MicroRNA-Deficient Murine NK Cells Exhibit Impaired Development and Survival but Enhanced IFN-γ Production In Vitro and In Vivo

Abstract 357

NK cells are innate immune lymphocytes important for early host defense against infectious pathogens and malignant transformation. MicroRNAs (miRNAs) are small regulatory RNAs that control a wide variety of cellular processes by specific targeting of mRNA 3'UTRs. The Dicer1 gene encodes a conserved enzyme essential for miRNA processing, and Dicer1 deficiency leads to a global defect in miRNA biogenesis. While miRNA expression and regulation of adaptive T and B lymphocytes are well established, their role in the regulation of NK cell biology remains unclear. We postulated that miRNAs serve an essential role in orchestrating NK cell development and activation. To test this hypothesis, we combined lymphocyte-restricted hCD2-Cre transgenic, Rosa26-YFP-Cre-reporter, and Dicer1 ‘floxed' mice. In this model, 25–50% of Dicer1 wt/wt NK cells are YFP⁺ marking expression of Cre. As expected, YFP+ NK cells from Dicer1 fl/fl and fl/wt mice were confirmed to excise Dicer1, and exhibit decreased total miRNA content based on Nanostring profiling and real-time qPCR (Dicer1 fl/fl: P<0.001, fl/wt: P<0.01). MiRNA-deficient Dicer1 fl/fl mice exhibited reduced YFP⁺ NK percentages (spleen Dicer1 fl/fl: 14±4%, fl/wt: 35±7%, wt/wt 36±7%, P<0.001) as well as reduced absolute numbers of YFP⁺ NK cells [spleen Dicer1 fl/fl: 3.4±0.6×10E5, fl/wt: 6.3±1.7×10E5, wt/wt 6.1±.99×10E5, P<0.01]. In addition, Dicer1 fl/fl mice had reductions NK cell precursors in the BM (stage 2–3 NK precursors mean decrease 70±14% in Dicer1 fl/fl compared to wt/wt, P <0.01). Further, Dicer1 fl/fl NK cells exhibited reduced survival ex vivo when cultured in medium (P<0.01), low dose- (P<0.01), or high dose-IL-15 (P<0.01). These data collectively indicate that Dicer1-dependent miRNAs regulate NK cell development and homeostasis, and the net effect of miRNA loss is impaired NK development and/or survival. However, in our model Dicer1-deficient mature NK cells exhibited enhanced functionality; a finding that contrasts to less NK selective miRNA-deficient NK cell models (Bezman et al. J Immunol 185:3835, 2010). Degranulation (CD107a⁺, a surrogate for cytotoxicity) was enhanced in vitro in response to YAC-1 tumor target cells (P<0.05) and activating NK cell receptor ligation (P<0.001). This was unlikely due to alteration in activating NK cell receptor expression since the surface density of NKG2D and NKp46 were not affected by miRNA deficiency. Moreover, interferon-gamma (IFN-γ) production was enhanced in vitro in miRNA-deficient NK cells in response to IL-12+IL-15 (P<0.01), YAC-1 tumor target cells (P<0.01), and activating NK cell receptor ligation (P<0.001). Further, evaluation of NK cells 36 hours after infection with MCMV resulted in significantly increased IFN-γ production (% NK YFP⁺IFN-γ⁺) in Dicer1 fl/fl (64± 4.9%) vs. fl/wt (52±11%, p <0.01) or vs. wt/wt (41±6%, p <0.001) in vivo. MiRs-15/16 were identified as abundant miRNAs in NK cells that had reduced expression in Dicer1 fl/fl NK cells, and are predicted to target the murine IFN-γ 3'UTR. This targeting was validated in vitro, by transfecting 293T cells with miRNA-15/16 or control over-expression vectors and a sensor plasmid that places luciferase under the control of the murine IFN-γ 3'UTR (34% decrease, P<0.01). Moreover, the targeting was direct, since miR-15/16 targeting of IFN-γ was abrogated after mutation of two predicted binding sites in the IFN-γ 3'UTR. These data indicated that miR-15/16 may regulate IFN-γ translation by resting NK cells. Thus, our study suggests that the function of miRNAs in NK cell biology is complex, with an important role in NK cell development, survival and/or homeostasis, while tempering peripheral NK cell activation. Further study of individual miRNAs in an NK cell specific fashion will provide insight into these complex miRNA regulatory effects in NK cell development/survival and effector function.