Targeting 3D chromosomal architecture at the RANK loci to suppress myeloma-driven osteoclastogenesis.

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.

S100A8 & S100A9: Alarmin mediated inflammation in tendinopathy.

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.

Alarmins in Frozen Shoulder: A Molecular Association Between Inflammation and Pain.

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.

Targeting danger molecules in tendinopathy: the HMGB1/TLR4 axis.

OBJECTIVES: To seek evidence of the danger molecule, high-mobility group protein B1 (HMGB1) expression in human tendinopathy and thereafter, to explore mechanisms where HMGB1 may regulate inflammatory mediators and matrix regulation in human tendinopathy. METHODS: Torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing 'early pathology') biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from patients undergoing arthroscopic stabilisation surgery. Markers of inflammation and HMGB1 were quantified by reverse transcriptase PCR (RT-PCR) and immunohistochemistry. Human tendon-derived primary cells were derived from hamstring tendon tissue obtained during hamstring tendon anterior cruciate ligament reconstruction and used through passage 3. In vitro effects of recombinant HMGB1 on tenocyte matrix and inflammatory potential were measured using quantitative RT-PCR, ELISA and immunohistochemistry staining. RESULTS: Tendinopathic tissues demonstrated significantly increased levels of the danger molecule HMGB1 compared with control tissues with early tendinopathy tissue showing the greatest expression. The addition of recombinant human HMGB1 to tenocytes led to significant increase in expression of a number of inflammatory mediators, including interleukin 1 beta (IL-1ß), IL-6, IL-33, CCL2 and CXCL12, in vitro. Further analysis demonstrated rhHMGB1 treatment resulted in increased expression of genes involved in matrix remodelling. Significant increases were observed in Col3, Tenascin-C and Decorin. Moreover, blocking HMGB1 signalling via toll-like receptor 4 (TLR4) silencing reversed these key inflammatory and matrix changes. CONCLUSION: HMGB1 is present in human tendinopathy and can regulate inflammatory cytokines and matrix changes. We propose HMGB1 as a mediator driving the inflammatory/matrix crosstalk and manipulation of the HMGB1/TLR4 axis may offer novel therapeutic approaches targeting inflammatory mechanisms in the management of human tendon disorders.

MicroRNA29a Treatment Improves Early Tendon Injury.

Tendon injuries (tendinopathies) are common in human and equine athletes and characterized by dysregulated collagen matrix, resulting in tendon damage. We have previously demonstrated a functional role for microRNA29a (miR29a) as a post-transcriptional regulator of collagen 3 expression in murine and human tendon injury. Given the translational potential, we designed a randomized, blinded trial to evaluate the potential of a miR29a replacement therapy as a therapeutic option to treat tendinopathy in an equine model that closely mimics human disease. Tendon injury was induced in the superficial digital flexor tendon (SDFT) of 17 horses. Tendon lesions were treated 1 week later with an intralesional injection of miR29a or placebo. miR29a treatment reduced collagen 3 transcript levels at week 2, with no significant changes in collagen 1. The relative lesion cross-sectional area was significantly lower in miR29a tendons compared to control tendons. Histology scores were significantly better for miR29a-treated tendons compared to control tendons. These data support the mechanism of microRNA-mediated modulation of early pathophysiologic events that facilitate tissue remodeling in the tendon after injury and provides a strong proof of principle that a locally delivered miR29a therapy improves early tendon healing.

Brief Report: Proatherogenic Cytokine Microenvironment in the Aortic Adventitia of Patients With Rheumatoid Arthritis.

OBJECTIVE: Patients with rheumatoid arthritis (RA) are at increased risk of developing cardiovascular disease (CVD) via mechanisms that have not yet been defined. Inflammatory pathways, in particular within the vascular adventitia, are implicated in the pathogenesis of primary CVD but could be amplified in RA at the local tissue level. The aim of this study was to examine the aortic adventitia of coronary artery disease (CAD) patients with or without RA to determine the cytokine profile contained therein. METHODS: Aortic adventitia and internal thoracic artery biopsy specimens obtained from 19 RA patients and 20 non-RA patients undergoing coronary artery bypass graft surgery were examined by immunohistochemistry. RESULTS: Interleukin-18 (IL-18), IL-33, and tumor necrosis factor (TNF) were expressed in aortic adventitia biopsy specimens from both groups, and expression of these cytokines was significantly higher in RA patients. In RA patients, IL-33 expression in endothelial cells correlated positively with the number of swollen joints, suggesting a link between the systemic disease state and the local vascular tissue microlesion. CONCLUSION: The presence of the proinflammatory cytokines IL-18, IL-33, and TNF may play a role in the inflammatory process within the adventitia that contributes to plaque formation and destabilization. In theory, the amplified expression of these cytokines may contribute to the known increased occurrence and severity of CAD in patients with RA.

Co-opting endogenous immunoglobulin for the regulation of inflammation and osteoclastogenesis in humans and mice.

OBJECTIVE: Cells of the monocytic lineage play fundamental roles in the regulation of health, ranging from the initiation and resolution of inflammation to bone homeostasis. In rheumatoid arthritis (RA), the inflamed synovium exhibits characteristic infiltration of macrophages along with local osteoclast maturation, which, together, drive chronic inflammation and downstream articular destruction. The aim of this study was to explore an entirely novel route of immunoglobulin-mediated regulation, involving simultaneous suppression of the inflammatory and erosive processes in the synovium. METHODS: Using in vivo and in vitro studies of human cells and a murine model of RA, the ability of staphylococcal protein A (SPA) to interact with and modulate cells of the monocytic lineage was tested. In addition, the efficacy of SPA as a therapeutic agent was evaluated in murine collagen-induced arthritis (CIA). RESULTS: SPA showed a capacity to appropriate circulating IgG, by generating small immunoglobulin complexes that interacted with monocytes, macrophages, and preosteoclasts. Formation of these complexes resulted in Fcγ receptor type I-dependent polarization of macrophages to a regulatory phenotype, rendering them unresponsive to activators such as interferon-γ. The antiinflammatory complexes also had the capacity to directly inhibit differentiation of preosteoclasts into osteoclasts in humans. Moreover, administration of SPA in the early stages of disease substantially alleviated the clinical and histologic erosive features of CIA in mice. CONCLUSION: These findings demonstrate the overarching utility of immunoglobulin complexes for the prevention and treatment of inflammatory diseases. The results shed light on the interface between immunoglobulin complex-mediated pathways, osteoclastogenesis, and associated pathologic processes. Thus, therapeutic agents designed to harness all of these properties may be an effective treatment for arthritis, by targeting both the innate inflammatory response and prodestructive pathways.

GPCR production in a novel yeast strain that makes cholesterol-like sterols.

The activities of many mammalian membrane proteins including G-protein coupled receptors are cholesterol-dependent. Unlike higher eukaryotes, yeast do not make cholesterol. Rather they make a related molecule called ergosterol. As cholesterol and ergosterol are biologically non-equivalent, the potential of yeast as hosts for overproducing mammalian membrane proteins has never been fully realised. To address this problem, we are trying to engineer a novel strain of Saccharomyces cerevisiae in which the cholesterol biosynthetic pathway of mammalian cells has been fully reconstituted. Thus far, we have created a modified strain that makes cholesterol-like sterols which has an increased capacity to make G-protein coupled receptors compared to control yeast.

IP(3)R-mediated Ca(2+) release is modulated by anandamide in isolated cardiac nuclei.

Cannabinoids (CBs) are known to alter coronary vascular tone and cardiac performance. They also exhibit cardioprotective properties, particularly in their ability to limit the damage produced by ischaemia reperfusion injury. The mechanisms underlying these effects are unknown. Here we investigate the intracellular localisation of CB receptors in the heart and examine whether they may modulate localised nuclear Ca(2+) release. In isolated cardiac nuclear preparations, expression of both the inositol 1,4,5-trisphosphate receptor type 2 (IP(3)R) and CB receptors (CB(1)R and CB(2)R) was demonstrated by immunoblotting. Both receptors localised to the nucleus and purity of the nuclear preparations was confirmed by co-expression of the nuclear marker protein nucleolin but absence of cytoplasmic actin. To measure effects of IP(3)R and CBR agonists on nuclear Ca(2+) release, isolated nuclei were loaded with Fluo5N-AM. This dye accumulates in the nuclear envelope. Isolated nuclei responded to IP(3) with rapid and transient Ca(2+) release from the nuclear envelope. Anandamide inhibited this IP(3)-mediated release. Preincubation of nuclear preparations with either the CB(1)R antagonist (AM251) or the CB(2)R antagonist (AM630) reversed anandamide-mediated inhibition to 80% and 60% of control values respectively. When nuclei were pre-treated with both CBR antagonists, anandamide-mediated inhibition of IP(3)-induced Ca(2+) release was completely reversed. These results are the first to demonstrate the existence of cardiac nuclear CB receptors. They are also the first to show that anandamide can negatively modulate IP(3)-mediated nuclear Ca(2+) release. As such, this provides evidence for a novel key mechanism underlying the action of CBs and CBRs in the heart.

Expression and alternative processing of IL-18 in human neutrophils.

Interleukin-18 (IL-18), a member of the IL-1 cytokine superfamily, is an important regulator of both innate and acquired immune responses. We demonstrate here constitutive expression of IL-18 by human neutrophils. Unexpectedly, we observed that neutrophils from peripheral blood or rheumatoid synovial compartments contained not only pro and mature IL-18, but also several novel smaller-molecular-weight IL-18-derived species. Using specific protease inhibitors, and serine protease gene-targeted mice, we demonstrate that these IL-18-derived products arose through caspase-independent cleavage events mediated by the serine proteases, elastase and cathepsin G. Moreover, we report that the net effect of elastase treatment of mature recombinant IL-18 was to reduce its IFN-gamma-inducing activity. Thus, human neutrophils contain IL-18 and IL-18-derived molecular species that can arise through novel enzymatic processing pathways. Through cytosolic, membrane or secretory expression of such processing enzymes, together with generation of IL-18 itself, neutrophils likely play a critical role in regulating IL-18 activities during early innate immune responses.

Temporal changes in the populations of immune cells at the site of experimental Dermatophilus congolensis infection in mice and sheep.

Abstract The temporal patterns of dermal immune cell influx were compared in mice and sheep, species reputedly resistant and susceptible, respectively, to infection with Dermatophilus congolensis. In both species, the response involved early mast cell degranulation, vasodilatation and an influx of dendritic cells which accumulated and apparently differentiated beneath the infected epidermis. A concomitant dermal invasion by neutrophils and T and B lymphocytes led to epidermal infiltration, particularly by neutrophils and thence to the formation of the surface scab. Hypertrophy of the epidermis also indicates keratinocyte involvement in the host response. The duration of the response, however, was considerably shorter in the mouse (about 5 days) and B cells' were the predominant lymphocyte under and adjacent to the lesion. During the more protracted response in the sheep (> 21 days), T cells, including T19 antigen +γδ T cells, outnumbered B cells. Résumé- Des modifications dans les populations des cellules immunes dans le derme sont comparées chez la souris et le mouton, espèces respectivement résistantes et sensibles à Dermatophilus congolensis. Dans ces deux espèces, la réponse fait intervenir d'abord la dégranulation des mastocytes, la vasodilatation et l'arrivée de cellules dendritiques qui s'accumulent et se différencient apparemment sous l'épiderme infecté. Une invasion concomittente dermique par des neutrophiles, des lymphocytes T et B est observée et aboutit à l'infiltration épidermique, particulièrement par les neutrophiles et pour cette raison, à la formation de croûtes superficielles. L'hypertrophie de l'épiderme indique aussi l'implication du kératinocyte dans la réponse de l'hôte. Cependant, la durée de cette réponse, est bien plus courte chez la souris (environ 5 jours) et les lymphocytes B sont les lymphocytes prédominants, sous-jacents à la lésion. Chez le mouton, la réponse est prolongée (plus de 21 jours) et les lymphocytes T, incluant les lymphocytes CD 19 et γδ surpassent en nombre les lymphocytes B. [Sasiak, A.B., Lloyd, D.H., McEwan Jenkinson, D., Kitson, S., Elder, H. Y. Temporal changes in the populations of immune cells at the site of experimental Dermatophilus congolensis infection in mice and sheep (Particularités de la population cellulaire immunitaire aux sites d'infections expérimentales à Dermatophilus congolensis chez la souris et le mouton). Veterinary Dermatology 1996; 7: 59-66.] ResumeAn Se compararon los patrones temporales de llegada de células inmunitarias en el ratón y en la oveja, especies consideradas resistentes y susceptibles, respectivamente a la infección por Dermatophilus congolensis. En ambas especies la respuesta implicó degranulación temprana de mastocitos, vasodilatación y llegada de células dendríticas que se acumulaban y aparentemente diferenciaban bajo la epidermis infectada. La invasión dérmica concomitante por netrófilos y linfocitos T y B llevó a una infiltración epidérmica, especialmente por neutrófilos y consecuentemente a la formación de una costra superficial. La hipertrofi de la epidermis también indica la implicación de queratinocitos en la respuesta del huésped. Sin embargo, la duración de la respuesta fue considerablemente más corta en el ratón (unos 5 dias) y los linfocitos B fueron la célula predominante en la zona adyacente a la lesión y debajo de ella. Durante la respuesta más prolongada en la oveja (> 21 días), las células T, incluyendo el antigeno T19, células T γδ, superaban en número las células B. [Sasiak, A.B., Lloyd, D.H., McEwan Jenkinson, D., Kitson, S., Elder, H. Y. Temporal changes in the populations of immune cells at the site of experimental Dermatophilus congolensis infection in mice and sheep (Alteraciones temporales en las poblaciones de celulas inmunitarias en zonas cutaneas de infeccion experimental por Dermatophilus congolensis en raton y en la oveja). Veterinary Dermatology 1996; 7: 59-66.] Zusammenfassung- Die temporären Muster des Influx dermaler Immunzellen wurden bei Maus und Schaf verglichen, Tierarten, die angeblich resistent und empfänglich speziell für die Infektion mit Dermatophilus congolensis sind. Bei beiden Spezies bezog die Reaktion eine frühe Mastzelldegranulation ein, weiterhin Vasodilatation und einen Influx dendritischer Zellen, die sich unter der infizierten Epidermis anhäuften und offensichtlich differenzierten. Eine begleitende Invasion der Dermis durch Neutrophile, T- und B-Lymphozyten führte zu einer epidermalen Infiltration, besonders durch Neutrophile und von dort an zur Bildung von Oberflächenschorf. Die Hypertrophic der Epidermis zeigt außerdem eine Beteiligung der Keratinozyten bei der Antwort des Wirtstieres. Die Dauer der Reaktion jedoch war bei der Maus beträchtlich kürzer (etwa 5 Tage) und die B-Zellen waren die vorherrschenden Lymphozyten unter und angrenzend an die Läsion. Während der mehr protrahierten Reaktion beim Schaf (> 21 Tage) übertrafen die T-Zellen einschließlich T19-Antigen und gamma-delta-T-Zellen die Anzahl der B-Zellen. [Sasiak, A. B., Lloyd, D. H., McEwan Jenkinson, Kitson, S., Elder, H. Y. Temporal chnges in the populations of immune cells at the site of experimental Dermatophilus congolensis infection in mice and sheep (Temporäre Veränderungen in den Populationen von Immunzellen an der Stelle experimenteller Infektionen mit Dermatophilus congolensis bei Maus und Schaf). Veterinary Dermatology 1996; 7: 59-66.].