scholarly journals The Recombinant Protein EphB4-Fc Changes the Ti Particle-Mediated Imbalance of OPG/RANKL via EphrinB2/EphB4 Signaling Pathway and Inhibits the Release of Proinflammatory Factors In Vivo

2020 ◽  
Vol 2020 ◽  
pp. 1-15
Author(s):  
Yu-Wei Ge ◽  
Kai Feng ◽  
Xiao-Liang Liu ◽  
Hong-Fang Chen ◽  
Zhen-Yu Sun ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Inflammatory Response ◽  
Signaling Pathway ◽  
Osteoclast Differentiation ◽  
Wear Particle ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tartrate Resistant Acid Phosphatase ◽  
Wear Particles ◽  
Proinflammatory Factors

Aseptic loosening caused by wear particles is one of the common complications after total hip arthroplasty. We investigated the effect of the recombinant protein ephB4-Fc (erythropoietin-producing human hepatocellular receptor 4) on wear particle-mediated inflammatory response. In vitro, ephrinB2 expression was analyzed using siRNA-NFATc1 (nuclear factor of activated T-cells 1) and siRNA-c-Fos. Additionally, we used Tartrate-resistant acid phosphatase (TRAP) staining, bone pit resorption, Enzyme-linked immunosorbent assay (ELISA), as well as ephrinB2 overexpression and knockdown experiments to verify the effect of ephB4-Fc on osteoclast differentiation and function. In vivo, a mouse skull model was constructed to test whether the ephB4-Fc inhibits osteolysis and inhibits inflammation by micro-CT, H&E staining, immunohistochemistry, and immunofluorescence. The gene expression of ephrinB2 was regulated by c-Fos/NFATc1. Titanium wear particles activated this signaling pathway to the promoted expression of the ephrinB2 gene. However, ephrinB2 protein can be activated by osteoblast membrane receptor ephB4 to inhibit osteoclast differentiation. In in vivo experiments, we found that ephB4 could regulate Ti particle-mediated imbalance of OPG/RANKL, and the most important finding was that ephB4 relieved the release of proinflammatory factors. The ephB4-Fc inhibits wear particle-mediated osteolysis and inflammatory response through the ephrinB2/EphB4 bidirectional signaling pathway, and ephrinB2 ligand is expected to become a new clinical drug therapeutic target.

scholarly journals The Role of TLR and Chemokine in Wear Particle-Induced Aseptic Loosening

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Qiaoli Gu ◽  
Qin Shi ◽  
Huilin Yang
Keyword(s):  
Inflammatory Response ◽  
Aseptic Loosening ◽  
Osteoclast Differentiation ◽  
Adaptor Protein ◽  
Wear Particle ◽  
Cellular Migration ◽  
Cellular Interaction ◽  
Wear Particles ◽  
Orthopaedic Implants ◽  
Inhibitory Protein

Wear particle-induced periprosthetic osteolysis remains the principal cause of aseptic loosening of orthopaedic implants. Monocytes/macrophages phagocytose wear particles and release cytokines that induce inflammatory response. This response promotes osteoclast differentiation and osteolysis. The precise mechanisms by which wear particles are recognized and induce the accumulation of inflammatory cells in the periprosthetic tissue have not been fully elucidated. Recent studies have shown that toll-like receptors (TLRs) contribute to the cellular interaction with wear particles. Wear particles are recognized by monocytes/macrophages through TLRs coupled with the adaptor protein MyD88. After the initial interaction, wear particles induce both local and systemic migration of monocytes/macrophages to the periprosthetic region. The cellular migration is mediated through chemokines including interleukin-8, macrophage chemotactic protein-1, and macrophage inhibitory protein-1 in the periprosthetic tissues. Interfering with chemokine-receptor axis can inhibit cellular migration and inflammatory response. This paper highlights recent advances in TLR, and chemokine participated in the pathogenesis of aseptic loosening. A comprehensive understanding of the recognition and migration mechanism is critical to the development of measures that prevent wear particle-induced aseptic loosening of orthopaedic implants.

scholarly journals Inhibition of RANKL-Induced Osteoclastogenesis by Novel Mutant RANKL

2021 ◽  
Vol 22 (1) ◽  
pp. 434
Author(s):  
Yuria Jang ◽  
Hong Moon Sohn ◽  
Young Jong Ko ◽  
Hoon Hyun ◽  
Wonbong Lim
Keyword(s):  
Nuclear Translocation ◽  
Critical Role ◽  
Osteoclast Differentiation ◽  
Point Mutations ◽  
Tartrate Resistant Acid Phosphatase ◽  
Mice Model ◽  
Gsk 3Β ◽  
Rank Signaling

Background: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK–RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. Results: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3β phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.

scholarly journals Role of Synovial Exosomes in Osteoclast Differentiation in Inflammatory Arthritis

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 120
Author(s):  
Ji Eun Song ◽  
Ji Soo Kim ◽  
Ji Hye Shin ◽  
Ki Won Moon ◽  
Jin Kyun Park ◽  
...  
Keyword(s):  
Synovial Fluid ◽  
Inflammatory Arthritis ◽  
Mononuclear Cells ◽  
Osteoclast Differentiation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tartrate Resistant Acid Phosphatase ◽  
Healthy Donors ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

This study aimed to investigate the characteristics of exosomes isolated from synovial fluid and their role in osteoclast differentiation in different types of inflammatory arthritis. Exosomes isolated from synovial fluid of rheumatoid arthritis (RA), ankylosing spondylitis (AS), gout, and osteoarthritis (OA) patients were co-incubated with CD14+ mononuclear cells from healthy donors without macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Osteoclast differentiation was evaluated via tartrate-resistant acid phosphatase (TRAP) staining and activity and F-actin ring formation. RANKL expression on synovial exosomes was assessed using flow cytometry and an enzyme-linked immunosorbent assay (ELISA). Synovial exosomes were the lowest in OA patients; these induced osteoclastogenesis in the absence of M-CSF and RANKL. Osteoclastogenesis was significantly higher with more exosomes in RA (p = 0.030) than in OA patients, but not in AS or gout patients. On treating macrophages with a specified number of synovial exosomes from RA/AS patients, exosomes induced greater osteoclastogenesis in RA than in AS patients. Synovial exosomal RANKL levels were significantly higher in RA (p = 0.035) than in AS patients. Synovial exosome numbers vary with the type of inflammatory arthritis. Synovial exosomes from RA patients may bear the disease-specific “synovial signature of osteoclastogenesis.”

Cells in regenerating deer antler cartilage provide a microenvironment that supports osteoclast differentiation

2001 ◽  
Vol 204 (3) ◽  
pp. 443-455
Author(s):  
C. Faucheux ◽  
S. Nesbitt ◽  
M. Horton ◽  
J. Price
Keyword(s):  
Type I Collagen ◽  
Mononuclear Cells ◽  
Osteoclast Differentiation ◽  
Cathepsin K ◽  
Collagen Matrix ◽  
Tartrate Resistant Acid Phosphatase ◽  
Type I ◽  
Deer Antler

Deer antlers are a rare example of mammalian epimorphic regeneration. Each year, the antlers re-grow by a modified endochondral ossification process that involves extensive remodelling of cartilage by osteoclasts. This study identified regenerating antler cartilage as a site of osteoclastogenesis in vivo. An in vitro model was then developed to study antler osteoclast differentiation. Cultured as a high-density micromass, cells from non-mineralised cartilage supported the differentiation of large numbers of osteoclast-like multinucleated cells (MNCs) in the absence of factors normally required for osteoclastogenesis. After 48 h of culture, tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells (osteoclast precursors) were visible, and by day 14 a large number of TRAP-positive MNCs had formed (783+/−200 per well, mean +/− s.e.m., N=4). Reverse transcriptase/polymerase chain reaction (RT-PCR) showed that receptor activator of NF κ B ligand (RANKL) and macrophage colony stimulating factor (M-CSF) mRNAs were expressed in micromass cultures. Antler MNCs have the phenotype of osteoclasts from mammalian bone; they expressed TRAP, vitronectin and calcitonin receptors and, when cultured on dentine, formed F-actin rings and large resorption pits. When cultured on glass, antler MNCs appeared to digest the matrix of the micromass and endocytose type I collagen. Matrix metalloproteinase-9 (MMP-9) may play a role in the resorption of this non-mineralised matrix since it is highly expressed in 100 % of MNCs. In contrast, cathepsin K, another enzyme expressed in osteoclasts from bone, is only highly expressed in resorbing MNCs cultured on dentine. This study identifies the deer antler as a valuable model that can be used to study the differentiation and function of osteoclasts in adult regenerating mineralised tissues.

scholarly journals A simple method of suspending implant wear particles for more physiological modelling of cell-particle interactions in vitro

2021 ◽  
Author(s):  
Christine Poon
Keyword(s):  
Wear Debris ◽  
Culture Media ◽  
Polymeric Materials ◽  
Wear Particle ◽  
Physicochemical Characteristics ◽  
Wear Particles ◽  
Simple Method ◽  
Implant Wear

AbstractArthroplasty implants e.g. hip, knee, spinal disc sustain relatively high compressive loading and friction wear, which lead to the formation of wear particles or debris between articulating surfaces. Despite advances in orthopaedic materials and surface treatments, the production of wear debris from any part of a joint arthroplasty implant is currently unavoidable. Implant wear debris induces host immune responses and inflammation, which causes patient pain and ultimately implant failure through progressive inflammation-mediated osteolysis and implant loosening, where the severity and rate of periprosthetic osteolysis depends on the material and physicochemical characteristics of the wear particles. Evaluating the cytotoxicity of implant wear particles is important for regulatory approved clinical application of arthroplasty implants, as is the study of cell-particle response pathways. However, the wear particles of polymeric materials commonly used for arthroplasty implants tend to float when placed in culture media, which limits their contact with cell cultures. This study reports a simple means of suspending wear particles in liquid medium using sodium carboxymethyl cellulose (NaCMC) to provide a more realistic proxy of the interaction between cells and tissues to wear particles in vivo, which are free-floating in synovial fluid within the joint cavity. Low concentrations of NaCMC dissolved in culture medium were found to be effective for suspending polymeric wear particles. Such suspensions may be used as more physiologically-relevant means for testing cellular responses to implant wear debris, as well as studying the combinative effects of shear and wear particle abrasion on cells in a dynamic culture environments such as perfused tissue-on-chip devices.

scholarly journals Integrated Transcriptional Profiling Analysis and Immune-Related Risk Model Construction for Intracranial Aneurysm Rupture

2021 ◽  
Vol 15 ◽  
Author(s):  
Dezhi Shan ◽  
Xing Guo ◽  
Guozheng Yang ◽  
Zheng He ◽  
Rongrong Zhao ◽  
...  
Keyword(s):  
Immune Response ◽  
Inflammatory Response ◽  
Signaling Pathway ◽  
Immune Cell ◽  
Risk Model ◽  
Enrichment Analysis ◽  
Cell Infiltration ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immune Cell Infiltration ◽  
Related Risk

Intracranial aneurysms (IAs) may cause lethal subarachnoid hemorrhage upon rupture, but the molecular mechanisms are poorly understood. The aims of this study were to analyze the transcriptional profiles to explore the functions and regulatory networks of differentially expressed genes (DEGs) in IA rupture by bioinformatics methods and to identify the underlying mechanisms. In this study, 1,471 DEGs were obtained, of which 619 were upregulated and 852 were downregulated. Gene enrichment analysis showed that the DEGs were mainly enriched in the inflammatory response, immune response, neutrophil chemotaxis, and macrophage differentiation. Related pathways include the regulation of actin cytoskeleton, leukocyte transendothelial migration, nuclear factor κB signaling pathway, Toll-like receptor signaling pathway, tumor necrosis factor signaling pathway, and chemokine signaling pathway. The enrichment analysis of 20 hub genes, subnetworks, and significant enrichment modules of weighted gene coexpression network analysis showed that the inflammatory response and immune response had a causal relationship with the rupture of unruptured IAs (UIAs). Next, the CIBERSORT method was used to analyze immune cell infiltration into ruptured IAs (RIAs) and UIAs. Macrophage infiltration into RIAs increased significantly compared with that into UIAs. The result of principal component analysis revealed that there was a difference between RIAs and UIAs in immune cell infiltration. A 4-gene immune-related risk model for IA rupture (IRMIR), containing CXCR4, CXCL3, CX3CL1, and CXCL16, was established using the glmnet package in R software. The receiver operating characteristic value revealed that the model represented an excellent clinical situation for potential application. Enzyme-linked immunosorbent assay was performed and showed that the concentrations of CXCR4 and CXCL3 in serum from RIA patients were significantly higher than those in serum from UIA patients. Finally, a competing endogenous RNA network was constructed to provide a potential explanation for the mechanism of immune cell infiltration into IAs. Our findings highlighted the importance of immune cell infiltration into RIAs, providing a direction for further research.

scholarly journals Promotion of osteoclastogenesis by IL-26 in rheumatoid arthritis

2019 ◽  
Vol 21 (1) ◽  
Author(s):  
Kyung-Ann Lee ◽  
Kyoung-Woon Kim ◽  
Bo-Mi Kim ◽  
Ji-Yeon Won ◽  
Hong Ki Min ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Peripheral Blood ◽  
Osteoclast Differentiation ◽  
Joint Damage ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tartrate Resistant Acid Phosphatase ◽  
Dependent Manner ◽  
Receptor Subunit ◽  
Blood Monocytes ◽  
Peripheral Blood Monocytes

Abstract Background The inflammatory cascade in the rheumatoid arthritis (RA) synovium is modulated by a variety of cytokine and chemokine networks; however, the roles of IL-26, in RA pathogenesis, are poorly defined. Here, we investigated the functional role of interleukin-26 (IL)-26 in osteoclastogenesis in RA. Methods We analyzed levels of IL-20 receptor subunit A (IL-20RA), CD55, and receptor activator of nuclear factor kappaB (NF-κB) ligand (RANKL) in RA fibroblast-like synoviocytes (FLSs) using confocal microscopy. Recombinant human IL-26-induced RANKL expression in RA-FLSs was examined using real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Human peripheral blood monocytes were cultured with macrophage colony-stimulating factor (M-CSF) and IL-26, after which osteoclastogenesis was evaluated by counting the number of tartrate-resistant acid phosphatase-positive multinucleated cells. Additionally, osteoclastogenesis was evaluated by monocytes co-cultured with IL-26-prestimulated FLSs. Results The expression of IL-20RA in RA-FLSs was higher than that in osteoarthritis-FLSs. Additionally, in IL-26-pretreated RA-FLSs, the expression of IL-20RA (but not IL-10 receptor subunit B) and RANKL increased in a dose-dependent manner, with IL-26-induced RANKL expression reduced by IL-20RA knockdown. Moreover, IL-26-induced RANKL expression was significantly downregulated by inhibition of signal transducer and activator of transcription 1, mitogen-activated protein kinase, and NF-κB signaling. Furthermore, IL-26 promoted osteoclast differentiation from peripheral blood monocytes in the presence of low dose of RANKL, with IL-26 exerting an additive effect. Furthermore, co-culture of IL-26-pretreated RA-FLSs with peripheral blood monocytes also increased osteoclast differentiation in the absence of addition of RANKL. Conclusions IL-26 regulated osteoclastogenesis in RA through increased RANKL expression in FLSs and direct stimulation of osteoclast differentiation. These results suggest the IL-26/IL-20RA/RANKL axis as a potential therapeutic target for addressing RA-related joint damage.

Glia Maturation Factor-Gamma Negatively Modulates TLR4 Signaling In Macrophages Induced by Lipopolysaccharide (LPS).

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1482-1482
Author(s):  
Wulin Aerbajinai ◽  
Kyung Chin ◽  
Hyun Woo Lee ◽  
Jianqiong Zhu ◽  
Griffin P. Rodgers
Keyword(s):  
Immune Response ◽  
Inflammatory Response ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Luciferase Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Glia Maturation Factor ◽  
Tlr4 Signaling ◽  
Maturation Factor ◽  
Nf Kappab

Abstract Abstract 1482 Toll-like receptor 4 (TLR4) plays a critical role in innate immunity that recognize pathogenic molecules and trigger inflammatory response. However, excessive activation of TLR4 activation may contribute to pathogenesis of autoimmune and inflammatory diseases. Therefore, the negative regulation of TLR4-triggered inflammatory response attracts much attention in recent years. Activation of TLR4 signaling pathways by lipopolysaccharide (LPS) leads to the production of a broad array of cytokines and mediators that coordinate the immune response in macrophages. Glia maturation factor gamma (GMFG), a member of the ADF/cofilin family of proteins that regulate actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in macrophages immune response remains unclear. In this study, we investigated whether GMFG participates in the molecular events underlying the inflammatory reaction to LPS in macrophages by knockdown of GMFG using small-interfering RNA approach. We show here that knockdown of GMFG significantly enhanced LPS-induced production of proinflammatory cytokines and chemokines, including TNF-alpha, IL-1beta, IL-8, and MCP-1 in human peripheral blood monocytes-derived macrophage as determined by quantitative real time-PCR and confirmed by enzyme-linked immunosorbent assay. Silencing of GMFG expression potentiates LPS-induced activation of p38, ERK1/2 and NF-kappaB signaling pathways by Western blot analysis. Moreover, luciferase assay revealed that gene silencing of GMFG promoted LPS-induced NF-kappaB activity for ∼2.5- to 4-fold. Furthermore, we found that TLR4 protein expression level were higher in GMFG-silenced macrophage compared with that of the control siRNA-transfected macrophages after stimulated with LPS for 1 hour. These results suggest that GMFG negatively regulation of TLR4 signaling-induced inflammatory cytokines by modulation of TLR4 expression levels and its down-stream NF-kappaB and p38 MAPK signaling pathway. In summary, we report that GMFG, in macrophage, function as a novel negative regulator that participates in the regulation of TLR4-signaling pathway, implicating that macrophage-specific modulation of GMFG may be beneficial in the treatment of inflammation as well as autoimmune disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effect of Shikonin on Spinal Cord Injury in Rats Via Regulation of HMGB1/TLR4/NF-kB Signaling Pathway

2017 ◽  
Vol 43 (2) ◽  
pp. 481-491 ◽  
Author(s):  
Yihui Bi ◽  
Yapeng Zhu ◽  
Mingkai Zhang ◽  
Keke Zhang ◽  
Xingyi Hua ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Inflammatory Response ◽  
Signaling Pathway ◽  
Motor Function ◽  
Tissue Repair ◽  
Underlying Mechanism ◽  
Molecular Signaling ◽  
Cord Injury

Background/Aims: Shikonin, a compound extracted from Zicao, has been demonstrated to hold anti-bacterial, anti-inflammatory, and anti-tumor activities in various diseases and it has been shown to protect human organs from injuries. However, the effect of shikonin on the recovery of spinal cord injury (SCI) remains unknown. This study was designed to estimate the potential therapeutic effect and underlying mechanism of shikonin on SCI in vivo. Methods: In the study, we used HE staining, ELISA assay, transfection assay, TUNEL assay, real time PCR and Western blot to detect the effects of shikonin on spinal cord injury in rats. Results: we showed that shikonin could promote the recovery of motor function and tissue repair after SCI treatment in rats SCI model. Moreover, we demonstrated that shikonin inhibited the spinal cord edema in SCI model of rats. According to further investigation, shikonin induced the reduction of inflammatory response through decreasing the expression levels of HMGB1, TLR4 and NF-κB after SCI injury. In addition, we also found that shikonin could suppress the apoptosis and expression of caspase-3 protein in SCI model of rats. Conclusion: Our results demonstrated that shikonin induced the recovery of tissue repair and motor function via inactivation of HMGB1/TLR4/NF-κB signaling pathway in SCI model of rats. Meanwhile, shikonin regulated the inflammation response in SCI by suppressing the HMGB1/TLR4/NF-κB signaling pathway. The described mechanism sheds novel light on molecular signaling pathway in spinal cord injury and secondary injury including inflammatory response.

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