gbb12109

Genes, Brain and Behavior (2014) 13: 69–86 doi: 10.1111/gbb.12109

Review

Microbial genes, brain & behaviour – epigeneticregulation of the gut–brain axis

R. M. Stilling†, T. G. Dinan†,‡ and J. F. Cryan†,§,∗

†Alimentary Pharmabiotic Center, ‡Department of Psychiatry,and §Department of Anatomy and Neuroscience, UniversityCollege Cork, Cork, Ireland*Corresponding author: Prof J. F. Cryan, Department of Anatomy& Neuroscience, Western Gateway Building, University CollegeCork, Cork, Ireland. E-mail: [email protected]

To date, there is rapidly increasing evidence for

host–microbe interaction at virtually all levels of com-

plexity, ranging from direct cell-to-cell communication

to extensive systemic signalling, and involving various

organs and organ systems, including the central nervous

system. As such, the discovery that differential microbial

composition is associated with alterations in behaviour

and cognition has significantly contributed to establish-

ing the microbiota–gut–brain axis as an extension of the

well-accepted gut–brain axis concept. Many efforts have

been focused on delineating a role for this axis in health

and disease, ranging from stress-related disorders such

as depression, anxiety and irritable bowel syndrome to

neurodevelopmental disorders such as autism. There is

also a growing appreciation of the role of epigenetic

mechanisms in shaping brain and behaviour. However,

the role of epigenetics in informing host–microbe inter-

actions has received little attention to date. This is

despite the fact that there are many plausible routes

of interaction between epigenetic mechanisms and the

host-microbiota dialogue. From this new perspective we

put forward novel, yet testable, hypotheses. Firstly, we

suggest that gut-microbial products can affect chromatin

plasticity within their host’s brain that in turn leads to

changes in neuronal transcription and eventually alters

host behaviour. Secondly, we argue that the microbiota

is an important mediator of gene-environment interac-

tions. Finally, we reason that the microbiota itself may be

viewed as an epigenetic entity. In conclusion, the fields

of (neuro)epigenetics and microbiology are converging

at many levels and more interdisciplinary studies are

necessary to unravel the full range of this interaction.

Keywords: Anxiety, cognition, depression, epigenetics,germ-free, Gut, HDAC, histone modification, hologenome,learning, microbiome, microbiota, nucleomodulin, probiotic,stress

Received 21 October 2013 , revised 13 November 2013 ,accepted for publication 25 November 2013

Since their emergence, the evolution of multicellular eukary-otic organisms has taken place in the presence of prokaryotesand a plethora of diverse micro-organisms now colonizevirtually all body surfaces of animal hosts, residing asbeneficial symbionts, harmless commensals or pathogenicparasites (Dave et al. 2012; Schloissnig et al. 2013; Turn-baugh et al. 2007) most prominently within the gastroin-testinal tract. An understanding of the importance of theseinteractions is undergoing a renaissance with large-scale sci-entific projects like the Human Microbiome Project (HMP;Human Microbiome Project Consortium 2012; Turnbaughet al. 2007) designed to sample, determine and quantifyall human-associated microbiotic life. In parallel the Euro-pean HMP-counterpart MetaHIT focuses on the intestinal-tract microbiota in general (Qin et al. 2010) with theEldermet project centering on the elderly (Claesson et al.2012).

An estimated 90% of cells found in the human body arenot human after all but of mostly prokaryotic origin, derivedfrom at least 40 000 bacterial strains in 1800 genera (Forsythe& Kunze 2013; Frank & Pace 2008; Luckey 1972). Thoughconsiderably smaller in size, these approximately 100 trillioncells add up to a mass of almost 1–2 kg in an adult individual(Forsythe & Kunze 2013) – approximately the weight ofa full-grown human brain (ca. 1.5 kg, Parent & Carpenter1996).

There is a rapidly increasing amount of evidence impli-cating host–microbe interactions at virtually all levels ofcomplexity, ranging from direct cell-to-cell communication toextensive systemic signalling, and involving various organsand organ systems, including the central nervous system(CNS). The microbiome (see Box 1) critically supports hostmetabolism and yields a source of metabolites, many ofwhich would otherwise not be available to host cells.This is achieved by the huge diversity of genetic mate-rial that constitutes the microbiome. It is estimated thatthe human gut harbours more than 3.3 million non-humangenes (Zhu et al. 2010), making the 23 285 human protein-coding genes currently annotated in the ENSEMBL database(http://www.ensembl.org) appear almost negligible. Thus,the sole presence of micro-organisms as well as the specificcomposition of this microbiota has multiple, critical con-sequences for host physiological and metabolic processesranging from postnatal development and immunomodula-tion to, perhaps most surprisingly, behaviour and cognition(Sommer & Backhed 2013) which forms the basis of thisreview.

© 2013 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society 69

Stilling et al.

BOX 1: Glossary

Microbiota: The microbiota is the sum of all micro-organisms associated with a given host individual. Micro-organismscan be found on all body surfaces (including the lumen of gastro-intestinal organs, which strictly speaking belong to theoutside world) and cavities, such as the skin, nose, ears and genitals. The microbiota is not only limited to the bacterialdomain of life but also includes archaea as well as eukaryotes such as protozoa, fungi and (mostly parasitic) nematodes.Though not considered as organisms, one could even include viruses (host associated as well as bacteria associatedphages). However, the mammalian non-pathogenic virome and the complexity of phages are largely unexplored and willnot be addressed in this review.

Microbiome: The genome of a given organism is defined as the sum of its chromosomal genes and also includes the extra-chromosomal genetic information found in other organelles or endosymbionts (e.g. plasmids, chloroplasts, mitochondria).Analogously, the microbiome is defined as the entirety of all genes present in the micro-organisms colonizing a given host.The microbiome is also referred to as metagenome. Together, the genome and the associated metagenome make up anorganisms hologenome (Brucker & Bordenstein 2013), a term coined by Rosenberg et al. in 2007 (Rosenberg et al. 20072009).

Enterotype: The concept of enterotypes was introduced in 2011 to define inter-individual variation in gut-microbiotaspecies composition. More specifically, three distinct microbiomic clusters were identified that were mainly separatednot only by abundance of certain species but also by abundance of genes with shared molecular function (Arumugamet al. 2011).

Germ-free (GF): Also referred to as axenic (from the Greek ‘free from foreign[er]’), though axenic can have otherconnotations in microbiology. GF animals are defined to be free from any microbial colonization and are kept in isolatorsunder strictly sterile conditions. Also see Box 2.

Gnotobiotic: A system, in which all organisms are either defined by or known to the investigator, is referred to asgnotobiotic (from the Greek ‘known life’). Thus, a gnotobiotic animal is inhabited only by certain micro-organisms. Thestatus includes the absence of any colonization as in GF animals, since it can also be viewed as a known status. Oftenthe term is used to describe formerly GF animals that have be colonized by a defined set of micro-organisms such as the‘Schaedler Flora’ (Schaedler et al. 1965) or mono-association with just one strain of bacteria. For problems with definingthe gnotobiotic status see Box 2.Probiotic: Probiotics are living organisms that contribute to a host-beneficial microbial flora. Thus probiotic organisms canbe viewed as symbionts.

Prebiotic: Meant to contrast the term antibiotic, prebiotics are chemical compounds that influence the microbial flora ina host-beneficial way.

Psychobiotic: A concept recently introduced by Dinan, Stanton and Cryan defines psychobiotics – analogous toprobiotics – as live organisms that produce positive effects on mental health (Dinan et al. 2013). It can also be arguedthat psychobiotics may exhibit benefits to healthy individuals, for example as memory enhancers (Misra & Medhi 2013).

Dysbiosis and probiosis: A dysbiotic state is marked by disadvantageous alterations in microbial composition. Probiosis,on the other hand – following the definition of probiotics – rather describes a beneficial microbial status that supportsnormal host function, e.g. stress resilience.

Nucleomodulins: Several bacteria can influence their host’s transcriptome by secreting protein effectors directly targetingthe epigenetic machinery. So far these effectors have only been found in intracellular parasitic bacteria and viruses.Bierne and Cossart proposed to classify non-eukaryotic epigenetic effector proteins collectively as nucleomodulins(Bierne & Cossart 2012). We propose to apply this concept to potential transcriptional regulators that affect neuronalgene expression and alter host behaviour and could hence be termed neuro-nucleomodulins.

The microbiota–gut–brain axis

The discovery that differential microbial composition is asso-ciated with alterations in behaviour and cognition has sig-nificantly contributed to establish the ‘microbiota–gut–brainaxis’ as an extension of the well-accepted ‘gut–brain axis’concept. This concept is used to describe the bidirectionalcommunication between the CNS and intestinal organs andwas first introduced in terms of peripheral regulation ofemotions by William James and Carl Lange in the 1880sand further challenged and refined by Walter Cannon in the

1920s to account for the primacy of the brain in regulatinggastrointestinal function (see Mayer 2011). However, inthe light of new and intriguing data, mostly resultingfrom the study of rodents, the gut–brain axis has beenreviewed from a number of perspectives, focusing ondifferent aspects ranging from basic microbiology totranslational applications (e.g. see Bercik et al. 2012;Berer & Krishnamoorthy 2012; Collins et al. 2012b, 2013;Cryan & Dinan 2012; Cryan & O’Mahony 2011; Forsythe& Kunze 2013; Lyte 2011, 2013; Nicholson et al. 2012;Rhee et al. 2009; Sommer & Backhed 2013). In this

70 Genes, Brain and Behavior (2014) 13: 69–86

Microbial genes, brain & behaviour

review, we want to highlight our current understandingon the underlying mechanisms of microbiota–gut–braininteractions and associated behavioural alterations with anemphasis on a potential epigenetic contribution to thesemechanisms.

A new epoch is emerging with these findings in basicresearch and animal studies beeing translated into the clinic.Indeed, it is becoming clear that certain pathologies, whichare associated with an altered microbiome, are connectedto mood, stress, behaviour and/or cognition (for reviewsee Grenham et al. 2011; Shanahan 2012). In this regard,irritable bowel syndrome, which is highly comorbid withmood disorders such as depression, also leads to decreasedcognitive performance (Berrill et al. 2013; Kennedy et al.2013). Moreover, an important recent neuroimaging studyvalidated rodent studies (e.g. Bravo et al. 2011) in implicatingmicrobe-brain signalling in modulating resting brain activity inkey circuits involved in pain, emotion and cognition (Tillischet al. 2013).

Microbiota-associated phenotypes and behavioural

alterations

Tables 1 and 2 summarize the growing body of researchemanating from rodents that demonstrate a role formicrobiota in behaviour. At the centre of many of thesestudies are animals that have been raised in a sterileenvironment and thus without microbiota, referred toas GF (see Box 2). Additionally, behavioural studies onanimals with either defined infections, antibiotic treatmentor administration of probiotic bacteria have been carried out(for a review see Cryan & Dinan 2012; Foster & Neufeld2013, see Table 1). These studies showed reproducibleand largely consistent effects of the various microbialstates on behaviour in mice. The most commonly reportedphenotype was altered anxiety-related behaviour, which canbe assessed by a variety of tests (Table 1).

There is now an increasing number of studies focusing onthe positive behavioural effects of various bacterial strains,mostly Bifidiobacteria and Lactobacillus species (see Table 1and Bercik et al. 2010, 2011b) but also transient commensalssuch as Mycobacterium vaccae (Matthews & Jenks 2013).

Moreover, an increasing number of studies in animalmodels of stress, anxiety and depression also implicate arole for the microbiota in psychopathology (Bailey & Coe1999; Bailey et al. 2011; O’Mahony et al. 2011; Park et al.2013).

In almost all studies the authors also reported biochemicaland molecular changes (Table 2). In addition to thecommon finding that the microbial status is associatedwith altered corticosterone levels in the blood plasmaor serum of stressed as well as naïve mice, geneexpression changes in different brain regions could bedemonstrated (Table 2). Most commonly Bdnf and Fosexpression levels were analysed as a correlate for differentialneuronal activation. Interestingly, Bdnf is well-known for itsfunction in neuronal plasticity, learning and memory, anda number of psychiatric and neurodegenerative diseases(Cowansage et al. 2010; Tapia-Arancibia et al. 2008; Walkeret al. 2012). In addition, alterations in neurotransmitter

BOX 2: The germ-free animal

GF and other gnotobiotic animals have been generatedsince the early 20th century (for a historical introductionsee Reyniers 1959). To achieve a GF status in a mammal,a new-born needs to be delivered by Caesarean sectionand hand-reared with sterilized milk in a sterile, isolatedenvironment. Further generations can then be derivedby Caesarean section exclusively, since rearing can bearranged by an already GF foster mother. Future coloniesof GF animals can be maintained from interbreeding witheach other within a suitable Germ-Free Unit. However,it is important to bear in mind that increasing evidencesuggests that Caesarean-derived newborns are, againstlongstanding dogma, not sterile (for a recent reviewsee Funkhouser & Bordenstein 2013). Though severalstudies on GF animals monitored contamination usingculture methods, these methods are heavily biassedand bacteria, fungi, protozoans and viruses that are noteasily cultureable will not be detected. The only way ofdefinitely determining the microbial status of GF animalswould be using deep sequencing. Targeted amplificationof 16s rDNA by polymerase chain reaction would only beuseful to determine the status of bacterial colonization,and would not give information about fungi or viruses.Already in 1970 the first explicit behavioural observa-tions have been made on gnotobiotic piglets (Bahr 1970).Notably, it took another 34 years until the first studies onGF mice were published, that showed alterations in brainfunction (Sudo et al. 2004). Since 2011 a number of stud-ies demonstrated behavioural alterations in GF mice andthereby significantly extended the microbiota–gut–brainaxis concept. Thus, this almost century-old areaof research has still not lost its importance.

signalling, including neurotransmitters and associatedmetabolites and neurotransmitter receptors have beendescribed. Diaz Heijtz et al. (2011) took a genome-wideapproach to define the transcriptional profile of the GFmouse in five different brain regions. Further analysis showedthat genes associated with the functional categories ‘citratecycle’, ‘synaptic long-term potentiation’, ‘steroid hormonemetabolism’ and ‘cyclic adenosine 5′-phosphate (adenosinemonophosphate)-mediated signalling’ were enriched amongthe differentially regulated genes, which supports the phe-notypic observations. Interestingly, while in the cerebellumand hippocampus robust changes in gene expression werefound, the hypothalamus, the brain region involved in thestress-activated hypothalamuic-pituitary-adrenal axis (HPA-axis), showed almost no differential gene expression.

While certain behavioural and biochemical parameters(including anxiety, sociability, HPA-axis and tryptophanmetabolism) could be reversed by recolonization with aconventional microbiota or probiotic treatment, others wereunaffected by restoration of a normal microbiota (including5-HT concentration and social cognition; see Tables 1 and 2for details). Indeed, reversibility of the anxiolytic phenotype inGF mice is only guaranteed if recolonization happens during

Genes, Brain and Behavior (2014) 13: 69–86 71

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Stilling et al.

a critical time window during adolescence (Clarke et al. 2013;Foster & Neufeld 2013). However, since this hypothesis hasnot been tested systematically, it is unclear if this observationcan also be generalized to other GF-associated phenotypes.

Overall, accumulating evidence suggests that there is acorrelation between the microbiota composition and brainand behaviour. While intestinal probiosis is associated withincreased stress resilience and decreased basal anxiety,GF animals show increased basal corticosterone levelsalong with an increased stress-response, hyperlocomotionand decreased brain-derived neurotrophic factor (BDNF)levels but also reduced anxiety and social activity. It mayappear as a contradiction that the endocrinological stressparameters are elevated while anxiety on the behaviourallevel is reduced. However, these parameters do notnecessarily correlate (Neufeld et al. 2011) and may bedifferentially influenced by the microbiota. Dysbiosis ofthe enteric milieu on the other hand is marked by anincrease in anxiety, depressive-like behaviour and memoryimpairment along with decreased concentrations of keyneurotrophic factors involved in plasticity such as BDNF(Fig. 1). However, the underlying molecular mechanismsleading to these behavioural and biochemical alterations arenot well understood. Interestingly, there is now a growingappreciation of the role of epigenetic mechanisms in shapingbrain and behaviour.

Epigenetic mechanisms and neuroepigenetics

‘Epigenetics’ is one of the most overused words in thecurrent scientific vocabulary (Ledford 2008). Initially, it wasused to describe developmental programming but was laterredefined more specifically to refer to heritable changesin gene expression that do not originate from mutationsof the DNA sequence (Holliday 1987; Waddington 1953).The term is now commonly used in a broader sense,although it is associated with different connotations withinspecial scientific fields. While some disciplines focus on theaspect of sequence-independent transgenerational germ-line inheritance of a phenotypic trait, others, especiallyin the fields of neuroscience or biological psychiatry,emphasize early-life experiences that influence developmentand behaviour during later life and adulthood. However,in a more cellular view, transgenerational epigenetic traitsare interpreted in the context of somatic mitotic cellulardifferentiation within a multicellular organism. In molecularneuroscience, most often the term epigenetics is ratherused to take into account the multiple molecular eventsthat are involved in the dynamic regulation of neuronal geneexpression.

Irrespective of the interpretation one may adopt, the molec-ular machinery mediating these seemingly different effectsare indistinguishable in all interpretations of ‘epigenetics’. Ingeneral, this molecular machinery comprises plastic changesin regulation of nuclear architecture, chromatin structure andremodelling and gene expression. This epigenetic machineryincludes post-translational modifications of histone proteinsthat serve at least two, non-mutually exclusive functions:

regulatory factor recruitment and histone–DNA interaction.Acetylation of a lysine residue in the N-terminal tail region of ahistone reduces the electrostatic interaction of the positivelycharged lysine with the negatively charged DNA, therebymaking chromatin more accessible (Korolev et al. 2007).At the same time this modification serves as a recognitionsignal for bromodomain-containing proteins, which in turnrecruit factors of the transcriptional machinery to the nowaccessible genomic site (Chen et al. 2010; Hargreaves et al.2009; LeRoy et al. 2008). Apart from acetylation, an increas-ing variety of post-translational modifications of histoneproteins are known, including phosphorylation, methylation(three possible methylation states on lysine and arginineresidues), and ubiquitylation. In addition, the DNA itself canbe modified by cytosine methylation, which is generallyassociated with silencing of a genomic element (Jaenisch &Bird 2003). More recently also the different products duringactive demethylation of the DNA, especially TET1-mediatedcytosine hydroxymethylation, gained interest as they seemto have regulatory function that differs from the simpleabsence of methylation (Kaas et al. 2013; Mellen et al. 2012p. 2; Rudenko et al. 2013; Szulwach et al. 2011). Finally, asteadily increasing number of non-coding RNAs, which areoften transcribed from DNA sites previously thought to betranscriptionally inactive (‘junk DNA’), are assigned as part ofthe epigenetic machinery and can act on transcriptional aswell as translational activity (for some recent reviews on thediverse functions and biology of the increasingly complexRNA landscape see, e.g. Landry et al. 2013; Ng et al. 2013;O’Connor et al. 2012; Ørom & Shiekhattar 2013; Wang et al.2012 and references therein).

Interestingly, these processes have recently been identi-fied to play an important role in cognitive processes duringhealth and disease by regulating gene expression in thebrain (for recent review see Day & Sweatt 2011a,2011b;Graff & Tsai 2013a; Kosik et al. 2012; O’Connor et al. 2012).A connection between the two mechanisms of environ-mental information processing and epigenetics has beenestablished, often referred to as neuroepigenetics (Day &Sweatt 2010; Sweatt 2013). Concomitantly, the term chro-matin plasticity is used to acknowledge the fact that theneuronal nucleus is no exception to the general rule thatvirtually all components in the brain are able to undergoplastic changes (Dulac 2010). This rather new discipline triesto unravel the dynamic changes of transcriptional regula-tion in neurons upon stimulation or in disease. For example,dynamic regulation of gene expression in the hippocampusis critical for long-term memory consolidation and synapticplasticity (Da Silva et al. 2008; Igaz et al. 2002, 2004) anda specific role for all three mechanisms of the epigeneticmachinery has been demonstrated. Of these, histone acety-lation is probably the best-understood, since the catalysingenzymes (namely histone acetyltransferases, HATs and his-tone deacetylases, HDACs) are well-studied and can betargeted pharmacologically, e.g. by using HDAC inhibitors,which are currently discussed as cognitive enhancers (Graff& Tsai 2013a,2013b). Because of its activatory role, his-tone acetylation plays a permissive role in learning-inducedtranscription and the use of small-molecule HDAC inhibitorshas been shown to augment memory consolidation and to



Figure 1: The microbiota affects neurophysiological, biochemical and behavioural parameters. Accumulating evidence suggeststhat there is a correlation between the microbiota composition and brain and behaviour. Host-supporting species (probiosis) alterbehaviour and biochemical parameters of the host in a different direction as compared to pathogenic species (dysbiosis). The phenotypeof GF animals seems to represent a rather intermediate state, featuring characteristics of both conditions.

parallel the beneficial effects of environmental enrichmenton cognitive function mechanistically (Fischer et al. 2007).As such, HDAC inhibition has also been shown to amelio-rate cognitive decline during ageing and in mouse modelsof neurodegenerative diseases such as Alzheimer’s disease(for review see Graff & Tsai 2013b; Stilling & Fischer 2011).The presumed mechanism of action for elevated histoneacetylation to aid in procognitive processes is based on theidea that these molecules do not simply alter acetylationlevels at random genomic loci but rather support a givencell’s own regulatory program or facilitate it in cases whereit has gone out of balance (McQuown & Wood 2011; Peleget al. 2010; Stilling & Fischer 2011). The role of epigenet-ics in informing host–microbe interactions has received littleattention to date. In the following sections we will elaborateon potential convergences between epigenetic mechanismsand host-microbiota dialogue.

Microbe–brain interfaces: potential sites

for epigenetic regulation

Since microbes colonizing the host body are usually housedon the outer body surfaces, including the skin, mouth, lungs,gastrointestinal tract and vaginal mucosa, a large part of theinteraction between the microbiota and their host will be

mediated by host cells, which the microbes are in directcontact with. These are mainly epithelial cells but also cells ofthe immune system as well as peripheral neurons (Forsythe& Kunze 2013). However, several–mostly parasitic–bacterialspecies are capable of invading host tissues and can evenlive in intracellular vacuoles to manipulate their host cellsdirectly (Lievin-Le Moal & Servin 2013 and see below).In addition, host dendritic cells are able to engulf livingbacteria from the gut lumen and transport them through thebody, though it is unclear which of the many organ barriersdendritic cells may cross and whether bacterial cells are laterreleased or digested (Rescigno et al. 2001). Furthermore,surface-dwelling microbiota have means of signalling acrossthe epithelial border by secretion of bioactive moleculesthat penetrate the outer barriers and are carried to distanteffector organs, including the brain, through the bloodstream and the lymphatic system (Rhee et al. 2009).

The diverse possible routes for microbiota–gut–brainsignalling and diet-influenced gut–brain communication havepreviously been reviewed elsewhere (e.g. Collins et al.2012b; Cryan & Dinan 2012; Forsythe & Kunze 2013;Lyte 2013; Montiel-Castro et al. 2013) and will thus onlybe discussed with regard to potential sites of epigeneticregulation. It should be noted that to date there is apaucity of direct evidence for the role of epigenetics inshaping host–microbiota interactions. Yet, there are plenty


Stilling et al.

of indications as to how potential epigenetic mechanismscan modulate the biological interaction between hosts andmicro-organisms. Interestingly, epigenetic mechanisms havebeen recently been put forward as a central mechanismdriving host–pathogen interactions (Gomez-Díaz et al. 2012).Expanding this to non-pathogenic beneficial microbes is animportant goal of this review in the context of brain andbehaviour.

Interaction with the autonomous nervous system

As part of the peripheral nervous system, the autonomousnervous system is functionally subdivided into the enteric,sympathetic and parasympathetic nervous system. Since allof these systems are also involved in regulation of gastro-intestinal function, they provide the easiest target for aninteraction of the microbiota with nervous tissue. Of thesethe vagus nerve further offers a direct link between the largeintestine and the brain that makes it an interesting candidatefor the study of the gut–brain axis.

As such, several of the studies investigating behaviouraland neurophysiological changes investigated the contribu-tion of the vagus nerve. Indeed, vagotomy abolished someof the effects found in studies on mice fed with probiotics orpathogens (Bercik et al. 2011b; Bravo et al. 2011, Table 1).Other experiments, however, suggested that at least someof the observed effects are functionally independent of thevagus or other autonomous pathways (Bercik et al. 2011a).Together, these findings indicate that the vagus nerve isan important, though apparently not the only, mediator ofmicrobiota-gut–brain interaction and may depend on thebacterial strain used. The exact modalities of how the vagusinteracts with the microbiota to induce such effects remainunclear. As such, increased or decreased activation of vagalpathways, for examples as a result of altered gut motility orneuroactive signals secreted by bacteria (see below), maylead to the observed effects. It will be interesting to seewhether this altered vagal activation leads to sustainableepigenetic modifications in the respective cranial nucleusin the brain stem (dorsal nucleus of vagus nerve), which isfurther connected to the hypothalamus and the solitary tract.

Immunomodulation

The early colonization of the body with diverse micro-organisms offers a plethora of antigens, which are criticalfor appropriate maturation of the immune system (Cahenzliet al. 2013; Hooper et al. 2012). Consequently, GF animalsexhibit severely immature immune function (Cebra 1999).Early-life establishment of the acquired immune systemis heavily influenced by the presence of the microbiotaand it is well-documented that its establishment andmaintenance depend on epigenetic modifications that governthe expression of immune-related genes and transcriptionalprofiles of immune cells such as T cells (Stender & Glass2013; Weng et al. 2012). Interestingly, gut microbiota havevery recently been shown to modulate homeostasis andinflammatory response of the intestinal epithelium in anHDAC3-dependent manner (Alenghat et al. 2013), therebyestablishing a direct connection between microbiota andepigenetic gene regulation. Increasing evidence shows a

significant contribution of immune signalling in normal brainfunction as well as during ageing and in the context ofneurodegenerative diseases (Collins et al. 2012a; Czirr &Wyss-Coray 2012; Lampron et al. 2013; Rostene et al. 2007;Soliman et al. 2013; Villeda et al. 2011). However, we are onlybeginning to fully understand this widespread interaction ofthe immune system with the brain.

One other mechanism for inducing immunomodulatoryeffects in disorders of the brain gut axis is in the contextof a ‘leaky gut’ hypothesis. Indeed, chronic stress has beenshown to disrupt the intestinal barrier, making it leaky andincreasing the permeability to ions and bacterial peptides(Santos et al. 2001; Soderholm & Perdue 2001). Theseeffects can be reversed by probiotic agents (Ait-Belgnaouiet al. 2012; Zareie et al. 2006). In line with these find-ings, human studies indirectly suggest increased bacterialtranslocation in stress-related psychiatric disorders such asdepression (Maes et al. 2012).

Irritable bowel syndrome and visceral pain

Visceral hypersensitivity is a hallmark of pathologies ofthe gut–brain axis such as irritable bowel syndrome (IBS).The biological basis of IBS is poorly understood thoughthere is evidence for a contribution of genetic risk factorsas well as environmental stimuli such as early-life stress(Fukudo & Kanazawa 2011; Mayer et al. 2001). Moreover,we suggested that epigenetic mechanisms may be keyto the manifestation of IBS (Dinan et al. 2010). In linewith this Greenwood Van-Meerveld and colleagues lookedat how epigenetic modifications in the brain may affectvisceral sensitivity in rats. The authors locally injected anHDAC inhibitor Trichostatin A (TSA) into the brain after awater-avoidance stress and found an amelioration of thestress-induced increase in visceral pain sensitivity (Tran et al.2013). Though by itself this study does not rule out a CNS-based mechanism of differential pain processing in thestressed rats, it hints to a potentially beneficial effect ofHDAC inhibition in the treatment of stress-induced bowelsymptoms like visceral pain. Microbiota have been shownto be altered in IBS (Jeffery et al. 2012; Shankar et al.2013) and probiotic-based therapies have been shown toreverse visceral hypersensitivity in animal models (McKernanet al. 2010) and in human trials (Clarke et al. 2012).Future studies are needed to clarify what role microbesand microbial metabolites play in epigenetically modifyingpathways relevant to visceral pain.

Stress and depression

Depression-related behaviours are also altered in micetreated with probiotics or subclinical infection (Desbonnetet al. 2010; Bravo et al. 2011, Table 1). Interestingly,depressive-like symptoms in animal models have beenassociated with epigenetic mechanisms such as alteredHDAC activity. As such, chronic administration of theantidepressant drug imipramine induced HDAC5-mediateddifferential histone acetylation in the hippocampus of miceundergoing chronic defeat stress (Tsankova et al. 2006).HDAC5 activity and epigenetic regulation of gene expressionin the nucleus accumbens were also specifically associated



with chronic emotional stimuli such as cocaine exposureand social defeat stress (Renthal et al. 2007). Furthermore,Berton and colleagues demonstrated antidepressant-likeproperties of HDAC6-selective inhibitors and an HDAC6-dependent regulation of the behavioural stress responsein mice (Espallergues et al. 2012; Jochems et al. 2013).However, it should be noted that in neurons HDAC6 is notlocalized to the nucleus but rather deacetylates cytoplasmicproteins, including alpha-tubulin and HSP90.

Autism and neurodevelopmental disorders

It is increasingly acknowledged that the development ofautism-spectrum disorders (ASDs) is multifactorial withgenetic as well as environmental factors contributing totheir aetiology. The term ASD is used to collectivelydescribe a group of disorders that are characterized byclassical autistic symptoms, such as reduced sociabilityand social recognition. Recent evidence suggests, albeitin relatively small cohorts, that ASDs may be associatedwith alterations in microbiota composition and metabolism(Critchfield et al. 2011; de Theije et al. 2011; Douglas-Escobaret al. 2013; Gondalia et al. 2012; Louis 2012; Macfabe2012; Ming et al. 2012; Mulle et al. 2013). In addition tothese correlative findings in humans, GF mice have recentlybeen shown to have core social deficits and increasedrepetitive behaviours similar to that observed in ASD(Desbonnet et al. 2013). Together, these data suggest thatthe microbiota is a critical determinant for the developmentof social behaviour and the aetiology of ASD. Interestingly,also epigenetic mechanisms have been implicated in theaetiology of ASD, as comprehensively reviewed recently(Grafodatskaya et al. 2010) and it is currently unclear if theseare related to microbiota changes. Moreover, whether otherneurodevelopmental disorders such as schizophrenia areassociated with microbiota changes are not yet investigatedneither in animal models nor human populations.

Mediators of microbe–brain interactions

The gut microbiota helps to break down certain nutrients,which subsequently can be further metabolized by hostcells. Interestingly, several of these products are associatedwith neural function. As such, gut bacteria produce aminoacids, such as GABA and tryptophan, and monoamines, suchas serotonin, histamine and dopamine, that play importantroles in the brain as neurotransmitters or their precursors(Lyte 2011; Lyte & Freestone 2010; Thomas et al. 2012a;Wall et al. 2014). Though they may also target the CNS bytransport through the blood stream, it is likely that theseneuroactive products primarily affect the neurons in theenteric part of the peripheral nervous system.

Short-chain fatty acids

Apart from direct action on neurotransmission, gut-dwellingbacteria generate a number other chemicals that displayneuro-modulatory potential. For example, it was shown thatsome gut-dwelling bacteria produce spermidine (Noack et al.2000), a ubiquitous polyamine that has been shown to havebeneficial effects on ageing (Eisenberg et al. 2009) and age-associated memory impairment (Gupta et al. 2013), whichmay in part be mediated by an alteration in histone acetylation(Das & Kanungo 1979).

Moreover, it is well-known that gut bacteria are the keysource of short-chain fatty acids (SCFAs) such as butyricacid, propionic acid and acetic acid. While these moleculesdo not belong to the classic neuroactive substances theymay act on neuronal function in a more subtle way. The mostwell-known of these is probably butyrate, which acts as apotent inhibitor of HDACs (Candido et al. 1978; Davie 2003).Propionate and other SCFAs, as well as lactate and pyruvate,have HDAC-inhibitory functions as well, but to a much lesserdegree compared to butyrate (Latham et al. 2012; Thangarajuet al. 2006; Waldecker et al. 2008). In a similar IC50-rangeare certain polyphenol metabolites that are produced by gutbacteria (Waldecker et al. 2008, Table 3). Acetate on the

Table 3: In vitro IC50 concentrations for selected substances with HDAC-inhibitory function additional information

HDACi IC50 (mol/l) Source Reference Type

TSA 1.80E−09 Streptomyces platensis Selleck Chemicals AntimycoticSAHA 1.00E−08 Synthetic Selleck Chemicals Hydroxamic acidEtinostat (MS275) 5.00E−07 Synthetic Selleck Chemicals CarbamatePyruvate∗ 8.00E−05 All domains of life Thangaraju et al. (2006) Alpha-keto acidButyrate 9.00E−05 Gut microbiota Waldecker et al. (2008) SCFAp-Coumaric acid 1.90E−04 Gut microbiota Waldecker et al. (2008) PolyphenolPropionate 3.60E−04 Gut microbiota Waldecker et al. (2008) SCFA3-(4-OH-phenyl)-propionate 6.20E−04 Gut microbiota Waldecker et al. (2008) PolyphenolCaffeic acid 8.50E−04 Gut microbiota Waldecker et al. (2008) PolyphenolD-β-Hydroxybutyrate 4.00E−03 Mammalian cells Shimazu et al. (2013) SCFA derivativeD-Lactate 1.00E−02 Diet/dairy, gut microbiota Latham et al. (2012) Organic acidPyruvate∗ 3.00E−02 All domains of life Latham et al. (2012) Alpha-keto acidL-Lactate 4.00E−02 Diet/dairy, muscular tissue, gut microbiota Latham et al. (2012) Organic acid

Additional reference: Shimazu et al. (2013).SAHA, suberanilohydroxamic acid (Vorinostat); TSA, Trichostatin A.∗Pyruvate values greatly differ between cited publications.


Stilling et al.

other hand serves as a substrate for acyl-CoA synthetaseshort-chain family member 2 (ACSS2) in the synthesis ofacetyl-Coenzyme A (acetyl-CoA), the donor of acetyl groupsused for acetylation of histone tail lysine residues by HATs.Acetyl-CoA can also be derived from citrate via the enzymaticactivity of ATP citrate lyase (ACLY).

Thus, both processes, HDAC inhibition and increased avail-ability of HAT substrate may lead to increased histone acety-lation and thereby stimulate stimulus-driven transcription inactive neurons. This has been shown to facilitate long-termmemory consolidation and neuroprotection/-regeneration ina numerous in vitro studies and animal models for learn-ing and memory and neurodegenerative diseases (Fischeret al. 2010; Govindarajan et al. 2011; Graff & Tsai 2013b;Peleg et al. 2010). Though the effect of SCFAs that reachthe CNS may be rather subtle, cumulative chronic delivery ofSCFAs by the gut microbiota may result in long-lasting, stableeffects on gene expression. Indeed, intracerebroventricularadministration of relatively high doses of the SCFA propionicacid results in some autistic-like behaviours in rats (MacFabeet al. 2011; Thomas et al. 2012b).

Direct molecular interactions with the epigenetic

machinery

Several bacteria can influence their host’s transcription bysecreting protein effectors that mimic host-endogenoustranscriptional regulators and alter the epigenetic landscapeof the host cells (Bierne 2013; Bierne & Cossart 2012;Bierne et al. 2012; Eskandarian et al. 2013; Hamon &Cossart 2008; Pennini et al. 2010; Rolando et al. 2013). Alsocertain influenza viruses make use of the hosts epigeneticmachinery to replicate or hide within the hosts genome(Minarovits 2009) and the viral encoded histone-mimickingprotein NS1 has been described to mediate transcriptionalrepression of the host cell’s antiviral response (Marazzi et al.2012). In fact, Bierne and Cossart (2012) have proposedto classify these non-eukaryotic effectors collectively asnucleomodulins. However, such effectors have yet onlybeen shown to exist in intracellular parasites like Legionellapneumophilia that posses certain adapted secretion systemand have a more direct contact to the intracellular milieu tointeract with host signalling cascades. It is unclear whetherbrain-borne pathogens may have similar capabilities to altertranscription in neurons in a parasite-advantageous way,which in turn could have an effect on host behaviour (alsosee below).

While none of these mechanisms are explicitly describedfor gut-dwelling species or for parasites that live in braintissue, it shows the potentially versatile and manifoldways that are open to microbes to interact with the host’sepigenome. Undoubtedly, numerous parasites target for thebrain and have various means to do so (for a review seeKristensson 2011). Just recently a well known bacterium(Staphylococcus aureus) was shown to stimulate nociceptiveneurons directly (Chiu et al. 2013), which encourages us torethink what we know about gut bacteria and whether theymay stimulate neurons of the autonomous nervous systemby similar mechanisms, which could have implications forpathologies associated with visceral pain. Indeed, probiotic

Me

Me

PP

Me

Me

MeMeMe

Ac Ac

Ac

AcC C

Figure 2: Scheme of epigenetic mechanisms in the

microbiota–gut–brain axis. We propose to extend the classicpicture of the microbiota–gut–brain interaction by effects of theintestinal micro-organisms on epigenetic processes in the brainthrough diverse mechanisms such as neuroactive metabolitesecretion, immunomodulation and other yet unexplored neuro-nucleomodulins.

cell-wall components were shown to activate intestinalsensory neurons (Mao et al. 2013) and decreased excitabilitywas demonstrated for myenteric after-hyperpolarizationneurons prepared from GF mice (McVey Neufeld et al. 2013).However, the exact molecular signalling pathways inducingthese neurophysiological alterations remain elusive. In sum-mary, the microbiota has multiple ways of interaction withhost physiology and behaviour, some of which are potentiallymediated by alterations of the epigenetic landscape of dif-ferent host cells and tissues including neurons and glia. Wesuggest that the picture of the microbiota–gut–brain axisshould be extended by these mechanisms (Fig. 2), providedexperimental evidence will support our hypotheses.

The microbiota and gene–environment

interactions

Since epigenetic mechanisms are likely mediators ofgene–environment interactions as well as potential medi-ators for interactions between microbes and host, it isimportant to consider how the microbiota is linked togene–environment interactions. In the traditional view westart to become colonized with bacteria as we are deliv-ered through the birth canal of our mother. However, itis worth noting that there is an increasing body of evi-dence that the sterile-womb paradigm needs a criticalreview (Funkhouser & Bordenstein 2013) and that trans-mission of certain microbes already occurs in utero. Themammalian microbiota then becomes more complex during



Table 4: Environmental factors influencing the microbiotacomposition (Marques et al. 2010)

Factor

Mode of delivery (vaginal or caesarean section)Breast-feedingDietDiseaseStatus of the immune systemAgePharmacological treatments (especially antibiotics)Physical activity

delivery and is further established after birth. Hence, it isnot surprising that the microbiota has immense effects onpre- and postnatal development and that detrimental alter-ations in early-life stages may lead to undesirable pheno-types during adulthood. However, the microbial compo-sition is not fixed once and for all, as it is subject tochange through various environmental factors (see Mar-ques et al. 2010, Table 4). As such, the mode of deliv-ery shapes microbial composition not only in the gut butalso in multiple body surfaces (Dominguez-Bello et al.2010). In fact, the percentage of new-borns delivered byCaesarean section world wide is dramatically increasing[32.8% in the USA in 2010 (http://www.cdc.gov/nchs/fastats/delivery.htm)], which may be causally related to an increasein autoimmune diseases and allergies (Neu & Rushing2011).

In addition, the specific microbiota of an individual dependsalso on its behaviour. Montiel-Castro et al. (2013) haverecently argued that animals, apart from co-evolutionaryadaptation of the species-dependent microbiota, may havebehavioural means of selective colonization. For example,social behaviour in humans and non-human primates, includ-ing kissing, grooming and sexual intercourse may serveto enhance horizontal transfer of microbes (Montiel-Castroet al. 2013).

However, not only environmental factors seem to playa role in the establishment and variation of an individual’smicrobiota. In fact, the microbiota also depends on thespecies of an individual. Though mice harbour similar phyla ofbacteria in their guts, the species distribution is remarkablydifferent in humans and a host-specific microbiota is requiredfor proper immune system maturation (Chung et al. 2012).Moreover, differential microbial compositions have beenfound for closely related species, even when maintained onthe same diet (Brucker & Bordenstein 2012). Hence, alsogenetic factors must be critically determinants of the specificmicrobiota in an individual. Further evidence for this hypoth-esis comes from the fact that the genotype of a differentmouse strains is mainly responsible for variations in their gutmicrobiota (Kovacs et al. 2011) and adoptive transfer of phe-notypic strain differences by microbiota transplantations orinter-strain cross-fostering (Bercik et al. 2011a; Collins et al.2013; Francis et al. 2003). Moreover, ethnic affiliation is cor-related with vaginal and oral microbiota composition (Masonet al. 2013; Ravel et al. 2011). Indeed, monozygotic twins

seem to have a more similar gut microbiota composition thanmarital partners or unrelated persons (Zoetendal et al. 2001).

If genetic differences turn out to be commonly responsiblefor differences in microbiota composition, it would be excit-ing to find out what exactly these differences are. Studies onHydra sp. (a cnidarian) suggest specialized anti-microbial pep-tides may be involved in the process of selective colonization(Franzenburg et al. 2013). In animals with a more sophisti-cated immune system other molecules, especially moleculesthat establish the relationship between ‘self’ and ‘non-self’are likely to be involved. Furthermore, it would be intriguingto see whether there are also differences in the microbiomewithin a given species that can be attributed to host geneticvariation like single-nucleotide polymorphisms (SNPs) orcopy-number variations (CNVs) and thereby contribute togene–environment interactions (G × E, see Box 3).

BOX 3: Gene–environment interactions

(G×E)

The concept of G×E is often used to describe the effectof genetic risk factors on the development of certainpathologies that vary with environmental differences. Ina broader sense, however, G×E can also be used todescribe any effect of genetic variation on a phenotypictrait that is influenced by environmental conditions.G×E manifest when a changing environment leads todifferential gene expression. This is because a geneticvariation may remain undetectable as long as the geneis not expressed or silenced. Therefore, regulation ofgene expression is an important mediator of G×E. Askey regulators of gene expression, epigenetic processesintegrate signalling of environmental cues on the levelof transcriptional and translational regulation and canthereby reveal the effect of polymorphisms by exposingthem to function in a given environment.

If the microbiota composition were influenced by geneticvariation, in fact, interaction of the host genome andits microbiome would be an excellent demonstration ofG × E. To test this, studies, designed to find genome-wide associations between genetic variation and microbiotacomposition would be needed. Following this logic, it maybe possible to define new genetic risk factors with respectto microbial diversity/composition. Thus understanding thetemporal dynamics of microbiota composition in complexpsychiatric diseases, including ASDs, schizophrenia anddepression are needed to give credence to this hypothesis.

The existence of incompatible or adverse genome–microbiome combinations would have significant implica-tions for the success of faecal transplantation treatmentsand screening of donors and recipients of the transplant.Finally, genotype–enterotype (see Box 1) incompatibilitymay lie at the heart of enterotypic variation in humanpopulations – especially with ageing since it is associatedwith substantial reorganizations of the transcriptional profilein virtually all organs and body systems, most prominentlythe immune system.


Stilling et al.

The microbiota as a discrete epigenetic entity

The microbiota has been described as a ‘virtual endocrineorgan’ (Evans et al. 2013). In fact, the microbiota does notonly meet the definition of an endocrine organ but, as outlinedin this review, also features all characteristics of an epigeneticinstrument, independent of acting through other molecularepigenetic mechanisms as suggested in this article. In thefist instance, the critical role of maternal transmission in theestablishment of the offspring’s microbiota argues for a viewof the microbiota as an epigenetic entity (Fig. 3a).

As argued before, microbial composition is subjectto environmental changes and is likely to mediategene–environment interactions. These instances constituteanother commonality between the microbiota and classicepigenetic mechanisms (Fig. 3b). Furthermore, the micro-biota parallels epigenetic determination of gene expressionprograms in its ability to influence developmental regula-tion (Fig. 3c). While molecular epigenetic programs governdevelopmental processes like cellular identity and differen-tiation, the microbiotic influences on development includeimmune system maturation, energy metabolism and organmorphogenesis (reviewed by Sommer & Backhed 2013)

Finally, reversibility demonstrates another shared charac-teristic between classic epigenetic mechanisms and micro-bial colonization (Fig. 3d). As mentioned earlier, severalmolecular and behavioural phenotypes in studies of GF,infection and probiotic-treated animals were reversible byexternal intervention. Reversibility of a physiological param-eter is indicative of an acquired epigenetic contribution inregulation of this parameter. Irreversibility, however, pointsto a hard-wired effect on the parameter that is establishedduring development and is not likely reversed by external(e.g. pharmacological, environmental) intervention.

From this point of view, the concept of a ‘hologenome’(Rosenberg et al. 2007 2009, see Box 1), could be enhancedby the existence of the ‘holo-epigenome’, giving credit tothe fact that the diverse microbial genes act as an additionalinterface for environmental interaction and an dynamic andplastic resource for transgenerational phenotypic regulation,together affecting evolution and development. In fact, theholo-epigenome hypothesis could help to understand someof the fundamental questions in epigenetic and especiallyneuroepigenetic research (Bohacek et al. 2013; Sweatt 2013)regarding the heritability of experience-driven phenotypicchanges. To test whether the brain <−> gut <−> microbebidirectional communications are a part of a ‘soft-inheritance’paradigm (Sweatt 2013), careful experimental design isnecessary, including cross-fostering and in vitro-fertilizationstudies.

Friend or foe – symbionts or parasites?

Because of the long history of co-evolution it can be assumedthat the intimate connection between a host and its micro-biome is likely more extensive than exchanging metabolitesthat coincidently exhibit endocrine or neuroactive function. Infact, this co-evolutionary interdependence of microbes andtheir metazoan hosts poses a major challenge in classification

of a given host–microbe interaction as simply commensal,parasitic or mutualistic. Yet, in the light of finding new probi-otics or microorganisms with respect to psychiatric disease(psychobiotics see Box 1) this is an important question.

While it is debatable whether strict commensalism actuallyexists, in a truly mutualistic relationship, both species arerequired to benefit from each other. The benefits for micro-organisms to associate with a mammalian host are ratherobvious. For example, the gut microbiota benefits fromits host by a constant supply of high-quality nutrients anda relatively high, reaction-facilitating ambient temperature.The advantages of this association for the host, howeverare less clear. While the gut microbiota provides its hostwith additional enzymatic capacities to break down host-indigestible diet, this enhanced capacity may come atthe price of additional, potentially harmful, side productsand metabolites. Since the advantages of the relationshipwith certain micro-organisms may have outweighed thedisadvantages during evolution, negative side effects ofthe association may be easily overlooked or not perceivedas harmful in retrospect. Thus, it remains elusive whetherbehavioural alterations observed with a differential or absentmicrobiota are mere side products or represent anotherbeneficial effect on the side of the microbe. For example, ifwe view the decreased anxiety phenotype of GF mice as the‘standard behaviour’, and we imply that the microbiota aredriven towards supporting their host’s–and therefore theirown–survival, it stands to reason that the microbiota directtheir host to exhibit very specific behaviour. Ultimately wecannot ever fully appreciate whether a given micro-organismis rather symbiotic or parasitic until we know how evolutionand development would look without it.

The curious case of behaviour-manipulating

parasites

A more non-controversial case of rather unidirectionalhost–microbe interaction is presented by parasites thatremarkably manipulate their host in their very own favour. Thescientific literature describes a fascinating variety of micro-and macroparasites that significantly alter host behaviourto complete their sometimes complex life cycles by as yetelusive mechanisms (Berdoy et al. 2000; Cezilly et al. 2010;Libersat et al. 2009; Thomas et al. 2010). In appreciationof this growing field the Journal of Experimental Biologyrecently (January 2013) devoted a special issue to thetopic of ‘neural parasitology’ (Adamo & Webster 2013).One of the best known, and for humans the most relevant,example to date is the protozoan parasite Toxoplasma gondii.Infection with T. gondii is often latent or without serioussymptoms and is estimated to affect 25–30% of the worldpopulation (Flegr 2013). For replication, it is dependent onits terminal hosts, which are felines including house cats. Ithas been shown that rodents infected with T. gondii show aremarkable decrease in avoidance of cat urine odour, whichknown as the ‘fatal attraction phenomenon’ and is interpretedas a host-induced behavioural alteration that increases thechance of successful predation and ingestion by a cat (Berdoyet al. 2000; Vyas et al. 2007; Webster 2001). In humans T.gondii infection can occur by ingestion of uncooked meat



(a)

(b)

(c)

(d)Figure 3: The microbiota – a discrete

epigenetic entity. (a–d) Parallels betweenclassic epigenetic mechanisms and thefunctions and characteristics of the micro-biota argue for appreciating the micro-biota as a discrete epigenetic entity.We thus suggest, that the concept ofthe hologenome (see Box 1), could beenhanced by the existence of the ‘holo-epigenome’.

or direct contact with cat faeces and is associated with anincreased risk for the development of Schizophrenia andaltered risk assessment (Flegr 2013; Webster et al. 2013).Interestingly, T. gondii is an intracellular parasite and is well-known for infecting brain tissue (Berenreiterova et al. 2011).Thereby, it has very intimate contact to intracellular neuronalprocesses and may thus achieve host behaviour alteration bymanipulating the host-cell’s transcriptional machinery withthe use of neuro-nucleomodulins (see Box 1) in similar waysas discussed in this review.

Conclusions

In this review, we present a potential mechanism forhost–microbe interaction through means of interacting withmolecular epigenetic processes and provide multiple linesof evidence that alterations in microbiota and epigeneticmodifications are at least correlated in mediating effects of

the microbiota–gut–brain axis. We further suggest that somegut-microbial products can act as ‘neuro-nucleomodulins’ andthereby affect the epigenetic landscape of their host’s braincells which in turn has effects on host behaviour. Sucheffectors include regulators of enzymatic activity of histone-modifying enzymes by means of metabolic alterations andinteractions between bacterial-secreted molecules (such asbutyrate) and signalling pathways of the host’s neurons thatleads to a differential epigenetic landscape. However, atthe moment, it is unclear if any of the phenotypes thathave emerged in GF mice is due to absence of butyrateand/or other metabolites with epigenetic modifying potential.However, some parasites evidently highjack the host-cell’sepigenetic machinery and it might be worthwhile looking forsimilar effectors in symbiotic or commensal bacteria. Furtherinvestigation in this direction is needed to elucidate potentialsites for external intervention in experimental settings and inthe pursuit of more translatable applications.


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Moreover, we argue that the microbiota is an impor-tant mediator of gene–environment interaction and pro-pose the existence of gene–microbiota interactions asa special case of G×E. These interactions may lead togenotype–enterotype incompatibilities, which may have crit-ical implications for disease-associated genetic risk factorswith unidentified function and may limit the success of fae-cal transplantation as a tool for systemic treatment in somecases. Borody and colleagues suggested faecal transplantsmay be extended from the treatment of Clostridium dif-ficile infections to benefit patients suffering from multipleother pathological conditions, including neurodegenerativediseases such as Parkinson’s disease (Borody et al. 2000;Smits et al. 2013). In fact, the observed effects may beattributed to epigenetic gene regulation due to alteredmicrobiota composition since drugs targeting the epigeneticmachinery are currently investigated for such diseases.

Finally, we reason that the microbiota may even be viewedas an epigenetic entity itself as it exhibits similar featuresin its interaction with the host as compared to classicalepigenetic mechanisms such as histone modifications, DNAmethylation and ncRNA-mediated regulation. Thus, the fieldsof (neuro)epigenetics and microbiology have the potential toconverge at many levels and more interdisciplinary studiesare necessary to unravel the full range of this interaction.

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Acknowledgments

The authors are supported, in part, by Science Foundation Irelandin the form of a centre grant (Alimentary Pharmabiotic Centre)under Grant Number SFI/12/RC/2273 and by the Health ResearchBoard of Ireland. T.G.D. has until recently been on the Board ofAlimentary Health and both T.G.D. and J.F.C. have been on theSpeakers Bureau for Mead Johnson.


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