The interleukin-20 receptor axis in early rheumatoid arthritis: novel links between disease-associated autoantibodies and radiographic progression
Kragstrup et al. Arthritis Research & Therapy
The interleukin-20 receptor axis in early rheumatoid arthritis: novel links between disease-associated autoantibodies and radiographic progression
Tue Wenzel Kragstrup 0 1 2
Stinne Ravn Greisen 1
Morten Aagaard Nielsen 1
Christopher Rhodes 0
Kristian Stengaard-Pedersen 2
Merete Lund Hetland 5 6
Kim Hørslev-Petersen 4
Peter Junker 3
Mikkel Østergaard 5 6
Malene Hvid 1 7
Thomas Vorup-Jensen 1
William H. Robinson 0
Jeremy Sokolove 0
Bent Deleuran 1 2 7
0 Department of Immunology and Rheumatology, Stanford University , Stanford, CA , USA
1 Department of Biomedicine, Aarhus University , Aarhus , Denmark
2 Department of Rheumatology, Aarhus University Hospital , Aarhus , Denmark
3 Odense University Hospital , Odense , Denmark
4 University of Southern Denmark , Odense , Denmark
5 Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen , Denmark
6 Copenhagen Center for Arthritis Research, Center for Rheumatology and Spine Diseases, Rigshospitalet and Glostrup Hospital , Glostrup , Denmark
7 Department of Clinical Medicine, Aarhus University , Aarhus , Denmark
Background: Rheumatoid arthritis (RA) is often characterized by the presence of rheumatoid factor, anti-citrullinated protein antibodies, and bone erosions. Current therapies can compromise immunity, leading to risk of infection. The interleukin-20 receptor (IL-20R) axis comprising IL-19, IL-20, and IL-24 and their shared receptors activates tissue homeostasis processes but not the immune system. Consequently, modulation of the IL-20R axis may not lead to immunosuppression, making it an interesting drug target. We evaluated the role of the IL-20R axis in RA and associations between plasma cytokine levels and clinical disease. Methods: Plasma IL-19, IL-20, and IL-24 levels were measured in early RA patients during a treat-to-target strategy by enzyme-linked immunosorbent assays. The IL-20R1 and IL-22R1 levels in paired peripheral blood mononuclear cells and synovial fluid mononuclear cells from a different cohort of RA patients were evaluated by flow cytometry and confocal microscopy. Monocytes/macrophages were stimulated with heat-aggregated human immunoglobulin immune complexes and immune complexes containing citrullinated fibrinogen, and osteoclasts were incubated with IL-19, IL-20, and IL-24. Results: The plasma concentrations of IL-20 and IL-24 (but not IL-19) were increased in early RA patients compared with healthy controls (both P < 0.002) and decreased after 6 months of treatment (both P < 0.0001). The expression of IL-22R1 (but not IL-20R1) was increased on monocytes from RA synovial fluid compared with monocytes from both RA and healthy control peripheral blood. The plasma concentrations of IL-20 and IL-24 were increased in rheumatoid factor and anti-citrullinated protein antibody positive compared with negative early RA patients (all P < 0.0001). Immune complexes stimulated the production of the IL-20R cytokines by monocytes/macrophages. Increased baseline plasma concentrations of IL-20 and IL-24 were associated with Sharp-van der Heijde score progression after 24 months (Spearman's rho = 0.19 and 0.26, both P < 0.05) in the early RA patients. The IL-22R1 was expressed by osteoclast precursors and in multinucleated osteoclasts. IL-20 and IL-24 increased the secretion of monocyte chemoattractant protein 1 by these cells. Conclusions: This study suggests that IL-20 and IL-24 link RA-associated autoantibodies with radiographic progression via the IL-22R1. Modulation of this axis holds promise as feasible anti-erosive treatment modalities in seropositive RA.
Cytokines; Rheumatoid arthritis; Autoantibody(ies); Bone resorption; Monocytes/macrophages
Rheumatoid arthritis (RA) is a chronic immune-mediated
inflammatory disease. Current therapies are aimed at
halting erosive joint progression through sustained synovitis
suppression. However, these treatments can compromise
the normal immune response. This results in an increased
risk of infection, which is the main concern of
biological agents such as anti-tumor necrosis factor alpha
The interleukin (IL)-20 receptor (IL-20R) axis is
pivotal for epithelial tissue homeostasis, but is generally not
assumed to directly activate cells of the immune system
as a main function [
]. Consequently, modulation of the
IL-20R axis may not lead to immunosuppression, making
it an interesting treatment option [
]. The IL-20R axis
consists of the three cytokines IL-19, IL-20, and IL-24
(“the IL-20R cytokines”) and their two shared receptor
complexes. All three bind the receptor complex of
IL20R2/IL-20R1, while IL-20 and IL-24 also bind the
receptor complex of IL-20R2/IL-22R1 [
]. Recently, the
IL-20R2 subunit was identified as a novel risk locus for
the development of RA [
], suggesting the IL-20R axis
could be implicated in the pathogenesis of RA.
RA is often characterized by high levels of rheumatoid
factor (RF) and anti-citrullinated protein antibodies (ACPAs)
in serum [
]. RF and ACPAs are autoantibodies targeting
the Fc portion of immunoglobulin (Ig)G and citrullinated
proteins, respectively [
]. Both of these autoantibodies
contribute to the formation of immune complexes (ICs);
thus, they potentially contribute to the disease process in
RA, e.g. by stimulating monocytes/macrophages through
Fc receptors (FcRs) [
]. Furthermore, ICs containing
citrullinated fibrinogen (cFb) were recently shown to
costimulate macrophages via dual engagement of Toll-like
receptor 4 (TLR4) and the Fcγ receptor IIa (FcγRIIa) [
The hallmark of RA joint disease is the development
of bone erosions, which are mediated by osteoclasts
]. The OCs are tartrate-resistant acid
phosphatase positive (TRAP+) multinucleated cells capable of
resorbing bone [
]. The OC precursors can be found
among receptor activator of nuclear factor kappa-B
positive (RANK+) monocytes and also express the receptor
for the chemokine monocyte chemoattractant protein 1
(MCP-1) (also known as C–C chemokine ligand 2) [
In RA, OC formation and activity are closely associated with
the local cytokine milieu. In line with this, OCs can be
generated spontaneously in vitro from RA synovial cells [
The IL-20R axis has been associated with several
immune-mediated inflammatory diseases including psoriasis,
inflammatory bowel disease, and arthritis [
the context of arthritis, it is intriguing that IL-20 has also
been reported as a driver of osteoporosis [
] and that
both IL-19 and IL-24 had roles in a rat model of bone
]. Recently, neutralization of IL-20 in a phase
IIa trial was effective in treating seropositive RA patients,
suggesting a link between IL-20 and RF [
]. In expression
profile studies, production of the IL-20R cytokines was
found in TLR4-stimulated monocytes/macrophages,
while the receptor subunits were primarily identified on
epithelial cells of target organs (e.g., skin, gut, and bone)
2, 3, 29
]. However, the cellular sources and targets of the
IL-20R cytokines in RA are not well understood.
Previously, we found IL-20 and IL-24 protein in the
synovial membrane and synovial fluid of patients with
chronic RA, and that plasma IL-20 and IL-24 levels
correlated with MCP-1 [
]. The aim of this study was to
determine the role of the IL-20R axis in early RA, with a
focus on the associations of IL-19, IL-20, and IL-24 with
clinical disease parameters and prognosis.
Plasma samples from patients with early treatment naïve
RA were obtained from the OPERA study. A random set
of 152 samples at baseline and after 6 months of
treatment was used for measuring the plasma concentration
of IL-19, IL-20, and IL-24 (Table 1). A detailed study
design has been published elsewhere [
]. At the entry
of the double-blinded study, patients were randomized
to conventional therapy with methotrexate and
intraarticular steroid injections or conventional therapy
combined with adalimumab. Adalimumab was discontinued
after 1 year of treatment, but reinitiated when there was
disease relapse. Clinical disease parameters and test results,
including radiographs, were registered at the initiation of
treatment and after 6, 12, and 24 months. Radiographic
progression was scored according to the Sharp-van der
Heijde method . Radiographic outcomes were the
progression from baseline and the smallest detectable change
was 1.83 total Sharp score (TSS) units. The percentage of
progressors were the percentage of patients with change in
TSS ≥1.83 TSS units. No cells were collected from this
patient cohort. In another study population consisting of
chronic RA patients with at least one swollen joint (for
obtaining synovial fluid), paired synovial fluid mononuclear
cells (SFMCs) and peripheral blood mononuclear cells
(PBMCs) were analyzed by flow cytometry and OCs were
cultured from SFMCs (n = 15). Only diagnosis was
recorded for this study population, as these cells were only
used in vitro for proof of principle experiments.
Plasma samples from age- and gender-matched healthy
controls (HCs) from the Donor Bank at Aarhus University
Hospital (n = 88) were included for measuring the plasma
concentration of IL-19, IL-20, and IL-24 (Table 1). The
PBMCs or monocyte-derived macrophages from HCs
were included for flow cytometric analysis and IC
stimulation assays. Buffy coats were collected from the Donor
Data are expressed as the median with interquartile range unless otherwise indicated. CCP Cyclic citrullinated peptide, DAS28CRP disease activity score 28 based
on C-reactive protein, HC healthy control, IgM immunoglobulin M, RF rheumatoid factor, HAQ health assessment questionnaire, RA rheumatoid arthritis
Bank at Aarhus University Hospital (n = 10) or Stanford
Blood Center (n = 2).
All clinical samples were obtained after informed written
consent according to the Declaration of Helsinki and
approved by the Local Ethics Committee (De
Videnskabsetiske Komitéer for Region Midtjylland, project
numbers 20070008 and 20121329) and the Danish Data
Plasma samples were collected in
ethylenediaminetetraacetic acid (EDTA) tubes and kept at −80 °C until needed.
The PBMCs and SFMCs were isolated by conventional
Ficoll-Paque (GE Healthcare) density-gradient
centrifugation and cryopreserved at −135 °C until needed.
IL-19, IL-20, and IL-24 enzyme-linked immunosorbent assays
The plasma concentrations of IL-19, IL-20, and IL-24
were quantified as previously described [
the three cytokines were measured with commercially
available enzyme-linked immunosorbent assay (ELISA)
kits (R&D Systems). The ELISA systems used were
validated to prevent unspecific binding caused by RF and
heterophilic antibodies . The detection limit of the
IL-19, IL-20, and IL-24 ELISA systems were 62.5 pg/ml,
62.5 pg/ml, and 31.25 pg/ml, respectively. All plasma
samples were diluted 1:3 in blocking buffer; therefore,
the cut-offs for the plasma analyses were 187.5 pg/ml,
187.5 pg/ml, and 93.75 pg/ml, respectively.
Stimulation of PBMCs with ICs
The PBMCs were thawed and cultured in RPMI medium
supplemented with 10 % fetal calf serum (FCS), penicillin,
streptomycin, and glutamine at a density of 2 × 106 cells/ml.
Then two different experimental setups were performed to
stimulate the cells with ICs. First, ICs were generated by
heat-aggregating human Ig (Behring) for 30 minutes at
65 °C as previously described [
]. Then, 48-well
culture plates were coated with either increasing
concentrations of the heat-aggregated Ig ICs (haIg-ICs) or
with native Ig in phosphate-buffered saline (PBS). For
each type of experiment, an untreated (UT) cell culture
with the same number of cells in medium without
stimulants was used for comparison, and a culture
stimulated with lipopolysaccharide (LPS; Sigma-Aldrich) at a
concentration of 100 ng/ml was used as a positive control.
Cells were cultured for 48 hours at 37 °C in a humidified
incubator with 5 % CO2 without change of medium. Next,
ICs containing cFb were used as previously described [
Briefly, ICs were generated by incubating cFb (50 μg/ml)
with polyclonal anti-Fb antibody (75 μg/ml) for 45 minutes
at 37 °C. Human monocyte-derived macrophages from
two HC donors were pretreated for 30 minutes with
1 μg/ml of the TLR4 inhibitor CLI-095 (InvivoGen)
and/or 10 μg/ml FcγRIIa-blocking antibody (IV.3; Stem
Cell Technologies). Cells were added to 96-well plates
coated with the cFb-containing ICs (cFb-ICs) in triplicate
and incubated for 24 hours at 37 °C in a humidified
incubator with 5 % CO2, without change of medium. For
comparison, an UT cell culture of the same cells and medium
was used. After incubation, supernatants from the
triplicates from both donors were pooled. All supernatants
were stored at −80 °C until analysis of IL-19, IL-20, and
IL-24 content by ELISA.
IL-20R1 and IL-22R1 flow cytometry
Healthy control PBMCs and RA PBMCs and SFMCs
were transferred to FACS tubes (Nunc) in PBS with
0.5 % bovine serum albumin (BSA; Calbiochem) and
0.09 % NaN3 together with 100 μg/ml murine gamma
globulin (Jackson ImmunoResearch) and 100 μg/ml human
Ig (Behring) at room temperature (RT) for 30 minutes to
prevent unspecific binding. Receptor expression was
analyzed using anti-IL-20R1 PE (173714; R&D Systems),
antiIL-22R1 APC (305405; R&D Systems), live/dead near-IR
(Invitrogen), anti-CD14 V500 (MϕP9; BD Biosciences),
anti-CD16 FITC (3G8; Bechman Coulter), anti-CD33 PC7
(D3HL60.251; Bechman Coulter), and anti-RANK PerCP
(64C1385.1; Novus Biologicals). Fluorescence minus one
staining with isotype antibodies served as the negative
controls. Cells were incubated with antibodies at RT for
30 minutes. All samples were analyzed within 24 hours
using an LSR Fortessa (BD Biosciences) and FlowJo
software version 10.1 (Tree Star Inc.). First, cells were
gated according to the monocyte population on a plot
with forward scatter versus side scatter. Then, dead cells
were excluded followed by the selection of singlets. Finally,
the CD14-positive cells were selected for further analysis
of IL-20R1 and IL-22R1 expression. The IL-20R1 and
IL-22R1 positive cells were then studied for co-expression
of CD16, CD33, and RANK.
Generation of OCs from RA SFMCs
Osteoclasts were grown from RA synovial cells as
previously described [
]. Briefly, SFMCs were thawed and
cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10 % FCS, penicillin, streptomycin,
and glutamine at a density of 1 × 106 cells/ml at 37 °C in a
humidified incubator with 5 % CO2. The medium was
changed every 3–4 days. Differentiated OCs were used
after 19 days of culture for immunofluorescence or the
Immunofluorescence of OCs
OCs were grown from RA SFMCs on sterile glass slides
in 24-well cell culture plates and stained for confocal
microscopy as previously described [
]. Briefly, cells were
fixed with 4 % paraformaldehyde for 10 minutes at RT.
Non-specific binding was blocked by incubating in PBS
with 0.5 % BSA and 5 % goat serum for 30 minutes at
RT. Cells were stained with either anti-IL-20R1 IgG1
(173714; R&D Systems) or anti-IL-22R1 IgG1 (305405;
R&D Systems) in combination with goat anti-mouse
IgG1 Alexa 488 (Invitrogen). Cells were co-stained with
anti-TRAP IgG2b in combination with goat anti-mouse
IgG2b Alexa 647 (Invitrogen). Isotypes served as
negative controls. Glass slides were placed in Prolong
Gold Antifade Mountant with DAPI (Life Technologies)
and allowed to dry overnight. All micrographs were
collected using a Zeiss LSM-710 confocal microscope.
Stimulation of OCs
Two different experimental setups were carried out to
evaluate the effect of the IL-20R cytokines on OCs. First,
RA SFMCs were grown with 200 ng/ml IL-19, IL-20, or
IL-24 (all R&D Systems). The medium was changed and
cells were re-stimulated every 3–4 days to study the
effect of the three cytokines on osteoclastogenesis. Second,
OCs were generated from RA SFMCs in medium alone
for 19 days and then cultured with IL-19, IL-20, and IL-24
(200 ng/ml) at 37 °C in a humidified incubator with 5 %
CO2 for 48 hours. In all experiments UT cells were used
for comparison and cells stimulated with a combination of
macrophage-colony stimulating factor (M-CSF; 25 ng/ml)
and RANK ligand (RANKL; 50 ng/ml) were used as a
positive control. Supernatants were harvested after
centrifugation of the culture plates at 1200 rpm for 5 minutes
and analyzed for TRAP by enzymatic assay (B-bridge
International) and MCP-1 by ELISA (Biolegend).
Statistical analyses were performed using GraphPad Prism
6.0 for Mac (GraphPad Software) and Stata 11.1 for Mac
(StataCorp). The plasma concentrations of the IL-20R
cytokines, the percentage of receptor-positive monocytes,
and the secretion of IL-20R cytokines from PBMCs were
not normally distributed even after log transformation.
Therefore, these data were analyzed using non-parametric
statistics. Groups were compared using the Mann–
Whitney U test for non-paired data and the Wilcoxon
matched pairs test for paired data. The Friedman test
was used to compare three or more groups. Correlations
were made using Spearman’s Rho. Multiple regression
models were made with the baseline plasma cytokine levels
and radiographic progression correcting for age, gender,
and disease duration after checking the model assumptions
including the distribution of the residuals. The secretion of
TRAP and MCP-1 from SFMC-derived OC cultures was
calculated as ratios comparing stimulated cells with
untreated cells because of donor variation. The ratios were
log transformed and analyzed with paired t-test. In all tests,
the level of significance was a two-sided P value of less
Plasma concentrations of IL-20 and IL-24 were increased in early RA patients at baseline compared with HCs, and decreased after 6 months of treatment
Patients with early treatment naïve RA and HCs were
studied to assess alterations and changes in plasma levels
of the IL-20R cytokines before and after a treat-to-target
strategy (the OPERA regimen). The plasma
concentrations of IL-20 and IL-24 were significantly increased in
early RA patients compared with HCs (P = 0.0002 and
P = 0.0016, respectively) and decreased after 6 months
of treatment (both P < 0.0001) (Fig. 1a). After 6 months
of treatment, the plasma concentration of IL-20
decreased to the same level as HCs (P = 0.32), while IL-24
remained elevated (P = 0.042) (Fig. 1a). The addition of
adalimumab to conventional treatment did not further
decrease the concentration of IL-20 (P = 0.52) or IL-24
(P = 0.69). The plasma concentrations of IL-19 did not
differ between early RA patients and HCs, and did not
change after treatment (Fig. 1a). In summary, IL-20 and
IL-24 levels were increased in RA peripheral blood.
IL-22R1 expression was increased on monocytes from RA
SFMCs compared with RA PBMCs and HC PBMCs
Previously, IL-19, IL-20, and IL-24 were found to stimulate
monocytes, but the expression of the receptor subunits on
these cells has never been studied. Here, the expressions of
IL-20R1 and IL-22R1 on RA SFMCs and PBMCs and HC
PBMCs were studied by flow cytometry. Monocytes were
found among SFMCs and PBMCs using gates on live cells,
singlets, and CD14+ cells (data available on request). Small
subsets of monocytes from RA SFMCs, RA PBMCs, and
HC PBMCs expressed IL-20R1 and IL-22R1 (Fig. 1b). The
percentage of IL-22R1+ cells was increased among RA SF
monocytes compared with RA and HC PB monocytes (P =
0.016 and P = 0.0025, respectively) (Fig. 1c). Furthermore,
the percentage of IL-22R1+ cells was increased among RA
PB monocytes compared with HC PB monocytes (P =
0.018) (Fig. 1c). The monocyte expression of IL-20R1 did
not differ significantly between RA patients and HCs
(Fig. 1c). Our data indicate that the IL-22R1 subunit is
increased in RA and found only on a small subset of
Plasma concentrations of IL-20 and IL-24 associated with
IgM-RF and anti-CCP positivity in early RA patients
To further elucidate the role of the IL-20R cytokines in
RA, associations between plasma concentrations of the
three cytokines and the baseline characteristics of age,
gender, disease duration, IgM-RF positivity, and
anticyclic citrullinated peptide (anti-CCP) positivity were
analyzed. In early RA patients, the plasma concentrations
of IL-20 and IL-24 were significantly increased in IgM-RF
positive patients compared with negative patients (both
P < 0.0001) (Fig. 2a) and in anti-CCP antibody positive
patients compared with negative patients (both P < 0.0001)
(Fig. 2a). The plasma concentrations of IL-19, IL-20, and
IL-24 did not associate with age, gender, or disease
duration. These findings indicate that the IL-20R cytokines are
either involved in pathways leading to the production of
antibodies or autoantibodies can trigger the secretion of
the IL-20R cytokines.
ICs stimulated the production of the IL-20R cytokines in
Both RF and ACPAs can form ICs that can potentially
stimulate monocytes/macrophages through FcRs. We
tested whether ICs could induce the secretion of IL-19,
IL-20, and IL-24 in monocytes/macrophages using two
different types of ICs. First, ICs were generated by
haIgICs. These haIg-ICs stimulated the production of IL-19
and IL-24 (P = 0.018 and P = 0.0003, respectively)
(Fig. 2b). The positive control LPS increased the
production of IL-19, IL-20, and IL-24 (P = 0.016, P = 0.031, and
P = 0.016, respectively) (Fig. 2b). Then, ICs were
generated by combining citrullinated fibrinogen and
antifibrinogen antibodies (cFb-ICs). Previously, these ICs
stimulated macrophages through both TLR4 and
FcγRIIa. In line with this, the cFb-ICs increased the
secretion of all three cytokines and blockers of TLR4 and
the FcγRIIa diminished this induction (Fig. 2c). Taken
together, Ig aggregates and ICs containing cFb could to
some extent induce IL-19, IL-20, and IL-24 secretion in
Plasma concentrations of IL-20 and IL-24 at baseline were associated with radiographic progression after 12 and 24 months
Correlations between plasma concentrations of IL-19,
IL-20, and IL-24 and clinical disease parameters, response
rates, and test results obtained through 24 months of
follow-up were examined to understand the function of
the IL-20R cytokines. We analyzed the patient global,
physician global, health assessment questionnaire (HAQ),
and disease activity score 28 based on C-reactive protein
(DAS28CRP) scores as well as radiographic progression,
measured as changes in TSS, erosion score, and joint
space narrowing (JSN). The early RA patients with TSS
progression after 12 and 24 months had increased baseline
plasma concentrations of IL-20 (P = 0.0018 and P =
0.0047, respectively) and IL-24 (P = 0.0077 and P = 0.0057,
respectively) (Fig. 3a). The association between baseline
IL-20 and IL-24 levels and TSS progression did not
change after correction for age, gender and disease
duration (P = 0.07 without correction and P = 0.06 with
correction for IL-20 and P = 0.006 without correction
and P = 0.005 with correction for IL-24), and was also
seen when dividing the TSS into erosion score and joint
space narrowing (Table 2). There were no associations
between plasma concentrations of IL-19 and
radiographic progression (Fig. 3a and Table 2). There were
no significant associations between plasma concentrations
of the IL-20R cytokines and patient global, physician
global, HAQ, and DAS28CRP scores or response rates
(Table 2). Also, there were no associations between
changes in cytokine levels and disease activity or
Data were analyzed using the Spearman’s correlation. Numbers indicate
Spearmans Rho with P value in parenthesis. Bold numbers indicate P < 0.05.
aDisease activity scores at baseline. bResponse after 12 months of treatment.
cRadiographic progression after 12 months of treatment (12 months – baseline)
ACR American College of Rheumatology, DAS28CRP disease activity score 28
based on C-reactive protein, HAQ health assessment questionnaire,
disease activity improvement. The association between
IL-20 and IL-24 and radiographic progression suggests
these two cytokines could be involved in bone
destruction. In contrast, the IL-20R axis cytokines were not
associated with other measures of disease activity.
IL-22R1 was expressed by RANK+ OC precursors from RA
synovial fluid and in TRAP+ multinucleated OCs derived
from RA SFMCs
To study the function of IL-20 and IL-24 in bone
homeostasis, we first analyzed the expression of IL-20R1
and IL-22R1 on OC precursors and OCs. First, the
expression of the surface markers CD16, CD33, and RANK
on the IL-20R1+ and IL-22R1+ monocytes was
analyzed by flow cytometry. A large percentage of cells in
the IL-22R1+ monocyte subset from RA SFMCs
coexpressed CD33 and RANK (Fig. 3b). The percentage
of CD33+ and RANK+ monocytes were increased in
the IL-22R1+ monocytes compared with the IL-20R1+
monocytes and the IL-20R1− and IL-22R1− monocytes
(both P = 0.016) (Fig. 3b). In contrast, there was
increased co-expression of CD16 in the IL-20R1+
monocyte subset from PBMCs (data available on request).
Second, OCs were derived from SFMCs, stained for
the expression of IL-20R1 and IL-22R1, and examined
by confocal microscopy. Both TRAP+ multinucleated
OCs and TRAP+ single nucleated OC precursors
expressed the IL-22R1 subunit, but not IL-20R1
(Fig. 3c and d). These findings point to a role of IL-20
and IL-24 in OC function via the IL-22R1.
IL-20 and IL-24 increased MCP-1 secretion in OCs derived from RA SFMCs
The functional role of the IL-22R1 on OCs and OC
precursors was examined using two different experimental
strategies. First, SFMCs were cultured for 19 days with
IL-19, IL-20, and IL-24, or a combination of RANKL
and M-CSF to study osteoclastogenesis. Second, OCs
were generated from SFMCs in medium for 19 days and
then stimulated for 48 hours with IL-19, IL-20, and IL-24,
or a combination of RANKL and M-CSF. In culture
supernatants, IL-20 and IL-24 did not increase
osteoclastogenesis, measured by TRAP activity (Fig. 4a). In contrast,
IL-20 and IL-24 increased the secretion of MCP-1 in
already differentiated OCs (Fig. 4b). Our findings indicate
that the membrane-expressed IL-22R1 is part of a
functional receptor complex.
In RA, RF and ACPAs are well-known risk factors for
the progression of bone erosions [
], but the
mechanism for this association is not well understood .
Here, we provide evidence for a correlation between the
IL-20R axis and progression of structural damage by
showing the relationship between IL-20 and IL-24 and
RA-associated ICs and OC stimulation via the IL-22R1.
Unlike other pro-inflammatory interleukins, the IL-20R
cytokines are assumed not to be involved in direct
activation of leukocytes [
]. Rather, the IL-20R axis is
important for barrier functions and tissue homeostasis [
This could be interesting in the context of autoimmune
disease, because modulation of the IL-20R axis might
not result in the increased risk of infection seen with
targeting other inflammatory mediators, such as TNFα
]. The IL-20R axis has already been associated with
arthritis and the IL-20R2 subunit was recently
identified as a novel RA risk locus [
9, 18–23, 25
the cellular sources and targets of the three cytokines
and their specific role in disease remain unclear.
Expression of IL-19, IL-20, and IL-24 in both monocytes/
macrophages and fibroblast-like synovial cells has been
reported in arthritis [
]. In our study, two lines of
evidence suggest the three cytokines secreted from
monocytes/macrophages stimulated by RA-associated ICs could
contribute to RA. First, we identified an association
between IL-20 and IL-24 plasma levels and IgM-RF and
antiCCP antibody positivity. Second, the production of all three
cytokines increased in PBMCs when cultured with ICs
and was potentiated by RA-associated ICs capable of
costimulating TLR4 and FcγRIIa. This increase could be due
to both a direct effect of the ICs or an indirect effect of
other pro-inflammatory cytokines produced in response
to the ICs. Our findings are in line with results from a
recent phase IIa trial of anti-IL-20 in RA, where
autoantibody-positive patients showed a better response to the
anti-IL-20 antibody compared with seronegative
Knowledge about the function of the IL-20R axis in
RA is limited [
]. Our findings indicate a role of IL-20
and IL-24 in bone homeostasis. First, baseline plasma
levels of IL-20 and IL-24 were increased in early RA
patients with TSS progression at the follow-up visits at 12
and 24 months. Second, the expression of IL-22R1 was
increased on monocytes from the synovial joint and was
located to the CD33+ and RANK+ monocytes. This
surface expression pattern is consistent with the
IL-22R1positive cells being primarily OC precursors [
could explain the previous finding of increased MCP-1
secretion by SFMCs in response to IL-20 and IL-24 [
Third, IL-22R1 was found on TRAP+ multinucleated
OCs derived from RA SFMCs. Fourth, IL-20 and IL-24
increased MCP-1 secretion from the differentiated OCs.
Taken together, these findings indicate that IL-20 and
IL-24 are associated with radiographic progression and
that OC precursors and OCs express functional IL-22R1.
MCP-1 is a chemokine binding the chemokine receptor
CCR2, which is expressed by OC precursors [
Thus, we speculate that IL-20 and IL-24 induce MCP-1
production in IL-22R1+ OC precursors and OCs without
affecting other monocytes. This could attract more OC
precursors to sites of ongoing bone degradation. Our
findings are in line with the already identified role of IL-20 in
osteoporosis  and the established association between
the IL-22-IL-22R system and bone degradation in RA
]. However, in this study there was no direct
effect of the IL-20R cytokines on osteoclastogenesis. An
effect of the IL-20R cytokines on fibroblast-like synovial
cells and neutrophil granulocytes cannot be excluded.
These cells have been suggested to be one of the drivers
of joint space narrowing . In this way, secretion of
metalloproteinases from fibroblast-like synovial cells and
neutrophil granulocytes could contribute to the
association between IL-20 and IL-24 baseline levels and
progression in joint space narrowing found in this study.
There were no correlations between the two cytokines
and any disease activity score reflecting inflammation at
either baseline or follow-up after 6 months of treatment,
which suggests that the IL-20R cytokines are not closely
linked with inflammation. Also, the general expression
of the receptor subunits was rather low among the total
monocyte population, suggesting that these cytokines
might not contribute to the general activation of
monocytes. This is in agreement with a recent comprehensive
study of skin infections, where the IL-20R cytokines
actually had anti-inflammatory functions [
the IL-20R cytokines are expressed by both monocytes
and fibroblast-like synovial cells in response to
proinflammatory factors such as IL-1 and TLR4 agonists
]. Such pro-inflammatory cytokines and molecules
decrease with anti-inflammatory treatment. Also, the
formation of immune complexes and the expression of
fc receptors on monocytes seem to be altered with
antiinflammatory treatment . This could explain the
decrease in IL-20 and IL-24 plasma levels seen after
anti-inflammatory treatment in this study. In this way,
an association between the IL-20R cytokines and
inflammation cannot be excluded.
In this study, plasma IL-19 levels were not increased
in RA patients compared with HCs and did not associate
with radiographic progression. In addition, the
percentage of IL-20R1+ monocytes was not increased in RA
patients compared with HCs; IL-20R1 was not found
on OCs; and IL-19 did not activate these cells. Thus,
IL-19 might not be involved in bone destruction in RA.
We recently identified inverse correlations between
plasma IL-19 levels and disease activity in patients with
spondyloarthritis suggesting IL-19 and IL-20R1 have
antiinflammatory properties [
]. However, such significant
associations were not observed here.
Bone destruction is a key endpoint of arthritis and
inhibiting this process is pivotal in managing RA [
recent clinical study identified an association between
RF and radiographic progression that was independent
of disease activity [
]. This suggests that inflammatory
activity is not required for radiographic progression in
RA, with other possible mechanisms linking RA
pathogenesis and bone destruction. In line with this, it was
recently shown that ACPA can directly induce bone loss
]. Our findings indicate that IL-20 and IL-24 could be
another link between the presence of autoantibodies and
The aim of this study was to determine the role of the
IL-20R axis in early RA. In line with this, this study does
not provide information to draw conclusions regarding
the IL-20R cytokines as biomarkers of disease progression
or the IL-20R axis as a potential new therapeutic target.
The rather weak association between baseline cytokine
levels and radiographic progression indicates that
measuring the IL-20R cytokines in early RA might not have much
predictive value. However, the findings in this study could
help guide future drug development strategies. Anti-IL-20
has been tested in RA and the IL-20R axis is part of the
target of inhibitors of downstream signaling molecules
such as Janus kinase 1, Tyrosine kinase 2 and signal
transducer and activator of transcription 3 [
6, 28, 46
This study suggests that targeting the IL-20R axis could
be a treatment of bone destruction in rheumatic
disease. In particular, dual inhibition of IL-20 and IL-24 or
inhibition of IL-22R1 could be helpful in seropositive
In conclusion, we report that IL-20 and IL-24 link
RAassociated autoantibodies with radiographic progression
via IL-22R1. The similarities between IL-20 and IL-24
imply that dual inhibition of the two cytokines and
attenuation of IL-22R1 are potential anti-erosive treatment
modalities in seropositive RA.
ACPA: Anti-citrullinated protein antibody; BSA: Bovine serum albumin;
CCP: Cyclic citrullinated peptide; CD: Cluster of differentiation;
cFb: Citrullinated fibrinogen; DAS28CRP: Disease activity score 28 based on
C-reactive protein; ELISA: Enzyme-linked immunosorbent assay; FCS: Fetal calf
serum; haIg-IC: Heat-aggregating immunoglobulin immune complex;
HAQ: Health assessment questionnaire; HC: Healthy control; IC: Immune
complex; Ig: Immunoglobulin; IL: Interleukin; LPS: Lipopolysaccharide;
MCP-1: Monocyte chemoattractant protein 1; M-CSF: Macrophage-colony
stimulating factor; OC: Osteoclast; PB: Peripheral blood; PBMC: Peripheral
blood mononuclear cell; PBS: Phosphate-buffered saline; R: Receptor;
RA: Rheumatoid arthritis; RANK: Receptor activator of nuclear factor
kappa-B; RANKL: Receptor activator of nuclear factor kappa-B ligand;
RF: Rheumatoid factor; RT: Room temperature; SF: Synovial fluid;
SFMC: Synovial fluid mononuclear cell; TLR4: Toll-like receptor 4;
TNFα: Tumor necrosis factor alpha; TRAP: Tartrate-resistant acid phosphatase;
TSS: Total Sharp score; UT: Untreated.
The authors declare that they have no competing interests.
TWK helped to design the study, collect the SFMC and PBMC samples, carried
out the experiments, analyzed and interpreted the data, and drafted the
manuscript. SRG, MAN, CR, JS, and WHR helped to plan and carry out the
experiments. KSP, MLH, KHP, PJ, and MØ helped to collect samples and
information from the OPERA cohort. TVJ, MH, and BD helped to design the
study and supervised the project. All authors helped to analyze and interpret
the data, were involved in revising the manuscript, and read and approved the
We thank Karin Skovgård Sørensen and Bettina Grumsen (Department of
Biomedicine, Aarhus University) for technical assistance concerning the ELISA
assays, Charlotte Christie Petersen (FACS core facility, Aarhus University) for
advising us on the flow cytometry analyses, Professor Michael Vaeth
(Department of Biostatistics, Aarhus University) for help with the statistical
analysis, and Professor Julia Johansen (Department of Rheumatology, Herlev
Hospital) for assistance with handling the plasma samples from the OPERA
cohort. This work was supported by the Danish Rheumatism Association and
the Faculty of Health at Aarhus University.
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