S100A8/A9, a potent serum and molecular imaging biomarker for synovial inflammation and joint destruction in seronegative experimental arthritis

Edwin J W Geven, Martijn H J van den Bosch, Irene Di Ceglie, Giuliana Ascone, Shahla Abdollahi-Roodsaz, Annet W Sloetjes, Sven Hermann, Michael Schäfers, Fons A J van de Loo, Peter M van der Kraan, Marije I Koenders, Dirk Foell, Johannes Roth, Thomas Vogl, Peter L E M van Lent, Edwin J W Geven, Martijn H J van den Bosch, Irene Di Ceglie, Giuliana Ascone, Shahla Abdollahi-Roodsaz, Annet W Sloetjes, Sven Hermann, Michael Schäfers, Fons A J van de Loo, Peter M van der Kraan, Marije I Koenders, Dirk Foell, Johannes Roth, Thomas Vogl, Peter L E M van Lent

Abstract

Background: Seronegative joint diseases are characterized by a lack of well-defined biomarkers since autoantibodies are not elevated. Calprotectin (S100A8/A9) is a damage-associated molecular pattern (DAMP) which is released by activated phagocytes, and high levels are found in seronegative arthritides. In this study, we investigated the biomarker potential of systemic and local levels of these S100 proteins to assess joint inflammation and joint destruction in an experimental model for seronegative arthritis.

Methods: Serum levels of S100A8/A9 and various cytokines were monitored during disease development in interleukin-1 receptor antagonist (IL-1Ra)-/- mice using ELISA and multiplex bead-based immunoassay, and were correlated to macroscopic and microscopic parameters for joint inflammation, bone erosion, and cartilage damage. Local expression of S100A8 and S100A9 and matrix metalloproteinase (MMP)-mediated cartilage damage in the ankle joints were investigated by immunohistochemistry. In addition, local S100A8 and activated MMPs were monitored in vivo by optical imaging using anti-S100A8-Cy7 and AF489-Cy5.5, a specific tracer for activated MMPs.

Results: Serum levels of S100A8/A9 were significantly increased in IL-1Ra-/- mice and correlated with macroscopic joint swelling and histological inflammation, while serum levels of pro-inflammatory cytokines did not correlate with joint swelling. In addition, early serum S100A8/A9 levels were prognostic for disease outcome at a later stage. The increased serum S100A8/A9 levels were reflected by an increased expression of S100A8 and S100A9 within the ankle joint, as visualized by molecular imaging. Next to inflammatory processes, serum S100A8/A9 also correlated with histological parameters for bone erosion and cartilage damage. In addition, arthritic IL-1Ra-/- mice with increased synovial S100A8 and S100A9 expression showed increased cartilage damage that coincided with MMP-mediated neoepitope expression and in vivo imaging of activated MMPs.

Conclusions: Expression of S100A8 and S100A9 in IL-1Ra-/- mice strongly correlates with synovial inflammation, bone erosion, and cartilage damage, underlining the potential of S100A8/A9 as a systemic and local biomarker in seronegative arthritis not only for assessing inflammation but also for assessing severity of inflammatory joint destruction.

Keywords: Biomarker; Cartilage damage; IL-1Ra; Imaging; Inflammation; MMP; S100A8/A9; Seronegative arthritis.

Figures

Fig. 1
Fig. 1
Serum S100A8/A9 levels correlate with macroscopic score for joint swelling. a Starting at week 6, IL-1Ra–/– mice (n = 26) developed a spontaneous and highly heterogeneous joint swelling in the hind paws (combined score of left and right paw). b At end-point (week 15), serum levels of S100A8/A9 correlated to the combined macroscopic score for joint swelling. c Contrastingly, serum levels of the pro-inflammatory cytokines IL-1β, IL-6, and TNF did not correlate with the combined macroscopic score for joint swelling at week 15. IL interleukin, IL-1Ra interleukin-1 receptor antagonist, TNF tumor necrosis factor
Fig. 2
Fig. 2
Early serum S100A8/A9 levels are prognostic for development of increased joint swelling. a Serum levels of S100A8/A9 in IL-1Ra–/– mice (n = 37) were increased compared to BALB/c mice (n = 8) at week 8 of age (P = 0.029) and at weeks 10, 12, 14, and 16. b At week 8, serum levels of S100A8/A9 in IL-1Ra–/– mice correlated with the combined macroscopic score for joint swelling, and continued to be correlated with joint swelling at weeks 10, 12, 14, and 16 (data not shown). c IL-1Ra–/– mice were stratified for macroscopic joint swelling at week 16 in severe (score 2.25–4), mild (score 0.75–2), and low (0–0.5) joint swelling. Serum S100A8/A9 levels were increased in severe arthritic mice as early as week 10 compared to mild (P = 0.004) and low arthritic mice (P = 0.009). Serum levels of S100A8/A9 in severe arthritic mice continued to be increased compared to mild and low arthritic mice at weeks 12, 14, and 16. d Serum levels of S100A8/A9 at week 10 correlated with joint swelling at week 16, indicating that early serum S100A8/A9 levels are prognostic for disease outcome at a later stage. *P < 0.05, **P < 0.01, ***P < 0.001. IL-1Ra interleukin-1 receptor antagonist
Fig. 3
Fig. 3
Serum S100A8/A9 levels correlate with histological parameters of inflammation and local synovial S100A8 and S100A9 levels. a Increased joint swelling was associated with increased thickening of the synovium (black line) and serum levels of S100A8/A9 correlated with the cell influx in the synovium of 16-week-old IL-1Ra–/– mice. b IL-1Ra–/– mice with increased macroscopic score for joint swelling showed a clear increased S100A8 and S100A9 expression, with similar distribution, in the infiltrating cells of the ankle joint. Expression of S100A8 and S100A9 within the inflamed ankle joint of IL-1Ra–/– mice is co-expressed in monocytes and neutrophils. Isotype control IgG staining for S100A8 and S100A9 staining showed no staining in ankle joint sections of arthritic IL-1Ra–/– mice (inserts). c Intravenous injection of polyclonal anti-S100A8-Cy7 in 16-week-old arthritic IL-1Ra–/– mice (n = 6) led to a significantly increased fluorescent signal in the ankle joints compared with mice injected with irrelevant Rab-IgG-Cy7 (n = 6) (P = 0.0002) (macroscopic score for joint swelling in white). d Anti-S100A8-Cy7 targeting was imaged in ankle joints of IL-1Ra–/– mice with various degrees of joint swelling and the observed fluorescent signal correlated with the macroscopic score for joint swelling. ***P < 0.001. Ca calcaneus, Ig immunoglobulin, IL-1Ra interleukin-1 receptor antagonist, Nav navicular bone, Ta talus
Fig. 4
Fig. 4
Serum S100A8/A9 levels correlate with histological parameters of bone erosion and cartilage damage. a Increased bone erosion (*) was observed in arthritic IL-1Ra–/– mice, which correlated with serum levels of S100A8/A9. b–d Several parameters for cartilage damage were investigated. Proteoglycan depletion (b), chondrocyte death (c, arrow heads), and cartilage erosion (d, black line) were increased in arthritic IL-1Ra–/– mice, and all these parameters correlated to serum levels of S100A8/A9. Nav navicular, PG proteoglycan, Ta talus, Ti tibia
Fig. 5
Fig. 5
Increased S100A8 expression is associated with increased MMP activity in the inflamed ankle joints. a Increased expression of S100A8 in the inflamed ankle joints in 16-week-old IL-1Ra–/– mice was associated with increased VDIPEN staining around the chondrocytes and cartilage matrix of the articular cartilage. Isotype control IgG staining for VDIPEN staining showed no staining in ankle joint sections of arthritic IL-1Ra–/– mice (insert). b Intravenous injection of the MMP inhibitor AF-489 coupled to Cy5.5 led to an increased fluorescent signal in the inflamed ankle joints of arthritic IL-1Ra–/– mice and the observed fluorescent signal correlated to the macroscopic score for joint swelling (macroscopic score for joint swelling in white). Ig immunoglobulin, IL-1Ra interleukin-1 receptor antagonist, SNR signal-to-noise ratio, Ta talus, Ti tibia

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