Extracellular vesicle-derived miRNA as a novel regulatory system for bi-directional communication in gut-brain-microbiota axis

The gut-brain-microbiota axis (GBMAx) coordinates bidirectional communication between the gut and brain, and is increasingly recognized as playing a central role in physiology and disease. MicroRNAs are important intracellular components secreted by extracellular vesicles (EVs), which act as vital mediators of intercellular and interspecies communication. This review will present current advances in EV-derived microRNAs and their potential functional link with GBMAx. We propose that EV-derived microRNAs comprise a novel regulatory system for GBMAx, and a potential novel therapeutic target for modifying GBMAx in clinical therapy.


miRNA-biogenesis and function
A primary miRNA transcript (pre-miRNA) can be processed by several biogenesis procedures to form the miRISC (miRNA-associated RNA-induced silencing complex) [16][17][18][19]. miRISC is then guided to target mRNA by complementary base pairing between the target sequence (TS) of the miRNA (nucleotides 2-8 in the 5'-end of the miRNA) and its target site in the 3'-untranslated region (UTR) of target mRNAs [20,21]. Target gene expression can be down-regulated by either translational inhibition or mRNA degradation according to the extent of base pairing and the surrounding sequences of the TS [22]. Alternatively, some studies also demonstrate that miRNAs can up-regulate transcription of certain target mRNA [23][24][25]. Notably, a single miRNA can potentially target multiple mRNA, whereas one mRNA can be potentially targeted by multiple miR-NAs, reflecting the complex regulatory function of miR-NAs [16]. Recent methodological advances including miRNA profiling and loss-of-function studies enable high-fidelity analysis of bioinformation to better define the dynamic expression and functional link with various cellular process and biological pathways in diverse tissues and from diverse species [18,26,27]. MiRNAs have been identified as promising candidates for biomarkers and therapeutic targets in a variety of diseases [28].

Gut miRNA regulate gut microbiota
In 2016, Liu et al. first profiled miRNA expression patterns within feces and gut luminal contents from mice and humans [7]. Intestinal epithelial cells (IEC) and homeobox gene (Hopx)-positive cells were identified as the major cellular source of fecal miRNAs. In vitro studies with cultured Fusobacterium nucleatium and Escherichia coli demonstrated that fecal miRNA could regulate bacterial gene transcripts and growth [7]. Targeted deletion of the miRNA biogenesis enzyme Dicer in mice resulted in imbalanced gut microbiota and exacerbated dextran sulfate sodium-(DSS) induced colitis, which was reversed by fecal miRNA transplantation from wild-type littermates, strongly suggesting a critical role of fecal miRNA in shaping gut microbiota and maintaining intestinal homeostasis [7].
More recent studies support an essential role of gut miRNA in inducing dysbiosis related to various disease states. In ovariectomized (OVX) mice, intestinal and fecal miR155/let-7 g expression were increased and associated with altered gut microbiota and cardiovascular function [29]. In another mouse model of total abdominal irradiation (TAI), the expression level of miR-34a-5p was elevated in small intestine, which closely correlated with composition shifting of gut microbiota, possibly contributing to associated cognitive impairment [30]. Distinct fecal or intestinal miRNA expression profiles and their potential link with disease and the abundance of gut microbiota have been identified in inflammatory bowel disease and colorectal cancer, underlying their potential clinical relevance as biomarkers or therapeutic targets [31,32].

Gut microbiota regulate gut miRNA expression
The evidence regarding the impact of gut microbiota on host miRNA expression is primarily derived from miRNA expression profile studies comparing traditional mice with germ-free (GF), or colonized mice. Significant differences in miRNA expression profiles in the colon and ileum was detected between GF mice colonized with gut microbiota from GF mice and specific-pathogen free (SPF) colonized littermates [33]. Fecal miRNA expression patterns also exhibited apparent differences between conventional mice and GF mice [34]. Additionally, fecal miRNA profiles can be deferentially and specially regulated by various colitogenic and non-colitogenic microbiota [34]. The potential target mRNAs of those miRNAs may be involved in regulation of xenobiotic metabolism, intestinal barrier maintainance and regulation of immune system function [33,34].
Other studies reveal that gut microbiota regulate intestinal miRNA profiles in a highly cell type-specific manner [35]. The miRNA expression patterns of intestinal epithelial stem cell (IESC) are most significantly altered in response to gut microbiota among all intestinal epithelial cell types, with miR-375-3p identified as selectively sensitive to microbiota from IESC [35]. In addition to intestinal miRNA, the expression of fecal miRNA can also be influenced by gut microbiota. Higher abundance of fecal miRNA profiles is detected in GF mice than SPF colonized littermates, and alterations in fecal miRNA expression patterns can be induced by depleting gut microbiota with antibiotic in SPF mice [7,36].
In vitro studies demonstrate that commensal bacteria induce certain miRNA expression patterns in intestinal epithelial cells or dendritic cells,targeting mRNAs that regulate the innate immune response and barrier function [37,38]. Adherent-invasive E. coli (AIEC), a pathogen with high prevalence in Crohn's disease, has been shown to up-regulate miRNAs targeting genes responsible for the autophagy response (ATG5 and ATG16L) in mouse enterocytes, which may facilitate AIEC replication and exacerbation of intestinal inflammation [39] thereby altering intestinal immune regulation and barrier function [40][41][42][43].

Gut microbiota regulates brain miRNA expression
A large number of abnormal brain miRNAs implicated in anxiety-like behaviors have been detected in the region of amygdala and prefrontal cortex of GF mice or mice with microbiota depletion by an antibiotic cocktail [44]. Some dysregulated brain miRNAs in GF mice have been shown to be normalized by microbial colonization [44].
Gut microbiota have also been demonstrated to modulate hippocampal miRNA expression associated via kynurenine pathway enzymes which regulate hippocampal development and axon guidance pathway [45,46]. A more recent report describes that a microbial product, Bacteroides fragilis lipopolysaccharide (BF-LPS) can act as a neurotoxin via induction of a series of miRNAs targeting genes that regulate synaptic architecture and deficits, amyloidogenesis, and cerebral inflammatory signaling [47]. Some other microbial metabolites including tryptophan, butyrate, acetylcholine, norepinephrine, serotonin, dopamine may also influence miRNA biology indirectly via regulation of astrocyte function and bloodbrain-barrier integrity, or even by altering human behavior via disruption of normal neurotransmitter levels [48]. The gut microbiota-host miRNA interaction is summarized in Tables 1 and 2.

EV entrapment of fecal miRNA
In their study on fecal miRNA expression profiles, Liu et al. detected EVs in fecal samples and demonstrated that the most abundant fecal miRNAs were also contained within EVs, suggesting that EVs are the major extracellular source of fecal miRNAs [7]. EVs protect fecal miRNAs from degradation via a phospholipid bilayer comprising membrane proteins of EV which entrapping miRNA [96,97].

Brain-derived EVs
Recent studies describe a wide distribution of EV in the CNS, detected in oligodendrocytes, neurons, astrocytes, microglia, choroid plexus, and brain epithelial cells the interface of blood-brain barrier (BBB) and cerebrospinal fluid (CSF) [98][99][100]. Brain-derived EVs play a key role in cell-to-cell communication involved in neurogenesis, neural development, neuro-inflammation, synaptic communication and nerve regeneration [101][102][103][104]. Accumulating evidence suggest that brain-derived EVs, especially exosomes, play an important role in the pathogenesis of neurodegenerative diseases, infectious CNS diseases, neuroinflammation, psychiatric disease and brain tumors [105][106][107][108][109][110][111].Their output and cargo can be cell-specific and disease -specific and varied with different events during disease progress, features that provide strong potential for use as a biomarker for CNS disease [108,112,113]. Furthermore, several other key features of EVs including stability, low immunogenicity, facility of crossing the BBB, accurate cell targeting and specific delivery make them an attractive candidate for therapeutic delivery vehicles in treating CNS disease [114][115][116].
MiRNAs have been demonstrated to play an important active biological role within brain-derived EVs from astrocytes, neurons, macrophage/microglial cells, prefrontal cortices cells, glioma cells, glioblastoma cells, and glioblastoma stem-like cells, playing a critical role in neurogenesis, response to stress, virus induced neurotoxicity, schizophrenia and bipolar disorder, brain tumor progress, brain metastasis outgrowth [101,[117][118][119][120]. More recent research indicates that brain-derived EVs can be detectable in plasma, and astrocyte-derived exosomes are capable of transferring miRNA to metastatic tumor cells, suggesting that brain-derived EVs may transfer molecular information to tissues remote from the CNS [120][121][122]. Several recent studies have demonstrated that altered miRNA profiles in brain EVs from Alzheimer's patients, however the mechanisms and clinical significance underscoring these observations remain a focus of investigation [123][124][125]. Critically, the biological relevance for EV transfer from brain to gut has not been fully elucidated.

Microbiota-derived EV
Bacterial membrane vesicles, including outer-membrane vesicles (OMVs) derived from Gram-negative bacterium and membrane vesicles (MVs) derived from Gram-positive bacteria, parasites, fungi, mycobacteria, refer to a collection of nano-sized membrane vesicles released from bacteria into the extracellular environment [126,127]. Bacterial membrane vesicles are currently regarded as microbiota derived-EVs since they share characteristic similarities in size, structure and biological function with EVs derived from mammalian cells [128]. Microbiota-derived EVs can transfer a broad range of cargo including bioactive proteins, lipids, nucleic acids, and virulence factors to neighboring bacteria or host cells (epithelial cells, endothelial cells, immune cells). This bioinformatic transferring plays a critical role in cellular processes for both intra-kingdom (bacteria-bacteria) interactions and inter-kingdom (bacteria-host) communications [129,130]. The effect of microbiota derived EVs can be effectively differentiated from microbial metabolites or host by evaluating the effect of bacterial free microbiota-derived EVs isolated from bacterial cultures on fecal samples [131]. Recent advances in this field reveal that microbiotaderived EVs exhibit multiple regulatory functions central for bacterial survival and nutrient acquisition, bacterial virulence delivery, host colonization and invasion, microbial interactions, antimicrobial resistance, stress and inflammatory response, endothelial cell adhesion, and systemic inflammatory and metabolic response, which all play key roles in the pathogenesis of diverse infectious and inflammatory diseases [132][133][134][135][136][137][138][139]. Several key features of OMV including size, antigen stability, high immunogenicity, accurate host cell targeting, specific cargo delivery and host immune response make them a promising novel candidate for a vaccine target against bacterial infections, and as targeted drug delivery against cancer and other diseases [140][141][142]. Recent findings have focused on the modulatory effect of microbiota-derived EVs on intestinal barrier function and the immune response, two important components of GBMAx [143][144][145][146][147][148]. Furthermore, relevant studies also reveal that microbiota-derived EVs can be released into the systemic circulation and cross the BBB [8,149,150]. Staphylococcus aureus and Helicobacter pylori-derived EVs have been detected in the brain after oral administration or intramuscular injection via in vivo imaging procedures [151,152]. Additionally, LPS, a key virulence factor in porphyromonas gingivalis outer membrane vesicles has been found in glia and the major cerebral vessels of patients with Alzheimers disease (AD) by immunoblot [153]. It has been hypothesized that microbiota-derived EV may be absorbed into mesenteric veins, carried by the hepatic portal vein and liver, to finally enter the brain via the circulatory system [154]. These data strongly suggest that microbiota-derived EVs may exert a direct effect on the CNS and be an important central modulator for GBMAx.
Small RNA (SRNA) within microbiota derived EV can be internalized by host cells and play an important role in host-pathogen interaction. miRNA-sized sRNA and methionine transfer RNA (tRNA) secreted by bacterial OMV (periodontal pathogens and Pseudomonas aeruginosa) have been shown to enter host cells and modulate host immunity [155,156]. EV-contained miRNA secreted by gastrointestinal nematode has been detected in circulation, which can be internalized by small intestinal epithelial cells and modulate host innate response [157].
Microbiota derived RNA may act as ligands for Toll-like Receptor (TLR) and regulators for host innate immunity [158,159].
More recent research revealed that OMV may cross the blood-brain barrier and contribute to neuroinflammation and cognitive impairment linked with neurodegeneration disease such as Alzheimer's disease, Parkinson's disease and dementia. The possible mechanism may involve transfer of small RNA non-coding RNA elements contained within OMV into host cells, thereby regulating host gene expression [160][161][162][163][164][165].

EV derived miRNA in metabolic disease
Obesity, Metabolic Syndrome and diabetic mellitus are known risk factors for the development of CNS disorders including cerebrovascular disease, neurodegenerative diseases and dementia. Several lines of evidence have revealed that EV derived miRNA originated from gut microbiota, adipose tissue, steatotic hepatocytes, mesenchymal stem/stromal cells (MSC), and pancreatic islets play crucial role in the pathogenesis of those metabolic disease and associated target organ injury [166][167][168][169][170][171]. Their role and relevance to GBMAXs and cerebral disease remains an area of active investogatyion. The impact of EV derived miRNA on neurological and metabolic disease are summarized in Table 3.

Controversies and challenges
EV derived miRNA has gain great attention in the research of GBMAx. However, controversies and challenges remain in this fields.

EV classsification and miRNA extraction
The heterogeneity of EV may be far greater than we have recognized previously. A more complex classification system based on EV proteome, nucleic acid distribution and biological function (rather than only 3 subsets mentioned above) has been predicted1 [172][173][174]. Practical difficulty may exist in extraction of EV-derived miRNA including (1) tedious and costly procedures of ultracentrifugation and density gradient extraction, and purification; (2) lack of standardization with technological platforms and quantitative assays; (3) non-selective enrichment of specific EV subpopulations or differential cellular origins; and, (4) uncoupling from conventional reverse transcriptase quantitative PCR [175][176][177]. Novel extraction approach and technological improvements are warranted.

Environmental and human genetic factors
It must be acknowledged that the regulatory system of EV-derived miRNA on GBMAXs is not restricted to EV or miRNAs originating from gut microbiota, gut or brain. Environmental factors (e.g. diet, medications, smoking, environmental contaminants, stress) and human genetics also play a crucial modulatory role in GBMAXs via: (1) secreting miRNA containing EVs; (2) shaping gut microbiota; (3) stimulating microbial metabolic products; and, (4) regulation of miRNA expression and function of the host gut and brain [178][179][180][181][182][183][184]. The expression of host miRNA ( fecal miRNA or intestinal epithelial cellderived miRNA) and its associated function is impacted by these host genetic and environmental factors, which will ultimately modulate the composition of metabolite and function of gut microbiota [34,185,186]. Those environmental and genetic factors may be considered as an extension of the EV-derived miRNA system for GBMAXs, and should be taken into accounting novel drug development and therapeutic strategies targeting GBMAx.

Non-miRNA RNA biotypes and non-vesicle carriers
miRNA is the most studied extracellular RNA but only constitutes a minor composition of RNA biotype in the EV cargo. Other RNA biotypes including small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), long noncoding RNA (lncRNA), Y RNA may be more abundant in EV cargo [187][188][189]. EV is not the only RNA carrier for miRNA. Non-vesicular miRNAs presenting as ribonucleoprotein complex have been detected in various fluids and circulation, which are becoming candidates for biomarkers and therapeutic targets [190,191]. The regulatory systems consisting of non-miRNA RNA biotypes and non-vesicle carriers in GBMAx and their relationship with EV derived miRNA should be explored in further study (Table 4).

Conclusions
MiRNAs play a potentially critical role in gut microbiotagut interaction and gut microbiota-brain bi-directional communication. EVs can be derived from brain, gut and Table 3 The impact of EV derived miRNA on neurological and metabolic disease

Plasma
Unknown in Alzheimer's disease [123] miR-212 and miR-132 (Downregulated) Neurally derived plasma EV Unknown in Alzheimer's disease [124] miR-23a-3p, miR-223-3p, miR-190a-5p, miR-100-3p, Neurally Derived Plasma EV Unknown in Alzheimer's disease [125] miRNA cargo (periodontal bacteria) Aggregatibacter actinomycetemcomitans Neuroinflammation in Alzheimer's disease [165] miR-27b,miR-126 miR-130, miR-296 Pancreatic islets Beta cell-endothelium cross-talk in diabetes [165]     gut microbiota, coordinating cell-to-cell communication via transfer of miRNAs. We hypothesize that an EV-miRNA system throughout GBMAx could play a central role in exchange of molecular information among gut microbiota, gut and brain. This EV-miRNA based regulatory system is schematically outlined in Fig. 1. However, current research in this field remains in the early stages. Further investigations should be performed to elucidate: (1) the direct effect of brain-derived EVs on gut and gut micribota; (2) the precise regulatory mechanisms of EV miRNA transfer, and their biological function on GBMAx; (3) the functional link between EV-miRNA and other classical neuro-immune-endocrine pathways. Progress in this field will provide new insight into the comprehensive understanding of GBMAx and help advance the clinical development of novel biomarkers and therapeutic target for the variety of diseases associated with GBMAx imbalance. Fig. 1 Schematic presentation of EV derived miRNA acting as a novel regulatory system for bi-directional communication in gut-brain-microbiota axis. A proposed regulatory system consisting of extracellular vesicles (EVs) derived from the brain, gut and gut microbiota which modulate bi-directional communication in gut-brain-microbiota axis (GBMAx) via intercellular transfer of microRNAs (miRNAs). Brain-derived EVs may modulate the gut and gut microiota via a "top-down" manner by migrating from brain to gut and regulating the expression of gut miRNAs and fecal miRNAs. Fecal miRNAs entrapped within EVs can enter bacteria and shape gut microbiota via targeting bacterial nucleic acid sequences. Alternatively, or in parallel, microbiota derived-EVs (bacterial membrane vesicles) may modulate the brain via a "bottom-up" manner by crossing the blood brain barrier and exerting a direct effect on the central nervous system. Microbiota derived-EVs can also potentially modulate gut barrier function and the immune response directly