Blocking CCN2 Reduces Established Bone Loss Induced by Prolonged Intense Loading by Increasing Osteoblast Activity in Rats

AUTHORS

Alex G Lambi, Michele Y Harris, Mamta Amin, Patrice G Joiner, Brendan A Hilliard, Soroush Assari, Steven N Popoff, Mary F Barbe

ABSTRACT

We have an operant model of reaching and grasping in which detrimental bone remodeling is observed rather than beneficial adaptation when rats perform a high-repetition, high-force (HRHF) task long term. Here, adult female Sprague–Dawley rats performed an intense HRHF task for 18 weeks, which we have shown induces radial trabecular bone osteopenia. One cohort was euthanized at this point (to assay the bone changes post task; HRHF-Untreated). Two other cohorts were placed on 6 weeks of rest while being simultaneously treated with either an anti-CCN2 (FG-3019, 40 mg/kg body weight, ip; twice per week; HRHF-Rest/anti-CCN2), or a control IgG (HRHF-Rest/IgG), with the purpose of determining which might improve the trabecular bone decline. Results were compared with food-restricted control rats (FRC). MicroCT analysis of distal metaphysis of radii showed decreased trabecular bone volume fraction (BV/TV) and thickness in HRHF-Untreated rats compared with FRCs; responses improved with HRHF-Rest/anti-CCN2. Rest/IgG also improved trabecular thickness but not BV/TV. Histomorphometry showed that rest with either treatment improved osteoid volume and task-induced increases in osteoclasts. Only the HRHF-Rest/anti-CCN2 treatment improved osteoblast numbers, osteoid width, mineralization, and bone formation rate compared with HRHF-Untreated rats (as well as the latter three attributes compared with HRHF-Rest/IgG rats). Serum ELISA results were in support, showing increased osteocalcin and decreased CTX-1 in HRHF-Rest/anti-CCN2 rats compared with both HRHF-Untreated and HRHF-Rest/IgG rats. These results are highly encouraging for use of anti-CCN2 for therapeutic treatment of bone loss, such as that induced by chronic overuse. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Antibodies to sclerostin or G-CSF receptor partially eliminate bone or marrow adipocyte loss, respectively, following vertical sleeve gastrectomy

AUTHORS

Ziru Li, Kevin Qiu, Jingtong Zhao, Katrina Granger, Hui Yu, Alfor G. Lewis, Andriy Myronovych, Mouhamadoul H. Toure, Sarah J. Hatsell, Aris N. Economides, Randy J. Seeley, Ormond A. MacDougald

ABSTRACT

Vertical sleeve gastrectomy (VSG), the most utilized bariatric procedure in clinical practice, greatly reduces body weight and improves a variety of metabolic disorders. However, one of its long-term complications is bone loss and increased risk of fracture. Elevated circulating sclerostin (SOST) and granulocyte-colony stimulating factor (G-CSF) concentrations have been considered as potential contributors to VSG-associated bone loss. To test these possibilities, we administrated antibodies to SOST or G-CSF receptor and investigated alterations to bone and marrow niche following VSG. Neutralizing either SOST or G-CSF receptor did not alter beneficial effects of VSG on adiposity and hepatic steatosis, and anti-SOST treatment provided a further improvement to glucose tolerance. SOST antibodies partially reduced trabecular and cortical bone loss following VSG by increasing bone formation, whereas G-CSF receptor antibodies had no effects on bone mass. The expansion in myeloid cellularity and reductions in bone marrow adiposity seen with VSG were partially eliminated by treatment with Anti-G-CSF receptor. Taken together, these experiments demonstrate that antibodies to SOST or G-CSF receptor may act through independent mechanisms to partially block effects of VSG on bone loss or marrow niche cells, respectively.

Context-Dependent Roles for Toll-Like Receptors 2 and 9 in the Pathogenesis of Staphylococcus aureus Osteomyelitis

AUTHORS

Jenna R. Petronglo, Nicole E. Putnam, Caleb A. Ford, Virginia Cruz-Victorio, Jacob M. Curry, Casey E. Butrico, Laura E. Fulbright, Joshua R. Johnson, Sun H. Peck, Sana R. Fatah, James E. Cassat

ABSTRACT

Staphylococcus aureus is the major causative agent of bacterial osteomyelitis, an invasive infection of bone. Inflammation generated by the immune response to S. aureus contributes to bone damage by altering bone homeostasis. Increases in the differentiation of monocyte lineage cells into bone-resorbing osteoclasts (osteoclastogenesis) promote bone loss in the setting of osteomyelitis. In this study, we sought to define the role of Toll-like receptor (TLR) signaling in the pathogenesis of S. aureus osteomyelitis. We hypothesized that S. aureus-sensing TLRs 2 and 9, both of which are known to alter osteoclastogenesis in vitro, promote pathological changes to bone, including increased osteoclast abundance, bone loss, and altered callus formation during osteomyelitis. Stimulation of osteoclast precursors with S. aureus supernatant increased osteoclastogenesis in a TLR2-dependent, but not a TLR9-dependent, manner. However, in vivo studies using a posttraumatic murine model of osteomyelitis revealed that TLR2-null mice experienced similar bone damage and increased osteoclastogenesis compared to wild type (WT) mice. Therefore, we tested the hypothesis that compensation between TLR2 and TLR9 contributes to osteomyelitis pathogenesis. We found that mice deficient in both TLR2 and TLR9 (Tlr2/9−/−) have decreased trabecular bone loss in response to infection compared to WT mice. However, osteoclastogenesis is comparable between WT and Tlr2/9−/− mice, suggesting that alternative mechanisms enhance osteoclastogenesis in vivo during osteomyelitis. Indeed, we discovered that osteoclast precursors intracellularly infected with S. aureus undergo significantly increased osteoclast formation, even in the absence of TLR2 and TLR9. These results suggest that TLR2 and TLR9 have context-dependent roles in the alteration of bone homeostasis during osteomyelitis.

Inflammaging and bone loss in a rat model of spinal cord injury

AUTHORS

Dr. Corinne Metzger, Dr. Josephina Rau, Mr. Alexander Stefanov, Ms. Rose M Joseph, Dr. Heather C Allaway, Dr. Matthew R. Allen, and Dr. Michelle A Hook

ABSTRACT

Spinal cord injury (SCI) results in significant loss of sublesional bone, adding to the comorbidity of SCI with an increased risk of fracture and post-fracture complications. Unfortunately, the effect of SCI on skeletal health is also likely to rise as the average age of SCI has increased and there are well-known negative effects of age on bone. To date, however, the impact of age and age-associated inflammation (inflammaging) on skeletal health after SCI remains largely unknown. To address this, we compared bone parameters in young (3 month) and middle-aged (9 month) male and female rats with a moderate thoracic contusion injury, to age and sex matched sham-operated controls. Skeletal parameters, locomotor function and serum cytokine levels were assessed at both subchronic (30 days) and chronic (180 days) time points post injury. We hypothesized that SCI would lead to a dramatic loss of bone immediately after injury in all SCI-groups, with inflammaging leading to greater loss in middle-aged SCI rats. We also predicted that while younger rats may re-establish bone properties in more chronic phases of SCI, middle-aged rats would not. Supporting these hypothesis, trabecular bone volume was significantly lower in male and young female SCI rats early after injury. Contrary to our hypothesis, however, there was greater loss of trabecular bone volume, relative to age-matched shams, in young compared to middle-aged SCI rats with no effects of SCI on trabecular bone volume in middle-aged female rats. Moreover, despite recovery of weight-supported locomotor activity, bone loss persisted into the chronic phase of injury for the young rats. Bone formation rates were lower in young male SCI rats, regardless of the time since injury, while both young and middle-aged female SCI rats had lower bone formation in the subchronic but not chronic phase of SCI. In middle-aged rats, SCI-induced higher osteoclast surfaces, which also persisted into the chronic phase of SCI in middle-aged females. Neither age nor SCI-induced increases in inflammation seemed to be associated with bone loss. In fact, SCI had more dramatic and persistent effects on bone in male rats, while aging and SCI elevated serum cytokines only in female rats. Overall, this study demonstrates SCI-induced loss of bone and altered bone turnover in male and female rats that persists into the chronic phase post-injury. The sex and age dependent variations in bone turnover and serum cytokines, however, underscore the need to further explore both mechanisms and potential therapeutics in multiple demographics.

Toll-like receptor 9 deficiency induces osteoclastic bone loss via gut microbiota-associated systemic chronic inflammation

AUTHORS

Peng Ding, Qiyuan Tan, Zhanying Wei, Qiyu Chen, Chun Wang, Luyue Qi, Li Wen, Changqing Zhang & Chen Yao

ABSTRACT

Toll-like receptors (TLRs) play pivotal roles in inflammation and provide important links between the immune and skeletal systems. Although the activation of TLRs may affect osteoclast differentiation and bone metabolism, whether and how TLRs are required for normal bone remodeling remains to be fully explored. In the current study, we show for the first time that TLR9−/− mice exhibit a low bone mass and low-grade systemic chronic inflammation, which is characterized by the expansion of CD4+ T cells and increased levels of inflammatory cytokines, including TNFα, RANKL, and IL1β. The increased levels of these cytokines significantly promote osteoclastogenesis and induce bone loss. Importantly, TLR9 deletion alters the gut microbiota, and this dysbiosis is the basis of the systemic inflammation and bone loss observed in TLR9−/− mice. Furthermore, through single-cell RNA sequencing, we identified myeloid-biased hematopoiesis in the bone marrow of TLR9−/− mice and determined that the increase in myelopoiesis, likely caused by the adaptation of hematopoietic stem cells to systemic inflammation, also contributes to inflammation-induced osteoclastogenesis and subsequent bone loss in TLR9−/− mice. Thus, our study provides novel evidence that TLR9 signaling connects the gut microbiota, immune system, and bone and is critical in maintaining the homeostasis of inflammation, hematopoiesis, and bone metabolism under normal conditions.

Skeletal muscle mitoribosomal defects are linked to low bone mass caused by bone marrow inflammation in male mice

AUTHORS

Jingwen Tian, Hyo Kyun Chung, Ji Sun Moon, Ha Thi Nga, Ho Yeop Lee, Jung Tae Kim, Joon Young Chang, Seul Gi Kang, Dongryeol Ryu, Xiangguo Che, Je-Yong Choi, Masayuki Tsukasaki, Takayoshi Sasako, Sang-Hee Lee, Minho Shong, Hyon-Seung Yi

ABSTRACT

Background

Mitochondrial oxidative phosphorylation (OxPhos) is a critical regulator of skeletal muscle mass and function. Although muscle atrophy due to mitochondrial dysfunction is closely associated with bone loss, the biological characteristics of the relationship between muscle and bone remain obscure. We showed that muscle atrophy caused by skeletal muscle-specific CR6-interacting factor 1 knockout (MKO) modulates the bone marrow (BM) inflammatory response, leading to low bone mass.

Methods

MKO mice with lower muscle OxPhos were fed a normal chow or high-fat diet and then evaluated for muscle mass and function, and bone mineral density. Immunophenotyping of BM immune cells was also performed. BM transcriptomic analysis was used to identify key factors regulating bone mass in MKO mice. To determine the effects of BM-derived CXCL12 (C–X–C motif chemokine ligand 12) on regulation of bone homeostasis, a variety of BM niche-resident cells were treated with recombinant CXCL12. Vastus lateralis muscle and BM immune cell samples from 14 patients with hip fracture were investigated to examine the association between muscle function and BM inflammation.

Results

MKO mice exhibited significant reductions in both muscle mass and expression of OxPhos subunits but increased transcription of mitochondrial stress response-related genes in the extensor digitorum longus (P < 0.01). MKO mice showed a decline in grip strength and a higher drop rate in the wire hanging test (P < 0.01). Micro-computed tomography and von Kossa staining revealed that MKO mice developed a low mass phenotype in cortical and trabecular bone (P < 0.01). Transcriptomic analysis of the BM revealed that mitochondrial stress responses in skeletal muscles induce an inflammatory response and adipogenesis in the BM and that the CXCL12–CXCR4 (C–X–C chemokine receptor 4) axis is important for T-cell homing to the BM. Antagonism of CXCR4 attenuated BM inflammation and increased bone mass in MKO mice. In humans, patients with low body mass index (BMI = 17.2 ± 0.42 kg/m2) harboured a larger population of proinflammatory and cytotoxic senescent T-cells in the BMI (P < 0.05) and showed reduced expression of OxPhos subunits in the vastus lateralis, compared with controls with a normal BMI (23.7 ± 0.88 kg/m2) (P < 0.01).

Conclusions

Defects in muscle mitochondrial OxPhos promote BM inflammation in mice, leading to decreased bone mass. Muscle mitochondrial dysfunction is linked to BM inflammatory cytokine secretion via the CXCL12–CXCR4 signalling axis, which is critical for inducing low bone mass.