GBB 12109

Genes, Brain and Behavior (2014) 13: 6986 doi: 10.1111/gbb.
12109
Review
Microbial genes, brain & behaviour epigenetic
regulation of the gutbrain axis
R. M. Stilling
, T. G. Dinan
,
and J. F. Cryan
,,
Alimentary Pharmabiotic Center,

Department of Psychiatry,
and

Department of Anatomy and Neuroscience, University
College Cork, Cork, Ireland
*Corresponding author: Prof J. F. Cryan, Department of Anatomy
& Neuroscience, Western Gateway Building, University College
Cork, Cork, Ireland. E-mail: j.cryan@ucc.ie
To date, there is rapidly increasing evidence for
hostmicrobe 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 signicantly contributed to establish-
ing the microbiotagutbrain axis as an extension of the
well-accepted gutbrain 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 hostmicrobe 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 hosts 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 elds
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 modication, 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 prokaryotes
and a plethora of diverse micro-organisms now colonize
virtually all body surfaces of animal hosts, residing as
benecial symbionts, harmless commensals or pathogenic
parasites (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 these
interactions is undergoing a renaissance with large-scale sci-
entic projects like the Human Microbiome Project (HMP;
Human Microbiome Project Consortium 2012; Turnbaugh
et al. 2007) designed to sample, determine and quantify
all 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 the
Eldermet project centering on the elderly (Claesson et al.
2012).
An estimated 90% of cells found in the human body are
not human after all but of mostly prokaryotic origin, derived
fromat least 40000 bacterial strains in 1800 genera (Forsythe
& Kunze 2013; Frank & Pace 2008; Luckey 1972). Though
considerably smaller in size, these approximately 100 trillion
cells add up to a mass of almost 12kg in an adult individual
(Forsythe & Kunze 2013) approximately the weight of
a full-grown human brain (ca. 1.5kg, Parent & Carpenter
1996).
There is a rapidly increasing amount of evidence impli-
cating hostmicrobe interactions at virtually all levels of
complexity, ranging from direct cell-to-cell communication to
extensive systemic signalling, and involving various organs
and organ systems, including the central nervous system
(CNS). The microbiome (see Box 1) critically supports host
metabolism and yields a source of metabolites, many of
which 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 that
the human gut harbours more than 3.3 million non-human
genes (Zhu et al. 2010), making the 23285 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 specic
composition of this microbiota has multiple, critical con-
sequences for host physiological and metabolic processes
ranging from postnatal development and immunomodula-
tion to, perhaps most surprisingly, behaviour and cognition
(Sommer & B ackhed 2013) which forms the basis of this
review.
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-organisms
can be found on all body surfaces (including the lumen of gastro-intestinal organs, which strictly speaking belong to the
outside world) and cavities, such as the skin, nose, ears and genitals. The microbiota is not only limited to the bacterial
domain 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 associated
phages). However, the mammalian non-pathogenic virome and the complexity of phages are largely unexplored and will
not be addressed in this review.
Microbiome: The genome of a given organismis dened as the sumof 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 dened 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 an
organisms hologenome (Brucker & Bordenstein 2013), a term coined by Rosenberg et al. in 2007 (Rosenberg et al. 2007
2009).
Enterotype: The concept of enterotypes was introduced in 2011 to dene inter-individual variation in gut-microbiota
species composition. More specically, three distinct microbiomic clusters were identied that were mainly separated
not only by abundance of certain species but also by abundance of genes with shared molecular function (Arumugam
et al. 2011).
Germ-free (GF): Also referred to as axenic (from the Greek free from foreign[er]), though axenic can have other
connotations in microbiology. GF animals are dened to be free from any microbial colonization and are kept in isolators
under strictly sterile conditions. Also see Box 2.
Gnotobiotic: A system, in which all organisms are either dened by or known to the investigator, is referred to as
gnotobiotic (from the Greek known life). Thus, a gnotobiotic animal is inhabited only by certain micro-organisms. The
status includes the absence of any colonization as in GF animals, since it can also be viewed as a known status. Often
the term is used to describe formerly GF animals that have be colonized by a dened 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 dening
the gnotobiotic status see Box 2.
Probiotic: Probiotics are living organisms that contribute to a host-benecial microbial ora. Thus probiotic organisms can
be viewed as symbionts.
Prebiotic: Meant to contrast the term antibiotic, prebiotics are chemical compounds that inuence the microbial ora in
a host-benecial way.
Psychobiotic: A concept recently introduced by Dinan, Stanton and Cryan denes psychobiotics analogous to
probiotics as live organisms that produce positive effects on mental health (Dinan et al. 2013). It can also be argued
that psychobiotics may exhibit benets 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 denition of probiotics rather describes a benecial microbial status that supports
normal host function, e.g. stress resilience.
Nucleomodulins: Several bacteria can inuence their hosts transcriptome by secreting protein effectors directly targeting
the 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 neuronal
gene expression and alter host behaviour and could hence be termed neuro-nucleomodulins.
The microbiotagutbrain axis
The discovery that differential microbial composition is asso-
ciated with alterations in behaviour and cognition has sig-
nicantly contributed to establish the microbiotagutbrain
axis as an extension of the well-accepted gutbrain axis
concept. This concept is used to describe the bidirectional
communication between the CNS and intestinal organs and
was rst introduced in terms of peripheral regulation of
emotions by William James and Carl Lange in the 1880s
and further challenged and rened by Walter Cannon in the
1920s to account for the primacy of the brain in regulating
gastrointestinal function (see Mayer 2011). However, in
the light of new and intriguing data, mostly resulting
from the study of rodents, the gutbrain axis has been
reviewed from a number of perspectives, focusing on
different aspects ranging from basic microbiology to
translational applications (e.g. see Bercik et al. 2012;
Berer & Krishnamoorthy 2012; Collins et al. 2012b, 2013;
Cryan & Dinan 2012; Cryan & OMahony 2011; Forsythe
& Kunze 2013; Lyte 2011, 2013; Nicholson et al. 2012;
Rhee et al. 2009; Sommer & B ackhed 2013). In this
70 Genes, Brain and Behavior (2014) 13: 6986
Microbial genes, brain & behaviour
review, we want to highlight our current understanding
on the underlying mechanisms of microbiotagutbrain
interactions and associated behavioural alterations with an
emphasis on a potential epigenetic contribution to these
mechanisms.
A new epoch is emerging with these ndings in basic
research and animal studies beeing translated into the clinic.
Indeed, it is becoming clear that certain pathologies, which
are associated with an altered microbiome, are connected
to mood, stress, behaviour and/or cognition (for review
see Grenham et al. 2011; Shanahan 2012). In this regard,
irritable bowel syndrome, which is highly comorbid with
mood disorders such as depression, also leads to decreased
cognitive performance (Berrill et al. 2013; Kennedy et al.
2013). Moreover, an important recent neuroimaging study
validated rodent studies (e.g. Bravo et al. 2011) in implicating
microbe-brain signalling in modulating resting brain activity in
key circuits involved in pain, emotion and cognition (Tillisch
et al. 2013).
Microbiota-associated phenotypes and behavioural
alterations
Tables 1 and 2 summarize the growing body of research
emanating from rodents that demonstrate a role for
microbiota in behaviour. At the centre of many of these
studies are animals that have been raised in a sterile
environment and thus without microbiota, referred to
as GF (see Box 2). Additionally, behavioural studies on
animals with either dened infections, antibiotic treatment
or administration of probiotic bacteria have been carried out
(for a review see Cryan & Dinan 2012; Foster & Neufeld
2013, see Table 1). These studies showed reproducible
and largely consistent effects of the various microbial
states on behaviour in mice. The most commonly reported
phenotype was altered anxiety-related behaviour, which can
be assessed by a variety of tests (Table 1).
There is now an increasing number of studies focusing on
the positive behavioural effects of various bacterial strains,
mostly Bidiobacteria and Lactobacillus species (see Table 1
and Bercik et al. 2010, 2011b) but also transient commensals
such as Mycobacterium vaccae (Matthews & Jenks 2013).
Moreover, an increasing number of studies in animal
models of stress, anxiety and depression also implicate a
role for the microbiota in psychopathology (Bailey & Coe
1999; Bailey et al. 2011; OMahony et al. 2011; Park et al.
2013).
In almost all studies the authors also reported biochemical
and molecular changes (Table 2). In addition to the
common nding that the microbial status is associated
with altered corticosterone levels in the blood plasma
or serum of stressed as well as nave mice, gene
expression changes in different brain regions could be
demonstrated (Table 2). Most commonly Bdnf and Fos
expression levels were analysed as a correlate for differential
neuronal activation. Interestingly, Bdnf is well-known for its
function in neuronal plasticity, learning and memory, and
a number of psychiatric and neurodegenerative diseases
(Cowansage et al. 2010; Tapia-Arancibia et al. 2008; Walker
et al. 2012). In addition, alterations in neurotransmitter
BOX 2: The germ-free animal
GF and other gnotobiotic animals have been generated
since the early 20th century (for a historical introduction
see Reyniers 1959). To achieve a GF status in a mammal,
a new-born needs to be delivered by Caesarean section
and hand-reared with sterilized milk in a sterile, isolated
environment. Further generations can then be derived
by Caesarean section exclusively, since rearing can be
arranged by an already GF foster mother. Future colonies
of GF animals can be maintained frominterbreeding with
each other within a suitable Germ-Free Unit. However,
it is important to bear in mind that increasing evidence
suggests that Caesarean-derived newborns are, against
longstanding dogma, not sterile (for a recent review
see Funkhouser & Bordenstein 2013). Though several
studies on GF animals monitored contamination using
culture methods, these methods are heavily biassed
and bacteria, fungi, protozoans and viruses that are not
easily cultureable will not be detected. The only way of
denitely determining the microbial status of GF animals
would be using deep sequencing. Targeted amplication
of 16s rDNA by polymerase chain reaction would only be
useful to determine the status of bacterial colonization,
and would not give information about fungi or viruses.
Already in 1970 the rst explicit behavioural observa-
tions have been made on gnotobiotic piglets (B ahr 1970).
Notably, it took another 34 years until the rst studies on
GF mice were published, that showed alterations in brain
function (Sudo et al. 2004). Since 2011 a number of stud-
ies demonstrated behavioural alterations in GF mice and
thereby signicantly extended the microbiotagutbrain
axis concept. Thus, this almost century-old area
of research has still not lost its importance.
signalling, including neurotransmitters and associated
metabolites and neurotransmitter receptors have been
described. Diaz Heijtz et al. (2011) took a genome-wide
approach to dene the transcriptional prole of the GF
mouse in ve different brain regions. Further analysis showed
that genes associated with the functional categories citrate
cycle, synaptic long-term potentiation, steroid hormone
metabolism and cyclic adenosine 5
-phosphate (adenosine
monophosphate)-mediated signalling were enriched among
the differentially regulated genes, which supports the phe-
notypic observations. Interestingly, while in the cerebellum
and hippocampus robust changes in gene expression were
found, the hypothalamus, the brain region involved in the
stress-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 tryptophan
metabolism) could be reversed by recolonization with a
conventional microbiota or probiotic treatment, others were
unaffected by restoration of a normal microbiota (including
5-HT concentration and social cognition; see Tables 1 and 2
for details). Indeed, reversibility of the anxiolytic phenotype in
GF mice is only guaranteed if recolonization happens during
Genes, Brain and Behavior (2014) 13: 6986 71
Stilling et al.
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a critical time window during adolescence (Clarke et al. 2013;
Foster & Neufeld 2013). However, since this hypothesis has
not been tested systematically, it is unclear if this observation
can also be generalized to other GF-associated phenotypes.
Overall, accumulating evidence suggests that there is a
correlation between the microbiota composition and brain
and behaviour. While intestinal probiosis is associated with
increased stress resilience and decreased basal anxiety,
GF animals show increased basal corticosterone levels
along with an increased stress-response, hyperlocomotion
and decreased brain-derived neurotrophic factor (BDNF)
levels but also reduced anxiety and social activity. It may
appear as a contradiction that the endocrinological stress
parameters are elevated while anxiety on the behavioural
level is reduced. However, these parameters do not
necessarily correlate (Neufeld et al. 2011) and may be
differentially inuenced by the microbiota. Dysbiosis of
the enteric milieu on the other hand is marked by an
increase in anxiety, depressive-like behaviour and memory
impairment along with decreased concentrations of key
neurotrophic factors involved in plasticity such as BDNF
(Fig. 1). However, the underlying molecular mechanisms
leading to these behavioural and biochemical alterations are
not well understood. Interestingly, there is now a growing
appreciation of the role of epigenetic mechanisms in shaping
brain and behaviour.
Epigenetic mechanisms and neuroepigenetics
Epigenetics is one of the most overused words in the
current scientic vocabulary (Ledford 2008). Initially, it was
used to describe developmental programming but was later
redened more specically to refer to heritable changes
in gene expression that do not originate from mutations
of the DNA sequence (Holliday 1987; Waddington 1953).
The term is now commonly used in a broader sense,
although it is associated with different connotations within
special scientic elds. While some disciplines focus on the
aspect of sequence-independent transgenerational germ-
line inheritance of a phenotypic trait, others, especially
in the elds of neuroscience or biological psychiatry,
emphasize early-life experiences that inuence development
and behaviour during later life and adulthood. However,
in a more cellular view, transgenerational epigenetic traits
are interpreted in the context of somatic mitotic cellular
differentiation within a multicellular organism. In molecular
neuroscience, most often the term epigenetics is rather
used to take into account the multiple molecular events
that are involved in the dynamic regulation of neuronal gene
expression.
Irrespective of the interpretation one may adopt, the molec-
ular machinery mediating these seemingly different effects
are indistinguishable in all interpretations of epigenetics. In
general, this molecular machinery comprises plastic changes
in regulation of nuclear architecture, chromatin structure and
remodelling and gene expression. This epigenetic machinery
includes post-translational modications of histone proteins
that serve at least two, non-mutually exclusive functions:
regulatory factor recruitment and histoneDNA interaction.
Acetylation of a lysine residue in the N-terminal tail region of a
histone reduces the electrostatic interaction of the positively
charged lysine with the negatively charged DNA, thereby
making chromatin more accessible (Korolev et al. 2007).
At the same time this modication serves as a recognition
signal for bromodomain-containing proteins, which in turn
recruit factors of the transcriptional machinery to the now
accessible genomic site (Chen et al. 2010; Hargreaves et al.
2009; LeRoy et al. 2008). Apart from acetylation, an increas-
ing variety of post-translational modications of histone
proteins are known, including phosphorylation, methylation
(three possible methylation states on lysine and arginine
residues), and ubiquitylation. In addition, the DNA itself can
be modied by cytosine methylation, which is generally
associated with silencing of a genomic element (Jaenisch &
Bird 2003). More recently also the different products during
active demethylation of the DNA, especially TET1-mediated
cytosine hydroxymethylation, gained interest as they seem
to have regulatory function that differs from the simple
absence of methylation (Kaas et al. 2013; Mell en et al. 2012
p. 2; Rudenko et al. 2013; Szulwach et al. 2011). Finally, a
steadily increasing number of non-coding RNAs, which are
often transcribed from DNA sites previously thought to be
transcriptionally inactive (junk DNA), are assigned as part of
the epigenetic machinery and can act on transcriptional as
well as translational activity (for some recent reviews on the
diverse functions and biology of the increasingly complex
RNA landscape see, e.g. Landry et al. 2013; Ng et al. 2013;
OConnor et al. 2012; rom & Shiekhattar 2013; Wang et al.
2012 and references therein).
Interestingly, these processes have recently been identi-
ed to play an important role in cognitive processes during
health and disease by regulating gene expression in the
brain (for recent review see Day & Sweatt 2011a,2011b;
Gr aff & Tsai 2013a; Kosik et al. 2012; OConnor et al. 2012).
A connection between the two mechanisms of environ-
mental information processing and epigenetics has been
established, often referred to as neuroepigenetics (Day &
Sweatt 2010; Sweatt 2013). Concomitantly, the term chro-
matin plasticity is used to acknowledge the fact that the
neuronal nucleus is no exception to the general rule that
virtually all components in the brain are able to undergo
plastic changes (Dulac 2010). This rather new discipline tries
to unravel the dynamic changes of transcriptional regula-
tion in neurons upon stimulation or in disease. For example,
dynamic regulation of gene expression in the hippocampus
is critical for long-term memory consolidation and synaptic
plasticity (Da Silva et al. 2008; Igaz et al. 2002, 2004) and
a specic role for all three mechanisms of the epigenetic
machinery has been demonstrated. Of these, histone acety-
lation is probably the best-understood, since the catalysing
enzymes (namely histone acetyltransferases, HATs and his-
tone deacetylases, HDACs) are well-studied and can be
targeted pharmacologically, e.g. by using HDAC inhibitors,
which are currently discussed as cognitive enhancers (Gr aff
& Tsai 2013a,2013b). Because of its activatory role, his-
tone acetylation plays a permissive role in learning-induced
transcription and the use of small-molecule HDAC inhibitors
has been shown to augment memory consolidation and to
Figure 1: The microbiota affects neurophysiological, biochemical and behavioural parameters. Accumulating evidence suggests
that there is a correlation between the microbiota composition and brain and behaviour. Host-supporting species (probiosis) alter
behaviour and biochemical parameters of the host in a different direction as compared to pathogenic species (dysbiosis). The phenotype
of GF animals seems to represent a rather intermediate state, featuring characteristics of both conditions.
parallel the benecial effects of environmental enrichment
on 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 models
of neurodegenerative diseases such as Alzheimers disease
(for review see Gr aff & Tsai 2013b; Stilling & Fischer 2011).
The presumed mechanism of action for elevated histone
acetylation to aid in procognitive processes is based on the
idea that these molecules do not simply alter acetylation
levels at random genomic loci but rather support a given
cells own regulatory program or facilitate it in cases where
it has gone out of balance (McQuown & Wood 2011; Peleg
et al. 2010; Stilling & Fischer 2011). The role of epigenet-
ics in informing hostmicrobe interactions has received little
attention to date. In the following sections we will elaborate
on potential convergences between epigenetic mechanisms
and host-microbiota dialogue.
Microbebrain interfaces: potential sites
for epigenetic regulation
Since microbes colonizing the host body are usually housed
on the outer body surfaces, including the skin, mouth, lungs,
gastrointestinal tract and vaginal mucosa, a large part of the
interaction between the microbiota and their host will be
mediated by host cells, which the microbes are in direct
contact with. These are mainly epithelial cells but also cells of
the immune system as well as peripheral neurons (Forsythe
& Kunze 2013). However, several mostly parasiticbacterial
species are capable of invading host tissues and can even
live in intracellular vacuoles to manipulate their host cells
directly (Li evin-Le Moal & Servin 2013 and see below).
In addition, host dendritic cells are able to engulf living
bacteria from the gut lumen and transport them through the
body, though it is unclear which of the many organ barriers
dendritic cells may cross and whether bacterial cells are later
released or digested (Rescigno et al. 2001). Furthermore,
surface-dwelling microbiota have means of signalling across
the epithelial border by secretion of bioactive molecules
that penetrate the outer barriers and are carried to distant
effector organs, including the brain, through the blood
stream and the lymphatic system (Rhee et al. 2009).
The diverse possible routes for microbiotagutbrain
signalling and diet-inuenced gutbrain communication have
previously 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 only
be discussed with regard to potential sites of epigenetic
regulation. It should be noted that to date there is a
paucity of direct evidence for the role of epigenetics in
shaping hostmicrobiota interactions. Yet, there are plenty
Stilling et al.
of indications as to how potential epigenetic mechanisms
can modulate the biological interaction between hosts and
micro-organisms. Interestingly, epigenetic mechanisms have
been recently been put forward as a central mechanism
driving hostpathogen interactions (G omez-Daz et al. 2012).
Expanding this to non-pathogenic benecial microbes is an
important goal of this review in the context of brain and
behaviour.
Interaction with the autonomous nervous system
As part of the peripheral nervous system, the autonomous
nervous system is functionally subdivided into the enteric,
sympathetic and parasympathetic nervous system. Since all
of these systems are also involved in regulation of gastro-
intestinal function, they provide the easiest target for an
interaction of the microbiota with nervous tissue. Of these
the vagus nerve further offers a direct link between the large
intestine and the brain that makes it an interesting candidate
for the study of the gutbrain axis.
As such, several of the studies investigating behavioural
and neurophysiological changes investigated the contribu-
tion of the vagus nerve. Indeed, vagotomy abolished some
of the effects found in studies on mice fed with probiotics or
pathogens (Bercik et al. 2011b; Bravo et al. 2011, Table 1).
Other experiments, however, suggested that at least some
of the observed effects are functionally independent of the
vagus or other autonomous pathways (Bercik et al. 2011a).
Together, these ndings indicate that the vagus nerve is
an important, though apparently not the only, mediator of
microbiota-gutbrain interaction and may depend on the
bacterial strain used. The exact modalities of how the vagus
interacts with the microbiota to induce such effects remain
unclear. As such, increased or decreased activation of vagal
pathways, for examples as a result of altered gut motility or
neuroactive signals secreted by bacteria (see below), may
lead to the observed effects. It will be interesting to see
whether this altered vagal activation leads to sustainable
epigenetic modications in the respective cranial nucleus
in the brain stem (dorsal nucleus of vagus nerve), which is
further 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 critical
for appropriate maturation of the immune system (Cahenzli
et al. 2013; Hooper et al. 2012). Consequently, GF animals
exhibit severely immature immune function (Cebra 1999).
Early-life establishment of the acquired immune system
is heavily inuenced by the presence of the microbiota
and it is well-documented that its establishment and
maintenance depend on epigenetic modications that govern
the expression of immune-related genes and transcriptional
proles of immune cells such as T cells (Stender & Glass
2013; Weng et al. 2012). Interestingly, gut microbiota have
very recently been shown to modulate homeostasis and
inammatory response of the intestinal epithelium in an
HDAC3-dependent manner (Alenghat et al. 2013), thereby
establishing a direct connection between microbiota and
epigenetic gene regulation. Increasing evidence shows a
signicant contribution of immune signalling in normal brain
function as well as during ageing and in the context of
neurodegenerative diseases (Collins et al. 2012a; Czirr &
Wyss-Coray 2012; Lampron et al. 2013; Rost ` ene et al. 2007;
Soliman et al. 2013; Villeda et al. 2011). However, we are only
beginning to fully understand this widespread interaction of
the immune system with the brain.
One other mechanism for inducing immunomodulatory
effects in disorders of the brain gut axis is in the context
of a leaky gut hypothesis. Indeed, chronic stress has been
shown to disrupt the intestinal barrier, making it leaky and
increasing the permeability to ions and bacterial peptides
(Santos et al. 2001; S oderholm & Perdue 2001). These
effects can be reversed by probiotic agents (Ait-Belgnaoui
et al. 2012; Zareie et al. 2006). In line with these nd-
ings, human studies indirectly suggest increased bacterial
translocation in stress-related psychiatric disorders such as
depression (Maes et al. 2012).
Irritable bowel syndrome and visceral pain
Visceral hypersensitivity is a hallmark of pathologies of
the gutbrain axis such as irritable bowel syndrome (IBS).
The biological basis of IBS is poorly understood though
there is evidence for a contribution of genetic risk factors
as well as environmental stimuli such as early-life stress
(Fukudo & Kanazawa 2011; Mayer et al. 2001). Moreover,
we suggested that epigenetic mechanisms may be key
to the manifestation of IBS (Dinan et al. 2010). In line
with this Greenwood Van-Meerveld and colleagues looked
at how epigenetic modications in the brain may affect
visceral sensitivity in rats. The authors locally injected an
HDAC inhibitor Trichostatin A (TSA) into the brain after a
water-avoidance stress and found an amelioration of the
stress-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 the
stressed rats, it hints to a potentially benecial effect of
HDAC inhibition in the treatment of stress-induced bowel
symptoms like visceral pain. Microbiota have been shown
to be altered in IBS (Jeffery et al. 2012; Shankar et al.
2013) and probiotic-based therapies have been shown to
reverse visceral hypersensitivity in animal models (McKernan
et al. 2010) and in human trials (Clarke et al. 2012).
Future studies are needed to clarify what role microbes
and microbial metabolites play in epigenetically modifying
pathways relevant to visceral pain.
Stress and depression
Depression-related behaviours are also altered in mice
treated with probiotics or subclinical infection (Desbonnet
et al. 2010; Bravo et al. 2011, Table 1). Interestingly,
depressive-like symptoms in animal models have been
associated with epigenetic mechanisms such as altered
HDAC activity. As such, chronic administration of the
antidepressant drug imipramine induced HDAC5-mediated
differential histone acetylation in the hippocampus of mice
undergoing chronic defeat stress (Tsankova et al. 2006).
HDAC5 activity and epigenetic regulation of gene expression
in the nucleus accumbens were also specically associated
with chronic emotional stimuli such as cocaine exposure
and social defeat stress (Renthal et al. 2007). Furthermore,
Berton and colleagues demonstrated antidepressant-like
properties of HDAC6-selective inhibitors and an HDAC6-
dependent regulation of the behavioural stress response
in mice (Espallergues et al. 2012; Jochems et al. 2013).
However, it should be noted that in neurons HDAC6 is not
localized to the nucleus but rather deacetylates cytoplasmic
proteins, including alpha-tubulin and HSP90.
Autism and neurodevelopmental disorders
It is increasingly acknowledged that the development of
autism-spectrum disorders (ASDs) is multifactorial with
genetic as well as environmental factors contributing to
their aetiology. The term ASD is used to collectively
describe a group of disorders that are characterized by
classical autistic symptoms, such as reduced sociability
and social recognition. Recent evidence suggests, albeit
in relatively small cohorts, that ASDs may be associated
with alterations in microbiota composition and metabolism
(Critcheld et al. 2011; de Theije et al. 2011; Douglas-Escobar
et al. 2013; Gondalia et al. 2012; Louis 2012; Macfabe
2012; Ming et al. 2012; Mulle et al. 2013). In addition to
these correlative ndings in humans, GF mice have recently
been shown to have core social decits and increased
repetitive behaviours similar to that observed in ASD
(Desbonnet et al. 2013). Together, these data suggest that
the microbiota is a critical determinant for the development
of social behaviour and the aetiology of ASD. Interestingly,
also epigenetic mechanisms have been implicated in the
aetiology of ASD, as comprehensively reviewed recently
(Grafodatskaya et al. 2010) and it is currently unclear if these
are related to microbiota changes. Moreover, whether other
neurodevelopmental disorders such as schizophrenia are
associated with microbiota changes are not yet investigated
neither in animal models nor human populations.
Mediators of microbebrain interactions
The gut microbiota helps to break down certain nutrients,
which subsequently can be further metabolized by host
cells. Interestingly, several of these products are associated
with neural function. As such, gut bacteria produce amino
acids, such as GABA and tryptophan, and monoamines, such
as serotonin, histamine and dopamine, that play important
roles 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 by
transport through the blood stream, it is likely that these
neuroactive products primarily affect the neurons in the
enteric part of the peripheral nervous system.
Short-chain fatty acids
Apart from direct action on neurotransmission, gut-dwelling
bacteria generate a number other chemicals that display
neuro-modulatory potential. For example, it was shown that
some gut-dwelling bacteria produce spermidine (Noack et al.
2000), a ubiquitous polyamine that has been shown to have
benecial effects on ageing (Eisenberg et al. 2009) and age-
associated memory impairment (Gupta et al. 2013), which
may in part be mediated by an alteration in histone acetylation
(Das & Kanungo 1979).
Moreover, it is well-known that gut bacteria are the key
source of short-chain fatty acids (SCFAs) such as butyric
acid, propionic acid and acetic acid. While these molecules
do not belong to the classic neuroactive substances they
may act on neuronal function in a more subtle way. The most
well-known of these is probably butyrate, which acts as a
potent 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 lesser
degree compared to butyrate (Lathamet al. 2012; Thangaraju
et al. 2006; Waldecker et al. 2008). In a similar IC
50
-range
are certain polyphenol metabolites that are produced by gut
bacteria (Waldecker et al. 2008, Table 3). Acetate on the
Table 3: In vitro IC
50
concentrations for selected substances with HDAC-inhibitory function additional information
HDACi IC
50
(mol/l) Source Reference Type
TSA 1.80E09 Streptomyces platensis Selleck Chemicals Antimycotic
SAHA 1.00E08 Synthetic Selleck Chemicals Hydroxamic acid
Etinostat (MS275) 5.00E07 Synthetic Selleck Chemicals Carbamate
Pyruvate
8.00E05 All domains of life Thangaraju et al. (2006) Alpha-keto acid

Butyrate 9.00E05 Gut microbiota Waldecker et al. (2008) SCFA
p-Coumaric acid 1.90E04 Gut microbiota Waldecker et al. (2008) Polyphenol
Propionate 3.60E04 Gut microbiota Waldecker et al. (2008) SCFA
3-(4-OH-phenyl)-propionate 6.20E04 Gut microbiota Waldecker et al. (2008) Polyphenol
Caffeic acid 8.50E04 Gut microbiota Waldecker et al. (2008) Polyphenol
D--Hydroxybutyrate 4.00E03 Mammalian cells Shimazu et al. (2013) SCFA derivative
D-Lactate 1.00E02 Diet/dairy, gut microbiota Latham et al. (2012) Organic acid
Pyruvate
3.00E02 All domains of life Latham et al. (2012) Alpha-keto acid

L-Lactate 4.00E02 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 synthetase
short-chain family member 2 (ACSS2) in the synthesis of
acetyl-Coenzyme A (acetyl-CoA), the donor of acetyl groups
used for acetylation of histone tail lysine residues by HATs.
Acetyl-CoA can also be derived fromcitrate via the enzymatic
activity 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 in
active neurons. This has been shown to facilitate long-term
memory consolidation and neuroprotection/-regeneration in
a numerous in vitro studies and animal models for learn-
ing and memory and neurodegenerative diseases (Fischer
et al. 2010; Govindarajan et al. 2011; Gr aff & Tsai 2013b;
Peleg et al. 2010). Though the effect of SCFAs that reach
the CNS may be rather subtle, cumulative chronic delivery of
SCFAs by the gut microbiota may result in long-lasting, stable
effects on gene expression. Indeed, intracerebroventricular
administration of relatively high doses of the SCFA propionic
acid results in some autistic-like behaviours in rats (MacFabe
et al. 2011; Thomas et al. 2012b).
Direct molecular interactions with the epigenetic
machinery
Several bacteria can inuence their hosts transcription by
secreting protein effectors that mimic host-endogenous
transcriptional regulators and alter the epigenetic landscape
of 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). Also
certain inuenza viruses make use of the hosts epigenetic
machinery to replicate or hide within the hosts genome
(Min arovits 2009) and the viral encoded histone-mimicking
protein NS1 has been described to mediate transcriptional
repression of the host cells antiviral response (Marazzi et al.
2012). In fact, Bierne and Cossart (2012) have proposed
to classify these non-eukaryotic effectors collectively as
nucleomodulins. However, such effectors have yet only
been shown to exist in intracellular parasites like Legionella
pneumophilia that posses certain adapted secretion system
and have a more direct contact to the intracellular milieu to
interact with host signalling cascades. It is unclear whether
brain-borne pathogens may have similar capabilities to alter
transcription in neurons in a parasite-advantageous way,
which in turn could have an effect on host behaviour (also
see below).
While none of these mechanisms are explicitly described
for gut-dwelling species or for parasites that live in brain
tissue, it shows the potentially versatile and manifold
ways that are open to microbes to interact with the hosts
epigenome. Undoubtedly, numerous parasites target for the
brain and have various means to do so (for a review see
Kristensson 2011). Just recently a well known bacterium
(Staphylococcus aureus) was shown to stimulate nociceptive
neurons directly (Chiu et al. 2013), which encourages us to
rethink what we know about gut bacteria and whether they
may stimulate neurons of the autonomous nervous system
by similar mechanisms, which could have implications for
pathologies associated with visceral pain. Indeed, probiotic
Me
Me
P
P
Me
Me
MeMe
Me
Ac
Ac
Ac
Ac
C C
Figure 2: Scheme of epigenetic mechanisms in the
microbiotagutbrain axis. We propose to extend the classic
picture of the microbiotagutbrain interaction by effects of the
intestinal micro-organisms on epigenetic processes in the brain
through diverse mechanisms such as neuroactive metabolite
secretion, immunomodulation and other yet unexplored neuro-
nucleomodulins.
cell-wall components were shown to activate intestinal
sensory neurons (Mao et al. 2013) and decreased excitability
was demonstrated for myenteric after-hyperpolarization
neurons prepared from GF mice (McVey Neufeld et al. 2013).
However, the exact molecular signalling pathways inducing
these neurophysiological alterations remain elusive. In sum-
mary, the microbiota has multiple ways of interaction with
host physiology and behaviour, some of which are potentially
mediated by alterations of the epigenetic landscape of dif-
ferent host cells and tissues including neurons and glia. We
suggest that the picture of the microbiotagutbrain axis
should be extended by these mechanisms (Fig. 2), provided
experimental evidence will support our hypotheses.
The microbiota and geneenvironment
interactions
Since epigenetic mechanisms are likely mediators of
geneenvironment interactions as well as potential medi-
ators for interactions between microbes and host, it is
important to consider how the microbiota is linked to
geneenvironment interactions. In the traditional view we
start to become colonized with bacteria as we are deliv-
ered through the birth canal of our mother. However, it
is worth noting that there is an increasing body of evi-
dence that the sterile-womb paradigm needs a critical
review (Funkhouser & Bordenstein 2013) and that trans-
mission of certain microbes already occurs in utero. The
mammalian microbiota then becomes more complex during
Table 4: Environmental factors inuencing the microbiota
composition (Marques et al. 2010)
Factor
Mode of delivery (vaginal or caesarean section)
Breast-feeding
Diet
Disease
Status of the immune system
Age
Pharmacological treatments (especially antibiotics)
Physical activity
delivery and is further established after birth. Hence, it is
not surprising that the microbiota has immense effects on
pre- 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 xed once and for all, as it is subject to
change 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 but
also in multiple body surfaces (Dominguez-Bello et al.
2010). In fact, the percentage of new-borns delivered by
Caesarean section world wide is dramatically increasing
[32.8%in the USAin 2010 (http://www.cdc.gov/nchs/fastats/
delivery.htm)], which may be causally related to an increase
in autoimmune diseases and allergies (Neu & Rushing
2011).
In addition, the specic microbiota of an individual depends
also on its behaviour. Montiel-Castro et al. (2013) have
recently argued that animals, apart from co-evolutionary
adaptation of the species-dependent microbiota, may have
behavioural means of selective colonization. For example,
social behaviour in humans and non-human primates, includ-
ing kissing, grooming and sexual intercourse may serve
to enhance horizontal transfer of microbes (Montiel-Castro
et al. 2013).
However, not only environmental factors seem to play
a role in the establishment and variation of an individuals
microbiota. In fact, the microbiota also depends on the
species of an individual. Though mice harbour similar phyla of
bacteria in their guts, the species distribution is remarkably
different in humans and a host-specic microbiota is required
for proper immune system maturation (Chung et al. 2012).
Moreover, differential microbial compositions have been
found for closely related species, even when maintained on
the same diet (Brucker & Bordenstein 2012). Hence, also
genetic factors must be critically determinants of the specic
microbiota in an individual. Further evidence for this hypoth-
esis comes from the fact that the genotype of a different
mouse strains is mainly responsible for variations in their gut
microbiota (Kovacs et al. 2011) and adoptive transfer of phe-
notypic strain differences by microbiota transplantations or
inter-strain cross-fostering (Bercik et al. 2011a; Collins et al.
2013; Francis et al. 2003). Moreover, ethnic afliation is cor-
related with vaginal and oral microbiota composition (Mason
et al. 2013; Ravel et al. 2011). Indeed, monozygotic twins
seemto have a more similar gut microbiota composition than
marital partners or unrelated persons (Zoetendal et al. 2001).
If genetic differences turn out to be commonly responsible
for differences in microbiota composition, it would be excit-
ing to nd out what exactly these differences are. Studies on
Hydra 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 systemother molecules, especially molecules
that establish the relationship between self and non-self
are likely to be involved. Furthermore, it would be intriguing
to see whether there are also differences in the microbiome
within a given species that can be attributed to host genetic
variation like single-nucleotide polymorphisms (SNPs) or
copy-number variations (CNVs) and thereby contribute to
geneenvironment interactions (GE, see Box 3).
BOX 3: Geneenvironment interactions
(GE)
The concept of GE is often used to describe the effect
of genetic risk factors on the development of certain
pathologies that vary with environmental differences. In
a broader sense, however, GE can also be used to
describe any effect of genetic variation on a phenotypic
trait that is inuenced by environmental conditions.
GE manifest when a changing environment leads to
differential gene expression. This is because a genetic
variation may remain undetectable as long as the gene
is not expressed or silenced. Therefore, regulation of
gene expression is an important mediator of GE. As
key regulators of gene expression, epigenetic processes
integrate signalling of environmental cues on the level
of transcriptional and translational regulation and can
thereby reveal the effect of polymorphisms by exposing
them to function in a given environment.
If the microbiota composition were inuenced by genetic
variation, in fact, interaction of the host genome and
its microbiome would be an excellent demonstration of
GE. To test this, studies, designed to nd genome-
wide associations between genetic variation and microbiota
composition would be needed. Following this logic, it may
be possible to dene new genetic risk factors with respect
to microbial diversity/composition. Thus understanding the
temporal dynamics of microbiota composition in complex
psychiatric diseases, including ASDs, schizophrenia and
depression are needed to give credence to this hypothesis.
The existence of incompatible or adverse genome
microbiome combinations would have signicant implica-
tions for the success of faecal transplantation treatments
and screening of donors and recipients of the transplant.
Finally, genotypeenterotype (see Box 1) incompatibility
may lie at the heart of enterotypic variation in human
populations especially with ageing since it is associated
with substantial reorganizations of the transcriptional prole
in virtually all organs and body systems, most prominently
the immune system.
Stilling et al.
The microbiota as a discrete epigenetic entity
The microbiota has been described as a virtual endocrine
organ (Evans et al. 2013). In fact, the microbiota does not
only meet the denition of an endocrine organ but, as outlined
in this review, also features all characteristics of an epigenetic
instrument, independent of acting through other molecular
epigenetic mechanisms as suggested in this article. In the
st instance, the critical role of maternal transmission in the
establishment of the offsprings microbiota argues for a view
of the microbiota as an epigenetic entity (Fig. 3a).
As argued before, microbial composition is subject
to environmental changes and is likely to mediate
geneenvironment interactions. These instances constitute
another commonality between the microbiota and classic
epigenetic mechanisms (Fig. 3b). Furthermore, the micro-
biota parallels epigenetic determination of gene expression
programs in its ability to inuence developmental regula-
tion (Fig. 3c). While molecular epigenetic programs govern
developmental processes like cellular identity and differen-
tiation, the microbiotic inuences on development include
immune system maturation, energy metabolism and organ
morphogenesis (reviewed by Sommer & B ackhed 2013)
Finally, reversibility demonstrates another shared charac-
teristic between classic epigenetic mechanisms and micro-
bial colonization (Fig. 3d). As mentioned earlier, several
molecular and behavioural phenotypes in studies of GF,
infection and probiotic-treated animals were reversible by
external intervention. Reversibility of a physiological param-
eter is indicative of an acquired epigenetic contribution in
regulation of this parameter. Irreversibility, however, points
to a hard-wired effect on the parameter that is established
during 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 enhanced
by the existence of the holo-epigenome, giving credit to
the fact that the diverse microbial genes act as an additional
interface for environmental interaction and an dynamic and
plastic resource for transgenerational phenotypic regulation,
together affecting evolution and development. In fact, the
holo-epigenome hypothesis could help to understand some
of the fundamental questions in epigenetic and especially
neuroepigenetic research (Bohacek et al. 2013; Sweatt 2013)
regarding the heritability of experience-driven phenotypic
changes. To test whether the brain <>gut <>microbe
bidirectional communications are a part of a soft-inheritance
paradigm (Sweatt 2013), careful experimental design is
necessary, including cross-fostering and in vitro-fertilization
studies.
Friend or foe symbionts or parasites?
Because of the long history of co-evolution it can be assumed
that the intimate connection between a host and its micro-
biome is likely more extensive than exchanging metabolites
that coincidently exhibit endocrine or neuroactive function. In
fact, this co-evolutionary interdependence of microbes and
their metazoan hosts poses a major challenge in classication
of a given hostmicrobe interaction as simply commensal,
parasitic or mutualistic. Yet, in the light of nding 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 commensalismactually
exists, in a truly mutualistic relationship, both species are
required to benet from each other. The benets for micro-
organisms to associate with a mammalian host are rather
obvious. For example, the gut microbiota benets from
its host by a constant supply of high-quality nutrients and
a relatively high, reaction-facilitating ambient temperature.
The advantages of this association for the host, however
are less clear. While the gut microbiota provides its host
with additional enzymatic capacities to break down host-
indigestible diet, this enhanced capacity may come at
the price of additional, potentially harmful, side products
and metabolites. Since the advantages of the relationship
with certain micro-organisms may have outweighed the
disadvantages during evolution, negative side effects of
the association may be easily overlooked or not perceived
as harmful in retrospect. Thus, it remains elusive whether
behavioural alterations observed with a differential or absent
microbiota are mere side products or represent another
benecial effect on the side of the microbe. For example, if
we view the decreased anxiety phenotype of GF mice as the
standard behaviour, and we imply that the microbiota are
driven towards supporting their hostsand therefore their
ownsurvival, it stands to reason that the microbiota direct
their host to exhibit very specic behaviour. Ultimately we
cannot ever fully appreciate whether a given micro-organism
is rather symbiotic or parasitic until we know how evolution
and development would look without it.
The curious case of behaviour-manipulating
parasites
A more non-controversial case of rather unidirectional
hostmicrobe interaction is presented by parasites that
remarkably manipulate their host in their very own favour. The
scientic literature describes a fascinating variety of micro-
and macroparasites that signicantly alter host behaviour
to complete their sometimes complex life cycles by as yet
elusive mechanisms (Berdoy et al. 2000; C ezilly et al. 2010;
Libersat et al. 2009; Thomas et al. 2010). In appreciation
of this growing eld the Journal of Experimental Biology
recently (January 2013) devoted a special issue to the
topic 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 serious
symptoms and is estimated to affect 2530% of the world
population (Flegr 2013). For replication, it is dependent on
its terminal hosts, which are felines including house cats. It
has been shown that rodents infected with T. gondii show a
remarkable decrease in avoidance of cat urine odour, which
known as the fatal attraction phenomenon and is interpreted
as a host-induced behavioural alteration that increases the
chance of successful predation and ingestion by a cat (Berdoy
et 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. (ad) Parallels between
classic epigenetic mechanisms and the
functions and characteristics of the micro-
biota argue for appreciating the micro-
biota as a discrete epigenetic entity.
We thus suggest, that the concept of
the hologenome (see Box 1), could be
enhanced by the existence of the holo-
epigenome.
or direct contact with cat faeces and is associated with an
increased risk for the development of Schizophrenia and
altered risk assessment (Flegr 2013; Webster et al. 2013).
Interestingly, T. gondii is an intracellular parasite and is well-
known for infecting brain tissue (Berenreiterov a et al. 2011).
Thereby, it has very intimate contact to intracellular neuronal
processes and may thus achieve host behaviour alteration by
manipulating the host-cells transcriptional machinery with
the use of neuro-nucleomodulins (see Box 1) in similar ways
as discussed in this review.
Conclusions
In this review, we present a potential mechanism for
hostmicrobe interaction through means of interacting with
molecular epigenetic processes and provide multiple lines
of evidence that alterations in microbiota and epigenetic
modications are at least correlated in mediating effects of
the microbiotagutbrain axis. We further suggest that some
gut-microbial products can act as neuro-nucleomodulins and
thereby affect the epigenetic landscape of their hosts brain
cells which in turn has effects on host behaviour. Such
effectors include regulators of enzymatic activity of histone-
modifying enzymes by means of metabolic alterations and
interactions between bacterial-secreted molecules (such as
butyrate) and signalling pathways of the hosts neurons that
leads to a differential epigenetic landscape. However, at
the moment, it is unclear if any of the phenotypes that
have emerged in GF mice is due to absence of butyrate
and/or other metabolites with epigenetic modifying potential.
However, some parasites evidently highjack the host-cells
epigenetic machinery and it might be worthwhile looking for
similar effectors in symbiotic or commensal bacteria. Further
investigation in this direction is needed to elucidate potential
sites for external intervention in experimental settings and in
the pursuit of more translatable applications.
Stilling et al.
Moreover, we argue that the microbiota is an impor-
tant mediator of geneenvironment interaction and pro-
pose the existence of genemicrobiota interactions as
a special case of GE. These interactions may lead to
genotypeenterotype incompatibilities, which may have crit-
ical implications for disease-associated genetic risk factors
with unidentied function and may limit the success of fae-
cal transplantation as a tool for systemic treatment in some
cases. Borody and colleagues suggested faecal transplants
may be extended from the treatment of Clostridium dif-
cile infections to benet patients suffering from multiple
other pathological conditions, including neurodegenerative
diseases such as Parkinsons disease (Borody et al. 2000;
Smits et al. 2013). In fact, the observed effects may be
attributed to epigenetic gene regulation due to altered
microbiota composition since drugs targeting the epigenetic
machinery are currently investigated for such diseases.
Finally, we reason that the microbiota may even be viewed
as an epigenetic entity itself as it exhibits similar features
in its interaction with the host as compared to classical
epigenetic mechanisms such as histone modications, DNA
methylation and ncRNA-mediated regulation. Thus, the elds
of (neuro)epigenetics and microbiology have the potential to
converge at many levels and more interdisciplinary studies
are necessary to unravel the full range of this interaction.
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Acknowledgments
The authors are supported, in part, by Science Foundation Ireland
in the form of a centre grant (Alimentary Pharmabiotic Centre)
under Grant Number SFI/12/RC/2273 and by the Health Research
Board of Ireland. T.G.D. has until recently been on the Board of
Alimentary Health and both T.G.D. and J.F.C. have been on the
Speakers Bureau for Mead Johnson.

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Genes, Brain and Behavior (2014) 13: 6986 doi: 10.1111/gbb.

Alimentary Pharmabiotic Center,

8.00E05 All domains of life Thangaraju et al. (2006) Alpha-keto acid

3.00E02 All domains of life Latham et al. (2012) Alpha-keto acid

Pyruvate values greatly differ between cited publications.

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