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Osteoclasts (OC) originate from either bone marrow (BM)–resident or circulating myeloid OC progenitors (OCP) expressing the receptor CX3CR1. Multiple lines of evidence argue that OCP in homeostasis and inflammation differ. We investigated the relative contributions of BM-resident and circulating OCP to osteoclastogenesis during homeostasis and fracture repair. Using CX3CR1-EGFP/TRAP tdTomato mice, we found CX3CR1 expression in mononuclear cells, but not in multinucleated TRAP+ OC. However, CX3CR1-expressing cells generated TRAP+ OC on bone within 5 d in CX3CR1CreERT2/Ai14 tdTomato reporter mice. To define the role that circulating cells play in osteoclastogenesis during homeostasis, we parabiosed TRAP tdTomato mice (CD45.2) on a C57BL/6 background with wild-type (WT) mice (CD45.1). Flow cytometry (CD45.1/45.2) demonstrated abundant blood cell mixing between parabionts after 2 wk. At 4 wk, there were numerous tdTomato+ OC in the femurs of TRAP tdTomato mice but almost none in WT mice. Similarly, cultured BM stimulated to form OC demonstrated multiple fluorescent OC in cell cultures from TRAP tdTomato mice, but not from WT mice. Finally, flow cytometry confirmed low-level engraftment of BM cells between parabionts but significant engraftment in the spleens. In contrast, during fracture repair, we found that circulating CX3CR1+ cells migrated to bone, lost expression of CX3CR1, and became OC. These data demonstrate that OCP, but not mature OC, express CX3CR1 during both homeostasis and fracture repair. We conclude that, in homeostasis mature OC derive predominantly from BM-resident OCP, whereas during fracture repair, circulating CX3CR1+ cells can become OC.

Sexual dimorphism of the skeleton is well documented. At maturity, the male skeleton is typically larger and has a higher bone density than the female skeleton. However, the underlying mechanisms for these differences are not completely understood. In this study, we examined sexual dimorphism in the formation of osteoclasts between cells from female and male mice. We found that the number of osteoclasts in bones was greater in females. Similarly, in vitro osteoclast differentiation was accelerated in female osteoclast precursor (OCP) cells. To further characterize sex differences between female and male osteoclasts, we performed gene expression profiling of cultured, highly purified, murine bone marrow OCPs that had been treated for 3 days with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). We found that 125 genes were differentially regulated in a sex-dependent manner. In addition to genes that are contained on sex chromosomes, transcriptional sexual dimorphism was found to be mediated by genes involved in innate immune and inflammatory response pathways. Furthermore, the NF-κB-NFATc1 axis was activated earlier in female differentiating OCPs, which partially explains the differences in transcriptomic sexual dimorphism in these cells. Collectively, these findings identify multigenic sex-dependent intrinsic difference in differentiating OCPs, which results from an altered response to osteoclastogenic stimulation. In humans, these differences could contribute to the lower peak bone mass and increased risk of osteoporosis that females demonstrate relative to males. © 2021 American Society for Bone and Mineral Research (ASBMR).

Osteoclasts (OC) originate from either bone marrow (BM)-resident or circulating myeloid OC progenitors (OCP) expressing the receptor CX

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

We examined the effects that ovariectomy had on sclerostin mRNA and protein levels in the bones of 8-week-old mice that were either sham-operated (SHAM) or ovariectomized (OVX) and then euthanized 3 or 6 weeks later. In this model, bone loss occurred between 3 and 5 weeks postsurgery. In calvaria, ovariectomy significantly decreased sclerostin mRNA levels at 6 weeks postsurgery (by 52%) but had no significant effect at 3 weeks. In contrast, sclerostin mRNA levels were significantly lower in OVX femurs at 3 weeks postsurgery (by 53%) but equal to that of SHAM at 6 weeks. The effects of ovariectomy on sclerostin were not a global response of osteocytes because they were not mimicked by changes in the mRNA levels for two other relatively osteocyte-specific genes: DMP-1 and FGF-23. Sclerostin protein decreased by 83% and 60%, at 3 and 6 weeks postsurgery in calvaria, respectively, and by 38% in lumbar vertebrae at 6 weeks. We also detected decreases in sclerostin by immunohistochemistry in cortical osteocytes of the humerus at 3 weeks postsurgery. However, there were no significant effects of ovariectomy on sclerostin protein in femurs or on serum sclerostin at 3 and 6 weeks postsurgery. These results demonstrate that ovariectomy has variable effects on sclerostin mRNA and protein in mice, which are dependent on the bones examined and the time after surgery. Given the discrepancy between the effects of ovariectomy on serum sclerostin levels and sclerostin mRNA and protein levels in various bones, these results argue that, at least in mice, serum sclerostin levels may not accurately reflect changes in the local production of sclerostin in bones. Additional studies are needed to evaluate whether this is also the case in humans.

Runt-related transcription factor 1 (Runx1), a master regulator of hematopoiesis, is expressed in preosteoclasts. Previously we evaluated the bone phenotype of CD11b-Cre Runx1fl/fl mice and demonstrated enhanced osteoclasts and decreased bone mass in males. However, an assessment of the effects of Runx1 deletion in female osteoclast precursors was impossible with this model. Moreover, the role of Runx1 in myeloid cell differentiation into other lineages is unknown. Therefore, we generated LysM-Cre Runx1fl/fl mice, which delete Runx1 equally (∼80% deletion) in myeloid precursor cells from both sexes and examined the capacity of these cells to differentiate into osteoclasts and phagocytic and antigen-presenting cells. Both female and male LysM-Cre Runx1fl/fl mice had decreased trabecular bone mass (72% decrease in bone volume fraction) and increased osteoclast number (2–3 times) (P < .05) without alteration of osteoblast histomorphometric indices. We also demonstrated that loss of Runx1 in pluripotential myeloid precursors with LysM-Cre did not alter the number of myeloid precursor cells in bone marrow or their ability to differentiate into phagocytizing or antigen-presenting cells. This study demonstrates that abrogation of Runx1 in multipotential myeloid precursor cells significantly and specifically enhanced the ability of receptor activator of nuclear factor-κB ligand to stimulate osteoclast formation and fusion in female and male mice without affecting other myeloid cell fates. In turn, increased osteoclast activity in LysM-Cre Runx1fl/fl mice likely contributed to a decrease in bone mass. These dramatic effects were not due to increased osteoclast precursors in the deleted mutants and argue that inhibition of Runx1 in multipotential myeloid precursor cells is important for osteoclast formation and function.

Leukemia inhibitory factor (LIF) is a recently characterized glycoprotein with complex biologic activities on bone cells.We tested various rodent and human immortalized and malignant bone cell lines and primary osteoblast-enriched cell cultures from fetal rat calvarial digests for expression of LIF mRNA and LIF protein.Both human and rodent immortalized and malignant cells expressed a single 4.4 kb mRNA transcript that hybridized to a human LIF cDNA probe in Northern blots.LIF mRNA was undetectable in unstimulated rodent osteoblast-like cell lines MC3T3- E1 and Py1a.However, treatment with LPS (10ug/ml), TGF-beta (1ng/ml), TNF-alpha (100ng/ml) or inhibitors of protein synthesis (cycloheximide, emetine, puromycin and anisomycin) induced the expression of LIF message in these cells.In contrast, primary osteoblast-enriched cells did not express LIF mRNA in Northern blot assays either constitutively or after treatment with TNF-alpha or cycloheximide.The human osteosarcoma cell lines U-2 OS and Saos-2 constitutively expressed LIF mRNA and did not respond to LPS treatment.However, phorbol myristate acetate (PMA), an activator of protein kinase C, was a potent stimulator of LIF message in Saos-2 but not U-2 OS cells.The effects of PMA (0.5 ng/ml) on LIF mRNA in Saos-2 cells were detectable at 1h and maximal at 6h.TNF-alpha (100 ng/ml) and inhibitors of protein synthesis also increased LIF mRNA in both Saos-2 and U-2 OS cells.LIF protein was also detected constitutively in the conditioned medium from both Saos-2 and U-2 OS cells.IN adition, TNF (100 ng/ml) stimulated the release of LIF protein from both these cellc and PMA (2.5 ng/ml) stimulated LIF protein in Saos-2 cells.These results show that several different human malignant and rodent immortalized clonal bone cell lines can express and regulate steady-state LIF mRNA levels and produce LIF protein but that primary cultures of fetal rat osteoblastic cells do not express this cytokine.Hence, LIF may regulate malignant osteogenic cell growth and function in bone but may not be an important regulator of normal bone metabolism.

We examined the direct effects of IL-7 on osteoclastogenesis in murine bone marrow cultures, using cells from wild-type and IL-7- and IL-7 receptor (IL-7R)-deficient mice. IL-7 inhibited osteoclast-like cells (OCL) formation in macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL)-stimulated (both at 30 ng/ml) murine bone marrow cultures. Significant inhibitory effects were seen at 1 ng/ml (57%) and 10 ng/ml (86%). IL-7 also inhibited (P < 0.05) OCL formation in bone marrow cultures that were stimulated with vitamin D(3) (10(-8) M, 60%), bovine PTH (bPTH) (100 ng/ml, 54%), or RANKL alone (30 ng/ml, 50%). IL-7 (10 ng/ml) increased expression of the B lymphocyte marker B220 from 40-86% of total nonadherent cells in cultures treated with M-CSF and RANKL. Bone marrow cells from IL-7-deficient [IL-7 knockout (KO)] mice showed a significant (P < 0.05) increase in tartrate-resistant acid phosphatase(+) OCL numbers in cultures that were stimulated with vitamin D(3) (136 +/- 13.3%), bPTH (196 +/- 18.8%), or M-CSF and RANKL (160 +/- 7.2%). In contrast, in vitro osteoclast formation in bone marrow from IL-7R-deficient (IL-7R KO) mice showed a significant decrease in tartrate-resistant acid phosphatase(+) OCL numbers in cultures that were stimulated with vitamin D(3), PTH, RANKL, or M-CSF and RANKL. These results demonstrate that there are differences in the mechanisms regulating OCL formation between IL-7 KO and IL-7R KO cells. It seems that IL-7 is a direct inhibitor of OCL formation in vitro, based on results of adding IL-7 to wild-type cultures and the responses of IL-7 KO cells. It is unknown why IL-7R KO cells behave differently from IL-7 KO cells in vitro. However, it is possible that additional cytokines interact with IL-7R and that loss of these signals contributes to the responses of IL-7R KO cells. Alternatively, IL-7 may interact with multiple receptors.

We examined the ability of 1,25 (OH)2 vitamin D3 (Vit D) to stimulate osteoclast-like cell (OCL) formation in cocultures of spleen cells and primary calvarial osteoblasts from wild-type (WT) and IL-1R type 1-deficient (knockout; KO) mice. Vit D dose dependently increased OCL in cocultures containing WT osteoblasts. In contrast, there was a 90% reduction in OCL numbers in cocultures containing KO osteoblasts. In cocultures with either WT or KO osteoblasts, treatment with Vit D increased receptor activator of NF-κB ligand mRNA by 17-, 19-, or 3.5-fold, respectively. Vit D decreased osteoprotegerin mRNA to undetectable in all groups. Intracellular IL-1α protein increased after Vit D treatment in cocultures containing WT, but not KO osteoblasts. We also examined direct effects of Vit D, IL-1α, and their combination on gene expression in primary osteoblasts. In WT cells, Vit D and IL-1 stimulated receptor activator of NF-κB ligand mRNA expression by 3- and 4-fold, respectively, and their combination produced a 7-fold increase. Inhibition of osteoprotegerin mRNA in WT cells was partial with either agent alone and greatest with their combination. In KO cells, only Vit D stimulated a response. IL-1 alone increased IL-1α protein expression in WT osteoblasts. However, in combination with Vit D, there was a synergistic response (100-fold increase). In KO cultures, there were no effects of IL-1, Vit D, or their combination on IL-1α protein. These results demonstrate interactions between IL-1 and Vit D in primary osteoblasts that appear important in both regulation of IL-1α production and the ability of Vit D to support osteoclastogenesis.

available at http://www.ncbi.nlm.nih.gov/pubmed/25838546 Editorial Comment: Frailty is a geriatric syndrome that is associated with increased vulnerability in multiple clinical domains. The causes of frailty are complex and multifactorial. This study examined the potential link between nutritional status and frailty with an emphasis on dietary polyphenols. These compounds include carotenoids, vitamins E and D and bioflavonoids, and appear to have antioxidant and anti-inflammatory properties. Using a large, cross-sectional, population based study cohort, the levels of urinary polyphenols were compared with measures of frailty in 811 community dwelling people 65 years or older in Italy. The Mediterranean diet tends to be rich in polyphenols, and this group provided a unique population in which to study this question. The results demonstrated an inverse relationship between urinary polyphenol levels and degree of frailty. Sense of physical exhaustion and slowed gait were the frailty parameters most closely linked to decreased measured levels of urinary polyphenols. The cross-sectional study design precluded determination of absolute causality but the results raise interesting questions about the relationship between these variables. Future prospective nutritional research targeting this question will be required to enhance our understanding of this process, including the potential benefits of polyphenol rich foods as a mechanism to slow progression of the frailty phenotype. Abstract available at http://www.ncbi.nlm.nih.gov/pubmed/26215633 Editorial Comment: Delirium is a serious potential complication that often occurs postoperatively in geriatric patients. The etiology is generally thought to be multifactorial, and prevention is considered a key element in care of older adults. This study examined the potential relationship between inflammatory cytokines as biomarkers for risk of delirium in a cohort of 566 older adults undergoing major surgery. A nested, matched control study was performed to account for variables such as patient age and gender, type of surgery, baseline cognitive status, vascular comorbidity and apolipoprotein E, which is a genetic risk factor for dementia. The overall incidence of delirium was 24%, and those who demonstrated this condition by postoperative day 2 had significantly increased levels of interleukin-6 compared to controls. Patients with delirium also had increased interleukin-2 levels in the immediate postoperative period compared to controls, and this finding continued longitudinally through multiple subsequent time points. With time levels of other cytokines, including vascular endothelial growth factor and tumor necrosis factor-alpha, also differed between those who did and did not show delirium. These data suggest that there is a link between inflam- matory cytokines and the risk of delirium in the elderly surgical population. This finding may help lead to future development of preoperative or perioperative screening tests using these biomarkers to help reduce the incidence or severity of delirium postoperatively in older adults.

Receptor activator of NF-kappaB ligand (RANKL) and interleukin-1 (IL-1) individually plays a critical role in the differentiation and activation of osteoclasts in bone. In addition, both RANKL and IL-1 activate similar signal transduction pathways including p38 MAP kinase and c-Jun NH(2) terminal kinase (JNK). We examined if endogenously produced IL-1 influenced osteoclast-like cell (OCL) formation in murine bone marrow and bone marrow monocyte (BMM) cultures that were stimulated with M-CSF and RANKL. RANKL stimulated OCL formation in a dose-dependent manner in bone marrow cultures, and this response was significantly inhibited by IL-1 RA (100 ng/ml), a specific IL-1 antagonist. Interleukin-1 further increased OCL formation in BMM cultures that were treated with M-CSF (30 ng/ml) and RANKL (1, 3, 10 and 30 ng/ml). In addition, BMM cultures from IL-1 type I receptor-deficient mice, which do not respond to IL-1, demonstrated significantly less OCL formation compared to wild-type BMM cultures. We examined the time course and dose response of IL-1alpha protein expression by ELISA in BMM cultures that were treated with or without M-CSF and RANKL. RANKL dose dependently stimulated IL-1alpha protein significantly (up to 46%) in 6-day cultures. The interaction of RANKL and IL-1 on osteoclastogenesis did not appear significantly dependent on prostaglandin synthesis since PGE(2) expression in the conditioned medium of BMM cultures was nearly undetectable and the PGHS-2 specific inhibitor, NS-398, was without effect. We also investigated the effect of IL-1 on p38 MAP kinase and JNK in BMM cultures. The combination of RANKL and IL-1 had additive effects on JNK but not p38 MAP kinase compared to results in cultures treated with RANKL or IL-1 alone. In addition, SP600125, a specific JNK inhibitor, markedly reduced OCL formation in BMM cultures that were treated with RANKL or the combination of RANKL and IL-1. These findings demonstrate that endogenously produced IL-1 augments the response of bone marrow cells to RANKL, and this effect appears mediated by mechanisms that are associated with enhancement of JNK activity.

We examined the osteoclastogenic potential of murine bone marrow cells that were fractionated according to their expression of the surface antigen CD45R. Osteoclast-like cells (OCL) with many authentic osteoclast characteristics readily formed in purified CD45R(+) murine bone marrow cell cultures after treatment with receptor activator of nuclear factor kappaB ligand (RANKL) and M-CSF. Ovariectomy (Ovx) caused a 1.5- to 2-fold increase in OCL number in unfractionated and CD45R(+) murine bone marrow cell cultures without affecting OCL formation in CD45R(-) marrow cells. Limiting dilution assays confirmed that Ovx caused an increase in osteoclast precursor cell number in CD45R(+) but not CD45R(-) cells. Mice deficient in the type 1 IL-1 receptor (IL-1R1 KO) do not lose bone mass after Ovx. We found that unfractionated, CD45R(+), and CD45R(-) bone marrow cells from IL-1R1 KO mice showed no increase in OCL formation in vitro after Ovx. In both the wild-type (WT) and the IL-1R1 KO mice Ovx was associated with a 2-fold increase in pre-B-lymphocytes. About 1.3-3.5% of murine marrow cells expressed surface RANK (the receptor for RANKL) while about 11.9-15% of murine bone marrow cells expressed c-Fms (the receptor for M-CSF). There was little effect of Ovx on cells expressing either RANK or c-Fms. These results demonstrate that CD45R expression identifies a subset of murine bone marrow cells whose ability to form OCL in vivo is regulated by estrogen in WT but not IL-1R1 KO cells. The effects of estrogen on bone mass may be related to these responses.

Leukemia inhibitory factor (LIF) is a recently described cytokine with a variety of actions including a possible involvement in immune responses. We determined whether human dermal fibroblast cultures could produce LIF after they were treated with tumor necrosis factor alpha (TNF alpha), a cytokine that is produced as an early inflammatory response of activated monocytes. We found that treatment of the cultures with as little as 0.5 units/ml (1.5 pM) caused a detectable increase in both LIF message and protein as measured by Northern blot assay and ELISA, respectively. Furthermore, increasing concentrations of TNF alpha produced a dose-dependent increase in both steady-state LIF mRNA and protein levels up to a maximum response with 500 units/ml (1.5 nM). Increases in LIF mRNA levels were rapid and could be detected 1 hr after treatment with 500 units/ml of TNF alpha. However, this effect was transient. It reached a maximum at 2 hr and returned almost to baseline at 24 hr. In contrast, levels of LIF protein in the conditioned media of the cultures increased progressively over 24 hr. The LIF produced by these cultures was biologically active and was inhibited by a polyclonal antibody to human LIF in a bioassay. These results demonstrate that LIF is produced by human dermal fibroblasts in response to treatment with TNF alpha, a mediator of acute inflammation. Furthermore, they suggest that production of LIF by these cells may be involved in the development of both the local and generalized immune response.