Fig. 3

a Immunohistochemical staining of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and inducible nitric oxide synthase (iNOS) in ankle joint specimens from control collagen-induced arthritis (CIA) mice and mice treated with different dosages of soluble sialic acid-binding immunoglobulin-type lectin (sSiglec)-9 at low (×200 original magnification) and high (×1000 original magnification; insets) magnifications. Scale bars: black = 100 μm; red = 20 μm. Strong positive staining was observed in control CIA mice, and weaker staining was observed in mice treated with sSiglec-9. b Immunopositive cells were counted individually in three different areas under a light microscope at × 400 original magnification. The number of positively stained cells was then averaged. The proportion of immunopositive cells was determined by dividing the number of positive cells by the total number of cells within the field. sSiglec-9 treatment significantly decreased the proportion of cells positive for TNF-α, IL-6, and iNOS. Values are presented as mean ± SEM. *p < 0.05, **p < 0.01 versus control CIA mice (Bonferroni post hoc test). c Regulatory T (Treg) cell expansion in isolated CD4⁺ splenocytes. Representative results of flow cytometric analysis of Treg cells are shown. CD4⁺CD25⁺Foxp3⁺ expansion in isolated splenocytes of normal mice, control CIA mice, and CIA mice treated with 50 ng/g sSiglec-9. d Isolated CD4⁺ T cells from splenocytes were subjected to intracellular staining of CD25 and forkhead box P3 (Foxp3) for flow cytometric analysis. The number of CD25⁺Foxp3⁺ T cells was significantly reduced in control CIA mice compared with normal mice. This reduction was significantly recovered in mice treated with sSiglec-9. **p < 0.01 (Bonferroni post hoc test)