Eliminating key proteins of the miRNA biogenesis pathway can be used to study global miRNA function. Selective ablation of Dicer in Treg leads to a profound and lethal multi-organ autoimmune syndrome 18,19,20]. However, although DICER cleaves miRNA precursors, its activity is not entirely specific for canonical miRNAs 21]. The DROSHA - DGCR8 miRNA microprocessor complex is critical for the cleavage of primary miRNA transcripts and DGCR8 is thought to be specific for the processing of canonical miRNAs rather than other classes of nuclear (small) RNAs 21] although it remains controversial if and to what degree the microprocessor complex also cleaves other RNAs including snoRNAs 26,27]. Furthermore, the DROSHA - DGCR8 miRNA microprocessor complex recognizes and cleaves Dgcr8 mRNA in an autoregulatory loop mostly specific for the microprocessor itself 27,28,29] and DGCR8 stabilizes DROSHA through protein-protein interaction 28]. Thus, Dgcr8-deletion is mostly specific for canonical miRNAs. Herein, we demonstrate that Dgcr8 is essential for Treg function and lineage stability. Our results parallel the observation that DROSHA is essential for Treg function 19]. Our findings do not exclude that non canonical miRNAs like mirtrons or other small RNAs are functional in Treg but they suggest that ablation of the pool of canonical miRNAs is sufficient to cause Treg dysfunction which in turn leads to breakdown of immune homeostasis. In addition, freshly isolated Dgcr8-deficient Tregs expressed slightly reduced FoxP3 levels and were unable to maintain FoxP3 expression when cultured in vitro. Furthermore, the frequency of Dgcr8-deficient GFP expressing Treg was reduced in peripheral blood compared to control mice (data not shown). However, the relative reduction was more pronounced in peripheral blood than lymph nodes. Moreover, initial studies suggest a discrepancy between GFP and FoxP3 protein expression. This could indicate that FoxP3 transcription (read out as GFP) is terminated prematurely in the absence of miRNAs. However, further investigation is required to address this interesting observation. Together, these results confirm and extend our findings that Dicer-deficient Treg lose FoxP3 expression as revealed with a genetic lineage tracing system 18]. Importantly, the T cell receptor repertoire of Treg is skewed towards self-reactivity 30,31]. Hence, loss of FoxP3 could result in self-reactive pathogenic effector T cells. Indeed, miRNA-sufficient exFoxP3 cells can be pathogenic upon adoptive transfer 5]. However, it is unclear whether miRNA-deficient exFoxP3 cells would be pathogenic. Although miRNAs regulate expression of about 50% of all genes 32] and are important for proliferation, survival and differentiation of most cell types, cells devoid of miRNAs can still be functional. As an example, cancer cells can be functional despite absence of miRNAs 33]. Likewise, CD4-cre transgene mediated ablation of Dicer or Drosha in all CD4+ and CD8+ T cells results in a spontaneous multiorgan inflammatory syndrome, likely due to impaired Treg function 19,34]. Importantly, Dicer- and Dgcr8-deficient Tconv are prone to Th1 responses with a strongly increased relative number of Tconv secreting IFN-γ compared to control cells 35,36]. However, mice with a miRNA deficiency in both Treg and Tconv cells do not develop full-blown FoxP3-deficiency disease as seen with Treg-specific Dicer, Drosha or Dgcr8 ablation: disease starts much later and only affects some mice. This indicates that both Treg and Tconv cells are impaired functionally. Therefore, disease results from the net balance of miRNA-deficient effector T cells and miRNA-deficient Treg.
The rapid, fatal disease observed in mice with miRNA-deficient Treg likely reflects a complex collection of defects resulting from multiple missing individual miRNAs. miR-155 might stabilize Treg by facilitating IL-2 signaling 37] and miR-10a might stabilize the Treg lineage 38,39] but genetic deletion of either miRNA does not lead to substantial loss of FoxP3 indicating that other miRNAs or a combination of miRNAs must be stabilizing FoxP3. In addition, miRNAs are likely contributing to the characteristic absence of inflammatory cytokine production in Treg because Dicer-deficient Treg produce IFN-γ 18]. Since miR-29ab is a potent repressor of IFN-γ production in T cells 36,40] it might be involved in IFN-γ repression in Treg. Indeed, Treg express high levels of miR-29ab 41]. Since miR-29ab is induced by IFN-γ functioning in a negative feedback loop 42] it might be particularly important to prevent Treg from producing IFN-γ in an inflammatory setting. It will be interesting to test miR-29ab and other miRNA expression in human Treg expressing IFN-γ 43]. Finally, the availability of an increasing number of mouse models with genetic miRNA deficiencies will be important to study the complex miRNA functions in immune regulation in vivo 44,45].
In summary, canonical miRNAs are essential for normal FoxP3 expression and suppressive Treg function. Deciphering the function of individual miRNAs will be essential to define novel therapeutic targets. Furthermore, studying the function of individual miRNAs in Treg combined with a better understanding of the genetic networks regulated by those miRNAs holds the promise to yield new insight into Treg biology.