Figure 7. miR-155 expression by LPS-activated, GM-CSF-derived myeloid dendritic cells is necessary for proper production of Th17 cell relevant inflammatory cytokines.

A. CD11c⁺ DCs were derived using GM-CSF at 20 ng/ml. B. Expression of BIC (top) and mature miR-155 (bottom) before and after 20 hours of LPS stimulation (100 ng/ml) was assayed using qPCR. C. Total RNA was next used for a microarray analysis to determine mRNA expression differences between Mir155^+/+ and Mir155^−/− LPS treated DCs. Several selected targets of miR-155 were expressed in higher amounts in Mir155^−/− DCs, while a subset of selected proinflammatory cytokines were expressed at lower amounts. Red = higher expression and Green = lower expression in the Mir155^−/− vs. Mir155^+/+ DCs. D. Expression of SHIP1 and SOCS1 mRNAs was assessed by qPCR and by Western blotting (n=3). E. qPCR was also used to assay expression of IL-23 p19, IL-6, IL-12p40 and TNF-α mRNA amounts (n=3). Data represent two independent experiments. F. Concentrations of the cytokines from (E) in the culture supernatants were determined by ELISAs (n=3). Data represent two independent experiments. G. GM-CSF-derived DCs overexpressing miR-155, a miR-155 “seed” mutant or a control vector were stimulated with LPS for 20 hours and expression of IL-23 p19, IL-6, IL- 12p40 and TNF-α mRNA was assayed by qPCR. Data represent two independent experiments. H. Proliferation of 2D2 or OT2 CD4⁺ T cells in response to their respective antigens presented by WT or Mir155^−/− DCs was assessed by assaying ³[H] thymidine incorporation (n=3). Data represent two independent experiments. Error bars represent +/−SEM and * denotes statistical significance with a p value of <0.05 according to a student’s two-tailed t-test.