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.

Figure 1. Effects of plant miRNA treatment in human immune function.

(a,b) Effects of miRNA treatment on DC activation. DCs were exposed for 2 hr to FvmiR168 (10 ng/ml) and then exposed to LPS or PolyI:C (PIC, 10 ng/ml). After 24 hr IL-1β, TNFα, IL-10, IL-6 released on supernatant (a) and costimulatory markers’ expression on DC surface (b) have been measured. (c) Effects of treated DCs in stimulating T cells. T cells were exposed to DCs, in presence or not of miRNAs (10 ng/ml) and LPS or PolyI:C (10 ng/ml). After five days, proliferation has been measured as [³H]-Thy uptake by liquid scintillation [³H]-Thy. Data have been expressed as stimulation index: percentage of stimulation above background is determined for each stimulated sample, through comparison with results from an unstimulated sample. (d) Superntants have been collected, before addition of [³H]-Thy and INFγ release was assayed. In parallel, on the DCs used for the MLR assay IL-12p70 have been measured on 24 h supernatants. (e) In parallel, on the same set of T cells stimulated with LPS were not treated with [³H]-Thy and to characterize the T cell population present in the samples, intracellular staining was performed to evaluate the expression of Tbet, Rorγt and Gata3 by flow cytometry as well as the intracellular cytokine production for IL-10, IFNγ and IL-17. Data are presented as mean + SD, N = 5, *p < 0.05, **p < 0.01, Student t-test, pretreatment (+FvmiR168) vs no pretreatment (none, LPS or PIC alone). (f) CCR7 expression on DCs surface. DCs were exposed for 2 hr to miRNAs (10 ng/ml) and then exposed to LPS (10 ng/ml) for 24 hr. Thereafter, CCR7 expression was evaluated by flow cytometry on CD11c⁺ gated cells. Data are presented as mean + SD, N = 3, *p < 0.05, **p < 0.01, Student t-test, pretreatment (+FvmiR168) vs no pretreatment (none, LPS or PIC alone).

Figure 2. Plant miRNA interacts with TLR3.

(a,b) DCs enter in contact with plant miRNA. DCs were treated for 2 hr with 10 ng/ml FITC-labeled FvmiR68. Contact has been evaluated by assessing the number of green cells by flow cytometry on specifically gated population (a). Data are presented as mean + SD, N = 3 plus representative plots. (b) Representative images of green fluorescent cells after FvmiR168 treatment. (c) DCs bind FvmiR168 through TLR3. CD11c-APC–labeled DCs were incubated with FITC-labeled FvmiR168 in the presence or absence of LPS, LMW poly:IC (PIC), anti–TLR4 mAb, anti-TLR3 mAb, and anti–ALCAM Ab as control. Cells were then analyzed for positivity to FITC by flow cytometry. Basal binding (TSA) is set as 100%. Data are presented as mean ± SD (N = 4). **p-value < 0.01, Student t-test, blocked vs not-blocked cells (none). (d) PolyI:C competes with miRNA for TLR3 binding. CD11c-APC–labeled DCs were incubated with FITC-labeled PolyI:C in the presence or absence of FvmiR168. Cells were then washed and analyzed for positivity to FITC by flow cytometry. Basal binding (TSA) is set as 100%. Data are presented as mean ± SD (N = 7). *p-value < 0.05, **p-value < 0.01, Student t-test, blocked vs not-blocked cells (none). (e) Fluorescence microscopy reveals that TLR3 (in red) localizes with FvmiR168 (green) in DCs (nucleus staining in blue, DAPI) as indicated by white arrows.

Figure 3. Plant miRNA acts on TRIF-mediated signaling.

(a) DCs were treated or not for 30’ and 1 hr with 10 ng/ml FvmiR68. Cells were then collected, RNA extracted and levels of TRIF (TICAM1), IFNB, and IRF3 were detected by quantitative real-time PCR. (b,c) DCs were treated or not for 1 hr with 10 ng/ml FvmiR68, and then exposed for 30’, 1 hr and 4 hr to LPS (b) or PolyI:C (b, PIC). Cells were then collected, RNA extracted and levels of TRIF (TICAM1), IFNB, and IRF3 were detected by quantitative real-time PCR. Results are shown as means ± SD (N = 3). *p-value < 0.05, **p-value < 0.01, Student t-test treated (+FvmiR168) vs not-treated cells (none, LPS or PIC alone). (d) DCs were treated or not for 2 hr with 10 ng/ml FvmiR68 and then exposed for 20 hr to LPS (10 ng/ml) or PolyI:C (10 μg/ml). IDO expression has been analyzed by intracellular straining on gated CD11c⁺ DCs. Results are shown as means ± SD (N = 3). Above the graph a representative flow cytometry dot plot has been shown. *p-value < 0.05, Student t-test, pretreatment (+FvmiR168) vs not-pretreated cells (none).

Figure 4. Effects of different plant sRNA extracts on T cell proliferation.

(a–d) T cells were exposed to DCs, in presence or not of FvmiR146, bolmiR874 or osamiR168 (10 ng/ml, (a), F. vesca total sRNA (b) or human and B. taurus total sRNA (10 ng/ml, (c) or sRNA fractions obtained as described in Material and Methods section (10 ng/ml, (d) or B. taurus total and 10–60 nt sRNA fraction (e), and LPS (10 ng/ml). Fractioning of sRNA has been performed by polyacrylamide electrophoresis separation. Panel (d) refers to the results obtained using 10–60 nt sRNA, 70–100 nt sRNA and 100–150 nt sRNA. After five days of T cells-DCs co-cultures, proliferation has been measured as [³H]-Thy uptake by liquid scintillation [³H]-Thy. MLR results are shown. Proliferation is presented as stimulation index, Percentage Stimulation above background is determined for each stimulated sample, through comparison with results from an unstimulated sample. Mean ± SD, N = 6, *p < 0.05, **p < 0.01, Kruskal Wallis, pretreatment (+miRNA duplexes or sRNA fractions) vs not-pretreated cells (none).

Figure 5. Effect of plant miRNA 3′OH-methylation on T cell proliferation.

(a) T cells were exposed to DCs, in presence or not of 10 ng/ml methylated FvmiR168, bolmiR874 or their un-methylated analogs. After five days of T cells-DCs co-cultures, proliferation has been measured as [³H]-Thy uptake by liquid scintillation. MLR results are shown. Proliferation is presented as stimulation index, Percentage Stimulation above background is determined for each stimulated sample, through comparison with results from an unstimulated sample. In parallel, IFNγ was assayed on 5-days supernatants collected before [³H]-Thy addition. (b) Similarly, T cells were exposed to DCs, in presence or not of 10 ng/ml btamiR378 or the GFPs duplex and their methylated analog. MLR results and IFNγ production are shown. Mean ± SD, N = 4, *p < 0.05, **p < 0.01, Kruskal Wallis, pretreatment (+miRNA duplexes or sRNA fractions) vs not-pretreated cells (none). ^$p < 0.05, ^$$p < 0.01, Student t-test, methylated vs un-methylated miRNA pretreatment. um, un-methylated; meth, methylated.

Figure 6. Effect of plant sRNA in EAE.

(a) sRNA significantly ameliorated the clinical signs and disease progression of EAE. Analysis was performed by one way Anova test indicating that curves are statistically different (*p < 0.05). In particular clinical score is statistically significant in pool sRNA vs untreated mice and in pool sRNA vs vehicle treated mice at 8 and 15 days after immunization (d.p.i). (b) Effect of treatment with plant extract on inflammation and demyelination in the spinal cord of EAE mice. Sections were stained for Haematoxylin and Eosin (H&E) and LFB and examined to detect inflammatory infiltrates and demyelination, respectively, as indicated by arrows. Staining displayed a decrease of the number of infiltrates (arrows) in the white matter in the spinal cord of plant sRNA-treated mice compared to untreated or vehicle treated mice. Luxol Fast Blue (LFB) staining indicated a similar pattern after pool-sRNA treatment (arrows). Immunofluorescence analysis (IF) has also been performed. Section were stained with CD11c⁺ (RED) and Iba1 (green) to detect DCs and microglia respectively, and counterstained with DAPI (blue). Magnification of the black frames in upper panels are shown. CD11c infiltrates are absence in the spinal cord areas of immune cell invasion of plant sRNA treated mice. (c,d) Ex vivo analysis of T lymphocyte isolated from EAE lymphnodes. T cells were treated with ex vivo with MOG. To characterize the T cell population present in the samples, intracellular staining was performed to evaluate the expression of Tbet, Rorc, Gata3, Foxp3 and cMaf. Mean ± SD of two independent experiments (c). On cytokine supernatans, IL-10, IL-17, IFNγ, TNFα, IL-6, CXCL1 and CXCL2 were measured (d). *p < 0.05, **p < 0.01, Student t-test, treatment vs no treatment.