Figure 7. Ruling out iOPN in exacerbating T cell-mediated colitis.

(a) OPN secretion by T stimulated with anti-CD3/CD28 antibody for 3 days in tissue culture. CD4⁺ T cells obtained from the MLNs of Spp1^−/−Rag1^−/− mice 4 weeks after CD62L^hiCD4⁺ T cell transfer. Levels of sOPN were evaluated by ELISA. Error bars denote ± SEM. (b) The amount of iOPN per total cytoplasmic proteins in T cells (naive and activated) and BMMs. Cytoplasmic proteins were separated with the Pierce NE-PER reagent. (c–f) Colitis was induced by adoptively transferring WT, Spp1^−/− or LSL-iOPN CD62L^hiCD4⁺ T cells to Spp1^−/−Rag1^−/− recipients. Total MLN and CD4⁺ T cells (CD3⁺CD4⁺ of total leukocytes) were enumerated on day 6 (c, d) and day 28 (e). Apoptotic CD4⁺ T cells in MLNs on day 3 were labeled with Annexin-V (f). (g–i) Evaluation of the colon on day 28 after T cell transfer. Colon weight per length (g), CD45⁺ and CD4⁺ T cell (CD45⁺CD3⁺CD4⁺) numbers (h), and histology with scores (i) are shown. Scale bars indicate 100 µm (main images) or 1 mm (insets). (j, k) Mature B cells (CD19⁺B220⁺CD93⁻) (2×10⁶ per mouse) from WT, Spp1^−/− or LSL-iOPN mice were adoptively transferred to Rag1^−/− mice. Representative flow plots (j), and the frequencies and numbers of B cells (k) in the MLNs are shown. All datasets are representatives of two independent experiments. N.S.; Not significant. * p< 0.05. Data was analyzed by one-way ANOVA (d–i and k).