2020 12 09 417147v1 Full

bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.417147; this version posted December 9, 2020.
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Skeletal muscle mitochondrial dysfunction in mice is linked to bone loss via the bone marrow
immune microenvironment
JINGWEN TIAN1, Ji Sun Moon2, Ha Thi Nga1, Hyo Kyun Chung2, Ho Yeop Lee1, Jung Tae Kim1,2,
Joon Young Chang1,2, Seul Gi Kang1,2, Dongryeol Ryu3,4, Xiangguo Che5, Je-Yong Choi5, Masayuki
Tsukasaki6, Takayoshi Sasako7, Sang-Hee Lee8, Minho Shong1,2, and Hyon-Seung Yi1,2, *
1
Department of Medical Science, Chungnam National University School of Medicine, 266 Munhwaro,
Daejeon 35015, Republic of Korea.
2
Research Center for Endocrine and Metabolic Diseases, Chungnam National University School of
Medicine, 282 Munhwaro, Daejeon 35015, Republic of Korea.
3
Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 16419,
Republic of Korea.
4
Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, 06351, Republic of Korea.
5
Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, BK21 Plus KNU
Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu,
Republic of Korea.
6
Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University
of Tokyo, 7-3-1, Hongo, Bunkyo-ku, 113-0033 Tokyo, Japan
7
Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of
Tokyo, Tokyo, 113-8655, Japan
8
Bio-Electron Microscopy Research Center(104-Dong), Korea Basic Science Institute, Ochang,
Cheongju, Chungbuk, 28199, Republic of Korea.
Conflict of Interest Statement: The authors have declared that no conflict of interest exists.
1
bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.417147; this version posted December 9, 2020. The copyright holder for this
Key words: mitochondria, inflammation, bone marrow, bone loss
Word count: 6,091 (Introduction, Results, Discussion, and Methods)
Number of figures and tables: 8
*Corresponding author: Hyon-Seung Yi, Research Center for Endocrine and Metabolic Diseases,
Chungnam National University School of Medicine, Daejeon 35015, Korea. Phone: +82-42-280-6994;
Fax: +82-42-280-6990; E-mail: [email protected]
Abstract
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 caused by reduction of mechanical loading, questions remain about the biological characteristics
in the relationship between muscle and bone. Here, we have shown that muscle atrophy caused by
skeletal muscle-specific Crif1 knockout (MKO) modulates the bone marrow inflammatory response,
leading to bone loss. Transcriptome analysis of the extensor digitorum longus revealed that local
mitochondrial stress increased serum levels of fibroblast growth factor 21 (FGF21) in mice. However,
we have shown by Fgf21 knockout in MKO mice that FGF21 is dispensable for muscle atrophy-
mediated bone loss. RNA sequencing in MKO mice indicated that mitochondrial stress response in
skeletal muscles induces an inflammatory response and adipogenesis in the bone marrow. We also found,
using transcriptomic analysis of bone marrow, that the CXCL12–CXCR4 axis is important for T-cell
homing to the bone marrow, which is an immunological mediator of muscle-bone communication.
CXCR4 antagonism attenuated bone marrow inflammation and bone loss in MKO mice. Together, these
data highlight the role that muscle mitochondrial dysfunction plays in triggering bone marrow
inflammation via the CXCL12–CXCR4 signaling axis, which is critical for inducing bone loss.
2
Introduction
Mitochondria quality control pathways regulating mitochondrial morphology/function have been
implicated in the homeostatic control of muscle mass 1. Gradual loss of muscle mass and strength results
from an inflammatory state brought about by cytokines, apoptosis, alteration in mitochondrial
respiration, and oxidative stress 2. Decrease or dysfunction of skeletal muscle mitochondria is involved
in loss of muscle mass and muscle strength in humans 3. Progressive loss of muscle mass and function
has also been implicated as a critical risk factor for osteoporosis through reduction of bone strength
4,5
caused by decreasing mechanical loading on the skeleton . Moreover, mitochondrial DNA-mutator
mice showed premature aging-related phenotypes including muscle dysfunction and osteoporosis 6.
However, little information is available on the mechanical link between mitochondrial dysfunction-
mediated loss of muscle mass and strength, and bone loss.
Owing to their proximities, bone marrow (BM) immune cells contribute to the regulation of
osteoblasts and osteoclasts, leading to modulation of bone remodeling. For example, ovariectomy
induces T-cell activation and proliferation in BM, which promotes osteoclastogenesis 7 due to increases
in the populations of CD4+ and CD8+ T-cells, and in T-cell derived tumor necrosis factor alpha (TNF-
α). Additionally, ovariectomy-induced bone loss does not occur in mice lacking T-cells or lacking the
T-cell receptor CD40 ligand 8,9. Germ-free mice have a smaller population of CD4+ T-cells and reduced
levels of proinflammatory cytokines in the BM in combination with increased bone mass 10. Moreover,
BM CD4+ T-cells producing interleukin (IL)-17 and TNF-α, but not interferon (IFN)-γ, activate bone
resorption by inducing osteoclast differentiation in animal models of inflammatory bowel disease 11.
However, although previous investigations revealed several factors promoting BM inflammatory-
mediated bone loss, the role of mitochondrial function in skeletal muscle on the BM immune
microenvironment and bone mass remains to be elucidated.
3
CR6-interacting factor 1 (CRIF1), as a critical mitoribosomal protein, is essential for the
translation of mitochondrial oxidative phosphorylation (OxPhos) subunits and their insertion in the
mitochondrial inner membrane 12. Deficiency of CRIF1 produces impaired formation of the OxPhos
complex, leading to reduction of mitochondrial respiration. Consequently, both beta cell- and adipocyte-
specific Crif1 knockout mice show mitochondrial OxPhos dysfunction-associated metabolic

13,14
phenotypes such as glucose intolerance and adipose inflammation . On the other hand, we have
shown that OxPhos dysfunction in skeletal muscle-specific Crif1 knockout (MKO) mice is associated
with improved glucose tolerance and insulin resistance 15. However, in that study, we found that MKO
mice had markedly smaller gastrocnemius and extensor digitorum longus (EDL) muscles compared
with the controls 15. Thus, we hypothesized that MKO mice could be used as models for studying the
mechanisms underlying muscular mitochondrial dysfunction-induced bone loss.
Herein, we investigated whether skeletal muscle dysfunction caused by muscle-specific OxPhos
deficiency changes the population of inflammatory T-cells and the levels of proinflammatory cytokines
in the BM, thereby resulting in bone loss. We also examined the role of key mitokines, produced in
response to mitochondrial stress, in the regulation of bone mass and mineral density. In addition, the
present study identifies the mechanism underlying deterioration in bone mass and quality caused by
loss of muscular mitochondrial function and, consequently, a potential therapeutic target.
4
Results
Crif1 deficiency induces defective mitochondrial OxPhos function and stress response in skeletal
muscles. To assess the effect of skeletal muscle-specific Crif1 deficiency on the mitochondrial function
and physical performance of the mice, we generated MKO mice by crossing Mlc1f-cre mice with
Crif1flox/flox mice (Supplementary Fig 1a). CRIF1 protein expression was markedly lower in the EDL,
gastrocnemius, and soleus of the MKO mice than in the wild-type (WT) controls (Fig 1a,b, and
Supplementary Fig 1b,c). Consistent with the reduction in CRIF1 expression, the EDL and
gastrocnemius in MKO mice showed significantly lower expression of mitochondrial OxPhos complex
subunits, including complex II (SDHA), III (UQCRC2), and V (ATP5A), indicating that deficiency of
Crif1 induces abnormal mitochondrial proliferation and function in skeletal muscles (Fig 1a,b, and
Supplementary Fig 1b,c). Moreover, blue native polyacrylamide gel electrophoresis (BN-PAGE)
analysis revealed a decrease in assembly of complex I, III, and V in MKO mouse EDL and
gastrocnemius compared with the WT (Fig 1c). MKO mice also exhibited lower succinate
dehydrogenase (SDH) activity in the EDL, gastrocnemius, and soleus compared with the WT (Fig 1d,e,
and Supplementary Fig 1d). Electron microscopy also revealed accumulation of abnormal, swollen
mitochondria with disrupted cristae in gastrocnemius of MKO mice (Supplementary Fig 1d). In addition,
EDL from MKO mice expressed higher levels of mitochondrial stress response-related genes, such as
Lonp1, Clpp, Hspd1, and Atf4 (Fig 1f).
Next, we characterized the physical performance of MKO mice on a normal chow diet. MKO
mice showed a significant decrease in body mass relative to controls after 11 weeks of age (Fig 1g).
Motor coordination was assessed using rotarod apparatus. At fixed rotarod speed (10 rpm), a significant
difference between control and MKO mice was observed in latency to fall (Fig 1h). Furthermore, unlike
the controls, MKO mice showed a decline in grip strength and a higher drop rate in the wire hanging
test at 13 weeks of age (Fig 1i,j). Collectively, these findings indicate that Crif1 deficiency not only
adversely affects mitochondrial OxPhos function, but also reduces muscle strength and endurance in
5
mice.
Mitochondrial OxPhos dysfunction in muscle promotes bone loss in mice. Loss of muscle mass
and function is closely associated with increased bone loss and fracture incidence 16. Thus, to elucidate
the crosstalk between mitochondrial function of skeletal muscle and bone mass, at 14 weeks of age we
investigated the bone mass and mineral density (BMD) of control and MKO mice on a normal chow
diet. Analysis of femurs from euthanized mice by micro-computed tomography (micro-CT) revealed
that cortical bone thickness and BMD were significantly decreased in MKO mice (Fig 2a and
Supplementary Fig 2a). Femurs from MKO mice also showed a decrease in trabecular number,
trabecular thickness (Tb.Th.), and trabecular bone volume (BV), but exhibited an increase in trabecular
spacing (Tb.Sp.) compared with those from the controls (Fig 2b,c). Quantification and statistical
analysis of the bone phenotypes characterized by micro-CT are shown in Figure 2C.
Bone histomorphometry verified that MKO mice developed a low bone-mass phenotype in
cortical and trabecular bone, as shown by von Kossa staining (Fig 2d,e, and Supplementary Fig 2b,c),
but they showed a high number of osteoclasts per bone surface, as indicated by tartrate-resistant acid
phosphatase (TRAP)-stained sections of tibia (Fig 2f and Supplementary Fig 2d). Next, to quantify the
extent of increased osteoblast activity, calcein and alizarin red were sequentially given to control and
MKO mice 3 and 8 days prior to sacrifice. Analysis of undecalcified, unstained sections showed no
notable difference in vertebral bone formation rates between control and MKO mice (Fig 2g,h).
Additionally, measurement of the serum level of procollagen type I N-terminal propeptide (P1NP), a
marker for bone formation, and of C-terminal telopeptide of collagen (CTX), a marker for bone
resorption, indicated less bone formation and higher bone resorption in MKO mice compared with the
controls (Fig 2i). To exclude the effect of other endocrine hormones on bone loss, we checked the serum
levels of parathyroid hormone, testosterone, thyroxine, and triiodothyronine in the control and MKO
6
mice at 14 weeks of age. There was no significant difference in the serum level of each of these
hormones between the control and MKO mice (Supplemental Fig 3a-d). Taken together, these data
provide direct evidence that lower mitochondrial OxPhos-mediated muscle dysfunction induces bone
loss via activation of osteoclasts in mice.
Bone loss in MKO mice is independent of FGF21 action. The phenotypic changes observed so
far suggested that MKO mice express circulating factors involved in signaling from muscle to bone.
Therefore, to study the effects of muscular mitochondrial dysfunction-induced secretory factors on bone
loss in MKO mice, we performed RNA sequencing of EDL transcripts from control and MKO mice,
focusing on genes encoding secreted proteins. A large number of transcripts were altered in the EDL
from MKO mice (Fig 3a,b). In particular, we found that the expression of Fgf21, which is known as a
potent regulator of skeletal homeostasis, was much higher in the EDL of MKO mice than in that of the
controls (Fig 3b-d). As shown in Figure 3E, the serum levels of FGF21 were also markedly elevated in
the MKO mice.
Next, to investigate the role of FGF21 on the skeletal phenotype in MKO mice, we generated
global Fgf21 knockout mice and MKO mice with global Fgf21 deletion (MFKO), both on a C57BL/6J
background (Supplementary Fig 4a,b). Analysis of femurs by micro-CT revealed that cortical thickness,
Tb.Th., and trabecular number were remarkably lower in MKO mice compared with the controls, but
global Fgf21 knockout did not have any effect on the bone phenotype of WT control or MKO mice at
14 weeks of age (Fig 3f,g). These findings suggest that a reduction of BMD in MKO mice is independent
of FGF21 production caused by muscular mitochondrial OxPhos dysfunction.
MKO mice exhibit inflammation response in BM. In the next set of experiments, we asked
whether mitochondrial OxPhos dysfunction in muscle affects BM inflammation, which is an important

7
factor for instigating bone resorption. To address this issue, we investigated the expression of
proinflammatory cytokines in the BM cells from control and MKO mice. The expression of Tnf, Rankl,
Rorat, and Il17a was significantly increased in the BM from MKO mice compared with the controls
(Fig 4a). Seeking further evidence in support of the concept that mitochondrial OxPhos dysfunction in
skeletal muscle induces inflammation in BM, we measured the populations of diverse T-cells in the BM
from control and MKO mice using flow cytometry analysis. We found that at 14 weeks of age, mature
CD3+ T-cells were markedly elevated in the BM from MKO mice compared with those from the
controls (Fig 4b,c). Moreover, the population of TNF-α-producing CD4+ and CD8+ T-cells was
significantly larger in the BM of MKO mice (Fig 4d,e). The populations of CD44+ TNF-α+ and
CD44+IL-17A+ cells were also larger among the CD4+ and CD8+ T-cells of the BM of MKO mice
(Fig 4d-h). However, there was no difference in the size of the population of BM CD4+CD25+Foxp3+
regulatory T-cells between control and MKO mice (Fig 4i). Furthermore, to exclude systemic
inflammation underlying the bone loss in MKO mice, we measured TNF-α and IL-17A production in
CD4+ T-cells from the spleen of control and MKO mice at 14 weeks of age. The expression of
proinflammatory cytokines by CD4+ T-cells was not different in the spleens of control and MKO mice
(Fig 4j-l). In addition, there was no notable difference in the serum level of TNF-α or IL-17A between
control and MKO mice at 14 weeks of age (Supplementary Fig 5). Thus, these data revealed that
muscular mitochondrial dysfunction-mediated bone loss is associated with BM inflammation rather
than a systemic inflammatory response.
MKO mice show adipogenesis and an inflammatory response in the BM. Based on the results
from the flow cytometry analysis of the BM of control and MKO mice, we further investigated the
transcripts of whole BM cells using RNA sequencing. Given that macroscopically the long bones
dissected from MKO mice were more reddish than the controls (Fig 5a), we assumed that a more intense
inflammatory response had occurred in the BM of MKO mice. After hematoxylin and eosin (H&E)
8
staining, we also discovered a higher number of lipid droplets in the BM of MKO mice (Fig 5b). Thus,
we conducted RNA sequencing to identify genes in BM cells differentially expressed between control
and MKO mice at 14 weeks of age. Enrichment analysis with Network2Canvas revealed that genes
associated with myopathy, osteoporosis, signaling in the immune system, T-cells, and IL-12 and IL-17
pathways were enriched in the BM cells from MKO mice (Fig 5c, and Supplementary Fig 6a,b).
Moreover, gene set enrichment analysis (GSEA) indicated that the gene sets involved in adipogenesis
or adipocyte maturation was enriched in the BM cells of MKO mice compared with the controls (Fig
5d,e). In addition, consistent with the long bones appearing more reddish in MKO mice, genes related
to T-cell activation and co-stimulation were markedly increased in the BM cells of MKO mice, which
may be closely associated with the BM inflammation and bone loss observed (Fig 5f,g). A volcano plot
based on differential expression and statistical significance of the difference showed that, in addition to
that of the adipogenesis and T-cell activation gene sets, expression of Cxcl12 as well as of Fabp4,
Adipoq, and Cd3e was also changed in the BM cells of MKO mice (Fig 5h). We also found that CXCL12
protein expression was higher in the PDGFRβ+VCAM-1+ stromal cells (CXCL12-rich cells) among
BM cells from MKO mice using flow cytometry analysis (Fig 5i and Supplementary Fig 6c).
Furthermore, we searched genes potentially associated with Cxcl12 by applying Gene-Module
Association Determination (G-MAD) to mouse expression data sets using GeneBridge tools 17. As a
result, genes annotated in the inflammatory response and T-cell activation functional clusters were
strongly enriched (Supplementary Fig 6d). These results implicate the distinct effects of muscular
mitochondrial dysfunction on BM inflammation and bone loss in mice.
CXCR4 antagonism attenuates BM inflammation in MKO mice. CXCL12 is also strongly
expressed in the reticular cells located adjacent to sinusoids in BM, which express adiponectin and are
targetable with an adiponectin-Cre transgene 18. In addition, differentiated adipocytes induce CXCL12
secretion, which recruits proinflammatory macrophages into the adipose tissue of diet-induced obese
9
19
mice . The chemokine CXCL12 binds primarily to CXCR4, leading to activation of intracellular
20
signaling in multiple cell types including lymphocytes . Moreover, the CXCL12–CXCR4 axis is
involved in many physiological and pathological processes 20. Thus, we asked whether migration of
CXCR4+ immune cells to the MKO mouse BM contributes to the inflammatory response and
proinflammatory cytokine production therein. As shown in Figure 6A, G-MAD revealed that genes
annotated in the inflammatory response and T-cell activation functional clusters were strongly enriched.
Next, to reveal the function of the CXCL12–CXCR4 axis on BM inflammation, we administered the
CXCR4 receptor antagonist AMD3100 (5 mg/kg/day) intraperitoneally to 10-week-old control and
MKO mice for 3 weeks. To determine whether AMD3100 can reduce infiltration of proinflammatory
immune cells into the BM of MKO mice, we performed flow cytometry analysis to evaluate the
proportions of diverse types of immune cells in the BM. AMD3100 induced a significant decrease in
CD3+ T-cells of the BM in MKO mice, but it caused little change in the populations of BM CD4+ and
CD8+ T-cells in either WT or MKO mice (Fig 6b and Supplementary Fig 7a). Moreover, the production
of proinflammatory cytokines including IFN-γ, TNF-α, and IL-17A in CD4+ and CD8+ T-cells by BM
cells was significantly reduced in the MKO mice treated with AMD3100 (Supplementary Fig 7b-e).
Additionally, the expression of proinflammatory cytokines by the effector CD4+ and CD8+ T-cells was
significantly decreased in the BM cells from MKO mice treated with AMD3100 (Fig 6c-h and
Supplementary Fig 7f). These data indicate that CXCR4 antagonism attenuates BM infiltration by
inflammatory T-cells in the MKO mouse model of muscular mitochondrial dysfunction.
Treatment with CXCR4 antagonists prevents bone loss in MKO mice. To investigate the effect of
CXCR4 antagonism on bone loss in MKO mice, micro-CT analysis was conducted to assess BMD, BV,
and cortical and trabecular bone architecture in the control and MKO mice treated with or without
AMD3100. Consistent with previous results, MKO mice showed lower cortical and trabecular BV,
BMD, trabecular number, and Tb.Th. compared with the controls (Fig 7a-c). AMD3100 significantly
10
increased cortical and trabecular BV and trabecular number in MKO mice but not in the controls,
without any changes in markers indicative of liver injury or perturbed lipid metabolism (Fig 7a-c and
Supplementary Fig 8a-d). Taken together, these findings demonstrate that the CXCL12–CXCR4 axis is
important for the BM inflammatory response and bone loss in the MKO mouse model with muscle
dysfunction caused by reduced mitochondrial OxPhos.
Higher inflammatory response in the BM of hip fracture patients with lower body mass index.
Muscle mitochondrial impairment is an important feature of pre-frailty development in humans 21. It is
also well known that body mass index (BMI) has a positive correlation with muscle strength and mass
in humans 22. As shown in Supplemental Figure 9A, grip strength was significantly lower in hip fracture
patients with lower BMI compared with age- and gender-matched control subjects (Supplemental Table
1). We next investigated the immunophenotype of T-cells in the BM of hip fracture patients with lower
or normal BMI (Fig 8a). To compare the different subsets of T-cells in the BM of patients with a lower
BMI with those with a normal BMI, we evaluated the frequency of CD4+ and CD8+ BM T-cells
expressing naïve/memory markers (CD45RA+/RO+). The patients with lower BMI had larger and
smaller populations of CD8+ T-cells and CD4+ T-cells, respectively (Fig 8a and Supplementary Fig 9b).
In the subset analysis of CD4+ and CD8+ T-cells, in both populations the proportion of CD45RA-
CD45RO+ memory T-cells was significantly increased, while that of CD45RA+CD45RO- naïve T-cells
was decreased in patients with lower BMI (Fig 8b-c, and Supplementary Fig 9c,d). Similarly to the role
of T-cell senescence in chronic inflammatory conditions 23, the population of BM CD57+ senescent
CD4+ and CD8+ T-cells was also larger in the patients with lower BMI (Fig 8d,e, and Supplementary
Fig 9e). Furthermore, the expression of bone resorptive cytokines, including TNF-α and IL-17A, was
markedly increased in the BM CD4+ and CD8+ T-cells of patients with lower BMI (Fig 8f-i). The
expression of inflammation-related genes including CXCL12, CD44, TNF, and IL17A was also higher
in the BM of patients with lower BMI (Supplementary Fig 9f-i). Moreover, serum levels of FGF21 and
11
growth differentiation factor 15 (GDF15) trended higher in patients with lower BMI (Supplementary
Fig 10a,b). Collectively, these data suggest that lower BMI and muscle function predict a larger
population of proinflammatory and cytotoxic senescent T-cells in the BM of patients with hip fracture.
Discussion
In the present work, we investigated the impact of the mitochondrial OxPhos function in skeletal
muscle on the maintenance of bone mass in mice. Our observations indicate that lower mitochondrial
OxPhos function characterizes a reduction in muscle mass and physical activity, which contribute to the
bone fragility of MKO mice. In addition, we report that mitochondrial stress in skeletal muscle induces
BM inflammation, wherein the CXCL12–CXCR4 axis is involved in the migration and activation of T-
cells. Antagonism of CXCR4 protects against the bone loss of the MKO mouse model with muscular
mitochondrial dysfunction through attenuation of BM inflammation.
Mitochondria are important organelles regulating critical cellular processes in the physiology and
pathology of skeletal muscle 24. Consistent with this, our mouse model with skeletal muscle-specific
mitochondrial OxPhos dysfunction showed lower muscle mass and physical performance (Fig 1),
making it suitable for use as a model of muscle atrophy. Moreover, it has been suggested that
appendicular muscle mass and strength are closely associated with BMD 25. In line with this report, we
have demonstrated that MKO mice also exhibit lower bone mass, as well as a reduction in cortical and
trabecular BMD (Fig 2). Previous reports also indicate a positive correlation between mass and function
of skeletal muscle and bone mass, which may be due to reduced physical loading or a diminished
response to load. However, the biological mechanism of muscle dysfunction-induced bone loss and
fragility remains to be determined. To this end, in the current study, we showed through an
osteoimmunologic analysis of MKO mice that BM inflammation plays an essential role in muscle
dysfunction-induced low bone mass and structural deterioration of bone tissue. Thus, it is possible that
12
the improvement of muscle mitochondrial function by exercise training and/or diet may enhance bone
mass and quality.
Inflammation is regarded as playing a causal role in bone loss in humans and mice with sex
steroid deprivation. TNF-α-producing T-cells are increased in the BM of mice and humans through
26-29
natural or surgical menopause, contributing to bone loss and fragility . In addition, estrogen
deficiency induces high levels of serum IL-17 and promotes Th17 cell differentiation in ovariectomized
mice 30. IL-17 is increased in patients with primary hyperparathyroidism and also mediates parathyroid
hormone-induced bone loss 31. Moreover, systemic inflammation caused by sepsis produces osteoblast
ablation and bone loss without any change in osteoclast function 32. In the current work, we show that,
in MKO mice, muscular mitochondrial OxPhos dysfunction induces an increase in TNF-α- and IL-17A-
producing T-cells of the BM. The hip fracture patients with a markedly lower BMI also exhibited a
higher inflammatory response, as measured by proinflammatory cytokine production, in the BM.
Collectively, the present findings indicate that BM inflammation is required for loss of muscle mass
and function-mediated bone loss and fragility.
CXCR4 stimulates signal transduction for T-cell chemotaxis and gene expression via association
with the T-cell receptor 33. CXCR4 is required for the migration of pathogenic CD4+ and CD8+ T-cells
to the BM in a mouse model of aplastic anemia 34. In addition, CXCR4 overexpression by CD8+ T-cells
increases their migration toward the BM microenvironment, leading to memory T-cell differentiation
and production of effector cytokines including IFN-γ, TNF-α, and IL-2 35. Adoptively transferred central
memory T-cells accumulated more efficiently in BM cavities via their higher level of CXCR4
36
expression compared with naïve and effector T-cells . One of the most prominent features of the
present work is the observation that CXCR4 antagonism protected MKO mice against BM
inflammation and bone loss. Consistent with these findings, the CXCR4 antagonist AMD3100 has been
shown to improve ovariectomy-induced bone loss by facilitating mobilization of hematopoietic
progenitor cells 37. Moreover, treatment with vascular endothelial growth factor and AMD3100 can
13
mobilize mesenchymal stem cells toward fracture healing, leading to bone formation in a delayed union
osteotomy model 38. CXCL12, a chief ligand for CXCR4, is not only linked with disease severity of
39
postmenopausal osteoporosis , but also acts as a pro-inflammatory factor in the progression of
collagen-induced osteoarthritis by attracting inflammatory cells to joints and by activating osteoclasts

40
. On the other hand, genetic disruption of CXCR4 enhances osteoclastogenesis and leads to increased
41
osteolytic tumor growth in bone . Discrepancies in findings between different studies could be
attributed to the use of different mouse or disease models and/or to the distinct role of CXCR4 in many
different kinds of cell. Therefore, further investigation is required to establish the role of CXCR4 in the
diverse context of bone loss using a cell type-specific Cxcr4 knockout mouse model.
In this study, we analyzed two kinds of RNA sequencing data derived from EDL muscle and
whole BM cells from control and MKO mice. We observed that mitochondrial stress via OxPhos
dysfunction increases local expression and serum levels of FGF21, which do not have any effects on
bone mass and quality in MKO mice (Fig 3). On the other hand, GSEA indicated that BM cells from
MKO mice showed prominent expression differences in inflammatory response and adipogenesis gene
sets compared with the controls (Fig 5). Ectopic fat, defined as storage of triglyceride in tissues other
than adipose tissue, is associated with metabolic deterioration in humans as well as rodents. Although
the pathogenesis of ectopic fat deposition is largely unknown, free fatty acids released by adipocyte
hypertrophy and inflammatory response are important in the development of ectopic fat-induced organ
42
dysfunction in liver, skeletal muscle, and heart . Excessive fat deposition in non-adipose tissues
recruits immune cells including macrophages and activated T-cells, thereby promoting chronic
43
inflammation in the metabolic organs . Several investigations have revealed that BM is also
susceptible to fat deposition through old age, menopause, obesity, anorexia nervosa, and weight loss
44-47
surgery . Although multiple studies have investigated ectopic adipocyte accumulation in BM
cavities, our understanding of its role in the inflammatory response in BM is incomplete. Ectopic fat
accumulation in BM is associated with activation of immune cells and bone loss 48. Taken together,
14
these data suggest that muscular mitochondrial dysfunction-mediated bone loss is caused by the change
in the BM microenvironment rather than the change in mitokine secretion.
In conclusion, mitochondrial OxPhos function in skeletal muscles play a pivotal role in the
regulation of BM inflammation and bone loss in mice. Inhibition of BM inflammation by a CXCR4
antagonist attenuates bone loss in a mouse model with muscle mitochondrial dysfunction. However, the
human relevance of muscular mitochondrial OxPhos dysfunction and BM inflammation in the
regulation of skeletal homeostasis needs to be clarified.
Methods
Animals. To generate skeletal muscle-specific Crif1 deficiency in mice, floxed Crif1 mice were
crossed with Mlc1f-Cre mice on a C57BL/6 background (provided by S.J. Burden, New York University,
Baltimore, MD, USA). MKO mice were crossed with a global Fgf21 knockout on a C57BL/6
background (a kind gift from N. Itoh, Kyoto University Graduate School of Pharmaceutical Sciences,
Kyoto, Japan) to generate MKFO mice as a mitokine double knockout mouse model. All animal
experiments used male mice that were maintained in a controlled environment (12 h light/12 h dark
cycle; humidity, 50–60%; ambient temperature, 22 ± 2°C) and fed a normal chow diet. All experimental
procedures involving mice were conducted in accordance with the guidelines of the Institutional Animal
Care and Use Committee of Chungnam National University School of Medicine (CNUH-017-A0048,
Daejeon, Republic of Korea).
Human subjects. Patients with hip fracture were recruited from the Chungnam National
University Hospital between October 2019 and February 2020. The participants were divided into two
age- and gender-matched groups as follows: BMI, 22–25 (n = 7) and BMI <18 (n = 7). Patients with any
of the following conditions were excluded from the study: rheumatoid arthritis, neuromuscular disorder,
chronic kidney disease, and mineral and bone disorder. Patients with diseases that affect bone
15
metabolism (or those taking drugs that affect bone metabolism), history of any malignant or
inflammatory disease, and past hormone replacement therapy were also excluded. Hand grip strength
was measured using an electronic hand dynamometer (Lavisen, Namyangju, South Korea). Grip
strength of the dominant hand was measured only once in a sitting posture with 0° shoulder angle, 90°
elbow angle and neutral wrist angle. Lymphocytes were isolated from the BM cells of the enrolled
patients and stored at -180°C in liquid nitrogen prior to flow cytometry analysis. Whole BM cells were
used for real-time PCR analysis. This human study was reviewed and approved by the Institutional
Review Board of Chungnam National University Hospital (CNUH 2019-10-065), according to the
standards of the Declaration of Helsinki. Written and oral informed consent, documented by the
Department of Internal Medicine of Chungnam National University Hospital in South Korea, was
obtained from all of the participants prior to their inclusion in the study.
Flow cytometry analysis. Isolated BM cells were filtered through a 70 μm cell strainer, washed
in phosphate-buffered saline (PBS), and resuspended in a 40% Percoll (GE Healthcare, Chalfont St
Giles, UK) gradient. The cell suspension was centrifuged at 2,400 rpm for 30 min at 4°C. Then, the
cells were incubated with directly fluorochrome-conjugated monoclonal antibodies for 40 min at 4°C.
The antibodies used in this study are listed in Supplemental Table 2. For blocking non-specific antibody
binding, cells were pre-incubated with anti-mouse CD16/32 mouse Fc blocker (BD Biosciences, San
Jose, CA, USA) prior to staining with the antibodies. For intracellular staining, BM cells were
stimulated with Cell Stimulation Cocktail including phorbol myristate acetate, ionomycin, brefeldin A,
and monensin (eBioscience, San Diego, CA, USA) for 5 h. The cells were fixed and permeabilized
using a Fixation/Permeabilization Buffer kit (eBioscience, San Diego, CA, USA), and then washed and
resuspended in 1% formaldehyde, and further stained for intracellular cytokines with anti-IFN-γ-PE-
Cy7, anti-TNF-α-APC, or anti-IL-17A-APC. Multicolor flow cytometry was performed using a BD
LSRFortessa flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software
(Tree Star, Ashland, OR, USA). Results are expressed as cell frequency (%).
16
Immunoblots. Tissues and cells were lysed using 2% sodium dodecyl sulfate (SDS) with 2 M urea,
10% glycerol, 10 mM Tris-HCl (pH 6.8), 10 mM dithiothreitol, and 1 mM phenylmethylsulfonyl
fluoride. The lysates were centrifuged, and the supernatants were separated by SDS-polyacrylamide gel
electrophoresis (PAGE) and blotted onto a nitrocellulose (NC) membrane. After blocking with 5%
skimmed milk, the membrane was analyzed using specific antibodies and visualized by enhanced
chemiluminescence using WesternBright ECL Spray (Advansta, Menlo Park, CA, USA). Proteins were
detected by immunoblotting with antibodies listed in Supplemental Table 2. A horseradish peroxidase-
conjugated goat anti-rabbit IgG (Enzo Life Sciences, Farmingdale, NY, USA) secondary antibody was
used for visualization. Signals were obtained using an Odyssey imager and Image Studio Software (LI-
COR Biosciences, Lincoln, NE, USA). Serum levels of FGF21 were measured using an ELISA kit
(R&D Systems, Minneapolis, MN, USA).
BN-PAGE. To isolate mitochondria from the EDL and gastrocnemius of 14-week-old control and
MKO mice, samples were homogenized in isolation buffer (210 mM mannitol, 70 mM sucrose, 1 mM
EGTA, and 5 mM HEPES, pH 7.2) using a Teflon-glass homogenizer. The homogenized tissues were
centrifuged at 600 × g for 5 min at 4°C, and the supernatant was re-centrifuged at 17,000 × g for 10 min
at 4°C. The isolated mitochondrial fraction was supplemented with 0.5% (w/v) n-dodecyl-β-D-
maltoside and assessed for OxPhos complex content using a Native PAGE Novex Bis-Tris Gel system
(Invitrogen, Carlsbad, CA, USA). The separated proteins were transferred to polyvinylidene fluoride
membranes, which were incubated overnight at 4°C with an anti-OxPhos antibody cocktail (Invitrogen,
#45-8099, #45-7999), and were analyzed using the Western Breeze Chromogenic Western Blot
Immunodetection Kit (Invitrogen).
SDH staining. Transverse 12 μm muscle sections were mounted on Super Frost microscope slides
(Thermo Fisher Scientific, Waltham, MA, USA). Muscle fiber type-specific diameter measurements
were obtained using 12 μm-thick SDH-stained cross-sections at 14 weeks of age. Sections were outlined
with a PAP pen (Research Products International) and incubated in buffer solution (20 mM phosphate
17
buffer, 7.5% sucrose, 0.027% sodium succinate, and 10 mg nitrobluetetrazoleum) for 45 min at room
temperature. The sections were dehydrated, and then briefly rinsed in 30%, 60%, and 90% acetone in
distilled water in ascending and descending order, rinsed in distilled water, air-dried, and cover-slipped
using VectaMount (Vector Labs).
Micro-CT analysis. Micro-CT was performed on vertebrae and long bones using an eXplore
Locus SP scanner (GE Healthcare, London, Canada) with 8 µm resolution. All bone morphometric
parameters were calculated three‐dimensionally with eXplore MicroView version 2.2 (GE Healthcare),
which was used for measuring BV, total volume (TV), percent BV (BV/TV), bone surface (BS), bone
surface density (BS/TV), Tb.Th., and Tb.Sp. Bone parameters and density were analyzed at the region
between 0.7 and 2.3 mm below the growth plate of the distal femur. Cancellous bone was analyzed in
the distal area extending proximally 1.75 mm from the end of the primary spongiosa. The half the height
of the bone with half the width of the 5th lumbar vertebral body was used to analyze cancellous spine.
All bone micro-CT nomenclature follows the guidelines of the American Society for Bone and Mineral
Research (ASBMR).
Bone histological and morphological analysis. Mice were euthanized at 14 weeks and the bones
were removed and fixed in 4% paraformaldehyde (Biosesang, Seongnam, korea) at 4°C overnight. To
evaluate dynamic histomorphometry, mice were injected intraperitoneally with alizarin red (Sigma-
Aldrich, Dorset, UK, 30 mg/kg) and calcein (Sigma-Aldrich, Dorset, UK, 10 mg/kg) 8 and 3 days prior
to CO2 asphyxiation, respectively. For bone histological analysis, femurs and tibias of mice were
harvested, skinned, and fixed in 4% paraformaldehyde overnight at room temperature. The samples
were then dehydrated in ethanol solution and decalcified in 10% ethylenediaminetetraacetic acid
(Sigma-Aldrich, Dorset, UK) for 2 weeks at room temperature. The buffer was changed every 3–4 days
until complete decalcification. The tissues were embedded in paraffin, and 4 μm sagittal-oriented
sections were prepared and stained with H&E and TRAP for histological analysis using standard
protocols. For von Kossa staining, undecalcified bones were embedded in methyl‐methacrylate (Sigma)
18
and sectioned at a thickness of 6 µm as previously described 49. Bone histomorphometric analysis was
performed with the Bioquant Osteo II program (Bio‐Quant, Inc., Nashville, TN, USA). All bone
histomorphometry nomenclature follows the guidelines of the ASBMR 50.
Mouse grip strength and wire hanging test. Experiments were performed using a digital force-
gauging apparatus (GS 5000; Borj Sanat, Iran). Mice were allowed to grasp the pull bar with fore limbs,
and then they were gently pulled parallel away from the bar by the tail until the forelimbs released the
bar. Mice were not trained before testing. The maximum force prior to release of the mouse’s paw from
the bar was recorded. The test was repeated five times, and the average value of five consecutive
measurements was reported as the mouse’s grip strength. The wire hanging test was conducted to assess
motor function and neuromuscular grip strength. The mouse was placed on a cross-grip wire rack, which
was then turned upside down 20 cm above a cage filled with soft bedding, after which hanging time
was recorded. The average latency to fall of four trials was calculated for each animal. The maximum
hanging time was used in the analyses.
RNA sequencing. Total RNA was prepared from EDL and BM cells obtained from 10-week-old
control and MKO mice (n = 2 for each) using TRIzol reagent. The integrity of the total RNA was
assessed using an Agilent 2100 Bioanalyzer System (Agilent Technologies, Loveland, CO, USA) and
an Agilent RNA 6000 Nano Kit (Agilent Technologies, Loveland, CO, USA). The library was prepared
using a TruSeq 3000/4000 SBS Kit, v3. It preprocessed the raw reads from the sequencer to remove
results from low-quality RNA or artifacts such as adaptor sequences, contaminant DNA, and PCR
duplicates. The quality of the data produced is determined by the phred quality score of each base. The
FastQC quality control tool gives a box plot of average base quality per cycle, and a phred quality score
of 20 means that the assignment to that base is 99% accurate. Generally a phred score ≥20 is good
quality, and those in the present study were ≥30 was 97.19%. The obtained reads were mapped to a
reference Mus musculus (mm10) genome using HISAT2 v2.0.5. HISAT uses two types of index for
alignment (a global, whole-genome index and tens of thousands of small local indexes). These aligned
19
reads were then assembled from known genes/transcripts using a reference gene model in StringTie
v.1.3.3b. Transcript frequencies were quantified as normalized values, taking into account transcript
length and depth of coverage. Relative transcript abundance was expressed as fragments per kilobase
of transcript per million fragments mapped (FPKM), and FPKM values ≤0 were excluded. One was
added to each FPKM value for filtered genes, the filtered data were log2-transformed, and quantile
normalization was applied. Differentially expressed gene (DEG) analysis was performed using FPKM
value. Genes with a fold-change >2 and an independent t-test P-value <0.05 were extracted from the
results of the DEG analysis. A heatmap was produced by color-coding standardized log gene expression
levels (mean, zero; standard deviation, one) using R 3.5.1 available at http://www.r-project.org. Probe
sets are shown as hierarchically clustered by similarity, based on Euclidean distance and the Ward
aggregation algorithm.
RNA sequencing analysis using bioinformatics tools. DEGs were then subjected to hierarchical
clustering and phenotype ontology using Network2Canvas (http://maayanlab.net/N2C/). Phenotype
categories were visualized on the grid according to gene-list similarity, with enriched categories being
indicated by circles. GSEA (http://www.broadinstitute.org/gsea) was performed on transcriptome data
from the BM cells from control and MKO mice. Bioinformatic analysis was carried out with R package
v3.2.5, available at http://www.r-project.org. A heatmap was produced by color-coding standardized log
gene expression levels (mean, zero; standard deviation, one). Probe sets are shown as hierarchically
clustered by similarity, based on Euclidean distance and the Ward aggregation algorithm. We also used
G-MAD in GeneBridge tools (available at http://systems-genetics.org, an open resource), which uses
expression data from large-scale cohorts to propose potential functions of genes and allows the
annotation of gene function 17.
Treatment with AMD3100 in vivo. Control and MKO mice (9 weeks of age) were injected
intraperitoneally with 5 mg/kg PBS or AMD3100 (Sigma-Aldrich #A5602; St. Louis, MO, USA; Sigma
Aldrich.com) three times a week for 3 weeks. At the end of treatment, the mice were sacrificed, and
20
blood samples were collected for measuring proinflammatory cytokines and bone turnover markers.
Femurs and tibiae were removed, fixed with 4% paraformaldehyde in PBS solution (pH 7.4) for 16 hr,
and then stored at 4°C in 80% ethanol prior to measurement of BMD using micro-CT.
Statistical analysis. Results are expressed as mean values ± standard error of the mean (SEM).
Data were analyzed using Prism (version 8, GraphPad Software Inc., San Diego, CA, USA). A 2-tailed,
unpaired t-test with Welch’s correction was used to assess statistical significance between two groups.
A one-way analysis of variance (ANOVA) with Bonferroni’s correction for multiple comparisons was
used to examine differences between more than two groups. A P-value <0.05 was considered
statistically significant.
Author contributions
MS and HSY designed the studies. JWT, JSM, HTN, HYL, JTK, JYC, XC, and HSY performed the
research and analyzed the data. HKC, DR, JYC, MT, and TS analyzed the data. HSY and JWT wrote
the manuscript.
Acknowledgments
This work was supported by the Basic Science Research Program, through the National Research
Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning, Korea (NRF-
2018R1C1B6004439, NRF-2019M3E5D1A02068575) and the CNUH Research Fund, 2018. MS and
SKK were also supported by the NRF (NRF-2017K1A1A2013124 and NRF-2017R1E1A1A01075126).
21
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Figure 1. MKO mice show impairment in OxPhos and deterioration in physical performance. (a,
b) Representative western blots and band density measurements for OxPhos complex subunits and
CRIF1 in the EDL and gastrocnemius of chow-fed control and MKO mice at 14 weeks of age (n = 3).
(c) Representative blots showing BN-PAGE of the assembled OxPhos complex in EDL and
gastrocnemius from chow-fed control and MKO mice at 14 weeks of age. (d) Transverse EDL sections
were histochemically stained for SDH to identify oxidative muscle fibers at 14 weeks of age. Scale bar,
100 μm. (e) Quantification of unstained fibers in EDL of controls and MKO mice at 14 weeks of age
(each n = 5). (f) Relative mRNA expression of genes related to mitochondrial stress response from EDL
in 14-week-old control and MKO mice (each n = 4). (g) Body weight evolution of control and MKO
mice fed a chow diet for 8 weeks (n = 10 per group). (h) Latency to fall in rotarod test at 10 rpm (each
n = 7). (i) Forelimb grip strength and time to fall in the wire hanging assay for control and MKO mice
(each n = 10). (j) Grip strength normalized to body weight of control and MKO mice (each n = 10).
26
Data are expressed as mean ± SEM. Statistical significance was analyzed by unpaired t-tests. *, P <
0.05 and **, P < 0.01 compared with the indicated group.
27
Figure 2. Mitochondrial stress response in skeletal muscle promotes bone loss. (a,b) Representative
images of micro-CT of cortical and trabecular regions in the distal femur from control and MKO mice
at 14 weeks of age. (c) Measurement of Tb.Th., trabecular number (Tb.N.), BV/TV, BS/TV, BS/BV,
cortical volume (Ct.V.), Tb.Sp., and total bone volume (TBV) using micro-CT analysis. (d,e) Von Kossa
staining of undecalcified sections of vertebrae and femurs of control and MKO mice at 14 weeks of age.
Scale bars, 250 μm. (f) TRAP staining to reveal osteoclasts. Scale bars: 100 μm. (g) Histomorphometric
analysis of calcein/alizarin red double-stained sections was conducted to quantify bone formation in
vertebrae (n = 5/group). Green indicates calcein, and red indicates alizarin red. (h) Measurement of
endocortical MAR and MS/BS (%) of vertebrae in control and MKO mice. (i) Serum levels of P1NP
and CTX in 14-week-old control and MKO mice (each n = 7). Data are expressed as mean ± SEM.
28
Statistical significance was analyzed by unpaired t-tests. *, P < 0.05 and **, P < 0.01 compared with
the indicated group.
29
Figure 3. Bone loss is independent of FGF21 production caused by mitochondrial stress in skeletal
muscle of MKO mice. (a)Scatterplots of RNA sequencing data, displaying transcript levels in EDL of
control (x-axis) and MKO (y-axis) mice at 10 weeks of age. The text indicates mitokines showing much
higher fold-change in MKO mice among the secreted proteins. (b,c) Volcano plot and heat map showing
upregulated genes in EDL from normal chow diet-fed control and MKO mice at 10 weeks of age. (d)
Relative expression of mRNA encoding Fgf21 in EDL from 14-week-old control (n = 6) and MKO (n
= 6) mice. (e) Serum levels of FGF21 in 14-week-old control (n = 6) and MKO (n = 6) mice. (f) Micro-
CT images of the trabecular bone (Tr.b) near the distal femoral metaphyseal region from control (n = 4)
and MKO (n = 4) mice at 14 week of age. Scale bar for front view and 3D image Tr.b, 1000 and 500
μm, respectively. (g) Measurement of Tb.Th., Tb.N., BV/TV, BS/TV, BS/BV, Ct.V., Tb.Sp., and TBV
using micro-CT analysis. Data are expressed as mean ± SEM. Statistical significance was analyzed by
unpaired t-tests. **, P < 0.01 compared with the indicated group.
30
Figure 4. MKO mice exhibit local induction of TNF-α and IL-17 in BM without a systemic
inflammatory response. (a) BM cells isolated from control (n = 5) and MKO mice (n = 5) at 14 weeks
of age were subjected to real-time PCR analysis of osteoclastogenic genes. (b) Representative flow
cytometry contour plots are presented for CD3 expression by BM cells in control (n = 7) and MKO (n
= 7) mice at 14 weeks of age. (c) Statistical analysis of the population of CD3+ T-cells in BM cells in
the two groups. (d-h) The number of TNF-α- or IL-17A-secreting cells in the populations of BM
CD4+CD44+ and CD8+C44+ T-cells was compared between the two groups (each n = 7). (i) Population
of regulatory T-cells (CD4+CD25+FOXP3+) in the BM of 14-week-old control (n = 7) and MKO (n =
7) mice. (j-l) TNF-α- or IL-17A-producing cells among CD4+ and CD8+ T-cells from the spleens of
control (n = 7) and MKO (n = 7) mice at 14 weeks of age. Data are expressed as mean ± SEM. Statistical
significance was analyzed by unpaired t-tests. *, P < 0.05 and **, P < 0.01 compared with the indicated
group.
31
Figure 5. Transcriptome analysis shows adipogenesis and inflammatory response in the BM of
MKO mice. (a) Femur and tibia of a 14-week-old control and MKO mouse. BM is more visible through
the thin cortical bone of MKO mice. (b) A representative section of tibia from a 14‐week‐old control
and MKO mouse stained with H&E. Thin femoral bone and adipocyte-rich BM (arrowhead) are visible
in the MKO femur. Scale bars, 250 μm. (c) Using RNA sequencing data, genes that were significantly
upregulated in the BM cells of control and MKO mice were analyzed for gene-list enrichment with gene
set libraries created from level 4 of the MGI mouse phenotype ontology using Network2Canvas. (d-g)
The diagram shows the results of gene set (Adipogenesis, Adipocyte maturation, T-cell activation, and
Co-stimulators) enrichment analysis, including the enrichment scores. (h) Volcano plot based on the
differential expression and significance of the differences in the data from RNA sequencing of BM cells
of control and MKO mice at 14 weeks of age. Red dots represent genes associated with adipogenesis
and T-cell activation with a P-value <0.05 and a fold-change >1 log2. (i) In the flow cytometry analysis,
32
total BM cells were gated for a population negative for CD45, Ter119, CD31, and Sca1, and were
positive for VCAM and PDGFRβ, the phenotype of CXCL12-abundant reticular cells. Intracellular
levels of CXCL12 are shown as amount of protein in CAR cells in the BM of MKO mice relative to
CAR cells in the BM of the controls at 14 weeks of age (n = 4 mice/genotype). Scale bars: 50 μm (a)
and 10 μm (b). Data are expressed as mean ± SEM. Statistical significance was analyzed by unpaired t-
tests. *, P < 0.05 and **, P < 0.01 compared with the indicated group.
33
Figure 6. Treatment with CXCR4 antagonist reduces BM inflammation in MKO mice. (a) In the
G-MAD analysis, CXCR4 associates with T-cell migration and proliferation, and chemokine-binding
modules in mice. The threshold of significant gene-module association is indicated by the red dashed
line. Modules are organized by module similarities. Known modules connected to CXCR4 are
highlighted in red. GMAS, gene-module association score. (b) BM cells were subjected to flow
cytometry for mature T-cells, natural killer, and natural killer T-cell phenotypes. (c,d) Populations of
BM CD44+IFN-γ+ or CD44+ TNF-α+ among CD4+ T-cells from control and MKO mice treated with
AMD3100 or vehicle at 14 weeks of age. (e) IL-17A production by BM CD4+ T-cells was analyzed by
flow cytometry. (f,g) Populations of BM CD44+IFN-γ+ or CD44+ TNF-α+ among CD8+ T-cells from
control and MKO mice treated with AMD3100 or vehicle at 14 weeks of age. (h) Production of IFN-γ
or TNF-α by BM CD4+CD44+ or CD8+CD44+ T-cells of control and MKO mice treated with
34
AMD3100 or vehicle at 14 weeks of age. Data are expressed as mean ± SEM. Statistical significance
was analyzed by one-way ANOVA.
35
Figure 7. Treatment with AMD3100 attenuates bone loss in MKO mice. (a,b) At 9 weeks of age,
control and MKO mice were injected intraperitoneally with AMD3100 (5 mg/kg, three times per week)
for 3 weeks. Tibial and femoral trabeculae of control and MKO mice were measured by micro-CT. Scale
bars, 250 μm. (c) Measurement of Tb.N., Tb.Th., BV/TV, BS/TV, BS/BV, Ct.V., Tb.Sp., and TBV in
the tibiae from control and MKO mice using micro-CT analysis. Data are expressed as mean ± SEM.
Statistical significance was analyzed by one-way ANOVA. *, P < 0.05 and **, P < 0.01 compared with
the indicated group. DW, distilled water.
36
Figure 8. Immunophenotyping of BM T-cells in patients with hip fracture according to BMI. (a)
Representative flow cytometry contour plots are presented for CD4 and CD8 expression by CD3+ T-
cells in hip fracture patients with normal (22–25 kg/m2) and lower (<18 kg/m2) BMI. (b,c)
Representative plots of CD45RO and CD45RA expression among CD4+ or CD8+ T-cells in the BM
from hip fracture patients with a lower (<18 kg/m2) or normal (22–25 kg/m2) BMI (each n = 7). (d,e)
CD57+ senescent population in CD4+ and CD8+ T-cells of the BM from hip fracture patients with a
lower (<18) or normal (22–25) BMI (each n = 7). (f-h) Frequency of TNF-α- or IL-17A-producing cells
among the BM CD4+ and CD8+ cells were evaluated by flow cytometry (each n = 7). (i) Statistical
analysis of the population of TNF-α- or IL-17A-producing cells among the BM CD4+ and CD8+ cells
in the two groups (n = 5/group). Data are expressed as mean ± SEM. Statistical significance was
analyzed by unpaired t-tests. *, P < 0.05 and **, P < 0.01 compared with the indicated group.
37
Fig. 1
a b
Control MKO Control MKO EDL Gastrocnemius
SDHA (II) Control MKO

Control MKO
Fold change in band density

1.5 1.5
UQCRC2 (III) *
** ** **
** ** ** *
1.0 1.0
ATP5A (V)
CRIF1 0.5 0.5
GAPDH
0.0 0.0
EDL Gastrocnemius SDHA UQCRC2 ATP5A CRIF1 SDHA UQCRC2 ATP5A CRIF1
c d e f
Control MKO Control MKO SDH staining Weak Medium Strong Control MKO
5
100
**
Percent of total fibers

CI ** **
Control 4
**
Relative expression (AU)

CV **
*
3
50
CIV
MKO 2
CII
1
EDL 0
Gastrocnemius
Control MKO
0
Ppargc1a Lonp1 Clpp Hspd1 Tid1 Chop Atf4
g h i j
Control MKO Control MKO

**
Mean latency (sec, 10 RPM)
30 **
** 30 300 8
** **
** **
Grip strength (g/g)

Body weight (g)
25 200
6
20 100
20
40 4
Control 10
15
MKO 2
10
5 7 9 11 13 15 17 0 0 0
Age (week) Grip strength (g) Hanging time Control MKO
(sec)
sFig. 1
a b c
Crif1flox/flox MLC1f-cre
Control MKO
Crif1
X MLC Control MKO

1.5
SDHA (II) ** **
**
UQCRC2 (III) 1.0
ATP5A (V) 0.5

Crif1
GAPDH
0.0
MLC SDHA UQCRC2 ATP5A
Soleus
Crif1flox/flox;MLC1f-Cre
d Gastrocnemius
Control
MKO
a
Control Control
b f
TRAP staining Fig. 2
Control Control
MKO MKO
Control MKO
MKO MKO
g
c
Control MKO
(mm) Tb.Th. (mm-1) Tb.N. (%) BV/TV (%) BS/TV

0.15 2.5 ** 20 8
**
** **
2.0
15 6
0.10
1.5
10 4
1.0 Control MKO
0.05 h
5 2
0.5
Control MKO
0.00 0.0 0 0
1.5 40
(%) BS/BV (mm3) Ct.V. (mm) Tb.Sp. (mm) Ct.Th
90 1.6 ** 0.35 0.16 30
MS/BS(%)
MAR(μm/d)
** 1.0
80 ** 1.4
0.30 0.14 20
70 1.2
0.25 0.12 0.5
10
60 1.0
0.20 0.10
50 0.8 0.0 0
40 0.6 0.15 0.08 i

Control MKO
d e 80 **
60
40
20
0
Vertebrae Femur P1NP (ng/mL) CTX (ng/mL)
a
sFig. 2
Cortical bone
Control MKO
b Vertebrae
25 ** 30 ** 6 **
20
Tb.N (mm-1)
20 4
Tb.Th (μm)
BV/TV (%)
15
10
10 2
5
0 0 0
Control MKO Control MKO Control MKO
c d
Femurs Tibia
**
20 40 4 25
** **
**
20
15 30 3
OC surface (%)
Tb.N (mm-1)
Tb.Th (μm)
BV/TV (%)
15
10 20 2
10
5 10 1
5
0 0 0 0
Control MKO Control MKO Control MKO Control MKO
sFig. 3
a b c d
120 0.4 5 0.6

Serum iPTH (pg/mL)
Testosterone (ng/mL)
Serum T3 (ng/mL)
4
Serum T4 (μg/dL)
90 0.3
0.4
3
60 0.2
2
0.2
30 0.1
1
0 0.0 0 0.0
Control MKO Control MKO Control MKO Control MKO
Fig. 3
a b c d e
Control MKO EDL
Secretion Muscle Retn
2000 **
4
6 Adm2 **
Fgf21 expression (AU)

Fgf6
p-value (-log10 p)
3 1500
FGF21 (pg/mL)
Fgf11
4 Fgf21
Gdf15 1000
Ostn
2
Adm2
2 Adm2 Gdf15
Gdf9
Fgf21 Fgf20 1 500
Angptl6
0 Fgf5
-2 0 2 Fgf14 0 0
Log2FC Control MKO Control MKO
Z-score
-1 0 1
f g Control Fgf21 KO MKO MFKO
(mm) Tb.Th. (mm-1) Tb.N. (%) BV/TV (%) BS/TV

Control Fgf21 KO MKO MFKO
0.15 2.5 20 80
2.0
15 70
Front view
0.10
1.5
10 60
1.0
0.05
5 50
0.5
0.00 0.0 0 40
(%) BS/BV (mm3) Ct.V. (mm) Tb.Sp. (mm) Ct.Th

3D image Tr.b
2.0 250 0.35 0.16
200 0.14
1.5 0.30
150 0.12
1.0 0.25
100 0.10
0.5 0.20
50 0.08
0.0 0 0.15 0.06

sFig. 4
a b
MKO MFKO
1.5
**
300

Crif1f/f
200
1.0
500 Fgf21
0.5
200
(bp) Control Fgf21 KO MKO MFKO
0.0
Crif1 Fgf21
Fig. 4
a b c d
BM cells
5 WT MKO Control MKO (%) Control MKO

10
** **
4 ** 8
3.19% 13.2%
CD45+CD3+
3 6
*
2 ** 4
2.15% 7.58%
CD4
FSC
1 2
0 CD3 0 TNF-α
Tnf Rankl Rorαt Rorgt Il17a WT MKO
e f g h
CD3+CD4+
WT MKO (%)
WT MKO Control MKO
15 ** 10
**
CD4+CD44+IL-17A+
2.11% 6.16% 8
2.81% 5.11%
CD44+TNF-α+
*
10 6
IL-17A
5
2
CD8
0
TNF-α 0 CD44 Control MKO
CD4+ CD8+
i j k l
(%)
Ctrl MKO WT MKO Control MKO WT
10
MKO
8
4.86% 5.79% 5.95% 7.05% 1.63% 1.42%
6
4
IL-17A
Foxp3
2
CD4
0
CD25 TNF-α FSC TNF-α IL-17A
CD3+CD4+
sFig. 5
Control MKO
60
Serum level (pg/mL)

40
20
0
TNF-α IL-17A
a b c
Fig. 5
OMIM Diseases Reactome pathways
Myopathy
Signaling in
immune system
Osteoporosis
Integrin cell surface
interaction
d f
Z score T cell activation
Lat
-3 3 Cd3g
Stat3 NES= 1.64 NES= 2.07
Cd8b1
Adipogenesis Cebpd Nominal p-value= 0.006 Nominal p-value= 0.001
Cd3e
Cebpb FDR q-value= 0.006 FDR q-value= 0.001
Cebpa
Srebf1 Cd83
Smad3 Cd4
Tgfbr2 Cd247
Adipoq
Fgf1
g
Costimulators
Pparg
Tgfb3 Cd28
Icos
Control MKO
e h i
Adipocyte maturation Agpat2 4 CD45−Ter119−CD31−Sca1−
Fabp4
NES= 1.65
VCAM-1+PDGFRβ+ cells
Stat5b
Nominal p-value= 0.011 Isotype
Cd3e Fabp4
-log10(P value)
Cebpa FDR q-value= 0.011 2

Srebf1 Control
Lpl Cxcl12
Klf5
1 Adipoq MKO
Count
Foxo1
Pparg
Plin1 0.5
Medag
Control MKO
0.25
-4 -2 0 2 4
CXCL12
Fold changes (log2)
sFig. 6
a b
Mouse gene atlas IL12 pathway Biocarta pathways

T cytotoxic
pathway IL17 pathway
T-cells_CD8+
T-cells_CD4+
Bone marrow
Bone
TCR pathway
Osteoblast
c d
Cxcl12 Manhattan plot for modules associated with gene CXCL12
2.5
CXCR chemokine receptor binding Regulation of cellular extravasation
**
0.6 T cell proliferation
2.0
0.4
1.5
GMAS
0.2
1.0 0
-0.2
0.5
-0.4
0.0
Control MKO
Selected module Not selected modules
a b c Fig. 6
Manhattan plot for modules associated with gene CXCR4 CD4+ T cells
Lymphocyte activation T cell migration Veh AMD3100 Veh AMD3100

Chemokine binding
T cell proliferation
6.51% 6.02%
0.5
WT WT
GMAS
7.31% 5.32%
0 7.92% 3.84%
-0.2
MKO MKO
NK1.1
CD44
16.0% 12.0%
Selected module Not selected modules CD3 IFN-γ
d e f g
CD4+ T cells CD4+ T cells CD8+ T cells CD8+ T cells
Veh AMD3100 Veh AMD3100 Veh AMD3100 Veh AMD3100

8.93% 5.48% 6.16% 4.29%
6.76% 4.08%
WT WT WT WT
1.35% 0.84%
12.7% 6.77% 5.95% 2.32%

9.02% 3.47%
MKO MKO MKO MKO

CD44
CD44
CD44
FSC
2.00% 1.21%
TNF-α IL-17A IFN-γ TNF-α
h
CD4+CD44+ T cells CD4+CD44+ T cells CD8+CD44+ T cells CD8+CD44+ T cells
Control with vehicle
Control with AMD3100
MKO with vehicle

Count
Count
Count
Count
MKO with AMD3100
TNF-α IFN-γ TNF-α IFN-γ

a b
CD4+ T cells
c
CD4+ T cells
sFig. 7
Veh AMD3100 Veh AMD3100 Veh AMD3100
48.3% 47.6%
WT WT WT
35.0% 34.4% 5.95% 4.93% 20.4% 8.08%

54.4% 50.1%
MKO MKO
MKO
FSC
FSC
CD8
36.0% 36.8% 7.51% 4.74% 40.0% 26.8%

CD4 IFN-γ TNF-α
d e f Vehicle AMD3100
CD8+ T cells CD8+ T cells
(%) (%) (%)
20 10 15 **
Veh AMD3100 Veh AMD3100 *
**
CD4+CD44+TNF-α+
CD4+CD44+IFN-γ+
8
15
7.45% 4.51% 22.2% 9.98%
CD45+CD3+
WT WT 10
6
10
4
5
5
7.07% 3.25% 35.2% 22.6% 2
MKO MKO
FSC
FSC
0 0 0
IFN-γ TNF-α
(%) (%) (%)
2.5 * 8 15
**
CD4+CD44+IL-17A+
CD4+CD44+TNF-α+
CD4+CD44+IFN-γ+
2.0
6 **
10
1.5
4
1.0
5
2
0.5
0.0 0 0
Fig. 7
a c
Veh AMD3100
(mm3) Cortex_volume (mm) TBV (1/mm) Tb.N (mm) Tb.Th

1.4 0.4 2.5 0.15
WT
1.3 2.0
0.3
* 0.10
1.2 1.5
* 0.2
1.1 * 1.0
0.05
MKO 0.1
1.0 0.5
0.9 0.0 0.0 0.00
Tibia
b (mm) Tb.Sp (%) BV/TV (%) BS/BV (%) BS/TV
Veh 0.30 20 90 8
AMD3100
**
15 80 6
0.25
WT 10 * 70 4
0.20
5 60 2
0.15 0 50 0
MKO
WT+DW WT+AMD3100 MKO+DW MKO+AMD3100
Femur
sFig. 8
a b c d
AST ALT CHOL TG
(IU/L) (IU/L) (mg/dL) (mg/dL)
* *
200 80 140 160
*
150 60 120 140
100 40 100 120
50 20 80 100
0 0 60 80
WT+DW WT+AMD3100 MKO+DW MKO+ADM3100

a b c
Fig. 8
22-25 <18 22-25 <18 22-25 <18
31.6% 52.0% 38.0% 53.5% 42.1% 67.2%
CD45RO
CD45RO
CD8
60.2% 35.8% 45.7% 24.4% 39.1% 15.1%

CD4 CD45RA CD45RA
d e f
CD4+ T cells CD8+ T cells CD4+ T cells
22-25 <18 22-25 <18 22-25 <18

22.8% 28.2%
5.58% 7.52%
39.3% 47.0%
CD57
CD57
FSC
FSC FSC TNF
g h i
22-25 <18 22-25 <18 (%) (%) (%)

60 * 60 10
*
8
CD4+IL-17A+
CD4+TNF+
CD8+TNF+
40 40
CD4+TNF-α+
**
6
32.3% 41.7% 1.55% 5.20%
4
20 20
FSC
FSC
0 0 0
TNF IL-17A 22-25 <18 22-25 <18 22-25 <18
sFig. 9
a b c d
(%) 22-25 <18 (%) 22-25 <18 (%) 22-25 <18

40 80 80 80
* *
*
Grip strength (kg)
30 60 * 60 60
*
*
20 40 40 40
10 20 20 20
0 0 0 0
BMI (kg/m2) 22-25 <18 CD45RA-CD45RO+ CD45RA+CD45RO- CD45RA-CD45RO+
CD4+ T cells CD8+ T cells CD45RA+CD45RO-
e f g h i
22-25 <18 CXCL12 CD44 TNF IL17A

(%)
P = 0.056
30 3 2.5 2.5 ** 2.5
**

** **
2.0 2.0 2.0
20 2
1.5 1.5 1.5
P = 0.056 1.0 1.0 1.0

10 1
0.5 0.5 0.5
0 0 0.0 0.0 0.0

CD4+CD57+ T cells CD8+CD57+ T cells BMI (kg/m2) 22-25 <18 22-25 <18 22-25 <18 22-25 <18
sFig. 10
a b
500 800
*
P = 0.089
400
Serum GDF15 (pg/mL)

Serum FGF21 (pg/mL) 600
300
400
200
200
100
0 0
BMI (kg/m2) 22-25 <18 BMI (kg/m2) 22-25 <18

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bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.417147; this version posted December 9, 2020.

The copyright holder for this

Daejeon 35015, Republic of Korea.

Medicine, 282 Munhwaro, Daejeon 35015, Republic of Korea.

Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu,

of Tokyo, 7-3-1, Hongo, Bunkyo-ku, 113-0033 Tokyo, Japan

Tokyo, Tokyo, 113-8655, Japan

Cheongju, Chungbuk, 28199, Republic of Korea.

Key words: mitochondria, inflammation, bone marrow, bone loss

Word count: 6,091 (Introduction, Results, Discussion, and Methods)

Number of figures and tables: 8

Fax: +82-42-280-6990; E-mail: [email protected]

homing to the bone marrow, which is an immunological mediator of muscle-bone communication.

Mitochondria quality control pathways regulating mitochondrial morphology/function have been

from an inflammatory state brought about by cytokines, apoptosis, alteration in mitochondrial

mediated loss of muscle mass and strength, and bone loss.

However, although previous investigations revealed several factors promoting BM inflammatory-

microenvironment and bone mass remains to be elucidated.

CR6-interacting factor 1 (CRIF1), as a critical mitoribosomal protein, is essential for the

specific Crif1 knockout mice show mitochondrial OxPhos dysfunction-associated metabolic

mechanisms underlying muscular mitochondrial dysfunction-induced bone loss.

Herein, we investigated whether skeletal muscle dysfunction caused by muscle-specific OxPhos

loss of muscular mitochondrial function and, consequently, a potential therapeutic target.

Lonp1, Clpp, Hspd1, and Atf4 (Fig 1f).

loss via activation of osteoclasts in mice.

the MKO mice.

of FGF21 production caused by muscular mitochondrial OxPhos dysfunction.

whether mitochondrial OxPhos dysfunction in muscle affects BM inflammation, which is an important

muscular mitochondrial dysfunction-mediated bone loss is associated with BM inflammation rather

than a systemic inflammatory response.

Furthermore, we searched genes potentially associated with Cxcl12 by applying Gene-Module

mitochondrial dysfunction on BM inflammation and bone loss in mice.

CXCR4 antagonism attenuates BM inflammation in MKO mice. CXCL12 is also strongly

CXCR4 receptor antagonist AMD3100 (5 mg/kg/day) intraperitoneally to 10-week-old control and

inflammatory T-cells in the MKO mouse model of muscular mitochondrial dysfunction.

dysfunction caused by reduced mitochondrial OxPhos.

Muscle mitochondrial impairment is an important feature of pre-frailty development in humans 21. It is

mitochondrial dysfunction through attenuation of BM inflammation.

mass and quality.

higher inflammatory response, as measured by proinflammatory cytokine production, in the BM.

and function-mediated bone loss and fragility.

shown to improve ovariectomy-induced bone loss by facilitating mobilization of hematopoietic

collagen-induced osteoarthritis by attracting inflammatory cells to joints and by activating osteoclasts

in the BM microenvironment rather than the change in mitokine secretion.

regulation of BM inflammation and bone loss in mice. Inhibition of BM inflammation by a CXCR4

human relevance of muscular mitochondrial OxPhos dysfunction and BM inflammation in the

regulation of skeletal homeostasis needs to be clarified.

Daejeon, Republic of Korea).

Cy7, anti-TNF-α-APC, or anti-IL-17A-APC. Multicolor flow cytometry was performed using a BD

10% glycerol, 10 mM Tris-HCl (pH 6.8), 10 mM dithiothreitol, and 1 mM phenylmethylsulfonyl

detected by immunoblotting with antibodies listed in Supplemental Table 2. A horseradish peroxidase-

(R&D Systems, Minneapolis, MN, USA).

Immunodetection Kit (Invitrogen).

using VectaMount (Vector Labs).

histomorphometry nomenclature follows the guidelines of the ASBMR 50.

hanging time was used in the analyses.

clustering and phenotype ontology using Network2Canvas (http://maayanlab.net/N2C/). Phenotype