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Objective: IĸB protein B cell lymphoma 3‐encoded protein (BCL3) is a regulator of the NF‐κB family of transcription factors. NF‐κB signaling fundamentally influences the fate of bone‐forming osteoblasts and bone‐resorbing osteoclasts, but the role of BCL3 in bone biology has not been investigated. The objective of this study was to evaluate BCL3 in skeletal development, maintenance, and osteoarthritic pathology. Methods: To assess the contribution of BCL3 to skeletal homeostasis, neonatal mice (n = 6–14) lacking BCL3 (Bcl3−/−) and wild‐type (WT) controls were characterized for bone phenotype and density. To reveal the contribution to bone phenotype by the osteoblast compartment in Bcl3−/− mice, transcriptomic analysis of early osteogenic differentiation and cellular function (n = 3–7) were assessed. Osteoclast differentiation and function in Bcl3−/− mice (n = 3–5) was assessed. Adult 20‐week Bcl3−/− and WT mice bone phenotype, strength, and turnover were assessed. A destabilization of the medial meniscus model of osteoarthritic osteophytogenesis was used to understand adult bone formation in Bcl3−/− mice (n = 11–13). Results: Evaluation of Bcl3−/− mice revealed congenitally increased bone density, long bone dwarfism, increased bone biomechanical strength, and altered bone turnover. Molecular and cellular characterization of mesenchymal precursors showed that Bcl3−/− cells displayed an accelerated osteogenic transcriptional profile that led to enhanced differentiation into osteoblasts with increased functional activity, which could be reversed with a mimetic peptide. In a model of osteoarthritis‐induced osteophytogenesis, Bcl3−/− mice exhibited decreased pathological osteophyte formation (P < 0.05). Conclusion: Cumulatively, these findings demonstrate that BCL3 controls developmental mineralization to enable appropriate bone formation, whereas in a pathological setting, it contributes to skeletal pathology. [ABSTRACT FROM AUTHOR]

The development and progression of rotator cuff tendinopathy (RCT) is multifactorial and likely to manifest through a combination of extrinsic, intrinsic, and environmental factors, including genetics and epigenetics. However, the role of epigenetics in RCT, including the role of histone modification, is not well established. Using chromatin immunoprecipitation sequencing, differences in the trimethylation status of H3K4 and H3K27 histones in late-stage RCT compared to control were investigated in this study. For H3K4, 24 genomic loci were found to be significantly more trimethylated in RCT compared to control (p < 0.05), implicating genes such as DKK2, JAG2, and SMOC2 in RCT. For H3K27, 31 loci were shown to be more trimethylated (p < 0.05) in RCT compared to control, inferring a role for EPHA3, ROCK1, and DEFβ115. Furthermore, 14 loci were significantly less trimethylated (p < 0.05) in control compared to RCT, implicating EFNA5, GDF6, and GDF7. Finally, the TGFβ signaling, axon guidance, and regulation of focal adhesion assembly pathways were found to be enriched in RCT. These findings suggest that the development and progression of RCT is, at least in part, under epigenetic control, highlighting the influence of histone modifications in this disorder and paving the way to further understand the role of epigenome in RCT. [ABSTRACT FROM AUTHOR]

Bone disease represents a major cause of morbidity and mortality in Multiple Myeloma (MM); primarily driven by osteoclasts whose differentiation is dependent on expression of RANKL by MM cells. Notably, costimulation by ITAM containing receptors (i.e., FcγR) can also play a crucial role in osteoclast differentiation. Modeling the pathology of the bone marrow microenvironment with an ex vivo culture system of primary human multiple myeloma cells, we herein demonstrate that FcγR-mediated signaling, via staphylococcal protein A (SpA) IgG immune-complexes, can act as a critical negative regulator of MM-driven osteoclast differentiation. Interrogation of the mode-of-action revealed that FcγR-mediated signaling causes epigenetic modulation of chromosomal 3D architecture at the RANK promoter; with altered spatial orientation of a proximal super enhancer. Combined this leads to substantial down-regulation of RANK at a transcript, protein, and functional level. These observations shed light on a novel mechanism regulating RANK expression and provide a rationale for targeting FcγR-signaling for the amelioration of osteolytic bone pathology in disease. [ABSTRACT FROM AUTHOR]

Objective: To assess the involvement of the CCR6/CCL20 axis in psoriatic arthritis (PsA) and psoriasis (PsO) and to evaluate its potential as a therapeutic target. Methods: First, we quantified CCL20 levels in peripheral blood and synovial fluid from PsA patients and examined the presence of CCR6+ cells in synovial and tendon tissue. Utilizing an interleukin‐23 minicircle DNA (IL‐23 MC) mouse model exhibiting key features of both PsO and PsA, we investigated CCR6 and CCL20 expression as well as the preventive and therapeutic effect of CCL20 blockade. Healthy tendon stromal cells were stimulated in vitro with IL‐1β to assess the production of CCL20 by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay. The effect of conditioned media from stimulated tenocytes in inducing T cell migration was interrogated using a Transwell system. Results: We observed an up‐regulation of both CCR6 and CCL20 in the enthesis of IL‐23 MC–treated mice, which was confirmed in human biopsy specimens. Specific targeting of the CCR6/CCL20 axis with a CCL20 locked dimer (CCL20LD) blocked entheseal inflammation, leading to profound reductions in clinical and proinflammatory markers in the joints and skin of IL‐23 MC–treated mice. The stromal compartment in the tendon was the main source of CCL20 in this model and, accordingly, in vitro activated human tendon cells were able to produce this chemokine and to induce CCR6+ T cell migration, the latter of which could be blocked by CCL20LD. Conclusion: Our study highlights the pathogenic role of the CCR6/CCL20 axis in enthesitis and introduces the prospect of a novel therapeutic approach for treating patients with PsO and PsA. [ABSTRACT FROM AUTHOR]

Frozen shoulder is a common fibroproliferative disease characterized by the insidious onset of pain and restricted range of shoulder movement with a significant socioeconomic impact. The pathophysiological mechanisms responsible for chronic inflammation and matrix remodeling in this prevalent fibrotic disorder remain unclear; however, increasing evidence implicates dysregulated immunobiology. IL-17A is a key cytokine associated with inflammation and tissue remodeling in numerous musculoskeletal diseases, and thus, we sought to determine the role of IL-17A in the immunopathogenesis of frozen shoulder. We demonstrate an immune cell landscape that switches from a predominantly macrophage population in nondiseased tissue to a T cell-rich environment in disease. Furthermore, we observed a subpopulation of IL-17A-producing T cells capable of inducing profibrotic and inflammatory responses in diseased fibroblasts through enhanced expression of the signaling receptor IL-17RA, rendering diseased cells more sensitive to IL-17A. We further established that the effects of IL-17A on diseased fibroblasts was TRAF-6/NF-κB dependent and could be inhibited by treatment with an IKKμ inhibitor or anti-IL-17A antibody. Accordingly, targeting of the IL-17A pathway may provide future therapeutic approaches to the management of this common, debilitating disease. [ABSTRACT FROM AUTHOR]

Objectives: Increasing evidence suggests that inflammatory mechanisms play a key role in chronic tendon disease. After observing T cell signatures in human tendinopathy, we explored the interaction between T cells and tendon stromal cells or tenocytes to define their functional contribution to tissue remodelling and inflammation amplification and hence disease perpetuation.Methods: T cells were quantified and characterised in healthy and tendinopathic tissues by flow cytometry (FACS), imaging mass cytometry (IMC) and single cell RNA-seq. Tenocyte activation induced by conditioned media from primary damaged tendon or interleukin-1β was evaluated by qPCR. The role of tenocytes in regulating T cell migration was interrogated in a standard transwell membrane system. T cell activation (cell surface markers by FACS and cytokine release by ELISA) and changes in gene expression in tenocytes (qPCR) were assessed in cocultures of T cells and explanted tenocytes.Results: Significant quantitative differences were observed in healthy compared with tendinopathic tissues. IMC showed T cells in close proximity to tenocytes, suggesting tenocyte-T cell interactions. On activation, tenocytes upregulated inflammatory cytokines, chemokines and adhesion molecules implicated in T cell recruitment and activation. Conditioned media from activated tenocytes induced T cell migration and coculture of tenocytes with T cells resulted in reciprocal activation of T cells. In turn, these activated T cells upregulated production of inflammatory mediators in tenocytes, while increasing the pathogenic collagen 3/collagen 1 ratio.Conclusions: Interaction between T cells and tenocytes induces the expression of inflammatory cytokines/chemokines in tenocytes, alters collagen composition favouring collagen 3 and self-amplifies T cell activation via an auto-regulatory feedback loop. Selectively targeting this adaptive/stromal interface may provide novel translational strategies in the management of human tendon disorders. [ABSTRACT FROM AUTHOR]

Background: The use of the vancomycin wrap to pretreat the hamstring graft in anterior cruciate ligament reconstruction (ACLR) has grown in popularity since it was first described in 2012 and has significantly reduced rates of postoperative infection. However, it remains unknown if this antibiotic treatment affects the molecular composition of the graft. Purpose: To establish whether treatment with vancomycin at 5 mg/mL, the most commonly used concentration, alters the molecular function of the hamstring graft in ACLR. Study Design: Controlled laboratory study. Methods: Surplus hamstring tendon collected after routine ACLR surgery was used for in vitro cell culture and ex vivo tissue experiments. Vancomycin was used at 5 mg/mL in RPMI or saline diluent to treat cells and tendon tissue, respectively, with diluent control conditions. Cell viability at 30, 60, and 120 minutes was assessed via colorimetric viability assay. Tendon cells treated with control and experimental conditions for 1 hour was evaluated using semiquantitative reverse transcription analysis, immunohistochemistry staining, and protein quantitation via enzyme-linked immunosorbent assay for changes in apoptotic, matrix, and inflammatory gene and protein expression. Results: Vancomycin treatment at 5 mg/mL significantly reduced tenocyte viability in vitro after 60 minutes of treatment (P <.05); however, this was not sustained at 120 minutes. Vancomycin-treated tendon tissue showed no significant increase in apoptotic gene expression, or apoptotic protein levels in tissue or supernatant, ex vivo. Vancomycin was associated with a reduction in inflammatory proteins from treated tendon supernatants (IL-6; P <.05). Conclusion: Vancomycin did not significantly alter the molecular structure of the hamstring graft. Reductions in matrix protein and inflammatory cytokine release point to a potential beneficial effect of vancomycin in generating a homeostatic environment. Clinical Relevance: Vancomycin ACL wrap does not alter the molecular structure of the ACL hamstring graft and may improve graft integrity. [ABSTRACT FROM AUTHOR]

Introduction: Frozen shoulder is a common, fibro-proliferative disease characterised by the insidious onset of pain and progressively restricted range of shoulder movement. Despite the prevalence of this disease, there is limited understanding of the molecular mechanisms underpinning the pathogenesis of this debilitating disease. Previous studies have identified increased myofibroblast differentiation and proliferation, immune cell influx and dysregulated cytokine production. We hypothesised that subpopulations within the fibroblast compartment may take on an activated phenotype, thus initiating the inflammatory processes observed in frozen shoulder. Therefore, we sought to evaluate the presence and possible pathogenic role of known stromal activation proteins in Frozen shoulder, Methods: Shoulder capsule samples were collected from 10 patients with idiopathic frozen shoulder and 10 patients undergoing shoulder stabilisation surgery. Fibroblast activation marker expression (CD248, CD146, VCAM and PDPN, FAP) was quantified using immunohistochemistry. Control and diseased fibroblasts were cultured for in vitro studies from capsule biopsies from instability and frozen shoulder surgeries, respectively. The inflammatory profile and effects of IL-1β upon diseased and control fibroblasts was assessed using ELISA, immunohistochemistry and qPCR. Results: Immunohistochemistry demonstrated increased expression of fibroblast activation markers CD248, CD146, VCAM and PDPN in the frozen shoulder group compared with control (p < 0.05). Fibroblasts cultured from diseased capsule produced elevated levels of inflammatory protein (IL-6, IL-8 & CCL-20) in comparison to control fibroblasts. Exposing control fibroblasts to an inflammatory stimuli, (IL-1ß) significantly increased stromal activation marker transcript and protein expression (CD248, PDPN and VCAM). Conclusions: These results show that fibroblasts have an activated phenotype in frozen shoulder and this is associated with inflammatory cytokine dysregulation. Furthermore, it supports the hypothesis that activated fibroblasts may be involved in regulating the inflammatory and fibrotic processes involved in this disease. [ABSTRACT FROM AUTHOR]

Background: The pathophysiological mechanisms behind proliferation of fibroblasts and deposition of dense collagen matrix in idiopathic frozen shoulder remain unclear. Alarmins (also known as danger signals) are endogenous molecules that are released into the extracellular milieu after infection or tissue injury and that signal cell and tissue damage. Purpose: To investigate whether the presence of alarmins is higher in patients with idiopathic frozen shoulder than in control subjects. Study Design: Controlled laboratory study. Methods: Shoulder capsule samples were collected from 10 patients with idiopathic frozen shoulder and 10 patients with unstable shoulders (control). The samples were stained with hematoxylin and eosin (H&E) and analyzed by immunohistochemistry using antibodies against alarmin molecules including high-mobility group protein B1 (HMGB1), interleukin 33, S100A8, S100A9, and the peripheral nerve marker PGP9.5. Immunoreactivities were rated in a blinded fashion from “none” to “strong.” Immunohistochemical distribution within the capsule was noted. Before surgery, patient-ranked pain frequency, severity, stiffness, and the range of passive shoulder motion were recorded and statistically analyzed. Results: Compared with control patients, patients with frozen shoulder had greater frequency and severity of self-reported pain (P = .02) and more restricted range of motion in all planes (P < .05). H&E-stained capsular tissue from frozen shoulder showed fibroblastic hypercellularity and increased subsynovial vascularity. Immunoreactivity of alarmins was significantly stronger in frozen shoulder capsules compared with control capsules (P < .05). Furthermore, the expression of the alarmin molecule HMGB1 significantly correlated (r > 0.9, P < .05) with the severity of patient-reported pain. Conclusion: This study demonstrates a potential role for key molecular danger signals in frozen shoulder and suggests an association between the expression of danger molecules and the pain experienced by patients. [ABSTRACT FROM AUTHOR]

Metal-on-metal hip replacement implants generate wear debris and release ions both locally and systemically in patients. To investigate dissemination of metal, we determined blood and organ levels of cobalt (Co), chromium (Cr), and molybdenum (Mo) following the implantation of Co-Cr alloy wear debris in mice using skin pouches as a model system. We observed increased metal levels in blood for up to 72 h; the levels of Co were highest and remained elevated for 7 days. Co levels were elevated in all organs studied (liver, kidney, spleen, lung, heart, brain, and testes), with the peak at 48 h; highest levels were measured in liver and kidney (838.9 ± 223.7 ng/g in liver, and 938.8 ± 131.6 ng/g in kidney). Organ Cr levels were considerably lower than Co levels, for example, Cr in kidney was 117.2 ± 12.6 ng/g tissue at 48 h. Co is more mobile than Cr, reaching higher levels at earlier time points. This could be due to local tissue binding of Cr. Exposure to Co-Cr particles in vivo altered antioxidant enzyme expression and activities. We observed induction of catalase protein in the liver and glutathione reductase (GR) and peroxidase (GPx) proteins in the spleen. Activities of catalase and GPx in the liver were significantly increased while that of GR was decreased in the kidney. Organs of mice with Co-Cr particle implantation were exposed to increased metal levels capable of inducing reactive oxygen species scavenging enzymes, suggesting the tissue may be subjected to oxidative stress; however, the overall antioxidant defence system was not markedly disturbed. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012. [ABSTRACT FROM AUTHOR]

Cobalt-chromium (Co-Cr) alloy metal-on-metal hip resurfacing is increasingly common among younger more active patients suffering from osteoarthritis. Recent reports have increased awareness of metal ions leaching from metallic articulations; this ion exposure may have adverse effects on the immune system. As previous studies reported alterations in lymphocyte number and function in patients with Co-Cr implants, we investigated effects of clinically relevant concentrations of Cr6++ and Co2++ on primary human lymphocytes in vitro. Here, both resting and activated (anti-CD3 ±± anti-CD28 antibodies) primary human lymphocytes were exposed to Cr6++ or Co2++ (0.1--100 µµM). Following 24 or 48 h of exposure, cell viability, proliferation, cytokine [interferon-γγ (IFNγγ and interleukin-2 (IL-2)] release, and apoptosis (with and without pre-treatment of cells with a caspase-3 inhibitor) were assessed. Exposure to 10 and 100 µµM Cr6++ significantly decreased cell viability and increased apoptosis in both resting and activated lymphocytes. Cell proliferation and cytokine release were also significantly reduced in activated lymphocytes following exposure. The exposure of resting lymphocytes to 100 µµM Co2++ resulted in significant decreases in cell viability accompanied by a significant increase in apoptosis. Activated lymphocytes also showed this response after exposure to 100 µµM Co2++; in fact, activated cells were significantly more sensitive to Co2++ toxicity. Exposure to 10 µµM Co2++ led to significant decreases in cell proliferation and cytokine release, but no significant increase in apoptosis, in activated cells. The results indicate that exposure to high concentrations of metal ions initiate apoptosis that results in decreased lymphocyte proliferation. IL-2 release is inhibited by both metal ions at concentrations that are not overtly toxic. However, metal ion concentrations not directly cytotoxic to lymphocytes may affect events at a molecular level, thereby impeding lymphocyte proliferation. Hence, this may contribute to altered immune system function in patients with Co-Cr implants. [ABSTRACT FROM AUTHOR]

Inhibition of IKKβ/NF-κB in stromal cells prevents degeneration and improves healing, representing a potential therapeutic for chronic tendinopathy. Targeting tendinopathy: Inflammation and NF-κB signaling contribute to tendon degeneration and injury, such as rotator cuff injury. Abraham et al. investigated how this signaling pathway causes tendinopathy and potential therapeutic effects of inhibiting the pathway. Clinical tendon samples of human rotator cuff disease showed up-regulation of NF-κB, which was mimicked in mouse tendon fibroblasts overexpressing IKKβ. Genetic deletion of IKKβ in tendon in mice prevented maladaptive tendon remodeling in a treadmill running–induced overuse tendinopathy model and in a surgical model of tendon injury and repair, and human tendon stromal cells treated with an IKKβ inhibitor showed repressed NF-κB target gene transcription. These results suggest that there may be therapeutic potential in blocking IKKβ. Tendon disorders represent the most common musculoskeletal complaint for which patients seek medical attention; inflammation drives tendon degeneration before tearing and impairs healing after repair. Clinical evidence has implicated the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway as a correlate of pain-free return to function after surgical repair. However, it is currently unknown whether this response is a reaction to or a driver of pathology. Therefore, we aimed to understand the clinically relevant involvement of the NF-κB pathway in tendinopathy, to determine its potential causative roles in tendon degeneration, and to test its potential as a therapeutic candidate. Transcriptional profiling of early rotator cuff tendinopathy identified increases in NF-κB signaling, including increased expression of the regulatory serine kinase subunit IKKβ, which plays an essential role in inflammation. Using cre-mediated overexpression of IKKβ in tendon fibroblasts, we observed degeneration of mouse rotator cuff tendons and the adjacent humeral head. These changes were associated with increases in proinflammatory cytokines and innate immune cells within the joint. Conversely, genetic deletion of IKKβ in tendon fibroblasts partially protected mice from chronic overuse–induced tendinopathy. Furthermore, conditional knockout of IKKβ improved outcomes after surgical repair, whereas overexpression impaired tendon healing. Accordingly, targeting of the IKKβ/NF-κB pathway in tendon stromal cells may offer previously unidentified therapeutic approaches in the management of human tendon disorders. [ABSTRACT FROM AUTHOR]

Alarmins S100A8 and S100A9 are endogenous molecules released in response to environmental triggers and cellular damage. They are constitutively expressed in immune cells such as monocytes and neutrophils and their expression is upregulated under inflammatory conditions. The molecular mechanisms that regulate inflammatory pathways in tendinopathy are largely unknown therefore identifying early immune effectors is essential to understanding the pathology. Based on our previous investigations highlighting tendinopathy as an alarmin mediated pathology we sought evidence of S100A8 & A9 expression in a human model of tendinopathy and thereafter, to explore mechanisms whereby S100 proteins may regulate release of inflammatory mediators and matrix synthesis in human tenocytes. Immunohistochemistry and quantitative RT-PCR showed S100A8 & A9 expression was significantly upregulated in tendinopathic tissue compared with control. Furthermore, treating primary human tenocytes with exogenous S100A8 & A9 significantly increased protein release of IL-6, IL-8, CCL2, CCL20 and CXCL10; however, no alterations in genes associated with matrix remodelling were observed at a transcript level. We propose S100A8 & A9 participate in early pathology by modulating the stromal microenvironment and influencing the inflammatory profile observed in tendinopathy. S100A8 and S100A9 may participate in a positive feedback mechanism involving enhanced leukocyte recruitment and release of pro-inflammatory cytokines from tenocytes that perpetuates the inflammatory response within the tendon in the early stages of disease. [ABSTRACT FROM AUTHOR]