Individual Effector/Regulator T Cell Ratios Impact Bone Regeneration.

There is increasing evidence that T lymphocytes play a key role in controlling endogenous regeneration. Regeneration appears to be impaired in case of local accumulation of CD8+ effector T cells (TEFF), impairing endogenous regeneration by increasing a primary "useful" inflammation toward a damaging level. Thus, rescuing regeneration by regulating the heightened pro-inflammatory reaction employing regulatory CD4+ T (TReg) cells could represent an immunomodulatory option to enhance healing. Hypothesis was that CD4+ TReg might counteract undesired effects of CD8+ TEFF. Using adoptive TReg transfer, bone healing was consistently improved in mice possessing an inexperienced immune system with low amounts of CD8+ TEFF. In contrast, mice with an experienced immune system (high amounts of CD8+ TEFF) showed heterogeneous bone repair with regeneration being dependent upon the individual TEFF/TReg ratio. Thus, the healing outcome can only be improved by an adoptive TReg therapy, if an unfavorable TEFF/TReg ratio can be reshaped; if the individual CD8+ TEFF percentage, which is dependent on the individual immune experience can be changed toward a favorable ratio by the TReg transfer. Remarkably, also in patients with impaired fracture healing the TEFF/TReg ratio was higher compared to uneventful healers, validating our finding in the mouse osteotomy model. Our data demonstrate for the first time the key-role of a balanced TEFF/TReg response following injury needed to reach successful regeneration using bone as a model system. Considering this strategy, novel opportunities for immunotherapy in patients, which are at risk for impaired healing by targeting TEFF cells and supporting TReg cells to enhance healing are possible.

Human Immune System Increases Breast Cancer-Induced Osteoblastic Bone Growth in a Humanized Mouse Model without Affecting Normal Bone.

Bone metastases are prevalent in many common cancers such as breast, prostate, and lung cancers, and novel therapies for treating bone metastases are needed. Human immune system-engrafted models are used in immuno-oncology (IO) studies for subcutaneous cancer cell or patient-derived xenograft implantations that mimic primary tumor growth. Novel efficacy models for IO compounds on bone metastases need to be established. The study was performed using CIEA NOG (NOG) mice engrafted with human CD34+ hematopoietic stem cells (huNOG) and age-matched immunodeficient NOG mice. Bone phenotyping was performed to evaluate baseline differences. BT-474 human breast cancer cells were inoculated into the tibia bone marrow, and cancer-induced bone changes were monitored by X-ray imaging. Bone content and volume were analyzed by dual X-ray absorptiometry and microcomputed tomography. Tumor-infiltrating lymphocytes (TILs) and the expression of immune checkpoint markers were analyzed by immunohistochemistry. Bone phenotyping showed no differences in bone architecture or volume of the healthy bones in huNOG and NOG mice, but the bone marrow fat was absent in huNOG mice. Fibrotic areas were observed in the bone marrow of some huNOG mice. BT-474 tumors induced osteoblastic bone growth. Bone lesions appeared earlier and were larger, and bone mineral density was higher in huNOG mice. huNOG mice had a high number of human CD3-, CD4-, and CD8-positive T cells and CD20-positive B cells in immune-related organs. A low number of TILs and PD-1-positive cells and low PD-L1 expression were observed in the BT-474 tumors at the endpoint. This study reports characterization of the first breast cancer bone growth model in huNOG mice. BT-474 tumors represent a "cold" tumor with a low number of TILs. This model can be used for evaluating the efficacy of combination treatments of IO therapies with immune-stimulatory compounds or therapeutic approaches on bone metastatic breast cancer.

Bone Wasn't Built in a Day: Destruction and Formation of Bone in the Rheumatic Diseases.

The hallmark of rheumatoid arthritis is synovitis, or inflammation of synovial tissues lining joints. Synovitis in rheumatoid arthritis promotes destruction of articular bone by inducing the differentiation and function of osteoclasts, leading to significant patient morbidity. The cell types and pathways mediating articular bone destruction have now been elucidated and the critical role of receptor activator of nuclear factor-kappa B ligand has been recognized, leading to the identification of new targets for the protection of articular bone. Synovitis not only promotes bone destruction, but also inhibits the ability of bone-forming osteoblasts to repair bone. In stark contrast, inflammation in spondyloarthritis, including ankylosing spondylitis, promotes bone formation at periosteal sites, resulting in pain and decreased motion of the spine and joints. Local anatomic factors contribute to these distinct outcomes for bone and anabolic pathways regulating bone formation are now being investigated to identify novel targets for prevention of abnormal bone formation.

Regulation of bone mass by the gut microbiota is dependent on NOD1 and NOD2 signaling.

Germ-free (GF) mice have increased bone mass that is normalized by colonization with gut microbiota (GM) from conventionally raised (CONV-R) mice. To determine if innate immune signaling pathways mediated the effect of the GM, we studied the skeleton of GF and CONV-R mice with targeted inactivation of MYD88, NOD1 or NOD2. In contrast to WT and Myd88-/- mice, cortical bone thickness in mice lacking Nod1 or Nod2 was not increased under GF conditions. The expression of Tnfα and the osteoclastogenic factor Rankl in bone was reduced in GF compared to CONV-R WT mice but not in Nod1-/- or Nod2-/- mice indicating that the effect of the GM to increase Tnfα and Rankl in bone and to reduce bone mass is dependent on both NOD1 and NOD2 signaling.

Sclerostin Antibody Treatment Increases Bone Formation, Bone Mass, and Bone Strength of Intact Bones in Adult Male Rats.

We investigated the systemic effect of sclerostin monoclonal antibody (Scl-Ab) treatment on intact non-operated bones in an open osteotomy male Sprague Dawley (SD) rat model. Six-month-old male SD rats were subjected to transverse osteotomy at the right femur mid-shaft. Rats were injected subcutaneously with vehicle or Scl-Ab (25 mg/kg, 2 times per week) treatment for 9 weeks. Compared with vehicle control, Scl-Ab treatment significantly improved trabecular and cortical bone mass and microarchitecture at L5 vertebrae and left femora by micro-CT at week 6 and 9. Mechanical testing showed that Scl-Ab treatment resulted in significantly higher stiffness, energy to failure and ultimate load at the femora at week 9. Mineral apposition rate, mineralizing surface and bone formation rate on the trabecular bone in the distal femora was significantly increased in Scl-Ab group at week 6 and 9. The administered Scl-Ab was localized in the osteocytes and beta-catenin was strongly expressed in osteoblasts. Scl-Ab treatment significantly increased serum P1NP level and there was no between-group difference in serum level of CTX-1. In conclusion, Scl-Ab treatment could induce rapid and sustained increase in bone formation, bone mass and bone strength in non-operated bones. Sclerostin inhibition might be advantageous to prevent secondary fracture(s).

Skeletal consequences of RANKL-blocking antibody (IK22-5) injections during growth: mouse strain disparities and synergic effect with zoledronic acid.

High doses of bone resorption inhibitors are currently under evaluation in pediatric oncology. Previous works have evidenced transient arrest in long bone and skull bone growth and tooth eruption blockage when mice were treated with zoledronic acid (ZOL). The question of potential similar effects with a RANKL-blocking antibody (IK22.5) was raised. Sensitivity disparities in these inhibitors between mouse strains and synergic effects of zoledronic acid and a RANKL-blocking antibody were subsidiary questions. In order to answer these questions, newborn C57BL/6J and CD1 mice were injected every two or three days (4 injections in total so 7 or 10 days of treatment length) with high doses of a RANKL-blocking antibody. The consequences on the tibia, craniofacial bones and teeth were analyzed by µCT and histology at the end of the treatment and one, two and three months later. The results obtained showed that RANKL-blocking antibody injections induced a transient arrest of tibia and skull bone growth and an irreversible blockage of tooth eruption in C57BL/6J mice. In CD1 mice, tooth eruption defects were also present but only at much higher doses. Similar mouse strain differences were obtained with zoledronic acid. Finally, a synergic effect of the two inhibitors was evidenced. In conclusion as previously observed for bisphosphonates (ZOL), a RANKL-blocking antibody induced a transient arrest in long bone and skull bone growth and a blockage of tooth eruption with however disparities between mouse strains with regard to this last effect. A synergic effect of both bone resorption inhibitors was also demonstrated.

Adaptive immune response inhibits ectopic mature bone formation induced by BMSCs/BCP/plasma composite in immune-competent mice.

A combination of autologous bone marrow stromal cells (BMSCs) and biomaterials is a strategy largely developed in bone tissue engineering, and subcutaneous implantation in rodents or large animals is often a first step to evaluate the potential of new biomaterials. This study aimed at investigating the influence of the immune status of the recipient animal on BMSCs-induced bone formation. BMSCs prepared from C57BL/6 mice, composed of a mixture of mesenchymal stromal and monocytic cells, were combined with a biomaterial that consisted of biphasic calcium phosphate (BCP) particles and plasma clot. This composite was implanted subcutaneously either in syngenic C57BL/6 immune-competent mice or in T-lymphocyte-deficient Nude (Nude) mice. Using histology, immunohistochemistry, and histomorphometry, we show here that this BMSC/BCP/plasma clot composite implanted in Nude mice induces the formation of mature lamellar bone associated to hematopoietic areas and numerous vessels. Comparatively, implantation in C57BL/6 results in the formation of woven bone without hematopoietic tissue, a lower number of new vessels, and numerous multinucleated giant cells (MNGCs). In situ hybridization, which enabled to follow the fate of the BMSCs, revealed that BMSCs implanted in Nude mice survived longer than BMSCs implanted in C57BL/6 mice. Quantitative expression analysis of 280 genes in the implants indicated that the differences between C57BL/6 and Nude implants corresponded almost exclusively to genes related to the immune response. Gene expression profile in C57BL/6 implants was consistent with a mild chronic inflammation reaction characterized by Th1, Th2, and cytotoxic T-lymphocyte activation. In the implants retrieved from T-deficient Nude mice, Mmp14, Il6st, and Tgfbr3 genes were over-expressed, suggesting their putative role in bone regeneration and hematopoiesis. In conclusion, we show here that the T-mediated inflammatory microenvironment is detrimental to BMSCs-induced bone formation and shortens the survival of implanted cells. Conversely, the lack of T-lymphocyte reaction in T-deficient animals is beneficial to BMSCs-induced mature bone formation. This should be taken into account when evaluating cell/biomaterial composites in these models.

Effect of IL-15 and natural killer cells on osteoclasts and osteoblasts in a mouse coculture.

This study analyzes the effect of interleukin-15 (IL-15) on osteoclast formation using a coculture of mouse osteoblasts and bone marrow cells (BMCs) stimulated with prostaglandin E2 (PGE2), which both have important role in rheumatoid arthritis (RA) and periodontal disease (PD). BMCs isolate lacking T (BM(T-)) or NK (BM(NK-)) cells, BMCs with no cells removed (BM(T+NK+)), purified NK cells, and purified T cells were each cocultured with osteoblasts in the presence or absence of PGE2 and/or IL-15. The number of both osteoclasts and osteoblasts was decreased by IL-15 in a dose-dependent manner in BM(T+NK+), BM(T-). However, the reductions were improved in BM(NK-). The expression of caspase3 in osteoblasts cocultured with NK cells was increased in a dose-dependent manner by IL-15. IL-15 stimulates apoptosis of osteoblasts via activation of NK cells. Since osteoblasts have an important role in bone formation, IL-15 may be an inflammatory bone destructive factor in RA and PD.

Does complement play a role in bone development and regeneration?

The skeletal and the immune system are not two independent systems, rather, there are multifaceted and complex interactions between the different cell types of both systems and there are several shared cytokines. As a part of the innate immunity, the complement system was found to be an important link between bone and immunity. Complement proteins appear to be involved in bone development and homeostasis, and specifically influence osteoblast and osteoclast activity. This review describes the complex mutual regulation of the two systems, and indicates some of the negative side effects as a result of inappropriate or excessive complement activation.

Immune regulation of the tumor/bone vicious cycle.

The bone destruction attending skeletal metastasis is mediated by tumor-recruited osteoclasts (OCs). Hence, OCs are principal therapeutic targets in afflicted individuals. On the other hand, one-third of patients develop further skeletal-related events within two years of initiating antiresorptive therapies, suggesting that additional cells modulate bone tumor growth. Previous studies showing amelioration of bone metastases by targeting the OCs were performed in immune-compromised animals injected with human breast cancer cells. Consequently, the contribution of the immune system to bone tumor growth was unclear. Using genetic models of immune and OC modulation (PLCγ2 and Lyn), as well as pharmacological inhibition of OCs and T cells, we now demonstrate that a condition of immune deficiency can interfere with the antitumor effects of OC blockade. Thus, our findings expand the current tumor/bone vicious cycle model to include T cells as additional regulators of bone tumor growth, regardless of the OC status.

Preservation of femoral bone thickness in middle age predicts survival in genetically heterogeneous mice.

To see whether age-related changes in bone could predict subsequent lifespan, we measured multiple aspects of femur size and shape at 4, 15, and 24 months of age in genetically heterogeneous mice. Mice whose cortical bone became thicker from 4 to 15 months, associated with preservation of the endosteal perimeter, survived longer than mice whose endosteal cavity expanded, at the expense of cortical bone, over this age range. Femur size at age 4 months was also associated with a difference in life expectancy: mice with larger bones (measured by length, cortical thickness, or periosteal perimeter) had shorter lifespans. Femur length, midlife change in cortical bone thickness, and midlife values of CD8 T memory cells each added significant power for longevity prediction. Mice in the upper half of the population for each of these three endpoints lived, on average, 103 days (12%) longer than mice with the opposite characteristics. Thus, measures of young adult bone dimensions, changes as a result of bone remodeling in middle age, and immunological maturation provide partially independent indices of aging processes that together help to determine lifespan in genetically heterogeneous mice.

Osteoimmunology--the hidden immune regulation of bone.

Osteoimmunology is an emerging field of research dedicated to the investigation of the interactions between the immune and skeletal systems. These interactions are not only mediated by the release of cytokines and chemokines but also by direct cell-cell contact. Recently, it was proposed that immunoreceptors found in the immune cells are also an essential signal for osteoclasts activation, along with receptor activator NF-kappaB (RANK) ligand (RANKL) and macrophage-colony stimulating factor (M-CSF). In addition, adipose tissue also produces several factors (adipokines) that are known to interfere with the immune system and bone homeostasis. Chronic inflammation strongly influences osteoimmunology determining profound metabolic, structural and functional changes in bone.

Immunologic regulation of bone development.

A regulatory network comprised of transcription factors PU.1, Ikaros, E2A, EBF, and Pax5 control B cell fate specification and differentiation. Early B Cell Factor-1 (EBF-1) is essential for B cell fate specification while Pax5 is required for B cell development. Mice deficient in Pax5 or EBF-1 have a developmental arrest of B cell differentiation at the pro-B cell stage, which results in the absence of mature B cells. We analyzed the bone phenotype of Pax5 and EBF-1 wild-type (+/+) and homozygous mutant (-/-) mice to determine if the loss of these transcription factors regulated bone cell development. Bones from Pax5-/- mice were strikingly osteopenic 15 days after birth, with increased numbers of osteoclasts, and decreased trabecular number. The number of osteoblasts in Pax5-/- bones and their function in vitro were not different from controls. In addition, Pax5 was not expressed by wild-type osteoblasts. To investigate the origin of the in vivo increase in osteoclasts, Pax5-/- or +/+ spleen cells were cultured with M-CSF and RANKL and multinucleated, TRAP' cells counted. Cells from Pax5-/- spleen produced 5-10 times more osteoclasts than did controls. Tibia from EBF-1-/- mice had a striking increase in osteoblasts lining bone surfaces. Consistent with this was an increase in osteoid thickness and in the bone formation rate. This correlated with a 2-fold increase in serum osteocalcin. However, in vitro proliferation and ALP of mutant osteoblasts did not differ from control. In contrast, osteoclast number was similar in 4 week-old +/+ and -/- mice; however, at 12 weeks the number of osteoclasts was more than twice that of controls These data correlated with an increase in bone volume at 12 weeks of age. The most striking aspect of the EBF-1-/- bones was the presence of adipocytes, which filled the marrow space. The adipocytes in the marrow were present at both 4 and 12 weeks of age. Increased fat was also seen in the liver of mutant mice. However, subcutaneous fat was almost absent in EBF-1-/- mice. Importantly, EBF-1 mRNA was expressed in wild-type osteoblasts and in adipocytes. Loss of EBF-1 and Pax5 causes distinct, non-overlapping bone phenotypes. It is important to understand why this network of transcription factors, which are so important for B cell development, have such striking effects on bone cell growth and development.

Glucocorticoid-induced osteoporosis in children: impact of the underlying disease.

Glucocorticoids inhibit osteoblasts through multiple mechanisms, which results in significant reductions in bone formation. The growing skeleton may be especially vulnerable to adverse glucocorticoid effects on bone formation, which could possibly compromise trabecular and cortical bone accretion. Although decreased bone mineral density has been described in various pediatric disorders that require glucocorticoids, and a population-based study reported increased fracture risk in children who require >4 courses of glucocorticoids, some of the detrimental bone effects attributed to glucocorticoids may be caused by the underlying inflammatory disease. For example, inflammatory cytokines that are elevated in chronic disease, such as tumor necrosis factor alpha, suppress bone formation and promote bone resorption through mechanisms similar to glucocorticoid-induced osteoporosis. Summarized in this review are changes in bone density and dimensions during growth, the effects of glucocorticoids and cytokines on bone cells, the potential confounding effects of the underlying inflammatory-disease process, and the challenges in interpreting dual-energy x-ray absorptiometry results in children with altered growth and development in the setting of glucocorticoid therapy. Two recent studies of children treated with chronic glucocorticoids highlight the differences in the effect of underlying disease, as well as the importance of associated alterations in growth and development.

Osteoimmunology: shared mechanisms and crosstalk between the immune and bone systems.

Osteoimmunology is an interdisciplinary research field focused on the molecular understanding of the interplay between the immune and skeletal systems. Although osteoimmunology started with the study of the immune regulation of osteoclasts, its scope has been extended to encompass a wide range of molecular and cellular interactions, including those between osteoblasts and osteoclasts, lymphocytes and osteoclasts, and osteoblasts and haematopoietic cells. Therefore, the two systems should be understood to be integrated and operating in the context of the 'osteoimmune' system, a heuristic concept that provides not only a framework for obtaining new insights by basic research, but also a scientific basis for the discovery of novel treatments for diseases related to both systems.

Impaired skeletal development in interleukin-6-transgenic mice: a model for the impact of chronic inflammation on the growing skeletal system.

OBJECTIVE: To identify the mediator responsible for the impact of chronic inflammation on skeletal development in children (bone loss, defective peak bone mass accrual, stunted growth), we evaluated the effects of chronic interleukin-6 (IL-6) overexpression on the skeletons of growing prepubertal mice. METHODS: We studied IL-6-transgenic mice that had high circulating IL-6 levels since birth. Trabecular and cortical bone structure were analyzed by microcomputed tomography. Epiphyseal ossification, growth plates, and calvariae were studied by histology/histomorphometry. Osteoclastogenesis, osteoblast function/differentiation, and the effects of IL-6 on bone cells were studied in vitro. Osteoblast gene expression was evaluated by reverse transcriptase-polymerase chain reaction. The mineral apposition rate was evaluated dynamically in cortical bone by in vivo double fluorescence labeling. RESULTS: In prepubertal IL-6-transgenic mice, we observed osteopenia, with severe alterations in cortical and trabecular bone microarchitecture, as well as uncoupling of bone formation from resorption, with decreased osteoblast and increased osteoclast number and activity. Increased osteoclastogenesis and reduced osteoblast activity, secondary to decreased precursor proliferation and osteoblast function, were present. IL-6-transgenic mice also showed impaired development of growth plates and epiphyseal ossification centers. Intramembranous and endochondral ossification and the mineral apposition rate were markedly affected, showing the presence of defective ossification. CONCLUSION: Chronic overexpression of IL-6 alone induces a skeletal phenotype closely resembling growth and skeletal abnormalities observed in children with chronic inflammatory diseases, pointing to IL-6 as a pivotal mediator of the impact of chronic inflammation on postnatal skeletal development. We hypothesize that IL-6-modifying drugs may reduce skeletal defects and prevent the growth retardation associated with these diseases.