Rheumatology 2020;59:3070–3080

Rheumatology doi:10.1093/rheumatology/keaa192
Advance Access publication 17 May 2020

Original article
IL-37 inhibits M1-like macrophage activation to
ameliorate temporomandibular joint inflammation
through the NLRP3 pathway
Ping Luo1,2,3, Sisi Peng1,2,3, Yin Yan1,2,3, Ping Ji 1,2,3
and Jie Xu1,2,3

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Abstract
Objectives. IL-37 has been identified as an important anti-inflammatory and immunosuppressive factor. This study
was undertaken to explore how IL-37 affects M1/M2-like macrophage polarization and thus contributes to anti-
inflammatory processes in the temporomandibular joint.
Methods. Western blotting, quantitative real-time PCR (qRT-PCR) and immunofluorescence were used to verify the
IL-37-induced polarization shift from the M1 phenotype to the M2 phenotype, and the related key pathways were
analysed by western blotting. Human chondrocytes were stimulated with M1-conditioned medium (CM) or IL-37-
pretreated M1-CM, and inflammatory cytokines were detected. siRNA-IL-1R8 and MCC-950 were used to investigate
the mechanism underlying the anti-inflammatory effects of IL-37. Complete Freund’s adjuvant-induced and disc
perforation-induced inflammation models were used for in vivo studies. Haematoxylin and eosin, immunohistochemical
and safranin-O staining protocols were used to analyse histological changes in the synovium and condyle.
Results. Western blotting, qRT-PCR and immunofluorescence showed that IL-37 inhibited M1 marker expression
and upregulated M2 marker expression. Western blotting and qRT-PCR showed that pretreatment with IL-37 sup-
pressed inflammatory cytokine expression in chondrocytes. IL-37 inhibited the expression of NLRP3 and upregu-
lated the expression of IL-1R8. Si-IL-1R8 and MCC-950 further confirmed that the anti-inflammatory properties of
IL-37 were dependent on the presence of IL-1R8 and NLRP3. In vivo, IL-37 reduced synovial M1 marker expres-
sion and cartilage degeneration and increased M2 marker expression.
Conclusion. IL-37 shifting of the polarization of macrophages from the pro-inflammatory M1 phenotype to the
beneficial anti-inflammatory M2 phenotype seems to be a promising therapeutic strategy for treating temporoman-
dibular joint inflammation.
SC I E NC E

Key words: interleukin-37, macrophage, NLRP3, inflammation, cartilage

BASIC

Rheumatology key messages

. IL-37 shifts M1 macrophages into the M2 phenotype and has a protective effect on chondrocytes.
. IL-37 inhibits M1-like macrophage activation in complete Freund’s adjuvant-induced and disc-perforation-
induced temporomandibular joint inflammation models in synovium.
. IL-37 inhibits M1-polarized macrophages and partially ameliorates temporomandibular joint OA through NLRP3.

Introduction frequently affected by OA. Temporomandibular joint OA

(TMJOA), a prevalent chronic-pain disease, leads to syn-
The temporomandibular joint (TMJ), a synovial joint, ovial inflammation (synovitis), cartilage degeneration and
plays critical roles in complex jaw movements and is bone erosion [1, 2]. Over the past years, emerging evi-
dence has suggested that synovial inflammation is a
1
Department of Oral and Maxillofacial Surgery, College of driver of TMJOA progression [3, 4]. To date, there is nei-
Stomatology, Chongqing Medical University, 2Chongqing Key ther a preventive medicine to delay cartilage breakdown
Laboratory for Oral Diseases and Biomedical Sciences and
3
Chongqing Municipal Key Laboratory of Oral Biomedical nor a curative treatment.
Engineering of Higher Education, Chongqing, China In the TMJOA synovium, infiltrated macrophages con-
Submitted 26 December 2019; accepted 26 March 2020 tribute to synovial hyperplasia and secrete high levels of
Correspondence to: Jie Xu, Department of Oral and Maxillofacial inflammatory cytokines [5]. Macrophages play a critical
Surgery, The Affiliated Hospital of Stomatology, Chongqing Medical role in joint inflammation by creating an imbalance
University, No. 426, North Songshi Road, Yubei District, Chongqing between M1- and M2-polarized macrophages [6]. M1
401147, China. E-mail: xujie@hospital.cqmu.edu.cn

C The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com
V

macrophages, which are activated by IFN-c and lipo-
polysaccharide (LPS), are pro-inflammatory and are
characterized by the production of TNF-a, IL-1b, IL-6
and inducible NO synthase (iNOS), thus impairing the
TMJ [7]. In contrast, M2 macrophages are more likely to
play an anti-inflammatory role characterized by the pro-
duction of IL-10 and are beneficial in alleviating inflam-
mation [8, 9]. Thus, controlling the macrophage
phenotypic switch from M1 to M2 at specific time points
offers a promising solution for relieving inflammation.
The NACHT–LRR–PYD-containing protein 3 (NLRP3)
inflammasome, a multi-protein complex, contains Nod-
like receptor protein-containing pyrin domain (NLRP) and
caspase-1. This inflammasome induces the activation of
caspase-1, triggers inflammatory responses and stimu-
lates the secretion of pro-inflammatory cytokines, such
as IL-1b. Inhibition of caspase-1 activity is considered to
be a key therapeutic strategy for NLRP3 inflammasome-
associated diseases [10, 11]. IL-37, a member of the IL-1
family, has been reported to be a novel anti-inflammatory
cytokine. IL-37 can be secreted by innate immunocytes,
especially macrophages [12]. IL-37 has been reported to
play a critical role in regulating inflammatory and auto-
immune diseases [13, 14]. IL-37 has a caspase-1 cleav-
age site and translocates to the nucleus upon LPS
stimulation to down-regulate inflammatory cytokines,
and the nuclear translocation requires NLRP3-activated
caspase-1 cleavage [15]. It has been reported that IL-37
can inhibit inflammasome activation and disease severity
in murine aspergillosis [16]. IL-37 exerts anti-
inflammatory effects through suppression of the produc-
tion of inflammatory cytokines and promotion of the re-
lease of classic anti-inflammatory cytokines, such as IL-
10 [17]. In our previous study, IL-37 was demonstrated to
be expressed in the synovium and discs of patients with
TMJOA and in the cartilage of condylar fracture patients.
IL-37 was also highly expressed in the synovial fluid of
patients with synovitis [18].
However, to the best of our knowledge, no studies
have linked IL-37 to the function of macrophages in TMJ
inflammation. In addition, the mechanisms underlying the
effect of IL-37 on macrophage activation remain unclear.
In this study, we aimed to investigate whether IL-37 shifts
M1 macrophages into the M2 phenotype and explored
the effect of macrophage transformation on chondro-
cytes. We focused on the effect of this shift on complete
Freund’s adjuvant (CFA)- and disc perforation-induced in-
flammation in SD rats in vivo. This study may provide a
therapeutic target for the prevention and treatment of the
inflammation associated with TMJOA.
Methods
Ethical approval was obtained from the Committee of
Chongqing Medical University, and informed consent
was provided by all participants before human sample
collection. Tissue samples were collected and proc-
essed in accordance with the Declaration of Helsinki.
For the animal study, all animal experiments were
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IL-37 inhibits M1-like macrophage activation
approved by the Chongqing Medical University Ethics
Committee and performed according to institutional
guidelines.
Cell culture
A human monocyte cell line (THP-1) was cultured in
RPMI 1640 medium (Sigma-Aldrich, St Louis, MO, USA)
supplemented with 2 mM L-glutamine, 1% penicillin–
streptomycin, and 10% fetal bovine serum. Cells were
stimulated with 100 ng/ml phorbol 12-myristate-13-acet-
ate (Sigma-Aldrich) for 24 h. To induce the M1 pheno-
type in cells, cells were further incubated with 1 mg/ml
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lipopolysaccharide (LPS) (Sigma-Aldrich) and 50 ng/ml
IFN-c (Sinobiological, Beijing, China) in fresh medium.
For M2 phenotypic differentiation, acquired macro-
phages were pretreated with 0.1 ng/ml recombinant IL-
37 (hIL-37) (Sinobiological) for 2 h and then further incu-
bated with LPS and IFN-c in fresh medium. After 48 h,
the conditioned medium (CM) was collected by centrifu-
gation at 1000 g for 5 min and stored at 80 C.
Chondrocytes were isolated from patients with con-
dylar fractures, as described in our previous study [14].
The cells used in experiments were in passage 3. After
chondrocytes were seeded in culture plates, they were
treated with macrophage-CM (M0-CM, M1-CM, or
M1þIL-37-CM) diluted 1:1 with minimal essential me-
dium containing 10% fetal bovine serum. For gene ex-
pression analysis, samples were harvested after 24 h; for
protein expression analysis, samples were harvested at
48 h; and for a phosphokinase array, samples were har-
vested at 30 min.
Transfection of siRNA-IL-1R8
THP-1 cells were transiently transfected with a siRNA
targeting IL-1R8 (GenePharma, Shanghai, China) or a
negative-control siRNA (scrambled) (GenePharma) using
GP-siRNA-mate-plus transfection reagent (GenePharma)
in 2 ml of a-minimal essential medium according to the
manufacturer’s instructions. All experiments were per-
formed 48 h after transfection, and the most effective
single siRNA was used for further experiments.
Treatment with MCC-950 (an NLRP3 inhibitor)
THP-1 cells were incubated with the NLRP3 inhibitor
MCC-950 (10 mmol/l) (Selleck, Houston, Texas, USA) for
48 h and then further stimulated.
Western blotting
Cells were lysed with RIPA buffer containing phenylme-
thylsulfonyl fluoride (100:1). The total protein concentration
was measured using the Enhanced BCA Protein Assay Kit
(Beyotime, Shanghai, China). For western blot analysis,
30 lg of cell lysates were loaded for each sample and
separated by 10% SDS-PAGE. The proteins were trans-
ferred to polyvinylidene fluoride membranes (Millipore,
Billerica, MA, USA) and then incubated with antibodies
specific for CD86 (1:1000, Bioss, Shanghai, China), indu-
cible nitric oxide synthase (iNOS) (1:1000, Abcam,
3071
Ping Luo et al.
Cambridge, MA, USA), CD206 (1:1000, Abcam), MMP1
(1:1000, Bioss), MMP9 (1:800, Bioss), MMP13 (1:1000,
Proteintech, Wuhan, China), caspase-1 (1:1000, Genetex,
Irvine, CA, USA), NLRP3 (1:1000, Genetex), IL-1R8
(1:1000, Genetex), phospho (p)-extracellular signal-
regulated kinase (ERK) (1:1000, CST, Danvers, MA, USA)
and p-nuclear factor-jB (NF-jB) p65 (1:1000, CST,
Danvers, MA, USA) overnight at 4 C. Glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) (1:2000, Beijing,
Bioss) was blotted as the loading control. Images were
obtained and analysed with ImageJ software (NIH,
Bethesda, MD, USA).
Quantitative real-time PCR
For gene expression analysis, THP-1 cells and chondro-
cytes in six-well plates were collected. The cells were har-
vested and lysed in RNAiso plus (Takara, Nogihigashi,
Japan). Total RNA was collected and reverse transcribed
using the PrimeScriptTMRT Reagent Kit with gDNA Eraser
(Takara) to obtain cDNA. Quantitative real-time PCR (qRT-
PCR) was then performed with a PCR system (Bio-rad,
Hercules, California, USA) for 40 cycles using Power TB
green PCR Master Mix (Takara). GAPDH-specific primers
were used as an internal control. The relative expression of
target genes is reported using the 2DDCt method. The
sequences of the primers used for qRT-PCR in this study
are shown in (Supplementary Table S1, available at
Rheumatology online).
In vivo temporomandibular joint inflammation model
Sprague–Dawley (SD) rats were ordered from
Chongqing Medical University, housed in groups of four
and raised in a clean-grade room with a 12 h light–12 h
dark cycle. SD rats were kept in the animal facility for at
least 7 days before use and received food and water ad
libitum. Twenty-four SD rats (300–350 g) aged 12 weeks
were used in this study and divided into two sets for
CFA-induced and disc perforation-induced TMJ inflam-
mation modelling. The SD rats in each set were divided
into three groups: a sham-operation group (n ¼ 4), a
model group (n ¼ 4) and a treatment group (n ¼ 4). Then,
the treatment group was treated with a total volume of
50 ll of hIL-37 (Sino Biological, Beijing, China) at a dose
of 20 mg/ml (twice per week), and the model group was
treated with PBS. The next day, TMJ inflammation was
induced by injecting 50 ml of CFA (Sigma-Aldrich) into
the upper compartment of the unilateral TMJs. For the
disc perforation-induced TMJ inflammation model, disc
perforation was performed as previously described [18].
SD rats were randomly sacrificed at 1 week.
Postoperative complications did not occur. All sections
of this report adhere to the ARRIVE guidelines for
reporting animal research [19].
Histological staining
TMJ samples were harvested from all animals and fixed
in 4% paraformaldehyde overnight after sacrifice. After
decalcification in 10% EDTA (pH 7.2–7.4), the samples
3072
were embedded in paraffin and cut into 5-lm sections.
TMJ sections were stained with haematoxylin and eosin
(HE)(Solarbio, Beijing, China), safranin O and fast
green(Solarbio, Beijing, China) according to the manu-
facturer’s instructions.
Immunohistochemistry
Immunohistochemical staining for iNOS (1:200, Abcam),
CD206 (1:1000, Abcam), MMP9 (1:200, Bioss), and
MMP13 (1:200, Zen Bioscience, Chengdu, China) was
performed. Sections were incubated with the primary
antibody overnight at 4 C. The next day, horseradish
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peroxidase-conjugated secondary antibodies were used
for detection after incubating the slides in 0.3% H2O2 in
PBS for 15 min. PBS instead of the primary antibody
was added to negative controls. Diaminobenzidine was
used as the substrate for colour development. All slides
were counterstained with haematoxylin. TIFF images
were acquired with an Olympus microscope (Olympus,
Tokyo, Japan). An immunoreactivity scoring system was
applied as described elsewhere [20].
Immunofluorescence staining
Samples were fixed with 4% paraformaldehyde for 15 min.
After washing with PBS, sections were blocked with 10%
goat serum (Bioss) for 30 min and stained with an anti-
NLRP3 antibody (1:200, Genetex), anti-CD86 antibody
(1:200, Bioss), anti-CD 206 antibody (1:1000, Abcam), or
anti-iNOS antibody (1:250, Abcam) overnight at 4 C. The
sections were washed and incubated with a goat anti-
rabbit IgG antibody conjugated with Alexa Fluor 555
(Bioss,China) for 1 h, and 40 ,6-diamidino-2-phenylindole
(Bioss, China) was used as a counterstain to detect nuclei.
TIFF images were acquired using an Olympus
microscope.
Statistical analysis
GraphPad Prism 7.0 (GraphPad Software Inc., La Jolla,
CA, USA) was used for statistical analysis. Data are pre-
sented as the mean and S.D. The results were statistical-
ly analysed with Student’s t-test for two-group
comparisons and one-way ANOVA with Tukey’s post
hoc test for multigroup comparisons. For cell lines, three
experiments were performed with three replicates, and
for chondrocytes, experiments were performed from
three donors in triplicate, while animal experiments uti-
lized four animals in each. Data with P-values < 0.05
were considered significant.
Results
IL-37 promotes the polarization of LPS/IFN-c-treated
M1 macrophages towards the M2 phenotype
To examine M1 and M2 macrophage responses and the
role of IL-37 in this process, we pretreated macro-
phages with hIL-37 for 2 h, followed by stimulation with
LPS and IFN-c. The results showed that with LPS and
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 IL-37 inhibits M1-like macrophage activation

FIG. 1 IL-37 promotes LPS and IFN-c-induced M1 macrophage polarization towards the M2 phenotype

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(A) THP1-derived M1 macrophages were pretreated with or without IL-37 for 12 h. M1-macrophage markers and
M2-macrophage markers were detected at the mRNA level by qRT-PCR. (B) The protein levels of the M1-macro-
phage markers iNOS and CD86 and the M2-macrophage marker CD206 were analysed by western blotting. (C) The
protein levels of M1- and M2-macrophage markers were analysed by immunofluorescence. Data are expressed as
the mean (S.D.) (n ¼ 3). The results were analysed by one-way ANOVA followed by Tukey’s test. The significance levels
are indicated: ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. DAPI: 40 ,6-diamidino-2-phenylin-
dole; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; iNOS: inducible NO synthase; LPS: lipopolysaccharide;
qRT-PCR: quantitative real-time PCR.

IFN-c treatment, THP-1 cells highly expressed CD86,
iNOS, IL-1b, IL-6, TNF-a and CXCL-10 (M1 markers),
while IL-37 pretreatment elevated CD206 and IL-10
mRNA expression and decreased M1 marker mRNA ex-
pression (Fig. 1A). Western blot analysis (Fig. 1B) and
immunofluorescence staining (Fig. 1C) further supported
the occurrence of this phenomenon in macrophages.
(M1-CM), or IL-37þM1-derived CM (M1þIL-37-CM) was
used to treat chondrocytes. The results showed that the M1-
CM-treated chondrocytes highly expressed IL-1b,
IL-6, TNF-a, MMP1, MMP9 and MMP13. In contrast,
M1þIL-37-CM treatment significantly alleviated their expres-
sion (Fig. 2A). The same results were obtained by protein
analysis of IL-6, MMP1, MMP9, and MMP13 (Fig. 2B and C).

Macrophage-conditioned medium regulates IL-37 impairs inflammasome activation in M1

inflammatory reactions in chondrocytes macrophages
To examine the relationship between macrophages and To investigate the mechanism underlying macrophage
chondrocytes, M0-derived CM (M0-CM), M1-derived CM polarization, the protein levels of IL-1R8 and NLRP3

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Ping Luo et al.

FIG. 2 The effects of conditioned medium from M0, M1 macrophages or IL-37-pretreated M1 macrophages on
chondrocytes

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(A) The mRNA levels of inflammatory cytokines and MMPs detected by qRT-PCR. (B) The protein levels of inflam-
matory cytokines and MMPs detected by western blotting. (C) Semi-quantification of the western blotting results.
Data are expressed as the mean (S.D.) (n ¼ 3). The results were analysed by one-way ANOVA followed by Tukey’s
test. The significance levels are indicated: ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
GAPDH: glyceraldehyde 3-phosphate dehydrogenase; M0-CM: M0-derived conditioned medium; M1-CM: M1-
derived conditioned medium; M1þIL-37-CM: IL-37þM1-derived conditioned medium; qRT-PCR: quantitative real-
time PCR.

were verified by western blotting, and the results indi-
cated that IL-1R8 was highly expressed, whereas
NLRP3 expression was suppressed in the IL-37 treat-
ment groups. In addition, IL-37 inhibited LPS and IFN-c-
induced ERK and NF-jB activation in human M1 macro-
phages (Fig. 3A). To further investigate the effect of
NLRP3 on macrophage polarization, MCC-950, a
selective NLRP3 inhibitor, was used to study the role of
NLRP3 inflammasome activation in the pathogenesis of
inflammation (Fig. 3B). The results showed that with
MCC-950 treatment, the mRNA and protein levels of
NLRP3 were decreased (Fig. 3B and D). Furthermore,
MCC-950 treatment suppressed caspase-1 and IL-1b
expression at the mRNA and protein levels (Fig. 3C–E).

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 IL-37 inhibits M1-like macrophage activation

FIG. 3 IL-37 impairs inflammasome activation in THP-1-derived macrophages

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(A) Related pathways were detected by western blotting. (B) THP-1 cells were treated with the pharmacological in-
hibitor MCC-950 (a selective inhibitor of NLRP3), and the expression of NLRP3 was analysed by qRT-PCR. (C) The
mRNA expression of caspase-1 and IL-1b was assessed by qRT-PCR. (D) The protein expression of NLRP3, cas-
pase-1 and IL-1b was assessed by western blot analysis. (E) Semi-quantification of the western blotting results. Data
are expressed as the mean (S.D.) (n ¼ 3). The results were analysed by one-way ANOVA followed by Tukey’s test. The
significance levels are indicated: ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ERK: extracellu-
lar signal-regulated kinase; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; LPS: lipopolysaccharide; NLRP3:
NACHT–LRR–PYD-containing protein 3; NF-jB: nuclear factor-jB; p-: phospho-; qRT-PCR: quantitative real-time
PCR; SIGIRR: single Ig IL-1-related receptor.

These data suggest that IL-37 exerts its anti-
inflammatory effects by inhibiting NLRP3 inflammasome
activity.
IL-37 fails to inhibit inflammasome activation in the
context of IL-1R8 silencing
To explore whether IL-37 recruits IL-1R8 to exert anti-
inflammatory effects, we evaluated the impact of IL-37
on inflammasome activation and inflammation after
silencing IL-1R8. An IL-1R8-specific siRNA was con-
structed to knock down IL-1R8 gene expression in vitro.
qRT-PCR indicated that compared with control macro-
phages (transfected with a scrambled siRNA), macro-
phages transfected with siRNAs had decreased
expression of IL-1R8 (Fig. 4A). Decreased NLRP3 ex-
pression was observed in the context of IL-37 treatment,
but this expression increased upon silencing IL-1R8,
even with IL-37 treatment (Fig. 4B). Consistently, the
results demonstrated that IL-37 suppressed the expres-
sion of pro-inflammatory IL-1b, IL-6, TNF-a and CXCL-
10. In the absence of IL-1R8, the anti-inflammatory
effect of IL-37 was obviously weaker (Fig. 4C).
Moreover, the same results were found for the protein
levels of IL-1b and IL-6 (Fig. 4D and E).
IL-37 promotes the conversion of synovial
macrophages from the M1 phenotype to the M2
phenotype in complete Freund’s adjuvant-induced
temporomandibular joint inflammation
Since the previous results proved that IL-37 exerted a
significant anti-inflammatory effect on the polarity of
macrophages, we further assessed whether IL-37 con-
tributes to macrophage polarization in CFA-induced
arthritis in rats in vivo. Figure 5A shows that in the IL-
37-treated group, the tissue around the cartilage exhib-
ited less swelling and hyperplasia than that in the model
group (Fig. 5A). Histologically, HE staining showed more
inflammatory cell infiltration into the synovial tissue in
the model group than in the sham and treatment
groups. In contrast, in the IL-37 treatment group, there
was significantly less inflammatory cell recruitment
(Fig. 5B). Safranin O and fast green staining showed
that in the model group, proteoglycan loss was
remarkably observed. In contrast, the treatment

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Ping Luo et al.

FIG. 4 IL-37 fails to inhibit inflammasome activation in siRNA-IL-1R8-transfected THP-1 cells

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(A) Thp-1-derived macrophages were transfected with siRNA-IL-1R8 for 48 h, and the gene expression of IL-1R8 was
detected. The results were analysed by Student’s t test. (B) NLRP3 expression was evaluated by immunofluores-
cence staining with an anti-NLRP3 antibody. Nuclei were counterstained with DAPI. Scale bars, 100 mm.
(C) Inflammatory cytokines were detected by qRT-PCR. (D, E) Inflammatory cytokines were detected by western blot-
ting. Data are expressed as the mean (S.D.) (n ¼ 3). The results were analysed by one-way ANOVA followed by
Tukey’s test. The significance levels are indicated: ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001,
****P < 0.0001. DAPI: 40 ,6-diamidino-2-phenylindole; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; NLRP3:
NACHT–LRR–PYD-containing protein 3.

group showed limited proteoglycan loss (Fig. 5C).
Immunohistochemical staining showed that compared
with the CFA group, the IL-37 treatment group showed
significantly increased expression of the M2 marker
CD206 and dramatically decreased expression of the
M1 marker iNOS in the synovial lining (Fig. 5D). The acti-
vation of macrophages is associated with the pro-
inflammatory M1 phenotype, resulting in the release of
multiple pro-inflammatory cytokines and exacerbation of
TMJ injury. To detect the effect of synovial polarization
on cartilage, MMPs were detected. Compared with the
model group, the IL-37 treatment group exhibited a sig-
nificant cartilage-protective effect associated with
reduced MMP9 and MMP13 expression (Fig. 5E).
IL-37 attenuates disc perforation-induced inflamma-
tion through inhibition of M1-like macrophage acti-
vation in vivo
Macroscopic analysis showed that IL-37 treatment
reduced joint tissue swelling (Fig. 5F). As shown by HE
staining, the IL-37 treatment group showed less inflam-
matory cell recruitment than the model group (Fig. 5G).
Immunohistochemistry showed that synovial iNOS ex-
pression increased with CFA-induced inflammation but
decreased with IL-37 treatment. In addition, the expres-
sion of CD206 (an M2 marker) was enhanced in the
IL-37 treatment group (Fig. 5H).
Discussion
In this study, the experiments demonstrated that IL-37
inhibited M1-like macrophage polarization and promoted
M2-macrophage activation to ameliorate TMJ inflamma-
tion in vitro and in vivo. This study elucidated the role
and mechanism of IL-37 in the macrophage transition
and provided evidence for future investigations into the
potential therapeutic value of IL-37 in the treatment of
human TMJ inflammation (Fig. 6).
It has been reported that macrophages play an essen-
tial role in the pathogenesis of RA [18, 21, 22] and OA
[23]. Recently, studies have shown that the failure of
pro-inflammatory M1 macrophages to transition into the
anti-inflammatory M2 phenotype may contribute to
the initiation and progression of OA development in the

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 IL-37 inhibits M1-like macrophage activation

FIG. 5 IL-37 alleviates CFA-induced and disc-perforation-induced inflammation and promotes synovial macrophage
towards M2 polarization

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(A) Representative morphological features of the tissue surrounding the condyle. (B) HE staining. (C) Safranin O and
fast green staining. (D) M1 (iNOS) and M2 (CD206) macrophage markers detected by immunohistochemistry.
(E) MMP9 and MMP13 expression analysed by immunohistochemistry. (F) IL-37 shifted disc perforation-induced syn-
ovial macrophage polarization towards the M2 phenotype. Macroscopic analysis of the soft tissue surrounding con-
dylar cartilage. (G) HE staining. (H) M1 (iNOS) and M2 (CD206) macrophage markers detected by
immunohistochemistry. Data are expressed as the mean (S.D.) (n ¼ 4).The results were analysed by one-way ANOVA
followed by Tukey’s test. The significance levels are indicated: ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001,
****P < 0.0001. CFA: complete Freund’s adjuvant; HE: haematoxylin and eosin; iNOS: inducible NO synthase; NC:
negative control; P: perforation.

synovium [24–26]. IL-37, a novel anti-inflammatory cyto-
kine, has been reported to suppress inflammation in
periodontal inflammation [27], gout [28] and colitis [29].
Feng et al. reported that IL-37 can induce macrophages
to polarize towards an M2-like phenotype in liver injury
[30]. Therefore, we hypothesized that IL-37 can convert
M1 into M2 macrophages to attenuate TMJ inflamma-
tion. In line with these findings, our results showed that
in THP-1 cells, LPS and IFN-c induced high expression
of M1 markers, while pretreatment with IL-37 decreased
M1 marker expression and increased M2 marker ex-
pression, which revealed that IL-37 has the ability to
shift M1 macrophages towards the M2 phenotype.
Furthermore, there is increasing evidence that acti-
vated macrophages in the synovium secrete inflamma-
tory cytokines that alter chondrocyte function and
contribute to cartilage degradation during the progres-
sion of TMJOA [31]. Dreier R et al. reported that MMP-9
protein in coculture medium derived from macrophages
but activation required chondrocyte-derived factors. [32].
Limagne et al. confirmed pro-inflammatory paracrine
interactions between human primary chondrocytes and
macrophages [33]. Therefore, we were interested in
determining how macrophage polarization affects chon-
drocytes, as this was still unknown, and also explored
the effects of cytokines secreted by macrophages on
cartilage. This study was the first to explore the inter-
action between macrophage polarization and TMJ chon-
drocytes. Notably, consistent with expectations, the
results showed that M1-CM stimulated the expression
of inflammatory factors in human chondrocytes, while
IL-37-pretreated M1-CM inhibited inflammatory factor
expression. These results indicate that IL-37 attenuates
chondrocyte inflammation by promoting M1 macro-
phages to polarize into M2 macrophages to inhibit in-
flammatory factor secretion. Nevertheless, further study
is required to elucidate the mechanism involving IL-37 in
polarized M2-macrophage activation in inflamed joints.
Recently, accumulating studies have reported that the
anti-inflammatory effects of exogenous IL-37 require IL-
1R8 [34, 35]. Moretti et al. reported that the anti-
inflammatory effects of exogenous IL-37 relied on
NLRP3 in murine aspergillosis [16]. However, the effects
of IL-37 on macrophages and the associated

https://academic.oup.com/rheumatology 3077
Ping Luo et al.
FIG. 6 IL-37 inhibited M1-polarized macrophages and partially ameliorated TMJOA through IL-1R8 and NLRP3
NLRP3: NACHT–LRR–PYD-containing protein 3; TMJ: temporomandibular joint; TMJOA: temporomandibular joint OA.
mechanisms remain unclear, and the roles of the NLRP3
inflammasome in macrophages during the development
of OA have not yet been fully elucidated. Therefore, we
hypothesized that IL-37 induces M2-macrophage polar-
ization via a process that requires IL-1R8/NLRP3.
First, we investigated the essential role of NLRP3 in
anti-inflammatory IL-37 activity through the pharmaco-
logical inhibition of NLRP3 by MCC-950, which reduced
the production of IL-1b. These results suggest that the
protective effects of IL-37 against inflammation are
mediated via modulation of the inflammasome. We fur-
ther confirmed that the decoy receptor IL-1R8 was
required, as silencing IL-1R8, even with IL-37 pretreat-
ment, prevented NLRP3 downregulation, abolishing IL-
37-mediated protection against inflammation. In a recent
report, Rudloff et al. reported that IL-37 inhibited IL-1b
production by NLRP3 and IL-18 production by the
NLRP3 inflammasome in bone marrow-derived macro-
phages, consistent with our results [36]. The molecular
mechanisms underlying the regulation of IL-37 need to
be further explored and validated.
We expanded these findings in models of CFA- and
disc perforation-induced TMJ inflammation. It has been
reported that macrophages together with other specific
cells (i.e. T cells and fibroblasts) lead to synovial hyper-
plasia and reflect synovial tissue inflammation, which is
responsible for joint swelling [8]. This knowledge is
3078
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consistent with our results that CFA-induced inflamma-
tion leads to synovial hyperplasia and inflammatory cell
infiltration, while IL-37 treatment inhibits synovial hyper-
plasia and inflammatory cell recruitment. Zhang et al.
reported that synovial M1-macrophage polarization
exacerbates experimental OA [37]. Macrophages polar-
ized into pro-inflammatory M1 macrophages provoke
tissue inflammation, while differentiation into the anti-
inflammatory M2 phenotype resolves the inflamed milieu
[38]. Therefore, we hypothesized that IL-37 promotes
M2-type activation of synovial cells to suppress inflam-
mation. The results showed that in CFA- and disc
perforation-induced TMJ inflammation, infiltrated mono-
nuclear cells accumulated in the synovium, which was
consistent with the results that the inflamed synovial tis-
sue accumulated a higher number of macrophages than
healthy joint tissue [39], and the accumulated macro-
phages were mainly M1-like macrophages (iNOSþ). In
contrast, in the IL-37 group, M1-like macrophages bare-
ly appeared in the inflamed TMJ, and relatively few
mononuclear cells were present, while M2-like macro-
phages (CD206þ) were positively correlated with treat-
ment, which suggested that IL-37 mainly inhibited M1
polarization in CFA- and disc perforation-induced acute
joint inflammation.
To the best of our knowledge, this is the first study to
define IL-37-potentiated polarized M2-like macrophage
https://academic.oup.com/rheumatology

activation in CFA- and disc perforation-induced TMJ
acute inflammation rat models. In addition, M1 macro-
phages promote the inflammatory microenvironment
through interactions with synovial fibroblasts and chon-
drocytes, thus increasing the secretion of MMPs [24].
The upregulation of the expression of catabolic enzymes
in the cartilage matrix, including MMPs, is involved in the
pathology of TMJOA [40]. In this study, treatment with IL-
37 ameliorated the destruction of articular cartilage.
In conclusion, these results suggest that IL-37-
mediated inhibition of inflammatory M1 macrophage ac-
tivation may offer a novel strategy for limiting cartilage
degeneration and TMJOA progression.
Funding: This work was supported by the National
Natural Science Foundation of China (Grant No.
81800999), the Scientific and Technological Research
Program of the Chongqing Municipal Education
Commission (Grant No. KJQN201800414), the China
Postdoctoral Science Foundation (Grant No. 220812),
the Natural Science Foundation of Chongqing, China
(Grant No. cstc2019jcyj-msxmX0150) and the Program
for Innovation Team Building at the Institutions of Higher
Education in Chongqing in 2016.
Disclosure statement: The authors have declared that
they have no conflicts of interest.
Supplementary data
Supplementary data are available at Rheumatology online.
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