Figure 4.

MiR-155 induction is not mediated through host cytokines, but rather by soluble bacterial factors. (A) PBM were infected with F.n. at an MOI of 50 for 16 h in the presence of Brefeldin A (BFA) or DMSO vehicle control. RNA was isolated and mature miR-155 expression was assayed by qRT-PCR. (B) Extracellular TNFα levels were measured by ELISA. (C) Intracellular TNFα levels were measured by ELISA from the cell lysate. (D) WT and MyD88^−/− BMMs were infected for 24 h at an MOI of 50 and then sterile filtered to remove bacteria to generate conditioned media (CM). RNA was collected from the infected cells for qRT-PCR (Bars 1, 2, 7, 8). The conditioned media was then placed on new wild-type BMM for 24 h and RNA was subsequently collected for qRT-PCR (Bars 3, 4, 5, 6). These data are representative of three independent experiments. *Designates a p value < 0.05. (E) F.n. was suspended in RPMI-1640 with 10% heat-inactivated fetal bovine serum at a concentration of 3.5 × 10⁸ bacteria/ml (comparable to 5 × 10⁶ PBM infected at MOI 50 in 1 ml) and incubated at 37°C for 24 h. The media was sterile filtered to remove bacteria. PBM were directly infected with F.n. at an MOI of 50 or treated with the filtered media that previously contained F.n. for 16 h. RNA was isolated and miR-155 expression assayed by qRT-PCR. These data are representative of three independent experiments. *Designates a p value < 0.05.