The interleukin-20 receptor axis in early rheumatoid arthritis: novel links between disease-associated autoantibodies and radiographic progression

Abstract

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.

Keywords: Autoantibody(ies); Bone resorption; Cytokines; Monocytes/macrophages; Rheumatoid arthritis.

Fig. 1

Plasma concentrations of interleukin (IL)-19, IL-20, and IL-24 and monocyte expression of IL-20R1 and IL-22R1. a The plasma concentrations of IL-20 and IL-24 were increased in early rheumatoid arthritis (RA) patients (n = 152) at baseline (0) compared with after 6 months of treatment (6) and healthy controls (HCs) (n = 88). Data were analyzed using the Wilcoxon signed rank test for paired data and the Mann–Whitney U test for unpaired data. b, c The expression of the IL-22R1 subunit is increased on monocytes from synovial fluid (SF) of RA patients compared with the peripheral blood (PB) of RA patients and HCs. b Representative plots of cell membrane expression of IL-20R1 and IL-22R1 on monocytes from RA SF (n = 7), RA PB (n = 7), and HC PB (n = 5). c The percentage of IL-20R1 single positive cells, IL-22R1 single positive cells, and IL-20R1/IL-22R1 double positive cells among monocytes. Monocytes were gated using forward/side scatter and then selecting single cells, live cells, and CD14⁺ cells. Data were analyzed using the Mann–Whitney U test for unpaired data and the Wilcoxon signed rank test for paired data. Boxes indicate the median and interquartile range, and whiskers indicate 10–90 percentiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 2

Association of baseline plasma concentrations of interleukin (IL)-19, IL-20, and IL-24 with immunoglobulin M-rheumatoid factor (IgM-RF) and anti-cyclic citrullinated peptide antibody (αCCP) positivity in early rheumatoid arthritis (RA) patients and immune complex (IC) stimulation of the three cytokines in monocytes/macrophages. a The plasma concentrations of IL-20 and IL-24 were increased in IgM-RF positive (n = 108) compared with negative (n = 44) RA patients and in anti-CCP antibody positive (n = 98) compared with negative RA patients (n = 54). Data were analyzed using the Mann–Whitney U test for unpaired data. Boxes indicate the median and interquartile range and whiskers indicate 10–90 percentiles. b Secretion of IL-19, IL-20, and IL-24 from HC PBMCs stimulated with heat -aggregated immunoglobulin immune complexes (haIg-ICs) at 0.01–1 μg/ml, lipopolysaccharide (LPS) at 100 ng/ml, or immunoglobulin (Ig) at 1 μg/ml (n = 7). Data were analyzed using the Friedman test for dose–response data and the Wilcoxon signed rank test for paired data. c Secretion of IL-19, IL-20, and IL-24 from monocyte-derived macrophages stimulated with citrullinated fibrinogen immune complexes (cFb-ICs) with and without the Toll-like receptor 4 inhibitor CLI-095 (TLR), Fcγ receptor IIa-blocking antibody (Fc), or both inhibitors (n = 2 in triplicates, supernatants pooled). Bars indicate the median and whiskers indicate interquartile range. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

Association of baseline plasma concentrations of interleukin (IL)-19, IL-20, and IL-24 and total Sharp score (TSS) of radiographic progression in early rheumatoid arthritis (RA) patients and IL-20R1 and IL-22R1 expression on osteoclasts (OCs) and their precursors. a The median baseline values of IL-20 and IL-24 were higher in plasma from patients with TSS progression compared with no progression at 12 and 24 months (see % progressors in Table 1). Data were analyzed using the Mann–Whitney U test. b Co-expression of osteoclast precursor markers and IL-20R1 and IL-22R1 on RA synovial fluid (SF) monocytes. Representative plots of receptor activator of nuclear factor kappa-B (RANK) and CD33 expression on the three monocyte subsets (n = 7). The median percentage of RANK⁺/CD33⁺ cells was increased among IL-20R1⁻/IL-22R1⁺ monocytes compared with IL-20R1⁻/IL-22R1⁻ and IL-20R1⁺/IL-22R1⁻ monocytes. Data were analyzed using the Wilcoxon signed rank test. c Representative confocal microscopy images of IL-20R1 and IL-22R1 staining on tartrate-resistant acid phosphatase positive (TRAP ⁺) cells derived from SFMCs (n = 5). No IL-20R1 expression (20×), and co-expression of TRAP and IL-22R1 in permeabilized cells (20× and 64×, respectively). d Surface expression of IL-22R1 on non-permeabilized cells (20× and 64×, respectively). Boxes indicate the median and interquartile range and whiskers indicate 10–90 percentiles. *P < 0.05, **P < 0.01

Fig. 4

Effect of interleukin (IL)-19, IL-20, and IL-24 on osteoclastogenesis and osteoclast (OC) MCP-1 secretion. a No effect of continuous IL-19, IL-20, and IL-24 stimulation on osteoclastogenesis in cultures of rheumatoid arthritis synovial fluid mononuclear cells (RA SFMCs), as measured by tartrate-resistant acid phosphatase (TRAP) secretion. b IL-20 and IL-24 increased monocyte chemoattractant protein 1 (MCP-1) secretion by OCs generated from RA SFMCs prior to stimulation. Positive controls were stimulated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) in all experiments. The secretion of TRAP and MCP-1 was calculated as ratios comparing stimulated cells with untreated cells (UT). The ratios were then log transformed and analyzed with paired t-test. Bars indicate the median and whiskers indicate the interquartile range. *P < 0.05