Gastroenterology 2021;160:1486–1501

REVIEWS IN BASIC AND CLINICAL GASTROENTEROLOGY

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AND HEPATOLOGY
Douglas J. Robertson and Vincent W. Yang, Section Editors

The Microbiota-Gut-Brain Axis: From Motility to Mood

Kara G. Margolis,1 John F. Cryan,2 and Emeran A. Mayer3
1
Department of Pediatrics, Morgan Stanley Children’s Hospital, Columbia University Irving Medical Center, New York, New
York; 2Department of Anatomy & Neuroscience, University College Cork, Ireland, APC Microbiome Ireland, University College
Cork, Cork, Ireland; and 3G. Oppenheimer Center for Neurobiology of Stress and Resilience, Vachte and Tamar Manoukian
Division of Digestive Diseases, David Geffen School of Medicine at UCLA, Los Angeles, California

The gut-brain axis plays an important role in maintaining increasing number of central nervous system (CNS) disorders
homeostasis. Many intrinsic and extrinsic factors influence and, as in the case of Parkinson’s disease, GI dysfunction
signaling along this axis, modulating the function of both might occur even before central neurological symptoms
the enteric and central nervous systems. More recently the become evident.3 Similarly, GI symptoms are an important
role of the microbiome as an important factor in modu- component of disorders of brain-gut interactions such as ir-
lating gut-brain signaling has emerged and the concept of a ritable bowel syndrome (IBS), which is commonly associated
microbiota-gut-brain axis has been established. In this with psychological symptoms and psychiatric diagnoses.
review, we highlight the role of this axis in modulating Moreover, with the advent of brain imaging, the reciprocal
enteric and central nervous system function and how this interactions can be visualized for the first time, demon-
may impact disorders such as irritable bowel syndrome strating that gut stimuli can activate key brain regions
and disorders of mood and affect. We examine the over- involved in emotion regulation.2
lapping biological constructs that underpin these disor-
Most aspects of GI physiology are under neural control,
ders with a special emphasis on the neurotransmitter
which is exerted via a vast network of intrinsic enteric
serotonin, which plays a key role in both the gastrointes-
neurons and glia that span throughout the enteric nervous
tinal tract and in the brain. Overall, it is clear that although
animal studies have shown much promise, more progress system (ENS), GI smooth muscle, and the lamina propria of
is necessary before these findings can be translated for the mucosa, as well as extrinsic innervation from primary
diagnostic and therapeutic benefit in patient populations. afferent and autonomic fibers that connect the intestine to
the spinal cord and the brain.4,5 Although the ENS can
Keywords: Irritable Bowel Syndrome; Microbiota-Gut-Brain regulate GI peristalsis largely independent of CNS input, GI
Axis; Mood Disorders; Motility; Brain Gut Axis. motility is also modulated by factors extrinsic to the ENS,
including the brain6 and other divisions of the autonomic
nervous system (ANS), the gut-associated immune system,

T he understanding that the brain and gut participate in

continuous, bidirectional communication was recog-
nized as remotely as in Ancient Greece where philosophers
and the gut microbiome. The influence on the gut is not
unidirectional, because the gut also sends information to
these various systems through complex pathways that
such as Hippocrates, Plato, and Aristotle postulated that the function as bidirectional conduits for homeostasis, and al-
brain and the rest of the body are intrinsically connected. This terations in this communication are associated with disease.
notion led to the understanding that to study disease pro- Adequate gut function is thus critical for not only long-term
cesses, the whole person must be considered rather than an survival but also for brain-gut homeostasis. Precisely how
isolated organ system.1 It was not until the 1840s, however, gut-brain communication occurs in health and disease in
that William Beaumont experimentally showed that humans, however, remains an active area of investigation.
emotional status affected the rate of digestion and, thus, that More recently, the gut microbiome (the trillions of mi-
the brain affects the gut and that there is a brain-gut axis. croorganisms that reside in the gut) has emerged as an
Although this concept was subsequently recognized by the
greats of modern biology including Darwin, Pavlov, James,
Bernard, and Cannon,2 it took until the early to mid– 20th Abbreviations used in this paper: 5-HT4R, 5-HT4 receptor; Ahr, aryl hy-
century for the first scientifically recorded observations to be drocarbon receptor; ANS, autonomic nervous system; CNS, central ner-
made that correlated gut physiology changes with changes in vous system; ECC, enterochromaffin cell; ENS, enteric nervous system;
FMT, fecal microbiota transplantation; FODMAPs, fermentable oligosac-
emotion. These studies were limited, however, because of charides, disaccharides, monosaccharides, and polyols; GF, germ-free;
simple techniques and the lack of study of the reciprocal ef- GI, gastrointestinal; IBS, irritable bowel syndrome; MGB, microbiota-gut-
brain; MM, muscularis macrophage; PGNs, peptidoglycans; SCFA, short-
fects of changes in gut physiology on mental function. chain fatty acid; TLR, Toll-like receptor.
Emerging data has confirmed connections between brain and
Most current article
gut health and has further suggested several mechanistic
© 2021 by the AGA Institute
underpinnings. Alterations in gastrointestinal (GI) function 0016-5085/$36.00
and GI symptoms have been reported to accompany an https://doi.org/10.1053/j.gastro.2020.10.066
April 2021 Microbiota-Gut-Brain Axis 1487

integral player in gut-brain communication and a gut-brain (MGB) interactions is currently unknown. There
microbiome-gut-brain axis has been proposed.7–10 Although are large interindividual differences in the microbial compo-

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mechanistic studies on how this expansive community of sition and we are only beginning to understand the factors that
microorganisms influences human ENS and CNS develop- influence this in health and disease.16 Moreover, there is a
ment,11 GI motility,12 mood,10 cognition, and learning13 are growing appreciation from cross-sectional human studies that
still in their infancy, it offers itself as a potentially important changes in the diversity and relative abundances of the
site for future therapeutic interventions (Figure 1). Gut mi- microbiota and microbial metabolites are associated with a
crobes communicate to the CNS through neural, endocrine, wide array of neurologic and psychiatric disorders, including
and immune signaling channels. Conversely, the CNS can Parkinson’s disease, Alzheimer’s disease, Autism spectrum
affect the gut microbiota directly via stress mediator-induced disorders, and depression.18 Results from these studies,
virulence gene expression and indirectly through ANS- however, have been inconsistent and without evidence
mediated control of gut function (eg, motility, immune mod- establishing causality for the gut microbiome. Further, exam-
ulation, and secretion) (Figure 1).14 In addition, the ENS can ination of the MGB axis in clinical populations has been mostly
directly modulate microbial composition through changes in restricted to cross-sectional studies demonstrating associa-
secretion, motility, permeability, and immunologic defense. tions of gut microbes with brain architecture in healthy sub-
These parallel and interacting pathways are thus emerging to jects or disease states.19 There is thus a large gap in our
investigators as a complex communication matrix, which also understanding of the underlying mechanisms involved in the
has been referred to as the gut connectome.15 MGB dialogue. Based largely on results obtained in preclinical
In addition to the contributions of the microbiome, studies animal models, the more well-studied routes of communica-
in animal models have provided evidence that some GI tion thus far include the immune system,20 metabolites, and
dysfunction in neurologic conditions may also be due to ge- neurotransmitters and vagal nerve activation.21,22
netic defects and/or environmental influences that can
simultaneously impact gut and brain development and/or MGB Axis in Early Life
function. Supportive of this notion are the demonstrations that Whether microbial colonization occurs in utero is not yet
the ENS, often described as a "second brain," shares many fully understood.23 Maternal diet24 and maternal stress
likenesses with the CNS. Their shared structure, develop- exposure during pregnancy,25,26 however, have been shown
mental patterns, and neurochemistry have formed the basis for to influence the infant microbiome, and it is clear that the
research in understanding how pathogenic mechanisms that maternal microbiome can play a key role in shaping infant
give rise to CNS disorders might also lead to ENS dysfunction host development and physiology.27–30 Intriguingly, the
and vice versa. For example, one of the key transmitters in the periods of major change in the developing microbiota
CNS and the intestine, serotonin (5-HT), can act in neuroen- overlap partially with the timeframes for development of
docrine, endocrine, and/or paracrine fashions to impact the other bodily systems and particularly the brain31 and the
development and long-term functions of both the ENS and CNS. ENS.11 This parallel development is likely to be biologically
Given the critical involvement of the ENS, the CNS, and relevant, and these periods may correspond with sensitive
the microbiome in brain and gut development and function, periods in the development of the MGB axis that will be
a greater understanding of the relationships between these critical for establishing appropriate communication along
systems is likely to enable the development of novel ther- the axis throughout the lifespan.
apeutic targets for some of the most common yet poorly The postnatal microbiota is relatively volatile, gaining
understood medical conditions. The current state of stability and diversity across maturation.32 Most coloniza-
research, although impressive, leaves many vital unan- tion of the infant gut starts at birth when delivery exposes
swered questions that need to be addressed to facilitate the infant to a complex microbiota that are dependent on
novel, effective, therapeutic development. In this review, we many elements, including mode of delivery, breastfeeding,
address current evidence supporting the ways in which the prematurity, the environment, host genetics, antibiotic
brain, the gut, and the gut microbiota interact and the exposure, and maternal factors such as infection status,
emerging data supporting its contribution to human disease. stress, and/or obesity.
After the first several days of life, there is a shift toward
a microbiota population focused on extracting nutrients to
The Microbiota-Gut-Brain Axis support the rapid development of the brain and body of the
Driven by the development of next-generation sequencing host.33 A key component of gut microbiota differences may
technologies in tandem with large cohort studies, the past be dependent on whether an infant is breast or formula fed.
decade has seen a dramatic increase in our understanding of While heterogeneity exists among the population charac-
the microbiome in many aspects of health and disease.16 In teristics and study techniques, most studies show that both
humans, the greatest abundance of microbes is found in the diversity and richness of the microbiome are lower in
distal gut that hosts approximately 3$1013 microbes from breastfed than formula fed infants, with higher levels of
more than 60 genera.17 Although bacteria are the most abun- Proteobacteria and Bifidobacteria and lower levels Bacter-
dant and best-studied gut microorganisms, the multitude of oidetes and Firmicutes found in breastfed infants compared
archaea, yeasts, single-celled eukaryotes, helminth parasites, with those who are formula fed.34,35 However, these dif-
and viruses are also more recently being considered, although ferences do not appear to be linked to infant behavioral
the role that these other microorganisms play in microbiota- distinctions such as those associated with colic.35
 1488 Margolis et al Gastroenterology Vol. 160, No. 5
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Figure 1. Pathways of communication between microbiota and brain. A growing body of research is implicating different
pathways of communication between the microbiome and brain in disorders of both mood and motility. Multiple direct and
indirect (via systemic circulation) pathways exist through which the gut microbiota can modulate the gut-brain axis. They
include endocrine (cortisol), immune (cytokines), and neural (vagus, ENS, and spinal nerves) pathways. Several gut microbes
are capable of synthesizing neurotransmitters (ie, g-amino butyric acid [GABA], noradrenaline, and dopamine) locally, which
can act on target cells in the gut and act as an important avenue of communication. Neuroactive microbial metabolites can
modulate brain and behavior through a number of ways that are still being elucidated. These include affecting epithelial cells to
impact gut barrier function and enteroendocrine cells (EECs) to release GI hormones, as well as dendritic cells (DCs) to
modulate immune function. Specialized structures on EECs and ECCs, known as neuropods, have been shown to transduce
sensory signals from the intestinal milieu to the brain through forming synapse-like connections to afferent nerves, including
the vagus nerve. The ENS is perfectly poised to be an integral hub for microbial signals and can communicate with the brain via
vagal and spinal pathways. However, the exact molecular signaling pathways of all these pathways involved remain to be
defined.

The last major change takes place at weaning because composition of the microbiota.41–50 The strongest evidence
the infant shifts from breastmilk or formula to a solid diet for a role of the microbiota in neurodevelopment comes
with a pattern observed across species, including humans from research in germ-free (GF) mice.41,51 Studies where GF
and rodents.36,37 Although there are continuous changes rodents have been recolonized with “normal” microbiota (ie,
well into adolescence,32 these alterations are more gradual from specific pathogen-free animals) at different ages have
and geared toward an adult-like profile.38 In the adult, diet shown that postweaning recolonization is more effective at
has the greatest life-long influence on microbiota composi- restoring GF deficits than recolonization later in life, at least
tion39 although antibiotic use is also a key factor in dis- for specific aspects of brain or immune function and
rupting the microbiota across the lifespan.40 behavior.52–56 Still other functions in GF animals, such as
those affecting CNS serotonergic neurotransmission, cannot
be restored by recolonization by the age of weaning, sug-
Microbiota and CNS Development gesting that the window for microbial influence on these
Overall, fundamental central neural processes including functions is already closed.56
development, myelination, neurogenesis, and microglia Although these studies in GF mice have been important
activation have been shown to be dependent on the in providing evidence supporting the concept that the
April 2021 Microbiota-Gut-Brain Axis 1489

microbiome is involved in brain processes involved in stress receptor-mediated signaling pathways,80 5-HT, short-chain
hormone signaling, neural function, and neuroprotection,51 fatty acids (SCFAs),78 microbial-epithelial interactions,79

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there are significant limitations to human translation of and the transcription factor, aryl hydrocarbon receptor
these findings including, but not limited to, the presence of (Ahr).
defects in the immune system development, ENS formation, Ahr is a recognized biosensor for intestinal epithelial-
and CNS maturation of GF animals.41–50,57 The mechanistic and immune-cell homeostasis in the gut. As such, enteric
underpinnings of these relationships are also little neuron-specific Ahr may serve as a node that integrates
understood. signals from the luminal microbiota environment with the
The human studies that have sought to evaluate the physiological output of intestinal neural circuits to maintain
relationship between the microbiota and CNS development gut homeostasis.81 Interestingly, in a recent study it was
remain limited; most have been conducted in infants and shown that neuron-specific deletion of Ahr, or constitutive
are largely cross-sectional. Studies that have extended overexpression of its negative feedback regulator, CYP1A1,
follow-up to 2 years of age, however, have continued to results in reduced peristaltic activity of the colon, similar to
show connections. Antibiotic exposure in infancy has been that observed in microbiota-depleted mice.76 Moreover,
reported to have a negative impact on cognitive develop- expression of Ahr in the enteric neurons of mice treated
ment.58 Another study linked cognitive function at 2 years with antibiotics partially restores intestinal motility. These
of age with microbiota composition assessed 1 year studies suggest that the ENS has an ability to monitor the
earlier.59 More recently, the same research group demon- luminal microbial environment and adjust neuronal activity
strated that microbiota alpha diversity was also related to and motility accordingly and thus provides a further basis
cognitive outcomes at 2 years of age and, further, was for studies that examine the microbial detection mecha-
associated with functional connectivity between the sup- nisms used to alter GI or gut-brain physiology. Overall, more
plementary motor area and the inferior parietal lobule in research is needed to understand the mechanisms by which
infancy. Importantly, this connectivity was also related to the microenvironment of the gut lumen influences ENS
cognitive outcomes at 2 years of age.60 plasticity.
Finally, reverse regulation in which the ENS contributes
to the shaping of the microbiome is also possible and has
ENS Development been addressed by several preclinical studies, including
In the early postnatal period, enteric neuro- and glio- several in which alterations in the composition of colonic
genesis is accompanied by a functional maturation of in- and/or fecal microbiota were observed in murine or
testinal neural circuits.33,61 This evolution has been shown zebrafish models of congenital aganglionosis,82,83 It still
to continue beyond the postnatal period in preclinical needs to be determined. However, whether these abnor-
models; enteric gliogenesis is maintained at low levels malities represent direct effects of ENS circuits on micro-
throughout life,62–64 enteric neuronal turnover may occur biota or are merely the consequences of abnormal
even more rapidly than that of glial cells,65 and changes in peristalsis.
synaptic contacts within the enteric circuitry are seen in
mice through adolescence.66
To date, ENS development has been examined primarily Mechanisms of Microbiota to Gut-Brain
from its molecular and genetic origins.67 An increase in the
Signaling
understanding of the importance of postnatal ENS devel-
opment, however, has led to an emergence of literature Vagus Nerve
focusing on factors that are contained within the postnatal As a major bidirectional highway of brain-gut connec-
gut microenvironment, including the presence of a complex tion, the afferent branch of the vagus nerve has been the
gut microbiota and immune system.11,68–70 focus of multiple studies examining its effects on brain-gut
Gut microbiota-driven effects on ENS development and communication in health and disease.14,84 Although the
function have been exemplified in studies on GF mice. These sensory vagus nerve and ENS are intrinsically linked, the
mice harbor reduced numbers and subtype distributions of mechanisms underpinning this interaction and the role of
enteric neurons that are associated with deficits in gut vagal signalling from the ENS to the brain remain incom-
motility68–70 as well as attenuated excitability of intrinsic pletely understood.
primary afferent neurons, a key component of gut-brain The afferent branch of the vagus nerve is the main
neural pathways.71,72 Conventionalization of adult GF mice neural conduit connecting the GI tract to the nucleus of the
reduces the deficit in intestinal transit time,69,73 restores solitary tract and higher emotion-regulating networks in the
neuronal excitability,71 alters the chemical coding of enteric mammalian brain.85 Although it does not appear to interact
neurons, and normalizes enteric glial cell density and gut with the gut microbiota directly, evidence suggests that the
physiology,64,69 demonstrating an important role for the vagus nerve can sense microbial signals in the form of
microbiota in ENS plasticity. Similar effects have been noted bacterial metabolites, or be influenced via microbiota-
after bacterial exposure through probiotics or specific bac- mediated modulation of enteroendocrine and enterochro-
terial strains.74–76 Moreover, studies have also provided maffin (ECCs) cells in the gut epithelium (Figure 2).86 For
insight as to which microbial mechanisms may affect enteric example, gut bacteria produce SCFA metabolites (eg, buty-
nerve activity.77–79 These include G-protein–coupled rate, propionate, acetate, and valerate) that regulate
 1490 Margolis et al Gastroenterology Vol. 160, No. 5
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Figure 2. Serotonin (5-HT) as a critical regulator of gut-brain-microbiome axis signaling. Gut bacteria in the intestinal
microbiome produce SCFAs that directly stimulate tryptophan hydroxylase 1 (TPH1), resulting in 5-HT synthesis in and
secretion from intestinal ECCs. 5-HT released from the basal membrane of intestinal EC cells then interacts with receptors
from neurons in the ENS to modulate motility and, during development, neuronal development and differentiation. Vagal af-
ferents signal to the nucleus of the solitary tract (NTS) and the dorsal raphe nucleus (DRN), the latter of which houses the
majority of the brain’s 5-HT neurons. These areas then interact with emotion-regulating brain networks that influence mood. Of
note, SCFAs produced by gut bacteria can also directly stimulate free fatty acid receptors on multiple cell types, including
epithelial cells, ECCs, immune cells, and nerve cells, including the vagus nerve and primary afferent neurons. This signaling
can also modulate downstream regulation of motility, secretion, and gut-brain signaling. Abbreviations: Trp, tryptophan; SERT,
serotonin reuptake transporter; MAO, monoamine oxidase.

physiological intestinal functions, including those involving neurobehavioral disorders. For example, in mice, vagotomy
motility, secretion, and inflammation (as described later in has been shown to block central signaling of Lactobacillus
this article), through their cognate free fatty acid re- and Bifidobacterium species, resulting in the besiegement of
ceptors.87 Further, other receptors on vagal nerve fibers their mood-modifying effects.55,89,90
such as those for serotonin (5-HT3, 5-HT4) and other gut A bidirectional communication system between diet, the
peptide receptors may also facilitate these messenger gut microbiome, ECCs, and the vagus nerve has recently
pathways.86,88 Vagotomy studies in mice also highlight been reported. ECCs contain more than 90% of the body’s
possible roles for the vagus nerve in CNS-microbiota serotonin (5-HT) and 5-HT synthesis and release in ECCs is
communication, which may translate to human mood and modulated by SCFAs and 2BAs produced by spore-forming
April 2021 Microbiota-Gut-Brain Axis 1491

Clostridiales.12,91 These microbes increase their stimulatory microbial presence and deliver messages to the ENS that
actions on ECCs with increased dietary tryptophan avail- result in changes in gut nervous system development and

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ability.92 ECCs also communicate with afferent nerve fibers function. Enteric neurons and glia have the machinery
through synaptic connections of neuropod-like extensions of necessary to detect the gut microbiota; they both express
ECCs.93 On the other hand, the ANS can activate ECCs to TLR2 and TLR4.68,103 Further, antibiotic depletion of the
release 5-HT into the gut lumen, where it can both be taken microbiota alters TLR expression in mice and also results in
up by serotonin transporter-like mechanisms and influence concomitant alterations in GI motility and sensitivity to
gut microbial function.94 acetycholine.104 These effects, at least in part, may be
mediated by TLR4 and/or TLR2. TLR4-deficient mice have
Immune Mechanisms for Microbiota to Gut-Brain decreased numbers of nitrergic neurons and reduced
motility, a similar phenotype to that observed in GF and
Signaling antibiotic-treated mice.68 Mice deficient in TLR2 signaling
In the gut, an intact immune system is critical for
exhibit abnormalities in the neurochemical coding of the
maintaining the careful balance between homeostatic
ENS that is accompanied by gut dysmotility and attenuated
tolerance of commensal organisms and the simultaneous
chloride production in intestinal explants.103 Further, anti-
protection of the body against pathogenic microbial inva-
biotic treatment of wild-type mice leads to ENS abnormal-
sion. In addition, immunity also serves a critical role in
ities that can be reversed after supplementation with a
mediating communication between the gut microbiota, the
TLR2 agonist, further confirming the idea that the gut
ENS, and the brain. Toll-like receptors (TLRs) and peptido-
microbiota-TLR2 axis is important for ENS morphology and
glycans (PGNs) mediate the immune response to microbes
function.103
by acting as sensors of microbial components.95,96 An intact
These data suggest that the ENS has the capacity to
gut barrier also prevents the inappropriate activation of
respond to stimuli from distinct types of microbes affecting
immune cells and the development of systemic immune
its physiology. Precisely how microbe-TLR communication
activation.
affects ENS structure and function, and how these changes
Bacteria can release immune agonists, such as lipo-
relate to gut-brain signaling, have yet to be determined. For
polysaccharide and PGN, into the circulation where they can
example, it would be important to determine how alter-
gain access to the brain. TLRs have been found in the brain
ations in TLR activation during early life, including those
of mouse disease models and especially the microglia,
induced by infections and/or antibiotics, can affect ENS
where they have been studied in the development of Alz-
development and long-term function of brain-gut in-
heimer’s disease,97 Parkinson’s disease,98 visceral pain,99
teractions. The plasticity of the ENS demonstrated in these
and depression.100 GF and antibiotic-treated mice also
studies and others makes this likely to be a high-yielding
both display a reduction in the expression of several of the
area of therapeutic investigation.
receptors that detect PGN in the striatum, which suggests
Macrophages are present throughout the gut where they
that gene expression in the brain is sensitive to microbiota
play an essential role in the reparative response to intes-
manipulation.101 Moreover, knockdown of a PGN-sensing
tinal injury.105,106 Monocyte-derived and tissue-resident
receptor, PGN-recognition protein 2, resulted in an in-
macrophages are decreased in quantity in GF mice or in
crease in sociability in mice, indicating that loss of the ability
mice that are microbiota-depleted with antibiotics, implying
to sense peptidoglycan results in host behavioral
a role for the microbiota in intestinal macrophage recruit-
changes.101 More research is needed, however, to unravel
ment and differentiation.107 Further, muscularis macro-
the functional consequences of such immune signaling
phages (MMs) engage in a bidirectional relationship with
across the lifespan in health and disease
enteric neurons that appears to be regulated by the gut
Diet-induced changes in the gut microbiome can lead to
microbiota; MM activation by the cytokine, bone morpho-
a compromised mucus layer, allowing access of luminal
genetic protein 2, results in alterations in GI motility and
microbes to extensions of dendritic cells, resulting in acti-
production of macrophage colony-stimulating factor 1, a
vation of these cells by both pathogens and commensals.
critical mediator of MM development, and both colony-
This local immune activation can lead to increased perme-
stimulating factor 1 and bone morphogenetic protein 2
ability of the epithelial tight junctions that further com-
production are decreased after antibiotic treatment.107
promises the intestinal barrier. The diet-induced release of
Microbiota-neuron-macrophage interactions have also
immune mediators into the systemic circulation is referred
been exemplified in studies showing that sympathetic
to as metabolic endotoxemia, which can lead to immune
ganglia activation elicited by Salmonella typhimurium may
activation in different organs, including the brain.102 This
influence macrophages to protect the ENS by stimulating
low-grade immune activation has been implicated in the
processes that both limit enteric neuronal damage and
pathophysiology of some forms of depression and neuro-
enhance gut motility.108 The ENS may also protect itself
degenerative disorders such as Alzheimer’s and Parkinson’s
from invasive S typhimurium infection by producing inter-
disease.
leukin-18, a cytokine that both drives goblet cell antimi-
crobial peptide production and maintains the mucosal
Immune Signaling and the ENS barrier.109 It has most recently been demonstrated that the
TLRs and other components of the innate immune sys- gut microbiota may influence gut-extrinsic sympathetic
tem (eg, macrophages) may serve as sensors of gut activation through a gut-brain circuit.110
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Microbial Metabolites has also been shown to induce maturation of the adult ENS
via activation of 5-HT4 receptors (5-HT4R); GF mice retain a
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The gut microbiota generate metabolites that are impli-

cated in the modulation of both CNS and ENS physiology higher degree of nestin-expressing neuronal stem cells and
and behavior. Although in vitro studies have shown that exhibit slower intestinal transit and both factors normalize
specific bacteria can produce neurotransmitters (eg, after bacterial colonization that is dependent on 5-HT4R
noradrenaline, dopamine, and g-amino butyric acid),111,112 signaling.73 Finally, the gut microbiota may also act through
whether these neurotransmitters are capable of reaching neurotransmitter precursors; it can influence serotonergic
specific targets within the CNS, and/or in sufficient con- neurotransmission through regulating the availability of the
centrations, is unknown. The short half-lives of most neu- 5-HT precursor, tryptophan. Circulating tryptophan con-
rotransmitters and their limited ability to cross the blood centrations are significantly higher in male GF mice relative
brain barrier, however, make this possibility unlikely. The to conventional controls56 and these altered tryptophan
way this gut-brain communication may happen has been levels correspond with an increase in hippocampal seroto-
studied most extensively for serotonin and other tryptophan nin, and its metabolite, 5-hydroxy-indole acetic acid.56
metabolites (as discussed later in this article), which have Whether this has any bearing on the social deficits
been shown to exert major influences on ENS and CNS observed in these animals, however, requires further
development and functions including GI motility, mood, and investigation.
behavior.113–118 Tryptophan Metabolism: Beyond Serotonin. The
Short-Chain Fatty Acids. Gut bacteria produce SCFA main physiological pathway for tryptophan metabolism is
metabolites that can regulate motility, secretion, and gut- along the kynurenine pathway. GF mouse studies have
brain signaling by acting through free fatty acid receptors shown an increased availability of tryptophan in GF animals
on epithelial cells, enteroendocrine cells, ECCs, immune as a consequence of a reduction in peripheral kynurenine
cells, and intrinsic and extrinsic neurons (Figure 2).12,91,119 pathway activation.56 Moreover, in a rodent model of
Centrally, administration of acetate, propionate, and buty- chronic variable stress, a stress-induced reduction of Lac-
rate is capable of restoring morphologic deficits of microglia tobacilli reduced hydrogen peroxide–mediated inhibition of
in GF mice120 and reversing the behavioral and physiolog- Indoleamine 2, 3-Dioxygenase 1. This inhibition resulted in
ical effects of chronic stress.121 Moreover, SCFAs may in- an increase in the conversion of tryptophan to kynurenine, a
fluence the production of neurotransmitters in the brain feature that was linked to depression-like behavioral alter-
through regulating the expression of enzymes involved in ations in mice exposed to chronic stress.127 In contrast to
their biosynthesis; administration of propionate and buty- serotonin, kynurenine can traverse the blood brain barrier
rate to PC12 cells (neuroblastic cells that differentiate to and negatively impact brain health by inducing neuro-
neuron-like cells) in vitro increased the expression of inflammation and neurodegeneration.128
tyrosine hydroxylase, the rate-limiting enzyme involved in Indole production, which is limited to the gut microbiota,
noradrenaline and dopamine synthesis.122 Whether these is catalyzed by the microbial enzyme, tryptophan hydroxy-
microbial metabolites are capable of regulating neuro- lase, and indole has been detected in blood, brain, and the GI
transmission in vivo, however, is not clear. More evidence is tract.92 There is a growing literature supporting the concept
also needed to determine the extent to which physiologi- that the microbial processing of tryptophan to indole affects
cally relevant concentrations of SCFAs are capable of gut-brain axis function.129 Indoles exert many beneficial ac-
reaching the brain given their relatively short half-life (25 tions on intestinal and systemic homeostasis.130 Yet, adverse
minutes to 3 hours). To date, studies investigating the effect effects on the gut-brain axis are also evident after absorption
of exogenous SCFA administration on brain physiology and from the gut and host processing, where some indole de-
behavior have typically used concentrations that far exceed rivatives have been shown to exert neurodepressive-like ef-
what is microbially derived.123 fects on behavior, at least in preclinical studies.131
Serotonin: A Key Regulator of the MGB Axis in the Tryptamine. Bacteria express tryptophan decarbox-
Gut and Brain. There is growing appreciation that the ylase and are capable of producing tryptamine from dietary
neurotransmitter, 5-HT is one of the key players in MGB axis tryptophan. It has recently been demonstrated that
signaling in the brain, the gut, and in gut to brain commu- bacterial-derived tryptamine can act via the 5-HT4R to
nication. Indeed, recent studies have shown that specific impact GI motility.88 The capacity of the gut microbiota to
spore-forming bacteria from both humans and mice in- produce metabolites like tryptamine, which can influence
crease colonic and serum 5-HT levels in GF mice and host physiology by activation of G protein–coupled re-
ameliorate GF-associated gut dysmotility by producing ceptors, is an important facet of host-microbe interactions
SCFAs,124 which increase 5-HT production by up-regulating that warrants increased attention.132,133 It is currently un-
Tph1 expression in ECCs (Figure 2).12,91 Recent evidence clear, however, if microbially derived tryptamine reaches
suggests that 5-HT released from ECCs communicates with the CNS and whether it acts there to control behavior.
the gut microbiota, Turicibacter sanguinis, which has sero-
tonin uptake mechanisms that are involved in its coloniza-
tion and host physiology.125 This may account for some of The MGB Axis in Healthy Subjects
the bidirectional influences that occur between some psy- There are a number of studies that demonstrate the ef-
chotropic drugs, including selective serotonin reuptake in- fects of microbiota-targeted interventions in healthy in-
hibitors, and the intestinal microbiota.126 Gut microbiota dividuals.19 Four weeks of probiotic consumption by
April 2021 Microbiota-Gut-Brain Axis 1493

healthy females led to changes in the functional connectivity in the central and peripheral regulation of brain-gut in-
of an emotion recognition network in the brain.134 In a teractions in IBS.154

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different cohort of female healthy control subjects, white
and gray matter imaging parameters were associated with
IBS and the Microbiome
two bacterial genus-based clusters that each differed in the
A causal role for altered gut microbiota in IBS symptoms
structures associated with emotional, attentional, and sen-
remains to be determined, although a number of cross-
sory processing and also in fMRI-measured emotional
sectional studies have reported alterations in fecal micro-
reactivity.135 Several trials involving the administration of a
bial community composition in IBS subjects, based on
Lactobacillus rhamnosus (JB-1) supplement to healthy males
disease subtype (IBS-diarrhea, IBS-constipation, IBS-mixed),
showed inconsistent outcomes in stress-related behaviors
age (pediatric vs adult), and/or compartment (mucosa vs
with either a positive136 or no137 difference in those
stool).155 Recent evidence suggests the presence of IBS
receiving the supplement. In another examination of healthy
subgroups based on gut microbial community structure,
subjects, however, probiotics improved emotional decision-
with groups not differing from healthy controls despite GI
making and affect relative to placebo with concomitant
symptoms.156,157 In one study, a dysbiotic IBS subgroup
changes in specific taxa.138 In contrast to preclinical models,
differed in regional brain volumes from a group with normal
there is currently no solid evidence that probiotic ingestion
gut microbiota,157 suggesting a relationship between mi-
in healthy individuals can modulate behavior because re-
crobial community composition and brain structure. How-
ported studies on probiotic consumption in small clinical
ever, as both microbiota-defined subgroups met the IBS
studies have produced contradictory outcomes.139–143
diagnostic criteria and did not differ in any clinical param-
eters, these findings put into question a causative role for
dysbiosis in IBS symptoms. The absence of group differ-
Disorders of Brain-Gut Interactions: IBS ences in the microbial composition between healthy con-
IBS is the most common disorder of brain-gut interac-
trols and individuals with IBS has been reproduced
tion, occurring in up to 4.8% of the population world-
elsewhere, although IBS symptom severity was found to be
wide.144 Based on the current symptom criteria,145 IBS is
correlated with dysbiosis.158 A more recent cross-sectional
defined by chronically recurring abdominal pain associated
study revealed significant differences among IBS subtypes
with altered bowel habits in the absence of detectable
in the distribution of Clostridiales. Relative Clostridiales
organic disease. This gut-restricted definition overlooks the
abundance was correlated with significant differences in the
findings that up to 50% of individuals who meet diagnostic
level of fecal SCFAs, which together were associated with
criteria for an anxiety disorder have IBS, and that in-
altered fecal cytokine levels.159 Although this study aimed to
dividuals with IBS have a greater than 3-fold risk of meeting
identify mechanistic pathways in gut microbe-host in-
diagnostic criteria for an anxiety disorder.146 Although in
teractions, the findings need to be confirmed in a study with
the majority of patients with IBS, CNS-related precipitants in
a control population and a larger sample size.
early and adult life (eg, psychological trauma, stress, abuse,
The diverse findings from the IBS microbiota studies
and maternal neglect)147 have been identified, about half of
have been attributed to the extensive range of technologies
patients with IBS present after an intestinal trigger.148 The
used for microbiota study, sample source, differences in IBS
bidirectional nature of brain-gut involvement in IBS was
subtype, differential effects of the ANS on other aspects of
illustrated in a 1-year population-based prospective study
physiology (eg, mucus secretion, intestinal permeability, and
that evaluated individuals with anxiety þ/- depression and
mucosal immunity160), as well as the many other influences
IBS as well as control individuals with neither condition. At
that affect microbial composition and function (eg, age, diet,
the conclusion of the study, it was found that individuals
antibiotic exposure, geography, probiotic intake, and medi-
with higher baseline levels of anxiety and depression were
cation exposure).155,161 Further complicating this dynamic
significantly more likely to develop IBS and, conversely, that
is the need to factor in the CNS-mediated aspects of motility
those subjects with baseline IBS reported significantly
and gut physiology, including sleep quality and stress. The
higher levels of anxiety or depression. Interestingly, in 2/3
examination of much larger cohorts in longitudinal studies
of the comorbid cases, an IBS diagnosis preceded the mood
that also integrate clinical phenotypes and diet are thus
disorder, implying that in some patients primary gut
needed for a more comprehensive understanding of these
dysfunction might serve as a driver for mood disorders.149
populations.
Alterations in fMRI-detected brain activity are linked to
abdominal pain.150 Connections have been shown between
brain networks that mediate anxiety and ANS output, like IBS and Serotonin
the amygdala, with mechanisms that modulate colonic Serotonin is one of most highly studied neurotransmit-
sensitivity and gut motility. Increased and decreased acti- ters in IBS physiology. As a major determinant of ENS and
vation of endogenous pain excitatory and inhibitory path- CNS development and a modulator of IBS-related symptoms
ways, respectively, have also been observed in CNS locations (eg, motility, secretion, and visceral hypersensitivity) as well
that have been associated with visceral afferent processing as mood,113 serotonin may thus be an important develop-
and emotional arousal.151–153 Interestingly, these pathways mental modulator of the comorbid mood and IBS diagnoses
share significant homology to a stress circuit in rodents that made in some affected patients. Alterations in enteric
implicates the involvement of corticotropin-releasing factor mucosal and blood serotonin signaling have also been
 1494 Margolis et al Gastroenterology Vol. 160, No. 5

demonstrated in adults and children with IBS, potentially A different dietary approach has been taken in the
indicative of GI-initiated serotonergic dysregulation.162,163 treatment of depression that comes under the umbrella of
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Although serotonin can activate more than 15 receptors/ Nutritional Psychiatry. There is clinical evidence from both
receptor subtypes in the brain and intestine, the majority of epidemiologic and several interventional studies that a
IBS research has focused on the 5-HT3 and 5-HT4 receptors largely plant-based diet, such as the traditional Mediterra-
because both have been shown to have effects on mood, nean diet, has benefits in the adjuvant treatment of
motility, and abdominal pain.113,164–167 depression.172,173 There are many open questions that
Altogether, these data suggest a possible role of the gut remain, especially in regard to the mechanisms of how diet
microbiome in altered brain-gut interactions in IBS. They may impact mood and which components are the most
also provide the basis for larger, longitudinal interventional beneficial.
studies, both clinical and functional, to identify the roles of
specific microbiota in behavioral and gut dysfunction in IBS
Supplementing the Gut Microbiome to Improve
and also the utility of serotonin-based modulators as po-
tential therapeutic targets (Figure 2).
MGB Axis
Apart from dietary intervention, probiotics have also
been tried as a treatment meant to target the microbiome.
Gut Microbiome in Depression Although numerous preclinical and some clinical studies
In recent years, there have been an increasing number of have reported beneficial effects of specific probiotics on
studies showing that patients with Major Depressive Dis- mood and emotional behaviors, clinically significant effects
order have an altered gut microbiome composition when of probiotics in the treatment of psychiatric disorders have
compared with healthy controls, although the nature of the not been demonstrated.174 There is thus a need for high-
alterations in each study are diverse.168–170 This variation in quality randomized controlled clinical studies in human
outcomes is likely due to similar reasons as those noted for subjects to demonstrate that the beneficial effects observed
IBS. It is worth noting that studies have also shown that in preclinical models can be translated and confirmed in
transferring the microbiome of a depressed individual into a human settings.
Studies on probiotics, including strains of Bifidobacte-
healthy rodent can induce depressive-like behaviors in the
rium and/or Lactobacillus, have been shown to improve
murine recipient, suggesting the possibility of a causal role
symptom severity in adults and children with IBS.175–177
for the microbiota in pathophysiology of depression and
opening up the concept of targeting the microbiome for Due to the insufficient quality of these studies, however,
mental health benefit.19,169 the recently published American Gastroenterological Asso-
ciation Guidelines on probiotics do not recommend their use
in IBS, other than in controlled studies.176,177
Mood and IBS: Targeting the MGB Axis Interestingly, although some probiotic supplementation
in humans changes the gut microbiota composition, as
The effects of enteric microbial manipulations in
judged by 16S rRNA sequencing, others show no or only
controlled clinical trials in patients with depression and/or
transient modification of the collective microbiome tran-
IBS have been evaluated with probiotics, antibiotics, and the
scriptional state. These findings suggest that measurement
low–fermentable oligosaccharides, disaccharides, mono-
of probiotic intervention on gut microbial profiles must be
saccharides, and polyols (FODMAPs) diet. Several studies
accompanied by technologies assessing microbial function,
have demonstrated the effectiveness of a low-FODMAP diet
in the short-term treatment of IBS symptoms, and diet- such as metagenomics or metabolomics.
induced changes in the gut microbiome have been impli-
cated as an underlying mechanism. The low-FODMAP diet Fecal Microbiome Transplantation as a Therapy
results in a decreased production of gas and osmotically for IBS and Mood Disturbances
active metabolites, as a result of decreased microbial Clinical studies on fecal microbiota transplantation
fermentation, which is thought to lead to improvements in (FMT) remain limited and systematic review fails to show
bloating, flatulence, and pain.171 In line with this theory, any overall benefit.178,179 Two recent randomized control
adult randomized control trials demonstrate that intake of a trials studies showed alterations in gut microbial composi-
low-FODMAP diet improves IBS symptoms, regardless of tion in the group receiving a FMT. However, although one
subtype, as well as health-related quality of life, anxiety, and study showed a significant IBS symptom reduction 3
activity impairment in adults with IBS-D171. However, months after FMT, the other study demonstrated greater
although these study results indirectly support a role for the symptom improvement in the placebo-treated group.178–180
gut microbiome in some IBS symptoms and may be useful High-quality FMT studies in patients with depression are in
for short-term treatment of some IBS symptoms, the value the pipeline.
of a low-FODMAP diet for long-term treatment for IBS has
been questioned.171 This reduction efficacy may be because
of the reduction of oligosaccharides important for the di- Conclusions and Future Directions
versity and abundance of the gut microbiota and/or because Considerable progress has been made in the under-
of the low compliance rate associated with long-term standing of the MGB axis in preclinical models of human
adherence.171 brain disorders and in the potential translation of these
April 2021 Microbiota-Gut-Brain Axis 1495

origins. A causal role of the gut microbiome in these in-

teractions remains to be determined. This valuable knowl-

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REVIEWS AND
edge will inform the development of cross-disciplinary
therapeutic approaches well into the future.
Key areas that have not yet been examined extensively
are the roles that sex and race play in microbiome-gut-brain
axis development and disease. Accumulating evidence has
shown, however, that both sex and race may exert impor-
tant influences over the gut microbiota. The microbiome
may alter brain development in a sex-specific manner and
females are significantly more likely to suffer from stress-
related and functional GI disorders.56,181,182 On the other
hand, at least in animal models, the male brain appears to be
more susceptible to microbial disturbances in early life than
the female brain.183 These sex-specific effects have given
rise to justified calls for more intensive study of the
“microgenderome.”181,184 Although racial diversity of the
gut microbiome has been explored somewhat in cancer and
other medical conditions, there has been no in-depth
exploration of how race impacts microbial diversity and
its relationship with brain-gut axis conditions.185 The cur-
rent knowledge, however, emphasizes the need to under-
stand how the sexual dimorphism and racial diversity
impact the microbiome and contribute to brain-gut axis
disorders.
It is important to reiterate that translation of preclinical
findings into more effective therapies for human brain dis-
orders has largely been unsuccessful to date (Figure 3).
Moving forward, the development of live biotherapeutics or
substances whose beneficial effects on the brain are
bacteria-mediated (ie, psychobiotics) are currently being
investigated as direct and/or adjunctive therapies for brain
disorders, but this field is very much in its infancy.19 Until
identification of gut microbiome-related patient subtypes
becomes possible and new pharmacologic and specific
microbiome-targeted approaches emerge, the most effective
treatments for IBS and other disorders of MGB interactions
remain a combination of personalized diet approaches,
Figure 3. Challenges in translational research. Schematic behavioral therapies, and a limited number of pharmaco-
representation of research approaches aimed to identify a logic treatments aimed at improvement of bowel
causal role of the gut microbiome in human brain and brain- function.186
gut disorders. There is extensive evidence for cross-sectional
differences in the gut microbial composition between defined
disease populations and healthy control populations (top
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Correspondence
microbiota transplantation in irritable bowel syndrome: a Address correspondence to: Kara G. Margolis, MD, Columbia University
systematic review and meta-analysis. Am J Gastro- Medical Center; College of Physicians and Surgeons; 620 West 168th Street;
enterol 2019;114:1043–1050. New York, NY 10032 e-mail: [email protected].

179. Enck P. Primum non nocere: is faecal microbiota trans- Acknowledgments

plantation doing harm to patients with IBS? Gut 2019; The authors thank Narek Israelyan (Columbia University), Dr. Kenneth
O’Riordan (University College Cork), and Cathy Liu (UCLA) for their
68:1722–1723. contributions to the figures.
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Authorship Contributions
faecal microbiota transplantation for patients with irrita- Study concept and design: K.G.M., J.F.C., E.A.M.
ble bowel syndrome in a randomised, double-blind, Acquisition of data: K.G.M., J.F.C., E.A.M.
placebo-controlled study. Gut 2020;69:859–867. Drafting of the manuscript: K.G.M., J.F.C., E.A.M.
Critical revision of the manuscript for important intellectual content: K.G.M.,
181. Audet MC. Stress-induced disturbances along the gut J.F.C., E.A.M.
microbiota-immune-brain axis and implications for
Conflicts of interest
mental health: does sex matter? Front Neuroendocrinol Kara G. Margolis has been a consultant for Takeda. John F. Cryan has been an
2019;54:100772. invited speaker at meetings organized by Yakult, Alkermes, Ordesa &
182. Vemuri R, Sylvia KE, Klein SL, et al. The microgenderome Alimentary Health, has been a consultant for Alkermes & Nestle, and has
received research funding from Mead Johnson, Cremo, Nutricia, DuPont,
revealed: sex differences in bidirectional interactions be- Pharmavite, & 4D Pharma. Emeran A. Mayer is a scientific advisory board
tween the microbiota, hormones, immunity and disease member of Danone, Axial Biotherapeutics, Viome, Amare, Mahana
Therapeutics, Pendulum, Bloom Biosciences, and APC Microbiome Ireland.
susceptibility. Semin Immunopathol 2019;41:265–275.
183. Jaggar M, Rea K, Spichak S, et al. You’ve got male: sex Funding
and the microbiota-gut-brain axis across the lifespan. This research was supported by grants from the National Institutes of Health:
RO1 NS015547 (K.G.M.) and R01 DK048351 (E.A.M.); the Department of
Front Neuroendocrinol 2020;56:100815. Defense: PR160365 (K.G.M.); Science Foundation Ireland: SFI/12/RC/
184. Mulak A, Tache Y, Larauche M. Sex hormones in the 2273_P2 (J.F.C.); the Saks Kavanaugh Foundation (J.F.C.); and the EU
H2020 project DLV-848228 DISCOvERIE (development, diagnosis and
modulation of irritable bowel syndrome. World J Gas- prevention of gender-related somatic and mental comorbidities in irritable
troenterol 2014;20:2433–2448. bowel syndrome in Europe) (J.F.C.).