Figure 6: Reconstituting late sepsis Gr1⁺CD11b⁺ cells from the S100A9 knockout mice with S100A9 restores their immunosuppressive functions.

Gr1⁺CD11b⁺ cells were isolated from the bone marrow of S100A9 knockout mice during the late sepsis phase. The cells were transfected with S100A9 expression plasmid for 36 hr and then stimulated with 10 ng/ml of recombinant murine IL-10 for 6. (A) Effect of Gr1⁺CD11b⁺ cells on CD4⁺ T cell proliferation. Spleen CD4⁺ T cells were isolated from normal (naive) mice by positive selection and labeled with the fluorescent dye CFSE for 10 min at room temperature. Gr1⁺ CD11b⁺ cells were then cocultured (1:1 ratio) with T CD4⁺ cells. The culture was stimulated with anti-CD3 plus anti-CD28 antibodies (1 μ g/ml/each). After three days, CD4⁺ T cell proliferation was determined by the step-wise dilution of CFSE dye in dividing CD4⁺ T cells by flow cytometry. Representative dot plots (upper panels) of CFSE positive T cells gated on CD4 are shown. Percentages of cell proliferation (lower) were calculated as follow: % cell proliferation = 100 × (count from T cell + Gr1⁺CD11b⁺ cell culture/count from T cell culture). *p < 0.001 vs. sham or early sepsis; **p < 0.001 vs. late sepsis WT. (B) ELISA determined levels of IFNγ in the culture supernatants. Cells from sham and sepsis wild-type mice, and without transfection were used as controls. Cells were pooled from 2–3 mice per group. Data are means ± s.d. (n = 3–4 mice per group). *p < 0.05 vs. sham wild-type; **p < 0.05 vs. late sepsis + vector or late sepsis + S100A9. The results are representative of three independent experiments. KO, knockout.