RESUMO
Bone resorption is driven through osteoclast differentiation by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-Β ligand (RANKL). We noted that a disintegrin and metalloproteinase (ADAM) 10 and ADAM17 are downregulated at the expression level during osteoclast differentiation of the murine monocytic cell line RAW264.7 in response to RANKL. Both proteinases are well known to shed a variety of single-pass transmembrane molecules from the cell surface. We further showed that inhibitors of ADAM10 or ADAM17 promote osteoclastic differentiation and furthermore enhance the surface expression of receptors for RANKL and M-CSF on RAW264.7 cells. Using murine bone marrow-derived monocytic cells (BMDMCs), we demonstrated that a genetic deficiency of ADAM17 or its required regulator iRhom2 leads to increased osteoclast development in response to M-CSF and RANKL stimulation. Moreover, ADAM17-deficient osteoclast precursor cells express increased levels of the receptors for RANKL and M-CSF. Thus, ADAM17 negatively regulates osteoclast differentiation, most likely through shedding of these receptors. To assess the time-dependent contribution of ADAM10, we blocked this proteinase by adding a specific inhibitor on day 0 of BMDMC stimulation with M-CSF or on day 7 of subsequent stimulation with RANKL. Only ADAM10 inhibition beginning on day 7 increased the size of developing osteoclasts indicating that ADAM10 suppresses osteoclast differentiation at a later stage. Finally, we could confirm our findings in human peripheral blood mononuclear cells (PBMCs). Thus, downregulation of either ADAM10 or ADAM17 during osteoclast differentiation may represent a novel regulatory mechanism to enhance their differentiation process. Enhanced bone resorption is a critical issue in osteoporosis and is driven through osteoclast differentiation by specific osteogenic mediators. The present study demonstrated that the metalloproteinases ADAM17 and ADAM10 critically suppress osteoclast development. This was observed for a murine cell line, for isolated murine bone marrow cells and for human blood cells by either preferential inhibition of the proteinases or by gene knockout. As a possible mechanism, we studied the surface expression of critical receptors for osteogenic mediators on developing osteoclasts. Our findings revealed that the suppressive effects of ADAM17 and ADAM10 on osteoclastogenesis can be explained in part by the proteolytic cleavage of surface receptors by ADAM10 and ADAM17, which reduces the sensitivity of these cells to osteogenic mediators. We also observed that osteoclast differentiation was associated with the downregulation of ADAM10 and ADAM17, which reduced their suppressive effects. We therefore propose that this downregulation serves as a feedback loop for enhancing osteoclast development.
Assuntos
Proteína ADAM10 , Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide , Diferenciação Celular , Regulação para Baixo , Proteínas de Membrana , Osteoclastos , Ligante RANK , Proteína ADAM17/metabolismo , Proteína ADAM17/genética , Proteína ADAM10/metabolismo , Proteína ADAM10/genética , Osteoclastos/metabolismo , Osteoclastos/citologia , Animais , Diferenciação Celular/genética , Camundongos , Regulação para Baixo/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Humanos , Ligante RANK/metabolismo , Células RAW 264.7 , Fator Estimulador de Colônias de Macrófagos/farmacologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Senile osteoporosis may be caused by an imbalance in intestinal flora and oxidative stress. Trimethylamine-N-oxide (TMAO), a metabolite of dietary choline dependent on gut microbes, has been found to be significantly increased in osteoporosis. However, the role of TMAO in bone loss during osteoporosis remains poorly understood. In this study, we examined the impact of TMAO on osteoclast differentiation and bone resorption in an in vitro setting. METHODS: Osteoclast differentiation was induced by incubating RAW 264.7 cells in the presence of Receptor Activator for Nuclear Factor-κB Ligand (RANKL) and macrophage-stimulating factor (M-CSF). Flow cytometry, TRAP staining assay, CCK-8, and ELISA were employed to investigate the impact of TMAO on osteoclast differentiation and bone resorption activity in vitro. For mechanistic exploration, RT-PCR and Western blotting were utilized to assess the activation of the NF-κB pathway. Additionally, protein levels of secreted cytokines and growth factors were determined using suspension array technology. RESULTS: Our findings demonstrate that TMAO enhances RANKL and M-CSF-induced osteoclast formation and bone resorption in a dose-dependent manner. Mechanistically, TMAO triggers the upregulation of the NF-κB pathway and osteoclast-related genes (NFATc1, c-Fos, NF-κB p65, Traf6, and Cathepsin K). Furthermore, TMAO markedly elevated the levels of oxidative stress and inflammatory factors. CONCLUSIONS: In conclusion, TMAO enhances RANKL and M-CSF-induced osteoclast differentiation and inflammation in RAW 264.7 cells by activating the NF-κB signaling pathway. These findings offer a new rationale for further academic and clinical research on osteoporosis treatment.
Assuntos
Diferenciação Celular , Metilaminas , NF-kappa B , Osteoclastos , Estresse Oxidativo , Ligante RANK , Transdução de Sinais , Animais , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Camundongos , Metilaminas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células RAW 264.7 , NF-kappa B/metabolismo , Ligante RANK/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Reabsorção Óssea/metabolismoRESUMO
This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.
Assuntos
Reabsorção Óssea , Células-Tronco Pluripotentes Induzidas , Humanos , Osteoclastos , Diferenciação Celular , Fator Estimulador de Colônias de Macrófagos/farmacologia , Osso e Ossos , Glicoproteínas de Membrana , Ligante RANKRESUMO
Structural and physiological cues provide guidance for the directional migration and spatial organization of endogenous cells. Here, a microchannel scaffold with instructive niches is developed using a circumferential freeze-casting technique with an alkaline salting-out strategy. Thereinto, polydopamine-coated nano-hydroxyapatite is employed as a functional inorganic linker to participate in the entanglement and crystallization of chitosan molecules. This scaffold orchestrates the advantage of an oriented porous structure for rapid cell infiltration and satisfactory immunomodulatory capacity to promote stem cell recruitment, retention, and subsequent osteogenic differentiation. Transcriptomic analysis as well as its in vitro and in vivo verification demonstrates that essential colony-stimulating factor-1 (CSF-1) factor is induced by this scaffold, and effectively bound to the target colony-stimulating factor-1 receptor (CSF-1R) on the macrophage surface to activate the M2 phenotype, achieving substantial endogenous bone regeneration. This strategy provides a simple and efficient approach for engineering inducible bone regenerative biomaterials.
Assuntos
Regeneração Óssea , Durapatita , Fator Estimulador de Colônias de Macrófagos , Osteogênese , Polímeros , Receptor de Fator Estimulador de Colônias de Macrófagos , Alicerces Teciduais , Regeneração Óssea/efeitos dos fármacos , Alicerces Teciduais/química , Animais , Camundongos , Durapatita/química , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/química , Polímeros/química , Diferenciação Celular , Quitosana/química , Indóis/química , Transdução de Sinais , Engenharia Tecidual/métodos , Macrófagos/metabolismo , Macrófagos/citologia , Células RAW 264.7RESUMO
BACKGROUND: A growing body of evidence suggests that immune dysfunction and inflammation in the peripheral tissues as well as the central nervous system are associated with the neurodevelopmental deficits observed in autism spectrum disorder (ASD). Elevated expression of pro-inflammatory cytokines in the plasma, serum, and peripheral blood mononuclear cells of ASD has been reported. These cytokine expression levels are associated with the severity of behavioral impairments and symptoms in ASD. In a prior study, our group reported that tumor necrosis factor-α (TNF-α) expression in granulocyte-macrophage colony-stimulating factor-induced macrophages (GM-CSF MΦ) and the TNF-α expression ratio in GM-CSF MΦ/M-CSF MΦ (macrophage colony-stimulating factor-induced macrophages) was markedly higher in individuals with ASD than in typically developed (TD) individuals. However, the mechanisms of how the macrophages and the highly expressed cytokines affect neurons remain to be addressed. METHODS: To elucidate the effect of macrophages on human neurons, we used a co-culture system of control human-induced pluripotent stem cell-derived neurons and differentiated macrophages obtained from the peripheral blood mononuclear cells of five TD individuals and five individuals with ASD. All participants were male and ethnically Japanese. RESULTS: Our results of co-culture experiments showed that GM-CSF MΦ affect the dendritic outgrowth of neurons through the secretion of pro-inflammatory cytokines, interleukin-1α and TNF-α. Macrophages derived from individuals with ASD exerted more severe effects than those derived from TD individuals. LIMITATIONS: The main limitations of our study were the small sample size with a gender bias toward males, the use of artificially polarized macrophages, and the inability to directly observe the interaction between neurons and macrophages from the same individuals. CONCLUSIONS: Our co-culture system revealed the non-cell autonomous adverse effects of GM-CSF MΦ in individuals with ASD on neurons, mediated by interleukin-1α and TNF-α. These results may support the immune dysfunction hypothesis of ASD, providing new insights into its pathology.
Assuntos
Transtorno do Espectro Autista , Citocinas , Feminino , Masculino , Humanos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Leucócitos Mononucleares/metabolismo , Interleucina-1alfa/metabolismo , Interleucina-1alfa/farmacologia , Transtorno do Espectro Autista/metabolismo , Células Cultivadas , Sexismo , Macrófagos/metabolismo , Granulócitos/metabolismo , Dendritos/metabolismoRESUMO
The ubiquitination of transmembrane receptors regulates endocytosis, intracellular traffic, and signal transduction. Bone marrow-derived macrophages from myeloid Cbl-/- and Cbl-b-/- double knockout (DKO) mice display sustained proliferation mirroring the myeloproliferative disease that these mice succumb to. Here, we found that the ubiquitin ligases Cbl and Cbl-b have overlapping functions for controlling the endocytosis and intracellular traffic of the CSF-1R. DKO macrophages displayed complete loss of ubiquitination of the CSF-1R whereas partial ubiquitination was observed for either single Cbl-/- or Cbl-b-/- macrophages. Unlike wild type, DKO macrophages were immortal and displayed slower CSF-1R internalization, elevated AKT signaling, and a failure to transport the CSF-1R into the lumen of nascent macropinosomes, leaving its cytoplasmic region available for signaling. CSF-1R degradation depended upon lysosomal vATPase activity in both WT and DKO macrophages, with this degradation confined to macropinosomes in WT but occurring in distributed/tubular lysosomes in DKO cells. RNA-sequencing comparison of Cbl-/-, Cbl-b-/- and DKO macrophages indicated that while the overall macrophage transcriptional program remained intact, DKO macrophages had alterations in gene expression associated with growth factor signaling, cell cycle, inflammation and senescence. Cbl-b-/- had minimal effect on the transcriptional program whereas Cbl-/- led to more alternations but only DKO macrophages demonstrated substantial changes in the transcriptome, suggesting overlapping but unique functions for the two Cbl-family members. Thus, Cbl/Cbl-b-mediated ubiquitination of CSF-1R regulates its endocytic fate, constrains inflammatory gene expression, and regulates signaling for macrophage proliferation.
Assuntos
Receptor de Fator Estimulador de Colônias de Macrófagos , Ubiquitina , Camundongos , Animais , Ubiquitina/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Macrófagos/metabolismoRESUMO
CD115, the receptor for colony stimulating factor 1, is essential for survival and differentiation of monocytes and macrophages and is therefore frequently used to define monocyte subsets and their progenitors in immunological assays. However, CD115 surface expression and detection by flow cytometry is greatly influenced by cell isolation and processing methods, organ source, and disease context. In a systematic analysis of murine monocytes, we define experimental conditions that preserve or limit CD115 surface expression and staining by flow cytometry. We also find that, independent of conditions, CD115 surface levels are consistently lower in Ly6Clo monocytes than in Ly6Chi monocytes, with the exception of Ly6Clo monocytes in the bone marrow. Furthermore, in contrast to IL-34, the presence of colony stimulating factor 1 impairs CD115 antibody staining in a dose-dependent manner, which, in a model of ischemic kidney injury with elevated levels of colony stimulating factor 1, influenced quantification of kidney monocytes. Thus, staining and experimental conditions affect quantitative and qualitative analysis of monocytes and may influence experimental conclusions.
Assuntos
Monócitos , Receptor de Fator Estimulador de Colônias de Macrófagos , Camundongos , Animais , Monócitos/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Citometria de Fluxo , Macrófagos/metabolismo , Diferenciação CelularRESUMO
Macrophages regulate metabolic homeostasis in health and disease. Macrophage colony-stimulating factor (CSF1)-dependent macrophages contribute to homeostatic control of the size of the liver. This study aimed to determine the systemic metabolic consequences of elevating circulating CSF1. Acute administration of a CSF1-Fc fusion protein to mice led to monocytosis, increased resident tissue macrophages in the liver and all major organs, and liver growth. These effects were associated with increased hepatic glucose uptake and extensive mobilization of body fat. The impacts of CSF1 on macrophage abundance, liver size, and body composition were rapidly reversed to restore homeostasis. The effects of CSF1 on metabolism were independent of several known endocrine regulators and did not impact the physiological fasting response. Analysis using implantable telemetry in metabolic cages revealed progressively reduced body temperature and physical activity with no change in diurnal food intake. These results demonstrate the existence of a dynamic equilibrium between CSF1, the mononuclear phagocyte system, and control of liver-to-body weight ratio, which in turn controls systemic metabolic homeostasis. This novel macrophage regulatory axis has the potential to promote fat mobilization, without changes in appetence, which may have novel implications for managing metabolic syndrome.NEW & NOTEWORTHY CSF1 administration expands tissue macrophages, which transforms systemic metabolism. CSF1 drives fat mobilization and glucose uptake to support liver growth. The effects of CSF1 are independent of normal hormonal metabolic regulation. The effects of CSF1 are rapidly reversible, restoring homeostatic body composition. CSF1-dependent macrophages and liver size are coupled in a dynamic equilibrium.
Assuntos
Fator Estimulador de Colônias de Macrófagos , Macrófagos , Animais , Camundongos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Metabolismo dos Carboidratos , Glucose/metabolismo , LipídeosRESUMO
Immunometabolism has been unveiled in the last decade to play a major role in controlling macrophage metabolism and inflammation. There has been a constant effort to understand the immunomodulating properties of regulated metabolites during inflammation with the aim of controlling and re-wiring aberrant macrophages in inflammatory diseases. M-CSF and GM-CSF-differentiated macrophages play a key role in mounting successful innate immune responses. When a resolution phase is not achieved however, GM-CSF macrophages contribute substantially more towards an adverse inflammatory milieu than M-CSF macrophages, consequently driving disease progression. Whether there are specific immunometabolites that determine the homoeostatic or inflammatory nature of M-CSF and GM-CSF-differentiated macrophages is still unknown. As such, we performed metabolomics analysis on LPS and IL-4-stimulated M-CSF and GM-CSF-differentiated human macrophages to identify differentially accumulating metabolites. Adenine was distinguished as a metabolite significantly higher in M-CSF-differentiated macrophages after both LPS or IL-4 stimulation. Human macrophages treated with adenine before LPS stimulation showed a reduction in inflammatory gene expression, cytokine secretion and surface marker expression. Adenine caused macrophages to become more quiescent by lowering glycolysis and OXPHOS which resulted in reduced ATP production. Moreover, typical metabolite changes seen during LPS-induced macrophage metabolic reprogramming were absent in the presence of adenine. Phosphorylation of metabolic signalling proteins AMPK, p38 MAPK and AKT were not responsible for the suppressed metabolic activity of adenine-treated macrophages. Altogether, in this study we highlight the immunomodulating capacity of adenine in human macrophages and its function in driving cellular quiescence.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos , Fator Estimulador de Colônias de Macrófagos , Humanos , Adenina/metabolismo , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Células Cultivadas , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Inflamação/metabolismo , Interleucina-4/metabolismo , Lipopolissacarídeos/farmacologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , MacrófagosRESUMO
BACKGROUND: Macrophages (Mφ) can exist along a spectrum of phenotypes that include pro-inflammatory (M1) or anti-inflammatory (M2) immune cells. Mφ colony stimulating factor (M-CSF) and granulocyte Mφ colony stimulating factor (GM-CSF) are cytokines important in hematopoiesis, polarization and activation of Mφ. METHODS AND RESULTS: To gain a greater understanding of the relationship between GM-CSF and M-CSF, we investigated an in vitro model of differentiation to determine if GM-CSF and M-CSF can antagonize each other, in terms of Mφ phenotype and functions. We determined that Mφ cultured in mixed M-CSF: GM-CSF ratios exhibit M1-like GM-CSF-treated macrophage phenotype when the ratios of the two cytokines are 1:1 in culture. Moreover, GM-CSF is dominant over M-CSF in influencing Mφ production of proinflammatory cytokines such as IL-6, TNFα, and IL-12p40, and the anti-inflammatory cytokine IL-10. CONCLUSIONS: Our data established that GM-CSF is more dominant over M-CSF, triggering the Mφ to become pro-inflammatory cells. These findings provide insight into how GM-CSF can influence Mφ activation with implications in inflammatory diseases where the Mφ status can play a significant role in supporting the inflammatory conditions.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos , Fator Estimulador de Colônias de Macrófagos , Macrófagos , Anti-Inflamatórios/farmacologia , Diferenciação Celular , Células Cultivadas , Citocinas/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Hematopoese , Fator Estimulador de Colônias de Macrófagos/farmacologia , FenótipoRESUMO
Bone homeostasis is regulated by the balanced actions of osteoblasts that form the bone and osteoclasts (OCs) that resorb the bone. Bone-resorbing OCs are differentiated from hematopoietic monocyte/macrophage lineage cells, whereas osteoblasts are derived from mesenchymal progenitors. OC differentiation is induced by two key cytokines, macrophage colony-stimulating factor (M-CSF), a factor essential for the proliferation and survival of the OCs, and receptor activator of nuclear factor kappa-B ligand (RANKL), a factor for responsible for the differentiation of the OCs. Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs), p38, and c-Jun N-terminal kinases, play an essential role in regulating the proliferation, differentiation, and function of OCs. ERKs have been known to play a critical role in the differentiation and activation of OCs. In most cases, ERKs positively regulate OC differentiation and function. However, several reports present conflicting conclusions. Interestingly, the inhibition of OC differentiation by ERK1/2 is observed only in OCs differentiated from RAW 264.7 cells. Therefore, in this review, we summarize the current understanding of the conflicting actions of ERK1/2 in OC differentiation.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular , Osteoclastos , Osteoclastos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Osteoblastos/metabolismo , Diferenciação Celular , Citocinas/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Ligante RANK/metabolismoRESUMO
Dendritic cell-derived exosomes (Dex) have overcome the disadvantages associated with dendritic cell (DC) vaccines, such as cost effectiveness, stability, and sensitivity to the systemic microenvironment. However, in clinical trials, Dex failed to provide satisfactory results because of many reasons, including inadequate maturation of DC as well as the immunosuppressive tumor microenvironment (TME). Hence, culturing DCs in the presence of a maturation cocktail showed an induced expression of MHCs and co-stimulatory molecules. Additionally, targeting the colony stimulating factor-1 (CSF-1)/CSF-1 receptor (CSF-1R) signaling pathway by a CSF-1R inhibitor could deplete tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) which are responsible for immunosuppressive TME. Hence, in this study, mDexTA were isolated from bone marrow-derived DC cultured in the presence of a novel maturation cocktail and tumor antigen. mDexTA showed elevated expression of major histocompatibility complexes (MHCs) and co-stimulatory molecules and was found capable of activating naïve DC and T cells in vitro more efficiently when compared to imDexTA isolated from immature DCs. In addition, PLX-3397, a small molecule inhibitor of CSF-1/CSF-1R, was used in combination to enhance the antitumor efficacy of mDexTA. PLX-3397 showed dose-dependent toxicity against bone marrow-derived macrophages (BMDMs). In the B16-F10 murine melanoma model, we found that the combination treatment delayed tumor growth and improved survival compared to the mice treated with mDexTA alone by enhancing the CD8 T cells infiltration in TME. mDexTA when combined with PLX-3397 modulated the TME by shifting the Th1/Th2 toward a dominant Th1 population and depleting the TAMs and MDSCs. Interestingly, PLX-3397-induced FoxP3 expression was diminished when it was used in combination with mDexTA. Combination treatment also induced favorable systemic antitumor immunity in the spleen and lymph node. In conclusion, our findings provide insights into the synergy between mDexTA-based immunotherapy and PLX-3397 as the combination overcame the disadvantages associated with monotherapy and offer a therapeutic strategy for the treatment of solid tumors including melanoma.
Assuntos
Exossomos , Melanoma , Camundongos , Animais , Fator Estimulador de Colônias de Macrófagos/farmacologia , Microambiente Tumoral , Antígenos de Neoplasias , Células DendríticasRESUMO
Dendritic cell immunoreceptor (DCIR) is an inhibitory C-type lectin receptor that acts as a negative regulator in the immune system and bone metabolism. We previously revealed that DCIR deficiency enhanced osteoclastogenesis and antigen presentation of dendritic cells, and that asialo-biantennary N-glycan (NA2) functions as a ligand for DCIR. NA2 binding to DCIR suppressed murine and human osteoclastogenesis that occurs in the presence of M-CSF and RANKL. The DCIR-NA2 axis, therefore, plays an important role in regulating osteoclastogenesis in both mice and humans, although the underlying mechanisms remain unclear. Here we found that Dcir -/- bone marrow-derived macrophages (BMMs) exhibited greater proliferative and differentiation responses to M-CSF and RANKL, respectively, than wild-type (WT) BMMs. Moreover, Dcir -/- osteoclasts (OCs) increased resorptive activity and cell fusion more significantly than WT OCs. DCIR deficiency affects gene expression patterns in OCs, and we found that the expression of neuraminidase 4 was increased in Dcir -/- OCs. Furthermore, DCIR-NA2 interaction in WT BMMs, but not Dcir -/- BMMs, decreased Akt phosphorylation in response to M-CSF and RANKL. These data suggest that DCIR regulates osteoclastogenesis by downregulating M-CSF and RANKL signaling, and that DCIR-mediated signaling may contribute to the terminal modification of oligosaccharides by controlling the expression of glycosylation enzymes.
Assuntos
Reabsorção Óssea , Fator Estimulador de Colônias de Macrófagos , Animais , Humanos , Camundongos , Reabsorção Óssea/metabolismo , Proliferação de Células , Fator Estimulador de Colônias de Macrófagos/farmacologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Osteoclastos/metabolismo , Transdução de SinaisRESUMO
INTRODUCTION: This study aimed to understand the influence of periodontal fibroblasts (PDLFs) on clastic differentiation of macrophages (Mφ) in different resorptive environments. METHODS: PDLF-Mφ direct coculture (juxtacrine) was seeded on dentin, cementum, and polystyrene with/without lipopolysaccharide, macrophage colony-stimulating factor, and receptor activator of nuclear factor kappa beta ligand for 7 and 14 days and stained for tartrate-resistant acid phosphatase (TRAP) activity. PDLF-Mφ cocultured on polystyrene were immunostained for CD80, CD206, NFATc1, STAT6, and periostin, and cell culture supernatants were assessed for cytokines on days 2 and 7. Mφ grown in conditioned media of PDLFs (paracrine) and Mφ monoculture were used as controls. Data was analyzed using Student t test and one-way analysis of variance with the Tukey multiple comparisons test (P < .05). RESULTS: PDLF-Mφ coculture showed a higher number of TRAP-positive multinucleated cells than Mφ monoculture on dentin and polystyrene. No TRAP-positive multinucleated cells were observed in paracrine and cementum. The expression of CD80 and CD206 in PDLF-Mφ was similar at day 2, whereas CD206 was greater than CD80 at day 7. The expression of STAT6 was greater than NFATc1 at both days 2 and 7 (P < .05). Periostin expression in the presence of the lipopolysaccharide, macrophage colony-stimulating factor, and receptor activator of nuclear factor kappa beta ligand combination was down-regulated in PDLF monoculture, whereas it was up-regulated in PDLF-Mφ coculture. The cytokine profile of PDLF-Mφ on day 2 was predominated by interleukin (IL)-1ß, tumor necrosis factor alpha, and MMP9 and MMP2 on day 7. IL-6 and IL-8 showed steady expression at both days 2 and 7. CONCLUSIONS: The study highlights the juxtacrine effect of PDLFs on the clastic differentiation of Mφ with a difference in clastic activity between dentin and cementum. The study also emphasizes the temporal effect of tumor necrosis factor alpha, MMP2, MMP9, and IL-1ß on intercellular crosstalk in resorptive environments.
Assuntos
Fator Estimulador de Colônias de Macrófagos , Reabsorção da Raiz , Humanos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz/metabolismo , Lipopolissacarídeos/farmacologia , Ligantes , Poliestirenos/metabolismo , Poliestirenos/farmacologia , Reabsorção da Raiz/metabolismo , Macrófagos/metabolismo , Fibroblastos/metabolismo , Diferenciação Celular , Células CultivadasRESUMO
OBJECTIVES: Colony-stimulating factor-1 receptor (CSF1R) is vital for the recruitment of monocytes, and their proliferation and differentiation into functional osteoclasts. Mouse studies, where CSF1R and its cognate ligand are absent, have significant craniofacial phenotypes, but these have not been studied in detail. MATERIALS AND METHODS: Pregnant CD1 mice were fed diets laced with CSF1R inhibitor-PLX5622 starting at embryonic day 3.5 (E3.5) up to birth. Pups were collected at E18.5 to study CSF1R expression using immunofluorescence. Additional pups were studied at postnatal day 21 (P21) and P28 using microcomputed tomography (µCT) and Geometric Morphometrics, to evaluate craniofacial form. RESULTS: CSF1R-positive cells were present throughout the developing craniofacial region, including the jaw bones, surrounding teeth, tongue, nasal cavities, brain, cranial vault and base regions. Animals exposed to the CSF1R inhibitor in utero had severe depletion of CSF1R-positive cells at E18.5 and had significant differences in craniofacial form (size and shape) at postnatal timepoints. Centroid sizes for the mandibular and cranio-maxillary regions were significantly smaller in CSF1R-inhibited animals. Proportionally, these animals had a domed skull, with taller and wider cranial vaults and shortening of their midfacial regions. Mandibles were smaller vertically and anterio-posteriorly, with proportionally wider inter-condylar distances. CONCLUSIONS: Embryonic inhibition of CSF1R impacts postnatal craniofacial morphogenesis, with significant influences on the mandibular and cranioskeletal size and shape. These data indicate that CSF1R plays a role in early cranio-skeletal patterning, likely through osteoclast depletion.
Assuntos
Fator Estimulador de Colônias de Macrófagos , Crânio , Animais , Feminino , Camundongos , Gravidez , Fator Estimulador de Colônias de Macrófagos/farmacologia , Mandíbula/metabolismo , Morfogênese , Crânio/diagnóstico por imagem , Crânio/metabolismo , Microtomografia por Raio-XRESUMO
Osteoclasts are multinucleated bone resorbing cells that can be differentiated from human monocytes in vitro. There are few studies comparing osteoclastogenesis of different monocyte sources. We compared monocytes from human bone marrow (BM), peripheral blood (PB), and umbilical cord blood (CB) and their osteoclastogenic potential by culturing them with RANKL (20 and 80 ng/ml) and M-CSF (10 ng/ml) for 14 days. We also cultured cells without growth factors, as umbilical cord blood monocytes have been reported to be able to fuse spontaneously into osteoclasts. The data was analysed on d4, d8, d11, and d14. After culture with RANKL and M-CSF, all types of cell cultures developed TRACP -positive multinuclear cells that were able to form resorption pits on human bone slices. Only occasional multinuclear cells and small infrequent resorbed areas could be found in PB and CB-derived cultures without growth factors. BM-derived cells formed greater resorption areas than PB- and CB-derived monocytes. The greatest monocyte population in BM samples were intermediate (CD14++CD16+) and in PB and CB classical monocytes (76.3% and 54.4%, respectively). In conclusion, our data demonstrates that bone resorbing osteoclasts can be differentiated from BM, PB and CB. However, the osteoclast precursor origin can affect the osteoclast properties and function.
Assuntos
Distrofias de Cones e Bastonetes , Monócitos , Humanos , Medula Óssea , Sangue Fetal , Fator Estimulador de Colônias de Macrófagos/farmacologia , OsteogêneseRESUMO
Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14- mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.
Assuntos
Reabsorção Óssea , Osteoclastos , Humanos , Actinas , Diferenciação Celular/fisiologia , Células Cultivadas , Leucócitos Mononucleares , Fator Estimulador de Colônias de Macrófagos/farmacologia , Monócitos , Ligante RANKRESUMO
Disrupted osteoblastogenesis or aberrant activation of osteoclastogenesis usually results in the break of bone homeostasis thus causing bone-associated diseases like osteoporosis. Obacunone, as a natural compound present in citrus fruits, has been demonstrated for various biological activities including anti-cancer and anti-inflammatory properties. However, the role of obacunone in regulating osteoclastogenesis has not been elucidated so far. Here, using in vitro cell models of RANKL (Receptor activator of nuclear factor-kB ligand) and M-CSF (Macrophage-colony-stimulating factor)-induced osteoclastogenesis, we showed that obacunone inhibited osteoclast differentiation in RAW264.7 cells and bone marrow macrophages (BMMs), as evidenced by obacunone dose-dependent reduction in numbers of osteoclasts and downregulated expressions of osteoclastogenesis-associated key genes. The anti-osteoclastic properties of obacunone were associated with downregulated expressions of Integrin α1 and attenuated activation of Focal adhesion kinase (FAK) and Steroid receptor coactivator (Src) signaling. Functional Integrin α1 blockade or FAK-Src inhibition suppressed RANKL/M-CSF-induced osteoclastogenesis, while Integrin α1 overexpression or FAK/Src activation partially attenuated obacunone's effects on suppressing RANKL/M-CSF-induced osteoclast differentiation. Furthermore, in vivo administration of obacunone displayed super therapeutic effects in attenuating ovariectomy-induced bone loss in mice, as indicated by decreases in serum biomarkers of bone turnover, restoring of femur fracture maximum force, and reversing of the worsened bone-related parameters in ovariectomized animals. Taken together, these findings demonstrate that obacunone has pharmacological activities to suppress osteoclast differentiation through modulating the Integrin-FAK-Src pathway, and suggest that obacunone is a therapeutic candidate for the treatment and prevention of bone diseases such as osteoporosis.
Assuntos
Reabsorção Óssea , Osteoporose , Receptores de Esteroides , Feminino , Camundongos , Animais , Osteogênese , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Integrina alfa1/metabolismo , Diferenciação Celular , Osteoclastos/metabolismo , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Receptores de Esteroides/metabolismo , Ligante RANK/metabolismoRESUMO
Colony-stimulating factor-1 receptor (CSF-1R), also known as FMS kinase, is a type I single transmembrane protein mainly expressed in myeloid cells, such as monocytes, macrophages, glial cells, and osteoclasts. The endogenous ligands, colony-stimulating factor-1 (CSF-1) and Interleukin-34 (IL-34), activate CSF-1R and downstream signaling pathways including PI3K-AKT, JAK-STATs, and MAPKs, and modulate the proliferation, differentiation, migration, and activation of target immune cells. Over the past decades, the promising therapeutic potential of CSF-1R signaling inhibition has been widely studied for decreasing immune suppression and escape in tumors, owing to depletion and reprogramming of tumor-associated macrophages. In addition, the excessive activation of CSF-1R in inflammatory diseases is consecutively uncovered in recent years, which may result in inflammation in bone, kidney, lung, liver and central nervous system. Agents against CSF-1R signaling have been increasingly investigated in preclinical or clinical studies for inflammatory diseases treatment. However, the pathological mechanism of CSF-1R in inflammation is indistinct and whether CSF-1R signaling can be identified as biomarkers remains controversial. With the background information aforementioned, this review focus on the dialectical roles of CSF-1R and its ligands in regulating innate immune cells and highlights various therapeutic implications of blocking CSF-1R signaling in inflammatory diseases.
Assuntos
Inflamação , Terapia de Alvo Molecular , Fosfatidilinositol 3-Quinases , Receptor de Fator Estimulador de Colônias de Macrófagos , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Ligantes , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos , Fosfatidilinositol 3-Quinases/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/farmacologiaRESUMO
Although pituitary neuroendocrine tumors (PitNETs) are usually benign, some are highly invasive and recurrent. Recurrent PitNETs are often treatment-resistant and there is currently no effective evidence-based treatment. Tumor-associated macrophages (TAMs) promote tumor growth in many cancers, but the effect of TAMs on PitNETs remains unclear. This study investigated the role of TAMs in the incidence of recurrent PitNETs. Immunohistochemical analysis revealed that the densities of CD163- and CD204-positive TAMs tended to increase in recurrent PitNETs. Compared with TAMs in primary lesions, those in recurrent lesions were enlarged. To clarify the cell-cell interactions between TAMs and PitNETs, in vitro experiments were performed using a mouse PitNET cell line AtT20 and the mouse macrophage cell line J774. Several cytokines related to macrophage chemotaxis and differentiation, such as M-CSF, were elevated significantly by stimulation with macrophage conditioned medium. When M-CSF immunohistochemistry analysis was performed using human PitNET samples, M-CSF expression increased significantly in recurrent lesions compared with primary lesions. Although no M-CSF receptor (M-CSFR) expression was observed in tumor cells of primary and recurrent PitNETs, flow cytometric analysis revealed that the mouse PitNET cell line expressed M-CSFR. Cellular proliferation in mouse PitNETs was inhibited by high concentrations of M-CSFR inhibitors, suggesting that cell-to-cell communication between PitNETs and macrophages induces M-CSF expression, which in turn enhances TAM chemotaxis and maturation in the tumor microenvironment. Blocking the M-CSFR signaling pathway might be a novel therapeutic adjuvant in treating recurrent PitNETs.