Figure 5. Expression of miR-21 and miR-181 in C/EBPβ-deficient Gr1⁺CD11b⁺ cells from late septic mice switches them into the immunosuppressive phenotype.

Gr1⁺CD11b⁺ cells were isolated from the bone marrow of control and C/EBPβ conditional knockout mice during late sepsis. Cells were transfected with negative control, miR-21 or miR-181b precursor (50 nM final concentration) in the presence of 10 ng/ml of recombinant IL-10. (A) Cytokine production. Cells (1×10⁶) were cultured with 1 μg/ml LPS for 12 h. Supernatants were collected and levels of TNFα and IL-10 were determined by ELISA. (B) Effect on T cell proliferation. Gr1⁺CD11b⁺ cells were co-cultured (at 1:1 ratio) with spleen CD4⁺ T cells that have been isolated from normal (naive) mice and labeled with the fluorescent dye CFSE for 10 min at room temperature. The cells then were incubated in the presence of anti-CD3 plus anti-CD28 antibodies (1 μg/ml/each). After 3 days, CD4⁺ T cell proliferation was determined by the step-wise dilution of CFSE dye in dividing CD4⁺ cells by flow cytometry. Percentages of cell proliferation were calculated as follow: % cell proliferation = 100 x (count from T cell + Gr1⁺CD11b⁺ cell culture/count from T cell culture). Data are expressed as mean ± s.d. (*^/#p < 0.05) of 5 mice per group and represent one of two experiments. *, compared with cells isolated from control mice; ^#, compared with cells isolated from C/EBPβ cKO mice and transfected control precursor. cKO, conditional knockout.