Background/Purpose:  SKG mice exhibit features of spondyloarthropathy (SpA) and inflammatory bowel disease (IBD). Their SpA-like phenotype is induced by a beta-glucan, curdlan, via the Dectin-1 pathway, residing upstream of TNFα, IL23 and IL17. In SpA, the mechanism for enthesial bone formation and ankylosis is not well understood. Currently, no treatment can reverse this process, although recent evidence suggests that TNF inhibitors may prevent bone formation if initiated early. The goal of this study was to discover genes and pathways that lead to axial/enthesial inflammation and bone formation. SKG mice were chosen for this purpose as they were previously reported to develop axial bone formation within 12 weeks of curdlan injection.

Methods:  Nine week old SKG or control BALB/c mice were injected intraperitoneally with curdlan. Histology and microCT were perfomed at various time points to evaluate axial bone formation. Laser capture microscopy was performed on formalin-fixed paraffin-embedded (FFPE) tissue sections. Samples were obtained from entheseal sites near tail vertebrae at early inflammation, 3 weeks after injection. These were compared with identical anatomical sites in non-arthritic BALB/c mice. RNA collected from these tissues was extracted using the Qiagen RNeasy FFPE kit. Gene expression was analyzed using Affymetrix mouse transcriptome assay 1.0 (MTA 1.0).

Results:  SKG mice developed clinical inflammation scores similar to those reported in published studies, with findings of skin and eye inflammation, arthritis, enthesitis, colitis/ileitis and weight loss when compared to control mice. We found that inflammation at axial sites developed earliest at the base of the tail. Contrary to previous reports, we did not observe axial bone formation, but rather significant erosion of vertebral bodies, and laxity of tendons leading to tail base deformity, as late as 24 weeks post injection. Early axial inflammatory tissue captured via laser was found to express S100a8 and S100a9 at 23-fold and 35-fold higher, respectively, when compared to control. Upregulation of S100A8 protein was confirmed by immunohistochemistry. Pathway analysis demonstrated enrichment of bone related pathways including endochondral ossification, TGF-beta receptor signaling and the Wnt signaling pathway. S100A8/9 (or the heterodimer, calprotectin) are antimicrobial peptides (AMPs), secreted by activated phagocytes, that act as endogenous activators of TLR4, upstream of TNFα, to induce a proinflammatory response. Studies show that calprotectin serum levels are elevated in SpA, correlate with disease activity and decrease with TNF inhibition. Recently, it has been shown that Dectin-1 stimulation upregulates expression of S100A8. Our results suggest that S100a8/9 are highly upregulated, likely via Dectin-1 in axial sites, and may drive inflammation and axial erosion in SKG mice.

Conclusion:  Our whole transcriptome analysis revealed that S100a8/9 are the most highly upregulated genes in inflamed axial sites in SKG mice. We hypothesize that these play a role in axial inflammation and bone erosion in SKG mice.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/calprotectin-is-highly-upregulated-in-inflamed-axial-entheses-in-skg-mice/