Mitochondrial Complex I Activity Suppresses

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Mitochondrial Complex I Activity Suppresses
Cell Metabolism
Article
Mitochondrial Complex I Activity Suppresses
Inflammation and Enhances Bone Resorption
by Shifting Macrophage-Osteoclast Polarization
Zixue Jin,1 Wei Wei,1 Marie Yang,1 Yang Du,1 and Yihong Wan1,*
1Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.cmet.2014.07.011
SUMMARY
Mitochondrial complex I (CI) deficiency is associated with multiple neurological and metabolic disorders. However, its effect on innate immunity and
bone remodeling is unclear. Using deletion of the
essential CI subunit Ndufs4 as a model for mitochondrial dysfunction, we report that mitochondria
suppress macrophage activation and inflammation while promoting osteoclast differentiation and
bone resorption via both cell-autonomous and systemic regulation. Global Ndufs4 deletion causes
systemic inflammation and osteopetrosis. Hematopoietic Ndufs4 deletion causes an intrinsic lineage
shift from osteoclast to macrophage. Liver Ndufs4
deletion causes a metabolic shift from fatty acid
oxidation to glycolysis, accumulating fatty acids
and lactate (FA/LAC) in the circulation. FA/LAC
further activates Ndufs4 / macrophages via reactive oxygen species induction and diminishes
osteoclast lineage commitment in Ndufs4 / progenitors; both inflammation and osteopetrosis in
Ndufs4 / mice are attenuated by TLR4/2 deletion.
Together, these findings reveal mitochondrial CI as
a critical rheostat of innate immunity and skeletal
homeostasis.
INTRODUCTION
The nuclear-encoded protein NADH: ubiquinone oxidoreductase iron-sulfur protein 4 (Ndufs4) is critical for mitochondrial
complex I (CI) assembly (Karamanlidis et al., 2013; Kruse et al.,
2008) and identified as a hotspot for mutations (Tucker et al.,
2011). CI is the largest complex in mitochondrial respiratory
chain. Its dysfunction is frequently linked with neurological diseases such as Alzheimer, Parkinson, and Leigh syndrome
(Coskun et al., 2012), as well as metabolic defects such as
impairment of oxidative phosphorylation (OXPHOS; Kirby et al.,
1999). CI is also the site where the respiratory chain generates
reactive oxygen species (ROS; Kushnareva et al., 2002). Physiological changes in ROS production are associated with processes such as autophagy, differentiation, metabolic adaption
and immune cell activation (Sena and Chandel, 2012). However,
ROS deregulation is associated with many malfunctions (James
et al., 2012).
Macrophages are essential components of the innate immunity and play a key role in inflammation. Emerging evidence
reveal a tightly controlled crosstalk between metabolism and
inflammation (Tschopp, 2011). Toll-like receptors (TLRs) not
only recognize lipopolysaccharide (LPS) in bacterial cell wall
but also sense nutritional signals such as fatty acids (Baker
et al., 2011; Hotamisligil and Erbay, 2008; Kawai and Akira,
2007). Inflammation is also increasingly recognized as a key factor in the development of metabolic diseases including obesity,
insulin resistance, type 2 diabetes, and atherosclerosis (Rocha
and Libby, 2009; Schenk et al., 2008). Furthermore, our recent
studies reveal that there is also a delicate balance of metabolism
and inflammation during lactation: maternal genetic defects
such as peroxisome proliferator-activated receptor-g (PPARg)
or very low density lipoprotein receptor deletion, as well as
maternal dietary defects such as high-fat-diet can cause metabolic defects during lactation that lead to the production of ‘‘toxic
milk,’’ which triggers a systemic inflammation in wild-type (WT)
nursing neonates that is manifested as a transient alopecia (Du
et al., 2012a, 2012b; Wan et al., 2007b). We hypothesize that genetic programs also exist in the offspring that allow the suckling
neonates to cope with the lipid- and energy-rich milk produced
by normal lactation and to prevent inflammation. Using alopecia
as readout for neonatal inflammation, here we have identified the
mitochondrial Ndufs4 as part of a critical anti-inflammatory genetic program in the offspring.
Osteoclasts are also derived from the monocyte-macrophage
lineage. Upon binding of RANKL (receptor activator of nuclear
factor kappa-B ligand) to its receptor RANK, macrophage precursors undergo differentiation into mature osteoclasts that are
multinucleated, specialized, bone-resorbing cells. This process
can be enhanced by other signaling pathways and small molecules such as the nuclear receptor PPARg and its agonist rosiglitazone, a widely used diabetes drug (Lazarenko et al., 2007;
Sottile et al., 2004; Wan, 2010; Wan et al., 2007a; Zinman et al.,
2010). Osteoclasts are essential for physiological bone remodeling, deficiency of which can cause osteopetrosis. However,
excessive osteoclasts can cause osteoporosis (Zaidi, 2007). Nuclear receptor estrogen-related receptor-a and the transcription
coactivator PGC1b are critical for osteoclast function, implicating
that osteoclastogenesis demands high energy and intact mitochondria (Brown and Breton, 1996; Ishii et al., 2009; Wan,
2010; Wei et al., 2010). Here we have identified Ndufs4 as a key
mitochondrial component required for osteoclastogenesis.
Cell Metabolism 20, 483–498, September 2, 2014 ª2014 Elsevier Inc. 483
Cell Metabolism
Ndufs4 Regulation of Macrophages and Osteoclasts
WT
A
Ndufs4-/-
B
Ndufs4 in Pups
% Hair Loss
n=
KO
100
7
Het
4
27
WT
0
14
KO (WT foster mom)
100
6
* Pups were from Het x Het mating.
P22
CD11b
C
F4/80
Gr-1
C D1 1c
WT
Ndufs4-/-
Spleen
***
60
*
40
**
IL-6
IL-12p40
0
IL-12P70
20
IFN-g
*
I
****
5
***
0.15
0.025
WT
Ndufs4-/-
10
MMP-9
COX-2
Pro-Inflammatory Mf
15
CD11b+ Ly6Ch i%
Ly6C
3.01
1.30
*
*
2
0.020
0.10
0.015
0.05
0.010
0.005
0.01
****
0.000
0
BM
WT
Ndufs4-/-
*
**
Relative mRNA
10.3
4.78
Median Fluorescence
4
Ndufs4-/-
H
Bone
Marrow
6
0
MCP-3
MCP-1
MMP9
COX-2
IL-18
IL-12p40
IL-12p35
*
*
*
8
IL-1b
****
*
**
IL-12p35
**
80 Serum
F
Liver
10
TNFa
mRNA Fold (KO/WT)
***
WT
G
12
E
WT
Ndufs4-/-
****
TNFa
200 Skin
180
160
140
120
100
12
10
****
8
6
4
2
0
IL-1b
mRNA Fold (KO/WT)
D
Spleen
BM
Sp
10
BM-Mf
***
0.00
BM
SP
****
6
4
2
0
0
BM-Mf
WT
Ndufs4-/-
***
**
**
COX-2
20
****
8
iNOS
**
40
L
MMP9
WT
Ndufs4-/-
IL-12p35
CD11b
CD11b+ Ly6Ch i%
43.6
Ly6C
19.5
BM-Mf
Pro-Inflammatory Mf
60
Ndufs4-/-
IL-1b
K
WT
mRNA Fold (KO/WT)
CD11b
J
(legend on next page)
484 Cell Metabolism 20, 483–498, September 2, 2014 ª2014 Elsevier Inc.
Cell Metabolism
Ndufs4 Regulation of Macrophages and Osteoclasts
To enhance our understanding of the complex disorders associated with mitochondrial deficiency, it is pivotal to identify new
mechanisms for how mitochondria modulate cellular and physiological processes. In light of the critical roles of macrophage
and osteoclast in many aspects of physiology and disease, it is
also important to delineate new mechanisms underlying the
control of their lineage specification, differentiation, and function. Using Ndufs4 deletion as a model for mitochondrial CI
dysfunction, here we show that normal mitochondrial function
suppresses macrophage activation and inflammation while
promoting osteoclast differentiation and bone resorption via
dual mechanisms of both cell-autonomous and systemic
regulation.
RESULTS
Global Ndufs4 Deletion Causes Systemic Inflammation
Manifested as Alopecia
Global Ndufs4 loss in the Ndufs4 / mice results in mitochondrial CI deficiency and encephalomyopathy around postnatal
day 30 (P30), leading to lethality at 7 weeks of age (Kruse
et al., 2008). Interestingly, the Ndufs4 / pups also exhibited a
transient alopecia phenotype (Kruse et al., 2008). The first pelage
appeared normal until P16 when precocious hair loss
occurred; the hair loss peaked P20–P24 (Figure 1A), and then
recovered after weaning (P35) when a new coat of hair grew
back. To determine whether the alopecia was dependent on
offspring or maternal genotype, we performed the following experiments. First, when Ndufs4+/ females were bred with
Ndufs4+/ males, 100% of the Ndufs4 / pups and 4% of
Ndufs4+/ pups developed hair loss, whereas all the WT control
pups were normal (Figure 1B). Second, hair loss could not be
rescued when Ndufs4 / pups were fostered by WT lactating
dams (Figure 1B). These results indicate that the alopecia was
caused by the defects in the Ndufs4 / pups rather than in the
Ndufs4+/ dams.
To examine whether the alopecia is associated with excessive
recruitment of myeloid immune cells, we collected skin from
Ndufs4 / and WT littermate controls at P22 and performed
immunofluorescence staining with antibodies for myeloid cell
markers. The skin from Ndufs4 / pups displayed an infiltration
of CD11b+, Gr-1+, and F4/80+ cells, which are largely inflammatory monocytes and macrophages (Figure 1C). In contrast, there
was no overt accumulation of CD11c+ dendritic cells (Figure 1C).
Consistent with these observations, the mRNA levels for a set of
inflammatory markers were elevated in the skin of Ndufs4 /
pups (Figure 1D). These results indicate that the alopecia was
likely caused by an inflammatory response during suckling.
We next investigated whether the inflammation was systemic
or skin-specific. First, expression of inflammatory markers was
also elevated in the liver of Ndufs4 / pups (Figure 1E). Second,
serum levels of inflammatory cytokines including interleukin-6,
interferon gamma, and interleukin-12p70 were higher in
Ndufs4 / pups (Figure 1F). Third, the percentage of CD11b+
Ly6Chi proinflammatory monocytes/macrophages was increased in the bone marrow and spleen of Ndufs4 / pups (Figures 1G and 1H). Fourth, expression of inflammatory markers
was also elevated in primary bone marrow cells and splenocytes
of Ndufs4 / pups (Figure 1I). These results indicate that the
Ndufs4 / pups suffered from systemic inflammation.
To examine if Ndufs4 deletion in the myeloid progenitors confers an intrinsic activation of macrophages, we compared bone
marrow-derived macrophages (BM-Mfs) from Ndufs4 / mice
or WT controls that were differentiated with a defined concentration of cytokine. Both the percentage of CD11b+Ly6Chi macrophages and the expression of inflammatory genes were higher
in the Ndufs4 / cultures (Figures 1J–1L). These results indicate
that the systemic inflammation in Ndufs4 / pups is likely attributed to, at least in part, cell-intrinsic defects.
Ndufs4 / mice exhibited unaltered B cells and T cells, as well
as Th17 and Treg subpopulations (Figures S1A–S1D available
online). In addition to the increased proinflammatory M1 markers
in the GMCSF-differentiated Ndufs4 / macrophages and pup
skin (Figures 1D and 1L), there were also decreased anti-inflammatory M2 markers in the MCSF-differentiated Ndufs4 / macrophages and pup skin (Figure S1E). Serum corticosterone levels
were normal (Figure S1F). Mitochondrial CI activity was reduced
by >99% in Ndufs4 / macrophages (Figure S1G). Furthermore,
treatment with a CI inhibitor rotenone also elevated the expression of inflammatory genes in WT macrophages (Figure S1J).
These results further indicate that CI deficiency promotes inflammatory activation of macrophages.
Global Ndufs4 Deletion Decreases Bone Resorption and
Increases Bone Mass
In light of the potential roles of mitochondria in osteoclast differentiation and bone remodeling, we investigated the skeletal
phenotype in Ndufs4 / mice. Microcomputed tomography
(mCT) analysis of the proximal tibiae from P22 pups revealed
that Ndufs4 / mice exhibited a high-bone-mass phenotype
(Figures 2A–2G). Histomorphometry showed that osteoclast
numbers and surface were decreased, whereas osteoblast
numbers and surface were unaltered (Figures 2H and 2I). Consistently, serum bone resorption marker CTX-1 (C-terminal telopeptides of type I collagen) was 48% lower (Figure 2J), whereas
serum bone formation marker N-terminal propeptide of type I
Figure 1. Global Ndufs4 Deletion Causes Systemic Inflammation Manifested as Alopecia
(A and B) Ndufs4 / pups exhibit alopecia. (A) A representative image on postnatal day 22 (P22). (B) Alopecia could not be rescued by WT foster dams.
(C) Skin of Ndufs4 / pups were infiltrated with leukocytes such as macrophages (detected by anti-CD11b, anti-F4/80, and anti-Gr-1) but not dendritic cells
(detected by anti-CD11c) on P22. Scale bars represent 25 mm.
(D and E) Expression of inflammatory genes in the skin (D) and liver (E) on P22 (n = 6).
(F) Cytokine levels in the serum on P22 (n = 6).
(G and H) Percentage of CD11b+Ly6Chi proinflammatory macrophages in bone marrow (BM) and spleen on P22. (G) FACS 2D dot plots. (H) Quantification (n = 5).
(I) Expression of inflammatory genes in the primary bone marrow cells or splenocytes on P22 (n = 5).
(J and K) Percentage of CD11b+Ly6Chi proinflammatory macrophages in BM-Mf cultures on day 6. (J) FACS 2D dot plots. (K) Quantification (n = 5).
(L) Expression of inflammatory genes in BM-Mf cultures on day 6 (n = 5). Error bars represent SD.
Cell Metabolism 20, 483–498, September 2, 2014 ª2014 Elsevier Inc. 485
Cell Metabolism
Ndufs4 Regulation of Macrophages and Osteoclasts
B
Ndufs4-/-
D
0.20
***
***
8
Tb.N (1/mm)
BV/TV
Trabecular
0.15
0.10
0.05
6
4
2
WT
E
35
1000
500
20
15
10
Ndufs4-/-
WT
G
0.3
Tb.Sp (mm)
BS (mm2)
*
25
*
1500
0
WT
Ndufs4-/-
30
Proximal Tibia
2000
0
0.00
C
F
10
Conn. D. (1/mm3)
WT
0.2
**
0.1
Ndufs4-/-
0.4
Cortical BV/TV
A
***
0.3
0.2
0.1
5
5
0
WT Ndufs4-/-
WT Ndufs4-/-
WT
n.s.
6
4
2
Ndufs4-/-
1 ± 0.15
M
0.5
P
Relative mRNA
1.5
0.4
0.3
***
0.2
O
***
0.3
0.2
0.1
20
****
15
10
0.0
5
0
WT Ndufs4-/-
WT Ndufs4-/-
CAR2
0.12
0.03
****
****
Veh
RANKL
RANKL+Rosi
0.03
1.0
0.08
0.02
0.02
0.5
****
****
+++
WT
Ndufs4-/-
0.01
0.01
Ndufs4-/-
ATP6V0d2
**
+
++
0.5
0.02
Ndufs4-/-
WT
**
++
+
Ndufs4-/-
Veh
RANKL
RANKL+Rosi
+++
++
0.002
*
0.000
0.0
WT
0.003
0.004
0.002
++
0.00
**
***
0.006
1.0
+++
Ndufs4-/ACO2
0.004
0.008
1.5
***
+++
WT
Ndufs4-/-
0.010
***
0.06
++
0.00
WT
ATP5b
2.0
****
****
+++
0.00
WT
PGC1b
0.10
0.04
0.04
+++
++
0.00
0.08
****
++
+++
Relative mRNA
WT Ndufs4-/-
CALCR
****
0.0
Q
50
0
0.4
WT Ndufs4-/-
0.04
100
WT Ndufs4-/-
N
Resorptive
Activity
CTSK
****
n.s.
0
0.0
0.13 ± 0.06****
TRAP
**
50
WT Ndufs4-/-
0.1
Oc Size:
100
0
WT Ndufs4-/-
Calcium (uM)
L
10
8
Ndufs4-/-
150
AnnexinV 7-ADD (%)
0
n.s.
K
150
-
5
J
10
WT
Ndufs4-/-
+
0
*
WT
Serum CTX-1 (ng/ml)
5
10
15
Ob.N/B.Ar (%)
***
10
15
Ndufs4-/-
BrdU Incorporation (OD450)
I
20
Oc.N/B.Ar (%)
Oc.S/B.S (%)
15
Ob.S/B.S (%)
H
0.0
0.0
WT
Serum P1NP (ng/ml)
0
0.001
0.000
WT
Ndufs4-/-
WT
Ndufs4-/-
(legend on next page)
486 Cell Metabolism 20, 483–498, September 2, 2014 ª2014 Elsevier Inc.
Cell Metabolism
Ndufs4 Regulation of Macrophages and Osteoclasts
procollagen (P1NP) was normal (Figure 2K). These data indicate
that the high-bone-mass in Ndufs4 / mice was mainly caused
by a compromised osteoclast function.
Ex vivo bone marrow osteoclast differentiation assay using
defined cytokine concentration showed that RANKL-mediated
and rosiglitazone-stimulated osteoclastogenesis was severely
impaired by Ndufs4 deletion. Ndufs4 / cultures exhibited
decreased number and size of multinucleated TRAP+ (tartrateresistant acid phosphatase+) mature osteoclasts (Figure 2L),
lower bone resorptive activity (Figure 2M), elevated precursor
proliferation (Figure 2N), and increased apoptosis (Figure 2O).
Moreover, induction of osteoclast differentiation markers, such
as TRAP, CTSK, CALCR, and CAR2, were diminished (Figure 2P);
and induction of osteoclast function genes that promote mitochondria biogenesis and oxidative phosphorylation, such as
PGC1b, ATP5b, ATP6V0d2, and ACO2, were severely blunted
(Figure 2Q). CI activity was reduced by >99% in Ndufs4 / osteoclasts (Figure S1G). Osteoclast and osteoblast coculture
showed that the osteoclastogenic defects were observed only
when knockout (KO) osteoclast progenitors were cocultured
with WT osteoblasts, but not when WT osteoclast progenitors
were cocultured with KO osteoblasts (Figures S2A and S2B).
Moreover, osteoblast differentiation from Ndufs4 / bone
marrow mesenchymal stem cells was normal (Figure S2C).
These results indicate that CI deficiency causes an intrinsic
defect in osteoclastogenesis, leading to a decreased bone
resorption and an increased bone mass.
Hematopoietic Ndufs4 Deletion Enhances Macrophage
Activation and Inflammation
To further investigate whether CI plays a cell-autonomous role in
macrophage activation and osteoclastogenesis, we generated
hematopoietic Ndufs4 conditional KO mice using Ndufs4flox/flox
mice and Tie2-Cre mice. Tie2-Cre deletes flox’d alleles in all hematopoietic lineages but not in mesenchymal lineages; thus in
macrophages and osteoclasts but not in osteoblasts (Wan
et al., 2007a, 2007b; Wei et al., 2010). Tie2-Ndufs4 KO mice appeared healthy and survived to adulthood. Interestingly, Tie2Ndufs4 KO pups did not exhibit alopecia (Figure 3A). Similar to
Ndufs4 / macrophages, Tie2-Ndufs4 KO macrophages also
showed a >99% reduction in CI activity (Figure S1H), leading
to a higher expression of inflammatory markers (Figure 3B).
These results indicate that CI indeed plays a cell-autonomous
role in macrophages to suppress inflammation.
In contrast to the Ndufs4 / global KO mice, the percentage
of CD11b+Ly6Chi proinflammatory macrophages in the bone
marrow and spleen of Tie2-Ndufs4 KO pups were unaltered (Fig-
ures 3C and 3D). This observation and the absence of the alopecia phenotype in Tie2-Ndufs4 KO mice indicate that other tissues
outside of the hematopoietic lineages may also contribute to the
systemic inflammation caused by global CI deficiency.
Ndufs4 Deletion Shifts Metabolism from Fatty Acid
Oxidation to Glycolysis
Mitochondrial CI plays a central role in energy metabolism by
promoting fatty acid b-oxidation and TCA (tricarboxylic acid)
cycle. Because milk provides the suckling neonates rich nutrients such as fat and sugar, we hypothesize that Ndufs4 /
pups also exhibit whole body metabolic defects. We found that
serum levels of triglyceride and nonesterified fatty acid (NEFA)
were both 50% higher in Ndufs4 / pups at P22 (Figures 3E
and 3F); at the same time, serum lactate was also 88% higher
(Figure 3G). BM-Mfs from Ndufs4 / mice also produced more
lactate into the culture supernatant, leading to a more acidic
environment (Figure 3H). These results (Figures 3E–3H) indicate
a metabolic shift in Ndufs4 / pups from fatty acid oxidation to
glycolysis, leading to decreased fatty acid catabolism and
increased triglyceride accumulation, and at the same time,
a compensatory elevated glucose catabolism, and lactate
accumulation.
Interestingly, serum levels of triglycerides and lactate were not
significantly altered in Tie2-Ndufs4 KO pups on P22 (Figure 3I),
suggesting that tissues other than the hematopoietic population
were largely responsible for the global metabolic changes. Liver
is a major metabolic tissue in suckling neonates, along with muscle and fat. We found that indeed expression of the proglycolysis
genes Hif1a and Pfkfb2 were upregulated in the liver of
Ndufs4 / pups on P22 (Figure 3J). Thus, we generated liverspecific Ndufs4 KO using Albumin-Cre mice. CI activity in the
liver was reduced by 84% in Alb-Ndufs4 KO compared to controls (Figure S1I). Serum triglycerides and lactate were 39%
and 30% higher, respectively, in Alb-Ndufs4 KO pups than controls on P22 (Figure 3K). Although the metabolic defects in AlbNdufs4 KO pups were less severe than in Ndufs4 / pups, these
results indicate that the liver is the major tissue that mediates CI
regulation of neonatal metabolism; nonetheless, additional tissues such as heart, skeletal muscle, and fat may also contribute
to the global phenotype.
Fatty Acids and Lactate Potentiate the Activation of
Ndufs4–/– Macrophage
We next asked whether the excessive fatty acids as the result of
decreased b-oxidation could potentiate the proinflammatory
phenotype of Ndufs4 / macrophages. BM-Mfs from Ndufs4 /
Figure 2. Global Ndufs4 Deletion Decreases Bone Resorption and Increases Bone Mass
(A–G) Micro-CT analyses of tibiae on P22 (male, n = 8). (A) Representative images of the trabecular bone of the tibial metaphysis (top; scale bar represents 10 mm)
and the entire proximal tibia (bottom; scale bar represents 1 mm). (B–F) Quantification of trabecular bone volume and architecture. (B) BV/TV, bone volume/tissue
volume ratio. (C) BS, bone surface. (D) Tb.N, trabecular number. (E) Tb.Sp, trabecular separation. (F) Conn.D., connectivity density. (G) Cortical BV/TV.
(H and I) Bone histomorphometry (P22, male, n = 8). (H) Osteoclast number (Oc.N/B.Ar) and osteoclast surface (Oc.S/B.S; n = 8). (I) Osteoblast number
(Ob.N/B.Ar) and osteoblast surface (Ob.S/B.S; n = 8). B.Ar, bone area.
(J) Serum CTX-1 (P22, male, n = 8).
(K) Serum P1NP (P22, male, n = 8).
(L–Q) Bone marrow osteoclast differentiation assay. (L) Representative images of differentiation cultures on day 12. Mature osteoclasts were multinucleated
(>3 nuclei) TRAP+ (purple) cells. Scale bar represents 25 mm. (M) Osteoclast activity (n = 8). (N) Osteoclast precursor proliferation (n = 8). (O) Osteoclast apoptosis
(n = 8). (P and Q) Expression of osteoclast differentiation markers (P) and osteoclast function genes (Q; n = 4). (P and Q) * Compares each treatment with vehicle
(Veh) control, + compares Ndufs4 / with WT control in the same treatment group. Error bars represent SD.
Cell Metabolism 20, 483–498, September 2, 2014 ª2014 Elsevier Inc. 487
Cell Metabolism
Ndufs4 Regulation of Macrophages and Osteoclasts
B
Ctrl
Tie2-Ndufs4
5.09
BM
IL-1b
1.52
1.70
BM
0.4
WT
10
5
BM-Mf
WT
Ndufs4-/-
J
n.s.
100
50
15
n.s.
10
5
0
0
Ndufs4-/-
Hif1α
0.010
Relative mRNA in Liver
150
0.008
0.04
0.006
0.03
0.004
0.02
0.002
0.01
0.000
0.00
***
0.03
WT
Ndufs4-/-
50
0
+
PA
WT Ndufs4-/-
15
*
10
5
Ctrl Alb-Ndufs4
++
***
0.0006
0.0004
*
+
+
0.0002
PA
n.s.
0.0015
0.04
Veh
LA
P
IL-18
*
**
+
0.0000
Veh
iNOS
****
0
IL-12p40
***
LA
IL-6
0.0003
5
0.0008
0.0000
Veh
10
0
+
0.0002
***
*
LA
****
Ctrl Alb-Ndufs4
++
0.0004
++++
0.0005
COX-2
100
0.0008
0.0000
0.00
***
0.0006
0.0010
+
M
Spleen
15
***
***
0.0015
0.01
150
IL-12p35
0.0010
++++
++++
PA
WT Ndufs4-/-
IL-6
0.0020
Veh
**
WT Ndufs4-/-
0.0025
0.03
0.02
K
Pfkfb
0.05
**
Ctrl Tie2-Ndufs4
COX-2
++++
WT
****
Ndufs4-/-
Ndufs4-/-
****
0
WT
**
PA
LA
Pup Genotype
Ctrl
Alb-Ndufs4
Ctrl
Alb+Tie2
-Ndufs4
*
****
0.03
0.02
0.0002
0.0010
*
0.02
0.0001
0.0005
*
LAC
Veh
4
2
****
****
^^^^
++++
^^^^
++++
^^^^
++++
TNFa
MMP9
****
****
6
^^^^
++++
****
8
^^^^
++++
****
10
***
****
2
Veh
***
***
4
0.00
LAC
^^^^
++++
****
****
**
Fold mRNA
BM CD11b+ Ly6Ch i%
Veh
O
8
6
0.0000
LAC
LAC
^^^^
++++
****
0.0000
Veh
****
0.00
0.01
*
Ctrl
Alb-Ndufs4
Tie2-Ndufs4
Alb+Tie2-Ndufs4
***
***
**
****
0.01
****
****
Relative mRNA
H
15
0.0
Ndufs4-/-
****
****
Relative mRNA
****
0.8
Ctrl Tie2-Ndufs4
N
G
Supernatant Lactate (mmol/dL)
0
0.04
n.s.
2
Serum Lactate (mmol/L)
50
L
4
0
Serum Triglycerides (mg/dL)
100
Serum Lactate (mmol/L)
Serum Triglycerides (mg/dL)
I
1.2
Serum Lactate (mmol/L)
**
WT
CD11b+ Ly6Chi%
Ly6C
***
*
Spleen
****
2
Ctrl
Tie2-Ndufs4
CD11b
F
Serum NEFA (mEq/L)
Serum Triglycerides (mg/dL)
150
6
n.s.
P22
E
D
Pro-Inflammatory Mf
4
0
Tie2-Ndufs4
4.43
****
6
COX-2
Tie2-Ndufs4
mRNA Fold (KO/WT)
Ctrl
Ctrl
C
8 BM-Mf
TNFa
Pup Genotype
iNOS
A
0
0
Ctrl Alb-Ndufs4
COX-2
IL-6
IL-12p40
MCP-3
IL-1b
P23
(legend on next page)
488 Cell Metabolism 20, 483–498, September 2, 2014 ª2014 Elsevier Inc.
Cell Metabolism
Ndufs4 Regulation of Macrophages and Osteoclasts
mice or controls were treated for 15 hr with either palmitic acid
(PA), a representative saturated fatty acid that is abundant in
milk; or linoleic acid (LA), a representative unsaturated fatty
acid. Expression of proinflammatory genes was stimulated by
PA and to a lesser extent by LA in WT macrophages (Figure 3L).
This effect was more pronounced in Ndufs4 / macrophages,
which already exhibited higher basal expression than WT macrophages (Figure 3L). Similarly, lactate also increased the expression of proinflammatory genes in WT macrophages, and more
dramatically in Ndufs4 / macrophages (Figure 3M).
The percentage of CD11b+Ly6Chi proinflammatory macrophages in the bone marrow of Alb-Ndufs4 KO pups was higher
than controls (Figure 3N). Moreover, the expression of inflammatory genes in the spleen was increased in Alb-Ndufs4 KO pups
and Tie2-Ndufs4 KO pups, and further potentiated in Alb+Tie2Ndufs4 double KO (DKO) pups (Figure 3O). These results indicate that the combination of the intrinsic defects in Ndufs4 /
macrophages and the systemic metabolic defects of FA/LAC
accumulation in the circulation of the Ndufs4 / pups lead to
an exacerbated macrophage activation and systemic inflammation compared with WT macrophages in the normal environment.
Alb-Ndufs4 KO pups or Alb+Tie2-Ndufs4 DKO pups also did not
exhibit alopecia (Figure 3P), suggesting that Ndufs4 deletion in
other metabolically active tissues, such as heart, skeletal muscle, and fat, may be also required to replicate metabolic and inflammatory defects as severe as in the global Ndufs4 / pups.
ROS Production Is Elevated in Ndufs4–/– Macrophages
and Exacerbated by Fatty Acids
Mitochondria-derived ROS is critical for macrophage activation
(West et al., 2011). Thus, we next investigated if the exacerbated
inflammation by Ndufs4 deletion is contributed by excessive
ROS production in vivo and in vitro. Quantified by MitoSOX staining (Misawa et al., 2013; Zhou et al., 2011), ROS was found to be
more abundant in the skin, bone marrow, and spleen of
Ndufs4 / pups compared with WT controls (Figures 4A and
4B). The ROS level was much higher in Ndufs4 / BM-Mfs and
was further increased by PA treatment (Figure 4C). Mitochondrial
membrane potential was lower in Ndufs4 / BM-Mfs and further
decreased by PA (Figure 4D). In line with the increased ROS production and oxidative stress, the expression of the stress sensor
genes Gadd45a/b, as well as apoptosis, was also higher in
Ndufs4 / macrophages and further potentiated by PA (Figures
4E and 4F). Importantly, PA induction of ROS (Figure 4G) and
proinflammatory genes (Figure 4H) was attenuated by the mitochondrial ROS inhibitor 2R,4R-APDC (2R,4R-aminopyrrolidine
dicarboxylate). Mito-TEMPO, another mitochondria-targeted
antioxidant and a specific scavenger of mitochondrial ROS,
also attenuated PA induction of ROS (Figure 4G), inflammatory
genes (Figure 4I), and stress sensor genes (Figure 4J). These
findings indicate that increased mitochondrial ROS production
represents an important mechanism underlying the systemic
inflammation in Ndufs4 / suckling neonates.
Inflammation and Alopecia in Ndufs4–/– Pups Can Be
Rescued by TLR4/2 Deletion
Fatty acids and lactate induce inflammation and insulin resistance via TLR4/2 (Samuvel et al., 2009; Senn, 2006; Shi et al.,
2006). To further investigate the significance of the excessive
FA/LAC in the systemic inflammation in Ndufs4 / pups in vivo,
we examined whether the alopecia could be rescued by TLR4/2
deletion. Ndufs4/TLR2/TLR4 triple KO (TKO) mice were completely resistant to hair loss (Figure 5A). Consistent with this
observation, the excessive macrophage infiltration (Figure 5B)
and proinflammatory gene expression (Figure 5C) in the skin of
Ndufs4 / pups were ameliorated in Ndufs4/TLR2/TLR4 TKO
pups. Moreover, PA-stimulated inflammatory gene expression
(Figure 5D) and ROS production (Figures 5E and 5F) in Ndufs4 /
macrophages was also dampened by TLR2/4 deletion. Serum
NEFA, triglyceride, and lactate were unaffected by TLR2/4 deletion because they were elevated to a similar extent in both
Ndufs4-KO and Ndufs4/TLR2/4 TKO (Figure 5G), indicating
that the metabolic defects remained, but the response to this
metabolic shift was abolished by TLR2/4 deletion.
Further genetic dissection revealed that although TLR2/4
double deletion has the strongest rescue effects, TLR4 deletion alone, but not TLR2 deletion alone, also conferred significant attenuation of the alopecia, indicating that TLR4 is the
major mediator of the inflammation in the Ndufs4 / neonates
(Figure 5H).
This genetic evidence promoted us to investigate whether
pharmacological inhibition of TLR4 can ameliorate the inflammation in Ndufs4 / neonates, using alopecia as a convenient
readout. The results showed that Ndufs4 / pups treated with
Figure 3. Ndufs4 Deletion Shifts Metabolism to Potentiate Macrophage Activation
(A) Representative image showing that Tie2-Ndufs4 KO mice had no alopecia.
(B) Expression of inflammatory genes in Tie2-Ndufs4 KO BM-Mfs was increased on day 6 (n = 3).
(C and D) Percentage of CD11b+Ly6Chi proinflammatory macrophages in the bone marrow (BM) and spleen was normal in Tie2-Ndufs4 KO pups on P22.
(C) FACS 2D dot plots. (D) Quantification (n = 5).
(E–G) Serum triglycerides (E), nonesterified fatty acid (NEFA) (F), and lactate (G) were increased in Ndufs4 / pups (n = 8).
(H) Ndufs4 / macrophages secrets more lactic acid. Left, images showing the medium of Ndufs4 / BM-Mf cultures was more acidic (yellow). Right, quantification of lactate in culture medium (n = 6).
(I) Serum triglycerides (left) and lactate (right; n = 8).
(J) Expression of Hif1a and Pfkfb was higher in the liver of Ndufs4 / mice (n = 5).
(K) Serum triglycerides (left) and lactate (right) in Alb-Ndufs4 KO mice and controls (n = 8).
(L and M) Fatty acids (L) and lactate (M) potentiate the activation of inflammatory genes in Ndufs4 / macrophages (n = 5). BM-Mfs were treated with palmitic acid
(PA) or linoleic acid (LA; 400 mM) or lactate (15 mM) for 15 hr.
(N) Percentage of CD11b+Ly6Chi proinflammatory macrophages in the bone marrow (BM) was increased in Alb-Ndufs4 KO pups on P22 (n = 5).
(O) Expression of inflammatory genes in the spleen was increased in Tie2-Ndufs4 KO and Alb-Ndufs4 KO, and further potentiated in Alb+Tie2-Ndufs4 DKO mice
on P22 (n = 5).
(P) Alb-Ndufs4 and Alb+Tie2-Ndufs4 DKO mice had no alopecia.
Error bars represent SD.
Cell Metabolism 20, 483–498, September 2, 2014 ª2014 Elsevier Inc. 489
Cell Metabolism
Ndufs4 Regulation of Macrophages and Osteoclasts
A
DAPI
Mito-SOX
B
Merge
Ndufs4-/-
Relative Mito-SOX
WT
3
WT
Ndufs4-/-
***
2
****
1
0
Bone Marrow
PA
44.3
24.2
Ndufs4-/-
20.5
4
13.3
23.7
13.1
D
LA
Veh
PA
LA
Membrane Potential: Deep Red (%)
WT
Mito-SOX
Veh
Macrophage Mito-SOX
C
Spleen
++
**
3
++
n.s.
2
++
*
n.s.
1
0
WT
100
Veh
PA
LA
80
n.s.
+++
n.s.
+++
60
****
40
+++
20
****
0
Ndufs4-/-
WT
Ndufs4-/-
FL1
Relative mRNA
0.005
F
Gadd45b
++++
WT
Ndufs4-/-
0.15
80
**
****
0.004
0.10
0.003
++
0.002
0.05
++
*
*
0.001
0.000
0.00
Veh
Veh
Relative mRNA in Mf
****
40
***
20
+
5
4
***
**** ***
3
2
1
WT
PA
COX-2
IL-12p35
****
Veh
****
PA
**** ****
PA+APDC
PA+Mito-TEMPO
0
Veh
0.15
Veh
PA
PA+APDC
Ndufs4-/-
IL-12p40
**
0.002
0.002
**
****
0.10
*
*
0.04
+++
60
PA
IL-1b
0.06
WT
Ndufs4-/-
0
PA
H
0.08
G
++
Macrophage Mito-SOX
Gadd45a
AnnexinV+ 7AAD- (%)
E
0.001
0.001
*
****
0.05
0.02
0.00
0.00
WT
0.000
WT
Ndufs4-/-
Ndufs4-/-
0.000
WT
Ndufs4-/-
I
WT
Ndufs4-/-
J
IL-1b
Relative mRNA in Mf
0.06
0.04
Veh
PA
PA+Mito **
-TEMPO
COX-2
0.10
IL-12p35
***
0.04
0.02
Gadd45a
0.002
0.003
Gadd45b
0.25
0.002
*
***
**
0.08
0.002
0.06
**
IL-12p40
0.001
*
***
0.15
*
0.10
**
0.001
0.05
0.00
WT
Ndufs4-/-
*
0.001
0.02
0.00
**
0.20
0.000
WT
Ndufs4-/-
0.000
WT
Ndufs4-/-
0.000
WT
Ndufs4-/-
0.00
WT
Ndufs4-/-
WT
Ndufs4-/-
(legend on next page)
490 Cell Metabolism 20, 483–498, September 2, 2014 ª2014 Elsevier Inc.
Cell Metabolism
Ndufs4 Regulation of Macrophages and Osteoclasts
the TLR4 inhibitor TAK-242 were spared from hair loss (Figure 5I).
These findings uncover TLR4 as an essential mediator of the systemic inflammation in the Ndufs4 / pups, and highlight TLR4 inhibitors as an effective treatment of this mitochondrial disorder.
Hematopoietic Ndufs4 Deletion Impairs
Osteoclastogenesis and Bone Resorption
We next examined the hematopoietic-intrinsic regulation of
osteoclastogenesis by CI using the Tie2-Ndufs4 KO mice. Osteoclast differentiation and function from Tie2-Ndufs4 KO bone
marrow were severely impaired (Figures 6A–6D), causing a
41% lower serum CTX-1 (Figure 6E) and a higher bone mass
(Figures 6F and 6G). We have also generated myeloid-specific
Ndufs4 KO mice using the Lysozyme-Cre mice (Lyz-Ndufs4). A
time course analysis of osteoclast differentiation revealed that
Ndufs4 deletion was later and less complete in Lyz-Ndufs4
cultures than Tie2-Ndufs4 cultures, leading to a less severe osteoclast differentiation defect (Figures S5A–S5E). Nonetheless,
Lyz-Ndufs4 KO mice also showed a similar phenotype as Tie2Ndufs4 KO mice with lower bone resorption, higher bone
mass, and elevated inflammatory gene expression in the spleen
(Figures S5F–S5M). These results indicate that CI plays a cellintrinsic role in the osteoclast lineage to promote osteoclastogenesis and decrease bone mass.
Interestingly, Ndufs4 deletion in the liver also significantly
elevated the bone mass in the Alb-Ndufs4 KO mice (Figures 6F
and 6G). Moreover, Ndufs4 deletion in both osteoclast and liver
as in the Alb+Tie2-Ndufs4 DKO mice further increased bone
mass compare to individual single KO (Figures 6F and 6G).
Osteoclast surface (Figure 6H) and serum CTX-1 (Figure 6I)
were also lower in Alb-Ndufs4 KO mice and further decreased
in Alb+Tie2-Ndufs4 DKO mice; whereas serum P1NP was unaltered (Figure 6J). These results indicate that CI promotes osteoclastogenesis and bone resorption through both cell-intrinsic
and systemic metabolic mechanisms.
In light of the dual regulation of bone resorption and inflammation by CI, we next examined an LPS-induced inflammatory
arthritis model (Chang et al., 2008; Jimi et al., 2004; Takayanagi
et al., 2000; Zhao et al., 2009). LPS induction of bone resorption
and bone loss was abolished in Alb-Ndufs4 KO, Tie2-Ndufs4 KO,
and Alb+Tie2-Ndufs4 DKO mice (Figures S3A–S3C), despite the
exacerbated inflammation (Figure S3D). This indicates that
osteoclastogenic defects are dominant over inflammatory defects in the context of inflammatory arthritis, and mitochondrial
CI inhibition may confer resistance to inflammation-induced
bone loss.
Fatty Acids and Lactate Exacerbate the
Osteoclastogenic Defects in Ndufs4–/– Cells
Macrophage activation and osteoclast maturation represent two
divergent and competing differentiation outcomes for monocyte
precursors. The findings that the excessive FA/LAC in Ndufs4 /
mice potentiate macrophage activation, and liver Ndufs4 deletion also decrease bone resorption, prompted us to investigate
whether FA/LAC also suppress osteoclast differentiation and
lineage allocation. Osteoclast differentiation from WT bone
marrow was suppressed by fatty acids such as PA and LA (Figures 7A–7C); in contrast, osteoblast differentiation was unaffected (Figure S2D). Moreover, fatty acids exacerbated the
osteoclastogenic defects in Ndufs4 / cells, leading to a further
reduction in osteoclast differentiation (Figures 7A–7C). Lactate
had similar effects (not shown). RANK and FMS are two receptors that define osteoclast precursors and are functionally
required for osteoclastogenesis in response to RANKL and
MCSF. The percentage of FMS+RANK+ osteoclast precursors
was lower in the bone marrow of Ndufs4 / mice and Alb-Ndufs4
KO mice (Figures 7D and S6A). Similarly, the percentage of
FMS+RANK+ osteoclast precursors was also reduced in
Ndufs4 / osteoclast differentiation cultures (Figure 7E). The
osteoclastogenic defects in Ndufs4 / cultures could not be
rescued by higher RANKL concentrations (Figure S4A), further
supporting the severely compromised RANK level. Decreased
FMS level in Ndufs4 / cultures was only observed during osteoclast differentiation after RANKL treatment but not during proliferation (Figure S4B), in agreement of the specific impairment
of osteoclast differentiation but not precursor proliferation (Figures 2L–2N). Because the osteoclastogenic defects in Ndufs4 /
cultures were associated with reduced expression of not only
FMS and RANK, but also NFATc1 and c-fos, two key transcription factors for osteoclast differentiation, we examined the
importance of each factor in this regulation. The results show
that overexpression of FMS, RANK and NFATc1, but not c-fos,
was able to partially rescue the osteoclastogenic defects (Figures S4E–S4L).
Interestingly, the percentage of FMS+RANK+ osteoclast precursors in WT and Ndufs4 / cultures was further diminished
by PA, LA, or lactate (Figure 7E). The antiosteoclastogenic
effects of PA, LA, or lactate remained when the treatment was
restricted to only the first 3 days after RANKL treatment and
then removed (Figure S4C), further supporting that the regulation
resides mainly in the precursor stage. In line with this in vitro
finding, WT mice treated with PA and LA for 2 weeks displayed
a decreased bone resorption and an increased bone mass
Figure 4. ROS Level Is Elevated in Ndufs4–/– Macrophages and Exacerbated by Fatty Acids
(A) Representative images of Mito-SOX staining of skin on P22. Scale bars represent 25 mm.
(B) FACS quantification of Mito-SOX levels in bone marrow and spleen on P22 (n = 5).
(C) Mito-SOX levels in BM-Mfs treated with PA or LA (400 mM). Left: FACS 2D dot plots. Right: quantification (n = 5).
(D) Mitochondrial membrane potential was decreased in Ndufs4 / macrophages and further reduced by PA, but not LA (n = 5), quantified by percentage of
MitoTracker deep red via FACS.
(C and D) * and n.s. compare treatment with vehicle control in the same genotype; + compares Ndufs4 / with WT in the same treatment group.
(E and F) Expression of the stress sensor genes Gadd45a/b (E) and apoptosis (F) were higher in Ndufs4 / macrophages and further potentiated by PA
(n = 3). * compares Ndufs4 / with WT under the same treatment; + compares PA with vehicle in the same genotype.
(G) PA induction of mitochondrial ROS in macrophages, measured by Mito-SOX, was abolished by ROS inhibitors 2R,4R-APDC (50 mM) or Mito-TEMPO (50 mM).
(H and I) ROS inhibitors 2R,4R-APDC (H) or Mito-TEMPO (I) partially rescued the PA-activated inflammatory gene expression in WT and Ndufs4 / macrophages.
(J) Mito-TEMPO partially rescued the PA-activated stress sensor gene expression in WT and Ndufs4 / macrophages. BM-Mfs were treated with PA (400 mM),
with or without APDC or Mito-TEMPO (50 mM). Error bars represent SD.
Cell Metabolism 20, 483–498, September 2, 2014 ª2014 Elsevier Inc. 491
Cell Metabolism
Ndufs4 Regulation of Macrophages and Osteoclasts
****
0.0004
0.015
**
E
Mito-SOX
WT
Ndufs4-/24.8
R
2/
4/-
TL
N
du
du
f
N
**** ****
0.10
**** ****
**** ****
0.04
0.003
WT
Ndufs4-/Ndufs4-/- TLR2/4-/-
0.05
0.002
0.02
0.001
0.00
0.000
Ndufs4-/-TLR2/4-/39.3
0.06
0.00
F
2.0
Macrophage Mito-SOX
CD11b
0.004
COX-2
IL-6
IL-1b
0.005
n.s.
fs
4/-
s4
-/s4
-/-
Nd
uf
N
du
f
C
Relative mRNA in Skin
B
****
0.00
T
0.0000
0.01
n.s.
W
0.000
***
N
du
0.0001
fs
4/R2
/4
-/-
0.005
W
0.02
0.0002
n.s.
TL
0.010
****
0.03
0.0003
fs
4/-
0.020
COX-2
0.04
0.0005
s4
-/ -
KO
IL-6
Veh
PA **
W
T
KO
IL-1b
0.025
N
du
WT
D
KO
T
Ndufs4:
WT
R2
/4
-/-
WT
TL
TLR2/4:
Relative mRNA in Mf
A
11.6
****
****
WT
Ndufs4-/Ndufs4-/- TLR2/4-/-
1.5
1.0
0.5
0.0
Serum NEFA (mEq/L)
2.0
****
n.s.
1.5
1.0
0.5
0.0
H
200
100
KO
WT
KO
TLR4:
WT
KO
KO
KO
KO
n.s.
150
TLR2:
Ndufs4:
****
Serum Lactate (mmol/L)
G
Serum Triglycerides (mg/dL)
FL1
50
0
I
KO
15
****
n.s.
10
WT
Ndufs4-/Ndufs4-/- TLR2/4-/-
5
0
Rx:
Veh
TAK-242
Ndufs4:
KO
KO
Figure 5. Inflammation and Alopecia in Ndufs4–/– Pups Can Be Rescued by TLR2/4 Deletion
(A–F) Comparison of Ndufs4/TLR4/TLR2 triple KO, Ndufs4 / , and WT controls on P22 (n = 4).
(A) Representative images showing alopecia phenotype.
(B) CD11b immunostaining of skin. Scale bars represent 25 mm.
(C) Expression of inflammatory genes in the skin.
(D) Expression of inflammatory genes in BM-Mfs treated with PA or vehicle (Veh).
(legend continued on next page)
492 Cell Metabolism 20, 483–498, September 2, 2014 ª2014 Elsevier Inc.
Cell Metabolism
Ndufs4 Regulation of Macrophages and Osteoclasts
(Figure S4D). TLR2 and TLR4 expression in Ndufs4 / macrophage precursors was normal (Figure S6B). TLR2/4 deletion
partially rescued the bone resorption defects in Ndufs4 /
mice both in vivo (Figure 7F) and in vitro (Figure 7H), whereas
bone formation was unaffected (Figure 7G). Consequently, the
high bone mass in Ndufs4 / mice was attenuated in Ndufs4/
TLR2/4-TKO mice (Figure S6C). These results provide critical
mechanisms for the low bone resorption and high bone mass
in mitochondrial dysfunction: CI deficiency reduces bone
resorption by inhibiting both osteoclast differentiation and osteoclast lineage allocation via both cell-intrinsic defects and systematic metabolic defects.
DISCUSSION
Our study uncovers a critical yet previously unrecognized role of
mitochondrial CI in macrophage-osteoclast polarization to suppress systemic inflammation but enhance bone resorption. This
regulation involves mechanisms at both cell-intrinsic and systemic levels (Figure 7I). First, CI in the myeloid precursors
impedes inflammatory responses in macrophages but activates
osteoclast differentiation by functioning in a cell-autonomous
manner. Second, CI in metabolically active tissues such as liver
shifts metabolism from glycolysis to fatty acid oxidation, leading
to less accumulation of fatty acids and lactate in circulation,
which in turn further dampen macrophage activation and
enhance osteoclast differentiation via TLR4/2 signaling. Furthermore, CI favors osteoclastogenesis over macrophage activation
at several stages by modulating lineage allocation, differentiation, and activity. Therefore, mitochondrial CI orchestrates an
intricate regulatory program to synergistically control physiology
and disease.
Using primary bone marrow osteoclast differentiation assays,
we found that fatty acids, such as PA and LA, suppress osteoclastogenesis by inhibiting osteoclast differentiation and diminishing FMS and RANK expression in osteoclast precursors.
The antiosteoclastogenic effects of fatty acids have also been reported in other studies (Cornish et al., 2008). Importantly, our
ex vivo observations were also supported by in vivo findings
that the increased serum levels of fatty acids in the Ndufs4 /
and Alb-Ndufs4-KO mice, as well as PA+LA treated mice, led
to lower bone resorption and higher bone mass. Using
RAW264.7 macrophage cell line, a recent report shows that PA
may enhance osteoclast differentiation by increasing tumor
necrosis factor alpha (Drosatos-Tampakaki et al., 2014). This
discrepancy may be explained by the differences in the culture
systems, for example, PA treatment of an immortalized cell line
may not reflect the full effects of PA on the entire osteoclast differentiation process.
Inflammation-induced bone erosion such as in arthritis is a
highly prevalent disorder. In line with the report that rotenone
treatment attenuates LPS-induced bone loss (Kwak et al.,
2010), our genetic dissection reveals that Ndufs4 deletion con-
fers resistance to inflammation-induced bone erosion via both
osteoclast-intrinsic and metabolic/systemic regulation. These
findings highlight the exciting therapeutic potential of mitochondrial CI inhibition in treating bone degenerative diseases.
In our previous studies, we identified maternal genetic and dietary factors as key regulators of lactation, milk quality, and
neonatal inflammation (Du et al., 2012a, 2012b; Wan et al.,
2007b). In this study, we identified mitochondrial CI as a critical
neonatal genetic program that is essential for the postnatal
metabolic adaptation to prevent inflammation and ensure normal
bone remodeling. Ndufs4 deletion in mice causes early postnatal
lethality with metabolic defects including increased serum triglycerides and lactic acid; similarly, human mitochondrial CI
deficiency also leads to disorders with early childhood onset
(Distelmaier et al., 2009) that are manifested as metabolic abnormalities including fatal infantile lactic acidosis (FILA; Loeffen
et al., 2000; Smeitink et al., 2004). Similar to the alopecia in
Ndufs4 / mice, human patients with mitochondrial mutation
also often have skin or hair problems, which are already
accepted as an indicator of mitochondrial defects in clinic (Bodemer et al., 1999; Kubota et al., 1999; Silengo et al., 2003). This
in vivo study reveals systemic inflammation as a key etiology for
mitochondrial diseases in a unique natural metabolic context of
lactation and suckling neonates. Several prevalent and devastating infantile disorders, such as necrotizing enterocolitis, still
have no known causes or effective treatments. Future clinical investigations will further examine whether inflammation and mitochondrial dysfunction contribute to these newborn diseases.
Importantly, our findings not only elucidate the mechanisms
underlying the neonatal defects in systemic inflammation and
low bone resorption caused by mitochondrial CI deficiency,
but also uncover TLR4 inhibition as a potential treatment of infantile disorders and mitochondrial diseases.
EXPERIMENTAL PROCEDURES
Mice
Ndufs4 / and Ndufs4flox/flox mice (Kruse et al., 2008; backcrossed to C57BL/6
mice for >15 and 7 generations, respectively) were provided by Dr. Richard
Palmiter (University of Washington). Tie2-Cre (Kisanuki et al., 2001), Lysozyme-Cre, and Albumin-Cre (Jackson Laboratory) transgenic mice were maintained on a pure C57BL/6 background. To generate hematopoietic-, myeloid-,
and liver-specific Ndufs4 KO mice, Ndufs4flox/flox mice were bred with Tie2Cre+/ , Lyz-Cre+/ or Alb-Cre+/ mice, respectively; the Ndufs4flox/+Cre+/
F1 mice were bred with Ndufs4flox/flox mice to obtain Ndufs4flox/floxCre+/ F2
mice, which were bred with Ndufs4flox/flox mice to generate Ndufs4flox/flox
Cre+/ mice and littermate Ndufs4flox/floxCre / controls. TLR2/4 DKO mice
on a pure C57BL/6 background (Hoshino et al., 1999; Takeuchi et al., 1999)
were bred with Ndufs4+/ mice to generate Ndufs4/TLR4/TLR2 triple KO
mice, Ndufs4/TLR4 or Ndufs4/TLR2 double KO mice, and Ndufs4 KO controls.
All experiments were conducted using littermates. Mice were fed ad libitum
with standard chow (Harlan Laboratories). Serum triglyceride and lactate
levels were measured using a Vitros-250 chemistry analyzer at UTSW Metabolic Phenotyping Core. Serum nonesterified fatty acids were measured using
the NEFA-HR assay (Wako). The same panels of cytokines were analyzed for
expression, and only the ones with significant changes are shown to be
(E and F) Mito-SOX levels in BM-Mfs treated with PA (400 mM). (E) FACS 2D dot plots. (F) Quantification (n = 5).
(G) TLR2/4 deletion did not alter the metabolic defects in Ndufs4 / mice (n = 5).
(H) Alopecia in Ndufs4 / pups was rescued by TLR4 deletion but not TLR2 deletion (P22).
(I) Alopecia in Ndufs4 / pups was rescued by TLR4 inhibitor TAK-242 (P22). Ndufs4 / pups were gavaged with TAK-242 (10 mg/kg/day) or vehicle control
starting P1. Error bars represent SD.
Cell Metabolism 20, 483–498, September 2, 2014 ª2014 Elsevier Inc. 493
Cell Metabolism
Ndufs4 Regulation of Macrophages and Osteoclasts
Veh
RANKL
RANKL+Rosi
0.08
0.04
1.0
0.02
0.5
PGC1b
0.06
Relative mRNA
Relative mRNA
B
CTSK
TRAP
1.5
0.00
0.0
Ctrl
C
0.06
1.0
0.02
0.5
++
++++
++++
0.00
Ctrl
Tie2-Ndufs4
Ctrl
D
Tie2-Ndufs4
Tie2-Ndufs4
****
50
Veh
RANKL+Rosi
0.4
1 ± 0.12
++++
n.s.
0.2
*
20
10
Ctrl
Serum CTX-1 (ng/ml)
Alb+Tie2-Ndufs4
Tie2-Ndufs4
100
I
Tie2-Ndufs4
30
Tie2-Ndufs4
F
Alb-Ndufs4
40
0
Ctrl
0.16 ± 0.02****
Ctrl
Tie2-Ndufs4
E
Resorptive Activity
0.6
Ctrl
0.0
Oc Size:
++++
0.0
Tie2-Ndufs4
Calcium (uM)
Ctrl
1.5
0.04
++++
++++
++++
++++
ATP5b
2.0
Veh
RANKL
RANKL+Rosi
Serum CTX-1 (ng/ml)
A
**
80
60
***
***
*
****
40
20
A
lb Ctr
l
-N
d
Ti
e2 ufs
A
4
lb
+T Nd
i e uf s
24
Nd
uf
s4
0
2
0.00
0
0.01
*
*
8
6
*
**
***
4
2
0.00
0
40
n.s.
30
n.s.
n.s.
n.s.
n.s.
20
10
0
Al
Ct
br
Nd l
Ti
uf
e
s
A
lb 2-N 4
+T
du
ie
f
s
2N 4
du
fs
4
4
*
0.02
J
10
Serum P1NP (ng/ml)
****
6
0.05
A
**** ***
H
Oc.S/B.S (%)
8
****
****
****
*
A
lb Ctr
-N
l
du
Ti
Al e2- fs 4
N
b+
Ti du
fs
e2
-N 4
du
fs
4
0.10
lb Ctr
-N
l
d
Ti
e 2 ufs
A
4
lb
+T Nd
uf
ie
s
24
Nd
uf
s4
BV/TV
**** ****
0.03
*
A
lb Ctr
-N
l
d
Ti
e 2 ufs
A
4
lb
+T Nd
uf
ie
s
2N 4
du
fs
4
****
0.20
0.15
*
10
*
Tb.Th (mm)
*
Tb.N (1/mm)
0.25
Al
C
tr
bNd l
Ti
u
Al e2 - fs4
N
b+
Ti du
fs
e2
-N 4
du
fs
4
G
Figure 6. Hematopoietic and Liver Ndufs4 Deletion Inhibit Bone Resorption
(A–D) Ex vivo bone marrow osteoclast differentiation assay of Tie2-Ndufs4 KO mice. (A) Expression of osteoclast differentiation markers (n = 4). (B) Expression of
osteoclast function genes (n = 4). (C) Images of differentiation cultures on day 12. Scale bar represents 25 mm. (D) Osteoclast activity (n = 8).
(E) Serum CTX-1 (3 months, male, n = 6).
(F–J) Comparison of Alb-Ndufs4 KO, Tie2-Ndufs4 KO, Alb+Tie2-Ndufs4 DKO pups, or controls (P22, male, n = 6). (F and G) mCT. (F) Images of the trabecular bone
of the tibial metaphysis. Scale bar represents 10 mm. (G) Trabecular bone volume and architecture. (H) Osteoclast surface. (I) Serum CTX-1. (J) Serum P1NP. Error
bars represent SD.
concise. All mRNA expression was normalized by L19. Serum cytokines were
measured using Cytometric Bead Array Mouse Inflammation Kits (BD
Biosciences), and the ones that were detectable and significantly different
are reported. Sample size estimate was based on power analyses performed
using SAS 9.3 TS X64_7PRO platform at the UTSW Biostatistics Core. With
the observed group differences and the relatively small variation of the in vivo
measurements, n = 4 and n = 3 will provide >90% and >80% power at type I
error rate of 0.05 (two-sided test), respectively. All protocols for mouse
494 Cell Metabolism 20, 483–498, September 2, 2014 ª2014 Elsevier Inc.
Cell Metabolism
Ndufs4 Regulation of Macrophages and Osteoclasts
TRAP
A
++
0.02
++++
++++
0.05 ± 0.01****
0.00 ± 0.00****
0.01 ± 0.00****
0.00 ± 0.00****
0.00
Veh
PA
Veh
WT
PA
Veh
Ndufs4-/-
PA
2.0
PA
Ndufs4-/-
PA
CTSK
Veh
RANKL
RANKL+Rosi
1.5
Veh
WT
TRAP
0.06
0.04
1.0
++
++++
LA
Veh
WT
Veh
E
30
8
**
6
LA
4
2
++++
10
****
Veh
G
**
200
Serum P1NP (ng/ml)
***
80
60
40
20
0
****
++++++++
0
Veh
++++
****
0
Veh
LAC
I
FAO
Glycolysis
Fatty Acids / Lactate
W
N
T
du
fs
fs
44/
KO
TL
R
2/
4TK
O
W
T
f
fs
s4
4/
-K
TL
O
R
2/
4TK
O
N
du
N
du
1.0
TLR4/2
Macrophage
n.s. n.s.
n.s.
***
++++
****
5
LA
50
Veh
PA
LA
LAC
** *** **
10
****
0
0.5
15
100
1.5
Fold (TRAP mRNA)
PA
n.s.
10
20
150
Nd
u
H
n.s.
20
++++
****
Ndufs4-/-
25
30
WT
Ndufs4-/-
0
100
LA
Ndufs4-/-
20
0
WT
Veh
WT
FMS+ RANK+ (%)
FMS+ RANK+ % in BM
LA
Ndufs4-/-
10
Serum CTX-1 (ng/ml)
LA
0.00
Veh
F
+++
++++
0.0
D
+++
0.02
+++
0.5
FMS+ RANK+ (%)
Relative mRNA
0.09 ± 0.01****
+++
++++
0.0
B
1 ± 0.11
Veh
0.04
++++
Ndufs4-/-
0.06
1.0
0.5
WT
C
CTSK
Veh
RANKL
RANKL+Rosi
FMS+ RANK+ (%)
Relative mRNA
1.5
*** ***
Ndufs4
Osteoclast
Ndufs4
Inflam
Diff
ROS
Mito Bio
Inflammation
Bone Resorption
0.0
WT
Ndufs4-KO Ndufs4/TLR2/4-TKO
(legend on next page)
Cell Metabolism 20, 483–498, September 2, 2014 ª2014 Elsevier Inc. 495
Cell Metabolism
Ndufs4 Regulation of Macrophages and Osteoclasts
experiments were approved by the Institutional Animal Care and Use Committee of UTSW.
oclast activity was measured as calcium release using CalciFluo ELISA assay
(Lonza).
Immunofluorescence Staining and Fluorescence-Activated Cell
Sorting
Frozen sections were stained with FITC-CD11b, FITC-Gr-1, FITC-CD11c (BD
Biosciences), or FITC-F4/80 (Serotec), washed twice, and mounted with
medium containing DAPI (Vector Laboratories). Fluorescence-activated cell
sorting (FACS) was conducted with FACSCalibur using FITC-Ly6C, PECD11b (BD Biosciences), PE-RANK, and APC-FMS (eBioscience).
Statistical Analyses
All statistical analyses were performed with Student’s t-test and represented
as mean ± SD unless noted otherwise. The p values were designated as
*p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001; and n.s., nonsignificant
(p > 0.05).
Mitochondrial ROS, Membrane Potential, and CI Activity
To detect mitochondrial-generated ROS by FACS, cells were detached from
culture plates with Cell Dissociation Buffer (Life Technologies) and washed
in cold PBS. Detached cells or primary bone marrow and spleen cells were
incubated with Mito-SOX red mitochondrial superoxide indicator (Life Technologies) at 37 C for 20 min, washed twice with PBS, and then analyzed
with flow cytometry. To detect mitochondrial ROS in skin, frozen skin sections
were incubated with Mito-SOX at 37 C for 20 min, washed, and counterstained with DAPI before image acquisition. To quantify mitochondrial membrane potential, cells were stained with MitoTracker deep red (a fluorescent
probe sensitive to the membrane potential) and MitoTracker green (a probe
that stains mitochondrial membrane lipids independently of membrane potential), and the percentage of deep red-positive cells was quantified (Nakahira
et al., 2011; Tal et al., 2009). CI enzyme activity was analyzed with a microplate
assay (Abcam, ab109721) using mitochondria isolated from tissue (Abcam,
ab110168) or cells (Abcam, ab110170).
Supplemental Information includes six figures and can be found with this
article online at http://dx.doi.org/10.1016/j.cmet.2014.07.011.
Bone Analyses
Micro-CT was performed using a Scanco mCT-35 instrument (SCANCO Medical) as described (Wei et al., 2010). Histomorphometry were performed as
described (Wan et al., 2007a; Wei et al., 2011). Serum CTX-1 and P1NP
were measured with RatLaps enzyme immunoassay (EIA) kit and Rat/Mouse
PINP EIA kit (Immunodiagnostic Systems). For LPS-induced bone loss, PBS
or LPS (5 mg/kg) was injected intraperitoneally into the 6- to 8-week-old
mice at day 0 and day 4, and the mice were killed on day 7.
Ex Vivo Macrophage and Osteoclast Differentiation
Macrophage and osteoclasts were differentiated from bone marrow cells as
described (Wan et al., 2007a). For macrophages, the cells were differentiated
with 20 ng/ml of mouse GM-CSF (R&D Systems) in minimum essential medium
alpha (a-MEM) containing 10% FBS for 6 days. For FA/LAC stimulation,
cells were treated with 400 mM PA or LA (Sigma; Wen et al., 2011) or 15 mM
L-(+)-lactic acid (Sigma; Samuvel et al., 2009) for 15 hr on day 6 unless indicated otherwise. To inhibit mitochondrial ROS, macrophages were treated
with 50 mM 2R,4R-APDC, or Mito-TEMPO (Sigma) 24 hr before the addition
of fatty acids. For osteoclasts, the cells were differentiated with 40 ng/ml of
mouse M-CSF in a-MEM containing 10% FBS for 3 days (day 1–3), then
with 40 ng/ml of mouse MCSF and 100 ng/ml of mouse RANKL (R&D Systems)
for 3–9 days (day 4–12), with or without rosiglitazone (1 mM). Mature osteoclasts were identified as multinucleated (>3 nuclei) TRAP+ cells on day 12.
Osteoclast differentiation was quantified by the RNA expression of osteoclast
markers on day 6 using RT-quantitative PCR analysis. Osteoclast precursor
proliferation was quantified by bromodeoxyuridine incorporation (GE Healthcare). Osteoclast apoptosis was quantified with FACS analysis of AnnexinV+7AAD cells (BD Biosciences). To quantify osteoclast function, osteoclast
differentiation was conducted on OsteoAssay bone plates (Lonza), and oste-
SUPPLEMENTAL INFORMATION
AUTHOR CONTRIBUTIONS
Z.J. and Y.W. conceived the project and designed the experiments. Z.J. performed the majority of the experiments and data analyses. W.W. assisted with
microcomputed tomography, ELISA, and histomorphometry. Y.D. and M.Y.
assisted with the initial phenotype characterization of the Ndufs4 / mice.
Y.W. and Z.J. wrote the manuscript.
ACKNOWLEDGMENTS
We thank Dr. Richard Palmiter (University of Washington) for Ndufs4 / and
Ndufs4flox/flox mice, UT Southwestern Mouse Metabolic Phenotyping Core
and Biostatistics Core for their assistance in our studies, and Drs. Paul Dechow
and Jerry Feng (Baylor College of Dentistry) for assistance with microcomputed tomography and histomorphometry. Y.W. is a Virginia Murchison Linthicum Scholar in Medical Research. This work was supported in part by the
March of Dimes (#6-FY13-137 to Y.W.), The Welch Foundation (I-1751 to
Y.W.), NIH (R01 DK089113 to Y.W.), CPRIT (RP130145 to Y.W.), DOD BCRP
Idea Award (W81XWH-13-1-0318 to Y.W.), and UTSW Endowed Scholar
Startup Fund (to Y.W.).
Received: March 4, 2014
Revised: May 27, 2014
Accepted: July 10, 2014
Published: August 14, 2014
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498 Cell Metabolism 20, 483–498, September 2, 2014 ª2014 Elsevier Inc.
Cell Metabolism, Volume 20
Supplemental Information
Mitochondrial Complex I Activity Suppresses Inflammation and Enhances Bone Resorption by
Shifting Macrophage-Osteoclast Polarization
Zixue Jin, Wei Wei, Marie Yang, Yang Du, and Yihong Wan
Jin Fig. S1
n.s.
n.s.
10
20
5
n.s.
0
BM
0
Spleen
BM
Spleen
0.0006
0.0004
0.0002
n.s.
BM
n.s.
0.006
0.004
0.002
n.s.
0.000
Spleen
0.02
****
0.00
G
****
Ym1
Ym2
Macrophage
0.010
****
0.005
0.000
Arginase
1.5
15
2
1.0
10
1
0.5
5
0
WT
Ndufs4-/-
J
0.0
n.d.
WT
Ndufs4-/-
IL-6
BM
Spleen
0.005
****
0.003
0.002
0.002
0.001
0.001
Veh
40
20
20
10
0
WT
Rotenone
0.000
H
****
0
WT
Ndufs4-/-
Tie2-N4 Alb+Tie2-N4
I
Macrophage
Liver
8
4
3
2
1
0
n.s.
Alb-N4
5
n.d.
Ctrl
Tie2-Ndufs4
6
4
2
0
****
Ctrl
IL-12p35
****
Veh
Ctrl
Ndufs4-/-
Rotenone
0.0015
*
*
0.0008
0.0010
0.0004
0.0005
0.0000
Alb-Ndufs4
IL-12p40
0.0012
0.004
0.003
0.000
30
0
n.s.
n.s.
40
60
COX-2
0.005
0.004
n.s.
80
Heart
Osteoclast
3
**
50
100
CI activity (mOD/min)
0.04
WT
Ndufs4-/-
CI activity (mOD/min)
0.06
0.015
Serum Corticosterone (ng/ml)
WT
Ndufs4-/-
Relative mRNA in skin (P22)
Relative mRNA in
MCSF BMDM
n.s.
0.0008
F
0.08
CI activity (mOD/min)
Treg Cell
0.008
0.0000
E
Relative mRNA
D
Th17 Cell
0.0010
15
CD3+%
40
n.s.
C
T Cell
20
Relative Foxp3 mRNA
WT
Ndufs4-/-
60
B220+%
B
B Cell
80
Relative Rorc mRNA
A
Veh
Rotenone
0.0000
Veh
Rotenone
Figure S1 Mitochondrial Complex I deficiency activates inflammation in macrophages, related to Figure 1.
(A-D) Ndufs4-/- mice exhibited unaltered populations of B cell (A), T cell (B), Th17 cell (C) and Treg cell (D) in bone marrow (BM) and
spleen (n=6). (E) Ndufs4 deletion decreased the expression of M2 markers in MCSF-differentiated bone marrow macrophages (left)
and pup skin (right) (n=6). (F) Ndufs4 deletion does not alter serum corticosterone levels. Left, 22-day-old Ndufs4-/- pups vs. littermate
WT controls (n=4). Right, 6-week-old Alb-Ndufs4 KO, Tie2-Ndufs4 KO, Alb+Tie2-Ndufs4 DKO vs. WT controls (n=4). N4, Ndufs4.
Quantified by ELISA (Enzo Life Sciences ADI-900-097). (G) Complex I activity was diminished in the macrophage, osteoclast and heart
from Ndufs4-/- mice (n=6, P22). (H) Complex I activity was diminished in the macrophage from Tie2-Ndufs4 KO mice (n=6, 6-8 week
old). (I) Complex I activity was diminished in the liver of Alb-Ndufs4 KO mice (n=6, 6-8 week old). G-I, mitochondria were isolated from
tissue (Abcam, ab110168) or cells (Abcam, ab110170), and Complex I (CI) enzyme activity was analyzed with a microplate assay
(Abcam, ab109721); n.d, not detected. (J) Complex I inhibition by rotenone (10nM) increased the expression of pro-inflammatory genes
in GMCSF-differentiated bone marrow macrophages (n=3). Error bars, SD.
Jin Fig. S2
A
Oc:
WT
WT
Ndufs4-/-
Ndufs4-/-
Ob:
WT
Ndufs4-/-
WT
Ndufs4-/-
Oc
Oc
Oc
Oc
Ob
Relative TRAP mRNA
0.4
0.2
0.1
C
n.s.
OcWT+ObWT
OcWT+ObKO
OcKO+ObWT
Osterix
Relative mRNA
0.5
WT
Ndufs4-/-
n.s.
5
4
0.3
3
0.2
2
0.0
1
n.s.
-ObDiff
+Obdiff
0
WT
Ndufs4-/-
0.004
n.s.
0.002
OcWT+ObWT
D
OcWT+ObKO
OcKO+ObWT
Osterix
4
n.s.
OcKO+ObKO
Col1a1
0.4
n.s.
****
****
0.006
0.000
OcKO+ObKO
Col1a1
0.4
0.1
n.s.
0.008
0.3
0.0
Ob
****
****
Relative CTSK mRNA
n.s.
Ob
Relative mRNA
B
Ob
n.s.
n.s.
0.3
3
0.2
2
0.1
1
n.s.
n.s.
-ObDiff
+Obdiff
0.0
Veh
PA
LA
0
Veh
PA
LA
Figure S2 Ndufs4 does not affect osteoblastogenesis, related to Figure 2 and 7.
(A-B) Osteoblast and osteoclast co-cultures show that osteoclast differentiation was only impaired when osteoclast progenitors were
Ndufs4-/-, regardless whether osteoblasts were WT or Ndufs4-/-, indicating an osteoclast-autonomous defect. To expand mesenchymal
stem cells (MSC), bone marrow cells were cultured in MSC medium for 4 days; osteoblast differentiation was then induced with 5 mM
β-glycerophosphate and 100 μg/mL ascorbic acid for 6 days; bone marrow osteoclast progenitors were then added to the osteoblast
cultures and differentiated into osteoclasts in the presence of 1, 25 dihydroxyvitamin D3 (10−8 M) for 4-6 days. (A) Representative
images of TRAP staining of co-cultures. Ob, osteoblast; Oc, osteoclast. Scale bars, 25 μm. (B) Quantification of osteoclast
differentiation by the expression of marker genes (n=3). (C) Osteoblast differentiation from Ndufs4-/- bone marrow was normal,
quantified by osteoblast marker gene expression (n=3). ObDiff, osteoblast differentiation cocktail. (D) Osteoblast differentiation was not
affected by palmitic acid (PA) or linoleic acid (LA) treatment, quantified by osteoblast marker gene expression (n=3). Error bars, SD.
Jin Fig. S3
A
Ctrl
Alb-Ndufs4
Tie2-Ndufs4
Alb+Tie2-Ndufs4
PBS
LPS
B
****
80
60
++++
+ n.s.
40
++ ++++
++++
n.s.
++++ n.s.
0.05
****
Al
b+
++++
Spleen
4
Ctrl
Alb-Ndufs4
Tie2-Ndufs4
Alb+Tie2-Ndufs4
^^^
++++
****
^^^^
****
****
++++
+++
****
Ti
e2
-N
4
Ti
e2
-N
4
N4
A
lb
-
C
trl
Ti
e2
-N
4
A
lb
+
^^^
^^^
IL-12p35 IL-12p40 TNFa
IL-6
**
*
*
*
*
*
COX-2
***
****
***
****
****
^
**
*
IL-6
+++
+++
****
*
***
***
****
****
***
***
**
*
***
2
1
0
COX-2
^^^^
3
*
***
*
***
***
****
++++
++++
****
+++
^^^^
****
+++
^^^
2
0
0.10
^^^^
5
BM
+
****
***
Relative mRNA
Ti
e2
-N
4
N
4
Al
b-
Ct
rl
^
6
4
++++
++++
n.s. ++++++++
n.s.
0.00
***
8
++++
++++ n.s.
PBS
LPS
20
0
D
ANOVA p<0.0001
0.15
PBS
LPS
BV/TV
Serum CTX-1 (ng/ml)
C
ANOVA p<0.0001
100
MCP-1
MCP-3
IL-1b
IL-12p35 IL-12p40
TNFa
MCP-1
MCP-3
IL-1b
Figure S3 Ndufs4 deletion by Alb-cre, Tie2-cre or both prevents inflammation-induced bone loss, related to Figure 6.
(A-C) Alb-Ndufs4 KO, Tie2-Ndufs4 KO, Alb+Tie2-Ndufs4 DKO or control mice (n=4) were injected with LPS (i.p., 5mg/kg) at day 0 and
day 4, and then sacrificed on day 7. (A) Representative images of H&E staining of distal femurs showing that the LPS-induced bone
loss in control mice was abolished in all 3 strains of KO mice. Scale bars, 25 μm. (B) Serum CTX-1 resorption marker. (C) Trabecular
BV/TV of proximal tibiae by uCT. N4, Ndufs4. Black * and n.s. compare LPS with PBS in each genotype; red and blue + compare each
KO genotype with control (ctrl) in each treatment. (D) Alb-Ndufs4 KO, Tie2-Ndufs4 KO and Alb+Tie2-Ndufs4 DKO mice showed
exacerbated inflammation upon LPS treatment compared to controls (n=4). Mice were injected with LPS (i.p., 5mg/kg) and then
sacrificed 2 hrs later. Inflammatory gene expression in bone marrow (BM) and spleen was quantified by RT-QPCR. Blue * compares
each KO genotype with control; red + compares DKO with Alb-Ndufs4; green ^ compares DKO with Tie2-Ndufs4. Error bars, SD.
Jin Fig. S4
0.4
0.005
0.2
1.5
WT Ndufs4-/-
0.000
n.s.
***
1.0
0.5
0.0
0.0
n.s.
WT Ndufs4-/-
d3
d4
Pro
d5
PA
LA
WT
FMS
1.5
1.0
0.5
0.0
I
****
NFATc1
0.00
**
mRNA in Ndufs4-/- Mf
Relative mRNA
1.0
0.02
0.5
Vec
FMS
0.10
Vec
0.02
0.05
0.01
30
0.15
****
****
20
10
Veh
****
Vec NFATc1
Vec NFATc1
0.04
0.5
0.02
L
0.5
0.00
****
Vec RANK
TRAP
n.s.
0.02
0.5
0.01
Vec c-fos
****
0.03
1.0
0.0
WT Ndufs4-/-
Vec RANK
c-fos
1.5
1.5
1.0
****
PA+LA
TRAP
0.06
1.0
0.0
WT Ndufs4-/c-fos
Veh
RANK
1.5
0.2
0.00
PA+LA
H
0.4
0.0
0.10
0.05
**
0.00
d6
Diff
0.20
0.6
K
**
0.03
d5
40
RANK
0.0
FMS
TRAP
0.04
****
0.00
WT Ndufs4-/-
0.00
NFATc1
0.15
0.10
0.05
0.04
1.5
J
0.15
****
d4
Pro
0.8
2.0
0.0
WT Ndufs4-/-
****
G
Relative mRNA
2.0
TRAP
0.06
Relative mRNA
mRNA in Ndufs4-/- Mf
Relative mRNA
2.5
d3
D
LAC
Ndufs4-/-
F
FMS
2.5
200
0
0
E
***
400
d6
Serum CTX-1 (ng/ml)
Veh
WT
Ndufs4-/-
n.s.
600
Diff
Withdrawal after 3 days
C
***
***
800
***
BV/TV
RANKL
100ng/ml
150ng/ml
250ng/ml
0.010
2.0
mRNA in Ndufs4-/- Mf
0.015
mRNA in Ndufs4-/- Mf
0.6
B
CTSK
FMS mean intensity
Relative mRNA
0.8
TRAP
Relative FMS mRNA
A
0.00
Vec c-fos
Figure S4 Additional analyses of Ndufs4 regulation of osteoclastogenesis, related to Figure 2 and 7.
(A) Osteoclast differentiation defects in Ndufs4-/- cultures were not rescued by higher RANKL concentration (n=3). (B) A reduction in
FMS mRNA expression (left) and FMS mean intensity by FACS (right) in Ndufs4-/- cultures only occurred during differentiation (Diff)
stage (after RANKL addition on day 3), but not during proliferation (Pro) stage. (C) The inhibitory effects of palmitic acid (PA), linoleic
acid (LA) and lactate (LAC) on osteoclast differentiation remained when removed after 3 days of treatment. WT osteoclast
differentiation cultures were treated during the first 3 days after RANKL addition, and then changed to fresh medium without
compounds for 6 more days. Representative images for TRAP staining are shown. Scale bars, 25 μm. (D) PA and LA treatment
suppresses bone resorption and increases bone mass in vivo. C57BL/6 mice (8-week-old males, n=5) were injected with vehicle (Veh)
or PA+LA mixture (i.p. 20mg/kg/day each) daily for 14 days. Left, serum CTX-1 bone resorption marker. Right, trabecular BV/TV of
proximal tibiae by µCT. (E, G, I, K) Expression of FMS (E), RANK (G), NFATc1 (I) and c-fos (K) was decreased in Ndufs4-/- osteoclast
differentiation cultures (n=3). (F, H, J, L) Osteoclast differentiation defect in Ndufs4-/- cultures, quantified by TRAP mRNA, was partially
rescued by over-expression of FMS, RANK, NFATc1, but not c-fos (n=3). Error bars, SD.
Jin Fig. S5
0.001
d1
d2
d3
d4
d5
D
Ndufs4
0.004
WT
Lyz-Ndufs4-/-
0.003
0.002
0.001
0.000
F
d2
d3
d4
d5
0.006
0.004
0.2
0.002
0.1
d1
d2
d3
d4
d5
d6
0.000
0.5
0.010
0.4
0.3
0.006
0.2
0.004
0.1
0.002
Lyz-Ndufs4
d1
d2
G
d3
d4
d6
0.000
H
*
0.15
0.10
Relative mRNA
in Spleen (P22)
0.004
Ctrl
d5
d6
Oc Size:
1 ± 0.21
Lyz-Ndufs4
d2
d3
d4
d5
Oc Size:
d6
I
35
6
20
15
10
0.31 ± 0.05***
**
****
25
0
Lyz-Ndufs4
K
0.04
*
Ctrl
4
2
0.02
0.01
L
**
0.15
0.10
0.05
Ctrl
Lyz-Ndufs4
0.00
0
Lyz-Ndufs4
0.25
0.20
0.03
0.00
d4
5
Tb.Sp (mm)
Tb.Th (mm)
Proximal Tibia
J
d1
30
BS (mm2)
BV/TV
Trabecular
d5
0.25
0.00
Ctrl
Lyz-Ndufs4
d3
WT
Lyz-Ndufs4-/-
0.008
0.05
0.005
d2
CTSK
WT
Lyz-Ndufs4-/-
0.20
M
d1
TRAP
d6
CT
Ctrl
Ctrl
WT
Tie2-Ndufs4-/-
0.3
0.0
d1
WT
Tie2-Ndufs4-/-
0.4
0.0
d6
0.008
Tb.N (1/mm)
Relative mRNA
0.002
Relative mRNA
Relative mRNA
Relative mRNA
WT
Tie2-Ndufs4-/-
E
CTSK
TRAP
0.5
0.003
0.000
C
B
Ndufs4
0.004
Ctrl
Lyz-Ndufs4
Serum CTX-1 (ng/ml)
A
Ctrl
Lyz-Ndufs4
50
40
30
*
20
10
0
Ctrl
Lyz-Ndufs4
***
0.003
0.002
0.001
0.000
**
**
COX-2
****
IL-6
IL-12p40
MCP-3
Figure S5 Ndufs4 deletion by lysozyme-cre reduces bone resorption but increases inflammation, related to Figure 3 and 6.
(A-D) Comparison of an osteoclast differentiation time course in Tie2-Ndufs4 and Lyz-Ndufs4 cultures. (A, C) Ndfus4 deletion was
earlier and more complete in Tie2-Ndufs4 cultures than Lyz-Ndufs4 cultures (n=3). (B, D) Osteoclast differentiation defect, measured by
osteoclast marker expression, was more severe in Tie2-Ndufs4 cultures than Lyz-Ndufs4 cultures (n=3). (E) Representative images of
differentiation cultures on day 12 showing reduced number and size of mature osteoclasts in Lyz-Ndufs4 cultures. Mature osteoclasts
were identified as multinucleated (>3 nuclei) TRAP+ (purple) cells. Scale bar, 25 µm. (F-L) Comparison of Lyz-Ndufs4 KO or littermate
controls (3 month old, male, n=6). (F-K) µCT analysis of proximal tibiae. (F) Representative images of the trabecular bone of the tibial
metaphysis (top) (scale bar, 10 µm) and the entire proximal tibia (bottom) (scale bar, 1 mm). (G-K) Quantification of trabecular bone
parameters. (G) BV/TV, bone volume/tissue volume ratio. (H) BS, bone surface. (I) Tb.N, trabecular number. (J) Tb.Th, trabecular
thickness. (K) Tb.Sp, trabecular separation. (L) Serum CTX-1. (M) Expression of inflammatory markers in spleen (n=6). Error bars, SD.
Jin Fig. S6
Ctrl Alb-Ndufs4
0.02
n.s.
0.00
8
***
n.s.
0.05
n.s.
0.00
**
R2 WT
/4
N
du
Nd DK
O
fs
u
4/
f
TL s4R2 KO
/4
-T
KO
0
Liver
n.s.
3
***
*
2
n.s.
4
1
2
0
TL
****
n.s.
1500
**
0.3
0.2
0.1
0.0
Conn. D. (1/mm3)
n.s.
Mf
6
0.4
*
Tb.Sp (mm)
***
5
TL
0.05
TL
R 2 WT
/4
N
du
N d DK
O
fs
uf
4/
TL s4 KO
R
2/
4TK
O
R2 WT
/4
N
du
Nd DK
O
fs
u
4/
f
TL s4R2 KO
/4
-T
KO
TL
BS (mm2)
****
0.10
0
15
10
Liver
n.s.
W
R
T
2/
4
Nd
-D
N
KO
uf
s4 duf
s4
/T
LR - K
O
2/
4TK
O
0.00
20
Mf
R2 WT
/4
N
du
Nd DK
O
fs
4/ ufs
4TL
R2 KO
/4
-T
KO
0.10
****
0.04
Tb.N (1/mm)
BV/TV
0.15
0.06
0.15
****
*
1000
500
n.s.
0
R2 WT
/4
N
-D
du
N
KO
f s du
4/
fs
TL
4
R2 -KO
/4
-T
K
O
2
WT
Ndufs4-/-
TL
4
n.s.
SMI
**
0
C
Relative TLR2 mRNA
FMS+ RANK+ % in BM
8
6
0.08
Relative TLR4 mRNA
B
10
TL
A
Figure S6 TLR2/4 deletion partially rescues the bone phenotype of Ndufs4-/- mice, related to Figure 7.
(A) FACS analysis of percentage of FMS+RANK+ osteoclast precursors in the bone marrow of Alb-Ndufs4 KO mice and littermate
controls (n=6). (B) Expression of TLR2 and TLR4 was unaltered in the macrophages (Mf) or liver from Ndufs4-/- mice (n=3). (C) µCT
analysis of the trabecular bone in the proximal tibiae from WT, TLR2/4-DKO, Ndufs4-KO and Ndufs4/TLR2/4-TKO mice (n=4). BV/TV,
bone volume/tissue volume ratio; BS, bone surface; Tb.N, trabecular number; Tb.Sp, trabecular separation; SMI, structure model index,
which quantifies the relative amount of plates (SMI=0, strong) and rods (SMI=3, fragile); Conn.D., Connectivity Density. Error bars, SD.

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