EXTENDED REPORT
Autophagy regulates TNFα-mediated joint
destruction in experimental arthritis
Neng-Yu Lin,
1
Christian Beyer,
1
Andreas Gießl,
2
Trayana Kireva,
1
Carina Scholtysek,
1,3
Stefan Uderhardt,
1,3
Luis Enrique Munoz,
1
Clara Dees,
1
Alfiya Distler,
1
Stefan Wirtz,
4
Gerhard Krönke,
1,3
Brian Spencer,
5
Oliver Distler,
6
Georg Schett,
1
Jörg H W Distler
1
▸ Additional supplementary
figures are published online
only. To view these files please
visit the journal online (http://
dx.doi.org/10.1136/
annrheumdis-2012-201671)
1
Department of Internal
Medicine III and Institute for
Clinical Immunology, University
of Erlangen-Nuremberg,
Erlangen, Germany
2
Department of Biology,
Animal Physiology, University
of Erlangen-Nuremberg,
Erlangen, Germany
3
Nikolaus Fiebiger Center of
Molecular Medicine, University
Hospital Erlangen, University of
Erlangen-Nuremberg, Erlangen,
Germany
4
Department of Internal
Medicine I, University of
Erlangen-Nuremberg, Erlangen,
Germany
5
Department of Neurosciences,
University of California, San
Diego, La Jolla, California, USA
6
Center of Experimental
Rheumatology and Zurich
Center of Integrative Human
Physiology, University Hospital
Zurich, Zurich, Switzerland
Correspondence to
Dr Jörg H W Distler,
Department of Internal
Medicine III and Institute for
Clinical Immunology, University
of Erlangen-Nuremberg,
PO Box Glücksstr.4a, Erlangen
D-91054, Germany;
joerg.distler@uk-erlangen.de
Accepted 9 August 2012
Published Online First
12 September 2012
ABSTRACT
Objectives Autophagy is a homeostatic process to
recycle dispensable and damaged cell organelles.
Dysregulation of autophagic pathways has recently been
implicated in the pathogenesis of various diseases. Here,
we investigated the role of autophagy during joint
destruction in arthritis.
Methods Autophagy in osteoclasts was analysed in
vitro and ex vivo by transmission electron microscopy,
Western blotting and immunohistochemistry for Beclin1
and Atg7. Small molecule inhibitors, LysMCre-mediated
knockout of Atg7 and lentiviral overexpression of Beclin1
were used to modulate autophagy in vitro and in vivo.
Osteoclast differentiation markers were quantified by real-
time PCR. The extent of bone and cartilage destruction
was analysed in human tumour necrosis factor α
transgenic (hTNFα tg) mice after adoptive transfer with
myeloid specific Atg7-deficient bone marrow.
Results Autophagy was activated in osteoclasts of
human rheumatoid arthritis (RA) showing increased
expression of Beclin1 and Atg7. TNFα potently induced
the expression of autophagy-related genes and activated
autophagy in vitro and in vivo. Activation of autophagy by
overexpression of Beclin1-induced osteoclastogenesis and
enhanced the resorptive capacity of cultured osteoclasts,
whereas pharmacologic or genetic inactivation of
autophagy prevented osteoclast differentiation. Arthritic
hTNFα tg mice transplanted with Atg7
fl/fl
×LysMCre
+
bone marrow cells (BMC) showed reduced numbers of
osteoclasts and were protected from TNFα-induced bone
erosion, proteoglycan loss and chondrocyte death.
Conclusions These findings demonstrate that
autophagy is activated in RA in a TNFα-dependent
manner and regulates osteoclast differentiation and bone
resorption. We thus provide evidence for a central role of
autophagy in joint destruction in RA.
INTRODUCTION
Rheumatoid arthritis (RA) is a chronic inflamma-
tory disease that results in severe destruction of
articular cartilage and bone.
1
Osteoclasts are the
major cellular mediators of bone degradation in
RA, which results in articular erosions and sys-
temic osteoporosis. In this context, osteoclast pre-
cursors and mature osteoclasts are abundant at
sites of arthritic bone erosions.
23
The differenti-
ation of osteoclasts from its precursors, so-called
osteoclastogenesis, depends on the presence of
macrophage colony-stimulating factor (M-CSF)
and receptor activator of NF-κB ligand (RANKL).
4–6
In addition, proinflammatory cytokines, such as
tumour necrosis factor α (TNFα) can enhance the
osteoclast differentiation.
7
Indeed, mice overexpres-
sing human TNFα (hTNFα tg) show increased
osteoclastogenesis and destructive arthritis closely
resembling RA.
89
Autophagy describes a general mechanism, by
which cells degrade unnecessary or dysfunctional
cellular organelles through the lysosomal machin-
ery.
10
The degradation of cellular organelles can
maintain cellular energy levels and guarantee cell
survival during starvation, but may turn into cell
death if nutrient supply cannot be restored.
10
Proper regulation of autophagy guarantees the fine
balance of synthesis, degradation and recycling of
cellular components. These functions render autop-
hagy essential for proper cell differentiation and
organ development.
11
Autophagy is initiated by the formation of an iso-
lation membrane, which then elongates to enclose
the target organelles and to form the so-called autop-
hagosome. The autophagosome subsequently fuses
with lysosomes to degrade the intravesicular compo-
nents. Several key regulators of autophagy have been
identified in the last years. Beclin1 is essential for
the initiation of autophagy.
12–15
Atg7 mediates
the elongation of the isolation membrane, which
culminates in conversion of a soluble form of
microtubule-associated protein one light chain three
(LC3-I) to phosphatidylethanolamine-conjugated
membrane-bound form (LC3-II), a process often
used to demonstrate active autophagy.
16
Although autophagy is tightly regulated to limit
uncontrolled activation, deregulated autophagy
may contribute to the pathogenesis of various dis-
eases including infections, cancer, neurodegenera-
tive and heart disease.
17
Of particular interest, a
most recent study showed that autophagy regu-
lates the release of lysosomal components by
osteoclasts.
18
This finding may have profound
impact on understanding the mechanisms of bone
damage in arthritis, and in finding new strategies
for prevention and treatment.
In this study, we aimed to elucidate the role of
autophagy in human RA and in murine inflamma-
tory arthritis, and the function of autophagy in
TNFα-mediated bone destruction. We demon-
strated that autophagy is activated in RA in a
TNFα-dependent manner and stimulates osteoclast
differentiation. By contrast, selective inhibition of
autophagy in monocytes strongly reduces
Ann Rheum Dis 2013;72:761–768. doi:10.1136/annrheumdis-2012-201671 761
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osteoclast differentiation and joint destruction in hTNFα tg
mice. Together, these findings provide evidence for a central role
of autophagy in joint destruction in RA.
MATERIALS AND METHODS
Mice and bone marrow transplantation
For bone marrow transplantation, 4-week-old recipient hTNFα
tg mice (strain Tg197; C57BL/6 background)
8
were irradiated
with 10.5 Gy (Stabilipan; Siemens, Erlangen, Germany). After
24 h, bone marrow was reconstituted by intravenous injection
of either 5×10
6
Atg7
fl/fl
× LysMCre
−
BMCs, or Atg7
fl/fl
×
LysMCre
+
BMCs, or BMCs transduced with lentiviruses encod-
ing Beclin1 (LVBeclin1), or lentiviral control vectors
(LVscramble).
19 20
The animal studies were approved by the
local animal welfare committee.
Isolation of osteoclast precursors and osteoclast
differentiation assay
Bone marrow-derived osteoclasts were prepared as previously
described.
21
In brief, donor mice were sacrificed at the age of
6–8 weeks. BMCs from tibia and femur were flushed with PBS
and filtered through a 0.4 μm filter. The cells were cultured in
α-MEM (Invitrogen, Carlsbad, California, USA) containing 10%
fetal bovine serum. One day after plating, floating cells were har-
vested in complete medium with 20 ng/ml murine M-CSF
(rmM-CSF) and 1–30 ng/ml murine RANKL (R&D Systems,
Ambington, UK) for 3 days. Medium and cytokines were
replaced after 72 h. Osteoclast differentiation was evaluated by
staining cells for tartrate-resistant acid phosphatase (TRAP)
using a Leukocyte Acid Phosphatase Kit (Sigma-Aldrich, St
Louis, Missouri, USA).
Lentiviral infection
After 72 h of stimulation with M-CSF and RANKL, bone
marrow-derived osteoclasts were infected with either
LV-Beclin1 or LVscramble
19
at a multiplicity of infection of 10
for 24 h.
In vitro bone resorption assay
BMCs were plated on bone slices (IDS, London, Great Britain)
at a density of 5×10
4
cells/slice in 96-well culture plates with
200 ml culture medium per well. The cultured medium was
exchanged every third day. After 14 days, bone pits were
stained with 1% toluidine blue O (Sigma-Aldrich).
CLINICAL AS SESSMENT
Clinical evaluation was performed weekly and was performed
in a blinded manner as described.
22
Briefly, paw swelling was
examined in all four paws, and a clinical score of 0–3was
assigned as follows: 0=no swelling, 1=mild swelling, 2=mod-
erate swelling and 3=severe swelling of the toes and ankle. In
addition, grip strength was examined in each paw, using a
3 mm diameter wire, and was scored on a scale from 0 to −4
as follows: 0=normal grip strength, −1=mildly reduced,
−2=moderately reduced, −3=severely reduced, and −4=no grip
strength. Mice were weighed weekly.
Histomorphometric analysis
Decalcified, paraffin-embedded hind paws were cut in 5 μm sec-
tions and stained with toluidine blue, haematoxylin (Merck,
Darmstadt, Germany) or TRAP, using a leukocyte acid phos-
phatase staining kit (Sigma-Aldrich). Bone erosion, osteoclast
number, and cartilage destruction were quantified with a Zeiss
Axioskop 2 microscope (Carl Zeiss AG, Oberkochen, Germany)
equipped with a digital camera and image analysis system
(OsteoMeasure; Osteometrics, Decatur, Georgia, USA).
23
Immunofluorescence staining
Synovial tissue sections from patients with RA and osteoarth-
ritis (OA) were incubated with polyclonal rabbit anti-Atg7
(AnaSpec, San Jose, California, USA) or Beclin1 (Abcam,
Cambridge, UK). Irrelevant isotype antibodies were used as
negative controls. Polyclonal goat anti-rabbit antibodies labelled
with fluorescent dye Alexa Fluor 488 (Invitrogen, Darmstadt,
Germany) were used as secondar y antibodies. Quantification
was performed with ImageJ software (V.1.41; National
Institutes of Health) and the intensity of immunofluorescence
was analysed under equal conditions for all samples within the
experiments. This includes equal exposure times and light
intensity. The fluorescence intensity of 5–10 randomly selected
osteoclasts per sample was quantifi ed by Image J.
Western blot analysis
Whole-cell lysates were prepared as previously described.
24
Polyvinylidene fluoride membranes were incubated with poly-
clonal rabbit anti-Atg7 (AnaSpec), Beclin1 (Abcam) or LC3
(Novus, Jena, Germany). Horseradish peroxidase-conjugated
polyclonal goat anti-rabbit or monoclonal rabbit anti-mouse
antibodies (DAKO, Hamburg, Germany) were used as second-
ary antibodies. Equal loading of proteins was confirmed by
β-actin (Sigma-Aldrich).
Quantitative real-time PCR
Gene expression was quantified by SYBR Green real-time PCR
as described.
24
The following primer pairs were used: mouse
cathepsin K: 5
0
-GGAAGAAGACTCACCAGAAGC-3
0
and 5
0
-GT
CATATAGCCGCCTCCACAG-3
0
; mouse TRAP 5
0
-CGACCATT
GTTAGCCACATACG-3
0
and 5
0
-TCGTCCTGAAGATACTGCAG
GTT-3
0
; mouse OSCAR 5
0
-TCGCTGATACTCCAGCTGTC-3
0
and 5
0
-ATCCCAGGAGTCACAACTGC-3
0
; mouse nuclear factor
of activated T cells c1 (NFATc1), 5
0
-CAACAAGCGCAAGTA
CAGTCTC-3
0
and 5
0
-CAGGTATCTTCGGTCACACTGA-3
0
;
mouse β -actin, 5
0
-TGGCATTGTGGAAGGGCTCATGAC-3
0
and
5
0
-ATGCCAGTGAGCTTGCCGTTCAGC -3
0
. Samples without
enzyme in the reverse transcription reaction (Non-RT-controls)
were used as negative controls to exclude genomic contamin-
ation. β-actin was used to normalise for the amounts of loaded
cDNA. Differences were calculated with the threshold cycle
(Ct) and the comparative Ct method for relative quantification.
MicroCT analysis
Hind paws were analysed by microCT (μCT) (μCT35;
SCANCO Medical AG, Brüttisellen, Switzerland). The follow-
ing acquisition parameters were used: voltage: 40 kV; x-ray
current: 250 μA; exposure time: 5000 ms/projection, 720 projec-
tions; matrix: 1024×1024; and voxel size in reconstructed
image: 9 μm.
Transmission electron mic roscopy
For transmission electron microscopy, cells were harvested and
fixed with 2.5% glutaraldehyde in cacodylate buffer solution
(ph 7.4, containing sucrose). Thereafter, cells were washed,
postfixed with 2% osmium tetroxide in cacodylate buffer and
embedded in 2% agarose. During dehydration by ethanol, the
cells were fixed with 1.0% uranyl acetate in 70% ethanol, fol-
lowed by embedding in araldite and cutting with an ultrami-
crotome (Reichert Ultracut, Vienna, Austria). Images were
captured with a Zeiss EM10 electron microscope (Carl Zeiss
762 Ann Rheum Dis 2013;72:761–768. doi:10.1136/annrheumdis-2012-201671
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AG) and a Gatan SC1000 OriusTM CCD camera in combin-
ation with the Digital Micrograph TM software (Gatan GmbH,
Munich, Germany).
Statistical analysis
All data are presented as median with IQR using the GraphPad
Prism 4.0 Software. Differences between the groups were tested
for their statistical significance by non-parametric Mann–
Whitney U test. A p value of less than 0.05 was considered
statistically significant.
RESULTS
Atg7 and Beclin1 are overexpressed in RA osteoclasts
We first analysed whether autophagy is activated in human
osteoclasts of RA patients. Indeed, the expression of Atg7,
which is essential for the elongation of the isolation membrane
during autophagy, was increased in TRAP-positive cells of RA
patients compared with samples from OA patients (figure 1A).
The expression of Beclin1, which stimulates the initiation of
autophagy, was also increased in TRAP-positive cells of RA
patients (figure 1B). The average fluorescence intensity was
increased by 77% for Atg7 and by 284% for Beclin1 (p=0.002
and 0.004; figure 1C). Together, these results suggest that
autophagy is activated in osteoclasts in RA.
TNFα stimulates autophagy in murine osteoclasts in vitro and
in vivo
Given the key role of TNFα in inflammatory bone resorption in
arthritis, we hypothesised that TNFα might stimulate autop-
hagy in RA osteoclasts. Indeed, we found that TNFα upregu-
lated the expression of Atg7 and Beclin1 with increased mRNA
and protein levels of both autophagy markers in murine
osteoclasts, in particular, under conditions with lower concen-
trations of RANKL (1 ng/ml) that may mimic onset of inflam-
mation or less active disease. The stimulating effect of TNFα
on Atg7 and Beclin1 was less pronounced in the presence of
very high concentrations of RANKL (30 ng/ml) (figure 2A–C).
Consistent with the induction of Atg7 and Beclin1, TNFα
also stimulated the conversion of LC3 I into LC3 II in osteo-
clasts, which is another hallmark of active autophagy. To
confirm the stimulatory effects of TNFα on autophagy in vivo,
we analysed the expression of Atg7 and Beclin1 in osteoclasts
of hTNFα tg mice and wildtype littermates. The expression
levels of Atg7 and Beclin1 were increased by 160% and 380% in
osteoclasts of hTNFα tg mice compared with the findings in
TRAP-positive cells in sections of control mice (p=0.007 and
0.009; figure 2E,F). Together, these results demonstrate that
TNFα activates autophagy in murine osteoclasts in vitro and in
vivo.
Autophagy regulates murine osteoclast differentiation and
bone resorption in vitro
To determine, whether autophagy plays a role for osteoclast dif-
ferentiation, we first performed electron microscopy (figure 3A).
We observed numerous autophagosomes containing mitochon-
dria and other organelles in differentiating osteoclasts generated
from BMCs of wildtype mice. In contrast, mature autophagocy-
tic vacuoles engulfing mitochondria and other organelles were
absent in Atg7-deficient osteoclasts, but electron-dense imma-
ture autophagosomes and lysosomes accumulated in the
cytoplasm of Atg7-deficient cells (figure 3A). We therefore
hypothesised that the impaired degradation of damaged orga-
nelles, together with the accumulation of immature autophago-
lysosome, might interfere with osteoclastogenesis. Indeed,
Figure 1 Atg7 and Beclin1 are overexpressed in osteoclasts of patients with RA. Overexpression of Atg7 and Beclin1 in osteoclasts indicates
activated autophagy at sites of bone erosions in RA patients. (A, B) Representative images of osteoclasts in synovial tissue from patients with RA
and OA double-stained for tartrate-resistant acid phosphatase (TRAP) and either Atg7 or Beclin1 are shown at 1000-fold magnification. TRAP
stainings (purple) are shown in the upper panels. Immunofluorescence staining for either Atg7 (A) or Beclin1 (B) (both green) are shown in the lower
panels. Scale bar: 10 μm, n=6 each. (C, D) Relative fluorescence intensity of Atg7 (C) or Beclin1 (D) was determined by Image J. **indicates
p<0.01. This figure is only reproduced in colour in the online version.
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inhibition of autophagy by genetic or pharmacologic approaches
prevented osteoclast differentiation. Osteoclast differentiation
was reduced in bone marrow cells derived from Atg7
fl/fl
×LysM
Cre
+
with decreased numbers of osteoclasts and decreased mean
size of osteoclasts as compared with Atg7
fl/fl
×LysM Cre
−
cells.
Similar results were obtained upon incubation of wildtype bone
marrow cells with bafilomycin, a chemical inhibitor of autop-
hagy (figure 3B,C).
To investigate whether activation of autophagy enhances the
potential of monocytes to differentiate into osteoclasts, we over-
expressed Beclin1 using a lentiviral construct. Overexpression of
Beclin1 potently induced autophagy in murine osteoclasts (see
online supplementary material figure S1A). Activation of autop-
hagy significantly increased the number of multinucleated,
TRAP-positive osteoclasts. In addition, the mean size of osteo-
clasts increased upon overexpression of Beclin1 (figure 3D).
We next evaluated whether modulation of autophagy alters
the expression of osteoclast-associated genes. Consistent with
the effects on osteoclast numbers and size, overexpression of
Beclin1 increased the mRNA levels of NFATc1, OSCAR, TRAP
and cathepsin K (figure 3E–G). By contrast, deficiency of Atg7,
or treatment with bafilomycin, a selective inhibitor of autop-
hagy, decreased the expression of osteoclast-associated genes.
To further elucidate the effects of autophagy on osteoclast
function, we performed in vitro bone resorption assays.
Activation of autophagy significantly enhanced the resorptive
capacity of murine osteoclasts, whereas genetic or pharmaco-
logic inhibition of autophagy dramatically reduced bone resorp-
tion in vitro (figure 4). To distinguish between effects mediated
by decreased osteoclast counts and effects of impaired osteo-
clast function, we increased the number of BMCs by twofold
to compensate for decreased osteoclast numbers due to
impaired osteoclastogenesis. After confirming equal numbers of
osteoclasts in all settings with this approach, we repeated the
bone resorption assays using those modified conditions. We
found that despite similar numbers of osteoclasts, bone respor-
tion was still decreased in Atg7
fl/fl
×LysM Cre
+
cells or cells
treated with Bafilomycin A1, respectively, as compared with
Atg7
fl/fl
×LysM Cre
−
and dimethyl sulfoxide-treated controls.
However, the differences were less pronounced then in previous
experiments, in which equal numbers of BMCs were seeded at
the beginning of the experiment. Together, those findings dem-
onstrate that decreased function and impaired differentiation
both contribute to the reduced bone resorbing capacity.
Local bone erosion and osteoclast numbe rs are decreased in
hTNFα tg mice transplanted with Atg7
fl/fl
×lysM Cre
+
BMCS
To determine the effects of autophagy on TNFα-induced arth-
ritis and bone destruction, hTNFα tg mice were transplanted
with bone marrow cells from Atg7
fl/fl
×LysMCre
+
mice to
selectively target monocytes in hTNFα tg model (Atg7
fl/fl
×
Cre
+
→hTNFα tg mice). We first confirmed that the depletion
of Atg7 occurred selectively in monocytes of Atg7
fl/fl
×LysM
Cre
+
, but not in other cell types, such as fibroblasts (see online
supplementary figure S1B). The effective inhibition of autop-
hagy in monocytic cells of Atg7
fl/fl
×LysM Cre
+
mice was
demonstrated by Western blots for Atg7, Beclin, LC 3 I and
LC3 II (see online supplementary figure S1C).
Figure 2 TNFα induces autophagy in osteoclasts in vitro and in vivo. (A) Representative Western blots of lysates from the mature osteoclasts
stimulated with receptor activator of NF-κB ligand (RANKL) (1 ng/ml), RANKL + TNFα (1 ng/ml, 40 ng/ml), RANKL + TNFα (1 ng/ml, 200 ng/ml) or
RANKL (30 ng/ml) using anti-Atg7, anti-Beclin1, anti-LC3 I, II, and anti-β-actin antibodies for detection (n = 3 for each). (B, C) Atg7 and Beclin1
mRNA levels in the osteoclasts were analysed by real-time PCR. n = 3 for each. (D, E) Representative images of joint sections from wildtype or
hTNFα tg mice double -stained with tartrate-resistant acid phosphatase (TRAP) and the autophagy markers Atg7 (D) or Beclin1 (E). TRAP staining for
osteoclasts ( purple) is shown in the upper panels of both figures. Immunofluorescence staining with Atg7 or Beclin1 (green) as indicated by arrows
is shown in the lower panels. Scale bar: 15 μm, n = 6 for each group. *indicates p 0.05 and **indicates p 0.01. This figure is only reproduced in
colour in the online version.
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Clinical signs of inflammation, such as paw swelling, grip
strength and body weight did not differ between Atg7
fl/fl
×
LysM Cre
+
→hTNFα tg mice and Atg7
fl/fl
×LysM Cre
−
→hTNFα
tg mice. We also did not observe differences in the inflamed
area by histomorphometry (figure 5), indicating that selective
inhibition of autophagy in monocytic cells does not affect joint
inflammation in TNFα-induced arthritis.
We next investigated whether targeting of autophagy affects
structural damage in hTNFα tg mice. Indeed, the osteoclast-
covered area and the number of osteoclasts was strongly
reduced in Atg7
fl/fl
×LysM Cre
+
→ hTNFα tg mice compared
with hTNFα tg mice reconstituted with Atg7
fl/fl
×LysM Cre
−
BMC (Atg7
fl/fl
×LysM Cre
−
→ hTNFα tg mice) (figure 6A,B).
The eroded area was also significantly reduced in Atg7
fl/fl
×LysM Cre
+
→ hTNFα tg mice (figure 6C). Consistently, mCT
demonstrated that Atg7
fl/fl
×LysM Cre
+
→ hTNFα tg were
largely protected from TNFα-induced bone destruction with
significantly reduced bone erosions (figure 6D).
We also analysed non-transgenic mice transplanted with
bone marrow from Atg7
fl/fl
LysM Cre
+
or Atg7
fl/fl
LysM Cre
−
mice. Consistent with the role of TNFα as a potent inducer of
autophagy, we did not observe differences in osteoclast
numbers or bone structure in non-TNFα transgenic mice (data
not shown).
Deficiency of Atg7 with subsequent inhibition of autophagy
prevented also TNFα-mediated proteoglycan loss as shown by
Toluidine blue staining and reduced numbers of empty lacunae
(figure 6E–H). To further elucidate the mechanism by which
LysM Cre driven knockdown of Atg7 may protect from cartil-
age loss, we analysed the expression levels of the monocyte/
macrophage-derived cytokines IL-1β and IL-6, both of which
have been implicated in cartilage destruction in arthritis. The
levels of IL-1β and IL-6 were both significantly decreased in
serum of Atg7
fl/fl
×LysM Cre
+
→ hTNFα tg mice compared
with Atg7
fl/fl
×LysM Cre
−
→ hTNFα tg mice (see online supple-
mentary figure S3). Thus, the decreased expression of IL-1β and
IL-6 may contribute to the observed protection of cartilage in
Atg7
fl/fl
×LysM Cre
+
→ hTNFα tg mice.
DISCUSSION
TNFα is a key player in the pathogenesis of RA that orches-
trates synovial inflammation and bone degradation. In the
Figure 3 Autophagy regulates receptor activator of NF-κB ligand-induced osteoclast differentiation. Activation of autophagy by lentiviral
overexpression of Beclin1 stimulates osteoclast differentiation, whereas, inhibition of autophagy, either by knockout of Atg7 or by treatment with
bafilomycin A1, prevents osteoclast differentiation. (A) Representative electron microscopy images of osteoclasts derived from Atg7
fl/fl
×LysMCre
−
BMCs or Atg7
fl/fl
×LysMCre
+
BMCs are shown at a 12 000-fold magnification. Scale bar: 0.5 μm. Note the mitochondrion engulfed in an
autophagosome with typical double membrane in osteoclasts derived from Atg7
fl/fl
×LysMCre
−
and electron-dense immature autophagic vacuoles in
BMCs derived from Atg7
fl/fl
×LysMCre
+
.(B–D) Representative images of tartrate-resistant acid phosphatase (TRAP) stainings from in
vitro-differentiated osteoclasts derived from BMCs derived from Atg7
fl/fl
×LysMCre
−
or Atg7
fl/fl
×LysMCre
+
mice (B), treated with either Bafilomycin
A1 or its solvent DMSO (C), or infected with lentiviral Beclin1, or the control scramble lentivirus (D), respectively, are shown (n=5 for each setting)
at a 100-fold magnifi cation. The relative number of osteoclasts defined as TRAP-positive cells with more than three nuclei in each setting are shown
in the graphs in the middle. The relative area covered by osteoclasts (mean from 100 osteoclasts per setting) is presented in the panels on the right.
(E–G) Quantification of mRNA levels of NFATc1, OSCAR, TRAP and CatK in indicated osteoclasts derived from BMCs as well as previously described
(B, C and D, respectively). n=5 For each group in each setting.
*
indicates p<0.05; **indicates p<0.01. This figure is only reproduced in colour in
the online version.
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