Irisin prevents trabecular bone damage and tumor invasion in a mouse model of multiple myeloma.

Bone disease associated with multiple myeloma (MM) is characterized by osteolytic lesions and pathological fractures, which remain a therapeutic priority despite new drugs improving MM patient survival. Antiresorptive molecules represent the main option for the treatment of MM-associated bone disease (MMBD), whereas osteoanabolic molecules are under investigation. Among these latter, we here focused on the myokine irisin, which is able to enhance bone mass in healthy mice, prevent bone loss in osteoporotic mouse models, and accelerate fracture healing in mice. Therefore, we investigated irisin effect on MMBD in a mouse model of MM induced by intratibial injection of myeloma cells followed by weekly administration of 100 µg/kg of recombinant irisin for 5 wk. By micro-Ct analysis, we demonstrated that irisin improves MM-induced trabecular bone damage by partially preventing the reduction of femur Trabecular Bone Volume/Total Volume (P = .0028), Trabecular Number (P = .0076), Trabecular Fractal Dimension (P = .0044), and increasing Trabecular Separation (P = .0003) in MM mice. In cortical bone, irisin downregulates the expression of Sclerostin, a bone formation inhibitor, and RankL, a pro-osteoclastogenic molecule, while in BM it upregulates Opg, an anti-osteoclastogenic cytokine. We found that in the BM tibia of irisin-treated MM mice, the percentage of MM cells displays a reduction trend, while in the femur it decreases significantly. This is in line with the in vitro reduction of myeloma cell viability after 48 h of irisin stimulation at both 200 and 500 ng/mL and, after 72 h already at 100 ng/mL rec-irisin. These results could be due to irisin ability to downregulate the expression of Notch 3, which is important for cell-to-cell communication in the tumor niche, and Cyclin D1, supporting an inhibitory effect of irisin on MM cell proliferation. Overall, our findings suggest that irisin could be a new promising strategy to counteract MMBD and tumor burden in one shot.

Dynamic YAP expression in the non-parenchymal liver cell compartment controls heterologous cell communication.

INTRODUCTION: The Hippo pathway and its transcriptional effectors yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are targets for cancer therapy. It is important to determine if the activation of one factor compensates for the inhibition of the other. Moreover, it is unknown if YAP/TAZ-directed perturbation affects cell-cell communication of non-malignant liver cells. MATERIALS AND METHODS: To investigate liver-specific phenotypes caused by YAP and TAZ inactivation, we generated mice with hepatocyte (HC) and biliary epithelial cell (BEC)-specific deletions for both factors (YAPKO, TAZKO and double knock-out (DKO)). Immunohistochemistry, single-cell sequencing, and proteomics were used to analyze liver tissues and serum. RESULTS: The loss of BECs, liver fibrosis, and necrosis characterized livers from YAPKO and DKO mice. This phenotype was weakened in DKO tissues compared to specimens from YAPKO animals. After depletion of YAP in HCs and BECs, YAP expression was induced in non-parenchymal cells (NPCs) in a cholestasis-independent manner. YAP positivity was detected in subgroups of Kupffer cells (KCs) and endothelial cells (ECs). The secretion of pro-inflammatory chemokines and cytokines such as C-X-C motif chemokine ligand 11 (CXCL11), fms-related receptor tyrosine kinase 3 ligand (FLT3L), and soluble intercellular adhesion molecule-1 (ICAM1) was increased in the serum of YAPKO animals. YAP activation in NPCs could contribute to inflammation via TEA domain transcription factor (TEAD)-dependent transcriptional regulation of secreted factors. CONCLUSION: YAP inactivation in HCs and BECs causes liver damage, and concomitant TAZ deletion does not enhance but reduces this phenotype. Additionally, we present a new mechanism by which YAP contributes to cell-cell communication originating from NPCs.

Iron homeostasis in mice: does liver lobe matter?

The liver plays a crucial role in maintaining systemic iron homeostasis through iron storage, sensing of systemic iron needs, and production of the iron-regulatory hormone hepcidin. While mice are commonly used as models for studying human iron homeostasis, their liver structure differs significantly from humans. Since the mouse liver is structured in six separated lobes, often, the analysis of a single defined lobe is preferred due to concerns over data reproducibility between experimental cohorts. In this study, we compared iron-related parameters in distinct liver lobes of C57BL/6 wild-type mice across different ages. We found that the non-heme iron levels, as well as the mRNA and protein expression of iron storage protein Ferritin and the iron importer Transferrin Receptor 1, were similar between liver lobes. Additionally, the mRNA expression of Hepcidin, as well as its regulators, Bmp2 and Bmp6, and iron importers Zip8 and Zip14 were comparable. Minor differences were observed in Ferroportin mRNA levels of 24-wk-old mice; however, this did not correlate with altered iron content. The findings in wild-type mice were reproduced in Hfe knock-out mice - a well-established genetic model of the most prevalent form of hemochromatosis. Overall, our results indicate that C57BL/6 mouse liver lobes can be used interchangeably for assessing iron content and expression of iron-related genes. Understanding if these findings are applicable to other mouse developmental stages, strains, or models of (iron-related) disorders will be key to promote reduction of experimental animal numbers and facilitate resource sharing among research groups studying liver iron homeostasis.NEW & NOTEWORTHY This study reveals that, despite being structurally separated, liver lobes from C57BL/6 wild-type and iron-overloaded mice can be used interchangeably for the evaluation of iron content and expression of iron-related genes.

Irisin Modulates Inflammatory, Angiogenic, and Osteogenic Factors during Fracture Healing.

Bone fractures are a widespread clinical event due to accidental falls and trauma or bone fragility; they also occur in association with various diseases and are common with aging. In the search for new therapeutic strategies, a crucial link between irisin and bone fractures has recently emerged. To explore this issue, we subjected 8-week-old C57BL/6 male mice to tibial fracture, and then we treated them with intra-peritoneal injection of r-Irisin (100 µg/kg/weekly) or vehicle as control. At day 10 post fracture, histological analysis showed a significant reduced expression of inflammatory cytokines as tumor necrosis factor-alpha (TNFα) (p = 0.004) and macrophage inflammatory protein-alpha (MIP-1α) (p = 0.015) in the cartilaginous callus of irisin-treated mice compared to controls, supporting irisin's anti-inflammatory role. We also found increased expressions of the pro-angiogenic molecule vascular endothelial growth factor (VEGF) (p = 0.002) and the metalloproteinase MMP-13 (p = 0.0006) in the irisin-treated mice compared to the vehicle ones, suggesting a myokine involvement in angiogenesis and cartilage matrix degradation processes. Moreover, the bone morphogenetic protein (BMP2) expression was also upregulated (p = 0.002). Taken together, our findings suggest that irisin can contribute to fracture repair by reducing inflammation and promoting vessel invasion, matrix degradation, and bone formation, supporting its possible role as a novel molecule for fracture treatment.

Hfe Is Highly Expressed in Liver Sinusoidal Endothelial Cells But Is Not Needed to Maintain Systemic Iron Homeostasis In Vivo.

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Core Cross-Linked Polymeric Micelles for Specific Iron Delivery: Inducing Sterile Inflammation in Macrophages.

Iron is an essential co-factor for cellular processes. In the immune system, it can activate macrophages and represents a potential therapeutic for various diseases. To specifically deliver iron to macrophages, iron oxide nanoparticles are embedded in polymeric micelles of reactive polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine). Upon surface functionalization via dihydrolipoic acid, iron oxide cores act as crosslinker themselves and undergo chemoselective disulfide bond formation with the surrounding poly(S-ethylsulfonyl-l-cysteine) block, yielding glutathione-responsive core cross-linked polymeric micelles (CCPMs). When applied to primary murine and human macrophages, these nanoparticles display preferential uptake, sustained intracellular iron release, and induce a strong inflammatory response. This response is also demonstrated in vivo when nanoparticles are intratracheally administered to wild-type C57Bl/6N mice. Most importantly, the controlled release concept to deliver iron oxide in redox-responsive CCPMs induces significantly stronger macrophage activation than any other iron source at identical iron levels (e.g., Feraheme), directing to a new class of immune therapeutics.

Regulation of iron homeostasis: Lessons from mouse models.

Iron is an essential micronutrient and a critical cofactor for proteins involved in fundamental processes such as oxygen transport, energy production and DNA synthesis. However, iron levels need to be tightly balanced to avoid pathological consequences of iron overload or deficiency. Genetically engineered mouse models with alterations in systemic or cellular iron handling advanced our knowledge how systemic and cellular iron homeostasis is maintained. Here, we prepared a comprehensive overview of mouse models that provide insight into mechanisms of iron regulation and/or rare or frequent iron-related disorders.

Shedding "LIGHT" on the Link between Bone and Fat in Obese Children and Adolescents.

Obesity may affect bone health, but literature reports are contradictory about the correlation of body mass index (BMI) and bone markers. LIGHT, one of the immunostimulatory cytokines regulating the homeostasis of bone and adipose tissue, could be involved in obesity. The study involved 111 obese subjects (12.21 ± 3.71 years) and 45 controls. Patients underwent the evaluation of bone status by quantitative ultrasonography (QUS). LIGHT amounts were evaluated in sera by ELISA, whereas its expression on peripheral blood cells was evaluated by flow cytometry. Osteoclastogenesis was performed by culturing peripheral blood mononuclear cells (PBMCs) with or without anti-LIGHT antibodies. Obese patients showed significant high BMI-standard deviation score (SDS), weight-SDS, and Homeostatic model assessment for insulin resistance (HOMA-IR) that negatively correlated with the reduced Amplitude Dependent Speed of Sound (AD-SoS)-Z-score and Bone Transmission Time (BTT-Z)-score. They displayed significantly higher serum levels of LIGHT compared with controls (497.30 ± 363.45 pg/mL vs. 186.06 ± 101.41 pg/mL, p < 0.001). LIGHT expression on monocytes, CD3+-T-cells, and neutrophils was also higher in obese patients than in the controls. Finally, in PBMC cultures, the addition of anti-LIGHT antibodies induced a significant osteoclastogenesis inhibition. Our study highlighted the high serum levels of LIGHT in obese children and adolescents, and its relationship with both the grade of obesity and bone impairment.

Total reflection X-ray fluorescence spectrometry for trace determination of iron and some additional elements in biological samples.

Trace elements are essential for life and their concentration in cells and tissues must be tightly maintained and controlled to avoid pathological conditions. Established methods to measure the concentration of trace elements in biological matrices often provide only single element information, are time-consuming, and require special sample preparation. Therefore, the development of straightforward and rapid analytical methods for enhanced, multi-trace element determination in biological samples is an important and raising field of trace element analysis. Herein, we report on the development and validation of a reliable method based on total reflection X-ray fluorescence (TXRF) analysis to precisely quantify iron and other trace metals in a variety of biological samples, such as the liver, parenchymal and non-parenchymal liver cells, and bone marrow-derived macrophages. We show that TXRF allows fast and simple one-point calibration by addition of an internal standard and has the potential of multi-element analysis in minute sample amounts. The method was validated for iron by recovery experiments in homogenates in a wide concentration range from 1 to 1600 µg/L applying well-established graphite furnace atomic absorption spectrometry (GFAAS) as a reference method. The recovery rate of 99.93 ± 0.14% reveals the absence of systematic errors. Furthermore, the standard reference material "bovine liver" (SRM 1577c, NIST) was investigated in order to validate the method for further biometals. Quantitative recoveries (92-106%) of copper, iron, zinc, and manganese prove the suitability of the developed method. The limits of detection for the minute sample amounts are in the low picogram range. Graphical abstract.

LIGHT/TNFSF14 regulates estrogen deficiency-induced bone loss.

Bone loss induced by ovariectomy is due to the direct activity on bone cells and mesenchymal cells and to the dysregulated activity of bone marrow cells, including immune cells and stromal cells, but the underlying mechanisms are not completely known. Here, we demonstrate that ovariectomy induces the T-cell co-stimulatory cytokine LIGHT, which stimulates both osteoblastogenesis and osteoclastogenesis by modulating osteoclastogenic cytokine expression, including TNF, osteoprotegerin, and the receptor activator of nuclear factor-κB ligand (RANKL). Predictably, LIGHT-deficient (Tnfsf14-/- ) mice are protected from ovariectomy-dependent bone loss, whereas trabecular bone mass increases in mice deficient in both LIGHT and T and B lymphocytes (Rag -/- Tnfsf14 -/- ) and is associated with an inversion of the TNF and RANKL/OPG ratio. Furthermore, women with postmenopausal osteoporosis display high levels of LIGHT in circulating T cells and monocytes. Taken together, these results indicate that LIGHT mediates bone loss induced by ovariectomy, suggesting that patients with postmenopausal osteoporosis may benefit from LIGHT antagonism. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

LIGHT/TNFSF14 Promotes Osteolytic Bone Metastases in Non-small Cell Lung Cancer Patients.

Tumor necrosis factor superfamily member 14 (TNFSF14), LIGHT, is a component of the cytokine network that regulates innate and adaptive immune responses, which promote homeostasis of lymphoid organs, liver, and bone. Metastatic tumors often disrupt the tissue microenvironment, thus altering the homeostasis of the invaded organ; however, the underlying mechanisms required further studies. We investigated the role of LIGHT in osteolytic bone disease induced by metastatic non-small cell lung cancer (NSCLC). Patients diagnosed with NSCLC bone metastasis show significantly higher levels of LIGHT expressed in monocytes compared with non-bone metastatic tumors and healthy controls. Serum LIGHT levels were also higher in patients with bone metastases than in controls, suggesting a role for LIGHT in stimulating osteoclast precursors. In bone metastatic patients, we also detected increased RNA expression and serum RANKL levels, thus by adding anti-LIGHT or RANK-fragment crystallizable region (RANK-Fc) in PBMC cultures, a significant inhibition of osteoclastogenesis was observed. To model this observation in mice, we used the mouse lung cancer cell line LLC-1. After intratibial implantation, wild-type mice showed an increased number of osteoclasts but reduced numbers of osteoblasts and decreased osteoid formation. In contrast, Tnfsf14-/- mice showed no significant bone loss or other changes in bone homeostasis associated with this model. These data indicate LIGHT is a key control mechanism for regulating bone homeostasis during metastatic invasion. Thus, LIGHT may be a novel therapeutic target in osteolytic bone metastases. © 2019 American Society for Bone and Mineral Research.

Effects of Sweet Cherry Polyphenols on Enhanced Osteoclastogenesis Associated With Childhood Obesity.

Childhood obesity is associated with the development of severe comorbidities, such as diabetes, cardiovascular diseases, and increased risk of osteopenia/osteoporosis and fractures. The status of low-grade inflammation associated to obesity can be reversed through an enhanced physical activity and by consumption of food enrich of anti-inflammatory compounds, such as omega-3 fatty acids and polyphenols. The aim of this study was to deepen the mechanisms of bone impairment in obese children and adolescents through the evaluation of the osteoclastogenic potential of peripheral blood mononuclear cells (PBMCs), and the assessment of the serum levels of RANKL and osteoprotegerin (OPG). Furthermore, we aimed to evaluate the in vitro effects of polyphenol cherry extracts on osteoclastogenesis, as possible dietary treatment to improve bone health in obese subjects. High RANKL levels were measured in obese with respect to controls (115.48 ± 35.20 pg/ml vs. 87.18 ± 17.82 pg/ml; p < 0.01), while OPG levels were significantly reduced in obese than controls (378.02 ± 61.15 pg/ml vs. 436.75 ± 95.53 pg/ml, respectively, p < 0.01). Lower Ad-SoS- and BTT Z-scores were measured in obese compared to controls (p < 0.05). A significant elevated number of multinucleated TRAP+ osteoclasts (OCs) were observed in the un-stimulated cultures of obese subjects compared to the controls. Interestingly, obese subjects displayed a higher percentage of CD14+/CD16+ than controls. Furthermore, in the mRNA extracts of obese subjects we detected a 2.5- and 2-fold increase of TNFα and RANKL transcripts compared to controls, respectively. Each extract of sweet cherries determined a dose-dependent reduction in the formation of multinucleated TRAP+ OCs. Consistently, 24 h treatment of obese PBMCs with sweet cherry extracts from the three cultivars resulted in a significant reduction of the expression of TNFα. In conclusion, the bone impairment in obese children and adolescents is sustained by a spontaneous osteoclastogenesis that can be inhibited in vitro by the polyphenol content of sweet cherries. Thus, our study opens future perspectives for the use of sweet cherry extracts, appropriately formulated as nutraceutical food, as preventive in healthy children and therapeutic in obese ones.

An update on the role of RANKL-RANK/osteoprotegerin and WNT-ß-catenin signaling pathways in pediatric diseases.

BACKGROUND: Bone remodeling is a lifelong process due to the balanced activity of osteoclasts (OCs), the bone-reabsorbing cells, and osteoblasts (OBs), and the bone-forming cells. This equilibrium is regulated by numerous cytokines, but it has been largely demonstrated that the RANK/RANKL/osteoprotegerin and Wnt/ß-catenin pathways play a key role in the control of osteoclastogenesis and osteoblastogenesis, respectively. The pro-osteoblastogenic activity of the Wnt/ß-catenin can be inhibited by sclerostin and Dickkopf-1 (DKK-1). RANKL, sclerostin and DKKs-1 are often up-regulated in bone diseases, and they are the target of new monoclonal antibodies. DATA SOURCES: The authors performed a systematic literature search in PubMed and EMBASE to June 2018, reviewed and selected articles, based on pre-determined selection criteria. RESULTS: We re-evaluated the role of RANKL, osteoprotegerin, sclerostin and DKK-1 in altered bone remodeling associated with some inherited and acquired pediatric diseases, such as type 1 diabetes mellitus (T1DM), alkaptonuria (AKU), hemophilia A, osteogenesis imperfecta (OI), 21-hydroxylase deficiency (21OH-D) and Prader-Willi syndrome (PWS). To do so, we considered recent clinical studies done on pediatric patients in which the roles of RANKL-RANK/osteoprotegerin and WNT-ß-catenin signaling pathways have been investigated, and for which innovative therapies for the treatment of osteopenia/osteoporosis are being developed. CONCLUSIONS: The case studies taken into account for this review demonstrated that quite frequently both bone reabsorbing and bone deposition are impaired in pediatric diseases. Furthermore, for some of them, bone damage began in childhood but only manifested with age. The use of denosumab could represent a valid alternative therapeutic approach to improve bone health in children, although further studies need to be carried out.

LIGHT/TNFSF14 as a New Biomarker of Bone Disease in Multiple Myeloma Patients Experiencing Therapeutic Regimens.

We have previously shown that through the production of high LIGHT levels, immune cells contribute to both osteoclastogenesis and bone destruction in Multiple Myeloma (MM)-related bone disease. With the aim of further exploring the mechanisms underlying the development of MM-related bone disease, here we focused on a possible role of LIGHT in MM patients with active bone disease despite the treatment received. We detected LIGHT over-expression by circulating CD14+ monocytes from MM patients still showing active bone disease, despite the treatment. In addition, we found over-expression of receptor activator of nuclear factor kappa-B ligand (RANKL), whose pro-osteoclastogenic role is well-known, in T-lymphocytes isolated from the same patients. Although the percentages of circulating osteoclast progenitors, CD14+CD16+ monocytes, were higher in all the MM patients than in the controls spontaneous osteoclastogenesis occurred only in the cultures derived from PBMCs of MM patients with unresponsive bone disease. Of note, in the same cultures osteoclastogenesis was partially or completely inhibited, in a dose-dependent manner, by the addition of RANK-Fc or anti-LIGHT neutralizing antibody, demonstrating the contribution of both LIGHT and RANKL to the enhanced osteoclast formation observed. In addition, high serum levels of TRAP5b and CTX, the two markers of osteoclast activity, were detected in MM patients with bone disease not responsive to treatment. In conclusion, our study indicates a prominent role of LIGHT in the crosstalk among osteoclasts and immune cells, co-involved together with RANKL in the pathophysiological mechanisms leading to MM-related bone disease. This TNF superfamily member may thus be a possible new therapeutic target in MM-related bone disease.

High expression of TRAIL by osteoblastic differentiated dental pulp stem cells affects myeloma cell viability.

Cells from dental tissues have a mesenchymal stem cell (MSC) phenotype, are multipotent and can differentiate into osteoblastic cells, as we have previously found. MSCs, due to their tumor­homing ability, are currently being used as cell­based delivery systems for cancer protein therapeutics, such as the TNF­related apoptosis­inducing ligand (TRAIL). In the present study we revealed that dental pulp stem cells (DPSCs) expressed TRAIL to a greater extent when they were differentiated into the osteoblastic lineage. TRAIL affected the viability of undifferentiated DPSCs, while osteoblastic differentiated DPSCs were not sensitive to TRAIL. The expression trend of TRAIL receptors underwent changes during the osteoblastic differentiation of DPSCs exhibiting low DcR2 and high DR5 levels in the undifferentiated DPSCs and an opposite scenario was presented in the differentiated cells. The sensitivity of the undifferentiated DPSCs to the TRAIL­apoptotic effect was also associated with low levels of intracellular anti­apoptotic proteins, such as c­FLIP, XIAP and the activation of caspase­8 and ­3. DPSC­differentiated osteoblasts expressing high TRAIL levels were capable to affect the cell viability of the human myeloma cell line H929, thus representing an effective anticancer therapeutic method.

Mechanisms of Enhanced Osteoclastogenesis in Alkaptonuria.

Alkaptonuria (AKU) is a rare disorder characterized by the deficiency of the enzyme homogentisate 1,2-dioxygenase and consequent homogentisate accumulation, which leads to progressive and severe osteoarthopathy starting from the second decade of life. Thus, in AKU patients, bone involvement represents an important clinical issue, which we investigated. Serum levels of receptor activator of NF-κB ligand (RANKL), osteoprotegerin, sclerostin, Dickkopf-1, and bone remodeling markers were measured in nine AKU patients (two children and seven adults) and 22 controls, together with lumbar spine bone mineral density (LS-BMD) and femoral-BMD. In the two AKU children, the average of LS-BMD and femoral-BMD Z-scores were within the normal range, but reduced with respect to the controls. Otherwise, in the adult AKU patients, LS-BMD T-score was inside the normal range, but femoral-BMD T-score reached osteopenic levels. Consistently, in AKU adults, higher RANKL and C-terminal telopeptide of collagen type 1 and lower osteoprotegerin levels were observed than in controls. Otherwise, spontaneous osteoclastogenesis was already evident in peripheral blood mononuclear cell cultures from AKU children, together with a high percentage of circulating osteoclast precursors. Osteoclastogenesis was sustained by the high levels of tumor necrosis factor-α, RANK, RANKL, and LIGHT. In conclusion, the altered osteoclastogenesis was observed already in AKU children, despite the absence of evident injury. Thus, a preventive approach in young patients, targeting osteoclast activity, may prevent the macroscopic bone disease that appears in adult AKU.

Impairment of Bone Remodeling in LIGHT/TNFSF14-Deficient Mice.

Multiple cytokines produced by immune cells induce remodeling and aid in maintaining bone homeostasis through differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts. Here, we investigate bone remodeling controlled by the tumor necrosis factor (TNF) superfamily cytokine LIGHT. LIGHT-deficient mice (Tnfsf14-/- ) exhibit spine deformity and reduced femoral cancellous bone mass associated with an increase in the osteoclast number and a slight decrease of osteoblasts compared with WT mice. The effect of LIGHT in bone cells can be direct or indirect, mediated by both the low expression of the anti-osteoclastogenic osteoprotegerin (OPG) in B and T cells and reduced levels of the pro-osteoblastogenic Wnt10b in CD8+ T cells in Tnfsf14-/- mice. LIGHT stimulation increases OPG levels in B, CD8+ T, and osteoblastic cells, as well as Wnt10b expression in CD8+ T cells. The high bone mass in Light and T- and B-cell-deficient mice (Rag- /Tnfsf14- ) supports the cooperative role of the immune system in bone homeostasis. These results implicate LIGHT as a potential target in bone disease. © 2017 American Society for Bone and Mineral Research.