Micro-RNA-155 inhibits IFN-gamma signaling in CD4+ T cells

Abstract

Micro-RNA (miR) are increasingly recognized as critical regulators of tissue-specific patterns of gene expression. CD4+ T cells lacking miR-155, for example, exhibit bias towards Th2 differentiation, indicating that the absence of individual miR could alter CD4+ T-cell differentiation. We now show that miR-155 is induced upon T-cell activation and that it promotes Th1 differentiation when over-expressed in activated CD4+ T cells. Antagonism of miR-155 leads to induction of IFN-gamma receptor alpha-chain (IFN-gammaRalpha), and a functional miR-155 target site is identified within the 3' untranslated region of IFN-gammaRalpha. These results identify IFN-gammaRalpha as a second miR-155 target in T cells and suggest that miR-155 contributes to Th1 differentiation in CD4+ T cells by inhibiting IFN-gamma signaling.

Figure 1

Analysis of miR-155, miR-146a and miR-150 expression by Northern blot. CD4⁺ T cells from BALB/c mice were stimulated in vitro, and cultured in unbiased, Th1-, or Th2-polarizing conditions. On the second, fourth and sixth days of culture cells were collected for RNA isolation and subsequent Northern blotting. The blot was probed for miR as indicated and for U6snRNA as a loading control. Data is representative of three independent experiments.

Figure 2

Over-expression of miR-155, miR-146a and miR-150 and antagonism of miR-155 in a CD4⁺ T cell Th1/Th2 differentiation assay. CD4⁺ T cells from C57BL/6 mice were stimulated and cultured in unbiased conditions. Cells were transduced with retrovirus encoding GFP and (A) miR-155, (B) miR-146a, or (C) miR-150 36 hours after plating. (D) Cells were cultured in the presence of an antagomir directed against miR-155 or vehicle control. On the fourth day of culture cells were re-stimulated with PMA and ionomycin and production of IL-4 and IFN-γ was measured by intracellular cytokine staining and flow cytometry. Plots shown in A, B, and C are gated on CD4⁺ GFP⁺ (transduced) cells. Plots in A, B, C, and D are representative of four independent experiments. Summary of data from A (E) and D (F), data show mean ± SEM from four independent experiments. *p<0.01 versus vector or **p<0.05 versus vehicle; Student's two-tailed t-test.

Figure 3

IFN-γRα is a target of miR-155 in Th1 cells. (A and B) CD4⁺ T cells from C57BL/6 mice were stimulated and cultured under Th1- or Th2-polarizing conditions with antagomir (black line) or vehicle control (gray filled line) (A) or transduced with retrovirus encoding GFP alone (vector, gray filled line) or with miR-155 (black line) (B). After four days, IFN-γRα surface expression was measured by flow cytometry. Plots in (B) are gated on GFP⁺ cells. (C) NIH 3T3 cells were transduced with retrovirus encoding miR-155 or miR-150 and then co-transfected with an unmodified firefly luciferase reporter and a renilla luciferase reporter either unmodified or modified to include three copies of the 27 base pair IFN-γRα 3′ UTR sequence containing the putative miR-155 target site. The ratio of renilla luciferase to firefly luciferase activity (RLU/FLU) is represented in the bar graph as mean ± SEM from three independent experiments. *p<0.01 versus miR-155 control; Student's two-tailed t-test. (D) CD4⁺ T cells from C57BL/6 mice were stimulated and cultured under unbiased conditions in the presence of antagomir or vehicle control, with or without anti-IFN-γ antibody. After four days cells were re-stimulated and production of IL-4 and IFN-γ was measured by flow cytometry. Data are representative of three independent experiments. (E and F) CD4⁺ T cells from C57BL/6 mice unlabeled (E) or CFSE labeled (F) were stimulated and cultured under unbiased conditions with anti-IFN-γ antibody (dashed line, E only), antagomir (gray filled line), or vehicle control (black line). (E) After 16 hours, CD4 and T-bet expression were analyzed by flow cytometry. (F) After three days cells were analyzed by flow cytometry for CFSE content. Histograms are gated on live CD4⁺ cells (E, F).