Regulation of the Immune System in Health and Disease by Members of the Bone Morphogenetic Protein Family.

Bone morphogenetic proteins (BMPs) are potent signaling molecules initially described as osteopromoting proteins. BMPs represent one of the members of the larger TGFß family and today are recognized for their important role in numerous processes. Among the wide array of functions recently attributed to them, BMPs were also described to be involved in the regulation of components of the innate and adaptive immune response. This review focuses on the signaling pathway of BMPs and highlights the effects of BMP signaling on the differentiation, activation, and function of the main cell types of the immune system.

Detection of GDF11 by using a Ti3C2-MXene-based fiber SPR biosensor.

In the research of resistant aging, the concentration of Growth differentiation factor-11(GDF11) is an indispensable parameter. So the accurate detection of GDF11 is very important in life science and medical cosmetology. Hereby, we proposed and demonstrated a simple method to detect low concentration GDF11 by using fiber surface plasmon resonance (SPR) sensor decorated with two-dimension (2D) material Ti3C2-MXene and gold nanosphere. The sensitivity of the fiber SPR sensor was increased to be 4804.64nm/RIU. After functionalized with GDF11 antibody, the fiber SPR sensor could specifically recognize GDF11, and the limit of detection (LOD) can reach 0.577pg/L which is 100 times lower than that of single-molecule ELISA method.

Secretome screening reveals immunomodulating functions of IFNα-7, PAP and GDF-7 on regulatory T-cells.

Regulatory T cells (Tregs) are the key cells regulating peripheral autoreactive T lymphocytes. Tregs exert their function by suppressing effector T cells. Tregs have been shown to play essential roles in the control of a variety of physiological and pathological immune responses. However, Tregs are unstable and can lose the expression of FOXP3 and suppressive functions as a consequence of outer stimuli. Available literature suggests that secreted proteins regulate Treg functional states, such as differentiation, proliferation and suppressive function. Identification of secreted proteins that affect Treg cell function are highly interesting for both therapeutic and diagnostic purposes in either hyperactive or immunosuppressed populations. Here, we report a phenotypic screening of a human secretome library in human Treg cells utilising a high throughput flow cytometry technology. Screening a library of 575 secreted proteins allowed us to identify proteins stabilising or destabilising the Treg phenotype as suggested by changes in expression of Treg marker proteins FOXP3 and/or CTLA4. Four proteins including GDF-7, IL-10, PAP and IFNα-7 were identified as positive regulators that increased FOXP3 and/or CTLA4 expression. PAP is a phosphatase. A catalytic-dead version of the protein did not induce an increase in FOXP3 expression. Ten interferon proteins were identified as negative regulators that reduced the expression of both CTLA4 and FOXP3, without affecting cell viability. A transcriptomics analysis supported the differential effect on Tregs of IFNα-7 versus other IFNα proteins, indicating differences in JAK/STAT signaling. A conformational model experiment confirmed a tenfold reduction in IFNAR-mediated ISG transcription for IFNα-7 compared to IFNα-10. This further strengthened the theory of a shift in downstream messaging upon external stimulation. As a summary, we have identified four positive regulators of FOXP3 and/or CTLA4 expression. Further exploration of these Treg modulators and their method of action has the potential to aid the discovery of novel therapies for both autoimmune and infectious diseases as well as for cancer.

Sclerostin antibody stimulates periodontal regeneration in large alveolar bone defects.

Destruction of the alveolar bone in the jaws can occur due to periodontitis, trauma or following tumor resection. Common reconstructive therapy can include the use of bone grafts with limited predictability and efficacy. Romosozumab, approved by the FDA in 2019, is a humanized sclerostin-neutralizing antibody (Scl-Ab) indicated in postmenopausal women with osteoporosis at high risk for fracture. Preclinical models show that Scl-Ab administration preserves bone volume during periodontal disease, repairs bone defects surrounding dental implants, and reverses alveolar bone loss following extraction socket remodeling. To date, there are no studies evaluating Scl-Ab to repair osseous defects around teeth or to identify the efficacy of locally-delivered Scl-Ab for targeted drug delivery. In this investigation, the use of systemically-delivered versus low dose locally-delivered Scl-Ab via poly(lactic-co-glycolic) acid (PLGA) microspheres (MSs) was compared at experimentally-created alveolar bone defects in rats. Systemic Scl-Ab administration improved bone regeneration and tended to increase cementogenesis measured by histology and microcomputed tomography, while Scl-Ab delivered by MSs did not result in enhancements in bone or cemental repair compared to MSs alone or control. In conclusion, systemic administration of Scl-Ab promotes bone and cemental regeneration while local, low dose delivery did not heal periodontal osseous defects in this study.

Bone Morphogenic Proteins Are Immunoregulatory Cytokines Controlling FOXP3+ Treg Cells.

Bone morphogenic proteins (BMPs) are members of the transforming growth factor ß (TGF-ß) cytokine family promoting differentiation, homeostasis, and self-renewal of multiple tissues. We show that signaling through the bone morphogenic protein receptor 1α (BMPR1α) sustains expression of FOXP3 in Treg cells in peripheral lymphoid tissues. BMPR1α signaling promotes molecular circuits supporting acquisition and preservation of Treg cell phenotype and inhibiting differentiation of pro-inflammatory effector Th1/Th17 CD4+ T cell. Mechanistically, increased expression of KDM6B (JMJD3) histone demethylase, an antagonist of the polycomb repressive complex 2, underlies lineage-specific changes of T cell phenotypes associated with abrogation of BMPR1α signaling. These results reveal that BMPs are immunoregulatory cytokines mediating maturation and stability of peripheral FOXP3+ regulatory T cells (Treg cells) and controlling generation of iTreg cells. Thus, we establish that BMPs, a large cytokine family, are an essential link between stromal tissues and the adaptive immune system involved in sustaining tissue homeostasis by promoting immunological tolerance.

Opposing and potentially antagonistic effects of BMP and TGF-ß in multiple sclerosis: The "Yin and Yang" of neuro-immune Signaling.

Bone Morphogenetic Proteins (BMP) and Transforming Growth Factor-beta (TGF-ß) are cytokines with similar receptors and messengers. They are important for immune cell function, with BMPs exerting mainly proinflammatory but also anti-inflammatory effects, and TGF-ß suppressing inflammation. Patients with Multiple Sclerosis exhibit BMP overactivity and suppressed TGF-ß signaling. This dysregulated signaling participates in the crosstalk between infiltrating immune cells and glia, where BMP inhibits remyelination. Reciprocal antagonism between the two pathways takes place via a variety of mechanisms. Although this antagonism has not been studied in the setting of Multiple Sclerosis, it could inform further research and treatment discovery.

Combined antisclerostin antibody and parathyroid hormone (1-34) synergistically enhance the healing of bone defects in ovariectomized rats.

Osteoporotic bones heal more slowly and ineffectively than normal bones. A combination of antibodies against sclerosing protein (Scl-Ab), and parathyroid hormone 1-34 (PTH 1-34) may improve healing. A standard osteoporotic rat model was established 12 weeks after bilateral ovarian resection (OVX). Bone defects were created in the right femora of 80 rats, which were randomly divided into 4 groups: control, Scl-Ab (25 mg/kg twice weekly), PTH (60 µg/kg of PTH 1-34 three times a week) and PTH plus Scl-Ab. After 12 weeks of treatment the rats were sacrificed and blood and the distal femora were harvested for biochemical evaluation, histology, microcomputed tomography and biomechanical testing. Compared to the control group, monotherapy and combination therapy with PTH and/or Scl-Ab promoted the formation of new bone, enhanced maximum femoral loading and increased the levels of procollagen type I N­terminal propeptide (PINP) and osteocalcin. The administration of PTH + Scl-Ab maximally enhanced bone defect healing. Combination treatment was better than either treatment alone, indicating a synergistic effect.

Noncoding RNAs Involved in the Pathogenesis of Ankylosing Spondylitis.

Ankylosing spondylitis (AS) is a form of arthritis that can lead to fusion of vertebrae and sacroiliac joints following syndesmophyte formation. The etiology of this painful disease remains poorly defined due to its complex genetic background. There are no commonly accepted methods for early diagnosis of AS, nor are there any effective or efficient clinical treatments. Several noncoding RNAs (ncRNAs) have been linked to AS pathogenesis and inflammation via selective binding of their downstream targets. However, major gaps in knowledge remain to be filled before such findings can be translated into clinical treatments for AS. In this review, we outline recent findings that demonstrate essential roles of ncRNAs in AS mediated via multiple signaling pathways such as the Wnt, transforming growth factor-ß/bone morphogenetic protein, inflammatory, T-cell prosurvival, and nuclear factor-κB pathways. The summary of these findings provides insight into the molecular mechanisms by which ncRNAs can be targeted for AS diagnosis and the development of therapeutic drugs against a variety of autoimmune diseases.

Pathway and network analysis of genes related to osteoporosis.

As a common degenerative disease, osteoporosis (OS) is characterized by reduced bone mass and microarchitectural deterioration of bone tissue. Both genetic and environmental factors are involved in OS development. To date, ~300 genes have been confirmed to be involved in the pathogenesis of OS, a large majority of which have been independently investigated. As OS is a polygenetic disease, a comprehensive analysis focusing on the biological functions and interactions of OS­related genes would provide valuable information. In this study, OS related research deposited in PubMed was retrieved and genes related to OS were catalogued. Pathways with an enriched biological function for these genes were extracted, and the crosstalk between the enriched pathways was analyzed. A comprehensive network was constructed, and a minimal network was extracted using the Steiner minimal network algorithm. In this study, a total of 294 genes in were retrieved from PubMed. Biological processes found to be enriched included those related to bone metabolism and the immune system. In total, 58 pathways were enriched. Furthermore, the comprehensive network consisting of 3,943 nodes and 7,976 edges was constructed, among which 631 nodes and 2,581 edges contributed to the OS­specific molecular network. In this network, in excess of 300 potential genes associated with OS and two modules were identified. Thus, this study provides a mechanistic insight into OS and suggests more than 300 potential OS­related genes for future research.

Sostdc1 Regulates NK Cell Maturation and Cytotoxicity.

NK cells are innate-like lymphocytes that eliminate virally infected and cancerous cells, but the mechanisms that control NK cell development and cytotoxicity are incompletely understood. We identified roles for sclerostin domain-containing-1 (Sostdc1) in NK cell development and function. Sostdc1-knockout (Sostdc1 -/-) mice display a progressive accumulation of transitional NK cells (tNKs) (CD27+CD11b+) with age, indicating a partial developmental block. The NK cell Ly49 repertoire in Sostdc1 -/- mice is also changed. Lower frequencies of Sostdc1 -/- splenic tNKs express inhibitory Ly49G2 receptors, but higher frequencies express activating Ly49H and Ly49D receptors. However, the frequencies of Ly49I+, G2+, H+, and D+ populations were universally decreased at the most mature (CD27-CD11b+) stage. We hypothesized that the Ly49 repertoire in Sostdc1 -/- mice would correlate with NK killing ability and observed that Sostdc1-/- NK cells are hyporesponsive against MHC class I-deficient cell targets in vitro and in vivo, despite higher CD107a surface levels and similar IFN-γ expression to controls. Consistent with Sostdc1's known role in Wnt signaling regulation, Tcf7 and Lef1 levels were higher in Sostdc1 -/- NK cells. Expression of the NK development gene Id2 was decreased in Sostdc1-/- immature NK and tNK cells, but Eomes and Tbx21 expression was unaffected. Reciprocal bone marrow transplant experiments showed that Sostdc1 regulates NK cell maturation and expression of Ly49 receptors in a cell-extrinsic fashion from both nonhematopoietic and hematopoietic sources. Taken together, these data support a role for Sostdc1 in the regulation of NK cell maturation and cytotoxicity, and identify potential NK cell niches.

TGFß/BMP immune signaling affects abundance and function of C. elegans gut commensals.

The gut microbiota contributes to host health and fitness, and imbalances in its composition are associated with pathology. However, what shapes microbiota composition is not clear, in particular the role of genetic factors. Previous work in Caenorhabditis elegans defined a characteristic worm gut microbiota significantly influenced by host genetics. The current work explores the role of central regulators of host immunity and stress resistance, employing qPCR and CFU counts to measure abundance of core microbiota taxa in mutants raised on synthetic communities of previously-isolated worm gut commensals. This revealed a bloom, specifically of Enterobacter species, in immune-compromised TGFß/BMP mutants. Imaging of fluorescently labeled Enterobacter showed that TGFß/BMP-exerted control operated primarily in the anterior gut and depended on multi-tissue contributions. Enterobacter commensals are common in the worm gut, contributing to infection resistance. However, disruption of TGFß/BMP signaling turned a normally beneficial Enterobacter commensal to pathogenic. These results demonstrate specificity in gene-microbe interactions underlying gut microbial homeostasis and highlight the pathogenic potential of their disruption.

Retention of osteocytic micromorphology by sclerostin antibody in a concurrent ovariectomy and functional disuse model.

Prolonged mechanical unloading in bedridden patients and concurrent hormonal dysregulation represents the cause of one of the severest forms of osteoporosis, a condition for which there are very few efficacious interventions available to date. Sclerostin, a Wnt antagonist, acts as a negative regulator of bone formation. Sclerostin antibody (Scl-Ab)-mediated blockade of sclerostin can dramatically enhance bone formation and reduce bone resorption. This study was designed to investigate the therapeutic effect of the Scl-Ab on severe bone loss induced by concurrent mechanical unloading and estrogen deficiency in a hindlimb-suspended and ovariectomized rat model, and to study the cellular mechanisms underlying severe osteoporosis and Scl-Ab action. Unloading and ovariectomy resulted in severe loss of trabecular and cortical bone mass and strength; Scl-Ab can significantly counteract the deterioration of bone in unloaded and/or ovariectomized rats, with noticeably increased cortical bone formation. Scanning electron microscopy analysis revealed that unloading and ovariectomy lead to multiple morphological and structural abnormalities of osteocytes in cortical bone and the abnormalities were abolished by Scl-Ab administration. This study extends our previous conclusion that Scl-Ab represents a promising therapeutic approach for severe bone loss that occurs after being exposed to estrogen deficiency and prolonged mechanical unloading.

Anabolic Therapies in Osteoporosis and Bone Regeneration.

Osteoporosis represents the most common bone disease worldwide and results in a significantly increased fracture risk. Extrinsic and intrinsic factors implicated in the development of osteoporosis are also associated with delayed fracture healing and impaired bone regeneration. Based on a steadily increasing life expectancy in modern societies, the global implications of osteoporosis and impaired bone healing are substantial. Research in the last decades has revealed several molecular pathways that stimulate bone formation and could be targeted to treat both osteoporosis and impaired fracture healing. The identification and development of therapeutic approaches modulating bone formation, rather than bone resorption, fulfils an essential clinical need, as treatment options for reversing bone loss and promoting bone regeneration are limited. This review focuses on currently available and future approaches that may have the potential to achieve these aims.

Role of Osteocytes in Myeloma Bone Disease: Anti-sclerostin Antibody as New Therapeutic Strategy.

Osteocytes are terminally differentiated cells of the osteoblast lineage. They are involved in the regulation of bone remodeling by increasing osteoclast formation or decreasing bone formation by the secretion of the osteoblast inhibitor sclerostin. Monoclonal antibody anti-sclerostin, Romosozumab, has been developed and tested in clinical trials in patients with osteoporosis. In the last years, the role of osteocytes in the development of osteolytic bone lesions that occurs in multiple myeloma, have been underlined. Myeloma cells increase osteocyte death through the up-regulation of both apoptosis and autophagy that, in turn, triggers osteoclast formation, and activity. When compared to healthy controls, myeloma patients with bone disease have higher osteocyte cell death, but the treatment with proteasome inhibitor bortezomib has been shown to maintain osteocyte viability. In preclinical mouse models of multiple myeloma, treatment with blocking anti-sclerostin antibody increased osteoblast numbers and bone formation rate reducing osteolytic bone lesions. Moreover, the combination of anti-sclerostin antibody and the osteoclast inhibitor zoledronic acid increased bone mass and fracture resistance synergistically. However, anti-sclerostin antibody did not affect tumor burden in vivo or the efficacy of anti-myeloma drugs in vitro. Nevertheless, the combination therapy of anti-sclerostin antibody and the proteasome inhibitor carfilzomib, displayed potent anti-myeloma activity as well as positive effects on bone disease in vivo. In conclusion, all these data suggest that osteocytes are involved in myeloma bone disease and may be considered a novel target for the use of antibody-mediated anti-sclerostin therapy also in multiple myeloma patients.

Anabolic Strategies to Augment Bone Fracture Healing.

PURPOSE OF REVIEW: The development of therapeutics that target anabolic pathways involved in skeletogenesis is of great importance with regard to disease resulting in bone loss, or in cases of impaired bone repair. This review aims to summarize recent developments in this area. RECENT FINDINGS: A greater understanding of how drugs that modulate signaling pathways involved in skeletogenesis exert their efficacy, and the molecular mechanisms resulting in bone formation has led to novel pharmacological bone repair strategies. Furthermore, crosstalk between pathways and molecules has suggested signaling synergies that may be exploited for enhanced tissue formation. The sequential pharmacological stimulation of the molecular cascades resulting in tissue repair is a promising strategy for the treatment of bone fractures. It is proposed that a therapeutic strategy which mimics the natural cascade of events observed during fracture repair may be achieved through temporal targeting of tissue repair pathways.

Treatment with anti-Sclerostin antibody to stimulate mandibular bone formation.

BACKGROUND: Anti-Sclerostin antibody (Scl-Ab) is a promising new bone anabolic therapy. Although anti-Scl-Ab stimulates bone formation and repair in the appendicular and axial skeleton, its efficacy in the craniofacial skeleton is still poorly understood. METHODS: Using an established model of Down syndrome-dependent bone deficiency, 10 Ts65Dn mice and 10 wild-type mice were treated weekly via i.v. tail vein injection with vehicle or anti-Sclerostin for 3 weeks and euthanized 1 week after. RESULTS: Wild-type mice treated with the anti-Scl-Ab had increased mandibular bone, trabecular thickness, and alveolar height compared with controls. Anti-Scl-Ab increased Ts65Dn mandibular bone parameters such that they were statistically indistinguishable from those in vehicle-treated wild-type mandibles. CONCLUSION: Treatment with anti-Scl-Ab significantly increased mandibular bone mass and alveolar height in wild type mice and normalized mandibular bone mass and alveolar height in Ts65Dn mice. The anti-Scl-Ab therapy represents a novel method for increasing mandibular bone formation.

Anabolic Therapy for the Treatment of Osteoporosis in Childhood.

PURPOSE OF REVIEW: Numerous forms of osteoporosis in childhood are characterized by low bone turnover (for example, osteoporosis due to neuromuscular disorders and glucocorticoid exposure). Anti-resorptive therapy, traditionally used to treat osteoporosis in the young, is associated with further reductions in bone turnover, raising concerns about the long-term safety and efficacy of such therapy. These observations have led to increasing interest in the role of anabolic therapy to treat pediatric osteoporosis. RECENT FINDINGS: While growth hormone and androgens appears to be relatively weak anabolic modulators of bone mass, emerging therapies targeting bone formation pathways (anti-transforming growth factor beta antibody and anti-sclerostin antibody) hold considerable promise. Teriparatide remains an attractive option that merits formal study for patients post-epiphyseal fusion, although it must be considered that adult studies have shown its effect is blunted when administered following bisphosphonate therapy. Mechanical stimulation of bone through whole body vibration therapy appears to be much less effective than bisphosphonate therapy for treating osteoporosis in children. New anabolic therapies which target important pathways in skeletal metabolism merit further study in children, including their effects on fracture risk reduction and after treatment discontinuation.

Sclerostin and Antisclerostin Antibody Serum Levels Predict the Presence of Axial Spondyloarthritis in Patients with Inflammatory Bowel Disease.

OBJECTIVE: The early diagnosis of inflammatory bowel disease (IBD)-associated spondyloarthritis (SpA/IBD) in patients affected by IBD represents a major topic in clinical practice; in particular, to date there are no available serum biomarkers revealing the presence of joint inflammation in these patients. Sclerostin (SOST), an antagonist of the Wnt/ß-catenin pathway, and antisclerostin-immunoglobulin G (anti-SOST-IgG) have been recently studied in patients with ankylosing spondylitis (AS) as a putative marker of disease activity. METHODS: SOST and anti-SOST-IgG serum levels were assayed in 125 patients with IBD, 85 with axial or peripheral SpA, and in control groups (patients with AS and rheumatoid arthritis, and healthy individuals). The diagnostic performance in discriminating the presence of SpA/IBD was assessed for both candidate biomarkers. RESULTS: Patients affected by SpA/IBD with axial involvement displayed significantly lower levels of SOST and higher levels of anti-SOST-IgG compared to patients with only peripheral arthritis, IBD, and controls. Moreover, SOST and anti-SOST-IgG serum levels were inversely correlated and were associated with the duration of articular symptoms. Both biomarkers showed good accuracy in predicting the presence of axial SpA in patients with IBD. CONCLUSION: We demonstrated that in patients with IBD, SOST and anti-SOST-IgG might represent novel biomarkers to assess the presence of axial joint involvement. Moreover, the development of anti-SOST-IgG and the subsequent decrease of SOST serum levels could play a role in the pathogenesis of SpA/IBD.

The effects of sclerostin antibody plus parathyroid hormone (1-34) on bone formation in ovariectomized rats.

Previous studies have demonstrated the effects of sclerostin antibody (Scl-Ab) and parathyroid hormone (1-34, PTH) on healing in osteoporosis; however, reports about the combined effects of Scl-Ab plus PTH on osteoporosis are limited. This study was designed to investigate the impact of combined treatment with Scl-Ab and PTH on osteoporosis healing in ovariectomized (OVX) rats. After bilateral ovariectomy, 12 weeks were allowed to pass for the establishment of standard conditions for osteoporosis in animal models. The rats then randomly received a vehicle (control), Scl-Ab (25 mg/kg body weight, twice weekly), PTH (60 µg/kg, three times per week) or PTH plus Scl-Ab until death at 12 weeks. The blood and distal femurs of the rats were harvested for evaluation. The results of treatment for osteoporosis were evaluated by serum analysis, histology, microcomputed tomography (micro-CT) and biomechanical tests. Results from this study indicated that PTH + Scl-Ab had stronger effects on the prevention and treatment of osteoporosis than either of the monotherapies in OVX rats. The PTH + Scl-Ab produced the strongest effects on bone volume fraction (BV/TV), bone trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular spacing (Tb.Sp), bone mineral density (BMD) and strength of distal femurs and increased the levels of procollagen type I N­terminal propeptide (PINP) and osteocalcin. In contrast, monotherapy with PTH or Scl-Ab showed no differences between treated groups in the assessment of the metaphysis of contralateral femurs by histology, serum, biomechanical tests and micro-CT. These results seem to indicate that Scl-Ab plus PTH has an additive effect on osteoporosis in OVX rats.

Sclerostin antibody enhances bone formation in a rat model of distraction osteogenesis.

Neutralizing monoclonal sclerostin antibodies are effective in promoting bone formation at a systemic level and in orthopedic scenarios including closed fracture repair. In this study we examined the effects of sclerostin antibody (Scl-Ab) treatment on regenerate volume, density, and strength in a rat model of distraction osteogenesis. Surgical osteotomy was performed on 179 Sprague Dawley rats. After 1 week, rats underwent distraction for 2 weeks, followed by 6 weeks for consolidation. Two treatment groups received biweekly subcutaneous Scl-AbIII (a rodent form of Scl-Ab; 25 mg/kg), either from the start of distraction onward or restricted to the consolidation phase. These groups were compared to controls receiving saline. Measurement modalities included longitudinal DXA, ex vivo QCT, and microCT, tissue histology, and biomechanical four-point bending tests. Bone volume was increased in both Scl-Ab treatments regimens by the end of consolidation (+26-38%, p < 0.05), as assessed by microCT. This was associated with increased mineral apposition. Importantly, Scl-Ab led to increased strength in united bones, and this reached statistical significance in animals receiving Scl-Ab during consolidation only (+177%, p < 0.01, maximum load to failure). These data demonstrate that Scl-Ab treatment increases bone formation, leading to regenerates with higher bone volume and improved strength. Our data also suggest that the optimal effects of Scl-Ab treatment are achieved in the latter stages of distraction osteogenesis. These findings support further investigation into the potential clinical application of sclerostin antibody to augment bone distraction, such as limb lengthening, particularly in the prevention of refracture. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1106-1113, 2018.