The RNAseIII enzyme Drosha is critical in T cells for preventing lethal inflammatory disease

Abstract

MicroRNAs (miRNAs) are implicated in the differentiation and function of many cell types. We provide genetic and in vivo evidence that the two RNaseIII enzymes, Drosha and Dicer, do indeed function in the same pathway. These have previously been shown to mediate the stepwise maturation of miRNAs (Lee, Y., C. Ahn, J. Han, H. Choi, J. Kim, J. Yim, J. Lee, P. Provost, O. Radmark, S. Kim, and V.N. Kim. 2003. Nature. 425:415-419), and genetic ablation of either within the T cell compartment, or specifically within Foxp3(+) regulatory T (T reg) cells, results in identical phenotypes. We found that miRNA biogenesis is indispensable for the function of T reg cells. Specific deletion of either Drosha or Dicer phenocopies mice lacking a functional Foxp3 gene or Foxp3(+) cells, whereas deletion throughout the T cell compartment also results in spontaneous inflammatory disease, but later in life. Thus, miRNA-dependent regulation is critical for preventing spontaneous inflammation and autoimmunity.

Figure 1.

Drosha deficiency abrogates miRNA but not rRNA processing. (A) CD4-cre efficiently deletes exon 9 from the conditional Drosha transcript from the DP thymocyte stage onwards. cDNA from the indicated populations was analyzed for expression of the Drosha exons 5/6 or exon 9/10 junction by quantitative RT-PCR. DN4, CD90⁺TCRβ^loCD4/8/44/25⁻; DP, CD4⁺8⁺; CD4SP, TCRβ^hiCD24^lo4⁺8⁻; CD8SP, TCRβ^hiCD24^lo4⁻8⁺. (B) Drosha protein expression in DP thymocytes, and peripheral TCRβ⁺ T cells and B220⁺ B cells from Drosha^F/Δ CD4-cre and control Drosha^F/+ CD4-cre mice. Molecular weights are shown. (C) Total RNA from DP thymocytes and peripheral T and B cells was resolved on a polyacrylamide gel and Northern blotted for expression of mature miRNAs. Also shown is the ethidium bromide gel for expression of small rRNA and transfer RNA species. (D) Total RNA was resolved on an agarose-formaldehyde gel and Northern blotted for expression of pri-miR-150. Also shown is the ethidium bromide gel for expression of the large rRNA subunits.

Figure 2.

Drosha deficiency at the DP thymocyte stage partially perturbs thymocyte output. (A) Total thymocyte and splenocyte numbers in Drosha^F/Δ CD4-cre and Drosha^F/+ CD4-cre (control) mice. (B) TCRβ versus TCRγδ expression on total thymocytes. (C) CD24 and CD69 expression on TCRβ^hi gated thymocytes. (D) CD4 versus CD8 expression on total, postselected (TCRβ^hiCD24^hi/69⁺) and mature (TCRβ^hiCD24^lo/69⁻) thymocytes. (E and F) FACS analysis of populations in total splenocytes (E) and TCRβ⁺ splenocytes (F). Also shown is the effect of Dicer deficiency. Percentages of cells are shown in B–F. (G) Absolute CD4⁺8⁺ DP, mature thymocyte, and TCRβ⁺ splenocyte numbers. (H) CD4/CD8 ratio in the mature thymocyte and TCRβ⁺ splenocyte compartments.

Figure 3.

T cell–specific Drosha deficiency results in spontaneous inflammatory disease and premature mortality. (A) Kaplan-Meyer survival plot of Drosha^F/Δ CD4-cre and control mice (Drosha^F/F, Drosha^Δ/+ , or Drosha^F/+ CD4-cre). (B) Aberrant activation of CD4⁺ T cells in lymphoid organs of moribund Drosha^F/Δ CD4-cre mice. (C and D) Cytokine expression by CD4⁺ (C) and CD8⁺ (D) T cells. (E) Frequent IFN-γ and IL-17A coexpression by CD4⁺ T cells. (F) Granulocyte (Gr-1⁺) and γδT cells populations in lymphoid organs. Percentages of cells are shown in B–F. (G) H&E-stained sections of the lung (i and iii) and liver (ii, iv, and v). Bars, 100 μm.

Figure 4.

Drosha- and Dicer-dependent pathways are required for efficient induction of Foxp3 expression and function of T reg cells. (A) Reduced TCRβ⁺Foxp3⁺ T reg cell populations in the thymus and spleen of Drosha^F/Δ CD4-cre and Dicer^F/F CD4-cre mice. (B) Foxp3 expression in peripheral T reg cell populations in the absence of Drosha or Dicer. (C and D) Drosha and Dicer are required for efficient differentiation of induced T reg cells. Naive CD62L^hi44^lo25⁻ CD4⁺ T cells from Drosha^F/Δ CD4-cre, Dicer^F/F CD4-cre, and control Drosha^F/+ CD4-cre mice were activated in vitro with anti-CD3/28 antibodies plus 10 U/ml IL-2, and TGF-β (C) or TGF-β plus 10 nM RA (D) for 3 d. The cells were restimulated with PMA/ionomycin plus GolgiStop for 3 h, and Foxp3 and IL-17A expression was analyzed by intracellular FACS. Percentages of cells are shown in A–D. (E) FACS-purified naive CD4⁺ cells from Cd45.1 mice were loaded with CFSE and mixed with purified CD4⁺25⁺ cells from Drosha^F/Δ CD4-cre or Drosha^F/+ CD4-cre (Cd45.2) mice at the indicated ratios. These were then incubated with inactivated splenocytes and anti-CD3 antibody. After 4 d, the CD45.1⁺ cells were analyzed for CFSE dilution.

Figure 5.

Drosha deficiency does not prevent polarization of naive CD4⁺ T cells into Th1 or Th2 cells. Naive CD62L^hi44^lo25⁻ CD4⁺ T cells from Drosha^F/Δ CD4-cre and control Drosha^F/+ CD4-cre mice were activated in vitro with anti-CD3/28 antibodies and 10 U/ml IL-2, plus (A) IL-12 and 1 μg/ml anti–IL-4 antibody or (B) IL-4 and 1 μg/ml anti-IFN-γ antibody for 4 d. The anti-CD3/28 antibodies were removed from the culture for the last 24 h, after which the cells were restimulated with PMA/ionomycin for 3 h in the presence of GolgiStop and analyzed for intracellular IFN-γ and IL-4 expression by FACS. Percentages of cells are shown.

Figure 6.

T reg cell–specific Drosha or Dicer deficiency recapitulates the scurfy phenotype. (A) Kaplan-Meyer survival plot of Drosha^F/Δ Foxp3^Cre/Cre(Y), Dicer^F/F Foxp3^Cre/Cre(Y), and control mice (Drosha^F/+ Foxp3^Cre/Cre(Y), Drosha^F/Δ Foxp3^Cre/+, Drosha^F/+ Foxp3^Cre/+, Dicer^F/+ Foxp3^Cre/Cre(Y), and Dicer^F/+ Foxp3^Cre/+). (B) Massive lymphadenopathy in a female Drosha^F/Δ Foxp3^Cre/Cre mouse. Shown are the cervical lymph nodes. (C) Cell counts of lymphoid organs from moribund Drosha^F/Δ Foxp3^Cre/Cre or Drosha^F/Δ Foxp3^Cre/Y mice and littermate controls. Data represent the mean ± SD of four mice at 2.5–3 wk of age. (D) Only marginal changes in the proportions of B (CD19⁺), γδT (TCRγδ⁺), myeloid (CD11b⁺), and T cells (TCRβ⁺) in the spleen and lymph nodes of Drosha^F/Δ Foxp3^Cre/Cre mice. (E) Aberrant T cell activation in the spleen and lymph nodes of moribund Drosha^F/Δ Foxp3^Cre/Cre mice. (F and G) Cytokine expression by CD8⁺ (F) and CD4⁺ (G) T cells from Drosha^F/Δ Foxp3^Cre/Cre mice. (H and I) Foxp3 expression in total splenocytes (H) and CD4⁺ T cells (I). Percentages of cells are shown in D–I. (J) H&E-stained sections of the lung (i and iii) and liver (ii and iv). Designation of Foxp3^Cre/Cre(Y) alleles indicates that both males and females were included in the experiments. Bars, 100 μm.