Plant microRNAs as novel immunomodulatory agents

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 years¹. The regulatory effects of miRNAs have been mainly elucidated studying their expression within a given cell type². 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 regulation^3,4,5,6,7. Yet, even still controversial, miRNAs have been shown also to act outside the cell from which they originate by long distance transport^{8,9,10,11,12,13}.

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 inflammation^15,16 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 cell^18,19 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 RNAs^23,24. 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 organisms^8,26,27, 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 animals^10,28,29,30. 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 regimes^28,31,32,33 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 relevant^28,59. We conclude that better understanding of the cross talk between exogenous miRNA and immune function will open novel opportunities to the development of an entirely new class of miRNA based anti-inflammatory drugs.

Methods

Ethics statement

The study was designed in conformity with the international recommendation (Dir. EU 2001/20/EC) and its Italian counterpart (DM 15 Luglio 1997; D.Lvo 211/2003; D.L.vo 200/2007) for clinical trial and following the Declaration of Helsinki, to assure protection and care of subjects involved. Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats provided by Careggi Hospital (Florence, ethical approval document n. 87/10), or by Centro Trasfusionale of Santa Chiara Hospital (Trento, ethical approval document n. 54896583) after obtaining ethical approval by their respective internal ethical committees, as indicated in brackets. Written informed consent has been obtained from all the adult subjects. All animal manipulations were performed in the animal facility at the CeSaL (University of Florence) according to the European Community guidelines for animal care (DL 116/92, application of the European Communities Council Directive 86/609/EEC) and the Guiding Principles for Research Involving Animals Beings. Experimental procedures were approved by the Committee for Animal Care and Experimental Use of the University of Florence.

Synthetic miRNA

Synthetic miRNA duplexes (Table 1) were synthesized by Sigma Genosys and purified by HPLC with the addition of the specific methyl modification at the 3′ termini. The annealing of small RNA strands was performed as described by Sigma oligo synthesis service. Upon arrival the small RNA duplexes were resuspended in RNAse-free water to the appropriate concentration. In each experiment miRNAs were complexed with DOTAP Liposomal Transfection Reagent (Roche) which facilitates nucleic acid entry into the cells^36,60. In each experiment, DOTAP is also added to the control cells to exclude cross reactivity.

Cell preparation

The PBMC fraction was obtained by density centrifugation of diluted blood over Ficoll-Paque (GE Healthcare). Monocytes were isolated from low density PBMCs by magnetic enrichment with anti-CD14 MicroBreads (Miltenyi Biotec) and cultured as previously described to allow DC differentiation⁶¹. DC activation was induced by LPS (10 ng/ml, Sigma Aldrich) or Low Molecular Weight (LMW) PolyI:C (10 μg/ml, InvivoGen). Total CD4⁺ T cells were obtained from PBMC by negative isolation with a combination of magnetic sorting (CD4⁺ T Cell Isolation II Kit). Depending on the experiment, supernatants were collected after 24 hr or 5 d and stored at 20 °C until the assays. Cells viability was assessed before each experiment by Trypan blue (Euroclone) exclusion. DC viability was also evaluated after each experiment by flow cytometry using the Annexin V-FITC kit (BD Pharmingen) (Supplementary Fig. 4). The viability was always higher than 95% in all the experiments performed.

Mixed lymphocyte reaction (MLR)

1 × 10⁵ CD4⁺ T cells were incubated, in 96-well U bottom plates, for 5 d in RPMI with 10% FBS together with allogeneic DCs, in presence or not of miRNA (10 ng/ml) and LPS or PolyI:C. At day 5, the proliferative response was measured by the [³H]-thymidine ([³H]-Thy, 1 μCi/mL, Amersham Bioscience) incorporation test. [³H]-Thy was added for the last 8 hr of incubation. Plates were harvested (Tomtec Mach III, Wallac) on glass fiber filters (PerkinElmer) and [³H]-Thy uptake was measured by liquid scintillation in a Microbeta 1450 Trimux counter (Wallac). The proliferative response is reported as a stimulation index (SI, mean cpm response/mean cpm background).

Human cytokine assay

At the indicated times, supernatants from human cell cultures were collected and cytokine detection was performed using the Milliplex^® MAP human cytokine/chemokine kit (Merck-Millipore), according to the manufacturer’s instructions. IL-6, TNFα, IL-1β, IL-10, IL-12p70 and IFNγ were assayed.

Human flow cytometry

For DC surface markers evaluation, cells were labeled with adequate concentrations of labeled antibodies in PBS with 1% FBS for 20 min at 4 °C, washed twice and analyzed immediately. T cell intracellular staining for Tbet, Rorγt, IL-17A, IL-10 and IFNγ were performed using the fixation/permeabilization buffer kit (Life Technologies) following the manufacturing recommendations. A minimum of 10000 events for each sample were acquired using a Guava easyCyte 8T flow cytometer (inCyte software,Merck-Millipore). The area of positivity was determined by using an isotype-matched control MAb. Antibodies used: Fluorescein isothiocyanate (FITC)–anti-IL17A, phycoerythrin (PE)–IL10, APC–anti-CD4, APC-anti-CD11c, PE-IL-10, FITC-Tbet (Merck-Millipore), PE-Cy7 Tbet, APC-Rorγt, PE-IFNγ, PE-CD11c, (BD Biosciences Pharmingen); FITC-CCR7 (eBioscience); FITC-CD80, PE-CD86, ECD-HLADR and PC5-CD83 (Beckman Coulter).

Binding assay

FvmiR168 was labeled with fluorescein using the 5′ End-Tag kit (Vendor Laboratories) following the manufacturer’s instruction. The labeled miRNA was then added at a concentration of 10 ng/ml to DCs. After 2 hr, cells were visualized for green-positivity by flow cytometry. In parallel, cells were visualized at fluorescence microscope. To test the effects of various reagents on ligand binding, the following concentrations were used: LPS (1 μg/ml), LMW poly:IC (10 μg/ml), anti–TLR4 mAb, anti-TLR3 mAb (both from InvivoGen) and anti–ALCAM Ab (30 μg/ml). Incubation was performed in 20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM CaCl₂, 2 mM MgCl₂ and 1% BSA. After 1 hr of incubation at 37 °C, cell–miRNA conjugates were analyzed by flow cytometry. DCs were labeled with anti-CD11c-APC to discriminate cells binding FITC-labeled miRNA from free miRNA-containing vesicles.

Quantitative real time PCR

Total RNA was extracted with the RNeasy Mini Kit (Qiagen, Valencia, CA). SuperScript^® VILO cDNA Synthesis Kit (ThermoFisher Scientific) was used for cDNA synthesis. Transcripts for TRIF/TICAM, IRF3 and IFNB1 were quantified with Applied Biosystems predesigned TaqMan Gene Expression Assays and reagents according to the manufacturer’s instructions. Quantification of the PCR signals was performed by comparing the cycle threshold value of the gene of interest with the cycle threshold value of the reference gene GAPDH. Values are expressed as fold increase of mRNA relative to that in not treated cells.

smallRNA preparation

Plant, epithelial (Caco2-derived) and beef derived-sRNAs were extracted with the cetyltrimethylammonium bromide (CTAB)-based method⁶² or using the mirPremier microRNA Isolation Kit (Sigma-Aldrich) according to the manufacturer’s protocol. The use of CTAB together with Polyvinylpyrrolidone (PVP 40) followed by three successive chlorophorm:isoamyl alcohol extractions - or, as in the case of the kit, the use of filtration columns - allowed to denature and eliminate the protein complex present in the sample. After each extraction, samples will be analyzed using the Agilent RNA 6000 Nano Kit through the BioAnalyzer instrument (Agilent Technologies), for checking purity from high molecular weight RNA. Furthermore each sRNA preparation were analyzed using the Agilent Small RNA kit, to characterized the size of the sRNA present in each fraction. Size determination has been assessed on the basis of the retention time with respect to the known sizes and timing of the ladder peaks. The sRNA content ranged between 10 and 150 nt for all the plant used.

Gel fractioning of sRNA

A standard 12% acrylamide gel [1X TBE, 12% acrylamide (19:1 acryl:bis-acryl)] was used to separate the plant sRNA. The gel was warmed to ~50 °C by running it for 10–15 minutes at 40 V in 1X TBE running buffer. 1 μg of sRNA was combined with an equal volume of Gel Loading Buffer II and then used for loading the gel. The gel was run until the leading dye travels about 4–5 cm down the gel, at 70 V. Gel was stained with SYBR Gold (Life Technologies) according to manufacturer’s extraction. Different gel slices (covering the range from 10 to 60 nt, 70–100 nt and 100–150 nt), were recovered according to the band profile and the two ladders used (the Low Range ssRNA Ladder and the microRNA Marker, both from New England BioLabs inc.) and crushed in 1 M NaCl and incubated overnight at 4 °C. The day after, the samples were centrifuged for 5 min at 2000 g and the supernatant transferred to a 50 ml tube. Crushed gel slices were re-elute with 2 ml of 1 M NaCl for 1 hr at room temperature and centrifuged again. The pooled supernatants were purified with MEGAclear Kit (Life Technologies). Obtained fractions were analyzed using the Agilent Small RNA kit, to check the sizing and purity of the fractionation performed as well as the miRNA (21 nucleotides) content, according to the manufacturer’s software.

Induction and clinical assessment of EAE

Six to eight-week old C57Bl/6 female mice were obtained from Harlan, Italy Srl. The mice were housed in macrocolon cages on a 12 hr light/dark cycle at 23 °C, with ad libitum access to food and water. Adequate measures were taken to minimize pain and discomfort. Mice were immunized subcutaneously in the flanks and at the base of the tail with a total of 200 μg of MOG_35–55 (synthesized by EspiKem Srl, Università di Firenze) per animal emulsified in complete Freund adjuvant (Sigma-Aldrich) supplemented with 4 mg/ml of Mycobacteriun tibercolosis (strain H37Ra; Difco Laboratories). Immediately thereafter and 48 hr later, the mice received an intraperitoneal injection of 500 ng pertussis toxin (Sigma-Aldrich) in 100 μl of phosphate buffer saline (PBS). The animal were examined daily for weight loss and disability and were clinically graded by investigators blind to group identity, as follows: zero indicate no signs, 0.5 partial loss of tail tonicity, 1 paralyzed tail, 2 ataxia and difficulty in righting, 3 paralysis of the hind limbs and/or paresis of the forelimbs, 4 tetraparalysis, 5 moribund or death.

Treatments

Plant sRNA (pool of three plants, total of 30 μg/mice), obtained as described above was freshly dissolved in PBS/DOTAP (60 μl/mice, stock solution 1 μg/μl before injection heated 20′ at 37 °C. 150 μl of PBS/DOTAP/plant extract or vehicle were intravenously injected every four days from 3 d.p.i. to 22 d.p.i. for a total of six injections. At 25 d.p.i. animals were sacrificed.

Neuropathological evaluations

At the time of sacrifice, the mice were anesthetized with pentobarbital (65 mg/kg, intraperitoneally). The spinal cord was removed from the column and fixed in 4% (v/v9 paraformaldehyde in PBS) and subsequently paraffin-embedded. Five micrometers thick transversal sections were cut and placed on glass slides. Serial sections were stained with Haematoxylin and Eosin (H&E), Luxol Fast Blue (LFB)-cresyl violet. Immunohistochemistry analysis was performed as reported⁵⁷ with anti-Iba1 (rabbit anti-mouse, Wako Chemicals) and anti-CD11c (hamster anti-mouse , eBioscience) as primary antibodies and anti-rabbit AlexaFluor 488 and anti-hamster AlexaFluor 546 (eBioscience) as secondary antibodies.

Immunological evaluations

Cells were isolated from draining lymphonodes of EAE animals and analyzed for T cell proliferative response, dendritic cell phenotype, T cell cytokines production and expression of transcription factors. Briefly, DCs phenotypes were investigated for surface marker expression by mean of flow cytometry: after staining with fluorescent monoclonal antibodies directed against MHC II, CD11c, CD80, CD86 and CD83 (all eBioscience), cells were analyzed on a four-color Epics XL cytometer (Expo32 software; Beckman Coulter). T lymphocytes of MOG_35–55 immunized mice treated or not were cultured in complete RPMI in 96 well plates (200000 cells/well) and stimulated with antigen. At day 3, the proliferative response was measured by ³H-thymidine incorporation test. Cytokines (IFNγ, IL1-7, TNFα, CXCL1, IL-6, CXCL2 and IL-10) were determined by Luminex (Kit Milliplex, Merck-Millipore) in cell supernatants harvested after 4 days of antigen stimulation (1 million cells/ml/well). Total RNA was extracted from lymphonodes’ cells isolated at sacrifice by using Qiazol following manufacturer’s protocol (Qiagen). cDNA was synthesized with RT quantitect (Qiagen). Real-time PCR was performed with an ABI Prism 7900HT Sequence Detection System (Applied Biosystems), according to manufacturer’s instructions. All PCR amplifications were performed with TaqMan Universal Master Mix and with Assay-on-demand (Applied Biosystems). Relative expression of mRNA levels was determined by comparing experimental levels with a standard curve generated with serial dilution of cDNA obtained from human PBMCs. Ubiquitin carboxy-terminal hydrolase L1 (Hprt1 Hypoxanthine phosphoribosyltransferase 1) was used as a housekee** gene for normalization. In each sample the level of the following mRNA level were evaluated: Gata3, Tbet, Foxp3, cMaf and Rorc. Ratio of transcription factors were calculated within each animal and means were compared.