The clinical significance of the glucocorticoid receptors: Genetics and epigenetics.

The impacts of glucocorticoids (GCs) are mainly mediated by a nuclear receptor (GR) existing in almost every tissue. The GR regulates a wide range of physiological functions, including inflammation, cell metabolism, and differentiation playing a major role in cellular responses to GCs and stress. Therefore, the dysregulation or disruption of GR can cause deficiencies in the adaptation to stress and the preservation of homeostasis. The number of GR polymorphisms associated with different diseases has been mounting per year. Tackling these clinical complications obliges a comprehensive understanding of the molecular network action of GCs at the level of the GR structure and its signaling pathways. Beyond genetic variation in the GR gene, epigenetic changes can enhance our understanding of causal factors involved in the development of diseases and identifying biomarkers. In this review, we highlight the relationships of GC receptor gene polymorphisms and epigenetics with different diseases.

Diversity of Vascular Niches in Bones and Joints During Homeostasis, Ageing, and Diseases.

The bones and joints in the skeletal system are composed of diverse cell types, including vascular niches, bone cells, connective tissue cells and mineral deposits and regulate whole-body homeostasis. The capacity of maintaining strength and generation of blood lineages lies within the skeletal system. Bone harbours blood and immune cells and their progenitors, and vascular cells provide several immune cell type niches. Blood vessels in bone are phenotypically and functionally diverse, with distinct capillary subtypes exhibiting striking changes with age. The bone vasculature has a special impact on osteogenesis and haematopoiesis, and dysregulation of the vasculature is associated with diverse blood and bone diseases. Ageing is associated with perturbed haematopoiesis, loss of osteogenesis, increased adipogenesis and diminished immune response and immune cell production. Endothelial and perivascular cells impact immune cell production and play a crucial role during inflammation. Here, we discuss normal and maladapted vascular niches in bone during development, homeostasis, ageing and bone diseases such as rheumatoid arthritis and osteoarthritis. Further, we discuss the role of vascular niches during bone malignancy.

The Role of NLRP3 Inflammasome Activities in Bone Diseases and Vascular Calcification.

Continuous stimulation of inflammation is harmful to tissues of an organism. Inflammatory mediators not only have an effect on metabolic and inflammatory bone diseases but also have an adverse effect on certain genetic and periodontal diseases associated with bone destruction. Inflammatory factors promote vascular calcification in various diseases. Vascular calcification is a pathological process similar to bone development, and vascular diseases play an important role in the loss of bone homeostasis. The NLRP3 inflammasome is an essential component of the natural immune system. It can recognize pathogen-related molecular patterns or host-derived dangerous signaling molecules, recruit, and activate the pro-inflammatory protease caspase-1. Activated caspase-1 cleaves the precursors of IL-1ß and IL-18 to produce corresponding mature cytokines or recognizes and cleaves GSDMD to mediate cell pyroptosis. In this review, we discuss the role of NLRP3 inflammasome in bone diseases and vascular calcification caused by sterile or non-sterile inflammation and explore potential treatments to prevent bone loss.

IL-23, but not IL-12, plays a critical role in inflammation-mediated bone disorders.

Interleukin-12 (IL-12) and IL-23 are thought to have central roles in inflammation and are critical to pathologies associated with inflammation-induced bone disorders. The deletion of IL-12p40 (a common subunit of IL-12 and IL-23) can improve bone regeneration. However, the relative roles of IL-12 and IL-23 in bone disorders are largely unknown. Methods: Ectopic bone formation and skull defect models were established to evaluate the relative roles of IL-12 and IL-23 in inflammatory bone disorders. Differences in bone mass among WT, IL-12p35-/-, and IL-12p40-/- mice (young and elderly) were detected by micro-CT. Osteogenic and osteoclastic activities were explored using ELISA, qRT-PCR, and histological analysis. Moreover, the mechanisms by which IL-12 and IL-23 regulated the differentiation of BMMSCs and RAW264.7 cells were explored using Alizarin Red and tartrate-resistant acid phosphatase staining in vitro. Apilimod was used to inhibit IL-12 and IL-23 production in vivo. Results: Mice deficient in IL-12p40 promoted bone formation and protected against aging-related bone loss. By contrast, bone loss was aggravated in IL-12-/- mice, suggesting that IL-23 may play a dominant role in inflammation-related bone disorders. Mechanistically, IL-12 and IL-23 coupled osteogenesis and osteoclastic activities to regulate bone homeostasis and repair. IL-23 deficiency increased bone formation and inhibited bone resorption. Finally, apilimod treatment significantly improved bone regeneration and calvarial defect repair. Conclusion: These data collectively uncover a previously unrecognized role of IL-23 in skeletal tissue engineering. Thus, IL-23 can act as a biomarker to predict diseases and treatment efficacy, and apilimod can be used as an effective therapeutic drug to combat inflammatory bone disorders.

Biochemical and imaging parameters in acid sphingomyelinase deficiency: Potential utility as biomarkers.

Acid Sphingomyelinase Deficiency (ASMD), or Niemann-Pick type A/B disease, is a rare lipid storage disorder leading to accumulation of sphingomyelin and its precursors primarily in macrophages. The disease has a broad phenotypic spectrum ranging from a fatal infantile form with severe neurological involvement (the infantile neurovisceral type) to a primarily visceral form with different degrees of pulmonary, liver, spleen and skeletal involvement (the chronic visceral type). With the upcoming possibility of treatment with enzyme replacement therapy, the need for biomarkers that predict or reflect disease progression has increased. Biomarkers should be validated for their use as surrogate markers of clinically relevant endpoints. In this review, clinically important endpoints as well as biochemical and imaging markers of ASMD are discussed and potential new biomarkers are identified. We suggest as the most promising biomarkers that may function as surrogate endpoints in the future: diffusion capacity measured by spirometry, spleen volume, platelet count, low-density lipoprotein cholesterol, liver fibrosis measured with a fibroscan, lysosphingomyelin and walked distance in six minutes. Currently, no biomarkers have been validated. Several plasma markers of lipid-laden cells, fibrosis or inflammation are of high potential as biomarkers and deserve further study. Based upon current guidelines for biomarkers, recommendations for the validation process are provided.

Steroid hormone-related polymorphisms associate with the development of bone erosions in rheumatoid arthritis and help to predict disease progression: Results from the REPAIR consortium.

Here, we assessed whether 41 SNPs within steroid hormone genes associated with erosive disease. The most relevant finding was the rheumatoid factor (RF)-specific effect of the CYP1B1, CYP2C9, ESR2, FcγR3A, and SHBG SNPs to modulate the risk of bone erosions (P = 0.004, 0.0007, 0.0002, 0.013 and 0.015) that was confirmed through meta-analysis of our data with those from the DREAM registry (P = 0.000081, 0.0022, 0.00074, 0.0067 and 0.0087, respectively). Mechanistically, we also found a gender-specific correlation of the CYP2C9rs1799853T/T genotype with serum vitamin D3 levels (P = 0.00085) and a modest effect on IL1ß levels after stimulation of PBMCs or blood with LPS and PHA (P = 0.0057 and P = 0.0058). An overall haplotype analysis also showed an association of 3 ESR1 haplotypes with a reduced risk of erosive arthritis (P = 0.009, P = 0.002, and P = 0.002). Furthermore, we observed that the ESR2, ESR1 and FcγR3A SNPs influenced the immune response after stimulation of PBMCs or macrophages with LPS or Pam3Cys (P = 0.002, 0.0008, 0.0011 and 1.97•10-7). Finally, we found that a model built with steroid hormone-related SNPs significantly improved the prediction of erosive disease in seropositive patients (PRF+ = 2.46•10-8) whereas no prediction was detected in seronegative patients (PRF- = 0.36). Although the predictive ability of the model was substantially lower in the replication population (PRF+ = 0.014), we could confirm that CYP1B1 and CYP2C9 SNPs help to predict erosive disease in seropositive patients. These results are the first to suggest a RF-specific association of steroid hormone-related polymorphisms with erosive disease.

Protease-Activated Receptor 1 Deletion Causes Enhanced Osteoclastogenesis in Response to Inflammatory Signals through a Notch2-Dependent Mechanism.

We found that protease-activated receptor 1 (PAR1) was transiently induced in cultured osteoclast precursor cells. Therefore, we examined the bone phenotype and response to resorptive stimuli of PAR1-deficient (knockout [KO]) mice. Bones and bone marrow-derived cells from PAR1 KO and wild-type (WT) mice were assessed using microcomputed tomography, histomorphometry, in vitro cultures, and RT-PCR. Osteoclastic responses to TNF-α (TNF) challenge in calvaria were analyzed with and without a specific neutralizing Ab to the Notch2-negative regulatory region (N2-NRR Ab). In vivo under homeostatic conditions, there were minimal differences in bone mass or bone cells between PAR1 KO and WT mice. However, PAR1 KO myeloid cells demonstrated enhanced osteoclastogenesis in response to receptor activator of NF-κB ligand (RANKL) or the combination of RANKL and TNF. Strikingly, in vivo osteoclastogenic responses of PAR1 KO mice to TNF were markedly enhanced. We found that N2-NRR Ab reduced TNF-induced osteoclastogenesis in PAR1 KO mice to WT levels without affecting WT responses. Similarly, in vitro N2-NRR Ab reduced RANKL-induced osteoclastogenesis in PAR1 KO cells to WT levels without altering WT responses. We conclude that PAR1 functions to limit Notch2 signaling in responses to RANKL and TNF and moderates osteoclastogenic response to these cytokines. This effect appears, at least in part, to be cell autonomous because enhanced osteoclastogenesis was seen in highly purified PAR1 KO osteoclast precursor cells. It is likely that this pathway is involved in regulating the response of bone to diseases associated with inflammatory signals.

The RANKL-RANK Axis: A Bone to Thymus Round Trip.

The identification of Receptor activator of nuclear factor kappa B ligand (RANKL) and its cognate receptor Receptor activator of nuclear factor kappa B (RANK) during a search for novel tumor necrosis factor receptor (TNFR) superfamily members has dramatically changed the scenario of bone biology by providing the functional and biochemical proof that RANKL signaling via RANK is the master factor for osteoclastogenesis. In parallel, two independent studies reported the identification of mouse RANKL on activated T cells and of a ligand for osteoprotegerin on a murine bone marrow-derived stromal cell line. After these seminal findings, accumulating data indicated RANKL and RANK not only as essential players for the development and activation of osteoclasts, but also for the correct differentiation of medullary thymic epithelial cells (mTECs) that act as mediators of the central tolerance process by which self-reactive T cells are eliminated while regulatory T cells are generated. In light of the RANKL-RANK multi-task function, an antibody targeting this pathway, denosumab, is now commonly used in the therapy of bone loss diseases including chronic inflammatory bone disorders and osteolytic bone metastases; furthermore, preclinical data support the therapeutic application of denosumab in the framework of a broader spectrum of tumors. Here, we discuss advances in cellular and molecular mechanisms elicited by RANKL-RANK pathway in the bone and thymus, and the extent to which its inhibition or augmentation can be translated in the clinical arena.

Osteoimmunology.

Bone is a crucial element of the skeletal-locomotor system, but also functions as an immunological organ that harbors hematopoietic stem cells (HSCs) and immune progenitor cells. Additionally, the skeletal and immune systems share a number of regulatory molecules, including cytokines and signaling molecules. Osteoimmunology was created as an interdisciplinary field to explore the shared molecules and interactions between the skeletal and immune systems. In particular, the importance of an inseparable link between the two systems has been highlighted by studies on the pathogenesis of rheumatoid arthritis (RA), in which pathogenic helper T cells induce the progressive destruction of multiple joints through aberrant expression of receptor activator of nuclear factor (NF)-κB ligand (RANKL). The conceptual bridge of osteoimmunology provides not only a novel framework for understanding these biological systems but also a molecular basis for the development of therapeutic approaches for diseases of bone and/or the immune system.

Inflammasomes in Bone Diseases.

Unresolved inflammation is harmful to any tissues in the organism. Bone in particular is vulnerable to inflammatory assaults because its integrity depends on the activity of osteoclasts, which arise from myeloid precursors. Osteoclasts are responsible for bone resorption in normal and disease conditions. Increased osteolysis is a common feature of inflammatory disorders and a risk factor for bone fractures. Thus, bone is impacted negatively not only by local and systemic inflammatory mediators, but also directly, by alterations affecting myelopoiesis and lineage allocations. Such perturbations are characteristics of dysregulated inflammasomes, which are key regulators of innate immunity. In this review, we discuss the role of inflammasomes in bone diseases caused by sterile or non-sterile inflammation.

C5aR1 interacts with TLR2 in osteoblasts and stimulates the osteoclast-inducing chemokine CXCL10.

The anaphylatoxin C5a is generated upon activation of the complement system, a crucial arm of innate immunity. C5a mediates proinflammatory actions via the C5a receptor C5aR1 and thereby promotes host defence, but also modulates tissue homeostasis. There is evidence that the C5a/C5aR1 axis is critically involved both in physiological bone turnover and in inflammatory conditions affecting bone, including osteoarthritis, periodontitis, and bone fractures. C5a induces the migration and secretion of proinflammatory cytokines of osteoblasts. However, the underlying mechanisms remain elusive. Therefore, in this study we aimed to determine C5a-mediated downstream signalling in osteoblasts. Using a whole-genome microarray approach, we demonstrate that C5a activates mitogen-activated protein kinases (MAPKs) and regulates the expression of genes involved in pathways related to insulin, transforming growth factor-ß and the activator protein-1 transcription factor. Interestingly, using coimmunoprecipitation, we found an interaction between C5aR1 and Toll-like receptor 2 (TLR2) in osteoblasts. The C5aR1- and TLR2-signalling pathways converge on the activation of p38 MAPK and the generation of C-X-C motif chemokine 10, which functions, among others, as an osteoclastogenic factor. In conclusion, C5a-stimulated osteoblasts might modulate osteoclast activity and contribute to immunomodulation in inflammatory bone disorders.

Bone Disease in Mastocytosis.

Systemic mastocytosis can give very different bone pictures: from osteosclerosis to osteoporosis. Osteoporosis is one of the most frequent manifestations particularly in adults and the most clinical relevant. It is often complicated by a high recurrence of mainly vertebral fragility fractures. The main factor of bone loss is the osteoclast with a relative or absolute predominance of bone resorption. The RANK-RANKL pathway seems of key importance, but histamine and other cytokines also play a significant role in the process. The predominance of resorption made bisphosphonates, as anti-resorptive drugs, the most rational treatment of bone involvement in systemic mastocytosis.

Obesity and bone metabolism.

The interaction between obesity and bone metabolism is complex. The effects of fat on the skeleton are mediated by both mechanical and biochemical factors. Though obesity is characterized by higher bone mineral density, studies conducted on bone microarchitecture have produced conflicting results. The majority of studies indicate that obesity has a positive effect on skeletal strength, even though most likely the effects are site-dependent and, in fact, obese individuals might be at risk of certain types of fractures. Mechanical loading and higher lean mass are associated with improved outcomes, whereas systemic inflammation, observed especially with abdominal obesity, may exert negative effects. Weight loss interventions likely lead to bone loss over time. Pharmacological treatment options seem to be safe in terms of skeletal health; however, the skeletal effects of bariatric surgery are dependent on the type of surgical procedure. Malabsorptive procedures are associated with higher short-term adverse effects on bone health. In this narrative review, we discuss the effects of obesity and weight loss interventions on skeletal health.

Complement involvement in bone homeostasis and bone disorders.

An integral part of innate immunity is the complement system, a defence system, consisting of fluid-phase and cell surface-bound proteins. Its role to ensure adequate responses to danger factors and thus promoting host defence against pathogens has been well described already for decades. Recently, numerous further reaching functions of complement have been discovered, among these are tissue homeostasis and regeneration, also with respect to the skeletal system. The influence of complement activation on bone was recognised first in pathological conditions of inflamed bone tissue and surrounding areas, observed, for example, in rheumatoid arthritis and osteoarthritis. Greatly enhanced levels of complement proteins were detected in synovial fluids and sera of arthritic patients compared to healthy individuals. Additionally, complement-mediated signalling was shown to modulate periodontitis disease development and progression. Periodontitis is an infectious condition of the periodontium, which involves severe bone loss. Moreover, the complement system critically modulates bone regeneration and healing outcome after fracture. This is seen in uneventful fracture healing, but particularly under severe inflammatory conditions induced by an additional traumatic injury. Therefore, complement activation plays an important role in both sterile and non-sterile inflammatory conditions of the bone, which will be addressed here in respect of findings in bone fractures, arthritides, periodontitis and osteomyelitis. Importantly, complement proteins are thought to be critical not simply in the states of an activated immune system, but also for bone growth during physiological development and bone homeostasis, given for example their presence in long-bone growth-plate cartilage. Furthermore, bone-cell development from precursor cells and bone-cell metabolism and communication, for example, between bone-forming osteoblasts and bone-resorbing osteoclasts, are dependent on or even critically influenced by the presence of complement proteins and complement-mediated signalling. The present review summarises the current view on the role of the complement cascade on bone, both under homeostatic physiological conditions and under inflammatory and infectious conditions, which strongly affect the bone and skeletal health. Furthermore, this review addresses the potential and the feasibility of therapeutic interventions involving the complement cascade, derived from experimental and clinical data. Modulating the complement system could help in the future to reduce bone infections, ensure a balanced bone turnover and to generally improve skeletal health.

Deficient invariant natural killer T cells had impaired regulation on osteoclastogenesis in myeloma bone disease.

Recent research showed that invariant natural killer T (iNKT) cells take part in the regulation of osteoclastogenesis. While the role of iNKT cells in myeloma bone disease (MBD) remains unclear. In our study, the quantity of iNKT cells and the levels of cytokines produced by them were measured by flow cytometry. iNKT cells and osteoclasts were induced from peripheral blood mononuclear cells after activation by α-GalCer or RANKL in vitro. Then, gene expressions and the levels of cytokines were determined by RT-PCR and ELISA, respectively. The results showed that the quantity of iNKT and production of IFN-γ by iNKT cells were significantly decreased in newly diagnosed MM (NDMM), and both negatively related with severity of bone disease. Then, the osteoclasts from healthy controls were cultured in vitro and were found to be down-regulated after α-GalCer-stimulated, while there was no significant change with or without α-GalCer in NDMM patients, indicating that the regulation of osteoclastogenesis by iNKT cells was impaired. Furthermore, the inhibition of osteoclastogenesis by iNKT cells was regulated by IFN-γ production, which down-regulated osteoclast-associated genes. In conclusion, the role of α-GalCer-stimulated iNKT cells in regulation of osteoclastogenesis was impaired in MBD, as a result of iNKT cell dysfunction.

Anti-citrullinated alpha enolase antibodies, interstitial lung disease and bone erosion in rheumatoid arthritis.

Objectives: RA is an articular chronic inflammatory disease that in a subgroup of patients can also present with extra-articular manifestations (EAMs). Despite intense investigation on this topic, reliable biomarkers for EAMs are lacking. In recent years several ACPAs, including those targeting anti-citrullinated alpha enolase peptide-1 (anti-CEP-1), have been identified in patients with RA. Data about the ability of anti-CEP-1 to predict the development of erosive disease are confliciting and no evidence concerning their possible association with EAMs in RA is currently available. The aim of this study was to investigate the prevalence and significance of anti-CEP-1 with regard to the association with erosive disease and EAMs in a large cohort of patients with RA. Methods: Anti-CCP and anti-CEP-1 antibodies have been assessed on serum samples of RA patients, healthy donors and patients with SpA using commercially available ELISA kits. Results: Anti-CEP-1 antibodies are detectable in over 40% of RA patients and are associated with erosive RA and with RA-associated interstitial lung disease (ILD). Conclusion: Anti-CEP-1 antibodies may represent a useful biomarker for RA-associated ILD and erosive disease to be employed in clinical practice.

Bone involvement in monogenic autoinflammatory syndromes.

Until recently the most common autoinflammatory diseases (AIDs) associated with bone disease in childhood included a few genetically complex (chronic non-bacterial osteomyelitis, synovitis, acne, pustulosis, hyperostosis and osteitis syndrome) and monogenic (Majeed syndrome, deficiency of IL-1 receptor antagonist, cherubism) AIDs. However, the spectrum of monogenic AIDs associated with bone manifestations has markedly expanded to include both recently identified diseases such as the type I interferonopathies and also newly recognized bone dysplasias in already established AIDs. In addition, we propose that some known bone dysplasia syndromes, especially those presenting with hyperostosis and associated systemic inflammation, be classified as AIDs. Collectively, we provide an overview of the diverse bone manifestations identified in the genetically defined AIDs, discuss the hypotheses of the underlying pathophysiological mechanisms and highlight potential novel therapeutic strategies.

Bone Disease in Axial Spondyloarthritis.

Axial spondyloarthritis is a chronic inflammatory skeletal disorder with an important burden of disease, affecting the spine and sacroiliac joints and typically presenting in young adults. Ankylosing spondylitis, diagnosed by the presence of structural changes to the skeleton, is the prototype of this disease group. Bone disease in axial spondyloarthritis is a complex phenomenon with the coexistence of bone loss and new bone formation, both contributing to the morbidity of the disease, in addition to pain caused by inflammation. The skeletal structural changes respectively lead to increased fracture risk and to permanent disability caused by ankylosis of the sacroiliac joints and the spine. The mechanism of this new bone formation leading to ankylosis is insufficiently known. The process appears to originate from entheses, specialized structures that provide a transition zone in which tendon and ligaments insert into the underlying bone. Growth factor signaling pathways such as bone morphogenetic proteins, Wnts, and Hedgehogs have been identified as molecular drivers of new bone formation, but the relationship between inflammation and activation of these pathways remains debated. Long-standing control of inflammation appears necessary to avoid ankylosis. Recent evidence and concepts suggest an important role for biomechanical factors in both the onset and progression of the disease. With regard to new bone formation, these processes can be understood as ectopic repair responses secondary to inflammation-induced bone loss and instability. In this review, we discuss the clinical implications of the skeletal changes as well as the underlying molecular mechanisms, the relation between inflammation and new bone formation, and the potential role of biomechanical stress.

[Elevated serum IL-25 levels in rheumatoid arthritis patients with bone erosion and interstitial lung disease].

Objective To detect the serum levels of IL-25 and IL-17 in rheumatoid arthritis (RA) patients and investigate the potential relationship with bone erosion and concomitant interstitial lung disease (ILD). Methods The study enrolled a total of 117 RA patients and 56 healthy subjects as controls. The serum levels of IL-25 and IL-17 were determined by ELISA, and rheumatoid factor (RF) was detected by turbidimetric immunoassay, anticyclic citrullinated peptide (anti-CCP) antibody as well as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were also tested. ILD was identified on high-resolution computed tomography (HR-CT), the degree of bone erosion was inspected by musculoskeletal ultrasonography, and radiographic grade was graded by Sharp-van der Heijde Score (SHS). Disease activity in RA was scored with the DAS28 and visual analogue scale (VAS). Correlation analysis was used to evaluate the correlations of IL-25 and IL-17 in different groups. Results Compared with healthy control group, the serum levels of IL-25 and IL-17 increased significantly in the patients with RA. Compared with bone erosion negative group, the serum level of IL-25 was higher significantly in bone erosion group. The level of IL-25 was higher in the ILD group of RA patients than the non-ILD group. In addition, there were positive correlations between the serum level of IL-25 and RF-IgG (r=0.285), RF-IgA (r=0.314), RF-IgM (r=0.380). Meanwhile, the serum level of IL-17 had the positive correlations with RF-IgG (r=0.198) and RF-IgM (r=0.273). Both of them had no correlations with anti-CCP antibody. Conclusion The serum level of IL-25 is raised in RA patients with bone erosion and ILD.