RESUMO
Endogenous nucleic acids and their receptors may be involved in the initiation of systemic autoimmune diseases including rheumatoid arthritis (RA). As the role of the DNA sensing Toll-like receptor (TLR) 9 in RA is unclear, we aimed to investigate its involvement in the pathogenesis of autoimmune arthritis using three different experimental models of RA. The data obtained revealed involvement of TLR9 in the T cell-dependent phase of inflammatory arthritis. In rats with pristane-induced arthritis (PIA), TLR9 inhibition before disease onset reduced arthritis significantly and almost completely abolished bone erosion. Accordingly, serum levels of IL-6, α-1-acid-glycoprotein and rheumatoid factor were reduced. Moreover, in TLR9-/- mice, streptococcal cell wall (SCW)-induced arthritis was reduced in the T cell-dependent phase, whereas T cell-independent serum-transfer arthritis was not affected. Remarkably, while TLR7 expression did not change during in vitro osteoclastogenesis, TLR9 expression was higher in precursor cells than in mature osteoclasts and partial inhibition of osteoclastogenesis was achieved only by the TLR9 antagonist. These results demonstrate a pivotal role for TLR9 in the T cell-dependent phases of inflammatory arthritis and additionally suggest some role during osteoclastogenesis. Hence, endogenous DNA seems to be crucially involved in the pathophysiology of inflammatory autoimmune arthritis.
Assuntos
Artrite Experimental/genética , Articulações/imunologia , Osteoclastos/imunologia , Osteogênese/genética , Receptor Toll-Like 9/genética , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/imunologia , Artrite Experimental/patologia , Cartilagem Articular/imunologia , Cartilagem Articular/patologia , Parede Celular/química , Misturas Complexas/administração & dosagem , Regulação da Expressão Gênica , Interleucina-6/genética , Interleucina-6/imunologia , Articulações/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Orosomucoide/genética , Orosomucoide/imunologia , Osteoclastos/patologia , Ratos , Fator Reumatoide/genética , Fator Reumatoide/imunologia , Transdução de Sinais , Streptococcus pyogenes/química , Terpenos/administração & dosagem , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologia , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/imunologiaRESUMO
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.
Assuntos
Artrite Experimental/patologia , Fibroblastos/patologia , Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologia , Membrana Sinovial/patologia , Animais , Artrite Experimental/metabolismo , Proliferação de Células , Citocinas/metabolismo , Modelos Animais de Doenças , Fibroblastos/metabolismo , Hiperplasia/genética , Hiperplasia/metabolismo , Inflamação/metabolismo , Metaloproteinases da Matriz/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Quinases S6 Ribossômicas 90-kDa/deficiência , Membrana Sinovial/metabolismo , Fator de Necrose Tumoral alfa/genéticaRESUMO
Tumor necrosis factor (TNF)-α is a key cytokine regulator of bone and mediates inflammatory bone loss. The molecular signaling that regulates bone loss downstream of TNF-α is poorly defined. Here, we demonstrate that inactivating the pro-osteoblastogenic ERK-activated ribosomal S6 kinase RSK2 leads to a drastically accelerated and amplified systemic bone loss in mice ectopically expressing TNF-α [human TNF transgenic (hTNFtg) mice]. The phenotype is associated with a decrease in bone formation because of fewer osteoblasts as well as a drastically increased bone destruction by osteoclasts. The molecular basis of this phenotype is a cell autonomous increased sensitivity of osteoblasts and osteocytes to TNF-induced apoptosis combined with an enhancement of their osteoclast supportive activity. Thus, RSK2 exerts a strong negative regulatory loop on TNF-induced bone loss.
Assuntos
Reabsorção Óssea/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Apoptose/genética , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: Wnt signaling plays a pivotal role in skeletal development and in the control of cartilage and bone turnover. We have recently shown that the secreted Wnt antagonist Wnt inhibitory factor 1 (WIF-1) is mainly expressed in the upper layers of epiphyseal and articular cartilage and, to a lesser extent, in bone. Nevertheless, WIF-1(-/-) mice develop normally. In light of these findings, we undertook this study to analyze the role of WIF-1 in arthritis. METHODS: Expression analyses for WIF-1 were performed by real-time reverse transcription-polymerase chain reaction (RT-PCR). WIF-1(-/-) and tumor necrosis factor (TNF)-transgenic mice were crossbred, and the progression of arthritis in TNF-transgenic WIF-1(-/-) mice and littermate controls was evaluated. Structural joint damage was analyzed by histologic staining, histomorphometry, and micro-computed tomography. Wnt/ß-catenin signaling was investigated by real-time RT-PCR and immunofluorescence on primary chondrocytes. RESULTS: WIF-1 expression was repressed by TNFα in chondrocytes and osteoblasts and down-regulated in experimental arthritis and in articular cartilage from patients with rheumatoid arthritis. WIF-1 deficiency partially protected TNF-transgenic mice against bone erosion and loss of trabecular bone, probably as a result of less osteoclast activity. In contrast, arthritis-related cartilage damage was aggravated by WIF-1 deficiency, while overexpression of WIF-1 attenuated cartilage degradation in TNF-transgenic mice. In chondrocytes, TNFα stimulated canonical Wnt signaling, which could be blocked by WIF-1, indicating a direct effect of TNFα and WIF-1 on Wnt signaling in this system. CONCLUSION: These data suggest that WIF-1 may take part in the fine-tuning of cartilage and bone turnover, promoting the balance of cartilage versus bone anabolism.
Assuntos
Artrite Experimental/metabolismo , Osso e Ossos/metabolismo , Cartilagem/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Artrite Experimental/genética , Artrite Experimental/patologia , Osso e Ossos/patologia , Cartilagem/patologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Condrócitos/metabolismo , Condrócitos/patologia , Progressão da Doença , Regulação para Baixo , Proteínas da Matriz Extracelular/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND: The increasing prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) incurs substantial morbidity, mortality and healthcare costs. Detection and clinical intervention at early stages of disease improves prognosis; however, we are currently limited by a lack of reliable diagnostic tests for population screening and monitoring responses to therapy. To address this unmet need, we investigated human invariant Natural Killer T cell (iNKT) activation by fat-loaded hepatocytes, leading to the discovery that circulating soluble CD46 (sCD46) levels accurately predict hepatic steatosis. METHODS: sCD46 in plasma was measured using a newly developed immuno-competition assay in two independent cohorts: Prospective living liver donors (n = 156; male = 66, female = 90) and patients with liver tumours (n = 91; male = 58, female = 33). sCD46 levels were statistically evaluated as a predictor of hepatic steatosis. FINDINGS: Interleukin-4-secreting (IL-4+) iNKT cells were over-represented amongst intrahepatic lymphocytes isolated from resected human liver samples. IL-4+ iNKT cells preferentially developed in cocultures with a fat-loaded, hepatocyte-like cell line, HepaRG. This was attributed to induction of matrix metalloproteases (MMP) in fat-loaded HepaRG cells and primary human liver organoids, which led to indiscriminate cleavage of immune receptors. Loss of cell-surface CD46 resulted in unrepressed differentiation of IL-4+ iNKT cells. sCD46 levels were elevated in patients with hepatic steatosis. Discriminatory cut-off values for plasma sCD46 were found that accurately classified patients according to histological steatosis grade. INTERPRETATION: sCD46 is a reliable clinical marker of hepatic steatosis, which can be conveniently and non-invasively measured in serum and plasma samples, raising the possibility of using sCD46 levels as a diagnostic method for detecting or grading hepatic steatosis. FUNDING: F.B. was supported by the Else Kröner Foundation (Award 2016_kolleg.14). G.G. was supported by the Bristol Myers Squibb Foundation for Immuno-Oncology (Award FA-19-009). N.S. was supported by a Wellcome Trust Fellowship (211113/A/18/Z). J.A.H. received funding from the European Union's Horizon 2020 research and innovation programme (Award 860003). J.M.W. received funding from the Else Kröner Foundation (Award 2015_A10).
Assuntos
Biomarcadores , Humanos , Masculino , Biomarcadores/sangue , Feminino , Pessoa de Meia-Idade , Células T Matadoras Naturais/metabolismo , Hepatócitos/metabolismo , Fígado Gorduroso/diagnóstico , Fígado Gorduroso/sangue , Fígado Gorduroso/metabolismo , Adulto , IdosoRESUMO
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. Proposed model of osteoclast fusion regulated by caspase-8 activation and PS exposure. RANK/RANK-L interaction. Activation of procaspase-8 into caspase-8. Caspase-8 activates caspase-3. Active capase-3 cleaves Xkr8. Local PS exposure is induced. Exposed PS is recognized by the fusion partner. FUSION. PS is re-internalized.
Assuntos
Caspase 8 , Fusão Celular , Osteoclastos , Fosfatidilserinas , Proteínas de Transferência de Fosfolipídeos , Caspase 8/metabolismo , Caspase 8/genética , Animais , Osteoclastos/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Reabsorção Óssea/genética , Diferenciação Celular , Ligante RANK/metabolismoRESUMO
IL-33 is a new member of the IL-1 family, which plays a crucial role in inflammatory response, enhancing the differentiation of dendritic cells and alternatively activated macrophages (AAM). Based on the evidence of IL-33 expression in bone, we hypothesized that IL-33 may shift the balance from osteoclast to AAM differentiation and protect from inflammatory bone loss. Using transgenic mice overexpressing human TNF, which develop spontaneous joint inflammation and cartilage destruction, we show that administration of IL-33 or an IL-33R (ST2L) agonistic Ab inhibited cartilage destruction, systemic bone loss, and osteoclast differentiation. Reconstitution of irradiated hTNFtg mice with ST2(-/-) bone marrow led to more bone loss compared with the chimeras with ST2(+/+) bone marrow, demonstrating an important endogenous role of the IL-33/ST2L pathway in bone turnover. The protective effect of IL-33 on bone was accompanied by a significant increase of antiosteoclastogenic cytokines (GM-CSF, IL-4, and IFN-γ) in the serum. In vitro IL-33 directly inhibits mouse and human M-CSF/receptor activator for NF-κB ligand-driven osteoclast differentiation. IL-33 acts directly on murine osteoclast precursors, shifting their differentiation toward CD206(+) AAMs via GM-CSF in an autocrine fashion. Thus, we show in this study that IL-33 is an important bone-protecting cytokine and may be of therapeutic benefit in treating bone resorption.
Assuntos
Reabsorção Óssea/metabolismo , Interleucinas/metabolismo , Macrófagos/metabolismo , Osteoclastos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Western Blotting , Células da Medula Óssea/metabolismo , Reabsorção Óssea/prevenção & controle , Diferenciação Celular/efeitos dos fármacos , Condrócitos/metabolismo , Humanos , Imuno-Histoquímica , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genéticaRESUMO
The proteasome inhibitor bortezomib is approved for the treatment of multiple myeloma and mantle cell lymphoma. We recently demonstrated that bortezomib eliminates autoreactive plasma cells in systemic lupus erythematosus mouse models, thereby representing a promising novel treatment for Ab-mediated diseases. In this study, we investigated the effects of bortezomib on the just developing and pre-existing T-dependent Ab response toward dinitrophenyl-keyhole limpet hemocyanin and the T-independent type 2 response toward (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP)-Ficoll in BALB/c mice. Bortezomib treatment strongly reduced T-dependent Ab titers mainly due to depletion of plasma cells. In contrast, the early T-independent type 2 response against i.v. administered NIP-Ficoll, which is predominantly dependent on marginal zone (MZ) B cells, resisted bortezomib. Upon bortezomib treatment, immunoproteasome subunits and the antiapoptotic unfolded protein response including NF-κB were induced in NIP-Ficoll-stimulated MZ B cells, but not in plasma cells and follicular B cells. In summary, bortezomib treatment decreases Ab titers arising from T-dependent immune responses predominantly by eliminating plasma cells. In contrast, the early T-independent type 2 response protecting the organism against blood-borne pathogens remains largely intact due to a remarkable resistance of MZ B cells against proteasome inhibition.
Assuntos
Formação de Anticorpos/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Plasmócitos/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Pirazinas/farmacologia , Animais , Subpopulações de Linfócitos B/efeitos dos fármacos , Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Bortezomib , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Plasmócitos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologiaRESUMO
OBJECTIVE: Immune activation triggers bone loss. Activated T cells are the cellular link between immune activation and bone destruction. The aim of this study was to determine whether immune regulatory mechanisms, such as naturally occurring Treg cells, also extend their protective effects to bone homeostasis in vivo. METHODS: Bone parameters in FoxP3-transgenic (Tg) mice were compared with those in their wild-type (WT) littermate controls. Ovariectomy was performed in FoxP3-Tg mice as a model of postmenopausal osteoporosis, and the bone parameters were analyzed. The bones of RAG-1(-/-) mice were analyzed following the adoptive transfer of isolated CD4+CD25+ T cells. CD4+CD25+ T cells and CD4+ T cells isolated from FoxP3-Tg mice and WT mice were cocultured with monocytes to determine their ability to suppress osteoclastogenesis in vitro. RESULTS: FoxP3-Tg mice developed higher bone mass and were protected from ovariectomy-induced bone loss. The increase in bone mass was found to be the result of impaired osteoclast differentiation and bone resorption in vivo. Bone formation was not affected. Adoptive transfer of CD4+CD25+ T cells into T cell-deficient RAG-1(-/-) mice also increased the bone mass, indicating that Treg cells directly affect bone homeostasis without the need to engage other T cell lineages. CONCLUSION: These data demonstrate that Treg cells can control bone resorption in vivo and can preserve bone mass during physiologic and pathologic bone remodeling.
Assuntos
Densidade Óssea/genética , Osso e Ossos/metabolismo , Diferenciação Celular/genética , Fatores de Transcrição Forkhead/genética , Osteoclastos/metabolismo , Animais , Densidade Óssea/imunologia , Reabsorção Óssea/genética , Reabsorção Óssea/imunologia , Reabsorção Óssea/metabolismo , Osso e Ossos/imunologia , Osso e Ossos/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Diferenciação Celular/imunologia , Células Cultivadas , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Osteoclastos/imunologia , Osteoclastos/patologia , Ovariectomia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologiaRESUMO
OBJECTIVE: During the course of different musculoskeletal diseases, joints are progressively damaged by inflammatory, infectious, or mechanical stressors, leading to joint destruction and disability. While effective strategies to inhibit joint inflammation, such as targeted cytokine-blocking therapy, have been developed during the last decade, the molecular mechanisms of joint damage are still poorly understood. This study was undertaken to investigate the role of the Wnt pathway modulator R-Spondin 1 (RSpo1) in protecting bone and cartilage in a mouse model of arthritis. METHODS: Tumor necrosis factor alpha (TNFalpha)-transgenic mice were treated with vehicle or Rspo1. Mice were evaluated for signs of arthritis, and histologic analysis of the hind paws was performed. Moreover, we determined the effect of Rspo1 on Wnt signaling activity and osteoprotegerin (OPG) expression in murine primary osteoblasts. RESULTS: The secreted Wnt pathway modulator RSpo1 was highly effective in preserving the structural integrity of joints in a TNFalpha-transgenic mouse model of arthritis by protecting bone and cartilage from inflammation-related damage. RSpo1 antagonized the Wnt inhibitor Dkk-1 and modulated Wnt signaling in mouse mesenchymal cells. In osteoblasts, RSpo1 induced differentiation and expression of OPG, thereby inhibiting osteoclastogenesis in vitro. In vivo, RSpo1 promoted osteoblast differentiation and bone formation while blocking osteoclast development, thereby contributing to the integrity of joints during inflammatory arthritis. CONCLUSION: Our results demonstrate the therapeutic potential of RSpo1 as an anabolic agent for the preservation of joint architecture.
Assuntos
Artrite Experimental/metabolismo , Osso e Ossos/metabolismo , Cartilagem/metabolismo , Inflamação/metabolismo , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/patologia , Western Blotting , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/patologia , Cartilagem/efeitos dos fármacos , Cartilagem/patologia , Imunofluorescência , Hibridização In Situ , Inflamação/tratamento farmacológico , Inflamação/patologia , Camundongos , Camundongos Transgênicos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Trombospondinas/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Pharmacological inhibitors have provided evidence for the key role of p38 MAPK in osteoclast differentiation and in inflammation-induced bone loss. However, these inhibitors block more than one of the four p38 isoforms, usually p38alpha and p38beta, and sometimes also other kinases such as JNK3. We show in this study that p38alpha is the main p38 isoenzyme expressed in the osteoclast precursors and in the mature osteoclasts. p38alpha as well as its downstream substrates were phosphorylated in osteoclast progenitors stimulated by TNF-alpha. Using Mx-cre-mediated conditional gene inactivation we demonstrated that mice lacking p38alpha were protected against TNF-alpha-induced bone destruction at the site of inflammation as well as against TNF-alpha-mediated systemic bone loss. The bone protection was associated to decreased osteoclast numbers in vivo as well as a decreased IL-1beta expression in the inflamed tissue and in the isolated monocytes. The phenotype was cell autonomous because, similarly to p38alpha-deficient cells, knockdown of p38alpha in monocytes resulted in a decreased osteoclast differentiation in vitro. It was not caused by major changes in RANKL-mediated ERK or JNK activation but rather associated to an increased NF-kappaB activation caused by a decrease in IkappaBalpha recovery. Thus, our data show that developing specific inhibitors of the alpha-isoenzyme of p38 would be beneficial for the treatment of inflammation-induced bone destruction as observed in rheumatoid arthritis.
Assuntos
Artrite Experimental/enzimologia , Reabsorção Óssea/enzimologia , Proteína Quinase 14 Ativada por Mitógeno/fisiologia , Animais , Artrite Experimental/patologia , Reabsorção Óssea/patologia , Reabsorção Óssea/prevenção & controle , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Células Cultivadas , Humanos , Isoenzimas/biossíntese , Isoenzimas/deficiência , Isoenzimas/genética , Isoenzimas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína Quinase 14 Ativada por Mitógeno/biossíntese , Proteína Quinase 14 Ativada por Mitógeno/deficiência , Proteína Quinase 14 Ativada por Mitógeno/genética , Osteoclastos/enzimologia , Osteoclastos/patologia , Especificidade por Substrato/imunologiaRESUMO
Osteoclasts are specialised bone resorbing cells that control both physiological and pathological bone turnover. Functional changes in the differentiation and activity of osteoclasts are accompanied by active metabolic reprogramming. However, the biological significance and the in vivo relevance of these events has remained unclear. Here we show that bone resorption of differentiated osteoclasts heavily relies on increased aerobic glycolysis and glycolysis-derived lactate production. While pharmacological inhibition of glycolysis did not affect osteoclast differentiation or viability, it efficiently blocked bone resorption in vitro and in vivo and consequently ameliorated ovariectomy-induced bone loss. Our experiments thus highlight the therapeutic potential of interfering with osteoclast-intrinsic metabolic pathways as possible strategy for the treatment of diseases characterized by accelerated bone loss.
Assuntos
Antimetabólitos/farmacologia , Reabsorção Óssea/metabolismo , Desoxiglucose/farmacologia , Glicólise , Osteoclastos/metabolismo , Osteoporose/metabolismo , Animais , Antimetabólitos/uso terapêutico , Reabsorção Óssea/tratamento farmacológico , Células Cultivadas , Desoxiglucose/uso terapêutico , Feminino , Ácido Láctico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Oxigênio/metabolismoRESUMO
Bone turnover, which is determined by osteoclast-mediated bone resorption and osteoblast-mediated bone formation, represents a highly energy consuming process. The metabolic requirements of osteoblast differentiation and mineralization, both essential for regular bone formation, however, remain incompletely understood. Here we identify the nuclear receptor peroxisome proliferator-activated receptor (PPAR) δ as key regulator of osteoblast metabolism. Induction of PPARδ was essential for the metabolic adaption and increased rate in mitochondrial respiration necessary for the differentiation and mineralization of osteoblasts. Osteoblast-specific deletion of PPARδ in mice, in turn, resulted in an altered energy homeostasis of osteoblasts, impaired mineralization and reduced bone mass. These data show that PPARδ acts as key regulator of osteoblast metabolism and highlight the relevance of cellular metabolic rewiring during osteoblast-mediated bone formation and bone-turnover.
Assuntos
Remodelação Óssea/fisiologia , Osteoblastos/metabolismo , Osteogênese/fisiologia , PPAR delta/genética , PPAR delta/metabolismo , Animais , Densidade Óssea/fisiologia , Diferenciação Celular , Células Cultivadas , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Osteoblastos/citologia , Osteoclastos/metabolismo , Fosforilação OxidativaRESUMO
This paper describes the development of a highly selective analytical method for the determination of deoxynivalenol (DON) in maize. The developed method is based on immuno-ultrafiltration (IUF) and is the first application of IUF as a clean-up strategy in food analysis. Quantification of DON was carried out by high-performance liquid chromatography with ultraviolet detection. In contrast to immunoaffinity chromatography, in IUF the antibodies are not bound to a solid support material but used in free form, thus making it possible to avoid the critical immobilisation step. Sample clean-up by IUF proved to be as selective as clean-up using commercially available immunoaffinity columns. The limit of detection (S/N=3) of the analytical method was found to be 74 ng DON/g maize. Repeated analysis of a certified maize reference material on four different days resulted in a mean recovery of 93% with a standard deviation of 10%.
Assuntos
Análise de Alimentos/métodos , Tricotecenos/análise , Zea mays/química , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Micotoxinas/análise , Reprodutibilidade dos Testes , Ultrafiltração/métodosRESUMO
NR4A1 (Nur77 or NGFI-B), an orphan member of the nuclear receptor superfamily, has been identified as a key regulator of the differentiation and function of myeloid, lymphoid, and mesenchymal cells. The detailed role of NR4A1 in bone biology is incompletely understood. Here, we report a role for NR4A1 as novel factor controlling the migration and recruitment of osteoclast precursors during bone remodeling. Myeloid-specific but not osteoblast-specific deletion of NR4A1 resulted in osteopenia due to an increase in the number of bone-lining osteoclasts. Although NR4A1-deficient osteoclast precursors displayed a regular differentiation into mature osteoclasts, they showed a hyper-motile phenotype that was largely dependent on increased osteopontin expression, suggesting that expression of NR4A1 negatively controlled osteopontin-mediated recruitment of osteoclast precursors to the trabecular bone. Pharmacological activation of NR4A1, in turn, inhibited osteopontin expression and osteopontin-dependent migration of osteoclast precursors resulted in reduced abundance of bone-resorbing osteoclasts in vivo as well as in an ameliorated bone loss after ovariectomy in mice. This study identifies NR4A1 as a crucial player in the regulation of osteoclast biology and bone remodeling and highlights this nuclear receptor as a promising target for therapeutic intervention during the treatment of osteoporosis. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.
Assuntos
Remodelação Óssea , Movimento Celular , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Animais , Reabsorção Óssea/patologia , Osso Esponjoso/metabolismo , Contagem de Células , Diferenciação Celular , Fusão Celular , Deleção de Genes , Homeostase , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/deficiência , Osteoblastos/metabolismo , Osteopontina/metabolismo , Ovariectomia , Proteínas Repressoras/metabolismoRESUMO
Knowledge concerning expression and function of Suppression of Tumorigenicity 2 (ST2) in chondrocytes is at present, limited. Analysis of murine growth plates and ATDC5 chondrocytes indicated peak expression of the ST2 transmembrane receptor (ST2L) and soluble (sST2) isoforms during the hypertrophic differentiation concomitant with the expression of the hypertrophic markers Collagen X (Col X), Runx2 and MMP-13. Gain- and loss-of-function experiments in ATDC5 and primary human growth plate chondrocytes (PHCs), confirmed regulation of ST2 by the key transcription factor Runx2, indicating ST2 to be a novel Runx2 target. ST2 knock-out mice (ST2-/-) exhibited noticeable hypertrophic zone (HZ) reduction in murine growth plates, accompanied by lower expression of Col X and Osteocalcin (OSC) compared to wild-type (WT) mice. Likewise, ST2 knockdown resulted in decreased Col X expression and downregulation of OSC and Vascular Endothelial Growth Factor (VEGF) in ATDC5 cells. The ST2 suppression was also associated with upregulation of the proliferative stage markers Sox9 and Collagen II (Col II), indicating ST2 to be a new regulator of ATDC5 chondrocyte differentiation. Runx3 was, furthermore, identified as a novel Runx2 target in chondrocytes. This study suggests that Runx2 mediates ST2 and Runx3 induction to cooperatively regulate hypertrophic differentiation of ATDC5 chondrocytes.
Assuntos
Condrócitos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Criança , Pré-Escolar , Condrócitos/patologia , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Subunidade alfa 3 de Fator de Ligação ao Core/fisiologia , Feminino , Humanos , Hipertrofia , Immunoblotting , Lactente , Proteína 1 Semelhante a Receptor de Interleucina-1/fisiologia , Masculino , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Few studies have addressed the effect of treatment of unruptured intracranial aneurysm (UIA) on cognitive function. OBJECTIVE: Neuropsychological assessment after UIA treatment is underreported, and prospective trials have repeatedly been demanded. In 2014, we conducted a prospective controlled study to evaluate the differences in cognitive processing caused by the treatment of anterior circulation UIAs. PATIENTS AND METHODS: Thirty patients were enrolled until September 2015. Ten patients received endovascular aneurysm occlusion (EV), 10 patients were treated microsurgically (MS), and 10 patients with surgically treated degenerative lumbar spine disease (LD) served as control. All patients underwent extended standardized neuropsychological assessment before (t1) and 6 weeks after treatment (t2). Tests included verbal, visual, and visuospatial memory, psychomotor functioning, executive functioning, and its subdomains verbal fluency and cognitive flexibility. We statistically evaluated intragroup and intergroup changes. RESULTS: Intragroup comparisons and group-rate analysis showed no significant impairment in overall neuropsychological performance, either postinterventionally or postoperatively. However, the postoperative performance in cognitive processing speed, cognitive flexibility, and executive functioning was significantly worse in the MS group than in the EV (P = 0.038) and LD group (P = 0.02). Compared with the EV group, patients with MS showed significant postoperative impairment in a subtest for auditory-verbal memory (Wechsler Memory Scale, Fourth Edition, Logical Memory II; MS vs. EV P = 0.011). The MS group trended toward posttreatment impairment in subtests for verbal fluency and semantic memory (Regensburg Word Fluency Test; MS vs. EV P = 0.083) and in auditory-verbal memory (Wechsler Memory Scale, Fourth Edition, Logical Memory II; MS vs. LD P = 0.06). CONCLUSIONS: Our preliminary data showed no effect of anterior circulation UIA treatment on overall neuropsychological function but impaired short-term executive processing in surgically treated patients.
Assuntos
Disfunção Cognitiva/epidemiologia , Aneurisma Intracraniano/cirurgia , Complicações Pós-Operatórias/epidemiologia , Adulto , Idoso , Cognição , Procedimentos Endovasculares , Função Executiva , Feminino , Humanos , Vértebras Lombares/cirurgia , Masculino , Microcirurgia , Pessoa de Meia-Idade , Testes Neuropsicológicos , Estudos Prospectivos , Desempenho Psicomotor , Memória Espacial , Processamento Espacial , Doenças da Coluna Vertebral/cirurgiaRESUMO
INTRODUCTION: Interleukin (IL)-36α is a newly described member of the IL-1 cytokine family with a known inflammatory and pathogenic function in psoriasis. Recently, we could demonstrate that the receptor (IL-36R), its ligand IL-36α and its antagonist IL-36Ra are expressed in synovial tissue of arthritis patients. Furthermore, IL-36α induces MAP-kinase and NFκB signaling in human synovial fibroblasts with subsequent expression and secretion of pro-inflammatory cytokines. METHODS: To understand the pathomechanism of IL-36 dependent inflammation, we investigated the biological impact of IL-36α signaling in the hTNFtg mouse. Also the impact on osteoclastogenesis by IL-36α was tested in murine and human osteoclast assays. RESULTS: Diseased mice showed an increased expression of IL-36R and IL-36α in inflamed knee joints compared to wildtype controls. However, preventively treating mice with an IL-36R blocking antibody led to no changes in clinical onset and pattern of disease. Furthermore, blockade of IL-36 signaling did not change histological signs of TNF-induced arthritis. Additionally, no alteration on bone homeostasis was observed in ex vivo murine and human osteoclast differentiation assays. CONCLUSION: Thus we conclude that IL-36α does not affect the development of inflammatory arthritis.
Assuntos
Artrite/induzido quimicamente , Artrite/prevenção & controle , Receptores de Interleucina-1/metabolismo , Transdução de Sinais , Animais , Artrite/metabolismo , Artrite/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1/farmacologia , Articulação do Joelho/efeitos dos fármacos , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Masculino , Camundongos , Camundongos Transgênicos , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Receptores de Interleucina-1/imunologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Peroxisome proliferator-activated receptors (PPARs) act as metabolic sensors and central regulators of fat and glucose homeostasis. Furthermore, PPARγ has been implicated as major catabolic regulator of bone mass in mice and humans. However, a potential involvement of other PPAR subtypes in the regulation of bone homeostasis has remained elusive. Here we report a previously unrecognized role of PPARß/δ as a key regulator of bone turnover and the crosstalk between osteoblasts and osteoclasts. In contrast to activation of PPARγ, activation of PPARß/δ amplified Wnt-dependent and ß-catenin-dependent signaling and gene expression in osteoblasts, resulting in increased expression of osteoprotegerin (OPG) and attenuation of osteoblast-mediated osteoclastogenesis. Accordingly, PPARß/δ-deficient mice had lower Wnt signaling activity, lower serum concentrations of OPG, higher numbers of osteoclasts and osteopenia. Pharmacological activation of PPARß/δ in a mouse model of postmenopausal osteoporosis led to normalization of the altered ratio of tumor necrosis factor superfamily, member 11 (RANKL, also called TNFSF11) to OPG, a rebalancing of bone turnover and the restoration of normal bone density. Our findings identify PPARß/δ as a promising target for an alternative approach in the treatment of osteoporosis and related diseases.
Assuntos
Osso e Ossos/metabolismo , PPAR delta/metabolismo , PPAR beta/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Alelos , Animais , Doenças Ósseas Metabólicas/metabolismo , Reabsorção Óssea , Feminino , Glucose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Fatores de Tempo , beta Catenina/metabolismoRESUMO
OBJECTIVE: To investigate the efficacy of a murine anti-interleukin-6 receptor (anti-IL-6R) antibody in directly blocking tumor necrosis factor (TNF)- and RANKL-mediated osteoclastogenesis in vitro and in vivo. METHODS: The efficacy of a murine antibody against IL-6R in blocking osteoclast differentiation of mononuclear cells stimulated with RANKL was tested. In addition, arthritic human TNFalpha-transgenic mice were treated with anti-IL-6R antibody, and osteoclast formation and bone erosion were assessed in arthritic paws. RESULTS: Blockade of IL-6R dose dependently reduced osteoclast differentiation and bone resorption in monocyte cultures stimulated with RANKL or RANKL plus TNF. In human TNFalpha-transgenic mice, IL-6R blockade did not inhibit joint inflammation, but it strongly reduced osteoclast formation in inflamed joints as well as bone erosion in vivo. Neither the cell influx into joints nor the synovial expression of IL-6 and RANKL changed with IL-6R blockade, while the synovial expression of IL-1 was significantly reduced. In contrast, TNF-mediated systemic bone loss was not inhibited by IL-6R blockade. CONCLUSION: These data suggest that blockade of IL-6R directly affects osteoclast formation in vitro and in vivo, suggesting a direct and specific effect of anti-IL-6R therapy on osteoclasts independently of its antiinflammatory effects. This effect adds significantly to the structure-sparing potential of pharmacologic blockade of IL-6R in arthritis.