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ARTICLE OPEN
IκB-ζsignaling promotes chondrocyte inflammatory
phenotype, senescence, and erosive joint pathology
Manoj Arra
1
, Gaurav Swarnkar
1
, Yael Alippe
2
, Gabriel Mbalaviele
2
and Yousef Abu-Amer
1,3
✉
Osteoarthritis is a joint disease characterized by a poorly-defined inflammatory response that does not encompass a massive
immune cell infiltration yet contributes to cartilage degradation and loss of joint mobility, suggesting a chondrocyte intrinsic
inflammatory response. Using primary chondrocytes from joints of osteoarthritic mice and patients, we first show that these cells
express ample pro-inflammatory markers and RANKL in an NF-κB dependent manner. The inflammatory phenotype of
chondrocytes was recapitulated by exposure of chondrocytes to IL-1βand bone particles, which were used to model bone matrix
breakdown products revealed to be present in synovial fluid of OA patients, albeit their role was not defined. We further show
that bone particles and IL-1βcan promote senescent and apoptotic changes in primary chondrocytes due to oxidative stress from
various cellular sources such as the mitochondria. Finally, we provide evidence that inflammation, oxidative stress and senescence
converge upon IκB-ζ, the principal mediator downstream of NF-κB, which regulates expression of RANKL, inflammatory, catabolic,
and SASP genes. Overall, this work highlights the capacity and mechanisms by which inflammatory cues, primarily joint
degradation products, i.e., bone matrix particles in concert with IL-1βin the joint microenvironment, program chondrocytes into
an “inflammatory phenotype”which inflects local tissue damage.
Bone Research (2022) 10:12 ; https://doi.org/10.1038/s41413-021-00183-9
INTRODUCTION
Osteoarthritis (OA) is the most prevalent joint disease worldwide,
causing pain, decreased quality of life, and reduced productivity
in those affected by this condition.
1–3
However, current OA
treatment regimens do not contain any disease-modifying
therapeutics but instead rely upon symptom-reducing com-
pounds, though promising advancements have been made in
the field. Given the prevalence of this disease, novel targets and
therapeutics are greatly needed to combat the impact of OA on
patients and society.
While OA was traditionally considered as non-inflammatory
arthritis, extensive work has displayed the role of inflammation in
OA.
4–7
Inflammation is induced by many processes, ranging from
mechanical stress to stimulation by inflammatory cytokines, all of
which play a role in the initiation and propagation of this
disease.
8,9
It is likely that OA is induced by various acute
inflammatory insults throughout life, such as mechanical injury,
which then contribute to a sustained chronic, inflammatory state
as patients grow older. The response of articular chondrocytes to
these inflammatory stimuli contributes to the development and
progression of OA by promoting the expression of catabolic
factors that break down ECM that normally gives cartilage its joint
protective properties. Furthermore, the role of senescent chon-
drocytes in articular cartilage as a source of pro-inflammatory
factors has been highlighted in recent years,
10–13
primarily due to
the production of senescence-associated secretory phenotype
(SASP) factors that are pathogenic. This can lead to a positive
feedback cycle of chronic inflammation through autocrine and
paracrine effects. Although it is still unclear what drives
chondrocyte senescence, some possibilities include factors such
as oxidative stress, microRNAs, and inflammatory pathways.
14–16
Oxidative and nitrosative stresses have especially gained interest
because they can arise from multiple components within the cell
and are intricately related to inflammation and cellular metabo-
lism.
17
Reactive oxygen species such as superoxide can damage
DNA, proteins, and lipids to alter cellular physiology and promote
pathology. It is predicted that prevention of senescence or
reduction of SASP expression from senescent chondrocytes, which
overlaps with the inflammatory response, may be important for
reducing the chronic inflammatory state of OA joints and prevent
OA progression.
18
The role of cartilage and bone degradative by-products in OA
disease pathology is also an area that requires further research.
The presence of calcium crystals and other by-products of bone
and ECM degradation has been noted in the synovial fluid of OA
joints, with some groups suggesting that these particles are pro-
pathogenic while others suggest that they are merely by-products
of disease.
19–21
The source of these particles is still unknown,
though it is predicted to be from a combination of cartilage
calcification by hypertrophic chondrocytes, bone erosion of juxta-
articular regions, or bone-on-bone erosions.
22
Even in the setting
of bone-on-bone erosion or internal knee injuries that release
bone fragments, cartilage remains present in the joint, and
understanding the function of these complex bone particles (BP)
on the remaining joint health needs to be elucidated, especially if
future therapies aim to focus on cartilage regeneration. One of the
major findings in OA patients is the alteration of subchondral
bone physiology and structure, leading to the prediction that
Received: 29 May 2021 Revised: 26 October 2021 Accepted: 29 October 2021
1
Department of Orthopaedic Surgery and Cell Biology & Physiology, Washington University School of Medicine, St. Louis, MO 63110, US;
2
Bone and Mineral Division, Department
of Medicine, Washington University School of Medicine, St. Louis, MO 63110, US and
3
Shriners Hospital for Children, St. Louis, MO 63110, US.
Correspondence: Yousef Abu-Amer (abuamery@wustl.edu)
www.nature.com/boneres
Bone Research
©The Author(s) 2022
1234567890();,:
interaction between articular cartilage and subchondral bone cells
is important for OA pathogenesis.
23–25
Subchondral bone sclerosis
and osteophyte formation late in OA is characteristic of the
disease, though it is unclear if early OA has similar findings and
should be further explored. It is possible that altered catabolism of
subchondral bone may be a contributor to the presence of crystals
in the synovial space, especially by osteoclast activity promoted by
chondrocytes.
26
Previous reports indicate that matrix fragments
and crystals can be pathological, with some animal studies
suggesting that these particles contribute to the chronic
inflammatory state of OA synovial joints.
27–31
The goal of this work is to explore the effects of pathogenic
molecular entities and associated inflammatory signals, such as
BP and IL-1β, on chondrocytes and the downstream mediators
involved. In this work, we display that chondrocytes can support
osteoclast formation in response to inflammatory stimuli through
increased Rankl production. Furthermore, we show that bone
matrix particles are highly pro-inflammatory and can synergize
with inflammatory cytokines to drive catabolic enzyme expres-
sion in an NF-κB dependent manner. We also display that
inflammatory stimuli can promote senescent and apoptotic
changes. Finally, we show that BP and IL-1βact via IκB-ζto
promote SASP expression in a manner dependent upon oxidative
and nitrosative stresses.
RESULTS
OA joints have increased osteoclast activity and Rankl production
by chondrocytes
It has been noted that joints likely undergo erosive changes
during the early stages of OA, followed by subchondral sclerosis in
advanced stages.
32
We surmised that the local joint microenvir-
onment supports bone and cartilage erosion through elevated
osteoclast activity, contributing to subchondral changes. Hence,
we set out to determine if OA chondrocytes in humans and
murine models display increased receptor activator of NF-κB
ligand (Rankl) expression, the critical factor required for osteo-
clastogenesis. We utilized the well-established meniscal ligamen-
tous injury (MLI) model to provoke experimental OA in mice
33
and
observed that cartilage from MLI joints after surgery had elevated
levels of Rankl (Tnfsf11) mRNA expression compared to sham
surgery joints (Fig. 1a). We also observed similar changes in
human OA cartilage when comparing more damaged medial
cartilage to relatively healthy lateral cartilage in patients with
medial compartment OA (Fig. 1b). Since we have recently shown
that inflammation is a significant driver of OA joint degradation,
34
we treated mouse-derived chondrocytes with IL-1β, a major
inflammatory mediator and marker of OA. We observed that
exposure of chondrocytes to IL-1βsignificantly increased the
expression of Rankl mRNA and Rankl protein secretion (Fig. 1c, d).
Furthermore, we have previously demonstrated in articular
chondrocytes that NF-κB activation, the principle inflammatory
response pathway, is a critical component of OA pathogenesis
34
and predicted that the increase in Rankl expression is mediated
via NF-κB activation. Confirming this proposition, we show that
NF-κB activation using the constitutively active form of IKK2
(IKK2ca) also drives Rankl expression mimicking inflammatory
stimuli (Supplementary Fig. S1a). This was further validated using
the IKK2 inhibitor, SC-514, which reduced Rankl expression in IL-
1β-treated chondrocytes (Fig. 1c). Since osteoprotegerin (Opg) is
also an important counterpart to Rankl in modulating osteoclas-
togenesis by negatively regulating osteoclast formation, we
sought to determine the Rankl:Opg ratio.
35
We observed that IL-
1βtreatment decreased Opg mRNA expression (Supplementary
Fig. S1b) and greatly increased the Rankl:Opg ratio but the
application of IKK2 inhibitor reduced the Rankl:Opg ratio through
both reduction in Rankl and increase in Opg mRNA expression
(Supplementary Fig. S1c).
Consistent with these observations, we predicted that chon-
drocytes may be an important source of Rankl near damaged
cartilage. We first tested whether the presence of Rankl in the
synovial fluid can promote subchondral osteoclast formation.
Recombinant Rankl was injected into the synovial space by intra-
articular injection, with PBS injection used as control. Rankl
injection into the synovial space led to a dramatic increase in
osteoclasts in the subchondral region (Supplementary Fig. S1d, e).
We then show that post-surgery MLI joints displayed increased
tartrate-resistant acid phosphatase (TRAP)-positive staining in the
anterior femoral subchondral bone region compared to contral-
ateral sham joints, indicating increased osteoclast activity and
altered bone resorption (Fig. 1e, f). However, we did not observe
statistically significant differences in osteoclast number in the
tibial regions or posterior femoral regions. When analyzed by μCT,
MLI joints in mice (Supplementary Fig. S1g) at 2 weeks post-
surgery have increased bone erosion and subchondral bone loss
compared to sham joints as measured by subchondral bone
volume (Fig. 1g, h, Supplementary Fig. S1f). We also utilized the
Aggrecan-ERT2-cre IKK2ca (Acan
IKK2ca
) mouse model, which
displays IKK2 activation in articular chondrocytes, that we have
previously shown can drive cartilage degradation and mimic an
OA-like phenotype.
34
These joints also have decreased joint
spacing and blunting of the bony surfaces in the subchondral
region similar to post-traumatic OA even in the absence of
mechanical injury (Supplementary Fig. S1i). This suggests that
chondrocyte-specific NF-κB activity can cause bone degradation
and modify the subchondral region, potentially via Rankl
expression and altered mechanical properties of the joint due to
cartilage degradation.
To substantiate our observations, we sought to determine if
chondrocytes can directly promote osteoclast formation and
regulate bone degradation. We utilized a chondrocyte-
macrophage co-culture system to allow for the interaction of
chondrocyte-secreted factors with bone marrow macrophages to
determine the effect on osteoclast differentiation. At basal
conditions, chondrocytes co-cultured with macrophages did not
increase the presence of TRAP positive cells compared to
macrophages in culture alone since chondrocytes produce
significant amounts of Opg under physiologic conditions.
26
Given
that chondrocytes treated with IL-1βhad increased expression of
Rankl, we then asked if under inflammatory conditions, chon-
drocytes could support and promote osteoclastogenesis. We show
that macrophage-chondrocyte co-cultures treated with IL-1βhad
increased TRAP positive cells compared to those without IL-1βin
the presence of permissive levels of exogenous Rankl, which was
added to prime commitment of monocyte/macrophage into OC
precursors (Fig. 2a, b). We confirmed this to be a chondrocyte-
mediated effect by comparing co-cultures using IKK2 deficient
chondrocytes treated with IL-1βand Rankl, which displayed
significantly less TRAP positive multi nucleated cells (Fig. 2a, b;
right panels). This finding suggests that IKK2 mediates the IL-1β
inflammatory response of chondrocytes to produce Rankl and
enhance osteoclastogenesis (Fig. 2a, b). Mirroring these findings,
we observed that chondrocyte-macrophage co-cultures had
significantly higher TRAP expression when treated with IL-1β
compared to macrophages treated with IL-1βalone, suggesting
that chondrocytes can support macrophage differentiation into
osteoclasts under inflammatory conditions (Fig. 2c).
Bone tissue particles are potent pro-inflammatory molecules that
act in an NF-κB dependent manner
We then proposed that joint pathology is exacerbated by cartilage
and bone mineral particles, released into the synovial space from
various factors such as cartilage calcification, osteoclast resorption,
and mechanical cartilage-bone erosion, which could act as pro-
inflammatory mediators. These inert particles likely act in a pro-
inflammatory manner to drive cartilage degradation based on
IκB-ζpromotes inflammatory chondrocytes and senescence
M Arra et al.
2
Bone Research (2022) 10:12
findings from prior work studying inert crystals.
27,36
Given the
principal role of NF-κB as the major inflammatory response
pathway, we predicted that bone matrix particles would activate
this pathway. Since mineralized bone degradation products
cannot be isolated from the joint fluid of mouse joints due to the
small volume, we used BP as surrogates for proof-of-concept
studies. BP were generated by grinding the cortical surface of
porcine long bones, rinsed repeatedly in sterile PBS, and stored in
10
a
e
gh
f
Subchondral bone osteoclasts
bc d
Sham
Sham
40 Pm
Anterior tibiaAnterior femur
BV/TV
Lateral Untreated
Untreated
IL-1E
IL-1E+ IKK2i
IKK2i
mRankI hRANKL RankI RankI
MLI
MLI
Sham posterior tibia
MLI posterior tibia
Sham anterior tibia
MLI anterior tibia
Sham posterior femur
MLI posterior femur
Sham anterior femur
MLI anterior femur
6
4
2
0
0.75
0.70
0.65
0.60
0.55
0.50
Sham
Sham MLI
MLI
Subchondral bone density Subchondral bone
Relative mRNA expression
Relative mRNA expression
Relative mRNA expression
Rankl expression/(pg·mL-1)
8
10 50 20
15
10
5
0
40
30
20
10
0
15
10
5
0
Number of osteoclasts
6
4
2
0
8
Medial IL-1E
*
*
**** ***
***
***
Fig. 1 Chondrocytes produce Rankl under inflammation. aMLI surgery was performed on 12-week-old mice. After 4 weeks, articular cartilage
was isolated from sham and MLI knee joints and gene expression of Rankl was measured (n=4, *P=0.050). Data are mean ± SEM. bMedial
and lateral knee articular cartilage was isolated from patients undergoing TKA with medial compartment OA. Gene expression of human
RANKL comparing more damaged medial cartilage to lateral cartilage (n=5, *P=0.031). Data are mean ± SEM. cPrimary chondrocytes were
cultured with IL-1β(10 ng·mL
−1
) ± SC-514 (10 μmol·L
−1
) for 24 h. Rankl gene expression was measured (Untreated vs IL-1β***P=0.000 2, IL-1β
vs IL-1β+IKK2i ***P=0.000 7), with data representing mean ± SEM of n=4 independent experiments. dRankl protein expression in IL-1β
treated (24 h) chondrocytes. Data are mean ± SD for n=2 representative replicates (***P=0.003 3). eTwo weeks post MLI, knee joints were
fixed and sectioned for TRAP staining to identify osteoclasts in the subchondral region (arrows). fTRAP positive cells were counted in anterior
and posterior compartments of femur and tibia in MLI and Sham joints (n=8). (Sham Anterior Femur vs MLI Anterior Femur ***P=0.002). Bars
represent mean ± S.D. gMLI and sham surgeries were performed on right and left mouse knees, respectively. After 2 weeks, mice were
sacrificed and joints were harvested and fixed. Bone volume/total volume of subchondral region was measured by uCT in same region of each
mouse tibia (n=5 mice) (*P=0.036 5). hRepresentative image of subchondral slice from sham and MLI mouse joints displaying decreased
bone volume (M and L indicate medial and lateral condyle, respectively)
IκB-ζpromotes inflammatory chondrocytes and senescence
M Arra et al.
3
Bone Research (2022) 10:12
70% ethanol. These BP were then washed again with PBS before
addition to cultures. We established that, similar to IL-1β, treatment
of chondrocytes with BP potently activates NF-κB(Fig.3a). BP were
also able to activate expression of pathological inflammatory
response genes including IL-6 and MMP13 (Fig. 3b, c). Furthermore,
we show that BP are able to synergize with constitutively active
IKK2 (IKK2ca) to increase the expression of Il-6,Mmp13 and Adamts4
(Supplementary Fig. S2a–c) akin to exacerbated disease conditions,
relative to IKK2ca alone. To substantiate our claim that BP act via
NF-κB, we tested if inhibition of NF-κB would be sufficient to
prevent the inflammatory response induced by BP. We showed that
genetic ablation of IKK2, the major NF-κB activating kinase, in
chondrocytes led to significant reduction in inflammatory gene
expression with BP treatment (Fig. 3b, c). We also utilized a kinase-
dead form of IKK2 (IKK2-KD) that has a dominant-negative effect to
show that it was able to prevent the inflammatory response
inducedbyBPbypreventingsignaltransductionthroughIKK2
(Supplementary Fig. S2d–f). Overall, these results suggest that BP
can promote a strong inflammatory response causing catabolic
changes in an NF-κB dependent manner.
Given the potent role of NF-κB in the IL-1βand BP inflammatory
response, we sought to define if the anti-inflammatory effect of
IKK2 inhibition observed in vitro could be translated in vivo to
protect against OA. We injected mice with IKK2 inhibitor (IKK2i)
systemically for 6 weeks post MLI surgery and observed significant
increase in cartilage integrity and improved OARSI scoring in MLI
joints of IKK2i-treated animals (Fig. 3d, e). In addition, there was
significantly decreased staining for MMP13 in the articular
cartilage of IKK2i-treated MLI joints, indicating decreased catabolic
activity (Fig. 3f; arrows).
WT
Chondrocytes
WT macrophages
alone + RL
WT macrophages
alone + RL + IL-1β
WT macrophages
+ IL - 1E + RL
WT macrophages
+ RL
IKK2-/-
Chondrocytes Osteoclastogenesis
***
**
Trap (Acp 5)
ab
c
50
MNC per well
40
30
20
10
0
WT
WT + IL-1β
IKK2-/-
IKK2-/- + IL-1β
200 Pm
200 Pm
6
4
2
0
Chondrocyte + Macrophage
Chondrocyte + Macrophage + IL-1E
Macrophage + IL-1E
Macrophage
Relative mRNA expression
Fig. 2 Chondrocytes support osteoclastogenesis under inflammatory conditions. aPrimary IKK2
f/f
chondrocytes were isolated and infected
with adenoviral-GFP or adenoviral-cre to delete IKK2. Bone marrow macrophages were then added to the culture. Permissive levels of Rankl
(5 ng·mL
−1
) was added ± IL-1β(10 ng·mL
−1
) for 3 days. Cultures were fixed and stained for TRAP positive osteoclasts (red arrows).
Macrophages alone cultures are displayed as control. bNumber of large, multi nucleated cells (MNC) in macrophage-chondrocyte co-culture
were quantified by counting. (WT +IL-1βvs IKK2
−/−
+IL-1β***P=0.000 2, n=4 wells from representative experiment). Bars represent mean
± S.D. cmRNA was isolated from co-cultures of chondrocytes and macrophages ± IL-1β. Gene expression analysis was performed for TRAP
(**P=0.001 7, n=4). Bars represent mean ± S.D.
IκB-ζpromotes inflammatory chondrocytes and senescence
M Arra et al.
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Bone Research (2022) 10:12
Bone particles activate NLRP3 inflammasome and IκB-ζin
chondrocytes
We sought to further characterize the mechanism(s) underlying
the response to BP exposure to identify potential therapeutic
targets. Given that BP are not a signaling protein per se, we
suspected that they may act as damage-associated molecular
pattern (DAMP) molecules to stimulate the expression of
inflammatory genes including Nlrp3, which assembles an inflam-
masome involved in the maturation of IL-1β, as well as the
regulation of various intracellular processes such as apoptosis.
37
We observed that BP treatment increases expression of Nlrp3 at
both the gene and protein level (Fig. 4a, b). Furthermore,
damaged human cartilage has elevated levels of NLRP3 gene
expression compared to healthier cartilage (Fig. 4c), suggesting
10 000 100
Relative mRNA expression
25
4
Treatment
Average OARSI score
Sham + Vehicle
Sham + IKK2i
MLI + Vehicle
MLI + IKK2i
3
2
1
0
20
15
10
5
0
80
60
40
20
0
Relative luminescence
Relative mRNA expression
NF-kB activity
SHAM
SHAM MLI
IHC:MMP13
VehicleIKK2 inhibitor
MLI
100 Pm
50 Pm
Tibial OARSI
abc
d
f
e
II-6 Mmp13
****
****
****
****
****
**
8 000
6 000
4 000
2 000
VehicleIKK2 inhibitor
0
Untreated
GFP
IKK2-/-
GFP
IKK2-/-
GFP + BP
GFP + BP
IKK2-/- + BP
IKK2-/- + BP
BP
IL-1E
IL-1E+ BP
Fig. 3 Bone particles drive the production of catabolic genes via NF-κB. aChondrocytes from NF-κB luciferase reporter mice were cultured
with BP or IL-1βfor 24 h. Luciferase assay was performed to measure NF-κB activation (Untreated vs IL-1β****P< 0.000 1, Untreated vs BP
**P=0.001, n=4 independent replicates). Results are mean ± SD from one of three representative experiments. b,cIKK2
−/−
chondrocytes
were transduced with adeno-GFP or adeno-cre. Cells were then cultured with BP for 24 h. Il-6 and Mmp13 gene expression was measured (IL-6:
GFP +BP vs IKK2
−/−
+BP
****
P< 0.000 1. MMP13: GFP +BP vs IKK2
−/−
+BP ****P< 0.000 1). Results are mean ± SD for n=4 replicates. dMLI
surgery was performed on 12-week-old mice. IKK2 inhibitor was injected I.P. for 6 weeks and joints were harvested for histology to perform
safranin-O staining, with most damage to tibial surface (arrows). Representative images are displayed. eBlinded OARSI scoring was performed
to grade OA severity on the tibial surface (Sham +Vehicle vs MLI +Vehicle ****P< 0.000 1, MLI +Vehicle vs MLI +IKK2i ****P< 0.000 1). Bars
represent mean ± SEM. from n=4 vehicle treated and n=3 inhibitor treated mice. fIHC was performed for Mmp13 in articular cartilage of
joint sections under the same conditions. Representative images are displayed
IκB-ζpromotes inflammatory chondrocytes and senescence
M Arra et al.
5
Bone Research (2022) 10:12
15
2.5 40
30
20
10
0
10
8
6
4
2
0
GFP
GFP + BP
IKK2ca
IKK2ca + BP
Relative mRNA expression
Relative mRNA expression
Relative mRNA expression
Relative mRNA expression
Relative mRNA expression
Relative mRNA expression
Relative mRNA expression
Relative mRNA expression
Relative mRNA expression
IL-1E/(pg·mL-1)
Untreated Untreated
2.0
1.5
1.0
0.5
0.0
60
**** ****
****
**
****
**** ****
****
**** ****
*
50 30
20
10
Untreated BP
BP
Actin
–
––
+
+
+
+
–
IkB-ζ
IL-1E
0
40
30
20
10
0
40
20
0
2 500 50 100
5
0
10
15
80
60
40
20
0
40
30
20
10
0
2 000
1 500
1 000
500
0
GFP
Nfkbiz-/-
GFP + BP
Nfkbiz-/- + BP
GFP + BP + IL-1E
Nfkbiz-/- + BP + IL-1E
GFP
Nfkbiz-/-
GFP + BP
Nfkbiz-/- + BP
GFP + BP + IL-1E
Nfkbiz-/- + BP + IL-1E
GFP
Nfkbiz-/-
GFP + BP
Nfkbiz-/- + BP
GFP + BP + IL-1E
Nfkbiz-/- + BP + IL-1E
GFP
Nfkbiz-/-
GFP + BP
Nfkbiz-/- + BP
GFP + BP + IL-1E
Nfkbiz-/- + BP + IL-1E
WT
WT + BP
NLRP3-/-
NLRP3-/- + BP
WT
WT + BP
NLRP3-/-
NLRP3-/- + BP
***
***
10
5
Untreated
Bone particles
0
Relative mRNA expression
Nlrp3
Actin
0 1 6 16 28 48/h
Lateral
4P=0.06
NLRP3
3
2
1
0
Medial
Relative mRNA expression
Il-1expression
II-6Mmp13 Nfkbiz
NIrp 3
II-6Mmp13 Nlrp3RankI
Il-1expression
ab c
de f
g
klmn
hij
IL-1EBP
Fig. 4 Bone particles promote inflammation via NLRP3 and IκB-ζactivity. aPrimary chondrocytes were cultured with BP for 24 h and Nlrp3 gene
expression was measured (***P=0.000 9). Bars represent mean ± SEM from n=4 independent experiments. bImmunoblotting was performed
for Nlrp3 protein using chondrocytes lysates under BP treatment time course, with actin loading used as control. The representative image is
shown. cMedial and lateral knee articular cartilage was isolated from patients undergoing TKA with medial compartment OA. Gene expression
analysis was performed for NLRP3. Bars represent mean ± S.D. for n=6 samples. d,eCells were treated with IL-1βor BP for 24 h and IL-1βgene
expression was measured. Bars are mean ± SD from three separate experiments. (*P=0.024 8). fCells were retrovirally transduced with GFP or
IKK2ca, then treated with bone particles for 24 h. Supernatant was collected and ELISA was performed for IL-1βlevels (GFP vs IKK2ca +BP **P=
0.001 8, n=4 replicates). g,hChondrocytes isolated from WT or Nlrp3-deficient mice and treated with BP for 24 h. Il-6 and Mmp13 gene
expression measured by qPCR (IL-6:WT+BP vs Nlrp3
−/−
+BP ****P< 0.000 1, Mmp13: WT +BP vs Nlrp3
−/−
+BP
****
P< 0.000 1). Bars are mean
±SDforn=4 replicates. iPrimary chondrocytes were treated with BP for 24 h. Nfkbiz gene expression was measured by qPCR (*P=0.018 7).
Bars are mean ± SEM for n=4 independent experiments. jChondrocytes were exposed to IL-1βand/or bone particles. Lysates were collected
and Immunoblotting was performed for IκB-ζ. Representative immunoblot is shown. k–nNfkbiz flox chondrocytes were transduced with adeno-
GFP (WT ) or adeno-cre (Nfkbiz
−/−
). Cells were then treated with BP ± IL-1β. Gene expression of Il-6,Mmp13,Nlrp3 and Rankl was measured by
qPCR (IL-6: GFP +BP vs Nfkbiz
−/−
+BP **P=0.002 9, GFP
+
BP +IL-1βvs Nfkbiz
−/−
+BP +IL-1β****P< 0.000 1. Mmp13: GFP +BP vs Nfkbiz
−/−
+
BP ****P< 0.000 1, GFP +BP +IL-1βvs Nfkbiz
−/−
+BP +IL-1β****P< 0.000 1. Nlrp3: GFP +BP vs Nfkbiz
−/−
+BP ****P< 0.000 1, GFP +BP +IL-1β
vs Nfkbiz
−/−
+BP
+
IL-1β****P< 0.000 1. Rankl:GFP+BP vs Nfkbiz
−/−
+BP
****
P< 0.000 1, GFP +BP +IL-1βvs Nfkbiz
−/−
+BP +IL-1β****P<
0.000 1). Bars represent mean ± S.D. from one representative experiment out of three
IκB-ζpromotes inflammatory chondrocytes and senescence
M Arra et al.
6
Bone Research (2022) 10:12
that the NLRP3 inflammasome may be involved in the human
disease.
We then displayed that IL-1βand BP treatment can both drive
gene expression of IL-1βitself (Fig. 4d, e). The ability of IL-1βto
induce its own expression suggests a feedforward pro-
inflammatory mechanism by which chondrocytes can propagate
inflammation in a paracrine or even autocrine manner. In
agreement with our hypothesis that BP can act as DAMP signals
that promote Nlrp3 activity, we observed that BP treatment of
chondrocytes expressing IKK2ca significantly increased chondro-
cyte secretion of IL-1β, whereas individual treatment of chon-
drocytes with BP or IKK2ca expression alone is not a potent
activator of IL-1βsecretion (Fig. 4f). This suggests that NF-κB
activation likely acts as a primary signal to promote Nlrp3
expression while BP act as the secondary signal for Nlrp3
assembly and activation. We also observe that Nlrp3-deficient
chondrocytes have reduced Il-6 and Mmp13 expression upon
bone particle treatment (Fig. 4g, h).
However, given that the inhibition of inflammatory gene
expression resulting from Nlrp3 deletion was only partial, we
sought to identify a factor further upstream that can be a more
potent activator of this inflammatory response. Our previous work
has displayed that incubation of chondrocytes with IL-1β
increased gene expression of Nfkbiz,
34
, which encodes for IκB-ζ,
a pro-inflammatory mediator.
38
Furthermore, we have recently
shown that MLI joints in mice and damaged cartilage from
humans have elevated expression of Nfkbiz in the articular
cartilage.
29
Our work, and that of others, has displayed that IκB-
ζis a prominent pro-inflammatory secretome in OA chondrocytes
and is necessary for the expression of various inflammatory and
catabolic genes.
39
To this end, treatment of chondrocytes with BP
increased the expression of Nfkbiz at the gene level and IκB-ζat
the protein level (Fig. 4i, j). Combined BP and IL-1βtreatment
further increased protein levels of IκB-ζ(Fig. 4j). Supporting its
direct role in this inflammatory response, treatment of Nfkbiz
−/−
chondrocytes with BP failed to elicit a meaningful inflammatory
response compared to wild type chondrocytes, even in the
presence of an intact NF-κB signaling pathway (Fig. 4k, l). In
addition, we observed that deletion of IκB-ζdecreased expression
of Nlrp3 (Fig. 4m), indicating that IκB-ζis upstream of inflamma-
some expression and activation. We also observed that deletion of
IκB-ζdecreased the expression of Rankl by BP or IL-1β-stimulated
chondrocytes (Fig. 4n). Collectively, these findings suggest that
under pathologic conditions, IκB-ζappears to centrally control the
expression of inflammatory, catabolic and osteoclastogenic factors.
Inflammatory stimuli promote chondrocyte senescence
Recent reports have proposed that chondrocyte senescence
plays a crucial part in OA pathogenesis
11,40,41
and we suspected
that inflammatory stimuli may contribute to chondrocyte
senescent transformation. Thus, we screened RNA sequencing
dataset from IL-1β-treated chondrocytes to determine how
inflammatory stimuli affect gene expression, wherein we
observed that apoptotic and senescence pathways are signifi-
cantly altered (Table 1).
Next, we examined if inflammatory stimuli were responsible for
promoting cellular senescence. BP or IL-1βtreatment increased
gene expression of cell cycle inhibitors p16 and p21, which are
well-established senescence markers (Fig. 5a–d). We focused
further on p16 since it has been shown to be important in
senescent OA chondrocytes, made evident by the protection of
p16-deficient mice against experimental OA development.
42,43
Gene expression findings were supported by increased protein
levels of p16 upon both IL-1βand BP treatment (Fig. 5e). We also
observed increased gene expression of P16 in damaged compared
to undamaged human OA cartilage (Fig. 5f), and increased staining
for p16 in OA cartilage (medial condyle) compared to healthy
cartilage (lateral condyle) (Fig. 5g). In addition, we show that IL-1β
and BP promote the expression of SASP genes Il-6, Lcn2, and
Mmp13 (Supplementary Fig. S3a–f). Furthermore, we show that
treatment with a well-established senolytic combination of
dasatinib and quercetin (D+Q)
44
was able to significantly reduce
expression of SASP factors Il-6, Mmp13, and Lcn2 induced by IL-1β
or BP treatment (Supplementary Fig. S3a–f). D +Qcombination
was also able to partially reverse the decreased expression of
aggrecan caused by inflammatory stimuli (Supplementary Fig. S3g).
Primers used for all genes are listed in Table 2.
Suspecting that the senescent shift was occurring due to NF-κB
activity, we show that chondrocytes expressing IKK2ca also had
increased expression of p16 (Fig. 5h). In addition, mice expressing
IKK2ca in chondrocytes (Acan
IKK2ca
) display increased p16 expres-
sion in the articular cartilage compared to wild type mice (Fig. 5i).
Validating these findings, inhibition of NF-κB through deletion of
IKK2 was able to decrease the expression of p16 and p21 in
response to BP stimulation (Fig. 5j, k). This effect was also seen at
the protein level, where deletion of IKK2 almost completely
inhibited expression of p16 (Fig. 5e). This suggests that NF-κB
activity is at least partially a driver of p16-mediated senescent
changes in addition to acting as a driver of SASP gene expression.
Since deletion of NF-κB is often detrimental to cell survival, we
compared the transcriptome of IL-1β-treated IκB-ζdeficient
(Nfkbiz
−/−
) chondrocytes to IL-1β-treated wild type chondro-
cytes
34
and observed that IκB-ζdeletion decreased expression of
many SASP factors associated with senescent chondrocytes
(Fig. 6a). We then validated these results by qPCR for SASP
genes such as Lcn2 and Nos2 to display that IκB-ζis the principal
mediator of inflammation-induced chondrocyte SASP expression
downstream of NF-κB(Fig.6b, c). However, IκB-ζdeletion did not
modulate expression of senescence markers, such as p16 and p21
(Supplementary Fig. S3h, i). Taken together, our data display that
IκB-ζis the central inflammatory mediator responsible for SASP,
while NF-κB separately regulates both SASP and senescent
marker changes.
Inflammatory stimuli promote oxidative stress in chondrocytes to
express IκB-ζand SASP
We then sought to identify a therapeutic target that was driving
the expression of IκB-ζ. We have previously shown that IκB-ζis
redox sensitive, with increased cellular ROS causing higher
protein expression of IκB-ζ
34
and sought to determine if IL-1β
Table 1. Chondrocytes were treated with IL-1βfor 24 h. RNA
sequencing was performed and significantly altered pathways are
displayed
Pathway P-value
Regulation of cell proliferation 1.55E-13
Cellular response to DNA damage stimulus 1.23E-12
Positive regulation of apoptotic process 2.63E-10
Apoptotic process 8.49E-09
Intrinsic apoptotic signaling pathway in response to
DNA damage
1.17E-06
Regulation of apoptotic process 3.66E-06
Intrinsic apoptotic signaling pathway in response to
oxidative stress
8.66E-05
Extrinsic apoptotic signaling pathway 2.46E-04
Positive regulation of cell death 1.63E-03
Positive regulation of cell cycle arrest 3.54E-03
Apoptotic mitochondrial changes 8.60E-03
Cellular senescence 1.49E-02
Replicative senescence 3.90E-02
IκB-ζpromotes inflammatory chondrocytes and senescence
M Arra et al.
7
Bone Research (2022) 10:12
5
a
ef
g
h
jk
i
-
bc d
**
*
*
***
** **
Relative mRNA expression
Relative mRNA expression
Relative mRNA expressionRelative mRNA expression
Relative mRNA expression
Relative mRNA expression
Relative mRNA expression
p16
p16
–––
––––
–– – –
–++
+++
++
+
++
++
BP
IL-1E
Actin
IKK2
-/-
p16
p16
p16 p21
p21 p21p16
10
2.5
2.0
1.5
1.0
0.5
0.0
3
2
1
0
GFP
IKK2-/-
GFP + BP
IKK2-/- + BP
GFP
IKK2-/-
GFP + BP
IKK2-/-+ BP
GFP IKK2ca
61.8
1.6
1.4
1.2
1.0
0.8
IHC:p16
WT Acan
IKK2ca
Lateral
Lateral
Medial
Medial
IB: p16-ink4a
4
5
4
3
2
2.5
2.0
1.5
1.0
0.5
0.0
1
0
2
0
Untreated
8
6
4
2
0
4
3
2
1
0
Untreated
Bone particles
Untreated
Bone particles
IL-1EUntreated IL-1E
100 Pm
Fig. 5 Inflammatory mediators promote senescent changes in chondrocytes. a–dChondrocytes were exposed to BP or IL-1βfor 24 h. Gene
expression analysis was performed for p16 or p21 (A: p16:*P=0.051 1. B: p21:*P=0.022 9. C: p16:**P=0.009 6. D: p21 **P=0.003 9). Bars
represent mean ± SEM for n=5 independent experiments.eAdeno-GFP or adeno-cre were transduced into IKK2
−/−
chondrocytes. Cells were
then treated with BP and/or IL-1βfor 24 h. After 24 h, protein lysates were collected and immunoblotting was performed for p16-Ink4a.
Representative immunoblot image is shown. fMedial and lateral knee articular cartilage was isolated from patients undergoing TKA with
medial compartment OA. P16 gene expression was measured by qPCR (P=0.053 0). Bars are mean ± SD for n=9 patients. gMedial and lateral
knee articular cartilage was isolated from patients undergoing TKA with medial compartment OA. Cartilage tissue was sectioned and IHC was
carried out for p16-Ink4a. Image is representative of n=4 healthy and diseases cartilage sections. hGFP or IKK2ca were retrovirally transduced
into chondrocytes. p16 expression was measured by qPCR (*P=0.012 2). Bars are mean ± SD from n=4 independent experiments. iIKK2ca
was expressed in chondrocytes of adult mice in vivo under the control of the tamoxifen inducible aggrecan-cre (Acan
IKK2ca
). Six weeks post
induction, mice were sacrificed and joints were embedded in paraffin and sectioned. IHC was performed for p16, with dark stain representing
p16 positive cells. Representative images are displayed. j,kIKK2
−/−
chondrocytes were transduced with adeno-GFP or adeno-cre. Cells were
then treated with BP for 24 h and p16 and p21 gene expression was measured (p16: *P=0.021 2, n=2. p21: **P=0.001 7, n=4). Bars
represent mean ± SD from representative experiment
IκB-ζpromotes inflammatory chondrocytes and senescence
M Arra et al.
8
Bone Research (2022) 10:12
and BP were drivers of oxidative stress. NOX enzymes are a major
cause of reactive oxygen species (ROS) upon inflammatory
stimulation of chondrocytes,
45,46
but unlikely to be the only
source. Mitochondria are major ROS producers,
47
especially when
there is mitochondrial dysfunction, and previous studies have
shown that chondrocytes treated with inflammatory stimuli
display defective mitochondria.
34,48
For the initial experiments
using fluorescent readouts of redox states, we primarily utilized
IL-1βsince BP interfere with fluorescent assays. We show that
chondrocytes treated with IL-1βhave increased mitochondrial
superoxide production (Fig. 7a), likely from electron transport
chain activity (ETC). This is further supported by increased
membrane permeability from proteins such as PUMA (Supple-
mentary Fig. S4a), which promotes mitochondrial membrane
permeability through the stabilization of Bax.
49
Together, these
changes can lead to ETC dysfunction. Supporting this paradigm,
antimycin A and rotenone, both ETC inhibitors, were able to
reduce overall cellular ROS levels that were elevated in response
to inflammation (Fig. 7b). Furthermore, previous studies have
displayed that high oxidative stress can contribute to cell
apoptosis as well. We utilized the Bax/Bacl2 ratio as an apoptotic
rheostat
50,51
and show that IL-1βand BP both pushed the cell
towards an apoptotic state (Fig. 7c, d). However, the use of NAC,
an antioxidant, was able to protect the cell against an apoptotic
shift. We then identified in vivo that MLI joints had higher
expression levels of cleaved caspase-3, which is an apoptosis
marker, as well as increased staining for γ-H2AX, which is a
marker of DNA damage that can be associated with oxidative
stress, suggesting that these in vitro findings likely extend in vivo
as well (Fig. 7e, f).
Given that IL-1βtreatment clearly caused mitochondrial dysfunc-
tion leading to oxidative stress, we wanted to determine if ETC
inhibitors were effective at blocking the IL-1βand BP-induced
inflammatory response by reducing oxidative stress. This would
validate our findings of inflammation-induced oxidative stress by
BP even if BP interfered with fluorescent assays. Mitochondrial ETC
inhibitors antimycin A and rotenone treatment reduced expression
of IκB-ζprotein and Il-6 and Mmp13 inflammatory gene expression
(Fig. 7g, h, Supplementary Fig. S4b, c) induced by both IL-1βand
BP, likely by decreasing ROS levels (Fig. 7e) in the cell. Likewise,
MitoTEMPO, a mitochondrial ROS scavenger, rotenone, and
antimycin A were also able to reduce IκB-ζprotein levels (Fig. 7i,
Supplementary Fig. S4d). This corroborates previous findings
wherein the use of an ETC inhibitor was able to reduce OA
development in an intra-articular fracture model of OA.
52
Finally,
since we suspected oxidative stress to be an important mediator of
catabolic gene expression in chondrocytes, we sought to determine
if activation of the Nrf2 antioxidant system can reduce catabolic
gene expression. We used an Nrf2 pathway activator, AI-1, to
display that inflammatory gene expression was reduced with
antioxidant gene expression (Fig. 7j). Furthermore, treatment with
AI-1 was able to decrease IκB-ζprotein levels (Fig. 7k). AI-1 activity
was confirmed by measuring the increased expression of Ho1 and
Nqo1 (Supplementary Fig. S4e, f). Overall, our data demonstrate
that NF-κBandIκB-ζmediated inflammatory and catabolic
responses in chondrocytes are regulated by mitochondria-
dependent oxidative stress.
Nitrosative stress can promote IκB-ζexpression but may not be
pro-inflammatory physiologically
After displaying the role of mitochondrial dysfunction as a source
of oxidative stress, we then wanted to explore the role of
nitrosative species such as nitric oxide (NO), which can combine
with ROS to form new damaging molecules.
53
Prior studies have
suggested NO is a pathological species in OA but its relation to
chondrocyte senescence and inflammatory response is not well
understood.
We first show that IL-1βand BP treatment increased iNOS
expression (Fig. 8a), an intracellular enzyme responsible for NO
production. We also display that IL-1βincreased NO levels in these
cells (Fig. 8b), as measured with a fluorescent probe. BP technically
interfered with the fluorescent assay used but were predicted to
cause an increase in NO based on the elevated iNOS expression.
However, using the Greiss assay to measure NO secretion from
Table 2. List of primers used in this study
Gene name Primer sequence
Actin (mouse) F- CTAAGGCCAACCGTGAAAAG
R- ACCAGAGGCATACAGGGACA
IL-6 (mouse) F- GAGGATACCACTCCCAACAGACC
R- AAGTGCATCATCGTTGTTCATACA
MMP13 (mouse) F- GCCAGAACTTCCCAACCAT
R- TCAGAGCCCAGAATTTTCTCC
IL-6 (human) F- CCAGCTATGAACTCCTTCTC
R- GCTTGTTCCTCACATCTCTC
MMP13 (human) F- AATATCTGAACTGGGTCTTCCAAAA
R- CAGACCTGGTTTCCTGAGAACAG
NFKBIZ (mouse) F- TCTCACTTCGTGACATCACC
R- GGTTGGTATTTCTGAGGTGGAG
NFKBIZ (human) F-CCGTTTCCCTGAACACAGTT
R- AGAAAAGACCTGCCCTCCAT
Rankl (mouse) F- TGAAGACACACTACCTGACTCCTG
R- CCCACAATGTGTTGCAGTTC
Rankl (human) F- CACTATTAATGCCACCGAC
R- GGGTATGAGAACTTGGGATT
OPG (mouse) F- GTTTCCCGAGGACCACAAT
R- CCATTCAATGATGTCCAGGAG
P16 (mouse) F- AATCTCCGCGAGGAAAGC
R- GTCTGCAGCGGACTCCAT
P16 (human) F- CCCAACGCACCGAATAGTTA
R- ACCAGCGTGTCCAGGAAG
P21 (mouse) F- TTGCCAGCAGAATAAAAGGTG
R- TTTGCTCCTGTGCGGAAC
P21 (human) F- TGGAGACTCTCAGGGTCGAAA
R- GGCGTTTGGAGTGGTAGAAATC
Lcn2 (mouse) F- CCATCTATGAGCTACAAGAGAACAAT
R- TCTGATCCAGTAGCGACAGC
Acan (mouse) F- CCAGCCTACACCCCAGTG
R- GAGGGTGGGAAGCCATGT
Nlrp3 (mouse) F- CCCTTGGAGACACAGGACTC
R- GAGGCTGCAGTTGTCTAATTCC
TRAP (mouse) F- CACTCCCACCCTGAGATTTGT
R- CATCGTCTGCACGGTTCTG
Adamts4 (mouse) F- CTTCCTGGACAATGGTTATGG
R- GAAAAGTCGCTGGTAGATGGA
Nqo1 (mouse) F- TTTAGGGTCGTCTTGGCAAC
R- GTCTTCTCTGAATGGGCCAG
Ho1 (mouse) F- GTCAAGCACAGGGTGACAGA
R- ATCACCTGCAGCTCCTCAAA
iNos (mouse) F- TGCATGGACCAGTATAAGGCAAGC
R- GCTTCTGGTCGATGTCATGAGCAA
NLRP3 (human) F- CTTCTCTGATGAGGCCCAAG
R- GCAGCAAACTGGAAAGGAAG
IκB-ζpromotes inflammatory chondrocytes and senescence
M Arra et al.
9
Bone Research (2022) 10:12
chondrocytes in vitro, we were able to show that IL-1β
significantly increased NO production (Fig. 8c) but BP were unable
to drive chondrocyte NO production (Fig. 8d), suggesting a
mechanistic difference between BP and IL-1βin mediating NO
production. Furthermore, we displayed that the effect observed
with IL-1βwas dependent upon the IKK2-NF-κB axis, since IKK2
−/−
chondrocytes had significantly lower NO production (Fig. 8c). To
confirm that the elevated NO is originating from iNOS, we used
L-NAME, a known iNOS inhibitor, to show that it is able to block
the IL-1βmediated increase in NO (Fig. 8b).
NO can pathologically combine with superoxide from the
mitochondria to form peroxynitrite, which is a known pathogenic
ROS.
53
Using a peroxynitrite-specificfluorescent probe, we display
that peroxynitrite levels increase with IL-1βtreatment in
chondrocytes (Fig. 8e). Peroxynitrite reacts with proteins to form
3-nitrotyrosine (3-NT), which is an indicator of oxidative stress and
damage that other groups have shown to be elevated in OA
cartilage.
54
We show that peroxynitrite and 3-NT levels elevated
by IL-1βdecreased with L-NAME treatment (Fig. 8e, Supplemen-
tary Fig. S5a), indicating that the NO arising from iNOS contributes
to damaging peroxynitrite formation. Furthermore, since we
suspected that NO combines with superoxide from the mitochon-
dria, we show that mitoTEMPO, a mitochondria-targeted antiox-
idant, is able to decrease peroxynitrite formation (Fig. 8e).
Nfkbiz
a
bc
Mmp2
Mmp3
Mmp10
Mmp12
Mmp13
Ccl7
Ccl20
Ccl25
Cxcl1
Cxcl2
Cxcl5
Saa3
Saa4
Nos2
Vegfc
Fas
II6
10
5
15
0
300
200
100
400
Nos2Lcn2
0
Lcn2
Areg
Serpinb2
Ereg
Fgf2
Timp1
WT
GFP
Nfkbiz-/-
Nfkbiz-/- + IL-1E
GFP + IL-1E
GFP
Nfkbiz-/-
Nfkbiz-/- + IL-1E
GFP + IL-1E
WT.1
WT.IL1b
WT.IL1b.1
NFKBIZ.KO
NFKBIZ.KO.1
NFKBIZ.KO.IL1b
NFKBIZ.KO.IL1b.1
Color key
–1.5 01
Relative mRNA expression
Relative mRNA expression
**** ****
****
****
Fig. 6 IκB-ζis a major driver of SASP expression in chondrocytes subject to inflammation. aWild type or Nfkbiz
−/−
chondrocytes were treated
with IL-1β(10 ng·mL
−1
) for 24 h. RNA sequencing was performed and significantly downregulated SASP genes with Nfkbiz deletion are
displayed. All samples were performed with biological duplicates. b,cWild type or Nfkbiz
−/−
chondrocytes were exposed to IL-1β(10 ng·mL
−1
)
for 24 h. Gene expression of Lcn2 and Nos2 was measured by qPCR. Bars are mean ± SD for three samples (****P< 0.000 1)
IκB-ζpromotes inflammatory chondrocytes and senescence
M Arra et al.
10
Bone Research (2022) 10:12
a
e
g
ijk
h
f
bcd
200
100
300
0
6
4
2
8
0
300
200
100
400
0
–+– +– + –+ –+
–
––
++
+
MitoSox DCFDA
IHC: cleaved caspase 3 IHC: J-H2AX
Bax/Bacl2 ratio Bax/Bacl2 ratio
600
400
200
800
0
Relative fluorescence
Relative fluorescence
****
****
*** ***
**
****
**** ****
***
****
*
****
Untreated
Untreated
Untreated
Untreated
BP
NAC
BP + NAC
NAC
IL-1E
IL-1E+ Antimycin A
IL-1E + Rotenone
IL-1E
Untreated
BP
BP + Rotenone
IL-1E
IL-1E + Rotenone
Rotenone
Untreated
Untreated
AI-1
IL-1E
AI-1 + IL-1E
BP
BP + Rotenone
IL-1E
IL-1E + Rotenone
Rotenone
IL-1E
IL-1E + NAC
Sham MLI 50 PmSham MLI 50 Pm
8
6
4
2
0
3
2
1
4
0
15 000
10 000
5 000
20 000
0
Relative mRNA expression
Relative mRNA expression
Relative mRNA expression
Relative mRNA expression
Relative mRNA expression
II-6
II-6
Mmp13
INB-]
Actin INB-]
Actin
0
0
100
0
500
010
0
100
0
IL-1E
IL-1E
AI-1
MitoT (µmol·L-1)
Rot (µmol·L-1)
Fig. 7 Inflammatory mediators promote apoptosis via mitrochondrial oxidative stress. aMitochondrial superoxide production was measured
by MitoSox fluorescence under similar conditions (****P< 0.000 1). Bars are mean ± S. for n=8 replicates from representative experiment.
bChondrocytes treated with IL-1β± antimycin A (10 μmol·L
−1
) or rotenone (100 μmol·L
−1
) for 24 h. ROS levels in the cell were measured by
DCFDA fluorescence (****P< 0.000 1). Bars represent mean ± SD for n=8 replicates from representative experiments. c,dChondrocytes
treated with IL-1βor BP ± NAC (3 mmol·L
−1
) for 24 h. Bax and Bcl2 gene expression normalized to actin expression. Bax and Bcl2 ratios were
then determined, normalizing to untreated cells (A: Untreated vs IL-1β***P=0.000 5, IL-1βvs IL-1β+NAC *P=0.041 9, B: Untreated vs BP
***P=0.000 4, BP vs BP +NAC *P=0.011 8). Bars are mean ± S.E.M from n=5 independent experiments. e,fMLI surgery was performed on
12-week-old mice. Control sham surgery performed on contralateral knee. Joints were collected, embedded in paraffin and sectioned. IHC for
cleaved caspase-3 and γ-H2AX was performed, with brown stain indicating positive cells. Representative images are displayed.
g, h Chondrocytes were exposed to IL-1βor BP for 24 h ± rotenone (100 μmol·L
−1
). Il6 and Mmp13 gene expression was measured by
qPCR (****P< 0.000 1). Bars are mean ± SD from representative experiment. iChondrocytes were treated with IL-1βin the presence or absence
of mitoTEMPO or Rotenone doses indicated. Western blotting was performed for IκB-ζwith actin used as control. Representative immunoblot
is displayed. jChondrocytes were exposed to IL-1β± AI-1 (40 μmol·L
−1
) for 24 h. Il6 gene expression was measured by qPCR. Bars represent
mean ± SD from n=4 replicates. kWestern blot was performed for IκB-ζunder the same conditions. Representative immunoblot is displayed
IκB-ζpromotes inflammatory chondrocytes and senescence
M Arra et al.
11
Bone Research (2022) 10:12
a
e
h
f
ij
g
bcd
Relative mRNA expression
Relative fluorescence
Relative fluorescence
Relative mRNA expression
Relative mRNA expression
Relative luminescence
Relative absorption
Relative absorption
*
***
****
*** *
*
****
**** **** **
iNos
II-6 II-6
4 000
3 000
2 000
2 000
200
150
100
50
0
250
–50
150
100
50
200
0
10 000
5 000
15 000
0
Peroxynitrite NF-NB activity
INB-]
IL-1E
Actin
0
–––+++
0.3
IHC: 3-Nitrotyrosine
Sham MLI 50 Pm
0.6 0 0.3 0.6
SNOG
(mmol·L-1)
1 500
1 000
500
0
1 000
5 000
0
0.4
0.2
0.6
0.0
0.3
0.2
0.1
0.4
0.0
DAF-FM-DA Greiss assay Greiss assay
4 000
3 000
2 000
1 000
5 000
0
Untreated
Bone particles
IL-1E
Untreated
Untreated
IL-1E
IL-1E + SNOG
IL-1E + SNOG IL-1E + L-NAME
BP + L-NAME
IL-1E + L-NAME
IL-1E
Untreated
WT
IKK2-/-
IKK2-/- + IL-1E
WT + IL-1E
WT
IKK2ca
IKK2ca + BP
WT + BP
L-NAME
L-NAME
L-NAME
SNOG
BP + SNOG
BP
BP
SNOG
MitoTEMPO
IL-1E + NL-NAME
IL-1E + L-NAME
L-NAME
IL-1E + MitoTEMPO
Untreated
Untreated
IL-1E
IL-1E
IL-1E
****
****
Fig. 8 Nitrosative stress has different effects on inflammatory response in vitro and in vivo. aChondrocytes were treated with IL-1βor BP for
24 h. Gene expression of iNos was measured by qPCR (F: Untreated vs IL-1β***P=0.000 6, Untreated vs BP *P=0.020 1). bChondrocytes were
treated with IL-1β± L-NAME (75 μmol·L
−1
) for 24 h. NO levels were measured using DAF-FM-DA fluorescent probe. Bars represent mean ± SD
of n=8 replicates from representative experiment (****P< 0.000 1). cWT or IKK2
−/−
chondrocytes were treated with IL-1βfor 24 h.
Supernatant was collected and Greiss assay was carried out to measure NO production. ( WT vs WT +IL-1β**P=0.002 6). Results are
representative of at least 3 biological replicates. dChondrocytes were transduced with pMX-GFP (WT) or pMX-IKK2ca. They were then treated
with BP for 24 h. Supernatant was collected and NO production was performed using Greiss assay. Results are representative of at least three
biological replicates. eChondrocytes treated with IL-1β± L-NAME (75 μmol·L
−1
) or MitoTEMPO (0.5 mmol·L
−1
) for 24 h. Peroxynitrite levels
were measured using fluorescent sensor dye (Untreated vs IL-1β****P< 0.000 1, IL-1βvs IL-1β+L-NAME ****P< 0.000 1, IL-1βvs IL-1β+
MitoTEMPO *P=0.045). Bars are mean ± SD from n=4-6 replicates. fNF-κB luciferase reporter chondrocytes treated with IL-1β± SNOG or
L-NAME for 24 h. Luciferase activity was measured as a readout of NF-κB activation (****P< 0.000 1). Bars are mean ± SD for n=8 replicates
from representative experiments. gPrimary chondrocytes treated with IL-1β± S-Nitrosoglutathione (0.6 mmol·L
−1
) for 24 h. Western blot was
performed for IκB-ζ. Representative immunoblot is displayed. hChondrocytes were treated with IL-1βor BP ± SNOG (0.6 mmol·L
−1
). Il6 gene
expression measured by qPCR (B: IL-1βvs IL-1β+SNOG ***P=0.000 6, BP vs BP +SNOG *P=0.04). Bars are mean ± SD of n=4 independent
experiments for Panel (b). iChondrocytes treated with IL-1βor BP ± L-NAME (75 μmol·L
−1
). Il6 gene expression was measured by qPCR. Data
represent mean ± SD for replicates from one representative experiment out of three. jMLI surgery was performed on 10-week-old mice.
Control sham surgery was done on contralateral knee. Joints were collected, embedded in paraffin and sectioned. IHC for 3-nitrotyrosine was
performed, with brown stain indicating positive cells. Representative images are displayed
IκB-ζpromotes inflammatory chondrocytes and senescence
M Arra et al.
12
Bone Research (2022) 10:12
After observing that oxidative stress can drive senescence and
SASP formation in an NF-κB-IκB-ζmediated manner, we sought to
interrogate the role of NO in this system. We treated chondrocytes
with an NO donor, S-nitrosoglutathione (SNOG), which we
confirmed was able to increase NO levels and 3-NT formation
(Supplementary Fig. S5b, c). We observed that SNOG was pro-
inflammatory, increasing NF-κBactivationbyIL-1βas well as
protein levels of IκB-ζ(Fig. 8g, h). Furthermore, it was able to
promote IL-1βand BP-induced expression of IL-6 (Fig. 8i). However,
the use of iNOS inhibitor, L-NAME, was unable to decreased IL-1β
and BP-induced expression of IL-6, suggesting that intrinsic NO
levels are unlikely to be pro-inflammatory (Fig. 8j).
Since we had shown that oxidative stress promotes senescence
and apoptosis, we wanted to determine if NO had similar pathologic
effects. SNOG treatment in the setting of inflammatory stimuli leads
to a significant increase in p21 expression, and a slight, although
non-significant increase in p16 expression (Supplementary Fig. S5d,
e). There was also a slight increase in Bax/Bcl2 ratio with SNOG
treatment, though it was not statistically significant (Supplementary
Fig. S5f). Furthermore, use of L-NAME, an iNOS inhibitor, was unable
to reduce Bax/Bcl2 ratio caused by IL-1βor BP as NAC was able to
(Fig. S5g, h), suggesting that a reduction of NO levels alone is
insufficient to prevent apoptotic changes since there is still
production of damaging oxidative species such as superoxide.
Overall, these results suggest that addition of extra-physiological
levels of NO with a donor can be pro-inflammatory but intrinsic NO
production is not in and of itself pro-inflammatory.
We then sought to determine how these findings translate to
the in vivo MLI model. Interestingly, we observed that MLI joints
had consistently decreased 3-NT staining in the articular cartilage
compared to sham joints, specifically with decreased staining in
articular chondrocytes. This finding suggests that contrary to its
role in other synovial joint components, 3-NT may not be a reliable
marker of inflammation in articular chondrocytes in OA and that
intrinsic NO may not be a pro-inflammatory mediator, as we
observed in vitro. These results indicate the need for further
research into the role of NO in the context of joint inflammation.
DISCUSSION
This work focuses on understanding the pathogenesis of OA through
the lens of inflammation, senescence, and oxidative stress. The
presence of mineral crystals and other matrix breakdown products in
the synovial fluid has been noted in patients with OA and
rheumatoid arthritis.
19,20,22,27,28
However, it has been difficult to
interpret the role of these particles in OA since it is unclear whether
they are a product or drivers of the disease. While still unclear, we
predict that they may arise from cartilage and bone erosions in part
due to osteoclast activity. It is well recognized that patients with late-
stage OA have subchondral sclerosis, though some groups have
demonstrated that early OA may actually present with decreased
subchondral bone volume.
32
We show that post-traumatic OA
(PTOA) joints display increased osteoclast numbers in the subchon-
dral region. We also display that early OA joints have decreased
subchondral bone, supporting the finding of increased bone
breakdown early in disease, likely by osteoclasts. This leads to a
dysregulation in bone remodeling, eventually leading to subchondral
sclerosis seen in late-stage OA. Furthermore, we propose that
chondrocyte-produced Rankl in response to inflammatory stimuli
may be a significant osteoclast promoting force in regions adjacent
to damaged cartilage in vivo. It is conceivable that “inflammatory
chondrocytes”may recruit OCs and their progenitors to cartilage and
adjacent bone matrices by Rankl secretion. Hence, subchondral
bone-cartilage interaction is likely to be an important factor in OA
disease progression, not simply a by-product of joint damage, as
several groups have identified increased osteoclast numbers in
subchondral bone,
55–57
leading to attempts at OA therapy through
modulation of subchondral bone remodeling.
58
Theactivityoftheseinflammatory chondrocytes is driven by
pathological mediators generated in the joint. Regardless of
their source, BP are an example of one such mediator that drive
a catabolic response in an NF-κB dependent manner, especially
when they synergize with other inflammatory stimuli such as IL-
1β.WhiletheBPweutilizedarenotthesamecrystalsfoundin
the synovial fluid of human OA joints, we suspect that they can
have similar pro-inflammatory effects as other inert crystal
compounds. Given that other groups have already studied the
effect of small crystals on the chondrocyte catabolic
response,
20,21,27,28
we wanted to take an approach that may
be more physiologically relevant to disease states in which there
is bony erosions or joint injuries leading to BP in the synovial
space. We utilized real BP that vary in size and shape, as well as
composed of diverse ECM compounds. Some physicians have
endorsed the use of synovial lavage for the treatment of OA,
although a meta-analysis displayed no benefitoverplacebo.
59
However, this technique may be useful in patients with
significant calcified compounds in their synovial space early in
the disease and should be further studied.
We observed that BP and IL-1βgenerate a feed forward cycle of
inflammation that contributes to the chronic inflammatory state
present in OA joints that cannot be targeted by biologics such as
anti-IL-1 or anti-TNF therapies, but can be addressed by targeting
downstream signaling pathways such as the NF-κB pathway,
which integrate signals downstream of several cytokines/recep-
tors. However, given the indispensable role of NF-κB in normal
physiologic pathways, sustained blockage of this pathway in
humans is counterproductive. Instead, we identified IκB-ζand
NLRP3 inflammasome as downstream mediators of BP-induced
inflammation, highlighting these as potential therapeutic targets.
We then provided evidence that inflammatory stimuli can
promote senescence via activation of the NF-κB signaling path-
way, which promotes various senescent programs such as the
p16-Ink4a pathway, and also SASP expression via the NF-κB-IκB-ζ
axis. We identified IκB-ζat the intersection of inflammation and
senescence given its ability to drive the expression of pro-
inflammatory SASP genes in response to inflammatory stimuli
such as BP or IL-1β. We also found that IκB-ζappeared to be
upstream of Nrlp3 and Rankl expression, further asserting its
importance in the control of inflammatory, degradative pathways
in the joint. Given that the contribution of senescent chondrocytes
to OA has been well-established, identifying factors such as IκB-ζ
to ameliorate SASP production may be highly beneficial for
preventing OA progression. In order to target IκB-ζ, we display
that its expression is driven by a combination of inflammatory
stimulation, oxidative and nitrosative stress to promote the
expression of SASP genes, providing multiple avenues for
reducing SASP expression.
Another interesting finding was that while IL-1βand BP are
both pro-inflammatory, they have different effects on ROS and
RNS. While IL-1βstrongly induces ROS and RNS production, BP
appear to be poor inducers of RNS, which may be explained by
differences in their signaling pathways. Both induce NF-κB
activation to drive inflammation, but they may have unique
alternative pathway activation regarding nitrosative stressors. We
identified the mitochondria as one source of oxidative stress
activated by both players that appears to be a key driver of the
inflammatory response through superoxide production. We
further found that ETC inhibitors may be effective for treatment
in the context of inflammation by blocking IκB-ζexpression, but
that iNOS inhibitors were not effective. Hence, the role of RNS in
the context of disease pathology requires further elucidation,
especially in vivo, where we unexpectedly saw decreased 3-NT
staining in the articular chondrocytes of OA joints. It is likely that
NO can have pro-inflammatory functions at supraphysiological
levels, which in conjunction with ROS species is likely to form
molecules such as peroxynitrite, consistent with our finding that
IκB-ζpromotes inflammatory chondrocytes and senescence
M Arra et al.
13
Bone Research (2022) 10:12
NO donors can exacerbate an inflammatory response in vitro by
stabilizing IκB-ζ. This would corroborate some studies that have
suggested that 3-NT levels are a pathological marker that is
elevated in OA.
52,58
However, we observed the opposite results in
the chondrocytes in our animal model, which may be a function of
the MLI mouse model of OA. Given that most of our work focuses
on chondrocytes, it will be important to further understand the
role of NO and 3-NT in other joint components such as
synoviocytes, synovial macrophages and subchondral bone, which
are intricately related to the articular cartilage, and may reconcile
our work with prior studies displaying elevated 3-NT in OA joint
synovial fluid, synovial cells and tissues. The lack of protection
with iNOS inhibition in vitro further indicates that ROS species
such as superoxide may be the primary inflammatory mediators
and NO by itself is less important for chondrocyte inflammation.
Another explanation is that exogenous NO donors produce NO
throughout the cell, while physiologically, there are unique
isoforms of iNOS that exist within subcellular compartments such
as mitrochondrial NOS, iNOS, eNOS, and nNOS.
60
Further
supporting our findings that NO may not be pathologic, other
groups have also displayed that NO can actually block mitochon-
drial ETC activity, leading to an antioxidant and protective effect
by reducing superoxide levels.
61,62
Given these findings, it is likely
that NO has unique spatial and temporal effects within
chondrocytes and needs to be better understood in the context
of the OA joint through deletion of specific NOS subtypes and
better NO detectors.
There are several experiments needed to further build upon
the foundation of this work. First, we seek to develop a
chondrocyte-specific Rankl knockout mouse to characterize the
role of Rankl in OA progression and better appreciate the
relationship between articular cartilage and subchondral bone,
an area in need of significant exploration. A shortcoming of this
project was the use of primarily murine chondrocytes, though
we did utilize human OA cartilage for studying gene expression
in order to validate our findings. The use of human synovial fluid
to measure BP and the factors present in the fluid can provide
insight into the role of these molecules in human disease.
Another limitation of this project was the performance of
in vitro experiments at 21% oxygen, which can influence the
understanding of oxidative and nitrosative pathways, though
we have displayed in a previous publication that inflammatory
responses are similar at 21% oxygen and 4% oxygen cultures.
34
Finally, it is important to use large animal models to validate our
findings regarding oxidative stressors in the joint. The thickness
of joint cartilage varies significantly from species to species,
which can affect the ability of oxidative species to travel and
affect adjacent cellular compartments. The use of pig and
bovine models to validate our results will provide greater
insight into the role of subchondral remodeling as well as
oxidative stress in the joint.
MATERIALS AND METHODS
Animal models
All animal models were bred on a C57/BL6 background. 10-
week-old wild type mice were used for PTOA models. For
isolation of primary chondrocytes, WT, IKK2
f/f
,Nfkbiz
f/f
,andNlrp3
KO pups aged P1-P3 were used. Recombination was induced in
Aggrecan-ERT2-cre mouse model by feeding tamoxifen chow for
2 weeks. IKK2ca flox/flox mice were crossed with Aggrecan-
ERT2-cre mice to express IKK2ca in chondrocytes in mature mice
at age 10 weeks. All experiments were performed using
littermate controls. Mice were housed at the Washington
University School of Medicine barrier facility. All experimental
protocols were carried out in accordance with the ethical
guidelines approved by the Washington University School of
Medicine Institutional AnimalCareandUseCommittee.Mice
were housed in barrier facility at five or less per cage at 24–26
degrees Celsius with humidity ranging between 30%–60% with
12 h light/dark cycles switching at 6 pm.
Meniscal ligamentous injury model
The MLI model was utilized to induce post-traumatic OA in mice
at 12 weeks of age. Medial ligament and meniscus were
transected in the right legs of mice, with sham surgery performed
on the contralateral knee joint. Sham surgery involves cutting skin
superficially and suturing without transecting ligaments in the
knee joint. Mice were sacrificed and limbs were harvested for
further analysis.
Cell culture
Primary chondrocytes were harvested from sterna and ribs of P1-
P3 mice using sequential digestion with pronase (2 mg·mL
−1
,
PRON-RO, Roche) at 37 degrees, collagenase D (3 mg·mL
−1
,
COLLD-RO, Roche) two times at 37 degrees, and cultured in
DMEM (Life Technologies, USA) containing 10% FBS and 1%
penicillin/streptomycin (Thermo Fisher). Primary chondrocytes are
not passaged and experiments are completed within 5 days of
isolation from mice. Primary macrophages (osteoclast precursors)
were harvested from long bones of WT mice and cultured in
M-CSF overnight before being added to chondrocyte cultures.
Cells were incubated with recombinant IL-1β(10 ng·mL
−1
), IKK2
inhibitor SC-514 (10 μmol·L
−1
), BP (1 mg·mL
−1
). Plat-E cells were
used to generate retroviral particles.
Bone particle generation
BP were obtained from porcine longbones.First,porcinebones
were obtained from butcher shops. Then bone surfaces were
extensively washed with PBS and 70% ethanol. The cortical bone
was then cut using an automatic low speed bone saw (Buehler
IsoMet) on the epiphyseal surfaces. The bone dust produced was
collected as BP. The BP were then thoroughly washed with sterile
PBS, autoclaved and stored in 70% ethanol at 4 degrees. Prior the
use, BP were washed in sterile PBS to remove ethanol and re-
suspendedinPBS.Aftershakingtubecontainingparticles,large
orifice pipette tips were used to pick up BP from the surface of the
solution and added to chondrocyte cell culture.
Retroviral and adenoviral infection
Adenovirus was utilized for deleting genes from cells with floxed
genes. Cells were infected with commercially obtained
adenoviral-GFP (VVC-U of Iowa 4) and adenoviral-cre (VVC-U of
Iowa 3554) at an MOI of 10 in the presence of 5 μg·mL
−1
of
polybrene (TR-1003, Sigma) in 5% FBS-containing-DMEM. After
24 h, media was switched to DMEM +10% FBS. Retrovirus was
utilized to infect chondrocytes with GFP, IKK2ca, or IKK2-KD.
Retrovirus was generated by transfecting PLAT-E cells with pMX-
GFP, pMX-IKK2ca or pMX-IKK2-KD constructs using X-tremegene
9(XTG9-RO, Roche) and media was changed after 24 h. After
48 h, supernatant containing retrovirus was used to infect
primary chondrocytes in the presence of 5 μg·mL
−1
of poly-
brene. After 24 h of infection, media was changed and
chondrocytes were treated as stated.
Western blotting
Cell lysates were collected in 1x cell lysis buffer (Cell Signaling
Technology, Danvers, MA, USA) with protease/phosphatase
inhibitor (Thermo Fisher Scientific). Samples were denatured in
1x sample buffer containing β-ME by boiling for 10 min. Samples
were analyzed using PAGE electrophoresis. Membranes were
incubated with desired primary and secondary antibodies. Signals
were captured using LiCor Odyssey reader (LI-COR Biosciences,
Lincoln, NE, USA). Primary antibodies used were anti-IκB-ζ(Cat#
14-16801-82, Invitrogen, 1:1 000), anti-p16, anti-PUMA and anti-
Actin (Cat# A228, Sigma, 1:5 000 dilution).
IκB-ζpromotes inflammatory chondrocytes and senescence
M Arra et al.
14
Bone Research (2022) 10:12
Quantitative PCR
Trizol (Sigma) and Chloroform at a ratio of 0.2:1 were added to
samples, followed by centrifugation at 12 000 × g for 15 min.
Aqueous layer was collected and equal amount of 70% ethanol
was added. RNA was then isolated from this fraction using
PureLink RNA mini kit (Cat# 12183025, Ambion, Grand Island,
NY, USA). cDNA was prepared using High Capacity cDNA
Reverse Transcription kit (Cat# 4368814, Applied Biosystems).
qPCR was carried out on BioRad CFX96 real time system using
iTaq universal SYBR green super-mix (Cat#1725120, BioRad,
Hercules, CA, USA). Actin was used to normalize mRNA
expression.
IL-1βELISA
Chondrocytes were cultured with BP and/or IL-1β.Supernatant
was collected for ELISA. Supernatant was added to IL-1β-coated
ELISA plates and incubated overnight at 4 degrees. Samples
were washed with PBST and biotin-conjugated detection anti-
body was added to plates for 1 h at room temperature. After
washing again, streptavidin-HRP was added to wells for 1 h,
washed, and TMB substrate was added. Reaction was stopped
prior to reading.
Histology
Knee joints were isolated from mice and fixed in 10% neutral
buffered formalin (HT501128, Sigma) for 24 h followed by
decalcification in Immunocal (StatLab, McKinney, TX) for 3 days.
Samples were then embedded in paraffin before cutting
5μmol·L
−1
sections. Unstained sections were utilized for IHC.
Remaining sections were stained with safranin-O/fast green stain.
Severity of OA was graded by OARSI scoring. Reviewers were
blinded to treatment conditions of slides being scores.
Immunohistochemistry
Sections were deparaffinized and rehydrated using three
changes of xylenes followed by ethanol gradient. Antigen
retrieval was performed using citrate solution pH 6.0 at 60 ℃
overnight, followed by blocking for 1 h using blocking solution
(10% goat serum in PBS, 1% tween-20, 1% BSA). Sections were
then incubated with primary antibody diluted in DAKO dilution
solution (S3022, Agilent, Santa Clara, CA) overnight at 4 degrees
then washed and incubated with biotin-conjugated secondary
antibody (BP-1100, Vector Biolabs, Burlingham, CA) at room
temperature for 2 h. Sections were washed and incubated with
streptavidin-HRP using Vectastain ABC-HRP (PK-4000, Vector
Laboratories, Burlington, CA) for 20 min. After washing, sections
were developed using DAB peroxidase kit (SK4100, Vector
Laboratories, Burlingham, CA). Anti-p16-ink4a (PA5-20379,
Thermo Fisher), anti-IκB-ζ(NBP-89835, Novus Biologicals,
Centennial, CO), anti-3-NT (06-284, Millipore Sigma, Burlington,
MA), cleaved caspase-3 (9661 S, CST, Danvers, MA), and Υ-H2AX
(2577 S, CST, Danvers, MA) were used in blocking solution
(1:100 dilution).
ROS and RNS assays
ROS species in the cell were measured using H2, DCF-DA
fluorescent dye (Cat#D6883, Sigma) and RNS species were
measured using H2-DAF-FM-DA fluorescent dye in 96 well plate
format. Cells were pre-treated 24 h with appropriate treatment
conditions in DMEM media. Next, cells were washed and
incubated with 5 μmol·L
−1
DCFDA or DAFFM-DA dissolved in
PBS for 30 min, washed several times with PBS and placed back in
the incubator. After 1 h, fluorescence was measured on microplate
reader at Ex/Em 495/518.
Peroxynitrite assay
Peroxynitrite was measured in the cell using Dax-J2 PON
fluorescent dye (Abcam ab233469) in 96 well plate format. Cells
were pre-treated for 24 h with appropriate treatment conditions in
DMEM media. Cells were incubated with 10 μmol·L
−1
Dax-J2 PON
in media for 30 min. Next, cells were washed several times with
PBS and placed back in the incubator. After 1 h, fluorescence was
measured on microplate reader at Ex/Em 495/518.
3-Nitrotyrosine ELISA
3-Nitrotyrosine adduct formation on proteins in the cell were
measured by ELISA kit (Abcam, ab116691tab). Cells were treated
with appropriate conditions for 24 h in six well plates. ELISA was
then performed according to protocol for the assay with no
modifications.
Rankl ELISA
Rankl secretion by chondrocytes in vitro was measured by
ELISA kit (Abcam, Ab100749). Chondrocytes were cultured with
IL-1βfor 24 h and 100 μL of supernatant was used undiluted in
the assay, performed in technical replicates for each biological
sample.
Greiss assay
Supernatant from chondrocyte culture was collected under
various conditions. Greiss assay was performed using assay kit
(G7921, Thermo Fisher) in microplate format, using 150 uL of
supernatant per well. Culture media was used as control for
background correction.
Micro computed tomography (μCT)
Intact knee joints were harvested, fixed overnight in 10% neutral
buffered formalin (HT501128, Sigma) then washing with Phos-
phate Buffer Saline (PBS) three times and transfered to 70%
ethanol (v/v). Bones were scanned at a resolution of 20 µm, slice
increment 10 µm, voltage 55 kV, current 145 µA and exposure time
of 200 ms and 3D images were constructed using Scanco Medical
micro-CT systems (Scanco, Wayne, PA, USA) at Washington
University in St. Louis (St. Louis, MO).
RNA sequencing
Primary chondrocytes were cultured ± IL-1β(10 ng·mL
−1
)for
24 h. RNA was collected and samples were prepared according to
library kit manufacturer’s protocol, indexed, pooled, and
sequenced on an Illumina HiSeq. Basecalls and demultiplexing
were performed with Illumina’s bcl2fastq software and a custom
python demultiplexing program with a maximum of one
mismatch in the indexing read. RNA-Seq reads were then
aligned to the Ensembl release 76 primary assembly with STAR
version 2.5.1a. Gene counts were derived from the number of
uniquely aligned unambiguous reads by Subread:featureCount
version 1.4.6-p5. Isoform expression of known Ensembl tran-
scripts were estimated with Salmon version 0.8.2. Sequencing
performance was assessed for the total number of aligned reads,
the total number of uniquely aligned reads, and features
detected. The ribosomal fraction, known junction saturation,
and read distribution over known gene models were quantified
with RSeQC version 2.6.2.
All gene counts were then imported into the R/Bioconductor
package EdgeR and TMM normalization size factors were
calculated to adjust for samples for differences in library size.
Ribosomal genes and genes not expressed in the smallest
group size minus one sample greater than one count-per-
million were excluded from further analysis. The TMM size
factors and the matrix of counts were then imported into the R/
Bioconductor package Limma. Weighted likelihoods based on
the observed mean-variance relationship of every gene and
sample were then calculated for all samples with the
voomWithQualityWeights. The performance of all genes was
assessed with plots of the residual standard deviation of every
gene to their average log-count with a robustly fitted trend line
IκB-ζpromotes inflammatory chondrocytes and senescence
M Arra et al.
15
Bone Research (2022) 10:12
of the residuals. Differential expression analysis was then
performed to analyze for differences between conditions and
the results were filtered for only those genes with Benjamini-
Hochberg false-discovery rate adjusted P-values less than or
equal to 0.05.
For each contrast extracted with Limma, global perturbations in
known Gene Ontology (GO) terms, MSigDb, and KEGG pathways
were detected using the R/Bioconductor package GAGE
8
to test
for changes in expression of the reported log 2-fold-changes
reported by Limma in each term versus the background log 2-
fold-changes of all genes found outside the respective term. The
R/Bioconductor package heatmap3 was used to display heatmaps
across groups of samples for each GO or MSigDb term with a
Benjamini-Hochberg false-discovery rate adjusted P-value less
than or equal to 0.05. Perturbed KEGG pathways where the
observed log 2-fold-changes of genes within the term were
significantly perturbed in a single-direction versus background or
in any direction compared to other genes within a given term with
P-values less than or equal to 0.05 were rendered as annotated
KEGG graphs with the R/Bioconductor package Pathview.
Statistical analysis
Experiments were routinely carried out in replicates, unless
otherwise stated. GraphPad Prism was employed for all experi-
ments using appropriate statistical test. Multiple treatments were
analyzed by One-way ANOVA followed by Tukey’s test multiple
comparisons test. Student’sTtest was used for comparing two
groups. Age and sex-matched mice were used. Values are
expressed as mean ± SD of representative experiment out of at
least three independent experiments. P-values are indicated
where applicable.
ACKNOWLEDGEMENTS
This work was supported by NIH/NIAMS R01-AR049192, R01-AR054326 (to YA),
Biomedical grant from Shriners Hospital for Children (YA), P30 AR074992 NIH Core
Center for Musculoskeletal Biology and Medicine (to YA) and NIH/NIAMS R01-
AR064755 and R01-AR068972 (to GM).
AUTHOR CONTRIBUTIONS
M.A., G.S., G.M., Y.A. participated in study design and troubleshooting; M.A., G.S.,
Y.A.l. performed experiments; M.A. and G.S. gathered, analyzed data, and
prepared figures; M.A. and Y.A. wrote the manuscript; G.S. and G.M. participated
in manuscript revisions; Y.A. guided the general outline and experimental
approach of the project.
ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41413-021-00183-9.
Competing interests: The authors declare no competing interests.
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IκB-ζpromotes inflammatory chondrocytes and senescence
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