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27
HTO changes macrophage polarization in knee OA
Figure legends
Figure 1. Comparing gene expression profiles in the synovium at initial HTO and plate removal
by microarray analysis (n = 3) and real-time reverse transcription–polymerase chain reaction
(qRT-PCR) (n = 31). The volcano plot (A) shows the differential expression of 477 genes (red:
upregulated and blue: downregulated genes >2-fold change) compared with the synovium at
HTO. Representative Gene Ontology (GO) processes of downregulated (B) genes at plate
removal. Representative Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of
upregulated (C) and downregulated (D) genes at plate removal. The proportion of cells in the
synovium (E) was analyzed by deconvolution analysis with MuSiC using single-cell
sequencing data (27). (F) Gene expression (relative to GAPDH) of catabolic factors, proinflammatory and anti-inflammatory cytokines, chemokines, and macrophage surface markers
in synovial tissues at HTO (Pre) and plate removal (Post). qRT-PCR was performed in duplicate
and repeated twice. The same patients were connected by lines pre- and post-HTO for all 31
patients, and the three cases used for the microarray were marked in red. Bars indicate medians
and interquartile ranges. *: P <0.05, **: P <0.01, and ***: P <0.001 (Wilcoxon matched-pairs
signed-rank tests). HTO, high tibial osteotomy; Pre, initial HTO; Post, plate removal
Figure 2. Synovitis scores and macrophage phenotypes at high tibial osteotomy (HTO) (Pre)
and plate removal (Post). (A) Representative images of synovial tissue (ST) samples stained
28
HTO changes macrophage polarization in knee OA
with hematoxylin and eosin showing all three features of the synovitis scoring system. Black
scale
bars,
200
μm.
(B)
Synovitis
scores.
Representative
images
of
double
immunofluorescence staining of CD68 and macrophage polarization markers CD80 (C) and
CD206 (D) on STs. White scale bars, 50 μm. Percentages of CD80 (E) and CD206 (F) positive
vs. total macrophages. P values were determined by the Wilcoxon matched-pairs signed-rank
test. Bars in graphs indicate medians and interquartile ranges.
Figure 3. Stimulation of peripheral blood mononuclear cell (PBMC) derived macrophages and
fibroblast-like synoviocytes (FLSs) by cartilage fragments. Representative images (A) of
CD80-positive macrophages stimulated with cartilage fragments and stained with
immunofluorescence. Scale bars, 20 μm. Mean signal intensity (B) of CD80 in macrophage
cytoplasm (n = 5). Gene expression in PBMC derived macrophages (n = 4) (C) and FLSs (n =
5) (E) stimulated with cartilage fragments determined by real-time polymerase chain reaction.
The expression of RNA was normalized to those of TBP for macrophages and GAPDH for
FLSs. The concentration of inflammatory cytokines in culture supernatants of macrophages (n
= 4) (D) and FLSs (n = 5) (F) stimulated with cartilage fragments. (−) no cartilage fragments;
(+) and (++) low- and high-density cartilage fragments, respectively. Bars in graphs indicate
the mean and standard deviations. The experiments were performed in duplicate and repeated
twice. *P <0.05, ** P <0.01, *** P <0.001, by one-way analysis of variance and Tukey post29
HTO changes macrophage polarization in knee OA
hoc tests.
Figure 4. Associations between clinical scores and macrophage-related gene expression.
Spearman correlations between gene expression in synovial tissue (ST) and knee injury and
osteoarthritis outcome scores (KOOS): total score at plate removal (Post-op) (A) and between
gene expression in ST and ΔKOOS: total score between before high tibial osteotomy (HTO)
and at plate removal (B). Δ, delta (difference).
Figure 5. Summarized schema of the study. A possible mechanism underlying the beneficial
effects of the high tibial osteotomy might be that reducing load-bearing in the damaged medial
cartilage decreases cartilage fragment production that results in decreased inflammatory
cytokine expression and polarization to M2 macrophages.
30
HTO changes macrophage polarization in knee OA
Table
Table 1. Demographics showing characteristics of the study population.
n = 31
Pre (at HTO)
Post (at plate removal)
P-value
Age, years
61.1 ± 7.5
Sex, Female (n, %)
19, 61%
Body mass index, kg/m2
27.0 ± 4.0
KL grade 1/2/3/4, (n, %)
3(10)/10(32)/13(42)/5(16)
1(3)/9(29)/15(48)/6(19)
0.18
HKA angle, degree
7.8 ± 4.0 varus
0.9 ± 2.8 valgus
<0.001*
WBL ratio, %
14.9 ± 16.7
53.4 ± 11.4
<0.001*
KOOS, point
60.1 (47.0–69.6)
86.3 (75.6–91.1)
<0.001
ICRS grade 2/3/4, (n, %)
2(6)/15(48)/14(45)
8(26)/14(45)/9(29)
0.001
Medial JSW, mm
2.3 ± 1.7
2.8 ± 1.2
<0.001*
Lateral JSW, mm
6.1 ± 1.8
5.8 ± 1.5
0.07*
HTO, high tibial osteotomy; KL, Kellgren–Lawrence; HKA, hip–knee–ankle; WBL, weightbearing line; KOOS, Knee injury and Osteoarthritis Outcome Score; ICRS, International
Cartilage Repair Society; JSW, joint space width. P values were calculated with the Wilcoxon
matched-pairs signed-rank test except for * (Paired t-test).
31
Figure 1
Neutrophil chemotaxis
SLN
MMP3
Cellular response to interleukin-1
CCL18
CCL3
IL6
Chemokine-mediated signaling pathway
IL1B
MFAP5
MMP1
Cellular response to tumor necrosis factor
USP34
FOS
Downregulated GO terms
SCAF11
JUN
p-value (-log10)
TSNAX
Inflammatory response
COMP
Monocyte chemotaxis
p=0.05
down regulation
up regulation
-4
-2
-Log10(FDR)
fold change(log2)
Alzheimer's disease
Toll-like receptor signaling pathway
Parkinson's disease
Rheumatoid arthritis
Ribosome
TNF signaling pathway
Non-alcoholic fatty liver disease (NAFLD)
Cytokine-cytokine receptor interaction
Huntington's disease
NF-kappa B signaling pathway
10
10
Pre
Post
Pre
Post
Post
Post
fibroblasts
Pre
Post
Post
Pre
Post
CD206
✱✱
Pre
Post
CD80
CD68
Pre
Post
Pre
TNFA
Pre
✱✱
synovial intimal fibroblasts
10
CCL18
synovial subintimal fibroblasts
Post
IL6
15
Post
Pre
endothelial cells
Pre
✱✱✱
CCL3
Relative expression
IL10/GAPDH
15
Pre
✱✱✱
✱✱
20
Post
IL10
IL1RA
25
10
Pre
Post
Relative expression
CCL3/GAPDH
Pre
20
Relative expression
IL1B/GAPDH
20
T cells
20
20
Mast cells
40
IL1B
10
Relative expression
MMP13/GAPDH
30
30
Relative expression
MMP3/GAPDH
Relative expression
MMP1/GAPDH
Relative expression
ADAMTS4/GAPDH
40
MMP13
Relative expression
CD68/GAPDH
50
Relative expression
IL1RA/GAPDH
MMP3
MMP1
Relative expression
CCL18/GAPDH
ADAMTS4
smooth muscle cells
60
-Log10(FDR)
-Log10(FDR)
inflammatory macrophages
Relative expression
IL6/GAPDH
immune regulatory macrophage
80
Relative expression
CD80/GAPDH
proliferating immune cells
100
Relative expression
CD206/GAPDH
Salmonella infection
Relative percent of population
Oxidative phosphorylation
Cell population
Downregulated KEGG Pathways
Upregulated KEGG Pathways
Relative expression
TNFA/GAPDH
Pre
Post
Pre
Post
Figure 2
Pre
Synovitis score
p✱✱✱✱
< 0.001
Post
Pre
Lining cell layer
Synovial stroma
Pre
Inflammatory infiltrate
M1/Total macrophage (%)
Post
CD80
CD68
CD80
DAPI
CD68
Pre
Post
CD80
DAPI
100
p <✱✱✱
0.001
80
60
40
20
Pre
Post
Post
CD68
CD206
DAPI
CD68
CD206
DAPI
M2/Total macrophage (%)
CD206
p < ✱✱✱
0.001
50
40
30
20
10
Pre
Post
Figure 3
✱✱
ns
✱✱
CD80
Mean signal intensity
26
none
+ (low density)
C.
24
22
20
18
16
++ (high density)
++
Cartilage fragments
PBMC derived macrophage
IL1B
IL6
TNFA
CD68
CD80
CD206
✱✱
✱✱
20
10
D.
1.0
0.5
0.0
++
Cartilage fragments
1.5
1.0
0.5
++
0.0
++
++
++
Cartilage fragments
Cartilage fragments
Cartilage fragments
Cartilage fragments
Relative expression
CD206/TBP
30
Relative expression
CD80/TBP
40
Relative expression
CD68/TBP
1.5
2.0
Relative expression
TNFA/TBP
Relative expression
IL6/TBP
Relative expression
IL1B/TBP
50
++
Cartilage fragments
PBMC derived macrophage
IL-1β
IL-6
TNFα
✱✱✱
✱✱
✱✱
25
✱✱✱
ns
✱✱✱
ns
✱✱
600
30
400
20
15
10
pg/ml
pg/ml
pg/ml
20
200
10
++
Cartilage fragments
++
Cartilage fragments
++
Cartilage fragments
FLSs
IL1B
IL6
FLSs
IL-1β
TNFA
IL-6
TNFα
✱✱
0.5
0.0
++
Cartilage fragments
++
Cartilage fragments
80
✱✱
60
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
++
Cartilage fragments
400
pg/ml
1.0
600
pg/ml
1.5
2.0
2.0
pg/ml
2.0
Relative expression
TNFA/GAPDH
Relative expression
IL6/GAPDH
Relative expression
IL1B/GAPDH
2.5
2.5
200
20
++
Cartilage fragments
40
++
Cartilage fragments
++
Cartilage fragments
Figure 4
50
0.0
0.5
1.0
1.5
2.0
70
60
50
2.5
Relative expression
Post-CD68/GAPDH
40
20
0.5
-20
60
⊿KOOS score
⊿KOOS score
60
r=0.22 (-0.16 to 0.54)
p=0.25
1.0
1.5
2.0
Relative expression
Post-CD68/GAPDH
60
50
90
80
70
60
50
0.0
0.5
1.0
Relative expression
Post-CCL3/GAPDH
r=-0.37 (-0.65 to -0.01)
p=0.04
r=-0.08 (-0.43 to 0.29)
p=0.67
r=-0.02 (-0.38 to 0.35)
p=0.92
Relative expression
Post-CD80/GAPDH
60
40
40
20
-20
Relative expression
Post-CD206/GAPDH
20
0.5
-20
1.0
Relative expression
Post-CCL3/GAPDH
90
80
70
60
50
Relative expression
Post-CCL18/GAPDH
Relative expression
Post-CD206/GAPDH
60
100
1.5
Relative expression
Post-CD80/GAPDH
20
-20
70
40
2.5
80
Post-op KOOS score
60
80
90
100
r=0.40 (0.04 to 0.66)
p=0.03
60
⊿KOOS score
70
90
100
⊿KOOS score
80
Post-op KOOS score
90
100
⊿KOOS score
100
r=-0.01 (-0.37 to 0.36)
p=0.97
r=0.36 (0.00 to 0.64)
p=0.047
r=-0.36 (-0.64 to 0.00)
p=0.046
Post-op KOOS score
r=0.20 (-0.18 to 0.52)
p=0.28
Post-op KOOS score
Post-op KOOS score
r=0.12 (-0.26 to 0.46)
p=0.54
40
20
1.5
-20
Relative expression
Post-CCL18/GAPDH
Figure 5
Osteoarthritis
Post realignment
Synovium
Synovium
Anti inflammatory mediators
IL-10 ↑
IL-1RA ↑
CCL18 ↑
M2
macrophages
M2
macrophages ↑
IL-1β ↓
Fibroblast like
synoviocytes
M1
macrophages
synovitis ↑
IL-1β ↑
IL-6 ↑
CCL3 ↑
Fibroblast like
synoviocytes
Cartilage fragments ↑
Cartilage degradation
Cartilage fragments ↓
Cartilage degradation ↓
Cartilage
Cartilage
Cartilage
M1
macrophages ↓
synovitis ↓
IL-1β ↓
IL-6 ↓
CCL3 ↓
...
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