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Summary Objective Osteoarthritis (OA) is characterized by articular cartilage loss, associated with synovial inflammation. We recently reported increased pro-inflammatory macrophages in murine post-traumatic OA (PTOA) joints, and blockade of the ubiquitin-proteasome system alleviates PTOA progression. However, the mechanisms whereby protein ubiquitination influences PTOA pathology are not well studied. We hypothesized that loss of the negative regulator of inflammation, E3 ligase Itch, in macrophages contributes to joint OA tissue damage by promoting pro-inflammatory polarization of macrophages. Methods Mice deficient Itch in macrophages (MΔItch) were generated by crossing Itchfl/fl mice with LysM-Cre mice. PTOA surgery was performed on global Itch knockout, Itch−/−, mice and MΔItch mice. Joint tissue damage and synovial macrophages were examined. Itch−/− cells were treated with IL-1 and pro-inflammatory polarization was determined. Expression of Itch protein and mRNA in PTOA synovium were assessed at different time points post PTOA. Results Similar to Itch−/− mice, MΔItch mice developed more severe joint damage than control mice following PTOA surgery (mean difference of OARSI score: 1.17 (95% CI 0.31–2.03) between MΔItch and Itchfl/fl mice), accompanied by increased the inflammatory macrophage infiltration in the synovium (mean difference of % F4/80 + CD86 + CD36-inflammatory macrophages: 14.81 (95% CI 8.90–20.73) between MΔItch and Itchfl/fl mice). Itch−/− macrophages exerted pro-inflammatory phenotype in response to IL-1β treatment. Itch protein, but not mRNA levels decreased during PTOA progression. Conclusion The negative regulator of inflammation, Itch, limits PTOA progression by inhibiting macrophage pro-inflammatory polarization. Itch protein degradation may contribute to PTOA pathology.

Background Skeletal muscle wasting (SMW) in cancer patients is associated with increased morbidity, mortality, treatment intolerance and discontinuation, and poor quality of life. This is particularly true for patients with pancreatic ductal adenocarcinoma (PDAC), as over 85% experience SMW, which is responsible for ~30% of patient deaths. While the established paradigm to explain SMW posits that muscle catabolism from systemic inflammation and nutritional deficiencies, the cause of death, and the cellular and molecular mechanisms responsible remain to be elucidated. To address this, we investigated the relationship between tumour burden and survival in the KCKO murine PDAC model. Methods Female C57BL/6J mice 6–8 weeks of age underwent orthotopic injection with KCKO‐luc tumour cells. Solid tumour was verified on Day 5, post‐tumour inoculation. In vivo, longitudinal lean mass and tumour burden were assessed via dual‐energy X‐ray absorptiometry and IVIS imaging, respectively, and total body weight was assessed, weekly. Animals were sacrificed at a designated end point of ‘failure to thrive’. After sacrifice, lower limb hind muscles were harvested for histology and RNA extraction. Results We found a strong correlation between primary tumour size and survival (r 2 = 0.83, P

Complex regional pain syndrome (CRPS) is a potentially devastating condition that can result in severe psychological and social morbidity. It is a diagnosis of exclusion, and other pathologic entities must be ruled out first. Glomus tumors are exquisitely painful benign vascular tumors that are most common in the hand and are rarely found in the lower extremity. Here, we present a case of a patient who developed a focus of severe anterior knee pain and tenderness a few months after a car accident that had been misdiagnosed as CRPS for 15 years. She coincidentally developed a sarcoma of her ipsilateral leg distal to this site. Magnetic resonance imaging of the sarcoma included the area of knee pain where, interestingly, it identified a separate small soft-tissue mass. A glomus tumor was diagnosed histologically in a needle biopsy specimen from this mass, which was resected along with the sarcoma. For the first time in 15 years, despite the additional sarcoma surgery, she reported relief of her pain and complete resolution of her "CRPS."

The most challenging issue for breast cancer (BC) patients is metastasis to other organs because current therapies do not prevent or eliminate metastatic BC. Here, we show that SM-164, a small molecule inhibitor, which degrades inhibitor of apoptosis proteins (IAPs), eliminated early-stage metastases and reduced progression of advanced BC metastasis from MDA-MB-231 BC cells in bones and lungs of nude mice. Mechanistically, SM-164-induced BC cell death is TNFα-dependent, with TNFα produced by IL-4-polarized macrophages triggering MDA-MB-231 cell apoptosis in combination with SM-164. SM-164 also inhibited expression of RANKL, which mediates interactions between metastatic BC and host microenvironment cells and induces osteoclast-mediated osteolysis. SM-164 did not kill adriamycin-resistant BC cells, while adriamycin inhibited SM-164-resistant BC cell growth, similar to parental cells. We conclude that SM-164 is a promising therapeutic agent for early stage bone and lung metastasis from triple-negative breast cancer that should be given prior to conventional chemotherapy.

Limited treatment options exist for cancer within the bone, as demonstrated by the inevitable, pernicious course of metastatic and blood cancers. The difficulty of eliminating bone-residing cancer, especially drug-resistant cancer, necessitates novel, alternative treatments to manipulate tumor cells and their microenvironment, with minimal off-target effects. To this end, bone-targeted conjugate (BP-Btz) was generated by linking bortezomib (Btz, an anticancer, bone-stimulatory drug) to a bisphosphonate (BP, a targeting ligand) through a cleavable linker that enables spatiotemporally controlled delivery of Btz to bone under acidic conditions for treating multiple myeloma (MM). Three conjugates with different linkers were developed and screened for best efficacy in mouse model of MM. Results demonstrated that the lead candidate BP-Btz with optimal linker could overcome Btz resistance, reduced tumor burden, bone destruction, or tumor metastasis more effectively than BP or free Btz without thrombocytopenia and neurotoxicity in mice bearing myeloma. Furthermore, pharmacokinetic and pharmacodynamic studies showed that BP-Btz bound to bone matrix, released Btz in acidic conditions, and had a higher local concentration and longer half-life than Btz in bone. Our findings suggest the potential of bone-targeted Btz conjugate as an efficacious Btz-resistant MM treatment mechanism. © 2021 American Society for Bone and Mineral Research (ASBMR).

Canonical nuclear factor κB (NF-κB) signaling mediated by homo- and heterodimers of the NF-κB subunits p65 (RELA) and p50 (NFKB1) is associated with age-related pathologies and with disease progression in posttraumatic models of osteoarthritis (OA). Here, we established that NF-κB signaling in articular chondrocytes increased with age, concomitant with the onset of spontaneous OA in wild-type mice. Chondrocyte-specific expression of a constitutively active form of inhibitor of κB kinase β (IKKβ) in young adult mice accelerated the onset of the OA-like phenotype observed in aging wild-type mice, including degenerative changes in the articular cartilage, synovium, and menisci. Both in vitro and in vivo, chondrocytes expressing activated IKKβ had a proinflammatory secretory phenotype characterized by markers typically associated with the senescence-associated secretory phenotype (SASP). Expression of these factors was differentially regulated by p65, which contains a transactivation domain, and p50, which does not. Whereas the loss of p65 blocked the induction of genes encoding SASP factors in chondrogenic cells treated with interleukin-1β (IL-1β) in vitro, the loss of p50 enhanced the IL-1β–induced expression of some SASP factors. The loss of p50 further exacerbated cartilage degeneration in mice with chondrocyte-specific IKKβ activation. Overall, our data reveal that IKKβ-mediated activation of p65 can promote OA onset and that p50 may limit cartilage degeneration in settings of joint inflammation including advanced age.

NF-κB and Notch signaling can be simultaneously activated in a variety of B-cell lymphomas. Patients with B-cell lymphoma occasionally develop clonally related myeloid tumors with poor prognosis. Whether concurrent activation of both pathways is sufficient to induce B-cell transformation and whether the signaling initiates B-myeloid conversion in a pathological context are largely unknown. Here, we provide genetic evidence that concurrent activation of NF-κB and Notch signaling in committed B cells is sufficient to induce B-cell lymphomatous transformation and primes common progenitor cells to convert to myeloid lineage through dedifferentiation, not transdifferentiation. Intriguingly, the converted myeloid cells can further transform, albeit at low frequency, into myeloid leukemia. Mechanistically, coactivation of NF-κB and Notch signaling endows committed B cells with the ability to self renew. Downregulation of BACH2, a lymphoma and myeloid gene suppressor, but not upregulation of CEBPα and/or downregulation of B-cell transcription factors, is an early event in both B-cell transformation and myeloid conversion. Interestingly, a DNA hypomethylating drug not only effectively eliminated the converted myeloid leukemia cells, but also restored the expression of green fluorescent protein, which had been lost in converted myeloid leukemia cells. Collectively, our results suggest that targeting NF-κB and Notch signaling will not only improve lymphoma treatment, but also prevent the lymphoma-to-myeloid tumor conversion. Importantly, DNA hypomethylating drugs might efficiently treat these converted myeloid neoplasms.

NF-κB signals through canonical transcription factor p65 (RelA)/p50 and noncanonical avian reticuloendotheliosis viral oncogene related B (RelB)/p52 pathways. The RelA/p50 is involved in basal and inflammatory lymphangiogenesis. However, the role of RelB/p52 in lymphatic vessel biology is unknown. Herein, we investigated changes in lymphatic vessels (LVs) in mice deficient in noncanonical NF-κB signaling and the function of RelB in lymphatic endothelial cells (LECs). LVs were examined in Relb(−/−), p52(−/−), or control mice, and the gene expression profiles in LECs with RelB knockdown. Relb(−/−), but not p52(−/−), mice exhibited multiple LV abnormalities. They include the following: i) increased capillary vessel diameter, ii) reduced smooth muscle cell (SMC) coverage of mature vessels, iii) leakage, and iv) loss of active and passive lymphatic flow. Relb(−/−) mature LVs had thinner vessel walls, more apoptotic LECs and SMCs, and fewer LEC junctions. RelB knockdown LECs had decreased growth, survival, and adhesion, and dysregulated signaling pathways involving these cellular events. These results suggest that Relb(−/−) mice have abnormal LVs, mainly in mature vessels with reduced SMC coverage, leakage, and loss of contractions. RelB knockdown in LECs leads to reduced growth, survival, and adhesion. RelB plays a vital role in LEC-mediated LV maturation and function.

Bortezomib (Btz) is a proteasome inhibitor approved by the FDA to treat multiple myeloma. It also increases bone volume by promoting osteoblast differentiation and inhibiting osteoclastogenesis in mice. However, Btz has severe systemic adverse effects, which would limit its use as a bone anabolic agent. Here, we designed and synthesized a bone-targeted form of Btz by conjugating it to a bisphosphonate (BP) with no antiresorptive activity. We report that BP-Btz inhibited osteoclast formation and bone resorption and stimulated osteoblast differentiation in vitro similar to Btz. In vivo, BP-Btz increased bone volume more effectively than Btz in three mouse models: untreated wild-type mice, mice with ovariectomy, and aged mice with tibial factures. Importantly, BP-Btz had significantly less systemic side effects than Btz, including less thymic cell death, sympathetic nerve damage, and thrombocytopenia, and it improved survival rates in aged mice. Thus, BP-Btz represents a novel anabolic agent to treat conditions, such as postmenopausal and age-related bone loss. Bone targeting is an attractive approach to repurpose approved drugs to treat skeletal diseases. © 2019 American Society for Bone and Mineral Research. © 2019 American Society for Bone and Mineral Research.

Background: The ligamentum teres (LT) is covered by synovium. It acts as a stabilizer of the hip and as such it has been compared to the ACL of the knee joint. Pathologic changes occur in the LT with aging and osteoarthritis (OA), including degeneration, occasional chondroid metaplasia, and synovial chondromatosis are well-recognized in the literature. However, there are no reports of intraligamentous synovial osteochondroma occuring in the LT. Methods: We reviewed the pathology reports of 542 osteoarthritic femoral arthroplasty specimens between January 2016 and December 2018. The LT was examined histologically in 55 cases because it was abnormal on gross examination. Results: A single synovial osteochondroma, ranging in size from 0.4-1.7 cm in diameter, was present in the body of the LT in 14 cases (9 males; 5 females, aged 34 to 81 years), representing 2.6% of 542 arthroplasty cases. Ten of the osteochondromas had bone marrow fat without hematopoietic elements, 1 had hematopoietic elements, and 3 had no marrow among the bony trabeculae. Radiographically, all cases had moderate to severe osteoarthritis with no mention of an abnormality of LT. Conclusion: To our knowledge, this is the first report of intraligamentous synovial osteochondroma in the LT in osteoarthritis patients undergoing hip arthroplasty. It provides further support for microscopic examination of arthroplasty specimens for histologic abnormalities. Further prospective study is needed to determine if this lesion contributes adversely to the development or progression of osteoarthritis and if it is a reactive or neoplastic process.

Key Points Common progenitor cells exist in clonally related concomitant chronic lymphocytic leukemia and acute myeloid leukemias. CLL cells dedifferentiated to clonally related myeloid cells posttransplantation.

The osteoclast is the unique type of cell that resorbs bone in vivo and it is required for normal skeletal development and postnatal homeostasis. Osteoclast deficiency impairs skeletal development during embryogenesis and results in osteopetrosis and impaired tooth eruption. In contrast, excessive osteoclast formation in adults results in bone loss in a number of conditions, including osteoporosis, rheumatoid arthritis, and metastatic bone disease. Osteoclasts are derived from monocytes/macrophages; they can be generated in vitro by treatment of these precursor cells with macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). This chapter describes procedures for generating osteoclasts from mouse bone marrow cells in vitro using M-CSF and RANKL and assessing their ability to form resorption lacunae on thin bone slices.

Osteomyelitis is a devastating complication of orthopaedic surgery and commonly caused by Staphylococcus aureus (S. aureus) and Group B Streptococcus (GBS, S. agalactiae). Clinically, S. aureus osteomyelitis is associated with local inflammation, abscesses, aggressive osteolysis, and septic implant loosening. In contrast, S. agalactiae orthopaedic infections generally involve soft tissue, with acute life-threatening vascular spread. While preclinical models that recapitulate the clinical features of S. aureus bone infection have proven useful for research, no animal models of S. agalactiae osteomyelitis exist. Here, we compared the pathology caused by these bacteria in an established murine model of implant-associated osteomyelitis. In vitro scanning electron microscopy and CFU quantification confirmed similar implant inocula for both pathogens (~105 CFU/pin). Assessment of mice at 14 days post-infection demonstrated increased S. aureus virulence, as S. agalactiae infected mice had significantly greater body weight, and fewer CFU on the implant and in bone and adjacent soft tissue (p < 0.05). X-ray, µCT, and histologic analyses showed that S. agalactiae induced significantly less osteolysis and implant loosening, and fewer large TRAP+ osteoclasts than S. aureus without inducing intraosseous abscess formation. Most notably, transmission electron microscopy revealed that although both bacteria are capable of digesting cortical bone, S. agalactiae have a predilection for colonizing blood vessels embedded within cortical bone while S. aureus primarily colonizes the osteocyte lacuno-canalicular network. This study establishes the first quantitative animal model of S. agalactiae osteomyelitis, and demonstrates a vasculotropic mode of S. agalactiae infection, in contrast to the osteotropic behavior of S. aureus osteomyelitis.

Advances in the design of potential bone-selective drugs for the treatment of various bone-related diseases are creating exciting new directions for multiple unmet medical needs. For bone-related cancers, off-target/non-bone toxicities with current drugs represent a significant barrier to the quality of life of affected patients. For bone infections and osteomyelitis, bacterial biofilms on infected bones limit the efficacy of antibiotics because it is hard to access the bacteria with current approaches. Promising new experimental approaches to therapy, based on bone-targeting of drugs, have been used in animal models of these conditions and demonstrate improved efficacy and safety. The success of these drug-design strategies bodes well for the development of therapies with improved efficacy for the treatment of diseases affecting the skeleton. LINKED ARTICLES: This article is part of a themed issue on The molecular pharmacology of bone and cancer-related bone diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.9/issuetoc.

Macrophages play critical roles in homeostasis and inflammation. Macrophage polarization to either a pro-inflammatory or anti-inflammatory status is controlled by activating inflammatory signaling pathways. Ubiquitination is a posttranslational modification that regulates these inflammatory signaling pathways. However, the influence of protein ubiquitination on macrophage polarization has not been well studied. We hypothesized that the ubiquitination status of key proteins in inflammatory pathways contributes to macrophage polarization, which is regulated by itchy E3 ubiquitin ligase (ITCH), a negative regulator of inflammation. Using ubiquitin proteomics, we found that ubiquitination profiles are different among polarized murine macrophage subsets. Interestingly, interleukin-1α (IL-1α), an important pro-inflammatory mediator, was specifically ubiquitinated in lipopolysaccharide-induced pro-inflammatory macrophages, which was enhanced in ITCH-deficient macrophages. The ITCH-deficient macrophages had increased levels of the mature form of IL-1α and exhibited pro-inflammatory polarization, and reduced deubiquitination of IL-1α protein. Finally, IL-1α neutralization attenuated pro-inflammatory polarization of the ITCH-deficient macrophages. In conclusion, ubiquitination of IL-1α is associated with increased pro-inflammatory polarization of macrophages deficient in the E3 ligase ITCH.

The skeleton is affected by numerous primary and metastatic solid and hematopoietic malignant tumors, which can cause localized sites of osteolysis or osteosclerosis that can weaken bones and increase the risk of fractures in affected patients. Chemotherapeutic drugs can eliminate some tumors in bones or reduce their volume and skeletal-related events, but adverse effects on non-target organs can significantly limit the amount of drug that can be administered to patients. In these circumstances, it may be impossible to deliver therapeutic drug concentrations to tumor sites in bones. One attractive mechanism to approach this challenge is to conjugate drugs to bisphosphonates, which can target them to bone where they can be released at diseased sites. Multiple attempts have been made to do this since the 1990s with limited degrees of success. Here, we review the results of pre-clinical and clinical studies made to target FDA-approved drugs and other antineoplastic small molecules to bone to treat diseases affecting the skeleton, including osteoporosis, metastatic bone disease, multiple myeloma and osteosarcoma. Results to date are encouraging and indicate that drug efficacy can be increased and side effects reduced using these approaches. Despite these successes, challenges remain: no drugs have gone beyond small phase 2 clinical trials, and major pharmaceutical companies have shown little interest in the approach to repurpose any of their drugs or to embrace the technology. Nevertheless, interest shown by smaller biotechnology companies in the technology suggests that bone-targeting of drugs with bisphosphonates has a viable future.

Background: Chordomas are rare malignant tumors of the spinal column often afflicting the upper cervical spine and sacrum. There are few large single center series on chordomas due to its rarity. The purpose of this study is to report a single center’s experience with the operative and non-operative management of chordomas. Methods: We evaluated our institution’s pathology database from 1994 to 2016 to identify patients diagnosed with chordomas. Inclusion criteria were chordomas of the mobile spine and sacrum. Exclusion criteria were chordomas of the clivus and resection performed at another institution. We collected patient demographics as well as the type of resection performed, intra-operative complications, wound complications and recurrence/prognosis. Results: We identified 18 patients diagnosed with chordomas at our institution, and 12 met our inclusion criteria. There were four females and eight males with an average age of 64 [32–87] years. All patients were Caucasian and 10 of the 12 had surgery. Of the two patients with sacral chordomas that did not have surgery, one received chemotherapy and the other did not elect for any treatment. One is alive 161 months following diagnosis and the second died 96 months following diagnosis. Five of the lesions were in the mobile spine (one cervical, two thoracic, two lumbar) and seven were in the sacrum. Six patients underwent an en bloc resection [two via total en bloc spondylectomy (TES)]. Average length of follow up is 60 [3–161] months and eight of 12 patients are alive at latest follow up. Intraoperative complications included cardiac arrest, pleural tear, and excessive blood loss (8 L). Two patients, with sacral chordomas, had wound complications. Recurrence occurred in one patient with piecemeal resection and one patient with incomplete resection had post-operative metastatic lesions to the liver and lung. Conclusions: We found less recurrence in patients managed with an en bloc resection as opposed to piece meal or intralesional resections. Sacral chordoma patients had higher wound complication rates as compared to chordomas of the mobile spine. The long life expectancy of non-surgically managed patients underscores the indolent nature of chordomas.

SUMMARY Canonical NF-κB signaling is constitutively activated in acute myeloid leukemia (AML) stem cells and is required for maintenance of the self-renewal of leukemia stem cells (LSCs). However, any potential role for NF-κB non-canonical signaling in AML has been largely overlooked. Here, we report that stabilization of NF-κB-inducing kinase (NIK) suppresses AML. Mechanistically, stabilization of NIK activates NF-κB non-canonical signaling and represses NF-κB canonical signaling. In addition, stabilization of NIK-induced activation of NF-κB non-canonical signaling upregulates Dnmt3a and downregulates Mef2c, which suppresses and promotes AML development, respectively. Importantly, by querying the connectivity MAP using up- and downregulated genes that are present exclusively in NIK-stabilized LSCs, we discovered that verteporfin has anti-AML effects, suggesting that repurposing verteporfin to target myeloid leukemia is worth testing clinically. Our data provide a scientific rationale for developing small molecules to stabilize NIK specifically in myeloid leukemias as an attractive therapeutic option., Graphical abstract

Expression levels of Notch signaling molecules are increased in synovium from patients with rheumatoid arthritis (RA). However, it is not known which cell type(s) in RA synovium have Notch activation or if they play a pathogenetic role in RA. Here, we used Hes1-GFP/TNF-transgenic (TNF-Tg) mice to investigate the role of cells with active Notch signaling (GFP+) in RA. The number of GFP+ cells was significantly increased in synovium in Hes1-GFP/TNF-Tg mice and about 60% of them were F4/80+ macrophages expressing the inflammatory macrophage (M1) marker. TNF-Tg mice transplanted with Hes1-GFP/TNF-Tg bone marrow (BM) had significantly more GFP+ cells in their synovium than in BM. Intraarticular injection of Hes1-GFP/TNF-Tg or Hes1-GFP+ BM macrophages into WT and TNF-Tg mice showed the highest synovial GFP+ cells in the TNF-Tg mice that received Hes1-GFP/TNF-Tg cells. Thapsigargin (THAP), a Notch inhibitor, decreased TNF-induced M1 and increased M2 numbers and reduced joint lesion, synovial M1s, and GFP+ cells in Hes1-GFP/TNF-Tg mice. THAP did not affect M1s from mice carrying a constitutively active Notch1. Thus, the main cells with activated Notch signaling in the inflamed synovium of TNF-Tg mice are M1s derived from BM and targeting them may represent a new therapeutic approach for patients with inflammatory arthritis. © 2017 American Society for Bone and Mineral Research.

Binding of RANKL to its receptor, RANK, on osteoclast precursors recruits TNF receptor-associated factors, which are adaptor proteins and ubiquitin ligases that regulate NF-κB, JNK, c-Fos, NFATc1 ERK, p38, NFATc1 and AKT signaling to control osteoclast formation and activity in normal and pathologic bone resorption. RANKL/RANK signaling also regulates immune cell functions and has been implicated in the development of breast cancer, cardiovascular disease and diabetes and can be inhibited by denosumab, a monoclonal antibody that binds to RANKL. Recent studies have indicated that RANK is expressed by osteoblast progenitors and that reverse signaling occurs through RANKL on osteoblastic cells can regulate osteoblast formation, raising the possibility that anabolic therapy might be possible through this pathway.

Metal ion toxicity is an underinvestigated and underappreciated issue in skeletal biology. With the continued accumulation of environmental and industrial chemicals in our ecosystem that may have an adverse effect on osseous tissues, it might be expected that this topic would be an important and well-studied area. However, it seems that among clinicians and scientists in the field there is a paucity of investigations related to how environmental agents affect bones and joints. As discussed in this chapter, the skeleton can be an avid reservoir for toxins, especially heavy metals. Two of these metals, lead and aluminum, are responsible for many of the pathologies associated with the skeleton.

During aging, muscle mass decreases, leading to sarcopenia, associated with low-level chronic inflammation (inflammaging), which induces sarcopenia by promoting proteolysis of muscle fibers and inhibiting their regeneration. Patients with a variety of pathologic conditions associated with sarcopenia, including rheumatoid arthritis (RA), have systemically elevated TNFα serum levels, and transgenic mice with TNFα overexpression (TNF-Tg mice, a model of RA) develop sarcopenia between adolescence and adulthood before they age. However, if and how TNFα contributes to the pathogenesis of sarcopenia during the normal aging process and in RA remains largely unknown. We report that TNFα levels are increased in skeletal muscles of aged WT mice, associated with muscle atrophy and decreased numbers of satellite cells and Type IIA myofibers, a phenotype that we also observed in adult TNF-Tg mice. Aged WT mice also have increased numbers of myeloid lineage cells in their skeletal muscles, including macrophages and granulocytes. These cells have increased TNFα expression, which impairs myogenic cell differentiation. Expression levels of TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase, which mediates signaling by some TNF receptor (TNFR) family members, are elevated in skeletal muscles of both aged WT mice and adult TNF-Tg mice. TRAF6 binds to TNFR2 in C2C12 myoblasts and mediates TNFα-induced muscle atrophy through NF-κB-induced transcription of the muscle-specific E3 ligases, Atrogen1 and Murf1, which promote myosin heavy-chain degradation. Haplo-deficiency of TRAF6 prevents muscle atrophy and the decrease in numbers of satellite cells, Type IIA myofibers, and myogenic regeneration in TRAF6+/- ;TNF-Tg mice. Our findings suggest that pharmacologic inhibition of TRAF6 signaling in skeletal muscles during aging could treat/prevent age- and RA-related sarcopenia by preventing TNFα-induced proteolysis and inhibition of muscle fiber regeneration. © 2020 American Society for Bone and Mineral Research.

Osteomyelitis is a devastating disease caused by microbial infection of bone. While the frequency of infection following elective orthopedic surgery is low, rates of reinfection are disturbingly high. Staphylococcus aureus is responsible for the majority of chronic osteomyelitis cases and is often considered to be incurable due to bacterial persistence deep within bone. Unfortunately, there is no consensus on clinical classifications of osteomyelitis and the ensuing treatment algorithm. Given the high patient morbidity, mortality, and economic burden caused by osteomyelitis, it is important to elucidate mechanisms of bone infection to inform novel strategies for prevention and curative treatment. Recent discoveries in this field have identified three distinct reservoirs of bacterial biofilm including: Staphylococcal abscess communities in the local soft tissue and bone marrow, glycocalyx formation on implant hardware and necrotic tissue, and colonization of the osteocyte-lacuno canalicular network (OLCN) of cortical bone. In contrast, S. aureus intracellular persistence in bone cells has not been substantiated in vivo, which challenges this mode of chronic osteomyelitis. There have also been major advances in our understanding of the immune proteome against S. aureus, from clinical studies of serum antibodies and media enriched for newly synthesized antibodies (MENSA), which may provide new opportunities for osteomyelitis diagnosis, prognosis, and vaccine development. Finally, novel therapies such as antimicrobial implant coatings and antibiotic impregnated 3D-printed scaffolds represent promising strategies for preventing and managing this devastating disease. Here, we review these recent advances and highlight translational opportunities towards a cure.

Gorham-Stout disease (GSD) is a rare bone disorder characterized by aggressive osteolysis associated with lymphatic vessel invasion within bone marrow cavities. The etiology of GSD is not known, and there is no effective therapy or animal model for the disease. Here, we investigated if lymphatic endothelial cells (LECs) affect osteoclasts (OCs) to cause a GSD osteolytic phenotype in mice. We examined the effect of a mouse LEC line on osteoclastogenesis in co-cultures. LECs significantly increased receptor activator of NF-κB ligand (RANKL)-mediated OC formation and bone resorption. LECs expressed high levels of macrophage colony-stimulating factor (M-CSF), but not RANKL, interleukin-6 (IL-6), and tumor necrosis factor (TNF). LEC-mediated OC formation and bone resorption were blocked by an M-CSF neutralizing antibody or Ki20227, an inhibitor of the M-CSF receptor, c-Fms. We injected LECs into the tibias of wild-type (WT) mice and observed massive osteolysis on X-ray and micro-CT scans. Histology showed that LEC-injected tibias had significant trabecular and cortical bone loss and increased OC numbers. M-CSF protein levels were significantly higher in serum and bone marrow plasma of mice given intra-tibial LEC injections. Immunofluorescence staining showed extensive replacement of bone and marrow by podoplanin+ LECs. Treatment of LEC-injected mice with Ki20227 significantly decreased tibial bone destruction. In addition, lymphatic vessels in a GSD bone sample were stained positively for M-CSF. Thus, LECs cause bone destruction in vivo in mice by secreting M-CSF, which promotes OC formation and activation. Blocking M-CSF signaling may represent a new therapeutic approach for treatment of patients with GSD. Furthermore, tibial injection of LECs is a useful mouse model to study GSD. © 2017 American Society for Bone and Mineral Research.

Notch signaling plays a critical role in maintaining bone homeostasis partially by controlling the formation of osteoblasts from mesenchymal stem cells (MSCs). We reported that TNF activates Notch signaling in MSCs which inhibits osteoblast differentiation in TNF transgenic (TNF-Tg) mice, a mouse model of chronic inflammatory arthritis. In the current study, we used Hes1-GFP and Hes1-GFP/TNF-Tg mice to study the distribution and dynamic change of Notch active cells in normal and inflammatory bone loss and mechanisms mediating their enhanced proliferation. We found that Hes1-GFP+ cells are composed of cells expressing mesenchymal, hematopoietic and endothelial surface markers. CD45−/Hes1-GFP+ cells express high levels of mesenchymal markers and form CFU-F and CFU-ALP colonies. Expansion of CFU-F colonies is associated with a rapid increase in Hes1-GFP+ cell numbers and their GFP intensity. The GFP signal is lost when a CFU-F colony differentiates into an ALP+ osteoblast colony. TNF increases the numbers of CD45−/Hes1-GFP+ cells, which are stained negatively for osteoblast marker osteocalcin and localized adjacent to endosteal and trabecular bone surfaces. CD45−/Hes1-GFP+ cells in Hes1-GFP/TNF-Tg mice have increased BrdU incorporation and PDGFRβ levels. TNF increases the number of proliferating Hes1-GFP+ cells, which is prevented by a specific PDGFRβ inhibitor. Notch inhibition blocks TNF-mediated PDGFRβ expression and cell proliferation. Thus, TNF-induced MSC proliferation is mediated by PDGFRβ signal, which works at downstream of Notch. Hes1-GFP mice can be used to assess the activation status of Notch in bone cells.

Skeletal health is maintained by bone remodeling, a process in which microscopic sites of effete or damaged bone are degraded on bone surfaces by osteoclasts and subsequently replaced by new bone, which is laid down by osteoblasts. This normal process can be disturbed in a variety of pathologic processes, including localized or generalized inflammation, metabolic and endocrine disorders, primary and metastatic cancers, and during aging as a result of low-grade chronic inflammation. Osteoclast formation and activity are promoted by factors, including cytokines, hormones, growth factors, and free radicals, and require expression of macrophage-colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) by accessory cells in the bone marrow, including osteoblastic and immune cells. Expression of TNF receptor-associated factor 6 (TRAF6) is required in osteoclast precursors to mediate RANKL-induced activation of NF-κB, which is also necessary for osteoclast formation and activity. TRAF3, in contrast is not required for osteoclast formation, but it limits RANKL-induced osteoclast formation by promoting proteasomal degradation of NF-κB-inducing kinase in a complex with TRAF2 and cellular inhibitor of apoptosis proteins (cIAP). TRAF3 also limits osteoclast formation induced by TNF, which mediates inflammation and joint destruction in inflammatory diseases, including rheumatoid arthritis. Chloroquine and hydroxychloroquine, anti-inflammatory drugs used to treat rheumatoid arthritis, prevent TRAF3 degradation in osteoclast precursors and inhibit osteoclast formation in vitro. Chloroquine also inhibits bone destruction induced by ovariectomy and parathyroid hormone in mice in vivo. Mice genetically engineered to have TRAF3 deleted in osteoclast precursors and macrophages develop early onset osteoporosis, inflammation in multiple tissues, infections, and tumors, indicating that TRAF3 suppresses inflammation and tumors in myeloid cells. Mice with TRAF3 conditionally deleted in mesenchymal cells also develop early onset osteoporosis due to a combination of increased osteoclast formation and reduced osteoblast formation. TRAF3 protein levels decrease in bone and bone marrow during aging in mice and humans. Development of drugs to prevent TRAF3 degradation in immune and bone cells could be a novel therapeutic approach to prevent or reduce bone loss and the incidence of several common diseases associated with aging.

Multiple myeloma (MM) is the most common cancer affecting the bone and bone marrow and remains incurable for most patients, novel therapies are therefore needed. Bortezomib (Btz) is an FDA-approved drug for the treatment of patients with MM. However, its severe side effects require a dose reduction or the potential discontinuation of treatment. To overcome this limitation, we conjugated Btz to a bisphosphonate (BP) residue lacking anti-osteoclastic activity using a novel chemical linker and generated a new bone-targeted Btz-based (BP-Btz) proteasome inhibitor. We demonstrated that BP-Btz, but not Btz, bound to bone slices and inhibited the growth of MM cells in vitro. In a mouse model of MM, BP-Btz more effectively reduced tumor burden and bone loss with less systemic side effects than Btz. Thus, BP-Btz may represent a novel therapeutic approach to treat patients with MM.

To define the cell populations in rheumatoid arthritis (RA) driving joint inflammation, we applied single-cell RNA-seq (scRNA-seq), mass cytometry, bulk RNA-seq, and flow cytometry to sorted T cells, B cells, monocytes, and fibroblasts from 51 synovial tissue RA and osteoarthritis (OA) patient samples. Utilizing an integrated computational strategy based on canonical correlation analysis to 5,452 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics together revealed cell states expanded in RA synovia: THY1+HLAhigh sublining fibroblasts (OR=33.8), IL1B+ pro-inflammatory monocytes (OR=7.8), CD11c+T-bet+ autoimmune-associated B cells (OR=5.7), and PD-1+Tph/Tfh (OR=3.0). We also defined CD8+ T cell subsets characterized by GZMK+, GZMB+, and GNLY+ expression. Using bulk and single-cell data, we mapped inflammatory mediators to source cell populations, for example attributing IL6 production to THY1+HLAhigh fibroblasts and naïve B cells, and IL1B to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.