paola de candia1, veronica de rosa2, maurizio casiraghi3 ...arabidopsis thaliana. uses extracellular...
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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: [email protected] or Dr. Paola de Candia, E-mail:
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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Role of extracellular RNAs in immune responses
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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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