Loss of microRNAs in thymus perturbs invariant NKT cell development and function

Abstract

microRNAs (miRNAs) are small noncoding RNAs that mediate RNA interference to suppress protein expression at the translational level. Accumulated evidence indicates that miRNAs play critical roles in various biological processes and disease development, including autoimmune diseases. Invariant natural killer T (iNKT) cells are an unusual CD1d-restricted subset of thymus-derived T cells that are potent regulators of diverse immune responses. Our previous studies with the mouse model of bone marrow-specific Dicer deletion suggest the involvement of Dicer-dependent miRNAs in the development and function of iNKT cells. In the present study, to further dissect the functional levels of Dicer-dependent miRNAs in regulating iNKT cell development, we generated a mouse model with the Dicer deletion in the thymus. Our data indicate that lack of miRNAs following the deletion of Dicer in the thymus severely interrupted the development and maturation of iNKT cells in the thymus and significantly decreased the number of iNKT cells in the peripheral immune organs. miRNA-deficient peripheral iNKT cells display profound defects in activation and cytokine production upon α-galactosylceramide (α-GalCer) stimulation. Our results demonstrate a critical role of the miRNA-dependent pathway in the thymus in the regulation of iNKT cell development and function.

Figure 1

Reduced number of iNKT cells in conditional thymic Dicer KO mice. (a) Reduced frequency of iNKT cells in Dicer^fl/flCD4cre⁺ mice. Thymocytes, splenocytes and hepatic leukocytes from 4- to 7-week-old Dicer^fl/flCD4cre⁺ KO and Dicer^flox/flox WT littermates were stained with either PBS-57-loaded CD1d tetramers to identify the iNKT population or unloaded CD1d tetramers to define nonspecific binding. Plots were gated on lymphocytes. In the case of spleen and liver, B220⁺ B cells have been gated out. Results are representative of at least five experiments. (b) Absolute number of iNKT cells. Thymus, spleen and liver mononuclear cells were labeled with PBS-57/CD1d tetramers and an anti-TCRβ monoclonal antibody. Using the total cell count obtained from each organ, absolute numbers of iNKT lymphocytes (gated as shown in a) were determined. Numbers shown are mean±SD. Total iNKT cells were enumerated from six experiments and are shown plotted on a logarithmic scale. The differences marked with one asterisk was statistically different with P<0.05 and marked with two asterisks was P<0.005 using a two-tailed Student's t-test. (c) Proliferation of thymic and splenic iNKT cells in response to α-GalCer. Thymocytes and splenocytes of Dicer^fl/flCD4cre⁺ KO and Dicer^flox/flox WT mice were cultured with 100 ng/ml α-GalCer for 48 h. Data are presented as mean±SD of one representative experiment of three. The difference marked with an asterisk was statistically different with P<0.01. (d) Reduced frequency of iNKT cells in Dicer^fl/flLCKcre⁺ mice. Thymocytes, splenocytes and hepatic leukocytes from 4- to 7-week-old Dicer^fl/flLCKcre⁺ KO and Dicer^flox/flox WT littermates were stained with PBS-57-loaded CD1d tetramers and an anti-TCRβ monoclonal antibody to identify the iNKT population. Results are representative of at least three experiments. c.p.m., count per minute; iNKT, invariant natural killer; KO, knockout; PBS, phosphate-buffered saline; TCR, T-cell receptor; WT, wild-type; α-GalCer, α-galactosylceramide.

Figure 2

iNKT cell homeostasis and CD1d expression in Dicer^fl/flCD4cre⁺ KO mice. (a) Four- to 7-week-old mice received BrdU (1 mg/mouse) i.p. and were killed after 24 h. Thymocytes were stained with PBS-57-loaded CD1d tetramers, anti-TCRβ and anti-BrdU-specific antibodies. Depicted are BrdU⁺ iNKT cells gated in CD1d tetramers⁺ iNKT cells. Results are representative of two experiments. (b) Apoptosis in iNKT cells. Dicer^fl/flCD4cre⁺ KO and Dicer^fl/fl WT iNKT cells from thymus were stained with Annexin V and 7-AAD. (c) Analysis of CD1d expression. Splenocytes or thymocytes from Dicer^fl/flCD4cre⁺ KO and Dicer^fl/fl WT mice were stained with anti-CD1d antibody or isotype control and analyzed by flow cytometry. Expression levels of CD1d on splenic B cells (B220⁺) and thymus DP cells (CD4⁺CD8⁺) are shown. Results are representative of at least three independent experiments (n=3–5 pairs). BrdU, 5-bromodeoxyuridine; DP, double positive; iNKT, invariant natural killer; i.p., intraperitoneal; KO, knockout; TCR, T-cell receptor; WT, wild-type; 7-AAD, 7-aminoactinomycin D.

Figure 3

The development of iNKT cells is altered in the absence of Dicer in thymus. (a) iNKT cell development in thymus. Thymocytes from Dicer^fl/flCD4cre⁺ KO and Dicer^fl/fl WT mice were stained and CD44 and NK1.1 expression shown in gated CD1d-tetramer⁺TCRβ ⁺ iNKT cells along with percentages of cells in the indicated quadrants. Data are representative of at least five experiments. (b) Absolute number of iNKT cell subpopulations (mean±SD) based on their CD44 and NK1.1 expression patterns. (c) Analysis of subsets of iNKT cells in the thymus. CD1d-tetramer⁺TCRβ ⁺ cells were gated on thymocytes from Dicer^fl/flCD4cre⁺ KO and Dicer^fl/fl WT mice and the relative frequencies of subsets defined by CD4 and CD8 expression were assessed. Graph shows summarized data from 5–7 mice. The difference marked with one asterisk was statistically different with P<0.05 and marked with two asterisks was P<0.005 using a two-tailed Student's t-test. iNKT, invariant natural killer T cells; KO, knockout; NK, natural killer; TCR, T-cell receptor; WT, wild-type.

Figure 4

The function of iNKT cells is altered in the absence of Dicer in the thymus. Dicer^fl/flCD4cre⁺ KO and Dicer^fl/fl WT littermate control mice were injected with 2 µg of α-GalCer or vehicle and subjected to the following analyses (a and b). (a) Serum was collected at 2 and 5 h for detection of IFN-γ by ELISA. The difference marked with one asterisk was statistically different with P<0.05 and with two asterisks was P<0.005. (b) The expression of CD69 by splenic B and CD8⁺ T cells was analyzed 5 h after injection. Results are representative of three independent experiments. (c) Splenocytes were collected at 40 min after injection with α-GalCer and then cultured in T-cell medium. Monesin was added to a final concentration of 3 µM, and the cells were incubated for an additional hour. The production of IFN-γ and IL-4 by splenic NKT cells was then analyzed with intracellular cytokine staining. (d) Splenocytes were left unstimulated or stimulated for 2 h in vitro with PMA and ionomycin. The production of IFN-γ and IL-4 by splenic NKT cells was then analyzed with intracellular cytokine staining. Results are representative of two independent experiments. IFN, interferon; iNKT, invariant natural killer; KO, knockout; PMA, phorbol myristate acetate; TCR, T-cell receptor; WT, wild-type; α-GalCer, α-galactosylceramide.