In the current study, we have provided data to demonstrate that miR-155 is induced in DCs upon maturation and that its expression in vivo negatively regulates the induction of antigen-specific T cells. Administration of bic
, a miR-155 expression vector, to epidermal DCs in vivo suppressed the generation of T cell-mediated immunity. This outcome was reversed by decreasing endogenous miR-155 levels in DCs with a partially antisense inhibitor, I
. These results altogether suggest that miR-155 has an inhibitory effect on DC-mediated T cell activation. In addition, our data also suggest that miRNA levels in DCs may be manipulated to modulate the activation of antigen-specific T cell-mediated immunity for therapeutic purposes.
Our findings reveal a new way in which miRNAs may exert control over the immune system. The role of miRNAs as key regulators of the innate and adaptive immune systems is becoming increasingly evident. Indeed, miRNAs have recently been implicated in a wide variety of immunological processes, including hematopoietic cell differentiation, lymphocyte development and function [6, 7, 15–17], as well as protective responses against bacterial  or viral  pathogens. The current study identifies a novel role of miRNA in the regulation of DC-mediated antigen-specific T cell priming.
In general, the expression of miRNAs is precisely controlled at the transcriptional level, enabling them to finely adjust cellular behavior as necessary. miR-155 production is believed to be transiently under the control of nuclear factor (NF)-κB and can be strongly induced during inflammation [18, 20, 21]. Recently, miR-155 has also been shown to play a role in the promotion of T cell-dependent tissue inflammation. It has been shown that basal miR-155 levels in murine macrophages are virtually undetectable but increase dramatically by 6 hrs after treatment with the TLR ligands polyriboinosinic:polyribocytidylic acid (poly(I:C)), LPS, CpG, and P3C, as well as the cytokines IFN-β and IFN-γ, in a c-Jun N-terminal kinase-dependent manner [18, 20]. We have demonstrated that this phenomenon is conserved in DCs, as stimulation of these cells with LPS strongly induced miR-155. Collectively, our findings suggest that miR-155 expression may be a global consequence of inflammation, although the precise role it plays in this process may vary depending on cell type.
It has been demonstrated that miR-155 is critical for the generation of robust T cell-mediated immunity in mice, partly through the action of this miRNA in DCs . In this context, we were surprised to find that coadministration of DNA encoding miR-155 with CRT/E7 led to a decrease in E7-specific CD8+ T cell-mediated immune responses in vivo. These results suggested to us that miR-155 may have a complicated function in DCs, in which its influence on T cell stimulatory capacity could vary depending on its expression level or the developmental stage of the cell. Alternatively, because our biolistic delivery method transfects a wide variety of epidermal cells in addition to DCs, it is conceivable that the phenomenon we observed is due to the effects of miR-155 on these other types of cells. We reasoned that if overexpression of miR-155 in DCs diminished antigen-specific T cell responses, then suppression of this miRNA should correspondingly augment these responses. Biolistic delivery to epidermal DCs of an efficient miR-155 repressor, I
, significantly amplified the number of IFN-γ-secreting E7-specific CD8+ T cells generated by the CRT/E7 vaccine. Therefore, manipulation of endogenous miR-155 levels in DCs alters the intensity of DC-induced T cell-mediated immunity in vivo in a systematic manner, consistent with the proposed immunosuppressive activity of this miRNA.
Several reported studies may support the observed function of miR-155 as an attenuator of the adaptive immune system and provide clues about the molecular mechanisms through which this miRNA exerts its effects. It was recently shown that the NF-κB signaling pathway may be repressed by miR-155 through the silencing of inhibitor of NF-κB (IκB) kinase (IKK) ε . In response to TLR signaling, the IKK family of proteins phosphorylates IκB, thereby enabling NF-κB to translocate into the nucleus and initiate transcription. IKKε has also been demonstrated to facilitate NF-κB nuclear import by phosphorylating its c-Rel subunit . In addition, IKKβ as well as a variety of other central immunological molecules--such as Fas-associated death domain protein (FADD) and receptor (TNFRSF)-interacting serine-threonine kinase 1 (Ripk1)--likely represent major direct targets of miR-155 . FADD is an adaptor molecule that is crucial for the development of normal immune responses [25, 26], and Ripk1 mediates tumor necrosis factor-induced activation of NF-κB . Therefore, since the maturation and T cell stimulatory capacity of DCs depends vitally on signaling through the NF-κB pathway, miR-155 could exert its inhibitory effects by suppressing this pathway on multiple levels. Furthermore, a recent study has reported that miR-155 produced by human monocyte-derived DCs attenuates the TLR/IL-1 pathway and silences transforming growth factor-β activated kinase 1 binding protein (TAB2), an important intermediate in the IL-1 cascade . This in vitro evidence directly supports our results which suggest that miR-155 expression in DCs is part of a negative feedback loop that suppresses the inflammatory response. While we believe, on the basis of its likely molecular targets, that miR-155 influences DC biology primarily at the level of antigen presentation and processing or costimulation, we cannot exclude the possibility that the in vivo effects we observed in this study were at least partially due to other factors, such as the potential ability of miR-155 to impair cellular migration to the peripheral lymphoid organs. These alternative mechanisms present an important area for future investigation.
On the basis of our current data and the work of others, it is intriguing to speculate about the purpose of miR-155 activity in DCs. We propose a dose-dependent paradigm in which a threshold miR-155 expression level must be maintained in DCs in order to generate effective T cell-mediated immunity against foreign pathogens. However, when environmental stimuli cause the concentration of cellular miR-155 to escalate beyond this basal level, miR-155 becomes sufficiently abundant to repress the translation of molecules involved in antigen presentation or T cell activation, and thus may form part of a negative regulatory pathway that restrains the magnitude of the adaptive immune response and perhaps evolved to protect against the onset of autoimmune pathologies.
The current study mainly focuses on an immature DC line, DC-1. It will be important to also consider performing experiments to interfere with miR-155 levels during different maturation stages of monocyte-derived DCs (moDCs) for a more comprehensive understanding of the effect of miR-155 on the biological function of moDCs . However, the potential limitations for such an experimental approach is the overexpression of miRNAs to non-physiological levels that may lead to altered target genes. Thus it is important to consider the expression levels of miRNAs in moDCs for gain- or loss-of-function studies to illustrate the influence of miR-155 on moDCs.
Finally, this study provides an impetus and framework for the development of miRNA-based therapeutics. To our knowledge, this is the first report of in vivo miRNA delivery for the modulation of adaptive immunity. We have demonstrated the general principle that miRNAs or their inhibitors can be biolistically administered into epidermal DCs to systematically manipulate T cell-mediated immune responses. It is exciting to envision the possibility that, in the near future, these small yet powerful RNA species may be incorporated into medicines for autoimmune disorders or into vaccines for cancers and viral infections.