Osteoclast heterogeneity: Lessons from osteopetrosis and inflammatory conditions

AbstractThe multinucleated osteoclast has a unique function: degradation of mineralized tissues. It is generally taken that all osteoclasts are alike, independent of the skeletal site where they exert their activity. Recent data, however, question this view as they show that osteoclasts at different bony sites appear to differ, for example in the machinery responsible for resorption. Support for the notion that there may be heterogeneity in osteoclasts is obtained from studies in which osteoclast activity is inhibited and from observations in osteopetrosis and inflammatory bone conditions. In this review we discuss the available evidence and propose the existence of bone-site-specific osteoclast heterogeneity

What Are the Peripheral Blood Determinants for Increased Osteoclast Formation in the Various Inflammatory Diseases Associated With Bone Loss?

Local priming of osteoclast precursors (OCp) has long been considered the main and obvious pathway that takes place in the human body, where local bone lining cells and RANKL-expressing osteocytes may facilitate the differentiation of OCp. However, priming of OCp away from bone, such as in inflammatory tissues, as revealed in peripheral blood, may represent a second pathway, particularly relevant in individuals who suffer from systemic bone loss such as prevalent in inflammatory diseases. In this review, we used a systematic approach to review the literature on osteoclast formation in peripheral blood in patients with inflammatory diseases associated with bone loss. Only studies that compared inflammatory (bone) disease with healthy controls in the same study were included. Using this core collection, it becomes clear that experimental osteoclastogenesis using peripheral blood from patients with bone loss diseases in prevalent diseases such as rheumatoid arthritis, osteoporosis, periodontitis, and cancer-related osteopenia unequivocally point toward an intrinsically increased osteoclast formation and activation. In particular, such increased osteoclastogenesis already takes place without the addition of the classical osteoclastogenesis cytokines M-CSF and RANKL in vitro. We show that T-cells and monocytes as OCp are the minimal demands for such unstimulated osteoclast formation. In search for common and disease-specific denominators of the diseases with inflammation-driven bone loss, we demonstrate that altered T-cell activity and a different composition—such as the CD14+CD16+ vs. CD14+CD16– monocytes—and priming of OCp with increased M-CSF, RANKL, and TNF- α levels in peripheral blood play a role in increased osteoclast formation and activity. Future research will likely uncover the barcodes of the OCp in the various inflammatory diseases associated with bone loss

Chronic exposure of gingival fibroblasts to TLR2 or TLR4 agonist inhibits osteoclastogenesis but does not affect osteogenesis

Chronic exposure to periodontopathogenic bacteria such as Porphyromonas gingivalis and the products of these bacteria that interact with the cells of the tooth surrounding tissues can ultimately result in periodontitis. This is a disease that is characterized by inflammation-related alveolar bone degradation by the bone-resorbing cells, the osteoclasts. Interactions of bacterial products with Toll-like receptors (TLRs), in particular TLR2 and TLR4, play a significant role in this chronic inflammatory reaction, which possibly affects osteoclastic activity and osteogenic capacity. Little is known about how chronic exposure to specific TLR activators affects these two antagonistic activities. Here, we studied the effect of TLR activation on gingival fibroblasts (GF), cells that are anatomically close to infiltrating bacterial products in the mouth. These were co-cultured with naive osteoclast precursor cells (i.e., monocytes), as part of the peripheral blood mononuclear cells (PBMCs). Activation of GF co-cultures (GF + PBMCs) with TLR2 or TLR4 agonists resulted in a weak reduction of the osteoclastogenic potential of these cultures, predominantly due to TLR2. Interestingly, chronic exposure, especially to TLR2 agonist, resulted in increased release of TNF-α at early time points. This effect, was reversed at later time points, thus suggesting an adaptation to chronic exposure. Monocyte cultures primed with M-CSF + RANKL, led to the formation of bone-resorbing osteoclasts, irrespective of being activated with TLR agonists. Late activation of these co-cultures with TLR2 and with TLR4 agonists led to a slight decrease in bone resorption. Activation of GF with TLR2 and TLR4 agonists did not affect the osteogenic capacity of the GF cells. In conclusion, chronic exposure leads to diverse reactions; inhibitory with naive osteoclast precursors, not effecting already formed (pre-)osteoclasts. We suggest that early encounter of naive monocytes with TLR agonists may result in differentiation toward the macrophage lineage, desirable for clearing bacterial products. Once (pre-)osteoclasts are formed, these cells may be relatively insensitive for direct TLR stimulation. Possibly, TLR activation of periodontal cells indirectly stimulates osteoclasts, by secreting osteoclastogenesis stimulating inflammatory cytokines

Mapping of DNA methylationsensitive cellular processes in gingival and periodontal ligament fibroblasts in the context of periodontal tissue homeostasis

Interactions between gingival fibroblasts (GFs) and oral pathogens contribute to the chronicity of inflammation in periodontitis. Epigenetic changes in DNA methylation are involved in periodontitis pathogenesis, and recent studies indicate that DNA methyltransferase (DNMT) inhibitors may protect against epithelial barrier disruption and bone resorption. To assess the impact of DNMT inhibition on GFs, cells were cultured with decitabine (5-aza-2’-deoxycytidine, DAC) for 12 days to induce DNA hypomethylation. We observed several potentially detrimental effects of DAC on GF biological functions. First, extended treatment with DAC reduced GF proliferation and induced necrotic cell death. Second, DAC amplified Porphyromonas gingivalis- and cytokine-induced expression and secretion of the chemokine CCL20 and several matrix metalloproteinases (MMPs), including MMP1, MMP9, and MMP13. Similar pro-inflammatory effects of DAC were observed in periodontal ligament fibroblasts. Third, DAC upregulated intercellular adhesion molecule-1 (ICAM-1), which was associated with increased P. gingivalis adherence to GFs and may contribute to bacterial dissemination. Finally, analysis of DAC-induced genes identified by RNA sequencing revealed increased expression of CCL20, CCL5, CCL8, CCL13, TNF, IL1A, IL18, IL33, and CSF3, and showed that the most affected processes were related to immune and inflammatory responses. In contrast, the genes downregulated by DAC were associated with extracellular matrix and collagen fibril organization. Our observations demonstrate that studies of DNMT inhibitors provide important insights into the role of DNA methylation in cells involved in periodontitis pathogenesis. However, the therapeutic potential of hypomethylating agents in periodontal disease may be limited due to their cytotoxic effects on fibroblast populations and stimulation of pro-inflammatory pathways.</p

Jaw and Long Bone Marrows Have a Different Osteoclastogenic Potential

Osteoclasts, the multinucleated bone-resorbing cells, arise through fusion of precursors from the myeloid lineage. However, not all osteoclasts are alike; osteoclasts at different bone sites appear to differ in numerous respects. We investigated whether bone marrow cells obtained from jaw and long bone differed in their osteoclastogenic potential. Bone marrow cells from murine mandible and tibiae were isolated and cultured for 4 and 6 days on plastic or 6 and 10 days on dentin. Osteoclastogenesis was assessed by counting the number of TRAP+ multinucleated cells. Bone marrow cell composition was analyzed by FACS. The expression of osteoclast- and osteoclastogenesis-related genes was studied by qPCR. TRAP activity and resorptive activity of osteoclasts were measured by absorbance and morphometric analyses, respectively. At day 4 more osteoclasts were formed in long bone cultures than in jaw cultures. At day 6 the difference in number was no longer observed. The jaw cultures, however, contained more large osteoclasts on plastic and on dentin. Long bone marrow contained more osteoclast precursors, in particular the myeloid blasts, and qPCR revealed that the RANKL:OPG ratio was higher in long bone cultures. TRAP expression was higher for the long bone cultures on dentin. Although jaw osteoclasts were larger than long bone osteoclasts, no differences were found between their resorptive activities. In conclusion, bone marrow cells from different skeletal locations (jaw and long bone) have different dynamics of osteoclastogenesis. We propose that this is primarily due to differences in the cellular composition of the bone site-specific marrow

Fibrodysplasia Ossificans Progressiva: what have we achieved and where are we now? follow-up to the 2015 Lorentz Workshop

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare progressive genetic disease effecting one in a million individuals. During their life, patients with FOP progressively develop bone in the soft tissues resulting in increasing immobility and early death. A mutation in the ACVR1 gene was identified as the causative mutation of FOP in 2006. After this, the pathophysiology of FOP has been further elucidated through the efforts of research groups worldwide. In 2015, a workshop was held to gather these groups and discuss the new challenges in FOP research. Here we present an overview and update on these topics

Gene Therapy for Fibrodysplasia Ossificans Progressiva: Feasibility and Obstacles

Fibrodysplasia ossificans progressiva (FOP) is a rare and devastating genetic disease, in which soft connective tissue is converted into heterotopic bone through an endochondral ossification process. Patients succumb early as they gradually become trapped in a second skeleton of heterotopic bone. Although the underlying genetic defect is long known, the inherent complexity of the disease has hindered the discovery of effective preventions and treatments. New developments in the gene therapy field have motivated its consideration as an attractive therapeutic option for FOP. However, the immune system\u27s role in FOP activation and the as-yet unknown primary causative cell, are crucial issues which must be taken into account in the therapy design. While gene therapy offers a potential therapeutic solution, more knowledge about FOP is needed to enable its optimal and safe application

Correction:How the COVID-19 pandemic highlights the necessity of animal research (vol 30, pg R1014, 2020)

(Current Biology 30, R1014–R1018; September 21, 2020) As a result of an author oversight in the originally published version of this article, a number of errors were introduced in the author list and affiliations. First, the middle initials were omitted from the names of several authors. Second, the surname of Dr. van Dam was mistakenly written as “Dam.” Third, the first name of author Bernhard Englitz was misspelled as “Bernard” and the surname of author B.J.A. Pollux was misspelled as “Pullox.” Finally, Dr. Keijer's first name was abbreviated rather than written in full. These errors, as well as various errors in the author affiliations, have now been corrected online

The Amsterdam Declaration on Fungal Nomenclature

The Amsterdam Declaration on Fungal Nomenclature was agreed at an international symposium convened in Amsterdam on 19–20 April 2011 under the auspices of the International Commission on the Taxonomy of Fungi (ICTF). The purpose of the symposium was to address the issue of whether or how the current system of naming pleomorphic fungi should be maintained or changed now that molecular data are routinely available. The issue is urgent as mycologists currently follow different practices, and no consensus was achieved by a Special Committee appointed in 2005 by the International Botanical Congress to advise on the problem. The Declaration recognizes the need for an orderly transitition to a single-name nomenclatural system for all fungi, and to provide mechanisms to protect names that otherwise then become endangered. That is, meaning that priority should be given to the first described name, except where that is a younger name in general use when the first author to select a name of a pleomorphic monophyletic genus is to be followed, and suggests controversial cases are referred to a body, such as the ICTF, which will report to the Committee for Fungi. If appropriate, the ICTF could be mandated to promote the implementation of the Declaration. In addition, but not forming part of the Declaration, are reports of discussions held during the symposium on the governance of the nomenclature of fungi, and the naming of fungi known only from an environmental nucleic acid sequence in particular. Possible amendments to the Draft BioCode (2011) to allow for the needs of mycologists are suggested for further consideration, and a possible example of how a fungus only known from the environment might be described is presented

Vitamin B-12 deficiency stimulates osteoclastogenesis via increased homocysteine and methylmalonic acid

The risk of nutrient deficiencies increases with age in our modern Western society, and vitamin B(12) deficiency is especially prevalent in the elderly and causes increased homocysteine (Hcy) and methylmalonic acid (MMA) levels. These three factors have been recognized as risk factors for reduced bone mineral density and increased fracture risk, though mechanistic evidence is still lacking. In the present study, we investigated the influence of B(12), Hcy, and MMA on differentiation and activity of bone cells. B(12) deficiency did not affect the onset of osteoblast differentiation, maturation, matrix mineralization, or adipocyte differentiation from human mesenchymal stem cells (hMSCs). B(12) deficiency caused an increase in the secretion of Hcy and MMA into the culture medium by osteoblasts, but Hcy and MMA appeared to have no effect on hMSC osteoblast differentiation. We further studied the effect of B(12), Hcy, and MMA on the formation of multinucleated tartrate-resistant acid phosphatase-positive osteoclasts from mouse bone marrow. We observed that B(12) did not show an effect on osteoclastogenesis. However, Hcy as well as MMA were found to induce osteoclastogenesis in a dose-dependent manner. On the basis of these results, we conclude that B(12) deficiency may lead to decreased bone mass by increased osteoclast formation due to increased MMA and Hcy levels