Abstract
Traumatic joint injuries often result in elevated proinflammatory
cytokine (such as IL-1β) levels in the joint cavity, which can increase
the catabolic activities of chondrocytes and damage cartilage. This
study investigated the early genetic responses of healthy in situ
chondrocytes under IL-1β attack with a focus on cell cycle and calcium
signaling pathways. RNA sequencing analysis identified 2,232
significantly changed genes by IL-1β, with 1,259 upregulated and 973
downregulated genes. Catabolic genes related to ECM degeneration were
promoted by IL-1β, consistent with our observations of matrix protein
loss and mechanical property decrease during 24-day in vitro culture of
cartilage explants. IL-1β altered the cell cycle (108 genes) and Rho
GTPases signaling (72 genes) in chondrocytes, while chondrocyte
phenotypic shift was observed with histology, cell volume measurement,
and MTT assay. IL-1β inhibited the spontaneous calcium signaling in
chondrocytes, a fundamental signaling event in chondrocyte metabolic
activities. The expression of 24 genes from 6 calcium-signaling related
pathways were changed by IL-1β exposure. This study provided a
comprehensive list of differentially expressed genes of healthy in situ
chondrocytes in response to IL-1β attack, which represents a useful
reference to verify and guide future cartilage studies related to the
acute inflammation after joint trauma.
Introduction
Interleukin 1β (IL-1β) is an essential mediator of acute joint
inflammation after traumatic injuries, one of the potential causes of
post-traumatic osteoarthritis (PTOA). Within 24 hours after trauma
injuries, the concentration of IL-1β in synovial fluid can increase up
to 70 times to 140 pg/mL in human^[38]1,[39]2 and 7 times to 6 ng/mL in
mice^[40]3. Overexpression of IL-1β protein is also observed in
chondrocytes of early osteoarthritic cartilage^[41]4,[42]5. High level
of IL-1β aggravates the catabolic activities of synovial cells and
chondrocytes^[43]6,[44]7 and stimulates chondrocytes to enter an
abnormal phenotypic shift, such as proliferation of pre-chondrocytes,
swelling of mature chondrocytes, and hypertrophic differentiation of
cells in deep zone^[45]8,[46]9. Associated with these changes are the
increased release of enzymes from chondrocytes, such as the MMP (matrix
metalloproteinases) and ADAMTS (a disintegrin and metalloproteinase
with thrombospondin motifs) families. Thus acute inflammatory attack
often results in the degeneration of healthy cartilage^[47]7,[48]10.
Due to its important role in OA pathology, IL-1β-treated articular
cells or tissues have been widely adopted as in vitro models to study
OA initiation or PTOA^[49]6,[50]11. For both isolated and in situ
chondrocytes, IL-1β has been observed to induce transient concentration
changes of intracellular calcium ([Ca^2+][i]) and small GTPases. The
coordination of Rho GTPases signaling and [Ca^2+][i] signaling plays a
fundamental role in cytoskeleton organization, regulating the
chondrocyte phenotypic shift and cartilage ECM homeostasis^[51]8,[52]9.
Many of these studies focused on specific genes/pathways in
chondrocytes. A systematic study to clarify the effects of IL-1β on the
entire gene expression profile and signaling transduction coordination
remains lacking. In this study, we put special focus on the healthy
chondrocytes under intensive inflammatory cytokine attack, which is a
common scenario during acute inflammation phase after traumatic
injuries, such as ACL rupture or meniscus tear in the knee joint.
The objective of this study is to obtain a complete list of gene
expression changes in the healthy chondrocytes that are subjected to
acute inflammatory attack. To maintain the natural environment of
chondrocytes and perform longitudinal evaluation of cells and ECM,
fresh cartilage explants were cultured in vitro and treated with IL-1β.
We performed RNA sequencing analysis on the treated chondrocytes, as
well as the enrichment analysis in KEGG curated pathways. To verify and
understand the changes in genetic profiles, we also tracked the loss
and synthesis of ECM components longitudinally, the poroelastic
properties of ECM, the proliferation of cells, the change of cell
volume and calcium signaling of in situ chondrocytes.
Results
Cartilage Degradation Induced by IL-1β
To assess the direct effects of IL-1β on cartilage structure integrity,
we tracked the ECM contents loss using an in vitro culture cartilage
explant model (Fig. [53]1a). Cylindrical cartilage explants were
harvested from femoral condyle head of calf knee joints and cultured in
chemically defined medium up to 24 days. IL-1β was supplemented into
the culture medium, inducing the loss of sGAG content from cartilage
explant in a dosage- and time-dependent manner (Fig. [54]1b). After
8-day treatment, IL-1β at concentration of 10 ng/mL and 25 ng/mL
resulted in greater than 90% sGAG loss from the cartilage respectively
(Fig. [55]1b). In contrast, 1 ng/mL IL-1β induced a stable, almost
linear sGAG loss accumulating up to 50% on day 8 (46.0 ± 6.4% vs.
13 ± 1.2% in the control group) (Fig. [56]1b). According to these data,
we adopted 1 ng/mL IL-1β treatment in the following tests. In mouse
knee joint, the concentration of IL-1β can reach 6 ng/mL after
traumatic damage^[57]3. IL-1β concentration of 1 ng/mL has been adopted
widely in previous studies using animal cartilage
samples^[58]12,[59]13. Loss of collagen content induced by 1 ng/mL
IL-1β remained at a low rate on the first 8 days and significantly
accelerated afterwards (Fig. [60]1c). After 24 days, collagen loss
accumulated to 32.7 ± 4.3% (control: 4.9 ± 0.5%). IL-1β at 1 ng/mL
showed no significant effects on the sGAG or collagen synthesis rate of
the in situ chondrocytes (Fig. [61]1d,e).
Figure 1.
[62]Figure 1
[63]Open in a new tab
Effects of IL-1β during the long-term in vitro culture of cartilage
explants. (a) Schematic diagram of experimental design. Cylindrical
cartilage explants (diameter = 3 mm, thickness = 2 mm) were harvested
from the central region of femoral condyle head of bovine knee joint
using a biopsy punch. (b) Accumulative loss of sGAG content from
cartilage explants during 8-day treatment of different doses of IL-1β.
The accumulative sGAG loss was defined as the total sGAG released into
the culture medium divided by total sGAG content in explant and culture
medium. (c) Accumulative collagen loss from cartilage explant in 24-day
treatment of 1 ng/mL IL-1β. (d) The total sGAG content of cartilage
explants, which includes the sGAG released into medium (white bar) and
sGAG left in explants (grey bar) at the end of 8-day culture. (e) The
total collagen content and distribution of collagen in medium and
explant after 24-day culture. (f) Safranin O staining of the
superficial zone of cartilage explant after 2- and 8-day culture. Scale
bar = 200 µm. (g) Mechanical properties of cartilage explants after
8-day IL-1β treatment, including equilibrium Young’s modulus, dynamic
modulus, and permeability. All data were shown as mean ± 95% confidence
intervals. * vs control, p < 0.001 if otherwise marked.
Safranin O staining revealed the spatial pattern of sGAG loss across
the cartilage explant (Fig. [64]1f). After mere 2-day IL-1β treatment,
sGAG loss can be noticed in the surrounding areas of the treated
explant. The difference between the control and treated samples became
even more evident on day 8. Loss of sGAG content showed a sharply
increasing gradient along the center to edge direction. Such
inhomogeneous sGAG loss pattern can significantly compromise the tissue
stiffness at small strain, as the outer layer with low sGAG content has
little resistance to compression and will be easily compressed under
small loading. Mechanical properties of cartilage explants reflected
the degradation and loss of ECM components. After 8-day IL-1β
treatment, both Young’s modulus (treated vs. control: 0.09 ± 0.02 vs.
0.21 ± 0.03 MPa) and dynamic modulus (4.14 ± 0.38 vs. 5.89 ± 0.7 MPa)
decreased significantly, while the hydraulic permeability of the tissue
increased by over 150% (treated vs. control: 3.71 ± 0.81 × 10^−15 vs.
1.43 ± 0.32 × 10^−15 m^4/N·s) (Fig. [65]1g). No significant differences
in mechanical properties were detected after 2-day IL-1β treatment
(Supplementary Fig. [66]1).
Differentially Expressed Genes (DEGs)
RNA sequencing was used to assess the early gene expression profile of
in situ chondrocytes under the stimulation of IL-1β (a full gene list
in Supplementary Data). Compared to the healthy control, IL-1β
treatment induced 2,232 DEGs (absolute value of fold change > 2 and
FDR < 0.05) in the chondrocytes, among which 1,259 genes were
upregulated (fold change =
[MATH: expressionlevelinIL−1βexpressionlevelincontrol :MATH]
) and 973 genes downregulated (fold change =
[MATH: −expressionlevelincontrolexpressionlevelinIL−1β :MATH]
). The heat map of DEGs is composed of four sharply isolated blocks
that indicated the prominent effects of IL-1β on chondrocyte
transcriptional profiles (Fig. [67]2a). Fold changes of all the genes
were analyzed in Fig. [68]2b. In total, 12,655 genes were mapped, with
17.6% DEGs. The fold changes of MMP-1, MMP-9, MMP-13, ADAMTS-4,
ADAMTS-5, ACAN, and COL2A1, determined by the qRT-PCR and RNA
sequencing were highly consistent, with a Pearson’s R value of 0.99 and
an adjusted R^2 of 0.97 (Fig. [69]2c). IL-1β-induced expression changes
in catabolic genes (MMPs and ADAMs) were hundreds of folds and much
higher than the changes (a few folds) of anabolic genes (COL2 and
ACAN)^[70]12,[71]14.
Figure 2.
[72]Figure 2
[73]Open in a new tab
Summary statistics of RNA sequencing data. (a) Unsupervised
hierarchical clustering was performed for differentially expressed
genes (absolute value of fold change >2 and FDR < 0.05) in the IL-1β
treated cartilage explants. (b) Distribution of gene expression fold
changes according to log[2] transformation. (c) Validation of RNAseq
results with qRT-PCR. Seven selected genes were tested and correlated
with the Pearson’s correlation analysis. (d) Enrichment analysis of the
protein family classification and Gene Ontology annotations in terms of
(e) molecular functions and (f) biological processes.
Matrix Protein Related DEGs
According to previous studies, we summarized the cartilage-specific
biological function of each gene from collagen, proteoglycan, MMP and
ADAMTS family in Supplementary Table [74]1. The expression levels of
collagen of type II (COL2A1), type IX (COL9A1, 2, 3), and type XI
(COL11A1, 2) were significant in the healthy cells with RPKM over
10,000 (for reference, RPKM of actin: 1137.52). Expression changes of
COL9 and COL11 were decreased by more than 2 folds by IL-1β, and COL2
was decreased by 1.82 folds (FDR < 0.05, RPKM of control: 224154.47).
The pericellular matrix (PCM) genes, COL6A1 (3.89 folds) and COL6A2
(4.03 folds) were promoted; while the perlecan (HSPG2) and COL6A3 (the
most abundant COL6 gene) were not significantly altered by IL-1β. Only
three proteoglycan related genes were significantly changed: aggrecan
(ACAN: −2.33 folds, RPKM of control: 55400.32), hyaluronan (HAS2: −2.49
folds, RPKM of control: 81.56) and decorin (DCN: 2.68 folds, RPKM of
control: 336.41). No significant changes were detected in other
proteoglycans, e.g., biglycan (BGN, RPKM: 6514.63), fibromodulin (FMOD,
RPKM: 7487.12), lumican (LUM, RPKM: 254.16) or perlecan (HSPG2, RPKM:
2399.10). The expression changes of cartilage growth factors that
regulate the chondrocyte anabolic activities were summarized in
Supplementary Table [75]2.
Expression levels of MMPs could vary between species^[76]15. Our data
suggested that IL-1β promoted the expression of MMP-3 (188.78 folds),
MMP-9 (6.77 folds), and MMP-13 (41.48 folds) in bovine chondrocytes.
MMP-1, which is essential for the pre-processing of pro-collagens, was
also changed by 317.73 folds, but its RPKM value in the control was as
low as 0.19. MMP-28, which maintains cell adhesion to ECM network, was
downregulated by 2.5 folds. MMP-2, 14 and 16, which maintain the
routine cartilage ECM remodeling, were highly expressed in bovine
chondrocytes but not changed by IL-1β (absolute value of folds change
< 2 or FDR > 0.05). The most upregulated ADAMTSs included ADAMTS-4
(16.37 folds), ADAMTS-5 (82.62 folds), and ADAMTS-7 (12.29 folds).
IL-1β decreased the expression of ADATMS-3 and ADAMTS-14, which play
essential roles in processing the precursor of type II collagen for
fibril formation^[77]16. ADAMTSL-4, a barely studied gene in
chondrocytes, was downregulated by 12.71 folds by IL-1β and showed the
highest mRNA level (RPKM: 106.97) among the ADAMTS family.
Enrichment Analysis of Gene Ontology and Pathway
To understand the functions and interconnections of the 2,232 DEGs,
enrichment analysis in terms of protein family classification and Gene
Ontology (GO) annotation was performed. Gene family definition and a
gene’s function are described using GO annotation based on the
experimental findings in literature. The enriched protein families
included microtubule binding protein, chemokine, G-protein coupled
receptor, intracellular calcium sensing protein, and cytokine receptor
(Fig. [78]2d). The enriched molecular functions, including G-protein
coupled receptor activity, calmodulin binding, and cytokine activity,
were promoted; while the motor activity was mainly inhibited
(Fig. [79]2e). In the enriched biological process families,
hemopoiesis, immune response, stress response, apoptotic process, and
cell communication were all significantly upregulated (Fig. [80]2f).
The pathway enrichment analysis identified 20 IL-1β-changed pathways.
For each pathway, the number of related DEGs and the associated P value
were reported (Table [81]1). The cell cycle pathway was interrupted by
IL-1β, which was confirmed by our experimental observations. In the H&E
staining images, proliferation of pre-chondrocytes was obvious in the
superficial zone, where many cells aggregated into cell clusters. In
the center of explant, IL-1β induced a significant cell volume increase
(Fig. [82]3a). Confocal imaging showed 68% cell volume increase in the
IL-1β treated group (control vs. IL-1β, 2.27 ± 0.59 × 10^3 vs.
3.83 ± 0.84 × 10^3 µm^3) (Fig. [83]3b). MTT assay showed that the
proliferation rate of the primary chondrocytes doubled in the
IL-1β-supplemented Medium (Fig. [84]3c). RNAseq revealed that IL-1β
suppressed the negative regulators of chondrocyte hypertrophy,
including insulin-like growth factor (IGF2: −3.36 folds), pappalysin
(PAPPA: −2.49 folds; PAPPA2: −4.52 folds), and IGF-binding protein
(IGFBP2: −5.41 folds). Plate derived growth factor (PDGF) pathway can
promote the chondrocyte proliferation^[85]17, among which 55 related
genes were significantly changed, such as PDGFC (16.76 folds) and PDGFD
(3.67 folds) (Table [86]1).
Table 1.
Pathway enrichment analysis identified 20 child pathways (sub level 2
to 3) that are significantly changed by IL-1β.
Group Reactome pathway Gene number FDR
Cell cycle Cell cycle, mitotic 108 0.00
Chromosome maintenance 23 0.00
G2/m checkpoints 21 0.02
Cell-cell communication Cell-cell communication 26 0.06
Cellular responses to stress Senescence-associated secretory phenotype
14 0.10
DNA replication DNA replication 26 0.03
ECM organization Degradation of the extracellular matrix 33 0.00
Collagen formation 28 0.00
Integrin cell surface interactions 19 0.02
Elastic fiber formation 13 0.02
ECM proteoglycans 15 0.03
Laminin interactions 9 0.05
Non-integrin membrane-ECM interactions 12 0.06
Immune system Toll-like receptors cascades 37 0.00
Cytokine signaling in immune system 95 0.00
Metabolism Glycosaminoglycan metabolism 26 0.03
Metabolism of proteins Regulation of IGF transport and uptake by IGF
binding proteins 8 0.03
Programmed cell death Caspase activation via extrinsic apoptotic
signaling 10 0.03
Signal transduction Rho GTPases signaling 72 0.00
PDGF signaling 55 0.08
[87]Open in a new tab
The child pathways of FDR < 0.1 were selected and grouped together if
belonging to one parent (top-level, left column) pathway.
Figure 3.
[88]Figure 3
[89]Open in a new tab
Chondrocyte swelling and proliferation. (a) H&E staining of cartilage
explants after 2- and 8-day IL-1β treatment. Cell swelling, also termed
as fattening, and proliferation can be observed after 8-day IL-1β
culture. Scale bar = 200 µm. (b) Cell volume of chondrocytes in
cartilage explants measured by 3D confocal imaging. Scale bar = 30 µm.
(c) Cell proliferation rate determined by MTT assay. (d) Visualization
of Ras superfamily signaling pathway regulated by IL-1β-treatment.
Flowchart was plotted using RNAseq data in Pathview (version 3.6). Each
node represented a gene, and the associated node color represented the
expression changes in log2 ratios. For better readability, multiple
genes with similar or redundant functional roles were pooled together
as a single node. Absolute value of the maximum of expression changes
of these genes was reflected by the node color. All data were shown as
mean ± 95% confidence intervals. * vs control, p < 0.001 if otherwise
marked.
The course of cell cycle is highly correlated with cytoskeleton
metabolism^[90]18. A summary of all cell cycle related DEGs was
provided in Supplementary Fig. [91]S2 (a). Rho/Ras GTPases are
essential “molecular switches” for the cytoskeleton organization of
chondrocytes^[92]8,[93]19. Expression change of Rho/Ras signaling
related genes was summarized in the flow chart of Fig. [94]3d, where
each node represents a gene, and node color indicates the expression
changes revealed by our RNAseq. If multiple genes with similar or
redundant functional roles were pooled together as a single node, the
node color was indicated by the absolute value of the maximum
expression changes of these genes. In Rho/Ras signaling, 72 genes were
changed significantly, such as Ras-related GTPase effector (RALGDS:
9.37 folds), Ras homolog family gene (RHOQ: 2.92 folds), Ras-Rab
interactor (RIN1: 5.28 folds), R-Ras gene (RRAS: 2.31 folds), and Rho
GTPase activating protein (ARHGAP10: 2.91 folds). These data revealed
the important role of Rho/Ras GTPases in the mediation of the catabolic
signaling pathways in chondrocytes.
IL-1β changed the expression of 95 (19.4%, 489 in total genes) genes in
cytokine signaling pathway and 37 (28.0%, 132 in total) genes in
toll-like receptors (TLRs) cascades pathway. The basal expression
levels of cytokine genes in healthy chondrocytes were low (RPKM:
0.03–5.24), while IL-1β significantly increased the expressions of
IL1B, IL6, IL34 by >30 folds. A central pro-inflammatory regulator,
NF-κB, was significantly promoted (NFKB2: 7.11 folds; and NFKB1: 4.46
folds). TLR2, 3, 4, 6, 7, and 10 showed similar basal expression levels
in healthy chondrocytes. TLR2 (160.66 folds, RPKM: 1.49) and TLR4 (6.14
folds, RPKM: 1.72) were upregulated by IL-1β, similarly as reported in
literature^[95]20,[96]21. In addition, eight essential pathway networks
(e.g., cell cycle, autophagy, chemokine signaling, and JAK-STAT
signaling) with gene expression changes were presented in Supplementary
Fig. [97]S2, as well as their specific roles in cartilage metabolism
and OA pathology.
Intracellular Calcium Signaling
Using our RNAseq data, we specifically looked into a fundamental
pathway, [Ca^2+][i] signaling pathway, which regulates a wide range of
biological processes, such as cell metabolism and morphology
(Fig. [98]4a). The expression changes of 24 genes related to 6
calcium-signaling pathways were listed in Table [99]2. Transient
receptor potential vanilloid 4 (TRPV4) was substantially expressed in
healthy chondrocytes and promoted by 1.31 folds by IL-1β (RPKM: 159.77,
FDR < 0.05), while TRPV1, 2, and 3 were barely expressed in bovine
chondrocytes (RPKMs < 1). Piezo-type mechanosensitive ion channel,
PIEZO1 and PIEZO2, had similar mRNA levels in chondrocytes (RPKM:
107.22 and 152.53). PIEZO1 was upregulated by 4.11 folds by IL-1β, with
PIEZO2 unchanged (−1.13 folds). Voltage-Gated Calcium Channels (VGCCs)
were significantly regulated by IL-1β, including N-type channel
(CACNA1B: −15.34 folds, RPKM: 2.44), R-type channel (CACNA1E: −3.13
folds, RPKM: 11.72), and L-type channel (CACNA1C: 5.2 folds, RPKM:
4.13). T-type VGCC (CACNA1H) had no significant change despite its
abundant mRNA level in chondrocytes (1.04 folds, 121.86 RPKM). A VGCC
auxiliary subunit, CACNA2D1, was the most expressed VGCC gene in
chondrocytes and suppressed by IL-1β (−1.72 folds, RPKM: 232.39).
Figure 4.
[100]Figure 4
[101]Open in a new tab
Calcium signaling of chondrocytes in cartilage explants. (a)
Visualization of calcium signaling pathway regulated by IL-1β. (b)
Illustration of calcium signaling. Half cartilage explant was dyed with
fluorescent indicator and imaged on a confocal microscope for
15 minutes. Due to the fluctuation of [Ca^2+][i] concentration, image
intensity of chondrocyte oscillates in recorded video (see
supplementary material for calcium signaling video). (c) Definition of
spatiotemporal parameters of [Ca^2+][i] peaks. (d) Representative
[Ca^2+][i] curves of in situ chondrocytes from the control group and
IL-1β treated group. (e) Parameters of [Ca^2+][i] peaks, including the
average number of [Ca^2+][i] peaks, magnitude of peaks, time to reach a
peak, and time between neighboring peaks. All data were shown as
mean ± 95% confidence intervals. * vs control, p < 0.05 if otherwise
marked.
Table 2.
Summary of key genes involved in six calcium-related pathways.
Calcium-related pathway Gene symbol RNAseq gene expression
Fold change (IL-1β/CTRL) RPKM (CTRL) RPKM (IL-1β) FDR
Mechano- sensitive channel TRPV4 + 1.31 159.77 209.80 0.09
PIEZO1 + 4.11 107.22 440.92 0.00
Ligand-gated ion channel GRIN2C + 2.19 0.67 1.47 0.06
GRIN2D + 2.29 10.32 23.61 0.00
GRINA + 2.59 21.95 56.82 0.00
Voltage-sensitive calcium channels CACNA1B — 15.34 2.44 0.16 0.00
CACNA1E — 3.13 11.72 3.75 0.03
CACNA2D1 — 1.72 232.39 135.42 0.00
CACNA1C + 5.20 4.13 21.44 0.00
Purinergic receptor P2RX6 — 3.69 1.88 0.51 0.00
P2RX4 — 1.43 20.30 14.21 0.03
P2RY2 — 1.36 12.66 9.28 0.06
P2RX5 + 1.97 4.95 9.73 0.00
PLC-IP[3] PLCH1 — 14.64 3.08 0.21 0.00
PLCD1 — 2.29 297.00 129.71 0.00
PLCE1 — 2.26 179.13 79.20 0.00
PLCD3 + 2.27 4.75 10.76 0.00
PLCH2 + 2.29 1.08 2.48 0.02
PLCH2 + 2.29 1.08 2.48 0.02
ITPRIP + 3.37 8.19 27.62 0.00
ITPR3 + 6.35 0.79 4.99 0.00
ER store STIM2 — 1.64 41.68 25.43 0.00
STIM1 + 1.31 14.45 18.89 0.10
ORAI2 + 1.95 2.96 5.76 0.01
[102]Open in a new tab
The “−” represents downregulation, and “+” represents upregulation.
To evaluate the ultimate effects of IL-1β on chondrocyte [Ca^2+][i]
signaling, we recorded the spontaneous [Ca^2+][i] signaling of in situ
chondrocytes after 2-day IL-1β treatment (Fig. [103]4b). Typical
[Ca^2+][i] oscillations of in situ chondrocytes were shown in
Fig. [104]4c,d and and videos in Supplemental Movie. 203 out of 661
(30.7 ± 0.14%) cells showed spontaneous [Ca^2+][i] oscillation in the
control explants, a significantly higher responsive rate than that in
the IL-1β treated samples (87 out of 621 cells, 14.0 ± 0.11%)
(Fig. [105]4e). For each responsive cell, the spatiotemporal parameters
of [Ca^2+][i] peaks were also significantly altered by IL-1β. The
magnitude of [Ca^2+][i] peaks was reduced (control vs. IL-1β:
5.78 ± 1.01 vs. 2.98 ± 0.87), and the number of multiple peaks was
decreased (control vs. IL-1β: 3.55 ± 0.3 vs. 2.19 ± 0.27). The time
between two neighboring peaks (control vs. IL-1β: 111.26 ± 5.12 vs.
175.70 ± 13.24) and the time to reach a peak from baseline (control vs.
IL-1β: 17.58 ± 2.78 vs. 33.2 ± 6.62) were both significantly prolonged
by IL-1β, indicating slow Ca^2+ transport (Fig. [106]4e).
Discussion
Previous studies have attempted to evaluate the large-scale gene
expression profile of chondrocytes using various OA models, including
monolayer chondrocytes^[107]13,[108]22, cartilage of human knee
joints^[109]23,[110]24, and cartilage from OA animal
models^[111]14,[112]25. An important finding of these studies is that
chondrocytes present declined transcriptomic changes along with OA
progression. Chondrocytes demonstrate drastic genetic changes before
cartilage shows visible degradation, whilst they adapt to the
degenerated extracellular environment with little chondrogenic features
at the late stage of OA^[113]5,[114]24. For instance, cartilage cells
at late OA stage barely express catabolic genes, such as MMPs and
ADAMTSs^[115]24. A detailed comparison between our RNAseq data and that
of late-stage OA cartilage found only 214 (9.26%) genes are sharing the
same trend (upregulation/downregulation)^[116]24. In contrast, high
consistency was observed between our RNAseq data and those from
chondrocytes at early-stage OA. Our study has 74% consistency with a
microarray study of healthy human chondrocytes subjected to 96-hour
IL-1β treatment^[117]13 and 67% consistency with a study of early
degenerative human cartilage^[118]26. A full comparison lists between
our RNAseq data and these two studies were provided in Supplementary
Tables [119]3,[120]4. There was 48% consistency with another gene
expression profile of a middle-stage OA model (rats at 4 weeks post ACL
rupture, 722 DEGs)^[121]25. To the best our knowledge, our RNAseq
outcome is also highly consistent with a number of specific chondrocyte
pathway studies, e.g., NFAT^[122]27, EGFR^[123]28,
IGF^[124]12,[125]19,[126]29, and TRPV^[127]30 pathways. Therefore, a
major contribution of our RNAseq study is to generate a complete list
of genes of chondrocyte in response to acute joint inflammation. This
study identified 2,232 DEGs and 10,423 non-DEGs, with information about
each gene’s basal expression level at healthy status and the relative
expression fold change in response to the IL-1β stimulation. The
expression changes identified by the RNAseq are highly consistent with
those reported in specific pathway or cartilage inflammation mechanism
studies. Thus the present RNAseq data set can serve as a unique source
to verify individual gene or pathway studies and provide guidance for
future PTOA research.
In cartilage ECM, aggrecan, the major proteoglycan, forms
supramolecular aggregates with hyaluronan and is entrapped in the
collagen II/IX/XI fibrillar network^[128]31. The nonaggregating
proteoglycans, i.e., fibromodulin, biglycan, decorin and lumican, can
bind with various types of collagens and regulate the formation of
fibril networks^[129]32. This specialized structure endows cartilage
with its biomechanical properties for joint loading. In our RNAseq
data, IL-1β significantly reduced the gene expression of aggrecan
(ACAN) and hyaluronan (HAS2); however, the expression of type II
collagen (COL2A1), fibromodulin (FMOD), biglycan (BGN), and lumican
(LUM) showed no significant changes. These results corroborate previous
observation that the degradation of proteoglycan aggregates is prior to
that of collagen network at the early stage of OA
initiation^[130]12,[131]32. Decorin, another important nonaggregating
proteoglycan, is known as an important regulator of collagen fibril and
proteoglycan assembly^[132]32. Upregulation of decorin was observed in
our RNAseq data as well as previous early-OA studies^[133]33. This
response has been regarded as an attempt by chondrocytes to increase
the adhesion between fragmented aggrecans, thereby delaying its loss
from cartilage. During OA progression, the PCM of chondrocytes, which
is mainly composed of type VI collagen and perlecan, also presents
aberrant remodeling process^[134]34. The drastic PCM degradation is
associated with and may be responsible for the phenotypic shift of
chondrocytes. In the early OA stage, type VI collagen is increasingly
expressed although its fibrils is disorganized with compromised
density^[135]35. Our RNAseq data also showed the upregulation of all
three collagen VI isoforms (COL6A1, 2, and 3) in the IL-1β-treated
cartilage samples. Perlecan, co-localized with type VI collagen in the
PCM, plays an important role in regulating chondrocyte anabolic
activities. The retention of growth factors by perlecan, such as
fibroblast growth factor (FGF2) and bone morphogenetic proteins
(BMP2/7), has been shown to promote chondrogenic differentiation and
matrix production^[136]35. WARP (von Willebrand factor A domain-related
protein), a newly identified component of chondrocyte PCM, can also
interact with perlecan to contribute to the assembly and maintenance of
cartilage structures during cartilage development^[137]36. In our
RNAseq result, neither perlecan (HSPG2) or WARP (VWA1) genes was
significantly changed by the IL-1β treatment, indicating no drastic
changes of their synthesis at the acute inflammation stage. Taken
together, these ECM and PCM proteins may play synergetic but unique
roles in regulating cartilage homeostasis, thus presenting distinct
responses to acute inflammation stimulation.
An important finding from the present study was that IL-1β can induce
significant changes in the [Ca^2+][i] signaling of chondrocytes. As
illustrated in Fig. [138]4a, [Ca^2+][i] signaling can be activated
partially through G-protein coupled receptors (GPCRs), an important
mediator regulating chondrocyte morphology. In both proliferating and
hypertrophic chondrocytes, the expression of GPCRs and regulators of
G-protein signaling (RGS) increases markedly^[139]22,[140]37. According
to RNAseq data (Fig. [141]4a), the GPCR- and RGS-family genes were
differentially regulated by IL-1β, such as GPR84 (133.07 folds), GPR68
(−4.81 folds), RGS8 (80.49 folds), and RGS22 (−5.31 folds), with
parallel changes of downstream calcium-related genes including PLC- and
adenylyl cyclase-(ADCY) family genes. Another initiation mechanism of
[Ca^2+][i] signaling is through the ion channels on plasma membrane. In
chondrocytes, TRPV4 and PIEZO1 were recently identified as two key
mechanosensitive ion channels. TRPV4 and PIEZO1 can be disturbed by
inflammatory mediators, further inducing ECM
degeneration^[142]30,[143]38,[144]39. According to our RNAseq data,
TRPV4 and PIEZO1 were two highly expressed genes in healthy bovine
chondrocytes, and both were changed by IL-1β with consistent trends as
reported previously^[145]30,[146]39. T-type VGCC, which exists mainly
in excitable cells (e.g., neurons and muscle cells) to facilitate
environmental calcium influx, plays an important role in regulating
[Ca^2+][i] signaling of chondrocytes under mechanical
stimulation^[147]40. Our previous study showed that inhibition of
T-type VGCCs can attenuate the OA-like phenotypes of chondrocytes by
reducing the expression of mechanical-stress responsive genes
Prostaglandin G/H synthase 2 (PTGS2) and osteopontin (SPP1)^[148]41. In
this study, RNAseq detected minor expression change in T-type VGCC gene
(CACNA1H), while both PTGS2 and SPP1 were promoted by 13.34 folds and
21.71 folds in the IL-1β-treated chondrocytes, respectively.
High level of IL-1β can shift chondrocytes towards an aberrant
phenotype, including cell swelling, proliferation or hypertrophy. Rho
GTPases, such as RhoA, Rac1, and Cdc42, are well recognized as crucial
regulators of chondrocyte cytoskeleton and cell cycle^[149]42. IL-1β
can affect the Rho family via [Ca^2+][i] signaling, leading to the
rearrangement of F-actin networks in chondrocytes^[150]8,[151]9.
Previous studies proved that overexpression of RhoA can suppress the
ECM synthesis and chondrogenic differentiation of the chondrogenic cell
line ATDC5^[152]42,[153]43. Insulin-like growth factors (IGFs), an
important anabolic factor in chondrocytes, can inhibit the abnormal Rho
GTPases activities and therefore protect the cytoskeleton
structure^[154]12,[155]19,[156]29. In our RNAseq data, IL-1β inhibited
the IGF-modulated signaling pathway via suppressing IGF2 and IGF2R,
with parallel decreases of PLCs genes (PLCH1, PLCD1, and PLCE1).
Another growth factor that can activate Rho signaling is called
platelet-derived growth factors (PDGFs), which is also involved in the
chondrocytes proliferative process^[157]17,[158]44. IL-1β promoted the
PDGFC by 17 folds and PDGFD by 3.67 folds. Taken together,
IL-1β-induced chondrocyte phenotypic shift is related to the Rho
GTPases signaling, whose regulatory effects are related to IGF and
PDGF.
A few limitations of this study should be noted. First, due to the
limited access to healthy young human cartilage, bovine cartilage
samples were used in this study. Nevertheless, this substitution can at
least be partially justified by the greatest evolutionary conservation
present between human and cattle^[159]45. Second, to avoid synergistic
and interactive effects between cytokines, IL-1β was employed as the
cytokine to simulate the inflammatory attack on cartilage during acute
joint inflammation. Despite playing a central role in cartilage
degeneration, IL-1β alone cannot recapitulate the complexity of
multiple pro-inflammatory cytokines. A large number of cytokines are
active in the joints, such as TNF-α, IL-1 and IL-6
families^[160]6,[161]7. The genetic responses of chondrocytes revealed
in the present study could vary from the actual situations in
inflammatory joints. Third, chondrocytes’ reaction to cytokines is a
highly time dependent behavior. This study focused on the effects of
IL-1β on cartilage gene expression after 2-day treatment, whereas acute
joint inflammation could last 2–4 weeks after traumatic injuries (e.g.,
meniscus tear). Fourth, investigation of either cell cycle or calcium
signaling of chondrocytes could be complicated and have attracted
tremendous efforts. Experiments in this study were designed as a
verification of the ultimate effects of transcriptional changes induced
by IL-1β. The experiments also functioned as case studies to illustrate
the potential application of the RNAseq data in the verification and
guidance of future OA studies related to the acute inflammation after
joint trauma. For example, RNAseq data revealed that ACTB and GAPDH are
proper reference genes for the genetic analysis of bovine chondrocytes
(see Supplementary Table [162]5).
This study performed a detailed transcriptional analysis of
IL-1β-treated healthy cartilage, which provides genetic evidence for
the (1) short-term transcriptional responses and signaling
transductions in chondrocytes during acute inflammation; and (2) the
dysregulation of cell cycle and [Ca^2+][i] signaling transductions of
chondrocytes, both of which may play critical roles in modulating cell
phenotypic shift under inflammatory attack. The comprehensive
transcriptional profile identified here is highly consistent with other
high-throughput studies and specific pathway studies in literature
and thus may serve as useful guidance and verification for future
chondrocyte research.
Methods
Effects of IL-1β on Cartilage Matrix and Chondrocytes
Cartilage explant harvest
Overall experimental design was outlined in Fig. [163]1a. Fresh young
bovine knee joints (3–6 months old) were obtained from a local
slaughter house (Green Village, NJ). Cylindrical cartilage explants
(diameter = 3 mm, thickness = 2 mm) were harvested from the central,
load-bearing region of femoral condyle head using a biopsy punch
(Fig. [164]1a). After harvesting, samples were cultured in the
chondrogenic medium (DMEM, 1% ITS + Premix, 50 μg/mL L-proline, 0.9 mM
sodium pyruvate, 50 μg/mL ascorbate 2-phosphate) at 37 °C for 72 hours
before further experiments^[165]46,[166]47. After the balance to in
vitro environment, cartilage explants were cultured in the medium
supplemented with 1, 10, or 25 ng/mL bovine IL-1β recombinant protein
(RBOIL1BI, Thermo Fisher) for 8 days. Matching samples from the same
region on the condyle head were cultured in regular medium and served
as the non-IL-1β treated control.
Loss of sGAG and collagen contents over time
During the in vitro culture of cartilage explants (n = 10 explants from
5 animals per group), the culture medium (500 µL/sample) was changed
and collected every other day. The sGAG and collagen assay were
performed as described previously^[167]12,[168]48. The accumulative
sGAG or collagen loss was calculated as the sGAG or collagen released
into the medium divided by the sum of sGAG or collagen contents in both
explant and culture medium^[169]12. According to the temporal features
of IL-1β-induced ECM degradation from cartilage explant^[170]12, the
sGAG loss was measured during the first 8-day culture, while the
collagen loss was tracked for 24 days. Different cartilage explants
were used to track the losses of sGAG and collagen, respectively.
Histology
After 2- and 8-day culture, histological analysis was performed on
cartilage explants from the IL-1β treated and control groups (n = 2
explants from 2 animals per group). Explants were cut into 5-µm thick
sections along depth direction and stained with Safranin O (Sigma) and
Hematoxylin and Eosin Y (H&E, Sigma).
Cell swelling and proliferation
To evaluate the effect of IL-1β on chondrocyte volume, cartilage
explants cultured in the regular medium and IL-1β-Supplemented Medium
(n = 4 explants from 2 animals per group) were dyed with red
fluorescent cell tracker (Red CMTPX Dye, Thermo Fisher) and imaged on a
confocal microscope (Zeiss 510) after 4-day culture. The fluorescent
image stacks were reconstructed into a 3D image in Image J^[171]49. The
volume of in situ chondrocytes was registered and quantified (n ≈ 30
cells from each explant). To estimate the cell proliferation rate,
primary chondrocytes were extracted from cartilage explants (n = 4
explants from 2 animals per group). MTT assay was then performed
following the previous instructions^[172]50.
Mechanical properties
Unconfined compression test was used to longitudinally measure the
mechanical properties of the cartilage explants at days 2 and 8 (n = 10
explants from 5 animals per group; samples cultured for mechanical
testing only)^[173]51. During the test, a 10% strain was applied on the
cartilage sample at a constant speed followed by a 20-min relaxation
period. After the reaction force reached an equilibrium state,
sinusoidal dynamic loading was applied on the sample for 15 minutes at
0.5 Hz with a magnitude of ±1%^[174]52. Equilibrium Young’s modulus and
dynamic modulus of the samples were determined using the recorded
force. Hydraulic permeability of the tissue was obtained by
curve-fitting the stress relaxation curve using a nonlinear poroelastic
model for cartilage^[175]53,[176]54.
RNA Sequencing Analysis and qRT-PCR of IL-1β Treated Cartilage
Cartilage explants were assigned into two groups and cultured in: 1)
regular medium, and 2) 1 ng/mL IL-1β supplemented medium for 48 hours
(n = 4 explants from 4 animals per group). The cellular RNA was
extracted^[177]55. RNA samples with mass >2 μg and RIN score >6.5 were
qualified for the following RNA sequencing and qRT-PCR tests. The
sample size and quality test threshold were determined according to
literature guidelines^[178]56,[179]57.
RNAseq library of each sample was constructed from 1 μg of RNA using
the TruSeq® Stranded mRNA Sample Preparation Kit (Illumina). Samples
were pooled and sequenced on the Illumina HiSeq. 2500, and at least 13
million reads (51 bp single-end read) were generated for each sample.
Data processing was performed using CLC Genomics Workbench v7.5
(QIAGEN). The low-quality sequence ends (>Q15), sequencing adapters,
read with ambiguous nucleotides (>1 nucleotide), and short sequences
(<40 bp after trimming) were removed before mapping to the bovine
genome (version 4.6.1). Gene expression value was calculated as the
total number of unique reads mapped to the exon sequence and normalized
to reads per kilo base of transcript per million mapped reads (RPKM),
and RPKM should correlate positively to the expression level of each
gene. The differential expression of each gene was determined by the
generalized linear model with animal as a random variable^[180]58. A
stringent cutoff of False Discovery Rate (FDR)<0.05 and absolute fold
change >2 was adopted to identify the differentially expressed genes
(DEGs) between the IL-1β treated and the control samples.
Enrichment analysis of protein families and Gene Ontology annotations
on DEGs data set was performed by Panther Classification
System^[181]59. Enrichment analysis of pathways was performed using
Reactome software^[182]60. To remove redundancy, only the enriched
pathways from levels two to three defined by Reactome were reported and
grouped by the level one parent pathway. The KEGG pathways generated by
Pathview (version 3.6) were used to visualize the changes in a specific
signaling pathway^[183]61,[184]62.
To verify the RNA sequencing data, qRT-PCR was performed on the same
RNA used for sequencing. Expression levels of seven major metabolic
genes in chondrocytes were quantified, including aggrecan (ACAN), type
II collagen (COL2A1), MMP-1, −9, −13 and ADAMTS-4, −5. Gene expression
fold change was calculated using the 2(-Delta Delta C(T)) method after
data normalized to the average of reference gene (β-actin, ACTB).
Calcium Signaling of Chondrocytes
Spontaneous [Ca^2+][i] signaling of in situ chondrocytes in cartilage
explant was analyzed after 48-hour IL-1β treatment and compared to that
of control. After the IL-1β treatment, cartilage explants (n = 5) were
halved axially and dyed with 5 μM Fluo-8AM for 40
minutes^[185]47,[186]52. Dyed sample was placed in an imaging chamber
and mounted on a confocal microscope (Zeiss LSM510) (Fig. [187]4b).
Calcium images of the in situ chondrocytes located in the center of
cross section area were recorded every 1.5 seconds for 15 minutes.
[Ca^2+][i] signaling of chondrocytes was analyzed as we described
previously^[188]40,[189]52. The responsive percentage of cells was
calculated as the fraction of cells with one or more [Ca^2+][i] peaks
over the total number of cells. The spatiotemporal parameters of the
[Ca^2+][i] peaks, including the number of multiple peaks, the magnitude
of peaks, the time to reach a peak and the time interval between
neighboring peaks, were also measured and compared.
Statistical Analysis
Tukey’s Honestly Significant Difference test was performed following
the one-way ANOVA to compare the sGAG loss induced by multiple IL-1β
dosage. The Chi-square test was utilized to compare the responsive rate
of [Ca^2+][i] signaling. Student’s t-test was performed to compare all
the other data between the IL-1β (1 ng/mL) and control groups. All data
were shown as mean ± 95% confidence intervals. Statistical significance
was indicated when P value < 0.05.
Electronic supplementary material
[190]Supplementary Information^ (667.2KB, pdf)
[191]Dataset 1^ (854.3KB, xlsx)
[192]Download video file^ (256.9KB, mp4)
A video of typical calcium signaling of healthy cartilage
[193]Download video file^ (557.1KB, mp4)
A video of typical calcium signaling of IL-1β-treated cartilage
Acknowledgements