Background
Rheumatoid arthritis (RA) is an autoimmune-mediated systemic arthritis. Treatment of RA aims to achieve the following: attainment of low disease activity or remission at the earliest, cessation of structural damage of the affected joints, and prevention of RA-related comorbidities [
1]. Pannus is a typical synovial hyperplasia of RA, which can invade into the adjacent articular structure, cartilage, and subchondral bone, thereby inducing erosive joint destruction and deformities [
2]. Prevention of such destructive bone erosion is important, primarily due to their irreversible nature. Activation of osteoclasts and secretion of proteases are the main mechanisms underlying cartilage and bone erosion [
2]. Maturation and activation of osteoclasts require interaction between receptor activator of nuclear factor κB (RANK) and RANK ligand (RANKL). RANKL can be produced by various cells, although fibroblast-like synoviocyte (FLS), the main cellular component of pannus, is the major source of RANKL in RA synovium [
3]. Suppression of RANKL is one of the treatment goals in RA to reduce osteoclastogenesis and eventually cease the irreversible articular damage in RA.
Many pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-17A, induce inflammatory processes and bone destruction in RA pathogenesis [
4]. Patients with RA, who do not respond to or tolerate conventional synthetic disease-modifying antirheumatic drugs (DMARDs), can use biologic DMARDs instead, which target specific pro-inflammatory cytokines or cell surface molecules [
4]. These biologic DMARDs have caused a marked improvement of treatment strategy and clinical remission in RA, although some patients still fail to respond to them, and joint destruction continues to progress. IL-22, a member of the IL-10 superfamily, has recently emerged as a pathological cytokine in animal models of RA [
5,
6]. IL-22-producing cluster of differentiation (CD) 4
+ T cell (Th22) population has been found to be elevated in patients with RA compared to that in healthy controls, and it is also correlated with the disease activity score [
7]. Furthermore, IL-22 has been reported to promote FLS proliferation and RANKL expression in FLS, and IL-22-pre-treated FLSs can upregulate osteoclastogenesis [
8,
9]. These findings propose a potential therapeutic approach in RA by suppressing IL-22.
IL-25, also called IL-17E, is one of the IL-17 superfamily cytokines, composed of six subtypes, IL-17A to IL-17F; they bind to the corresponding receptor, IL-17 receptor, which in turn is composed of five members, IL-17RA to IL-17RE [
10]. Although IL-17 family cytokines share approximately 50% of the amino acid sequence, their cellular responses vary. IL-17A, IL-17C, and IL-17F usually trigger host defense response and promote autoimmune inflammatory response, whereas IL-25 (IL-17E) induces Th2 polarization with allergic response [
10]. In recent studies, IL-25 has been shown to present anti-inflammatory response in RA by reducing Th17 differentiation and IL-17-mediated inflammation [
11,
12]. The aforementioned findings support an anti-inflammatory role of IL-25 in RA.
In this study, we investigated the expression levels of IL-22 and IL-25 in patients with RA and studied the role of IL-25 in IL-22-induced osteoclastogenesis. Furthermore, the underlying intracellular mechanisms of IL-25 with respect to osteoclastogenesis were evaluated in RA synoviocytes.
Methods
Patients
Samples of synovial tissue were isolated from 5 patients with RA (mean age 55.2 ± 3.8 years; range 44–64 years) and 5 with osteoarthritis (OA) patients (mean age 57.8 ± 3.0 years; range 50–68 years), who were undergoing total knee replacement surgery. Synovial fluid was obtained from patients with RA (N = 29), who fulfilled the revised criteria of the American College of Rheumatology, 1987 (formerly the American Rheumatism Association), and from patients who had symptomatic knee OA (N = 29). Additionally, a total 25 serum of healthy control were included. Informed consent was obtained from all patients, and the experimental protocol was approved by the Konkuk University School of Medicine Human Research Ethics Committee (KUH1010186).
Isolation of FLS
FLSs were isolated by enzymatic digestion of synovial tissues obtained from patients with RA, who were undergoing total knee replacement surgery, as described previously [
13].
Reagents
IL-22, IL-25, RANKL, and macrophage colony-stimulating factor (M-CSF) were obtained from R&D Systems (Minneapolis, MN, USA).
Enzyme-linked immunosorbent assay (ELISA) of IL-22, IL-25, and sRANKL
In brief, a 96-well plate (Nunc, Roskilde, Denmark) was coated with 4 μg/ml monoclonal antibodies against IL-22, IL-25, IL-1β, TNF-α, IL-6, IL-4, IL-13, and sRANKL (R&D Systems, Minneapolis, MN, USA) at 4 °C overnight. After blocking with phosphate-buffered saline/1% bovine serum albumin (BSA)/0.05% Tween 20 for 2 h at room temperature (22–25 °C), the test samples and the standard recombinant IL-22, IL-25, IL-1β, TNF-α, IL-6, IL-4, IL-13, and sRANKL (R&D Systems) were added to the 96-well plate and incubated at room temperature for another 2 h. The plates were washed four times with phosphate-buffered saline/Tween 20, and then incubated with 500 ng/ml biotinylated mouse monoclonal antibodies against IL-22, IL-25, IL-1β, TNF-α, IL-6, IL-4, IL-13, and sRANKL (R&D Systems) for 2 h at room temperature. After washing, streptavidin-alkaline phosphate-horseradish peroxidase conjugate (Sigma, St Louis, MA, USA) was incubated for 2 h, followed by another wash, and incubated with 1 mg/ml p-nitrophenyl phosphate (Sigma) dissolved in diethanolamine (Sigma) to develop the color reaction. The reaction was stopped by the addition of 1 M NaOH, and optical density of each well was measured at 405 nm. The lower limit of IL-22, IL-25, IL-1β, TNF-α, IL-6, IL-4, IL-13, and sRANKL detection was 10 pg/ml. Recombinant human IL-22, IL-25, IL-1β, TNF-α, IL-6, IL-4, IL-13, and sRANKL, diluted in culture medium, were used as calibration standards, ranging from 10 to 2000 pg/ml. A standard curve was drawn by plotting optical density against log of the concentration of recombinant cytokines, and the curve was used for determining IL-22, IL-25, IL-1β, TNF-α, IL-6, IL-4, IL-13, and sRANKL concentrations in test samples.
Immunohistochemistry of RA synovium
Immunohistochemical staining for IL-25 was performed with sections of synovium. Briefly, synovial samples were obtained from patients with RA and OA, fixed with 4% paraformaldehyde solution overnight at 4 °C, dehydrated with alcohol, washed, embedded in paraffin, and sectioned into 7-μm-thick slices. Sections were depleted of endogenous peroxidase activity by adding methanolic H2O2 and blocked with normal serum for 30 min. After overnight incubation with polyclonal anti-human IL-25 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C, the samples were incubated with a secondary antibody, biotinylated anti-rabbit IgG, for 20 min, and then with streptavidin-peroxidase complex (Vector Laboratories, Peterborough, UK) for 1 h, followed by a 5-min incubation with 3,3′-diaminobenzidine (Dako, Glostrup, Denmark). The sections were counterstained with hematoxylin. Samples were finally photographed using an Olympus (Tokyo, Japan) photomicroscope. The area of IL-25+ cell from samples was measured in samples using ImageJ software.
Expression of RANKL mRNA by real-time polymerase chain reaction (PCR)
FLSs were stimulated with various concentrations of IL-22 (0, 1, 10 ng/ml). They were incubated in the presence or absence of IL-25 (10, 50, 100 ng/ml) for 4 h before the addition of IL-22. After 72 h, mRNA levels were measured using real-time PCR, as reported previously [
14].
Western blot analysis
FLSs and PBMC were incubated with IL-22 in the presence or absence of IL-25. After incubation for 1 h, whole-cell lysates were prepared from approximately 2 × 105 cells, by homogenization in the lysis buffer, and then centrifuged at 14,000 rpm for 15 min. Protein concentration in the supernatant was determined using the Bradford method (Bio-Rad, Hercules, CA, USA). Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Uppsala, Sweden). For western blotting, the membrane was pre-incubated with 0.5% skim milk in 0.1% Tween 20 and Tris-buffered saline (TTBS) at room temperature for 2 h. The primary antibodies to phospho-stat3, stat3, phospho-p38, p38, phospho-IκB-α, and IκB-α (Cell Signaling Technology Inc., Danvers, MA, USA), diluted 1:1000 in 5% BSA–0.1% Tween 20/TBS, were added and incubated overnight at 4 °C. The membrane was washed 4 times with TTBS, followed by the addition of horseradish peroxidase-conjugated secondary antibody and incubation for an hour at room temperature. After TTBS washing, hybridized bands were detected using the ECL detection kit and Hyperfilm-ECL reagents (Amersham Pharmacia).
PBMCs were collected from healthy blood by density gradient separation, and monocytes (osteoclast precursors: pre-OC) were prepared from them. Human monocytes were seeded in 48-well plates at 5 × 10
4 cells/well with 1 ml of medium. Monocytes were cultured under α-minimum essential medium, 10% heat-inactivated FBS, and 25 ng/ml of recombinant human M-CSF (rhM-CSF) for 3 weeks. Then, monocytes were pre-treated with IL-25 and for 4 h, following which they were added to each well along with IL-22. RANKL was used as the positive control. On day 21, tartrate-resistant acid phosphatase (TRAP)-positive cells were identified, as described previously [
14].
Statistical analysis
All data are expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance and Bonferroni’s multiple comparisons test. Spearman’s correlated test was used to seek correlation between cytokine levels. In all analyses, P < 0.05 indicated statistical significance.
Discussion
IL-22 belongs to the IL-10 family and shares about 25% structural homology with the latter [
16]. It can evoke both tissue repair/host defense and inflammatory immune response depending on the organs and diseases [
17]. In addition to its diverse functions, it mainly acts on non-hematopoietic cells, such as epithelial cells and fibroblasts, and promotes epithelial cell regeneration. RA synovium has been reported to present high levels of IL-22 expression and is implicated in RA pathogenesis via FLS proliferation and production of monocyte chemoattractant protein 1 (MCP-1) [
15]. IL-22 can be expressed by many immune cells, and among the CD4
+ T cells, Th22 produces over 50% of IL-22 in the peripheral blood [
18]. Elevation of IL-22 and Th22 population in patients with RA has been adequately reported, and plasma IL-22 and Th22 levels have shown correlation with RA disease activity (DAS-28) [
19‐
21]. Elevated IL-22 levels in plasma can predict future bone erosion in RA [
22], and IL-22 produced by natural killer (NK) cells can induce FLS proliferation [
9]. Furthermore, IL-22 has been reported to promote osteoclastogenesis via the p38 MAPK/NF-κB and JAK2/STAT-3 signaling pathways [
8]; Th22 cells have been shown to play a crucial role in osteoclastogenesis by producing IL-22 [
23]. These findings collectively support the pathological roles of IL-22 in RA pathogenesis and progression. In the current study, besides re-confirming the pathological role of IL-22 in osteoclastogenesis, suppression of IL-22-induced osteoclastogenesis by IL-25 has been revealed for the first time.
IL-25, also called IL-17E, binds to the heterodimeric receptor composed of IL-17RA and IL17RB [
10]. It is known to induce Th2 dominant response and cause allergic reaction [
24]. Helminth-induced Th2 immune response can suppress inflammatory arthritis and bone loss via the IL-4/IL-13-induced STAT6 pathway [
25]. Similarly, IL-25 can attenuate Th17 differentiation in RA in an IL-13-mediated manner [
11]. IL-25 has been reported to be produced by synoviocytes in delayed phase after stimulation with IL-17A and TNF-α [
12]. Such delayed-phase generation of IL-25 suppresses the production of pro-inflammatory cytokines, IL-6 and IL-17A, in RA synoviocytes [
12]. IL-17RB is expressed in various cells, such as NKT, myeloid, Th9, mast, and dendritic cells, as well as basophils, eosinophils, and macrophages [
24]. Osteoclast precursor cells, monocytes, have been previously shown to express IL-17RB and IL-17RA in a mouse model [
26], as well as in human synoviocytes [
12]. In the present study, the novel antagonistic function of IL-25 on osteoclastogenesis induced by IL-22 has been presented.
Elevated plasma and synovial levels of IL-22 and IL-25 in RA have been revealed in previous studies [
7,
11,
15,
21,
22]. The regulatory role of IL-25 in RA has been introduced as antagonistic to IL-17A, at a delayed time point when stimulated by TNF-α and IL-17A, in RA [
12]. Here, we showed the correlation of IL-22 and IL-25 in the plasma and synovial fluid samples of patients with RA. Considering the regulatory function of IL-25 and the aforementioned correlation with IL-22, IL-25 might be upregulated in response to pathogenic cytokines, such as TNF-α, IL-17A, and IL-22, and antagonize the functions of the pro-inflammatory cytokines. Furthermore, IL-25 is produced by synoviocytes [
12], cornerstone component of pannus, and close proximity of synoviocytes with osteoclast precursors makes IL-25 an attractive treatment target.
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