Dr. Weeks’ Comment: Now science is catching up with us. It is exciting! Three years ago, I explained that part of the uniquely transformative power of eating seeds (crushed whole organic and non-GMO seeds, of course) is that in addition to nutrient density (seeds have 20-30x more nutrients than the the fruit) and the benefits of unadulterated seed oils (stop taking fish oil capsules!) but that we also get genetic spare parts to renovate your own tired old DNA double helix. Now we see the science supporting this claim. Thanks to the wise Dr. Jonathan Miller for sending me this revolutionary article. Eat the seeds – Nature’s own anti-aging genetic repair food!
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Plant microRNAs as novel immunomodulatory agents
Duccio Cavalieri, Lisa Rizzetto, Noemi Tocci, Damariz Rivero, Elisa Asquini, Azeddine Si-Ammour, Elena Bonechi, Clara Ballerini & Roberto Viola
Scientific Reports 6, Article number: 25761 (2016)
Published online:
11 May 2016
Abstract
An increasing body of literature is addressing the immuno-modulating functions of miRNAs which include paracrine signaling via exosome-mediated intercellular miRNA. In view of the recent evidence of intake and bioavailability of dietary miRNAs in humans and animals we explored the immuno-modulating capacity of plant derived miRNAs. Here we show that transfection of synthetic miRNAs or native miRNA-enriched fractions obtained from a wide range of plant species and organs modifies dendritic cells ability to respond to inflammatory agents by limiting T cell proliferation and consequently dampening inflammation. This immuno-modulatory effect appears associated with binding of plant miRNA on TLR3 with ensuing impairment of TRIF signaling. Similarly, in vivo, plant small RNAs reduce the onset of severity of Experimental Autoimmune Encephalomyelities by limiting dendritic cell migration and dampening Th1 and Th17 responses in a Treg-independent manner. Our results indicate a potential for therapeutic use of plant miRNAs in the prevention of chronic-inflammation related diseases.
Introduction
The discovery of microRNAs (miRNAs) and their interaction with the gene expression machinery in most organisms is one of the major scientific breakthroughs in recent years. The regulatory effects of miRNAs have been mainly elucidated studying their expression within a given cell type. Endogenous miRNAs are known to mediate gene expression of cognate messenger RNAs (mRNAs) in a sequence specific manner by affecting, via the RNA-induced silencing complex (RISC), fundamental processes ranging from cell development to cancer to immune regulation3,4,5,6,7. Yet, even still controversial, miRNAs have been shown also to act outside the cell from which they originate by long distance transport8,9,10,11,12,13.
The inter-cellular mode-of-action of miRNAs has led to suggestions for therapeutic applications14 in analogy with exogenously supplemented small interfering RNAs (siRNAs), which induce degradation of sequence-specific homologous mRNA via RNA interference. In the case of siRNAs, “off-targeting effects” and adverse reactions such as the promotion of inflammation15,16 have so far hampered their wider adoption as therapeutic application17. Exogenous double stranded RNA (dsRNA) as well as single stranded RNA (ssRNAs) are intercepted by the Pathogen Recognition Receptors (PRRs) of the innate immune system such as membrane-bound Toll-Like Receptors (TLRs) and cytoplasmic receptors abundant in the immune cell18,19 and activate an inflammatory response through the type I IFN system20. While TLR7 and TLR8 recognize ssRNAs21, TLR3 binds to both single stranded22 and double stranded viral RNAs23,24. TLR3 has also been shown to mediate siRNA non-sequence-specific immune suppression through the type I IFN system in cultured mammalian cells20. TLR7 and TLR8 also appear to be involved in siRNA pro-inflammatory effects25 and in miRNA-mediated paracrine loop between cancer cells and immune cells present in the tumor microenvironment with TLR-mediated pro-metastatic inflammatory response that ultimately may lead to tumor growth and metastasis26.
In addition to intercellular exchange within organisms8,26,27, inter-organismal miRNA exchanges are also known to occur including between taxonomic kingdoms as suggested by the recently reported presence of dietary plant microRNA in plasma and organs of humans and animals10,28,29,30. These latter findings raise paradigm-changing questions concerning our understanding of diet-health interactions. However, the validity of these claims has been challenged as some authors consider artefactual the detection of dietary plant miRNA sequences in the plasma after different feeding regimes28,31,32,33 while others do not question the presence of plant miRNAs in plasma or internal organs, but note that the reported copy number of individual sequences appears too low to be biologically relevant28,34. Plant-based dietary delivery of miRNA for therapeutic purposes has also been proposed10,35.
Since the immune function is the likely mediator of such interactions we have sought to explore the impact of plant miRNAs on dendritic cells (DCs), a component of the innate immune system present in the gut and responsible for instructing T cells to react and adapt to environmental challenges. Here we show that synthetic Fragaria vesca miR168 at physiological concentrations reduce inflammation mediated by TLR agonists via a TLR3-mediated mechanism. Indeed, this efficacy was not limited to miRNA from strawberry but was extended to sRNAs extracts from a wide variety of plant tissues and species. Furthermore, treatment with plant sRNAs could systemically reduce inflammation and prevent symptoms of multiple sclerosis in an Experimental Autoimmune Encephalomyelities (EAE) mouse model. These results provide a novel mechanistic explanation for known beneficiary effects exerted by fruit and vegetable in the prevention of inflammation associated disorders.
Results
Strawberry fruit FvmiR168 affects DC properties and their ability to respond to inflammatory stimuli by limiting T cell proliferation
For initial experiments we tested the immuno-modulatory efficacy of Fragaria vesca 3”² end methylated miR168 (Table 1), one of the most abundant miRNAs present in strawberry fruits, an example of edible miRNA rich plant tissue that is consumed unprocessed. When human monocyte-derived DCs were treated with FvmiR168 complexed with DOTAP at miRNA concentrations up to 10”‰Î¼g/ml,no effect was observed (Supplementary Fig. 1). However, when Fvmir168 pre-treated DCs were challenged with the inflammatory agents LPS or polyI:C, a significant reduction of their inflammatory response was observed (Fig. 1). FvmiR168 decreased LPS- and PolyI:C-induced production of IL-1β and TNFα (Fig. 1a, Student t-test, p”‰<”‰0.05). Fvmir168 also reduced the levels of CD80, CD86, CD83 and the class II immuno-histocompatibility complex (HLA-DR) (Fig. 1b, p”‰<”‰0.05). Significantly, these effects were observed at a concentration of 10”‰ng/mL, a level three order of magnitude lower than that required to induce a similar effect by human anti-inflammatory miRNAs36,37. FvmiR168 treated-DCs also showed a reduced ability to induce T cell proliferation in the presence of LPS or polyI:C (Fig. 1c, p”‰<”‰0.01). The amount of IFNγ produced by T cell was also reduced (p”‰<”‰0.01), in accordance with the reduced ability of DCs to secrete IL-12p70 (Fig. 1d, p”‰<”‰0.01). Th1 cell differentiation was indeed impaired by the FvmiR168 pre-treatment of DCs as revealed by reduction of both Tbet and IFNγ expression (p”‰<”‰0.01). On the other hand, as expected, Th2 differentiation was not affected (Fig. 1e). FvmiR168 treated-DCs also showed a significant reduction in the expression of CCR7 (Fig. 1f, p”‰<”‰0.01), a molecule known to guide DCs to and within lymphoid organs38, suggesting a defect in their migration ability. ` For the rest of the article READ IT HERE