The Effects of Sclerostin on the Immune System.

PURPOSE OF REVIEW: We reviewed recent progress on the role of sclerostin (SOST) and its effects on the immune system in order to summarize the current state of knowledge in osteoimmunology, in regard to hematopoiesis, lymphopoiesis, and inflammation. RECENT FINDINGS: Changes in sclerostin levels affect distinct niches within the bone marrow that support hematopoietic stem cells and B cell development. Sclerostin's regulation of adipogenesis could also be important for immune cell maintenance with age. Surprisingly, B cell development in the bone marrow is influenced by Sost produced by mesenchymal stem cells and osteoblasts, but not by osteocytes. Additionally, extramedullary hematopoiesis in the spleen and increased pro-inflammatory cytokine levels in the bone marrow are observed in global Sost-/- mice. In addition to changes in bone marrow density, sclerostin depletion affects B lymphopoiesis and myelopoiesis, as well as other changes within the bone marrow cavity that could affect hematopoiesis. It is therefore important to monitor for hematopoietic changes in patients receiving sclerostin-depleting therapies.

Effect of TNF-α-Induced Sclerostin on Osteocytes during Orthodontic Tooth Movement.

Osteocytes are abundant cells in bone, which contribute to bone maintenance. Osteocytes express receptor activator of nuclear factor kappa-B ligand (RANKL) and regulate osteoclast formation. Orthodontic tooth movement (OTM) occurs by osteoclast resorption of alveolar bone. Osteocyte-derived RANKL is critical in bone resorption during OTM. Additionally, tumor necrosis factor-α (TNF-α) is important in osteoclastogenesis during OTM. Sclerostin has been reported to enhance RANKL expression in the MLO-Y4 osteocyte-like cell line. This study investigated the effect of TNF-α on sclerostin expression in osteocytes during OTM. In vitro analysis of primary osteocytes, which were isolated from DMP1-Topaz mice by sorting the Topaz variant of GFP-positive cells, revealed that SOST mRNA expression was increased when osteocytes were cultured with TNF-α and that RANKL mRNA expression was increased when osteocytes were cultured with sclerostin. Moreover, the number of TRAP-positive cells was increased in osteocytes and osteoclast precursors cocultured with sclerostin. In vivo analysis of mouse calvariae that had been subcutaneously injected with phosphate-buffered saline (PBS) or TNF-α revealed that the number of TRAP-positive cells and the percentage of sclerostin-positive osteocytes were higher in the TNF-α group than in the PBS group. Furthermore, the level of SOST mRNA was increased by TNF-α. As an OTM model, a Ni-Ti closed-coil spring connecting the upper incisors and upper-left first molar was placed to move the first molar to the mesial direction in wild-type (WT) mice and TNF receptor 1- and 2-deficient (TNFRsKO) mice. After 6 days of OTM, the percentage of sclerostin-positive osteocytes on the compression side of the first molar in TNFRsKO mice was lower than that in WT mice. In this study, TNF-α increased sclerostin expression in osteocytes, and sclerostin enhanced RANKL expression in osteocytes. Thus, TNF-α may play an important role in sclerostin expression in osteocytes and enhance osteoclast formation during OTM.

Osteoimmunology: evolving concepts in bone-immune interactions in health and disease.

In terrestrial vertebrates, bone tissue constitutes the 'osteoimmune' system, which functions as a locomotor organ and a mineral reservoir as well as a primary lymphoid organ where haematopoietic stem cells are maintained. Bone and mineral metabolism is maintained by the balanced action of bone cells such as osteoclasts, osteoblasts and osteocytes, yet subverted by aberrant and/or prolonged immune responses under pathological conditions. However, osteoimmune interactions are not restricted to the unidirectional effect of the immune system on bone metabolism. In recent years, we have witnessed the discovery of effects of bone cells on immune regulation, including the function of osteoprogenitor cells in haematopoietic stem cell regulation and osteoblast-mediated suppression of haematopoietic malignancies. Moreover, the dynamic reciprocal interactions between bone and malignancies in remote organs have attracted attention, extending the horizon of osteoimmunology. Here, we discuss emerging concepts in the osteoimmune dialogue in health and disease.

Pro-inflammatory Cytokines: Cellular and Molecular Drug Targets for Glucocorticoid-induced-osteoporosis via Osteocyte.

Glucocorticoids are widely used to treat varieties of allergic and autoimmune diseases, however, long-term application results in glucocorticoid-induced osteoporosis (GIOP). Inflammatory cytokines: tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) play important regulatory roles in bone metabolism, but their roles in GIOP remain largely unknown. Osteocytes can modulate the formation and function of both osteoblasts and osteoclasts, directly via gap junctions, or indirectly by transferring molecule signaling. Apoptotic osteocytes release RANKL, HMGB1 and pro-inflammatory cytokines to stimulate osteoclastogenesis. Moreover, osteocytes can secrete FGF23 to regulate bone metabolism. Exposure to high levels of GCs can drive osteocyte apoptosis and influence gap junctions, leading to bone loss. GCs treatment is regarded to produce more FGF23 to inhibit bone mineralization. GCs also disrupt the vascular to decrease osteocyte feasibility and mineral appositional rate, resulting in a decline in bone strength. Apoptotic bodies from osteocytes induced by GCs treatment can enhance production of TNF-α and IL-6. On the other hand, TNF-α and IL-6 show synergistic effects by altering osteocytes signaling towards osteoclasts and osteoblasts. In addition, TNF-α can induce osteocyte apoptosis and attribute to a worsened bone quality in GCs. IL-6 and osteocytes may interact with each other. Therefore, we hypothesize that GCs regulate osteocyteogenesis through TNF-α and IL-6, which are highly expressed around osteocyte undergoing apoptosis. In the present review, we summarized the roles of osteocytes in regulating osteoblasts and osteoclasts. Furthermore, the mechanism of GCs altered relationship between osteocytes and osteoblasts/osteoclasts. In addition, we discussed the roles of TNF-α and IL-6 in GIOP by modulating osteocytes. Lastly, we discussed the possibility of using pro-inflammatory signaling pathway as therapeutic targets to develop drugs for GIOP.

Lenalidomide regulates osteocytes fate and related osteoclastogenesis via IL-1ß/NF-κB/RANKL signaling.

Osteolytic diseases are closely associated with osteocyte fate, indicating a more efficient and crucial role of osteocyte-targeting strategy in inhibiting osteoclastogenesis. Here, we investigated the effects of lenalidomide (Lena) on osteocyte fate in order to regulate osteoclastogenesis via effective cascade-controlling response. Our data revealed that lenalidomide treatment notably rescued IL-1ß induced loss of osteocyte viability by inhibiting osteocyte apoptosis with decreased osteoclast-related factors, RANKL and Sclerostin, as demonstrated by the restricted osteoclast formation and reduced bone resorption. Additionally, iTRAQ assay revealed that IL-1ß induced activation of NF-κB inhibitor α/ß were remarkably downregulated by lenalidomide, showing that lenalidomide impaired NF-κB signaling in osteocytes for inhibiting the expression of osteoclast specific genes in osteoclasts, which was further confirmed by KEGG pathway analysis and Western blot. More interestingly, the in vivo analysis of osteocyte apoptosis and osteoclastogenesis in osteoarthritis mice model indicated a role of lenalidomide in the regulation of osteocyte fate and the consequent inhibition of RANKL-induced osteoclastogenesis. Together, these results suggest that lenalidomide regulates osteocyte fate by attenuating IL-1ß/NF-κB signaling, thereby inhibiting RANKL expression for the attenuated osteoclastogenesis both in vitro and vivo, indicating a more efficient remedy among future anti-osteoclastogenesis approaches.

TGF­ß1 expression in adults with non­traumatic osteonecrosis of the femoral head.

Non-traumatic osteonecrosis of the femoral head (NONFH) is a common clinical osteoarthropathy. The present study aimed to investigate the association between transforming growth factor ß1 (TGF­ß1) and NONFH. Femoral head specimens were collected from patients with NONFH. Patients with traumatic osteonecrosis served as the control. Hematoxylin and eosin (H&E) staining was used to visualize the bone tissue architecture. Immunohistochemistry and densitometry were performed to quantify TGF­ß1 expression in tissues. Flow cytometry was used to detect cluster of differentiation (CD)3+, CD4+ and CD8+ cells, and the ratio of CD4+ to CD8+ T cells in the peripheral blood. H&E staining revealed osteonecrosis, disintegration of osteocytes with karyopyknosis and karyorrhexis, loss of osteocyte lacunae, aberrantly arranged circumferential lamellae, as well as dissolution of the lamellae and subtle osteogenesis in the experimental group, as opposed to the control group. Immunohistochemistry revealed that the expression of TGF­ß1 was significantly reduced in the experimental group (P<0.01). Further, the NONFH group had a decrease in the CD3+ and CD4+ cell populations (P<0.05 and P<0.01, respectively), an increase in the CD8+ cell population (P<0.05), as well as a reduction in the ratio of CD4+ to CD8+ cells (P<0.01). The present study indicated that TGF­ß1 expression was reduced in NONFH. This was associated with impaired repairing capacity of the femoral head and dysregulated subsets of T­lymphocytes and possible immune functions.

miR-200bc/429 cluster alleviates inflammation in IgA nephropathy by targeting TWEAK/Fn14.

Immunoglobulin A nephropathy (IgAN) is one of the most common glomerular diseases worldwide. Various studies have identified a host of microRNAs (miRNAs) abnormally expressed in IgAN and might affect the pathogenesis and progression of IgAN. However, miR-200bc/429 cluster in the pathopoiesis of IgAN remains poorly understood. For this study, we found that miR-200bc/429 cluster is downregulated in IgAN tissues and IgAN podocytes and HK2 cells compared with their matched controls respectively. In addition, overexpression of miR-200bc/429 cluster in IgAN podocytes and HK2 cells could attenuate the release of inflammatory cytokines MCP-1, IL-6 and RANTES. Moreover, the 3' untranslated region (UTR) of TNF-like weak inducer of apoptosis (TWEAK) was identified to be a direct target of miR-200bc/429 cluster. Furthermore, our results showed that miR-200bc/429 cluster can inhibit TWEAK mediated NF-κB pathway activation in IgAN. Overall, our findings revealed that miR-200bc/429 cluster alleviates inflammation in IgAN through TWEAK/Fn14 system and might serve as a biomarker as well as a promising therapeutic target for IgAN.

The role of stromal cells in inflammatory bone loss.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation, local and systemic bone loss and a lack of compensatory bone repair. Fibroblast-like synoviocytes (FLS) are the most abundant cells of the stroma and a key population in autoimmune diseases such as RA. An increasing body of evidence suggests that these cells play not only an important role in chronic inflammation and synovial hyperplasia, but also impact bone remodelling. Under inflammatory conditions FLS release inflammatory cytokines, regulate bone destruction and formation and communicate with immune cells to control bone homeostasis. Other stromal cells, such as osteoblasts and terminally differentiated osteoblasts, termed osteocytes, are also involved in the regulation of bone homeostasis and are dysregulated during inflammation. This review highlights our current understanding of how stromal cells influence the balance between bone formation and bone destruction. Increasing our understanding of these processes is critical to enable the development of novel therapeutic strategies with which to treat bone loss in RA.

Comparison of tissue transglutaminase 2 and bone biological markers osteocalcin, osteopontin and sclerostin expression in human osteoporosis and osteoarthritis.

Osteoporosis (OP) and osteoarthritis (OA) are the most common joint diseases, with a high incidence in the elderly population. OP is characterized by trabecular bone remodeling and reabsorption, whereas articular cartilage and subchondral bone remodeling are major features of OA. Although classically considered as independent or even conflicting processes, clinical coexistence of OP and OA was recently described. Transglutaminase 2 (TG2) expression is considered a biomarker of OA, but its role in osteoporotic bone remodeling is still uncertain. We investigated TG2 and bone biological markers (Osteocalcin, Osteopontin, and Sclerostin) in osteoporotic and osteoarthritic osteocartilagineous tissue (n = 54) and human chondrocyte cultures in vitro by immunohistochemistry, immunofluorescence and RT-PCR. Histomorphometric evaluation of bone trabecular remodeling was also performed. In cartilage, TG2 expression was faint in control and OP and significantly less than in OA and OP + OA chondrocytes; the opposite was found for Osteocalcin, whereas Osteopontin and Sclerostin expression was similar. In the subchondral trabecular bone, osteocytes/osteoblasts TG2 expression was slight and similar comparing control, OP, OA, and OP + OA group, whereas Osteocalcin and Osteopontin expression was lower in OP compared to control, OA and OP + OA. Increased TG2 and reduced Osteocalcin expression were maintained in human osteoarthritic chondrocytes in vitro. Histomorphometric analysis confirmed reduced trabecular bone mass in OP and OP + OA compared with OA patients. TG2 represented a suitable biomarker of osteoarthritic chondrocyte activation, whereas osteocalcin and osteopontin characterized osteoporotic osteocyte/osteoblast changes; differences were lost in OP + OA patients, suggesting careful consideration when coexistence of the two diseases occurs.

[Mechanisms of myeloma-induced bone disease].

Multiple myeloma(MM)develops and expands almost exclusively in the bone marrow, and generates devastating bone destruction. MM cells produce a variety of cytokines to stimulate RANKL-mediated osteoclastogenesis and suppress osteoblastic differentiation from bone marrow stromal cells, leading to extensive bone destruction with rapid loss of bone. Furthermore, osteocyte apoptosis has been demonstrated to be induced in parallel with enhanced osteoclast recruitment and osteoclastogenesis in myeloma bone lesions. Of note, osteocytes physically interact with myeloma cells to skew their signaling pathways and thereby production of mediators responsible for exacerbated bone resorption and suppressed bone formation in myeloma. The role of osteocytes in myeloma-induced bone lesions remains to be further clarified.

T cells, osteoblasts, and osteocytes: interacting lineages key for the bone anabolic and catabolic activities of parathyroid hormone.

Osteoimmunology is a field of research dedicated to the study of the interactions between the immune system and bone. Among the cells of the immune system that regulate bone turnover and the responsiveness of bone cells to calciothropic hormones are bone marrow T lymphocytes. T cells secrete osteoclastogenic cytokines such as RANKL and TNF-α, as well as factors that stimulate bone formation, one of which is Wnt10b. In addition, T cells regulate the differentiation and life span of stromal cells (SCs) and their responsiveness to parathyroid hormone (PTH) via costimulatory molecules expressed on their surface. The conditioning effect of T cells on SCs is inherited by the osteoblastic and osteocytic progeny of SCs. As a result, osteoblastic cells of T cell-deficient mice have functional characteristics different from corresponding cells of T cell-replete mice. These differences include the ratio of RANKL/OPG produced in response to continuous PTH treatment, and the osteoblastogenic response to intermittent PTH treatment. This article reviews the evidence indicating that the effects of PTH are mediated not only by osteoblasts and osteocytes but also by T cells.

Isolation of mesenchymal stromal cells from extraembryonic tissues and their characteristics.

We describe a method of isolation of human mesenchymal stromal cells from the umbilical cord (Wharton's jelly) and human placenta: amnion, placental villi, and trophoblast. Morphology, immunophenotypic characteristics, and differentiation potencies of isolated cells were studied. The capacity of mesenchymal stromal cells from extraembryonic tissues to osteogenic, adipogenic, and chondrogenic differentiation was demonstrated and the dynamics of this process was described. The isolated cells met the criteria for multipotent mesenchymal stem cells.

[Anti-sclerostin antibody : its bone formation effect and therapeutic potential for osteoporosis].

In a rare accident of nature, some families have been found to have dense and strong bones due to a recessive loss of function mutation in the SOST gene that encodes for sclerostin, a protein expressed by osteocytes that downregulates osteoblastic bone formation. Knowledge of this molecule and its actions led rather quickly to the development of anti-sclerostin antibodies that lead to marked increases in bone mass in both animals and human subjects. Blocking sclerostin action with anti-sclerostin antibodies is a promising new therapeutic approach to osteoanabolic therapy of osteoporosis.

Cells of the immune system orchestrate changes in bone cell function.

There is a complex interplay between the cells of the immune system and bone. Immune cells, such as T and NK cells, are able to enhance osteoclast formation via the production of RANKL. Yet there is increasing evidence to show that during the resolution of inflammation or as a consequence of increased osteoclastogenesis there is an anabolic response via the formation of more osteoblasts. Furthermore, osteoblasts themselves are involved in the control of immune cell function, thus promoting the resolution of inflammation. Hence, the concept of "coupling"-how bone formation is linked to resorption-needs to be more inclusive rather than restricting our focus to osteoblast-osteoclast interactions as in a whole organism these cells are never in isolation. This review will investigate the role of immune cells in normal bone homeostasis and in inflammatory diseases where the balance between resorption and formation is lost.

Effect of BMP-2 protein on the count and osteogenic properties of multipotent stromal cells and expression of cytokine genes in primary cultures of bone marrow and spleen cells from CBA mice immunized with bacterial antigens.

We studied the effect of BMP-2 added to the culture medium on osteogenic and proliferative properties of multipotent stromal cells (MSC) and on the expression of cytokine genes induced by immunization of experimental animals with bacterial antigens. It is shown that the presence of BMP-2 in the culture medium stimulates proliferation of bone marrow MSC and especially spleen MSC (which was seen from enlargement of MSC colonies); improves the efficiency of MSC cloning; increases osteogenic activity of mouse bone marrow MSC; induces osteogenic differentiation of splenic MSC (osteogenesis is normally not observed in the spleen); reduces the number of macrophages in cultures; inhibits synthesis of mRNA for proinflammatory cytokines (IL-1ß, IL-6, IL-8, TNF-α) that typically occurs in cultures of the bone marrow and spleen from animals immunized with S. typhimurium or group A streptococcus antigens. Bearing in mind that proinflammatory cytokines negatively affect osteogenic activity of the bone marrow, we can hypothesize that BMP-2 not only stimulates osteogenesis, but also provides optimal conditions for its realization by suppressing the expression of genes encoding these cytokines.

[Osteoimmunology: how inflammation influences bone metabolism]. / Osteoimmunologie: Wie die Entzündung den Knochenstoffwechsel beeinflusst.

Bone remodelling is characterized by a balance between bone resorption and bone formation. The osteoblasts are responsible for bone synthesis and the osteoclasts for bone resorption. A finely adjusted interaction between molecular mechanisms leads, via cytokines, hormones and growth factors, to a homeostasis of the bone metabolism. Here, the RANK/RANKL/OPG-system is actively involved in the differentiation and function of osteoclasts and seems to play a central role in most pathophysiological mechanisms. An increased osteoclast activity results in inflammatory destructive manifestations and/or osteoporosis whereas an increased osteoblast activity can result in osteopetrosis. The present overview describes the known pathophysiological relevant metabolic pathways in this remodelling process especially the effect of inflammation on bone metabolism, and presents the links from bench to bedside.

CD106 identifies a subpopulation of mesenchymal stem cells with unique immunomodulatory properties.

Mesenchymal stem cells (MSCs) reside in almost all of the body tissues, where they undergo self-renewal and multi-lineage differentiation. MSCs derived from different tissues share many similarities but also show some differences in term of biological properties. We aim to search for significant differences among various sources of MSCs and to explore their implications in physiopathology and clinical translation. We compared the phenotype and biological properties among different MSCs isolated from human term placental chorionic villi (CV), umbilical cord (UC), adult bone marrow (BM) and adipose (AD). We found that CD106 (VCAM-1) was expressed highest on the CV-MSCs, moderately on BM-MSCs, lightly on UC-MSCs and absent on AD-MSCs. CV-MSCs also showed unique immune-associated gene expression and immunomodulation. We thus separated CD106(+)cells and CD106(-)cells from CV-MSCs and compared their biological activities. Both two subpopulations were capable of osteogenic and adipogenic differentiation while CD106(+)CV-MSCs were more effective to modulate T helper subsets but possessed decreased colony formation capacity. In addition, CD106(+)CV-MSCs expressed more cytokines than CD106(-)CV-MSCs. These data demonstrate that CD106 identifies a subpopulation of CV-MSCs with unique immunoregulatory activity and reveal a previously unrecognized mechanism underlying immunomodulation of MSCs.

Immunomodulatory effects of human umbilical cord Wharton's jelly-derived mesenchymal stem cells on differentiation, maturation and endocytosis of monocyte-derived dendritic cells.

The Wharton's jelly of the umbilical cord is believed to be a source of mesenchymal stem cells (MSCs) which can be therapeutically applied in degenerative diseases.In this study, we investigated the immunomodulatory effect of umbilical cord derived-mesenchymal stem cells (UC-MSCs) and bone marrow-derived-mesenchymal stem cells (BM-MSCs) on differentiation, maturation, and endocytosis of monocyte-derived dendritic cells in a transwell culture system under laboratory conditions. Monocytes were differentiated into immature dendritic cells (iDCs) in the presence of GM-CSF and IL-4 for 6 days and then differentiated into mature dendritic cells (mDCs) in the presence of TNF-α for 2 days. In every stage of differentiation, immature and mature dendritic cells were separately co-cultured with UC-MSCs and BM-MSCs. The findings showed that UC-MSCs and BM-MSCs inhibited strongly differentiation and maturation of dendritic cells at higher dilution ratios (1:1). The BM-MSCs and UC-MSCs showed more inhibitory effect on CD1a, CD83, CD86 expression, and dendritic cell endocytic activity, respectively. On the other hand, these cells severely up-regulated CD14 marker expression. We concluded that UC-MSCs and BM-MSCs could inhibit differentiation, maturation and endocytosis in monocyte-derived DCs through the secreted factors and free of any cell-cell contacts under laboratory conditions. As DCs are believed to be the main antigen presenting cells for naïve T cells in triggering immune responses, it would be logical that their inhibitory effect on differentiation, maturation and function can decrease or modulate immune and inflammatory responses.

Human Wharton's jelly mesenchymal stem cells maintain the expression of key immunomodulatory molecules when subjected to osteogenic, adipogenic and chondrogenic differentiation in vitro: new perspectives for cellular therapy.

Rheumatoid arthritis and osteoarthritis are the main diseases that imply an inflammatory process at the joints involving the articular cartilage. Recently, mesenchymal stem cells (MSCs) derived from perinatal tissues were considered good candidates for cellular therapy of musculoskeletal and orthopaedic diseases, since they can differentiate into multiple cell types and are an easily accessible cellular source. Therefore, several protocols exist on the differentiation of mesenchymal stem cells of different origins into osteoblasts and chondrocytes. Another key feature of MSCs is their capacity to modulate the immune system responses in vitro and in vivo. This may have critical outcomes in diseases of the musculoskeletal system where an inflammatory or autoimmune process is at the basis of the main disease. In the present paper, after isolation of MSCs from Wharton's Jelly (WJ-MSCs), we performed the three standard differentiation protocols. The acquisition of the differentiated phenotype was demonstrated by the specific histological stains. As the main objective of this work, we determined the expression of immunomodulatory molecules (by immunohistochemistry and qualitative RT-PCR), both in undifferentiated cells and after differentiation. We demonstrated for the first time that immune-related molecules (as B7-H3/CD276 and HLA-E) which have been characterized in undifferentiated MSCs, are also expressed by the differentiated progeny. This strongly suggests that also after the acquisition of a mature phenotype, WJ-MSCs-derived cells may maintain their immune privilege. This evidence, which deserves much work to be confirmed in vivo and in other MSCs populations, may provide a formal proof of the good results globally achieved with WJMSCs as cellular therapy vehicle.