Role of extracellular RNAs in immune responses

1

Extracellular RNAs: a secret arm of immune system regulation

Paola de Candia1, Veronica De Rosa

2, Maurizio Casiraghi

3 and Giuseppe Matarese

1,4

From the: 1IRCCS MultiMedica, 20138 Milan, Italy

2Laboratorio di Immunologia, Istituto di Endocrinologia e Oncologia Sperimentale, Consiglio

Nazionale delle Ricerche (IEOS-CNR), Napoli, Italy; Unità di NeuroImmunologia, IRCCS

Fondazione Santa Lucia, Roma, Italy 3ZooPlantLab, Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-

Bicocca, Milano, Italy 4Dipartimento di Medicina Molecolare e Biotecnologie Mediche,

Università di Napoli “Federico II”, Napoli, Italy

Running Title: Role of extracellular RNAs in immune responses

To whom correspondence should be addressed: Prof. Giuseppe Matarese, Dipartimento di

Medicina Molecolare e Biotecnologie Mediche, Università di Napoli “Federico II”, Napoli, Italy,

Tel./Fax.: +39-0817464580, E-mail: giuseppe.matarese@unina.it or Dr. Paola de Candia, E-mail:

paola.decandia@multimedica.it

Keywords: Cell-free RNAs, extracellular vesicles, immune system, T cells, Treg cells

ABSTRACT

The immune system has evolved to protect

multicellular organisms from the attack of a

variety of pathogens. To exert this function

efficiently, the system has developed the capacity

to coordinate the function of different cell types

and the ability to down-modulate the response

when the foreign attack is over. For decades,

immunologists believed that these two

characteristics were primarily related to

cytokine/chemokine based communication and

cell-to cell direct contact. More recently, it has

been shown that immune cells also communicate

by transferring regulatory RNAs, microRNAs in

particular, from one cell to the other. Several

reports have suggested a functional role of

extracellular regulatory RNAs in cell-to cell

communication in different cellular contexts. This

minireview focuses on the potential role of

extracellular RNA transfer in the regulation of

adaptive immune response, also contextualizing it

in a broader field of what is known of cell free

RNAs in communication among different

organisms in the evolutionary scale.

Cell-free nucleic acids: a historical perspective

The first description of extracellular nucleic acids

in the blood of healthy and sick individuals goes

back to observations published by Mandel and

Metais in 1948 (1). After this first report, a series

of research groups described both DNA and RNA

circulating as extracellular molecules in the body.

These molecules were considered as potential

biomarkers mainly in cancer and for prenatal

diagnosis (2,3). More recently, microRNAs

(miRNAs), small RNA molecules (18-25

nucleotides) which in their mature single-stranded

form act as post-transcriptional repressors through

pairing with messenger RNAs (4), have been

shown to circulate in human blood associated with

either vesicles or protein complexes that protect

them from degradation (5-7). In the last seven

years, blood circulating miRNAs have become the

most promising clinical biomarkers for the

diagnosis, prognosis and therapeutic options of a

variety of pathological conditions such as cancer

(8-10), cardiovascular disorders (11,12), diabetes

(13) and liver diseases (14,15) among others (16).

While cell-free nucleic acids have the potential to

revolutionize medical diagnostics, the biological

significance of these molecules in the extracellular

environment remains still elusive. RNA release

http://www.jbc.org/cgi/doi/10.1074/jbc.R115.708842The latest version is at JBC Papers in Press. Published on February 17, 2016 as Manuscript R115.708842

Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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Role of extracellular RNAs in immune responses

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can be a passive phenomenon that results from

tissue damage, or an active secretory process of

healthy cells. Hence, is RNA simply discarded by

cells as waste or does it have a role in cell-to-cell

communication? Or both?

In immunology, transfer of immunity by

RNA has been first described more than 40 years

ago (17). Moreover, RNA extracted from the

thymus was shown to induce maturation and

expression of a T cell specific antigen in bone

marrow lymphocytes (18) and to activate the

proliferation of bone marrow plasma cells in vitro

(19). Then, it was first proposed that an analogous

mechanism in cell maturation in vivo might

involve RNA release by thymus cells. In the mid-

70s lymphocytes were shown to release double

stranded DNA in complex with low molecular

weight RNA. The information carried by

extracellular nucleic acids could be transferred

from one cell type to another in the course of an

immune response (20-22). The capability to

communicate through RNA molecule is possibly

very old and universally distributed in living

organisms.

Traveler RNA: a universal living language

The hypothesis of the so-called ‘RNA world’

suggests that RNA has been the first nucleic acid

in primordial cells around 3.8 billion years ago

(23,24) and may be the answer to a famous

‘chicken and egg’ problem: was there DNA or

protein first? RNA is the only molecule capable of

both storing genetic information and catalyzing

chemical reactions, being at the same time

genotype and phenotype. Around 3.6 billion years

ago, the more stable DNA molecule almost

completely replaced RNA for the storage of

genetic information, and thus allowed the passage

from a RNA world to a DNA world in which

RNAs, molecules, though, exert multiple

functions, participating to cellular chemical

reactions, regulating the transcription of genes and

modulating the activity of proteins. While the last

century of biological researches was dominated by

the idea that RNA is mostly devoted to the

translation of genes into proteins, and basic

biology textbooks focus on mRNAs, tRNAs and

rRNAs, nowadays RNA is recognized as the most

versatile biological macromolecule, with coding

RNA being only a continent of the RNA planet.

One of the best characterized non-coding

function of RNA is interference (RNAi), originally

defined as the capability of double stranded RNA

to induce sequence-specific degradation of

messenger RNAs. RNAi was first described in

plant biology in 1990 (25) and eight years later a

double stranded RNA was shown to trigger a

powerful cellular silencing mechanism in

Caenorhabditis elegans that could be transmitted

in the germ line and pass through several

generations, in addition to spreading from tissue to

tissue in the same individual (26). RNA-induced

gene-silencing mechanisms developed early at the

basal eukaryotic lineage possibly to control viruses

and transposable elements (27). RNAi may have

an even older origin, linked to the

replication/transcription pathways present in the

ancient RNA world (28). Importantly, RNA

molecules regulate biological mechanisms across

different species or even different kingdoms

pertaining to the interconnected conditions of

parasitism/infection/food intake. Endogenous

noncoding RNAs from Escherichia coli interfere

with gene expression and regulate physiological

conditions in C. elegans (29,30). A fungal

pathogen that infects Arabidopsis thaliana uses

extracellular small RNAs to hijack the host RNA

interference (RNAi) machinery by binding to plant

AGO1 and selectively silencing host genes, with

the effect of suppressing host immunity and

achieve infection (31). The protozoan parasite

Trypanosoma cruzi releases specific tRNA-

derived small RNAs that are transferred between

and from parasites to mammalian cells, eventually

altering their infection susceptibility (32). Plant

miRNAs present in food and acquired orally were

shown to use mammalian AGO2 to form AGO2-

associated RISC thus regulating the expression of

target genes in mammals (33); although later

studies reported notes of caution, suggesting that

plant miRNAs via dietary exposure may not be

universal in animals (34).

In conclusion, RNA is not the labile

molecule we were used to think about at the end of

the previous century. RNA molecules are involved

in several cellular activities and are even not

constrained inside single cells, but they are mobile

and travel between organisms of the same or

different species, with increasingly recognized

ecological roles. These RNA functions may be

extremely ancient, as RNA molecules are involved

in quorum sensing, a phenomenon of sociality

among bacteria, that can reach high level of

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Role of extracellular RNAs in immune responses

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regulation, including cheating and high

competition (35,36). If extracellular RNA-based

communication arose even before the origin of

eukaryotic cell, then we can hypothesize that it is a

widespread biological process, a powerful and

universal code that allows one organism/cell to

influence the behavior of others (37).

The need for a sophisticated type of

containment brought about the use of

phospholipidic bilayers with the formation of

vesicles at growing grade of complexity. Vesicles

give two major advantages to RNA messages:

protection from degradation and the possibility,

through the presence of proteins, to confer higher

level of specificity to the message.

Extracellular vesicles as cell-to-cell words with

a huge biological impact: the case of the

immune system

A growing body of evidence from the past years

has unveiled that not all extracellular vesicles are

created equal. There exist vesicles of nanometric

size (20-100 nm, often referred to as exosomes),

that are formed by the inward budding and

subsequent fusion to the plasma membrane of

multivesicular endosomes (38); and vesicles of

larger size (0.2-1micron) that bud directly from the

plasma membrane, are called microvesicles and

comprise also apoptotic and senescent bodies. An

exhaustive description of the biogenesis,

trafficking, morphology and isolation techniques

of extracellular vesicles goes beyond the scope of

the present review but it can be found elsewhere

(39).

The first accurate description of

extracellular vesicles (EVs) goes back to 1987,

when sheep reticulocytes cultured in vitro were

shown to release vesicles containing a number of

activities characteristic of the reticulocyte plasma

membrane, including acetylcholinesterase and the

transferrin receptor. It was thus suggested vesicle

externalization to be a mechanism for shedding

specific membrane functions (40). For quite some

time after those first observations, it was believed

that EVs are released by cells to eliminate

molecules that need to be down-modulated during

specific processes.

Starting from 1996, EVs started not to be

regarded as a mere garbage bin, as it was

discovered that both dendritic cells and B

lymphocytes release vesicles containing major

histocompatibility complex (MHC) class II that

are able to induce antigen-specific MHC class II-

restricted T cell responses, first showing a specific

role for vesicles in antigen presentation in vivo

(41,42). Also activated T lymphocyte-derived

exosomes, bearing TCR from the pool of activated

complexes, were shown to target cells bearing the

right combination of peptide/MHC complexes

(43). Since those first studies, many others have

contributed to discover a significant biological role

of EVs in controlling the immune response. The

general mechanism by which this control is

exerted is through the exchange of vesicles as

powerful vehicles to specifically deliver signals

from cell to cell in an autocrine, paracrine and also

endocrine fashion. The picture is very complex if

one thinks that most if not all cells of the immune

systems are known to release EVs; that vesicle

content can change according to activation status

and environmental clues of the releasing cells and

that the potential targets are both immune and non-

immune cells. EVs can help amplifying activation,

as in the case of exosomes derived from stimulated

T cells that cooperate with IL-2 to induce growth

of resting CD3+ T cells, by stimulating a

proliferative response and the secretion of more

cytokines and at higher levels than cells stimulated

by IL-2 alone (44). On the opposite side, EVs can

deliver death signals, as T cells have been

observed to kill target cells by CD95 engagement

through membrane CD95 Ligand bearing

exosomes (45).

Although no systematic studies have been

conducted yet, several cytokines and chemokines

have been found in association with EVs.

Examples are interleukin (IL)-1 (46,47); IL-6

(48); IL-18 (49); tumor necrosis factor (TNF)-

(50). To date, it is not known whether EV-

association imprints a different specificity

compared to free cytokines in vivo and what is the

effect of EV-association on our conventional

cytokine measurements. In 2001, EVs officially

entered the immune regulatory arena when

suppressing exosomes e.g. "tolerosomes" were

first described, as supra-molecular structures

assembled in and released from the small intestinal

epithelial cell, that carry MHC class II molecules

with bound gut lumen antigenic peptides, fully

capable of inducing antigen specific tolerance

(51). Tolerosomes were shown to induce donor-

specific allograft tolerance characterized by strong

inhibition of the anti-donor proliferative response

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and the delay of the appearance of chronic

rejection, thus suggesting that these vesicles can

induce regulatory responses able to modulate

allograft rejection (52). EVs can also mediate

regulatory T (Treg) cell differentiation: exosome-

like particles released from thymic cells were

shown to promote the conversion of naïve T cells

into forkhead-box-P (Foxp)3+ Treg cells, and may

be one of the endogenous drivers for inducing

Treg cells under physiological conditions via

vesicle-associated transforming growth factor

(TGF)- (53). It is conceivable that the thymus can

also communicate with peripheral organs,

including lymphoid and non-lymphoid sites, via

thymic particles to regulate host immune

tolerance. Importantly, tumor cells take advantage

of EV-based immune-suppressive system by

secreting vesicles that can either induce apoptosis,

impairment of T cell receptor (TCR) signaling and

cytotoxicity on T effector and naturel killer (NK)

lymphocytes or promote lymphocyte suppressive

activity through the release of Treg-promoting

exosomes (54). In particular, tumor-derived

vesicles can induce, expand and up-regulate

biological activities of Treg cells through an EV-

associated TGF--mediated mechanism (55).

Furthermore, although studies are still in their

infancy, it seems that also bacteria can produce

vesicles able to cross the epithelial barrier and

interact with cells of the host connective tissue,

primarily cells of the immune system (56).

In conclusion, to date extracellular

vesicles are fully recognized as communication

modules between and toward cells of the immune

system and are regarded by some authors as

important as cytokines and chemokines in

regulating microenvironment cues but also cell

targets at distance (57). Besides the immune

system, specific biological roles of EVs have been

described in pregnancy, embryonic development,

tissue repair, vascular biology, nervous system,

bone calcification and liver homeostasis and EVs

have been constantly detected in all mammalian

(in particular human) body fluids investigated:

blood, urine, saliva, synovial, bile, cerebrospinal

fluid, bronco-alveolar fluid, amniotic fluid, breast

milk and seminal plasma (58). When

characterizing EV composition, with the aim of

identifying the functional players associated with

these tiny messages, a component is invariably

found: microRNA.

Extracellular vesicle-associated microRNAs:

novel players of innate and adaptive immune

cell function

While microRNA (miRNA) role at the

intracellular level is recognized as a key player of

gene-expression regulation in eukaryotic cells (59)

and also in cells of the immune system specifically

(60,61), the biological role of miRNAs released at

the extracellular level has just started to be

explored (Fig. 1). Valadi et al. were the first to

show that exosomes contain messengers and

regulatory RNAs (among which miRNAs) inside

their membraneous structures while no DNA nor

ribosomal RNA (18S and 28S rRNA) is

detectable. Moreover, vesicle transfer was

described as specific, given that mast cell-derived

EVs were internalized by other mast cells but not

by CD4+ lymphocytes (62). Since that first report,

RNA (and miRNAs for that matter) has been

consistently found in vesicles released by most

cells investigated, from stem cells to neurons,

hepatocytes and blood cells, among others. Most

of the attention has been devoted to pathological

conditions, but several recent reports are also

pointing at a physiological relevance of miRNA

release. Some examples of miRNAs in EVs as a

cell-to-cell communication pathway are the

regulation of hematopoiesis in the bone marrow

(63), muscle cell differentiation (64) and the

crosstalk between neurons and astrocytes (65)

(Fig. 1).

An increasing number of reports are

demonstrating that EV-associated miRNAs have a

direct role in the immune system. During -

herpesvirus EBV infection, the passage of

functional viral miRNAs from infected to non-

infected B cells via EVs is an additional

mechanism adopted by the virus to control

immune cells of the host and gain persistent

infection (66). EBV may not be an isolated case.

HIV-encoded Trans-Activating Response (TAR)

element miRNA has been detected within the

exosomes of HIV-infected T lymphocytes,

together with the host miRNA machinery proteins

Dicer and Drosha. Notably, exposure of naïve T

cells to exosomes from infected cells increased

susceptibility of the recipient cells to HIV-1

infection and exosome-associated TAR RNA

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down-regulated Bim and Cdk9 proteins in

recipient cells, thus lowering apoptosis induction

(67). In inflammatory conditions, mature

macrophages produce and release EVs at high

concentrations that are able to induce phenotypic

differentiation and functional maturation of

monocytes into other macrophages. Importantly,

these EVs are enriched with miR-223, that is

necessary for vesicle-induced monocyte

maturation, and thus it is part of a feedback

mechanism that works to induce the differentiation

of recruited monocytes and the release of more

vesicle as a local response activating the native

immune system (68).

During the formation of an immune

synapse, EVs released by T cells contain miRNAs

that are transferred to the antigen presenting cells

(APCs) in an unidirectional and antigen-driven

fashion and are capable to modulate gene

expression in recipient cells (69,70) (Fig. 1). There

are two important features of this type of

communication: i) the miRNA repertoire of

vesicles differs significantly from that of parental

cells, suggesting that a selection of secreted

microRNAs may be highly regulated; ii) cell

cognate interaction that forms upon the

establishment of an immune synapse is necessary

to promote the release of vesicles on one side, and

to induce the fusion of these vesicles with the

plasma membrane of the recipient cell on the

other, demonstrating that the transfer is finely

controlled.

EV-associated miRNAs have also been

shown to be different in mature versus immature

dendritic cells and to actively participate in the

crosstalk of these cells for the fine-tuning of APCs

and the immune response (71). Dendritic cells do

communicate through vesicle exchange with

different miRNAs having different and specific

biological roles: in particular, EV-delivered miR-

155 enhances while miR-146a reduces

inflammatory response, by mediating target gene

repression and reprogramming the cellular

response to endotoxin (72) (Fig. 1).

This mechanism of cell communication is

envisaged to occur in a paracrine environment,

with vesicles traveling very short distances or even

passing from one cell to the other upon cell-to-cell

direct interaction. But EV-associated miRNAs can

also travel long distances and become endocrine

messages (73). In accordance with this hypothesis,

both B and T cells release EV-associated miR-150

that increases significantly in the blood of humans

after vaccination with influenza virus and

correlates with a higher immune response (74). In

contrast the level of extracellular miR-150

decreases in the blood of septic patients with an

unfavorable outcome (75). Therefore, the

modulation of circulating miR-150 may be an

important regulatory loop aimed at down-

modulating adaptive immune responses through

the transmission of extracellular messages to other

immune cells (also at distance) and the consequent

regulation of miR-150 target genes (Fig. 1).

Indeed, it has been demonstrated that vesicle-

packaged miR-150 specifically regulates target

gene expression and function in recipient cells

(76). The described examples of communication

via EV-associated miRNAs in the immune system

are reported in Table 1.

A long-distance miRNA-based

communication can occur also between mother

and child, with miRNAs traveling in milk during

the months of lactation. Tolerosomes had already

been described in breast milk, and shown to block

allergic responses or prevent allergy development

(77). More recently, human breast milk was found

to be enriched with immune-related miRNAs

(such as miR-17-92 cluster, known to be

fundamental for the development of the immune

system (78)) that may be transferred into the infant

body via the digestive tract, and play a critical role

in the development of the immune system in

infants (79) (Fig. 1). It has been specifically

hypothesized that milk miRNAs might induce

long-term thymic Treg lineage commitment and

down-regulate IL-4/Th2-mediated atopic

sensitization by controlling pivotal target genes

involved in the regulation of FoxP3 expression,

IL-4 signaling and immunoglobulin class

switching (80) (Fig. 1).

Treg cells and miRNAs: fighting the battle of

immune tolerance at both intra- and extra-

cellular level

miRNA expression is necessary for the

development of Treg cells in the thymus and the

efficient induction of Foxp3 by TGF- in a cell-

autonomous fashion (81). Moreover, ectopic

expression of the Treg cell-signature transcription

factor, FoxP3, in different cell lineages, results in

the acquisition of Treg cell-specific miRNA

profile, thus suggesting a key role for FoxP3 in the

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control of Treg cell intracellular miRNA content

(81). Genome-wide analysis of its transcriptional-

binding sites revealed that Foxp3 directly activates

several miRNAs, among which miR-155 that is

indispensable for Treg cells to normally respond to

their key survival and growth factor, IL-2, but

largely dispensable for Treg cell suppressor

function (82). The expression of other miRNAs is

necessary for Treg-mediated prevention of

inflammation and autoimmunity, because the

ablation of miRNA precursor-processing enzyme

Dicer in Treg cells results in a fatal early-onset

autoimmune pathology indistinguishable from that

of Foxp3 KO mice (83,84). In particular, Dicer-

deficient Foxp3+ T cells lose completely their

suppressive activity, despite their Foxp3

expression is comparable to that of control cells,

thus suggesting that the impairment is secondary

to a reduced expression of suppressor effector

molecules rather than to an altered Foxp3

expression (83,84). Strikingly, specific ablation of

miR-146a alone in Treg cells is able to cause fatal

lymphoproliferative diseases characterized by

sharply augmented Th1 responses, secondary to an

increased production of pro-inflammatory Th1

cytokines (IFNγ, above all) by both Treg and T

conventional cells, and to reduced suppressive

capability of miR-146a deficient Tregs (85).

miRNA expression is found altered in

different autoimmune conditions. Divekar et al.

recently demonstrated that Tregs of systemic lupus

erythematosus mice exhibit altered regulatory

phenotype, reduced suppressive capacity and

Dicer expression, together with a distinct miRNA

profile (86). Moreover, miR-26a, an IL-6-

associated miRNA that inhibits the generation of

Th17 and promotes the generation of Tregs in

vivo, thus functioning as regulator of the

Th17/Treg cell balance, has been found

dramatically decreased both in EAE mice and in

subjects affected by multiple sclerosis (87).

Notably, a recent study on T cell subsets

in mice demonstrated that Treg cells release EVs

containing miRNAs that differ from those released

by other effector cells and the release is tightly

regulated by distinct factors, such as IL-2,

amphiregulin, the calcium ionophore, monensin

and hypoxia. More importantly Treg cells

deficient for Dicer (necessary for miRNA

maturation) but also Treg deficient for Rab27

(necessary for vesicle release) are unable to

suppress Th1, demonstrating that non-cell-

autonomous gene silencing, mediated by miRNA-

containing EVs, is indeed a required mechanism

employed by Treg cells to suppress T-cell-

mediated disease. In particular the authors

identified Let-7d as preferentially packaged and

transferred to Th1 cells, halting Th1 cell

proliferation and IFNγ secretion and speculated

that local cytokine environment dictating

intracellular miRNA profile of Treg cells may also

shape the extracellular EV-associated miRNA

content (88). These findings suggest for the first

time that non-cell-autonomous gene silencing,

mediated by miRNA-containing EVs, is a

mechanism that cooperates with other known Treg

cell mechanisms to reinforce their suppressive

function and halt T cell-mediated diseases.

The analysis of Treg-specific miR-146a

KO mice described above has clearly suggested

that one miRNA can be crucial in maintaining a

certain optimal range of its targets, such as Stat1,

whose deregulated activation leads to a severe

failure of immunologic tolerance (85), but whether

miR-146a acts only at the intracellular or also at

the extracellular level, through EV-associated

passage from Treg cells to Th1 effector

counterpart has not been investigated yet.

Moreover, it is important to underline that

several cytokines and metabolic factors also

regulate EV release by Tregs (88), but whether

they can also shape their content is still unknown.

It is possible that, together with a dominant cell-

intrinsic role for miRNA secretion, sensitivity of

EV release to the microenvironment adds a further

mechanism of plasticity to fine-tune specific T cell

responses, thus ensuring immune tolerance.

Extracellular vesicle-associated miRNAs: how

much is enough?

Purified populations of EVs usually contain many

different miRNAs. Therefore, in line of principle,

EV-mediated miRNA transfer has the advantage

over classical intercellular communication

mediated by soluble factors that instead of one

single messenger, a single vesicle may suppress

functionally related genes in parallel, leading to a

very effective paracrine control over neighboring

cells (89). Unexpectedly, when a quantitative

assessment of EV content of miRNAs was

performed using a stoichiometric approach, it was

found that, regardless the biological source of the

vesicles, the ratio between the number of miRNA

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molecules and the number of vesicles is lower than

1, even for the most abundant miRNAs, meaning

that there are vesicles that do not contain any copy

of them (90). This report may have discouraged

people working in the field, because authors stated

that miRNAs may be individually unlikely to be

functional as communication vehicles. Instead, it

is clear that the absolute number of miRNAs per

vesicle is not the only parameter to be taken into

account when evaluating its cell-to-cell

communication potential. If vesicle uptake is an

infrequent but very selective event, the low-

occupancy/high-concentration occupancy model

(in which there are rare vesicles in the population

carrying many copies of a given miRNA) may be

consistent with vesicle-mediated communication

through miRNA-mediated silencing of mRNAs.

However, if cellular uptake of vesicles is rapid,

also miRNAs having a low-concentration/low-

occupancy (small fraction of vesicles carrying a

low concentration of miRNA) may accumulate

within the cell in physiologically relevant

quantities, given the minimum threshold in

mammalian cells to repress a target mRNA is

estimated to be 100 miRNA copies (91).

Recently, tumor-derived EVs were shown

to contain pre-miRNAs along with Dicer and to

possess cell-independent capacity to process

precursors into mature miRNAs (92). If this

capacity is not limited to tumor cell-derived

vesicles but is a more general feature of EVs, then

the quantification of mature miRNAs inside

vesicles is not giving us the correct idea of the

actual mRNA repression potential given by further

miRNA maturation in vesicles themselves.

Moreover, also very low quantities of miRNAs

may be compatible with recently proposed

nonconventional activities, such as binding to

Toll-like receptors as well as affecting DNA

transcription and/or epigenetic states, that

presumably require much lower concentrations of

miRNA delivery than conventional mRNA

targeting.

The majority of studies on the role of EVs

in mediating RNA-based cell-to-cell

communication (including the stoichiometric

evaluation of miRNA quantity (90)) are based on

the isolation of nanometric vesicles (50-200

nanometers in diameter), that excludes larger

vesicles, such as microvesicles and oncosomes,

that also contain miRNAs as well as longer

molecules of RNAs. Therefore, it is conceivable

that these larger EVs carry significantly higher

numbers of miRNA molecules and function as an

alternative vehicle for miRNA-based intercellular

communication. Last, but not least, it is not clear

what proportion of EV transcriptome consists of

fragments of mRNAs, tRNAs and other larger

RNA molecules, that may play regulatory function

similarly to miRNAs (93), in addition to long

interspersed elements and long terminal repeats

(94). All these complex perspectives need to be

further investigated.

The shared transcriptome: single cell identity

goes fuzzy

The most recent studies on symbiosis, defined as

the common living of organisms belonging to

different species, are shedding new lights on several

aspects of these interactions, and challenging the

view of unitary individuals, genomes, and, at the

end, life. Compartimentalization was an essential

pillar in the appearance of life organization as we

know it (95), but living organisms have never

forgotten the network environment in which they

appeared. We are facing a shift of paradigm, by

which our knowledge is progressing by abandoning

the idea that biological systems have precise

boundaries and embracing the concept of holobiont

(96) and hologenome (97). Here we have seen that

RNA is mobile and is exchanged among cells of the

same organisms or of different organisms of the

same species or of different species or even

kingdoms. It is possible to envisage two main

functions associated to sharing of the transcriptome:

i) regulation of target cell via non coding regulatory

RNAs (such as miRNAs); ii) buffering effect of

trasncriptome modulation in order to mount a

coordinate response in the case of an initially de-

synchronized cell population.

The growing knowledge on this ancient,

universal, widespread and possibly efficient

mechanism of communication will eventually lead

to embrace the new concept of

“holotranscriptome”, a sort of shared transcriptome

with RNA molecules crossing the strict borders we

impose to the biological unit of a cell.

Concluding remarks Networking is crucial for life since the first steps

around 4 billions years ago. Steps in which RNAs

may have played and may play a central role. A

deeper analysis of RNA-based cell-to-cell

communication will give us the tremendous

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8

opportunity to better understand relevant and broad

biological issues, such as the ecological and physio-

pathological relations between organisms and cells,

with insight into infections, symbioses, nutrition

and diseases. Moreover, if also human Treg cells

will be demonstrated to make use of EV-associated

miRNAs to regulate immune tolerance, then the

thorough analysis of this new potential pathogenic

mechanism of immune system dysregulation may

have an extraordinary impact on human health.

Acknowledgements: G.M. is supported by grants from the EFSD/JDRF/Lilly Programme 2015 and the

Italian Space Agency (ASI) RadioEBV n. 2014-033-RO. V.D.R. is also supported by the Ministero della

Salute grant n. GR-2010-2315414, the Fondo per gli Investimenti della Ricerca di Base (FIRB) grant n.

RBFR12I3UB_004 and the Fondazione Italiana Sclerosi Multipla (FISM) n. 2014/R/21. We thank

Dolores Di Vizio, Fabio Grassi and Nicola Manfrini for helpful discussions. We also thank Sara Ricciardi

for the continuous support.

Conflict of interest: the authors declare that they have no conflicts of interest with the contents of this

article.

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Role of extracellular RNAs in immune responses

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Figure Legend

Figure 1. Examples of EV-associated miRNAs relevant in the cross talk of immune cells.

(a) Both B and T lymphocytes release EV-associated miR-150 that increases significantly in blood upon

immune system activation. (b) Dendritic cell-derived miR-155 enhances while miR-146a reduces

inflammatory response. (c) Mature macrophages release EV-associated miR-223 that induces the

differentiation of recruited monocytes. (d) Regulatory T cells release Let-7d that affects Th1 cells, halting

their proliferation and IFNγ secretion. Whether EV-associated miRNAs functioning at a paracrine level

may be also released in the blood stream and which cells would be targeted is still largely unknown.

Abbreviations: T = T cells B = B cells; DC = dendritic cells; MC = monocytes; M= macrophage; Treg

= Regulatory T cells; Th1 = T helper1 cells.

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Table 1. Extracellular miRNAs with an identified role in immune cell communication

EXTRACELLULAR

miRNA RELEASING CELL

TARGET

CELL REFERENCE

miR-150-5p T and B cells Unknown (74)

Let-7d-5p Treg cell Th1 cell (88)

miR-150-5p Monocyte Endothelial cell (76)

miR-223 Macrophage Monocyte (68)

miR-146a-5p, miR-155-5p Dendritic cell Dendritic cell (72)

HIV-encoded trans-activation

response element miRNA HIV-infected T cell T cell (67)

EBV-encoded miR-BART15 EBV-infected B cell Dendritic cell (66)

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B

B

T T

B B

T T

miR-223

Treg

miR-150-5p

Th1

let-7d-5p

Target cells

Mf

MC

miR-146a-5p

miR-150-5p

miR-150-5p

a

b

c

DC

MC

MC

d

Blood Stream

DC

Figure 1. De Candia et al.

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Paola de Candia, Veronica De Rosa, Maurizio Casiraghi and Giuseppe MatareseExtracellular RNAs: a secret arm of immune system regulation

published online February 17, 2016J. Biol. Chem. 

  10.1074/jbc.R115.708842Access the most updated version of this article at doi:

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