Here we have shown identical phenotypes when either Dicer
gene was inactivated in NK cells. These results strongly suggest that these two molecules function in the same biological pathway in miRNA biogenesis, and demonstrate that the deficiency in miRNAs is the primary cause underlying the observed phenotypes. Although it is possible that Dicer-deficient cells also exhibit other more subtle phenotypes, such as the derepression of retrotransposons (24
), it appears that it is miRNAs, rather than other Dgcr8-independent, Dicer-dependent small RNAs, that are critical for NK cells.
Ablation of the miRNA biogenesis pathway, through deletion of Dicer or Dgcr8, led to increased apoptosis of peripheral NK cells. Similarly, Dicer deletion in developing B cells (17
), thymocytes (15
), or iNKT cells (19
) resulted in increased cell death. These results suggest that the miRNA pathway plays an important role in controlling cell survival. Potential mechanisms include mitotic defects due to centromere dysfunctions (43
), defects in heterochromatin maintenance (44
), and aberrant over expression of pro-apoptotic protein Bim (17
). In addition, our preliminary studies indicate that Bcl-2 mRNA level was consistently decreased in DicerΔ/Δ
NK cells (Suppl. Fig. 4
). Yamanaka et al.
recently showed that inhibition of miR21 in the human NKL cell line led to increased apoptosis associated with the up-regulation of pro-apoptotic miR21 targets PTEN, PDCD4, and Bim (38
). Although it is very likely that multiple miRNAs take part in the coordinate regulation of NK cell survival, it would be informative to test whether miR21 is a key regulator of NK cell survival.
Maturation of NK cells is characterized by a decrease in the expression of CD27, active proliferation, and a concomitant increase in the levels of CD11b and effector functions (35
). Analysis of DicerΔ/Δ
mice revealed a relative accumulation of more immature CD27hi
NK cells. This could be due to a selective loss of more mature CD27lo
NK cells or a maturation defect. We did not detect any preferential difference in the death or turnover in the CD27hi
NK cell subsets (unpublished observations). Furthermore, we found a similar accumulation of CD27hi
NK cells in CD45.2+
subsets of DicerΔ/Δ
mixed bone marrow chimeras (data not shown). These data indicate that impaired transition from CD27hi
of Dicer- and Dgcr8-deficient NK cells is hematopoietic cell intrinsic and cell autonomous, suggesting that miRNAs might regulate NK cell maturation. Interestingly, miR150 has been shown to regulate B cell development and this regulation occurs through the inhibition of Myb expression (46
). MiR150 is dynamically regulated during NK cell maturation (unpublished observations), and Chiossone et al.
reported that Myb is differentially up-regulated in CD27hi
NK cells (45
). Future studies will determine if NK cell development or maturation is regulated by miR150.
In contrast to having no significant effect on many of the activating and inhibitory NK cell receptors analyzed, Dicer and Dgcr8-deficiency had an impact on the level of NKG2D on the surface of NK cells. In addition, we found elevated surface levels of Rae-1 on NK cells from Dicer-deficient mice (). NKG2D is an activating receptor that binds to a diverse family of ligands that are distant relatives of MHC-class I molecules, including MICA and MICB in humans and Rae-1α-ε, MULT1, and H60 in mice (36
). Engagement of NKG2D by its ligands leads to the direct activation of killing and cytokine secretion by NK cells. Because of this potent killing ability, surface expression of NKG2D ligands is tightly regulated so that the immune response is not triggered inappropriately. Our findings suggest that miRNAs may regulate the expression of the mouse NKG2D ligand Rae-1. One possible mechanism would be a specific miRNA targeting Rae-1 mRNA. Several human miRNAs were recently identified to bind the 3’UTR of MICA and MICB and to repress their translation (48
). Alternatively, Dicer or Dgcr8 deficiency in NK cells might elicit a DNA damage response, which in turn results in the induction of Rae-1 expression (50
). Consistent with this hypothesis, silencing of Dicer in HEK293T and human hepatoma cells led to the upregulation of MICA and MICB (51
). In either of these scenarios, the increased levels of Rae-1 would likely lead to down modulation of NKG2D on NK cells, as has previously been demonstrated (36
We have shown that degranulation and IFNγ production were impaired in Dicer- and Dgcr8-deficient NK cells following stimulation via NK1.1, NKp46, or Ly49H. This impairment in IFNγ production might be caused by the reduction in the CD27lo
NK cell subset in DicerΔ/Δ
mice, because this subset has been demonstrated to play a dominant role in cytokine production (45
). To this end we co-stained NK cells with anti-CD27 and anti-CD11b antibodies and measured intracellular IFNγ levels. Both CD27hi
NK cell subsets from DicerΔ/Δ
mice produced less IFNγ compared to that in the corresponding NK cell subsets from the control mice (data not shown). Thus, a decline in CD27lo
NK cells in DicerΔ/Δ
mice does not explain the reduction in cytokine generation. Because stimulation with IL-12 and IL-18 induced comparable amounts of IFNγ in DicerΔ/Δ
, and control NK cells, we conclude that miRNAs are necessary for ITAM-based activation.
Li et al.
recently demonstrated that miR181a modulates signal strength of the T cell receptor (TCR) by down-regulating expression of several protein tyrosine phosphatases, including SHP-2, PTPN22, DUSP5, and DUSP6 (37
). These phosphatases are expressed in mouse NK cells (52
); with the exception of SHP-2, however, it is currently not known whether they regulate NK cell activation. In T cells, miR181a-mediated regulation of these phosphatases leads to enhanced basal activation of the TCR signaling molecules including Src kinase, Lck, and Erk (37
). In addition, Yamanaka et al.
showed that reducing levels of miR21 in the human NKL cell line resulted in the downregulation of phosphorylated AKTser473
). Thus, a potential explanation for the impaired function of ITAM-containing receptors in Dicer- and Dgcr8-deficient NK cells is that specific miRNA(s) might regulate signaling downstream of these ITAM-containing receptors.
NK cells have long been compared to effector and memory CD8+
T cells in phenotype and function (53
), and recently several groups have demonstrated that NK cells can become long-lived cells and mount secondary responses against viral antigens (31
). In this study, we determined the miRNA expression profile of mouse and human NK cells. Naïve NK cells and CD8+
T cells share a large part of their miRNA profile: miR142-3p, miR142-5p, miR150, miR16, miR23a, miR15b, miR29a, miR29b, miR30b, and miR26a are highly expressed in both naïve NK cells and CD8+
T cells (Suppl. Fig. 1
)). Similar to NK cells, the frequency and number of naïve CD8+
T cells were preferentially reduced in Dicer- and Dgcr8-deficient mice. Thus, miRNAs shared by naïve NK cells and CD8+
T cells might be implicated in the regulation of common molecular pathways, such as regulation of homeostasis.
NK cells also share a miRNA profile with effector and memory CD8+
T cells. MiR21, miR221, and miR222 are expressed in both NK and effector CD8+
T cells, whereas miR146a is found in both NK cells and memory CD8+
T cells (Suppl. Fig. 1
)). It is intriguing to speculate that the miRNAs shared by NK cells, effector CD8+
T cells, and memory CD8+
T cells might be implicated in the regulation of common pathways that lead to the acquisition of the effector phenotype and survival. Our identification of a requirement of Dicer and Dgcr8 for Ly49H+
NK cell expansion during MCMV infection predicts miRNAs might target factors that are important for maintaining the NK cell population during viral infection. In conclusion, our results present novel evidence for miRNAs regulating diverse aspects of NK cell biology, including basic processes such as turnover and survival, as well as the function of activating NK cell receptors during MCMV infection.