Figure 3.

F. novicida induction miR-155 expression is not mediated by TNFα or IFNβ. (A) PBM were treated with TNFα neutralizing antibody or isotype control antibody, then infected with F.n. at an MOI of 50 for 6 h. RNA was collected and assayed for miR-155 expression. (B) The media from the samples in (A) was assayed for human TNFα by ELISA. (C) Conditioned media (CM) was obtained as in Figures 2B. Conditioned media from monocytes uninfected (CM:R) or infected (CM:Fn) with F.n. at an MOI of 50 was sterile filtered and incubated with neutralizing antibody or isotype control antibody at 37°C for 1 h. New PBM were then cultured in the conditioned media conditions, uninfected (R), or direct infection (FN) for 6 h. RNA was collected and miR-155 expression was assayed by qRT-PCR. (D) Effective neutralization of IFNβ was demonstrated by pre-treating media with recombinant IFNβ (500 U/ml) with or without neutralizing antibody or isotype control antibody. Then PBM were cultured in media alone, IFNβ, IFNβ plus neutralizing antibody, or IFNβ plus isotype control for 15 min. Cells were lysed and subject to western blot for phosphorylated STAT1, followed by Actin re-probe.