MicroRNAs (miRNAs) are short ribonucleic acids, which consist of an average of 22 nucleotides, and bind to complementary sequences of target mRNAs to result in translational repression or target degradation, thus silencing gene expression. MiRNAs can be abundantly found in circulating blood, yet whether, as a class of regulatory molecules, they may interact with innate immune natural killer (NK) cells has not been explored.
Human NK cells were first enriched from the peripheral blood of healthy donors by negative selection using RosetteSep NK cell enrichment cocktail, followed by positive selection using anti-CD56 microbeads. After purity of ≥ 99% was confirmed by flow cytometry, NK cells were used for experiments. After being isolated from healthy donor serum by ExoQuick Exosome precipitation and verified by immunoblotting for CD9 expression, exosomes were assessed for miRNA content via real-time reverse-transcriptase (RT)-PCR using TaqMan miRNA assays. Purified NK cells were stimulated with either whole exosomes or miRNAs complexed with DOTAP, a liposomal transfection reagent. Downstream activation of Toll-like receptor (TLR) signaling by miRNAs was measured via immunoblotting for NF-kB, and its inhibition was similarly assayed in the presence of TLR blocking antibodies. Flow cytometry was used to assess NK cell activation (via CD69 surface expression) and NK cytotoxicity against tumor cells (in a CD107a degranulation assay). IFN-g production was measured via Real-time RT-PCR and enzyme-linked immunosorbent assay (ELISA). For in vivo stimulation, a complex consisting of miRNAs and Lipofectamine 2000 was administered by tail-vein injection. NK cell activation was then measured using the aforementioned in vitro assays. After in vivo stimulation with miRNAs, which was performed in the presence of NK cells or following NK depletion by TM-β1 (IL-2/15Rβ) mAb, development of implanted lymphoma tumor cells was monitored by bioluminescent imaging. NK cells purified from lymphoma patients and from healthy donors were assessed for expression of the NF-kB signaling component, p65, and TLRs via real-time RT-PCR. NK cell maturation was analyzed by flow cytometric staining for surface receptors, such as CD56 and CD94, indicative of NK cell maturation.
We found that, in the presence of a low dose IL-12, treatment of human NK cells with several mature miRNAs induced CD69 expression, IFN-g production, and expression of the degranulation marker, CD107a. MiRNA-containing exosomes freshly isolated from normal human donors were also able to activate NK cells, even in the absence of IL-12. In vivo, infusion of several miRNAs into the peripheral blood similarly activated murine NK cells, while T cells were not activated. Furthermore, miRNA administration significantly protected mice from developing tumors, and this occurred in an NK cell-dependent manner. Interestingly, miRNAs also augmented expression of surface markers associated with NK cell maturation, such as CD56 and CD94, suggesting that miRNAs may play a role in promoting NK cell maturation. Mechanistically, we found that stimulation with miRNAs led to downstream activation of NF-kB. This effect was blunted upon blockade of TLR (e.g. TLR1) signaling, and was attenuated in lymphoma patients.
Collectively, we provide the first evidence that extrinsic miRNAs, as a class of regulatory molecules, directly activate and may also promote the maturation of NK cells. These effects on NK cell activation and maturation are mediated, at least in part, by the TLR signaling pathway. This phenomenon may be important for normal host defense against infection and/or malignant transformation. Our studies indentify a new function of miRNAs with physiological relevance, and their potential for applications in preventing or treating cancer and infections either alone or as an adjuvant.
Jaglowski:Pharmacyclics: Research Funding.
Asterisk with author names denotes non-ASH members.