheumatoid arthritis, were shown to boost osteoclast differentiation. To date, the regulation of IgG-RF production in the absence of inflammatory triggers is unknown. Herein, we describe Fra1 as a key checkpoint that controls IgG-RF production by plasma cells and regulates autoimmune-mediated bone loss. Fra1 deficiency in B cells (Fra1ΔBcell ) led to increased IgG1-producing bone marrow plasma cells, enhanced IgG-RF production, and increased bone loss associated with elevated osteoclast numbers after immunization. The effect of IgG-RF on osteoclasts in vitro and on osteoclasts associated with bone loss in vivo was dependent on FcγR, especially FcγR3. Furthermore, immunization of WT mice with T-cell-dependent antigens induced a significant and robust decrease in Fra1 expression in bone marrow B cells, which was followed by increased IgG1 production and the induction of osteoclast-mediated bone loss. Overall, these data identify Fra1 as a key mediator of IgG-RF production and autoimmune-mediated bone loss. © 2019 American Society for Bone and Mineral Research.

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Rheumatoid arthritis (RA) models in mice induced by immunization enable the analyses of molecular mechanism involved in disease development, progression, and resolution of inflammation. Here, we describe three different models mimicking clinic symptoms of human rheumatoid arthritis, which could be transferable to any genetically modified mouse. We will present the protocol for collagen-induced arthritis (CIA), K/BxN serum-induced arthritis (SIA), and the antigen-induced arthritis (AIA) models. To do so, we will detail their way of induction but also how to analyze RA clinical score and pathology characterization. Finally, we will briefly discuss the main characteristics and disadvantages of all three models.

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Inflammatory responses require mobilization of innate immune cells from the bone marrow. The functionality of this process depends on the state of the bone marrow microenvironment. We therefore hypothesized that molecular changes in osteoblasts, which are essential stromal cells of the bone marrow microenvironment, influence the inflammatory response. Here, we show that osteoblast-specific expression of the AP-1 transcription factor Fra-2 (Fra-2Ob-tet) induced a systemic inflammatory state with infiltration of neutrophils and proinflammatory macrophages into the spleen and liver as well as increased levels of proinflammatory cytokines, such as interleukin-1ß (IL-1ß), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). By in vivo inhibition of osteopontin (OPN) in Fra-2Ob-tet mice, we demonstrated that this process was dependent on OPN expression, which mediates alterations of the bone marrow niche. OPN expression was transcriptionally enhanced by Fra-2 and stimulated mesenchymal stem cell (MSC) expansion. Furthermore, in a murine lung injury model, Fra-2Ob-tet mice showed increased inflammatory responses and more severe disease features via an enhanced and sustained inflammatory response to lipopolysaccharide (LPS). Our findings demonstrate for the first time that molecular changes in osteoblasts influence the susceptibility to inflammation by altering evasion of innate immune cells from the bone marrow space.

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Hypoxia-inducible factors (HIFs) are key elements for controlling immune cell metabolism and functions. While HIFs are known to be involved in T cells and macrophages activation, their functions in B lymphocytes are poorly defined. Here, we show that hypoxia-inducible factor-1α (HIF-1α) contributes to IL-10 production by B cells. HIF-1α regulates IL-10 expression, and HIF-1α-dependent glycolysis facilitates CD1dhiCD5+ B cells expansion. Mice with B cell-specific deletion of Hif1a have reduced number of IL-10-producing B cells, which result in exacerbated collagen-induced arthritis and experimental autoimmune encephalomyelitis. Wild-type CD1dhiCD5+ B cells, but not Hif1a-deficient CD1dhiCD5+ B cells, protect recipient mice from autoimmune disease, while the protective function of Hif1a-deficient CD1dhiCD5+ B cells is restored when their defective IL-10 expression is genetically corrected. Taken together, this study demonstrates the key function of the hypoxia-associated transcription factor HIF-1α in driving IL-10 expression in CD1dhiCD5+ B cells, and in controlling their protective activity in autoimmune disease.

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The role of AP-1 transcription factors in early B cell development and function is still incompletely characterized. Here we address the role of Fra-2 in B cell differentiation. Deletion of Fra-2 leads to impaired B cell proliferation in the bone marrow. In addition, IL-7-stimulated pro-B cell cultures revealed a reduced differentiation from large pre-B cells to small B cells and immature B cells. Gene profiling and chromatin immunoprecipitation sequencing analyses unraveled a transcriptional reduction of the transcription factors Foxo1, Irf4, Ikaros, and Aiolos in Fra-2-deficient B cells. Moreover, expression of IL7Rα and Rag 1/2, downstream targets of Irf4 and Foxo1, were also reduced in the absence of Fra-2. Pro-B cell proliferation and small pre-B cell differentiation were fully rescued by expression of Foxo1 and Irf4 in Fra-2-deficient pro-B cells. Hence, Fra-2 is a key upstream regulator of Foxo1 and Irf4 expression and influences proliferation and differentiation of B cells at multiple stages.

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OBJECTIVE: Arthritis is a chronic inflammatory disease characterised by immune cell infiltration and mesenchymal cell expansion in the joints. Although the role of immune cells in arthritis is well characterised, the development of mesenchymal cell hyperplasia needs to be better defined. Here, we analysed the role of the ribosomal S6 kinase Rsk2, which we found to be highly activated in joints of patients with arthritis, in the development of mesenchymal cell hyperplasia. METHODS: We genetically inactivated Rsk2 in the tumour necrosis factor (TNF)-α transgenic (TNFtg) mice, an animal model for human inflammatory arthritis. Clinical and histological signs of arthritis as well as molecular markers of inflammation and joint destruction were quantified. Fibroblast-like synoviocytes (FLS) were characterised in vitro and the effect of Rsk2 deletion on the pattern of gene expression was determined. RESULTS: Rsk2 deficiency in TNFtg mice results in earlier and exacerbated inflammation as well as increased bone and cartilage destruction. The production of inflammatory cytokines, matrix metalloproteinases and osteoclastogenic molecules was significantly increased in vivo upon Rsk2 inactivation. Bone marrow deficient in Rsk2 could not transfer this phenotype, indicating that Rsk2 expression in mesenchymal cells controls the course of arthritis. Indeed, Rsk2 deficiency was associated with a more activated phenotype and higher proliferative capacity of FLS, thereby increasing cytokines and production of matrix proteinases. CONCLUSIONS: Rsk2 emerges as a key regulator of mesenchymal cell numbers in the joint and thereby could be targeted to control the inflammatory and tissue-destructive feature of joints in arthritis.

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The cornerstone of humoral immunity is the differentiation of B cells into antibody-secreting plasma cells. This process is tightly controlled by a regulatory gene network centered on the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1). Proliferation of activated B cells is required to foster Blimp1 expression but needs to be terminated to avoid overshooting immune reactions. Activator protein 1 (AP-1) transcription factors become quickly up-regulated upon B cell activation. We demonstrate that Fra1, a Fos member of AP-1, enhances activation-induced cell death upon induction in activated B cells. Moreover, mice with B cell-specific deletion of Fra1 show enhanced plasma cell differentiation and exacerbated antibody responses. In contrast, transgenic overexpression of Fra1 blocks plasma cell differentiation and immunoglobulin production, which cannot be rescued by Bcl2. On the molecular level, Fra1 represses Blimp1 expression and interferes with binding of the activating AP-1 member c-Fos to the Blimp1 promoter. Conversely, overexpression of c-Fos in Fra1 transgenic B cells releases Blimp1 repression. As Fra1 lacks transcriptional transactivation domains, we propose that Fra1 inhibits Blimp1 expression and negatively controls plasma cell differentiation through binding to the Blimp1 promoter. In summary, we demonstrate that Fra1 negatively controls plasma cell differentiation by repressing Blimp1 expression.

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The bone marrow provides niches for early B cell differentiation and long-lived plasma cells. Therefore, it has been hypothesized that perturbing bone homeostasis might impact B cell function and Ab production. This hypothesis is highly relevant for patients receiving long-term treatment with antiresorptive drugs. We therefore analyzed the humoral immune response of mice chronically treated with ibandronate, a commonly used bisphosphonate. We confirmed the increased bone mass caused by inhibition of osteoclast activity and also the strongly reduced bone formation because of decreased osteoblast numbers in response to ibandronate. Thus, bisphosphonate drastically inhibited bone remodeling. When ibandronate was injected into mice after a primary immunization to mimic common antiosteoporotic treatments, the generation of the various B cell populations, the response to booster immunization, and the generation of plasma cells were surprisingly normal. Mice also responded normally to immunization when ibandronate was applied to naive mice. However, there, ibandronate shunted the homing of bone marrow plasma cells. Interestingly, ibandronate reduced the numbers of megakaryocytes, a known component of the bone marrow plasma cell niche. In line with normal Ab responses, increased plasma cell populations associated with increased megakaryocyte numbers were then observed in the spleens of the ibandronate-treated mice. Thus, although inhibition of bone remodeling disturbed the bone marrow plasma cell niche, a compensatory niche may have been created by relocating the megakaryocytes into the spleen, thereby allowing normal B cell responses. Therefore, megakaryocytes may act as a key regulator of plasma cell niche plasticity.

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