The gastrointestinal tract is one of the most colonized organs and harbors a large
microbial population (1014 bacteria) that has co-evolved with us establishing a finely
tuned symbiosis (Ley et al., 2008). As a result of their occurrence in complex mixtures,
their combined genomes or metagenome, contain 150-fold more genes respect to humans,
therefore providing us with novel functions (Qin et al., 2010). Increasing evidences
show that the disruption of this symbiosis may lead to pathologies such as obesity
or to an increased risk of developing inflammatory bowel disease (Ott et al., 2004).
Since the intestine is constantly exposed to an almost limitless number of foreign
antigens (i.e., food-derived materials, commensal microbes, pathogenic bacteria, viruses
and parasites), it is of fundamental importance that an appropriate immune homeostasis
in the gut mucosa is established and maintained. This requires a highly sophisticated
immunological regulatory systems achieved by the cooperative interaction of intestinal
epithelial cells (IECs) and mucosal cells of the immune response (Goto and Kiyono,
2011). The IECs comprise columnar epithelial cells, Paneth cells, endocrine cells
and goblet cells (van der Flier and Clevers, 2009) and consist in the first physical
barrier between the host and the external environment. Owing to many signals transmission
from epithelial cells to the various innate and acquired types of mucosal cells of
the immune response, these cells regulate each other resulting in intestinal immunological
homeostasis.
To gain information about the composition of gut microbiota communities, their molecular
interactions with the host and their impact on various host functional processes,
several studies have been carried out on germ-free animals by coupling genomics and
bioinformatics techniques (Hooper and Gordon, 2001; O'Hara and Shanahan, 2006). However,
little is known about the molecular mechanisms of such modulations and the host post-transcriptional
gene expression regulation by microRNAs.
MicroRNAs (miRNAs) are short (~22 nt) non-coding RNAs that control gene expression
by base pairing with 3′-untraslated regions (3′UTRs) of their regulated transcripts.
MiRNA biogenesis occurs through various steps in which are involved Drosha and Dicer,
two main RNase III endonucleases. Precursor miRNAs (pre-miRNAs) are ~70 nucleotide-long
RNA molecules with a characteristic hairpin structure. They originate in longer primary
transcripts (pri-miRNAs) that are cleaved in animals by the Drosha endonuclease in
the nucleus (Lee et al., 2003). Following the export of pre-miRNAs to the cytoplasm
by Exportin-5, the loop region of the hairpin is removed by the Dicer endonuclease
to produce a short, double-stranded RNA (dsRNA) (Cullen, 2004). Based on the thermodynamic
stability of each end of this duplex (O'Toole et al., 2006), one of the strands is
preferentially incorporated in the RNA-induced silencing complex (RISC), producing
a biologically active mature miRNA (generally the -5p miR) (Bartel, 2004), while the
inactive strand (the -3p miR) is degraded (Kim, 2005). The coupling of the active
miRNA to the 3′UTR of its target gene, facilitates mRNA degradation or translation
inhibition (Djuranovic et al., 2012). As a direct consequence, miRNAs regulate many
biological processes and have critical roles in cell proliferation, differentiation
and death (Shivdasani, 2006; Gomase and Parundekar, 2009).
However, the role of miRNAs in microbiota host interactions is beginning to be investigated
(Figure 1A) (Dalmasso et al., 2011; Kaser et al., 2011). Dalmasso et al. used germ-free
mice colonized with the microbiota from pathogen-free mice to study whether miRNAs
are involved in microbiota-mediated regulation of host gene expression (Dalmasso et
al., 2011). Their miRNA expression analysis revealed that nine miRNAs were differentially
expressed in the ileum and colon of colonized mice compared to germ-free mice. By
overlapping the predicted targets of deregulated miRNAs with DNA microarray gene expression
profiling, they found that the up-regulation of miR-665 induced a significant down-regulation
of the ATP-binding cassette sub-family C member 3 (Abcc3) gene (a target of miR-665).
Abcc3 belongs to the multidrug resistance-associated protein family, which mediates
the metabolism of xenobiotics and endogenous toxins (Hooper et al., 2001). A similar
study by Singh et al. emphasized the emerging interplay between endogenous microbiota
and caecal miRNA signature (Singh et al., 2012). In fact, intestinal miRNAs have been
proven experimentally to have roles in the regulation of neonatal nutrient metabolism
(Liao and Lonnerdal, 2010), in the control of intestinal fluid and electrolyte transport
(Sansom et al., 2010) and permeability (Zhou et al., 2010), affecting also intestinal
epithelial cell differentiation (Dalmasso et al., 2010) and maturation (Zeng et al.,
2009). By using germ-free and conventionally raised mice, the impact of the endogenous
microbiota on the global expression of caecal miRNAs in vivo has been investigated,
showing that the murine miRNA signature in the caecum is affected by the presence
of the microbiota (Singh et al., 2012). Moreover, authors found that 34 putative miRNA
target genes encode for proteins involved in the regulation of the intestinal barrier
function (i.e., glycosylation enzymes, junctional proteins, proteins found in the
mucus layers) and in the immune regulation (i.e., MHC I and II proteins). They found
that the expression of miRNAs depends on the endogenous microbiota and that 16 unique
miRNAs were deregulated between germ-free and conventional raised mice. By cross-matching
the list of intestinal barrier genes predicted to be modulated by differentially expressed
miRNAs, with genes already demonstrated to be deregulated in the jejunal mucosa of
intestinal-specific Dicer knock-out mice (McKenna et al., 2010), the authors supported
the hypothesis that gut commensals impact the intestinal barrier via miRNAs expression
modulation. Therefore, the miRNAs modulation by gut microbiota may potentially affect
the expression of a huge number of host genes, so far unexpected, especially in those
diseases where the microbiota composition is altered toward less desirable species.
In this context, the use of synthetic miRNAs could represent a potentially novel therapeutic
perspective.
Figure 1
(A) Research projects for the study of the interplays between gut microbiota and miRNAs.
(B) Intestinal epithelial differentiation and TH2 immune responses are regulated by
miRNAs: in wild type mice, Dicer1 and miR-375 inhibit KLF5, a known antagonist of
KLF4 that promotes the differentiation of goblet cells via KLF4. Helminth infection
induces TH2 cytokines, especially IL-13, which leads to epithelial expression of miR-375
and goblet-cell maturation via PI3K. Moreover, miR-375 also induces TSLP to accelerate
TH2 immune responses to parasite infections. In Dicer1
Δgut mice, depletion of Dicer1 or miR-375 results in fewer goblet cells and diminished
TH2 responses. The gut microbiota can be involved in the induction and regulation
of miRNA expression either for active or quiescent immunity. Likewise, other miRNAs
can be involved in the generation of optimal protective immunity to various pathogens.
This figure has been adapted from (Goto and Kiyono, 2011).
Another example of the role of miRNAs in affecting the intestinal epithelial monolayer,
has been provided by Biton et al. by using mice with an inducible intestinal epithelial
cell–specific deficiency in Dicer1 (Dicer1
Δgut) (Biton et al., 2011). They found that Dicer1 deletion in the mice gut lead to
goblet-cell depletion and that the regulation of goblet-cell differentiation is dependent
on the expression of miR-375. In fact, the expression of this miRNA is able to inhibit
the translation of KLF5, an antagonist of the goblet cell–differentiation factor KLF4,
supporting the differentiation of goblet cells. Moreover, they observed a lower expression
of IL-4, IL-5, and IL-13 in Dicer1
Δgut mice and an enhanced susceptibility to helminth parasite Trichuris muris infection
(Figure 1B). IL-13, presumably supplied by TH2 cells, induces miR-375 in IECs in vitro
and a down-stream production of the TH2-facilitating epithelial cytokine TSLP, indicating
an appropriately balanced TH2 feed-forward loop regulated by miR-375. On the basis
of their results, the authors suggested that miR-375 directs the differentiation of
goblet cells and the promotion of antiparasitic TH2 immune responses. As miR-375 expression
is very high in the human intestine (Wu et al., 2010), mucosal expression of this
particular miRNA might also be important in the regulation of intestinal homeostasis
and protection against parasite infection in humans (Goto and Kiyono, 2011). Further
investigation should allow answering to many open questions still remaining, such
as whether there are other miRNAs involved in this process or whether there are other
miR-375 targets relevant to the differentiation of goblet cells or in the maintenance
of gut immunological homeostasis. It is quite easily conceivable that in a near future
we will assist to the development of innovative mucosal miRNA–targeted treatments
and to the diagnosis of pathogenic mucosal conditions such as allergy, inflammatory
bowel diseases and colon cancer, as well as infection by bacteria, viruses and parasites
by employing specific miRNA-designed tests.
One of the most recently emerging and appealing concept is the role of toll-like receptors
(TLRs) as potential mediators between gut microbiota and miRNAs/mRNAs modulation in
humans. In fact, it has been recognized clearly that host gene expression is regulated
by gut microbiota along the length of the gut and that microorganisms recognition
is mediated by TLRs through the adaptor molecule MyD88 (Dalmasso et al., 2011; Larsson
et al., 2012). Therefore TLRs, localized at the interface between the microbiota and
the molecular machinery of host cells, may be key players in these relationships.
In a recent study, Chassin et al. found that the TLR-4-mediated transcriptional activation
of IECs observed in mice immediately after birth, was induced by oral ingestion of
environmental endotoxin and induced a post-transcriptional down-regulation of epithelial
IRAK1 protein expression, protecting further from bacteria-induced epithelial damages
(Chassin et al., 2010). According to evidences showing that IRAK-1 expression is regulated
by miR-146a (Taganov et al., 2006), miR-146a levels declined only in concomitance
to the increase of IRAK-1 protein level, whereas miR-146a silencing induced IRAK1
protein expression. Moreover, authors demonstrated that the oral treatment with anti-miRNAs
is a viable option to down-regulate the expression of miR-146a in intestinal epithelial
cells.
Another study focusing on the microbiota regulation of miRNAs expression and on the
maintenance of intestinal homeostasis, has been reported by Xue et al. who reported
a connection between the expression of miR-10a and of its target IL-12/IL-23p40, a
key molecule for innate immune responses to commensal bacteria (Xue et al., 2011).
They also found that commensal bacteria down-regulated dendritic cell miR-10a expression
via TLR–TLR ligand interactions through a MyD88-dependent pathway and that mice with
colitis expressed higher levels of IL-12/IL-23p40 and lower level of gut miR-10a,
compared to control mice, opening new perspectives for the study of miRNAs regulation
in intestinal diseases.
In the field of RNA silencing, very close to that of miRNAs for their common mechanism
of action, a novel approach exploiting engineered bacteria has been reported few years
ago (Xiang et al., 2006). This approach holds great promise for functional genomics
in mammalian systems and for other in vivo applications, since it demonstrates that
the trans-kingdom RNA interference (RNAi) process is feasible both in vitro and in
vivo. Authors employed E. coli engineered to produce short hairpin RNAs, by the use
of a plasmid that they termed TRIP (trans-kingdom RNAi plasmid). This vector contains
Inv and HlyA encoding for invasin, and listeriolysin O, respectively, enabling the
entry into β1-integrin-positive mammalian cells and the release of genetic materials
from internalized vesicles. By co-culturing human colon cancer cells (SW480) in vitro
with the engineered E. coli, a significant down-regulation of a specific target gene
has been observed, demonstrating the effectiveness of the trans-kingdom RNAi mechanism
in vitro. By oral or intravenous administration of the engineered E. coli, the authors
demonstrated also an efficient gene silencing in the intestinal epithelium and in
human colon cancer xenografts in mice, suggesting a clinically feasible approach to
the in vivo application of RNAi technology. Most interestingly, this trans-kingdom
RNAi approach not only can be exploited clinically to silence genes in the colonic
mucosa and in other organs colonized by bacteria (i.e., oral cavity, urinary bladder,
and female genital tract), but also suggest the speculative but intriguing possibility
that such RNAi mechanism may occur also in natural interactions such as infections,
commensal interaction and symbiosis.
In conclusion, I described two works (Dalmasso et al., 2011; Singh et al., 2012) dealing
with a differential expression of miRNAs in different areas of the intestinal tract
as a function of microbiota composition. In these cases, intestinal microbiota are
the “actors.” Conversely, we should also think to miRNAs as “actors” when, under proper
conditions, influence the regulation of goblet-cell differentiation (Biton et al.,
2011). Therefore, an interconnected cycle could be envisaged where miRNAs and gut
microbiota are the two main partners. Two interesting approaches (use of engineered
vectors and the oral delivery of anti-miRNAs) (Chassin et al., 2010; Xue et al., 2011)
have emerged as interesting possibilities to study experimentally the interplays between
miRNA and gut microbiota, and their mutual role in influencing host immune system
and related processes. Therefore, although still a few, the studies reported so far
emphasize that we cannot ignore that “our other genome” is intimately linked to “our
natural miRNome.”