REVIEWS

The mucosal immune system:

master regulator of bidirectional
gut–brain communications
Nick Powell1, Marjorie M. Walker2 and Nicholas J. Talley2
Abstract | Communication between the brain and gut is not one-way, but a bidirectional highway
whereby reciprocal signals between the two organ systems are exchanged to coordinate
function. The messengers of this complex dialogue include neural, metabolic, endocrine
and immune mediators responsive to diverse environmental cues, including nutrients and
components of the intestinal microbiota (microbiota–gut–brain axis). We are now starting to
understand how perturbation of these systems affects transition between health and disease.
The pathological repercussions of disordered gut–brain dialogue are probably especially
pertinent in functional gastrointestinal diseases, including IBS and functional dyspepsia.
New insights into these pathways might lead to novel treatment strategies in these common
gastrointestinal diseases. In this Review, we consider the role of the immune system as the
gatekeeper and master regulator of brain–gut and gut–brain communications. Although adaptive
immunity (T cells in particular) participates in this process, there is an emerging role for cells of
the innate immune compartment (including innate lymphoid cells and cells of the mononuclear
phagocyte system). We will also consider how these key immune cells interact with the specific
components of the enteric and central nervous systems, and rapidly respond to environmental
variables, including the microbiota, to alter gut homeostasis.

Globally, gastrointestinal diseases are major causes of
mortality and morbidity. Infectious diarrhoea remains a
major cause of death in the developing world, but chronic
gastrointestinal diseases are common ­diseases globally.
Chronic intestinal inflammatory diseases, including
Crohn’s disease and ulcerative colitis, the two major
manifesta­tions of IBD, affect nearly 1% of Western
­societies and the incidence of IBD continues to rise,
including increases in the number of newly reported cases
in the developing world1. However, a considerable burden
1
Experimental of disease is accounted for by functional gastro­intestinal
Immunobiology, Division of diseases (FGIDs), which are defined by character­istic
Transplant Immunology and
Mucosal Biology, Great Maze
patterns of symptoms in the absence of c­ lassic organic
Pond, King’s College London, pathology. FGIDs include IBS and functional dys-
SE1 9RT, UK. pepsia (FD), which are among the most c­ ommonly
2
University of Newcastle, ­encountered symptom complexes in most adult popula-
Faculty of Health and
tions. For instance, in Western societies, over one-third
Medicine, School of Medicine
& Public Health, Callaghan of the population has chronic gastrointestinal disorders,
NSW 2308, Australia. dominated by IBS and FD2–4. Accordingly, a pressing
Correspondence to N.P. need now exists to understand the patho­physiology of
nick.powell@kcl.ac.uk chronic gastrointestinal diseases and the key factors that
doi:10.1038/nrgastro.2016.191 influence their clinical course to improve patient out-
Published online 18 Jan 2017 comes. Although genetic factors have a role in chronic
gastrointestinal diseases such as IBD, coeliac disease
and FGID5–7, environ­mental vari­ables are domin­ant and
shape the development and clinical course of these dis-
eases. Furthermore, the digestive tract does not operate
independently, but instead requires functional integra-
tion with other organ systems. Crosstalk between the
gastrointestinal tract and the ­central nervous system
(CNS) and enteric nervous system (ENS) are impor-
tant examples of how signals originating in one of these
organ systems affect the function of the other. Of course,
­dialogue between these systems has been appreciated for
many years. Since the pioneering work of Ivan Petrovich
Pavlov at the turn of the nineteenth century it has been
unequivocally demonstrated that the brain directly influ-
ences gut function8. By forming fistulae in digestive tract
organs, including the salivary glands, stomach, duo­
denum and pancreas, Pavlov showed that gastro­intestinal
secretions could be triggered in response to auditory,
visual or painful stimuli (conditional reflexes), and by
ligating key peripheral nerves he confirmed neural con-
trol of digestive function. However, it is increasingly
recognized that brain–gut communication pathways are
­bidirectional. Indeed, signals originating in the gut can

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Key points
• Common gastrointestinal diseases, such as IBS, functional dyspepsia and IBD,
are closely linked to psychological morbidity
• This link is driven in part through bidirectional signalling between the brain and gut,
which reciprocally regulate each other
• Growing evidence implicates the importance of immune activation, which might
be overt (IBD) or more subtle (IBS, functional dyspepsia) in pathological gut–brain
interactions
• The composition of the intestinal microbiota affects behaviour and mood,
which could in part rely on selective activation of distinct host cytokine responses
• Therapeutic targeting of gut microorganisms, host immunity or psychological
symptoms could hold the key to uncoupling pathological interactions between
the gut and brain
also influence brain function, through endocrine, meta­
bolic, immune and neuronal mediators, which interface
with the ENS and the CNS9. The ENS is a complex net-
work of >1 × 108 neurons that can function independently
of the CNS, controlling gut motility, endocrine function,
trans­mucosal fluid movement and local blood flow10. The
ENS also directly communicates with cells of the intesti-
nal barrier, including neuroendocrine cells and mucosal
immune cells11. This neural network is composed of
enteric glia, neurons of peripheral ganglia and epithelial
sensors such as enteroendocrine cells (EECs), which have
been termed the gut connectome12. In ­normal digestion,
nutrients such as amino acids, peptides and glucose
stimulate EECs to produce hormones, which modulate
gut motility, hormone prod­uction (for example, insulin
secretion), satiety and meta­bolic ­status13,14. Although the
ENS can function as an autono­mous unit (especially in
the small and large ­intestine), it is also closely integrated
with, and reactive to, the CNS. Important anatomical
considerations of the ENS and its interaction with the
CNS are reviewed in BOX 1.
In this Review, we will consider the triggers, medi­
ators and consequences of bidirectional communication
between the gut and brain, and how these pathways
reciprocally affect organ physiology to shape patho­
logy. Emphasis will be directed at pathways regulated
by ­normal and aberrant host immune responses to
­exogenous stimuli, including the intestinal microbiota.
Gastrointestinal disease and the brain
Gastrointestinal disease is linked to disordered brain
function. The burden of psychiatric disturbance in
patients with gastrointestinal disease is substantial.
At one of end of the spectrum, chronic inflammation
and cancer in the gut is linked to so‑called sickness
behaviour, a characteristic behavioural response to
illness that is characterized by lethargy, social with-
drawal, reduced feeding and reduced sexual activity 15,16.
From an evolutionary perspective, sickness behaviour
has been speculated to have conferred a survival benefit
in the setting of acute infection by preserving energy,
avoiding dangerous encounters and limiting the spread
of infection to other community members16. At a popu-
lation level, host response to infection has always been
a key arbiter of survival; however, in the modern era,
and particularly in the developed world, there has been
a sharp decline in the prevalence of infectious diseases
and a simultaneous increase of chronic inflammatory
diseases17. Accordingly, benefits and costs of sickness
behaviour might have shifted. Indeed, unresolved
sickness behavioural responses driven by prolonged,
intractable gastrointestinal diseases such as IBD or FGID
might predispose to psychiatric manifestations, chiefly
in the form of depression18,19.
Chronic gut inflammation is linked to a consider-
able burden of psychological morbidity. Patients with
IBD have increased anxiety and depression20–24, which
is indepen­dently linked to worse outcomes, includ-
ing increased disease activity 25,26, increased relapse
rate27,28, reduced response to biological therapy 26 and
poor adherence to treatment 29. Experiments in rodents
have provided some important insights on the effect of
psycho­social stress on gut physiology and visceral pain
modulation, as well as how intestinal inflammation
influences these responses. Rodent models have proven
extremely valu­able in exploring mechanistic pathways
in these processes, and there are well established and/or
validated models of visceral pain (including colorectal
distension), visceromotor response monitoring, behav-
ioural assays and functional imaging 30. Experimental
stress induction (including acoustic stress, restraint stress,
water avoidance, maternal deprivation and p ­ redator con-
frontation) have demonstrated robustly how stressful
stimuli modulate visceral sensitivity, intestinal motility,
secretory function, intestinal permeability and vulner-
ability to intestinal inflammation31–38. Stress induction
in mice is associated with increased susceptibility for
experimental intestinal inflammation (or threshold for
re‑­induction) in different disease models, including dex-
tran sulfate sodium colitis, DNBS (2,4‑­dinitrobenzene
sulfuric acid) colitis and the spontaneous enterocolitis in
Il10−/− mice. Possible mechanisms include increased gut
leakiness (thereby exposing the mucosal immune s­ ystem
to luminal microorganisms), impaired tissue restitu­
tion and direct activation of intestinal ­immunity 35–38.
Inflammation has long-lasting effects on visceral hyper-
sensitivity with the potential to influence symptoms
long term. Even after recovery of acute, experimentally
induced colitis (including full mucosal and histological
resolution) there is prolonged alteration in visceral sen-
sitivity with reduced threshold for induction of viscero­
motor responses after colonic distension39. Increased
intestinal permeability and heightened susceptibility to
experimental intestinal inflammation observed in mice
with depressive behaviour (induced by maternal separ­
ation) is reversed by antidepressant therapy 37. Taken
together, these important preclinical data support the
concept that anxiety and depression directly influence
disease biology, not merely gastrointestinal symptoms.
FGIDs are also strongly linked to increased psycho­
logical morbidity, including anxiety, depression,
post-traumatic stress disorder and cognitive impair-
ment 40–44, as well as other ‘functional’ comorbidities,
including chronic pelvic pain, interstitial cystitis, chronic
fatigue syndrome and fibromyalgia45. Patients with IBS
also exhibit evidence of cognitive deficit, including

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impairment of visuospatial memory performance, which
is independent of psychological comorbidity 46. In com-
parison with healthy individuals as controls, patients with
IBS are more likely to be prescribed with ­antidepressants,
anxiolytics, antipsychotics and sedatives47.
Novel brain imaging approaches, including func-
tional MRI (fMRI) scanning is now starting to prov­
ide important new insights into neural activity and
circuitry in FGIDs. fMRI in patients with IBS demon-
strates that anxiety and depression are associated with
disturbed patterns in intrinsic brain function, including
decreased activity in the prefrontal cortex, posterior
cingulate ­cortex and bilateral inferior parietal cortices48.
fMRI studies indicate that cognitive processing of vis-
ceral pain, anticipation of pain and pain-related fear is
defective in patients with IBS, and is typically linked to
excessive engagement of emotional brain networks49–51.
FGIDs are bidirectionally associated with psycho-
logical morbidity. A strong association exists between
FGIDs and psychological morbidity, and the prevalence
of psychiatric symptoms. Anxiety disorders, depression
and somatization disorders are ~50% in these patient
groups40,52,53. Early-life stressors, including a history of
sexual, emotional and physical abuse, are also present
in 26–44% of patients with FGIDs, and is especially
over-represented in patients presenting to second-
ary care clinics54–56. Although FGIDs are undoubtedly
Box 1 | The central and enteric nervous systems
• Innervation of the gastrointestinal tract regulates secretions, sphincter control,
motility, blood flow and enteroendocrine function
• The enteric nervous system (ENS) is an autonomous neural network with a
comparable number of neurons to the spinal cord, and chiefly comprises myenteric
ganglia (between the longitudinal and circular muscle layers of the gut wall)
and submucosal ganglia
• The myenteric plexus circumferentially encases the gut from the upper oesophagus
to the internal anal sphincter, whereas the submucosal plexus is predominantly
present in the small and large intestine
• The three main types of neuron in the ENS are primary sensory neurons, interneurons
and primary motor neurons; these neurons directly synapse with each other to
mediate ENS intrinsic reflexes
• Although the small and large intestine are the key sites of autonomous ENS circuitry,
it is important to appreciate that the ENS is closely integrated and in extensive
communication with the central nervous system (CNS)
• The main lines of communication between the ENS and CNS are the vagus nerve
(proximal gastrointestinal tract) and the spinal nerves (including thoracolumbar and
lumbrosacral networks, which innervate the distal small bowel and colon), all of which
contain both sensory (afferent) and motor (efferent) pathways
• Vagal afferents detecting nutrients, chemicals or luminal contents (chemoreceptors),
and distention or movement (mechanoreceptors or tension receptors) project to the
nucleus tractus solitarius (NTS) via the dorsal and ventral vagal trunks
• Vagal efferents originating in the NTS and nucleus ambiguous directly innervate
striated muscle (e.g. oesophagus), but mostly synapse with enteric nerves to mediate
motor functions (e.g. motility, secretions, sphincter relaxation)
• The thoracolumbar spinal nerves are composed of sensory neurons with cell bodies in
the dorsal root ganglia and efferent sympathetic fibres, which innervate blood vessels
and synapse with myenteric and submucosal ganglia
• The pelvic spinal nerves innervate the rectum and distal colon, and include sensory
(including pain fibres) and motor pathways
linked to increased psychological morbidity, the direc-
tion of causality is uncertain. One possibility is that
psychological disturbance results from chronic and
debilitating gastrointestinal symptoms, reminiscent of
sickness behaviour. Another possibility is that FGIDs
might resemble somatiza­tion disorders, resulting from
primary problems with mood, abnormal emotional
responses and a dysregulated perception of pain, which
manifests with disturbed gut function. Growing evi-
dence supports the possibility that both scenarios exist
and that different FGID phenotypes could exist, with
some patients presenting first with mood disorders
(without gut symptoms) and then later developing gut
symptoms (brain driving gut), whereas others first pres-
ent with FGIDs and then emerge with psycho­logical dis­
turbance (gut driving brain). Convincing data to support
this notion have only just emerged, made possible by
study­ing well-defined study participants in longitudinal
series. In a landmark study, Koloski et al.41 examined a
population of 1,775 randomly selected Australian adults
who completed a validated questionnaire evaluating
gastro­intestinal symptoms and symptoms of anxiety
or depression (the Delusion Symptom States Inventory
tool). Patients were recruited in 1997 and a follow‑up
survey was conducted 12 years later. The key finding
was a clear population of patients with mood disorders
(­anxiety or depression) at inception, who were ­initially
free of FGID, but during follow‑up subsequently devel-
oped FGIDs. Conversely, individuals with FGIDs with-
out anxiety or depression at baseline had a markedly
increased chance of developing anxiety or depression
12 years later 41. The study concluded that the gut was
probably the driver of psychological morbidity in patients
in whom gastro­intestinal symptoms predated mood dis-
order, and the brain was probably the driver of FGIDs
in patients in whom anxiety or depression symptoms
manifested before gastrointestinal symptoms. In an
independent 1‑year prospective follow‑up study of 1,900
people, one-third of individuals had a mood disorder
that preceded FGID symptoms, whereas in two-thirds a
FGID preceded the mood dis­order 57. Although recogni-
tion of the directionality of this association might imply
the existence of distinct mechanisms of disease, it is also
possible that these different modes of presentation (that
is, brain driving gut, or gut driving brain) might instead
represent polar ends of the same disease process. A con-
dition manifests first in one organ system rather than the
other by chance.
Further support for the possibility that psychiatric
disturbance itself predisposes to the subsequent devel-
opment of FGIDs comes from a cohort of patients with
post-infectious IBS (PI‑IBS) who have been longitu-
dinally followed in Walkerton, a rural community in
Canada. PI‑IBS occured in ~10% of patients after an
acute episode of gastrointestinal infection, even after
clearance of the offending pathogen58. Heavy rainfall
in Walkerton in 2000 resulted in contamination of the
regional water source with faeces from grazing live-
stock, which resulted in a large outbreak of acute gastro­
enteritis predomin­antly with Campylobacter jejuni
and/or Escherichia coli O157:H7. The outbreak affected

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>2,000 local residents59. Walkerton residents, includ-
ing those affected by acute gastroenteritis, and other
­unaffected individuals were enrolled in a longitudinal
study to investigate long-term health outcomes in this
cohort. In comparison with unaffected residents, individ­
uals who developed acute infectious gastroenteritis were
subsequently more likely to develop functional gastro-
intestinal symptoms in long-term follow‑up, includ-
ing IBS59 and FD60. Importantly, at least in the case of
post-­infectious FD, gut symptoms were markedly more
likely to develop in patients with pre-­existing anxiety or
depression60, consistent with the possibility that mood
disorders predispose to the development of chronic
gut symptoms. These data are corroborated by other
indepen­dent cohorts of patients in whom IBS symp-
toms developed following acute bacterial gastro­enteritis,
whereby PI‑IBS was more likely to develop in individuals
with pre-­existing psychological morbidity and fatigue61,62.
However, some of this increased risk is explained by the
increased susceptibility of individuals with pre-existing
psychological ­disturbance to infectious gastroenteritis61.
Box 2 | The mucosal immune system63
• CD4+ effector T‑cell subsets are defined by the expression of specific transcription
factors and by the pattern of cytokines produced. Type 1 T helper (TH1) cells produce
IFNγ and TNF, which have many pro-inflammatory actions, including activation of
macrophages. Type 2 T helper (TH2) cells produce IL‑4, IL‑5 and IL‑13, which promote
activation, survival and maintenance of tissue mast cells and eosinophils; the TH2
response is implicated in host immunity to multicellular pathogens, such as helminths.
Type 17 T helper (TH17) cells produce IL‑17A, IL‑17F and IL‑22, which support the
recruitment and activation of neutrophils and have an important role in host
resistance to extracellular bacteria and fungi. Regulatory T cells expressing the
transcription factor FOXP3 serve to counterbalance overly exuberant effector
responses and dampen down inflammation; they secrete immunosuppressive
cytokines, such as IL‑10 and TGFβ, and are one of the main immunoregulatory
mechanisms in the gut (and beyond).
• Key cells of the mucosal innate immune compartment include cells of the mononuclear
phagocyte system (MPS), such as monocytes, macrophages and dendritic cells.
Intestinal macrophages, which are continually replenished by circulating monocytes,
are mostly anti-inflammatory cells in the steady state, producing immunoregulatory
cytokines such as IL-10; however, under inflammatory conditions they are a potent
source of inflammatory cytokines, including TNF, IL‑6 and IL‑1β. Tissue macrophages
exist as M1 (classically activated) and M2 (alternatively activated) subsets64.
M1 macrophages are activated by IFNγ (a major product of the TH1 response), but also
support TH1 activation or differentiation through IL‑12. IL‑4 is a major stimulus driving
alternative activation of M2 macrophages. M2 macrophages are also a source of IL‑4,
which supports TH2 cells65. Intestinal MPS cells are activated by bacterial products
(such as lipopolysaccharide) that trigger Toll-like receptors (TLRs).
• Innate lymphoid cells (ILCs) are a heterogeneous population of newly described innate
lymphocytes that resemble the effector CD4+ lineages with respect to the expression
of lineage defining transcription factors, cytokine production profiles and functional
roles. Key subsets are ILC1 (express T‑bet and produce IFNγ), ILC2 (express GATA3 and
produce IL‑5 and IL‑13) and ILC3 (express RORγt and produce IL‑22 and/or IL‑17A)66.
ILCs contribute to host defense against different mucosal pathogens, including
helminths (ILC2) and bacterial infections (ILC1 and ILC3)66,67. IL‑22‑producing ILC3
also serve to promote epithelial stem cell growth and recovery from injury, indicating
that these cells are crucial to long-term intestinal epithelial homeostasis68,69.
• Immune-cell trafficking to the gut is dependent on the expression of selective homing
molecules, including the chemokine receptor CCR9 and the integrin α4β7 (REF. 70).
The regulation of immune cell trafficking to the gut is now of major interest as
monoclonal antibodies blocking these pathways have emerged into clinical practice
to treat intestinal inflammation71,72.
Role of the immune system: two-way signals
Building on many decades of research, important
advances continue to be made in our understanding of
common pathways responsible for reciprocal modula­
tion of function in the brain and gut. Afferent and effer-
ent nerves and blood-borne humoral factors have the
potential to convey signals between these two organ
systems and, accordingly, factors that regulate these
pathways have the potential to impact on gut–brain
dialogue. The immune system integrates and modulates
bidirectional signals between the brain and the nervous
system and the gut, and consequently mechanistically
links alterations in function in both brain and gut. Many
gastrointestinal diseases are linked to inflammation.
In IBD, these changes are macroscopically evident, but
increasingly FGIDs are also being linked to subtle alter-
ations in mucosal immunity. Consequently, the concep-
tually attractive possibility exists that aberrant mucosal
immune activation directly regulates the effects of stress
and anxiety perception in the brain yet, at the same
time, stress responses originating in the higher cortical
centres have the potential to profoundly affect immune
system activity in the mucosal tissues and directly drive
gastrointestinal symptoms. To put these concepts into
context it is necessary to understand the dynamics of the
stress response and factors that modulate it (including
the immune system), but also to consider the immune
mechanisms operational in common gastrointestinal
diseases, including FGIDs.
Mucosal immune system, ENS and stress
Reciprocal interactions between mucosal immune
system, ENS and stress response. Even under homeo-
static conditions the gut is described to exist in a state
of ‘physio­logical inflammation’ as, in contrast to other
tissues, there is relative expansion of infiltrating lympho-
cytes in the lamina propria and intraepithelial compart-
ments of both the large and small intestine. These cells are
charged with the challenging task of remaining immuno-
logically restrained in the face of trillions of commensal
bacteria, but at the same time poised to defend the vast
surface areas of the gastrointestinal tract from invading
pathogens. Key cellular players of the mucosal immune
system include T cells, mononuclear phagocytes, innate
lymphoid cells and other cells of the innate immune
compartment (BOX 2; FIG. 1)63-72. Together, these cells com-
municate with the epithelium, intestinal microbiota and
ENS. Crucially, immune function is both responsive to,
and exerts influence upon, host stress responses.
The stress response is a rapidly mobilized, tightly
coordinated, multisystem programme invoked in
response to adverse ‘stressor’ conditions, summarized
in BOX 3 and reviewed elsewhere73. The stress response
regulates multiple aspects of immune function and, in
return, several immune mediators are involved in trigger­
ing the stress response and modulating its effects on dif-
ferent organ systems, including the gut. Neurons from
the paraventricular nuclei (PVN) activ­ate the sympa­
thetic arm of the autonomic stress response, includ-
ing direct innervation of immune t­ issue in the gut,
including mesen­teric lymph nodes and gut-associated

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Gut microbiota

Immunity Immunity Immunity to Epithelial

to bacteria to helminths bacteria and/or fungi resitution

Intestinal Histamine Eosinophils

epithelium TNF Neutrophils
Mast
cell Eosinophil
Macrophage granule
IL-4 proteins
IL-5
IFNγ IL-13 IL-5 IL-17 IL-22
TH1 ILC1 TH2 ILC2 TH17 ILC3 NCR+
ILC3
T-bet T-bet GATA3 GATA3 RORγt RORγt RORγt
IL-12

M2 macrophage

M1 macrophage

IFNγ IL-4

Monocyte
Figure 1 | Mucosal immune networks. The mucosal immune system has a crucial role in immunity to microorganisms
Nature
and in epithelial restitution. Distinct arms of host immunity can be mobilized to Reviews | Gastroenterology
defend against & Hepatology
specific microbial threats.
However, selective aspects of these immune response pathways can be inappropriately deployed in immune-mediated
diseases. T-Bet-expressing type 1 T helper (TH1) effector memory T-cells and type 1 innate lymphoid cells (ILCs) respond to
cytokine signals (including IL‑12) to trigger their effector responses, which includes production of IFNγ, which in turn
primes tissue mononuclear phagocytes, such as macrophages and monocytes, programming them for pro-inflammatory
activation. This pathway is especially important in host resistance to intracellular bacteria, including mycobacterial
infection. IL‑12‑producing M1 macrophages support the type 1 inflammatory response, and reciprocally IFNγ supports
M1 differentiation. The type 2 response is characterized by activation of GATA3‑expressing type 2 T helper (TH2) cells
and ILC2, which produce cytokines (such as IL‑4, IL‑5 and IL‑13) to support the activation, recruitment and survival of
eosinophils and mast cells. The type 2 response is implicated in host resistance to helminths. RORγt-expressing type 17
T helper (TH17) and ILC3 (especially natural cytotoxicity receptor (NCR)– ILC3) cells produce IL‑17 family cytokines,
which support the activation and recruitment of neutrophils and have an important role in host immunity to extracellular
bacteria and fungi. NCR+ ILC3 produce IL22, which supports the epithelial stem cell niche by promoting proliferation
of LGR5+ stem cells and also promote the production of antimicrobial peptides by epithelial cells. IL‑22‑producing ILC3
are implicated in host resistance to some mucosal pathogens.

lymphoid tissue (GALT)74. Sympathetic modulation of
immune t­ issue is focused around zones heavily popu­
lated by T cells, such as the cortical and subcortical
areas in lymph nodes, with relative sparing of B‑cell-
rich ­germinal centres74. Noradrenergic fibres extensively
arbor­ize in the interdomal regions of the lamina propria,
which is especially extensive around T‑cell zones75. This
direct line of communication permits rapid and focused
modulation of immune function, and especially T cells
through release of noradrenaline at this nerve-immune
‘synapse’. These data reinforce important work in rodents
that have shown a key role for CD4+ T cells in mediating
stress-induced gut dysfunction. Stress induction through
acoustic stimuli or restraint markedly increases colonic
permeability and reduces the threshold for reinduction of
colitis by administration of intrarectal DNBS38. Crucially,
this phenomenon could be recapitulated in CD8+ T‑cell-
deficient mice, but not in CD4+ T‑cell-deficient mice.
Moreover, adoptive transfer of the CD4+ compartment
to immunodeficient mice could reinstate colitis vulner-
ability in this stress model, confirming the key role of
CD4+ T cells in driving s­ tress-induced gut dysfunction38.
In human IBD, acute stress leads to rapid increases in
rectal immune activation, including increased mucosal
production of TNF and the generation of reactive oxygen
species76. Stress-induced mucosal immune activation
could in part explain the increased intestinal perme­
ability observed in individuals in whom public speak-
ing triggers increased cortisol production77. To enable
immune cells to respond to nerve-derived signals, they
express a variety of different receptors to neurotrans-
mitters, including dopamine78, serotonin (5‑HT)79,80,
neuropeptide Y81, substance P82 and vasoactive intesti-
nal peptide83, although work has mainly focused on the
role of catecholamines in modulating immune function,
mostly through the β2 adrenergic receptor (β2AR)84–87.

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Box 3 | The HPA axis and the stress response73
• Activation of the stress response affects both brain and gut function and profoundly
influences dialogue between these organ systems
• After exposure to perceived threat, sensory inputs are directed and processed
by limbic system structures in the forebrain, including the amygdala, hippocampus
and prefrontal cortex
• The amygdala directly innervates the hypothalamus stimulating release of
corticotropin-releasing hormone into the hypophyseal portal system, triggering
release of adrenocorticotropic hormone from the anterior pituitary gland
• Adrenocorticotropic hormone stimulates cortisol production by the zona fasciculata
of the adrenal cortex
• The amygdala also innervates the catecholaminergic locus coeruleus in the
brainstem, which is the principle source of noradrenaline in the brain, serving
to augment arousal, focus and attention
• Immediate sympathetic activation occurs after integration of stress signals
at the paraventricular nuclei (PVN) of the hypothalamus, which can be considered
the command centre of the autonomic stress response
• Neurons from the PVN project to sympathetic targets in the brain stem, spinal cord
and preganglionic noradrenergic nuclei to activate the sympathetic arm of the
autonomic stress response at target organs; direct and indirect (via synapses at the
nucleus tractus solitarius) projections exist to the locus coeruleus for induction of
centrally acting noradrenaline release
• Neurons from the PVN also directly innervate immune tissue in the gut, including
mesenteric lymph nodes and gut-associated lymphoid tissue
• The adrenal medulla is directly innervated by post-ganglionic sympathetic fibres,
stimulating catecholamine release into the bloodstream; the adrenal medulla
predominantly produces adrenaline and, to a lesser extent noradrenaline,
whereas the postsynaptic sympathetic fibres innervating the gut (and other organs)
are noradrenergic
HPA, hypothalamic–pituitary–adrenal.
Importantly, β2AR is only expressed by type 1 T helper
(TH1) cells but not other T‑cell lineages84 and β2AR liga-
tion in TH1 cells suppresses cytokine production and
activation markers88. Moreover, β2AR agonism impairs
in vitro and in vivo TH1 differentiation by suppressing the
production of the critical crucial TH1‑skewing cytokine
IL‑12 by dendritic cells and monocytes87. As TH1 cells
negatively regulate induction of other T‑cell effector sub-
sets, β2AR‑ligation-induced suppression of IL‑12, which
is mediated through inhibition of NF‑κB and tran-
scription factor AP‑1 (REF. 89), indirectly favours type 2
T helper (TH2)87 and type 17 T helper (TH17)89 differen-
tiation. Interestingly, single nucleotide polymorphisms
at the ADRA1D locus encoding the adrenergic 1D
receptor, which is also involved in sympathetic signal-
ling, are associated with severe IBS symptoms90, lending
further support to the possibility that ­catecholaminergic
­signalling is important in gut symptoms.
Data has also cast new light on the potential role of the
stress response and catecholamine signalling between
the ENS and the innate immune system that would also
favour type 2 immunity in the gut. Using 3D imaging of
intact intestinal tissue and in vivo imaging of mice with
fluorescent tags marking either macrophages or enteric-­
associated neurons, Gabanyi et al.91 demonstrated a
gradient in the functional specialization of intestinal
macrophage networks across the gut wall and their
interaction with enteric nerves. Macrophages in intes-
tinal muscle layers exhibited distinctive morphology
and resided in intimate contact with enteric nerves,
expressing an M2 transcriptional profile. By contrast,
lamina propria macrophages expressed an M1 gene
expression footprint. M2 differentiation of muscularis
macrophages was dependent on β2AR, highlighting the
importance of nerve-mediated instruction of immune
cell differentiation.
This process could be particularly relevant in FGIDs,
for which data continues to amass regarding possible
­activation of type 2 immunity, including tissue eosino­
philia in FD and mast cells in IBS (discussed later).
Noradrenergic promotion of intestinal muscularis
M2 macrophage differentiation or activation might
be ­capable of supporting type 2 immunity in the gut.
At other mucosal surfaces, M2 macrophages directly
promote type 2 immunity through activation of TH2
T cells, IL‑13‑producing innate lymphoid cell (ILC)2
and IL‑33‑dependent eosinophil recruitment 92,93.
Interestingly, duodenal eosinophilia observed in patients
with FD is also characterized by infiltration of eosino-
phils in the duo­denal submucosal plexus94. Additionally,
gastrointestinal pathogen exposure selectively drives
further activation of muscularis-resident M2 macro­
phages in mice, and it is tempting to speculate that
selective induction of type 2 immunity promoting M2
macrophages could potentially support mast cell (IBS) or
eosinophil (FD) expansion and/or activation as observed
in post-infection FGID. M2 macrophage expansion
and/or activation is observed at other mucosal surfaces
in the context of TH2‑mediated disease95. Catecholamines
could also affect other aspects of immune function,
including humoral immunity, l­eukocyte ­development
and leukocyte trafficking 74.
ILCs also form important interactions with enteric
glial cells. The outcome of this interaction has profound
implications for epithelial homeostasis in the gut, the
composition of microorganisms colonizing the gastro­
intestinal tract and host susceptibility to intestinal inflam-
mation. Neurotrophic factors are proteins prod­uced by a
variety of tissues to support development, differenti­ation,
maintenance and survival of neurons. The extended
family of neurotrophic factor proteins include glial-cell
derived neurotrophic factor (GDNF) and GDNF family
ligands (GFL), such as neurturin, artemin and persephin,
all of which are ligands for the tyrosine-protein kinase
receptor RET96. Surprisingly, using RET reporter mice,
intestinal ILC3s have been shown to also express RET97.
In the gut, enteric glial cells formed stellate projections
to interact with ILC3 in cryptopatches. These glial cells
were a major source of GFL, which was a power ­trigger
to induce IL‑22 by ILC3. Selective deletion of RET in
ILC3 resulted in severely diminished IL‑22 produc-
tion by ILC3, reduced production of antimicrobial
defences, dysbiosis, increased bacterial translocation
across the undefended gut wall and, ultimately, height-
ened susceptibility to gastro­intestinal infections and
intestinal inflammation97. Crucially, enteric glial cells
produce GFL in response to Toll-like receptor (TLR)
agonists or the alarmin cytokines IL‑1β and IL‑33 in a
MYD88‑dependent manner 97. These data indicate that
enteric nerves can respond to presence of microorganisms

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through TLR sensors, or early cytokine stimuli to trigger
ILC3 activation through production of GFL acting on the
RET receptor. Key interactions between the gut, immune
system and brain are depicted in FIG. 2.
Immune activation in FGID. Although FGIDs are con-
ventionally defined by the absence of clear-cut patho­
logical changes, such as overt macroscopic inflammation,
a shift in momentum has emerged to challenge this view-
point. Numerous studies, although not all, have identified
subtle immune changes in the gut of patients with FGIDs
(TABLE 1). Although ongoing debate remains regarding
the relevance of these findings as primary pathological
events, or bystander epiphenomena, the evidence in
favour of low-grade inflammation contributing to the
aetiology and symptomatology of FGIDs grows ever
more persuasive. Notably, there is a high prevalence of
functional gastrointestinal symptoms in patients with
recognized inflammatory diseases of the gut, including
IBD, infectious gastroenteritis, coeliac disease and micro-
scopic colitis even after sustained resolution of overt
inflammation58,98–101. Moreover, patients with FGIDs are
at increased risk of developing other immune-mediated
diseases102, consistent with a possible immune diathesis
in FGID. Numerous studies have described increased
pro-inflammatory cytokines (for example, IL‑6, TNF and
IL‑1β) in the serum or plasma of patients with FGID,
or increased production of these cytokines following

Stress response

Gut microbiota

AMP
Intestinal
epithelial
cell IL-22R IL-22 Mast Histamine
IL-4 cell
ILC3
Lamina RET
propria
GFL TH2 cell
IL-5
Enteric β2AR
neuron Eosinophil Eosinophil
NA
granule proteins
Circular
muscle

Myenteric ENS
plexus

M2 macrophage

Longitudinal
muscle

Systemic circulation
Cytokine-producing cells
(IL-1β, TNF, IL-6)
Figure 2 | Key brain–immune–gut interactions. There is complex crosstalk between the immune system, the brain
and the gut. Noradrenergic neurons in the gut release noradrenaline (NA),Nature which Reviews | Gastroenterology
ligates the & Hepatology
β2 adrenergic receptor (β2AR)
on macrophages in the myenteric plexus (supporting differentiation towards the M2 phenotype) and T cells, which limits
T helper (TH)1 differentiation (indirectly favouring TH2 and TH17 differentiation). Mast cells and eosinophils are found
degranulating next to enteric neurons, providing a mechanism for sensory excitation (which can be perceived by the
enteric nervous system (ENS) and central nervous system). Gut immune cells (e.g. γδ T cells) differentiating in the gut can
traffic to the brain under some circumstances (e.g. after brain injury). Blood-borne cytokines generated in the gut
can also signal in the brain. Enteric glial cells also produce glial-cell-derived neurotrophic factor family ligands (GFL),
which stimulate IL‑22 production by innate lymphoid cell (ILC)3 (acting on the specific receptor RET). IL‑22 acts on
an epithelial‑restricted receptor (IL‑22R) to stimulate epithelial proliferation, antimicrobial peptide production.
IL‑4 and IL‑5 (potentially generated locally by TH2 T cells) support activation of mast cells and eosinophils, respectively.
AMP, antimicrobial peptides.

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short-term culture of peripheral blood leukocytes iso-
lated from patients with FGID, often at concentrations
correlating with disease features or psychological mor-
bidity 103–108. Some studies have detected mucosal abnor-
malities in patients with FGID. Unsupervised profiling of
differentially expressed genes in colonic biopsy samples
from patients with IBS and healthy individuals shows
that the majority of differentially expressed genes localize
to the mucosal immune system109,110.
In IBS, arguably the most compellingly evidence
for a role of disordered immunity comes from studies
examining mucosal mast cells. Increased numbers of
mucosal mast cells have been observed in patients with
IBS62,111–116 (FIG. 3a), often correlating with key symptoms
such as abdominal pain111,115 or bloating 116, and might
be seen degranulating in close proximity to enteric
nerves111. Production of mast cell products, including
tryptase, histamine and prostaglandins, is increased in
gut tissue from patients with IBS111,117, and jejunal fluid
harvested from patients with diarrhoea-predominant
IBS have increased levels of tryptase112. In vivo admin-
istration of supernatants derived from explant cultures
from patients with IBS to the artery supplying a loop of
jejunum in experimental rats induced marked excitability
of visceral sensory nerve fibres and triggered excessive
calcium flux in dorsal root ganglia, effects which were
not observed following administration of explant culture
supernatants from control individuals117. Submucosal
nerves harvested from the distal colon of Guinea pigs also
exhibit exaggerated excitability when exposed to culture
supernatants derived from explants from patients with
IBS118. Consistent with mast cell degranulation driving
symptoms in IBS, histamine potentiates triggering of
colonic submucosal neurons with the TRPV1 agonist
capsaicin119. Histamine potentiated colonic nocicep-
tive afferent nerve fibres were mediated through the
histamine H1 receptor (HRH1), as the effect could
be blocked by HRH1 antagonists and were blunted in
­neurons harvested from Hrh1−/− mice119. Mast cells are
also implicated in loss of intestinal barrier function in
stress models. A marked expansion of colonic mast cells
and increased intestinal permeability is observed in rats
following stress induction by water deprivation; how-
ever, the permeability defect can be rescued when stress
is induced in m­ ast-cell-deficient animals (Ws/Ws rats)120.
A similar picture is emerging in FD, although rather
than primarily mast cells, a disordered homeostasis
of eosinophils in the proximal small bowel also seems
involved (FIG. 3b,c). In a landmark study of non-health-
care seeking individuals from Sweden who completed
validated gastrointestinal symptom questionnaires and
underwent upper gastrointestinal endoscopy, eosino-
phils were found to markedly increased in the proximal
duodenum in 40% of individuals reporting FD symp-
toms121. Subsequent work has corroborated these find-
ings and consistently links early satiety, a hallmark FD
symptom, with expansion of duodenal eosinophils122–125.
In patients with FD, accumulating duodenal eosinophils
are activated, degranulating in close proximity to enteric
nerve fibres.These observations have been extended,
showing that eosinophils and mast cells are massively
expanded in the duodenal submucosal plexus of patients
with FD94. In this elegant study the researchers were able
to quantify neuronal activity by measuring calcium flux
in submucosal nerve fibres. Although the number of
nerves and ganglia present in the submucosa of patients
with FD was comparable to healthy individuals, there was
­striking impairment of nerve excitability in FD. Moreover,
blunting of submucosal nerve fibre excitability correlated
with eosinophil and mast cell infiltration, highlighting
the potential importance of interactions between enteric
nerves and immune cells in the pathogenesis of FD.

Table 1 | Evidence for immune dysregulation in FGID

Focus Evidence
Epidemiology • High prevalence of ‘functional’ symptoms in patients with overt gastrointestinal inflammation,
including IBD (even in remission) and coeliac disease58,98–101
• Increased prevalence of other autoimmune diseases in patients with FGID102
Mast cells • Mast cells and their degranulation products (e.g. histamine, tryptase) are increased in colon or small
bowel of patients with IBS62,111–117
• Tissue mast cell numbers correlate with IBS symptoms111,115,116
• Antihistamines and mast cell stabilizers are effective in IBS119,204,205
Eosinophils Increased numbers of eosinophils in the duodenal mucosa and submucosal plexus, which correlate
with presence of functional dyspepsia symptoms94,121–125
Lymphocytes • Increased numbers of intraepithelial lymphocytes in IBS62,112,115,129
• Increased numbers of gut-homing lymphocytes in peripheral blood of patients with FGID,
which correlate with symptoms107,128
• Circulating lymphocyte numbers rapidly decrease in the postprandial phase in IBS130
Nerve/immune • Mast cells can be observed degranulating in close proximity to enteric nerves111
interactions • Histamine potentiates firing of colonic pain afferents119
Cytokines • Increased levels of cytokines in serum or plasma, or following short-term culture of peripheral blood
leukocytes from patients with FGID103–108
• Dysregulated T‑cell cytokine responses (shift to type 2 T-helper responses) in FGID127
Genomics • Immune genes are differentially expressed in gut biopsy samples from patients with IBS109,110
• Polymorphisms at loci encoding immune (IBD-associated) loci in IBS (e.g. TNFSF15) 216,217
FGID, functional gastrointestinal disorder.

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consistent with the possibility of increased gut homing

a b
c produces type 2 cytokines capable of supporting and
Figure 3 | Low-grade inflammation inNature | Increased
FGID. aReviews numbers of tryptase-positive
| Gastroenterology & Hepatology
mast cells (brown staining) in the colon of patients with IBS. b | Expansion of duodenal
eosinophils in the duodenal mucosa of patient with functional dyspepsia. c | Eosinophil
degranulation in duodenal mucosa of a patient with functional dyspepsia (carbol
chromotrope stain, arrow) adjacent to nerves (S100 immunostaining, arrowhead).
Inset: intact eosinophil. Magnification for parts a and b ×20, for part c ×40 and inset ×100.
FGID, functional gastrointestinal disorder.
The underlying factors responsible for initiating and
sustaining duodenal eosinophil and mast cell accumu­
lation in patients with FD have yet to be resolved,
although it has been hypothesized that dysregulated
CD4+ TH2 responses could be responsible, as these
cells are potent producers of key cytokines involved in
supporting the recruitment, survival and activation of
eosinophils and mast cells, such as IL‑4, IL‑5 and IL‑13
(REF. 126). Furthermore, there is experimental support
for this hypothesis, as polyclonal stimulation of T cells
isolated from patients with FD results in markedly
increased prod­uction of IL‑5 and IL‑13, with a recipro­
cal reduction in the production of the TH1 cytokine
IFNγ127. Consistent with a functionally important role
for T cells in FGIDs, T cells expressing gut-homing
integrins and chemokine receptors, including α4β7
and/or CCR9, are markedly increased in patients
with IBS128 or FD107. In the case of FD, the number of
α4β7+CCR9+CD4+ T cells is p ­ ositively corre­lated with
FD symptoms (such as abdominal pain, ­nausea and
vomit­ing) and with reduced gastric empty­ing, measured
by scinto­graphy 107. Similar to the situ­ation in acute ulcer­
ative colitis, MAdCAM1, the ligand for the gut-­homing
integrin α4β7, is markedly upregulated in the colon
of patients with IBS relative to healthy individ­uals128,
of pathological T‑cell lineages in IBS. Some studies have
also identified increased numbers of gut T cells in IBS,
particularly in the intra­epithelial compart­ment62,112,115,129.
Consistent with gut lymphocyte trafficking being
influenced by food intake, in patients with IBS a rapid
reduction in the number of circulating lymphocytes in
the post-­prandial phase is seen130. The emergence of
ILCs as mediators of intestinal inflammation66 raises
the ­intriguing possibil­ity that these cells might also be
involved in FGIDs, and especially the ILC2 subset that
activating mast cells and eosinophils. Notably, IL‑1β,
which is excessively produced by peripheral blood
mono­nuclear cells from patients with FD107, activates
ILC2 to produce IL‑5 (REF. 131). The potential role of
ILCs in FGIDs is currently being addressed.
Although local mucosal immune activation might
plausibly be expected to affect gastrointestinal physio­
logy (and ultimately symptoms) through interaction with
enteric nerves and gut smooth muscle, cytokines prod­
uced by immune cells might also act centrally to affect gut
function. For example, IL‑1β and TNF-mediated inhib­
ition of gastric emptying is achieved at much lower doses
when these cytokines were administered intra­cisternally
(compared with intravenously), is dependent on an intact
vagus nerve, and in the case of IL‑1β, is reversible by
intracisternal IL‑1β antagonists132,133.
Immune regulation of brain function
Extrinsic and intrinsic routes. Ostensibly, the immune
system has at least three avenues of communication
to conduct dialogue with the brain. Firstly, cytokines
(or hormones) generated by immune cells or other line­
ages in the gut (or elsewhere in the periphery) could be
transported in the blood to act on the brain (humoral
route). Other potentially relevant humoral factors
include bacterial cell wall products, such as lipopoly-
saccharide, as these ligands can trigger TLRs expressed
by microglial cells in the CNS (discussed later). Second,
immune cells in the gut could directly communicate
with afferent sensory fibres and/or enteric nerves, often
through production of cytokines or other immune
mediators (such as hista­mine) to trigger signals that
are ultimately relayed to the brain via neurons. Third,
circulating immune cells have the potential to ­traffic
to the brain, where they can liberate cytokines and
other inflammatory mediators in close proximity to
relevant brain structures. T‑cell trafficking from the
gut to the brain has been elegantly demonstrated using
KikGR33 mice that constitutively express Kikume green-
red (KikGR) fluorescent protein that converts from a
green to red fluorescent tag after exposure to ultraviolet
light. T cells in the intestine were photo­converted into
red-­fluorescent-tagged cells by exposing the small intes-
tine to UV light at laparo­tomy. However, red-­fluorescent-
tagged T cells that could only have been generated in
the intestine were also recovered from the meninges
and cervical lymph nodes after ischaemic injury to
the brain134. These data confirm that, under defined
inflammatory settings, T cells can traffic from the gut to

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the brain. Intriguingly, T cells destined to traffic to the
gut or the brain share expression of the tissue-homing
­integrin α4, although they have distinct β chains70.
Overall, the most evidence has been amassed regard-
ing how cytokine networks modulate brain function.
Cytokines are a family of >100 small protein molecules
that can access the brain through regions of the blood–
brain barrier (BBB) that are prone to leakiness. Access
occurs through active uptake, modulation of the vascular
endothelium of vessels supplying the brain and by acting
on peripheral vagal afferents in the gut, which in turn
relay signals to pertinent nuclei in the brain, including
the nucleus tractus solitarius (NTS) and hypothalamus135.
Cytokines might also act centrally to potentiate sensory
afferent inputs from the gut. Indeed, in animal models,
TNF activates the neurons at the NTS that are respon-
sive to gastric distension136. Although many different
cytokines have been shown to modulate brain activity,
IL‑6, TNF and IL‑1β might be regarded as the ‘unholy
trinity’ of cytokines that affect brain function at multi-
ple levels. These cytokines are also known to activate the
sympathetic nervous system and HPA axis, which in turn
have direct effects on the brain74.
In addition to these ‘extrinsic’ routes of immune regu­
lation of brain function, it is also important to realise
the importance of brain ‘intrinsic’ immune regulation.
Microglial cells are the key immune cells of the CNS.
These cells are distributed throughout the brain and spinal
cord, but are especially abundant in the grey matter, where
they extensively branch and communicate with neurons
and other cells of the CNS. Microglia differentiate from
the same immediate haematopoietic precursor as the
closely related monocyte–macrophage lineage. However,
unlike intestinal macrophages, which are continually
replenished by circulating monocytes, the microglial
compartment is mostly maintained by self-renewal or
in situ proliferation, although in overwhelming infection
or injury, microglial replenishment and/or expansion can
be supported by circulating monocytes137. As might be
expected, microglia share functional character­istics with
other cells of the mononuclear phagocyte system (MPS).
For example, they are phagocytic, express TLRs and
MHC class II molecules, and produce pro-­inflammatory
cytokines, particu­larly in the activated state138,139. Microglia
respond to TLR engagement by initiating potent cytokine
responses. Ligation of TLR3 triggers microglia to produce
TNF, IL‑6 and IL‑12 (REF. 139), highlighting the critical
role of brain immune cells in sensing threat and initiating
inflammatory ­cascades. Some of these responses might be
important for shaping neuronal repair following injury.
Indeed, consistent with a neuroprotective role of TNF,
genetic deletion of this cytokine (Tnfa−/− mice) results in
impaired regener­ation of oligo­dendrocytes after toxin-­
induced demyelin­ation140. In addition to the reparative
roles of microglial cells, innate immune sensing of patho­
gens through TLRs has a central role in host resistance
to brain infections, including pneumococcal meningitis
(TLR2 and TLR4)141, herpes simplex virus (HSV) and
encephalitis (TLR3)142. Indeed, otherwise healthy c­ hildren
carrying dominant-­negative TLR3 alleles are suscep­
tible to HSV infection142. Dysregulated TLR expression
and/or function and inappropriate activation of brain
myeloid cells, including microglia, plays a central part in
neuroinflamamtion and are strongly implicated in other
brain diseases, including multiple sclerosis (MS) and
Alzheimer disease (AD). In MS, the relative contribution
of microglia and other cells of the MPS remains contro-
versial. On the one hand they are key reparative agents in
the brain involved in restitution following injury (includ-
ing experimental demeyelin­ation), versus their potent
pro-inflammatory roles as producers of harmful effector
cytokines (for example, TNF), and cytokines involved in
the differentiation and maintenance of pathogenic TH17
and TH1 cells (for example, IL‑1, IL‑6, IL‑23 and IL‑12)143.
Microglia and other MPS cells also contribute to clearance
of extracellular amyloid‑β deposits, and disruption in the
balance between amyloid‑β accumulation and micro-
glial or MPS-mediated removal is probably a key patho­
logical event in the development of AD. CD14 (the TLR4
co‑­receptor) expressed by microglial cells directly inter-
acts with amyloid‑β and facilitates its clearance through
phagocytosis144. Why this process is impaired in AD is
currently under scrutiny, although impaired expression
of TLRs by microglia in AD and its potential link to defec-
tive phagocytosis of amyloid‑β is currently being actively
pursued. How the role of microglia and other immune
cells resident in the CNS behave and contribute to brain
dysfunction in systemic diseases, particularly to gut dis-
eases including FGIDs, is currently unknown. However,
it might be expected that TLR engagement could result
from impaired gut barrier function and bacterial trans-
location in numerous gut diseases. Increased intestinal
permeability is observed in IBD145 and in FGIDs146, which
is associated with increased concentrations of bacterial
products, including the TLR agonists LPS (TLR4) and
flagellin (TLR5) in the serum of these patients147,148.
Cytokines modulate mood. The role of cytokines in mood
disorders has been intensively studied, and even the
possibil­ity that these agents might serve as primary caus-
ative agents in depression has been debated149. Some have
shown dysregulated cytokine levels in biofluids, including
serum, plasma and cerebrospinal fluid of patients with
severe idiopathic depressive symptoms135. Other immune
changes include shifts in the numbers of monocytes,
neutro­phils and lymphocytes, as well as T‑cell activation
status and serum complement levels150,151. Patients treated
with cytokines for other disease states are also prone to
developing depression. For instance, IFNα administered
to treat hepatitis C frequently induces depressive symp-
toms152 and patients with increased baseline serum TNF
levels are especially susceptible153. These observations are
supported by mouse studies in which administration of
recombinant cytokines, or the immune activator LPS,
induces sickness behaviour in experimental animals with
features of depression154. For example, in rodents, intra­
cerebral injection of recombinant IL‑1β or TNF induces
pyrexia, sleepiness and hyperalgesia in a dose-­dependent
manner 155,156. IL‑6, TNF and IL‑1β in particular are also
potent activators of the stress response, activating the
HPA axis at multiple levels, including activ­ation of neu-
rons containing corticotropin-releasing hormone in the

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PVN of the hypothalamus, and even direct activation
of the adrenal cortex 157,158. Consistent with the prevail-
ing view that depression is closely linked to disordered
neurotransmitter homeostasis, cytokines (again, mostly
IL‑1β, IL‑6 and TNF) have been shown to regulate
multiple different neurotransmitters, including central
noradrenergic (predominantly IL‑1β), dopaminergic and
serotonergic systems149.
Cytokines disrupt blood–brain barrier function.
Maintenance of the microvascular endothelial barrier
has an important role in regulating brain function, and
cytokines play an important part in regulating this process.
TNF and IL‑6 increase the permeability of brain micro­
vascular endothelial cells, which culminates in disrup-
tion of the BBB159,160. Mechanistically, this process occurs
through induction of reactive oxygen species and NF‑κB,
which repress tight junction proteins, such as occludin,
claudin‑5 and zonula occludens‑1, at the transcriptional
and protein level in a dose-dependent ­manner 159,161,162.
High-dose TNF might even directly ­trigger brain micro-
vascular endothelial apoptosis in vitro160. Importantly, the
BBB itself is an immune-active compartment and astro-
cytes, which have a key role in the formation and mainten­
ance of the BBB, express TLRs, enabling them to sense
and respond to microbial exposure. Engagement of TLR3
­triggers astrocytes to produce cytokines, such as IL‑6,
IFNβ but also IL‑10 (REFS 139,163).
The microbiota–gut–brain axis
Molecular characterization of the microbial communi­
ties occupying the healthy human gastrointestinal
tract demonstrates that >90% of the species occupy-
ing this ecological niche belong to two main phyla, the
Firmicutes and Bacteroidetes, with minor contributions
from Actinobacteria and Proteobacteria164. However,
the composition of intestinal bacterial communities is
disrupted in IBD and FGIDs, and some aspects of the
gut microbiota crucially regulate selective immune
response networks.
The gut microbiota community structure is disrupted
in IBD and FGIDs. Interactions between the microbial
communities that colonize the human gastrointestinal
tract and their host have far-reaching consequences for
host health, including governing susceptibility to disease.
In a number of disease states, including gastro­intestinal
disease, there are shifts in the community structures of
the intestinal microbiota and growing evidence points to
the likelihood that these changes are not epi­phenomena
occurring secondary to the underlying disease pro-
cess, but instead have integral roles in disease patho-
genesis and/or clinical course. The dys­biosis present in
patients with IBD is character­ized by loss of bacterial
diversity, contraction of some phyla, such Bacetroidetes
and Firmicutes, and expansion of others (such as
Proteobacteria)165. Some s­ pecies, such as adherent-­
invasive Escherichia coli have been pathologically
implicated166. Other species, such as Faecalibacterium
prausnitzii and Roseburia ­hominis are selectively depleted
in patients with IBD167,168. Interestingly, F. prausnitzii
possesses anti-inflammatory properties in vitro and
in vivo, potentially explaining why its absence has been
linked to worse outcomes in patients with Crohn's dis-
ease167. Reduced intestinal microbiota diversity is also
a feature of IBS169–171 and has been linked to expansion
of gut lymphocytes and increased ­anxiety 170. Other
data support a shift in the relative proportion of major
bacterial phyla in some patients with IBS, character­
ized by a relative expansion of Firmicutes and contrac-
tion of Bacteroidetes172. Interestingly, F. prausnitzii, the
bacterium that seems to provide anti-­inflammatory
functions in IBD, is also depleted in patients with
diarrhoea-predominant IBS169.
Gut microbiota regulate key aspects of host immunity.
The composition of intestinal microbiota has an impor-
tant role in shaping host immunity, including influences
over cytokine expression, both in the gut and in sys-
temic tissues. Germ-free mice have numerous immune
abnormalities, including grossly enlarged caeca, failure
of second­ary lymphoid development (for example, lym-
phoid follicles), impaired antibody responses, diminished
numbers of T cells and B cells and defective production of
cytokines (such as TNF, IL‑6 and IL‑1β)173,174. Moreover,
selective constituents of the gut microbiota shape speci­
fic aspects of adaptive and innate immunity, including
the differentiation of particular effector T‑cell lineages.
For example, unless mice are colonized with bacteria that
can adhere to intestinal epithelial cells, such as segmented
fila­mentous bacteria Citrobacter rodentium or E. coli
0157, they fail to mount effective TH17 responses175,176.
Other bacteria, including Listeria, are permissive for host
TNF, IL‑1β and TH1 responses176–179. Conversely, some
bacteria promote immunosuppressive cytokines, such as
IL‑10 by regulatory T cells, which can be dependent on
bacterial prod­uction of short-chain fatty acids (SCFAs)
by ­clusters of Clostridia180,181, or polysaccharide A derived
from Bacteroides fragilis 182. Together, these data show
that the precise composition of the intestinal microbiota
can qualitatively and quantitatively alter host immune
responses, with clear implications for how this step might
influence the concentration and profile of cytokines
present in any given individual, which in turn has the
capacity to differentially affect brain function. Microbial
influences over T‑cell differentiation, including lineage
fate decisions, are illustrated in FIG. 4.
The intestinal microbiota modulates brain function.
Mounting evidence highlights the importance of the
intestinal microbiota in modulating multiple features
of brain function, including the HPA-axis-regulated
stress response, emotional responses, memory, motor
behaviour and aspects of social interaction. One of the
key areas of study to date concerns the reciprocal role
of the intestinal microbiota and the stress response.
Stress alters the community structure of the gut micro-
biota183, but experimentation with germ-free rodents has
again provided clear evidence that the intestinal micro­
biota directly affects stress responses and thresholds for
anxiety. Pioneering work by Sudo et al.184 convincingly
demonstrated that the intestinal microbiota substantially

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Gut microbiota In addition to reduced anxiety, other key behav-

SFB SFB Bacteroides fragilis
Listeria Citrobacter rodentium Clostridial clusters
Escherichia coli 0157
Intestinal
TH1 TH17 Treg epithelium
IFNγ IL-17A
TNF
M1 macrophage Neutrophils
Figure 4 | The gut microbiota directlyNature
influences T‑cell
Reviews differentiation. Specific
| Gastroenterology & Hepatology
bacterial constituents of the gut microbiota are required for lineage differentiation of
different T‑cell subsets. Bacteria, including segmented filamentous bacteria (SFB),
Citrobacter rodentium and Escherichia coli 0157 (which are all epithelial adherent
bacteria) are permissive for T helper (TH)17 differentiation. SFB and Listeria are also
implicated in the differentiation of TH1 cells. Regulatory T‑cell (Treg) numbers are boosted
by the presence of selective clostridial clusters and Bacteroides fragilis.
affected host responses to stressful stimuli. A twofold
increase in the magnitude of the HPA stress response,
as measured by increases in plasma adrenocorticotropic
hormone and corticosterone, was detected in germ-free
mice following restraint-induced stress, in comparison
with conventionally colonized mice. Reconstitution
of germ-free animals with probiotic bacteria, such as
Bifidobacterium infantis suppressed the stress response,
whereas monoassociation with enteropathogenic E. coli
(EPEC) further amplified the stress response, demon-
strating that it is not just the presence or absence of intes-
tinal bacteria that influence the stress response, but also
qualitative compositional changes with individual ­species
having profoundly different effects. Unlike B. infantis,
reconstitution with EPEC triggered a notable increase
in plasma IL‑6 and IL‑1β levels. Crucially, reconstitution
with a mutant strain of EPEC that cannot bind to the
intestinal epithelium failed to trigger the same magni-
tude of stress response and was unable to induce robust
IL‑6 and IL‑1β responses. Other work has corroborated
these findings and also investigated behavioural conse-
quences associated with the germ-free state. Curiously,
these experiments tend to show reduced anxiety in
germ-free animals185–187. Subsequent work has shown
that prolonged or short-term stressful stimuli disrupt
the community structure of bacteria present in both the
faecal stream and colonic mucosa183,184,188–190, and specific
changes induced include contraction or loss of beneficial
probiotic-like lactobacilli188–190 and expansion of particu-
lar genera, including Coprococcus and Pseudobutyrivibrio
— coloniza­tion of which was associated with more pro-
nounced production of proinflammatory cytokines, such
as IL‑6 (REF. 183). Stressful stimuli might also facilitate
colonization with overtly pathogenic bacteria, such as
C. rodentium, which can induce a s­ ubstantial local
inflammatory response and overt colitis191.
ioural and cognitive changes observed in germ-free
animals include increased motor activity, impaired
memory and defective social functioning, including
reduced time spent in social investigation and increased
engagement of repetitive grooming activity during
social interaction187,192,193. Data has demonstrated how
the intestinal microbiota influences brain injury, and
moreover, has identified the mucosal immune system
as the key driver of this phenomenon. By reducing
the complexity and diversity of the gut microbiota
with antibiotics, treatment for brain infarct volume is
markedly reduced in mice following transient middle
cerebral artery occlusion134. Crucially, protection from
ischaemic injury was conferred by defective differ-
entiation of microbially induced, pro-inflammatory
intestinal γδ T cells, and a failure of these cells to then
traffic to the brain after injury. In mice colonized with
conventional, diverse bacterial communities, robust
differentiation of IL‑17‑producing γδ T cells in the gut
was found. These cells were then observed to traffic to
the brain following ischaemic injury, where they propa­
gated a pro-inflammatory programme that exacer­
bated tissue damage. However, in mice with reduced
bacterial diversity induced by antibiotic treatment,
impaired differentiation of IL‑17‑producing‑γδ T cells
in the gut was found, with fewer of these cells migrat-
ing to the meninges or cervical lymph nodes, thereby
limiting post-ischaemic inflammatory tissue damage.
Intriguingly, work indicates that ischaemic or traumatic
brain injury itself can drive changes in the intestinal
microbiota in mice 194. These changes were mostly
attributed to activation of injury triggered noradrener­
gic stress responses. Noradrenaline was increased in
the caecum following stroke and, moreover, the shifts
in the composition of the microbial communities
observed following stroke could be recapitulated by
pharmacologically increasing noradrenaline release.
This work corroborates other data showing how
the stress response markedly affects composition of the
gut microbiota.
Gut microbiota direct enteric neurogenesis. The intes-
tinal microbiota serve to fine-tune the development of
a mature, functional ENS. The majority of the inter-
connecting neural circuits of the outer myenteric plexus
and the inner submucosal plexus are formed during
embryogenesis under the direction of specific transcrip-
tion factors (such as SOX10, FOXD3 and PHOX2B),
crucially coupled in a coordinated developmental pro-
gramme to the simultaneous formation of the other
key structures of the gut wall, including the epithelium,
muscle layers and lymphoid structures195. Integration
of these units into functional circuits continues post-
natally and signals from intestinal microorganisms,
which rapidly colonize the gastrointestinal tract in the
immediate postpartum period, help to shape the func-
tional maturation of this system. Germ-free mice have
reduced density and number of nerves in the myentric
plexus and diminished spontaneous smooth muscle
­contractility in the gut wall196.

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The gut microbiota also regulate changes in the CNS.
As already discussed (summarized in BOX 1), the ENS
and CNS are highly integrated and major changes in
the growth and development of both of these systems
occur at a time that coincides with de novo population
of neo­natal gut with bacteria. Experiments in germ-free
animals have again highlighted structural and func-
tional changes in the brain that are specifically linked to
bacterial coloniza­tion of the gastrointestinal tract. Gut-
microbiota-regulated CNS structural changes are focused
in the limbic system, at centres associated with m­ emory,
behaviour and emotional response. For ­e xample,
in germ-free mice, there is volumetric expansion of the
amygdala and hippocampus, and individual neurons
in these regions show striking morphological changes,
including dendritic hypertrophy of amygdala neurons198.
Indeed, early seminal work showed reduced expression
of key neutrophins, such as brain-derived neurotrophic
factor, in the hippocampus of germ-free animals184.
Stress response
• Psychotherapy
• CBT
• Antidepressants
• Anxiolytics
Therapeutic implications
The intimate, yet complex, interactions between the gut,
brain, immune system and intestinal microbiota have
far-reaching implications for host health. Looking for-
ward, the therapeutic horizon in FGID and IBD is likely to
see interventions targeting these systems as primary treat-
ments. These therapeutic paradigms and their p ­ otential
inter-relationships are represented in FIG. 5.
Psychological interventions. Psychological interventions,
including psychotherapy, hypnosis and anti­depressant
therapy, are effective in treating IBS197, although respon­
ses are variable, in keeping with the possibility that some
patients’ psychological distress precedes IBS (brain-gut
directed) and in others it follows on from IBS (gut-
brain directed). Cognitive behavioural therapy (CBT)
shows promise as a psychological intervention in IBS199
and large randomized controlled trials (ACTIB and
IBSOS) evaluating CBT in comparison with conventional
care or placebo are underway and might yet inform fur-
ther novel evidence-based practice200,201. Active screening
to enable early identification of psychological disturbance
in FGIDs will probably be key to considering which
psycho­therapeutic interventions are most likely to be
effective. Other strategies targeting stress alleviation are
also being considered, including self-­directed therapies
and mindfulness-based stress ­reduction, although robust
efficacy data are currently lacking.

Immune interventions. As inflammation could have a

role in driving gastrointestinal and psychological symp-
Disordered motility Immune activation
Visceral hypersensitivity • Anti-cytokine mAbs toms in FGIDs, it is tempting to speculate that targeted
• Antispasmodics • Antihistamines immunotherapies might be effective new treatments in
• Antidepressants • Topical steroids this arena. Notably, oral prednisolone had no effect on
• Autonomic blockade • Mast cell stabilizers PI-IBS symptoms (although it did reduce mucosal T‑cell
• Other
immunosuppressants
numbers)202. The efficacy of prolonged courses of high-
dose topical steroids remains to be seen. Likewise, it has
yet to be established whether selectively targeting individ-
ual cytokines with monoclonal antibodies will uncouple
the brain–gut axis and simultaneously treat inflam­
mation and inflammation-associated brain dysfunction.
Dysregulated
intestinal microbiota Biological agents targeting TNF, IL‑1β and IL‑6 among
• Antibiotics others, are already used in the clinic for a broad range of
• Probiotics inflammatory diseases63,203. Ongoing clinical trials inves-
• Prebiotics tigating blockade of individual mediators might provide
• FMT
• Dietary modification
insights into how they drive psychological aspects of
disease independently of their effects in resolving tissue
inflammation, and furthermore, might highlight the
effectiveness of these strategies at treating psychological
manifestations. Given the potential importance of mast
cells (IBS) and eosinophils (FD) in FGIDs, alternative
immune interventions targeting these cells might be
Figure 5 | Therapeutic paradigms for interfering with the brain–gut axis. expected to yield fruitful results. Historical studies have
Interventions directed at either the brainNature
or gutReviews
aspect of| Gastroenterology
the axis might be anticipated
& Hepatology shown possible efficacy of mast cell stabilizers in patients
to alleviate clinical features in both organ systems. For example, in patients in whom with IBS204,205, and a proof‑of‑principle pilot study in IBS
gut symptoms are primarily driving brain symptoms, modulation of the microbiota
antagonizing the histamine receptor HR1H has shown
(e.g. faecal microbiota transplantation (FMT) or probiotic therapy) would directly alter
host immune responses, alleviate local immune activation and simultaneously reduce promising results, including reduced abdominal pain
immune-driven brain dysfunction. Likewise, targeting the stress response and reduced visceral hypersensitivity in comparison with
(e.g. psychotherapy or antidepressants) would alleviate brain symptoms, but would placebo-treated patients119. In the future, antagonists that
also limit autonomic activation of host immunity to suppress aberrant immune-driven additionally target other histamine receptors respons­
gut symptoms. CBT, cognitive behavioural therapy; mAb, monoclonal antibody. ible for mediating central effects might confer additional

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REVIEWS

benefits. For instance, H3 receptor antagonists show
promise as novel anxiolytics in preclinical models206 and
reagents targeting both H1 and H3 receptors might be
expected to simultaneously modulate immune activation
and stress responses in patients with IBS. Trials evaluat-
ing more selective anti-allergy drugs, including mono-
clonal antibodies targeting IL‑5 or IL‑4–IL‑13, might be
expected to yield more impressive results, especially if
eosinophils are genuinely functionally important in FD.
Manipulation of the microbiota. Manipulation of the
microbiota through administration of probiotics, pre­
biotics, antibiotics (including highly selective agents
and nonabsorbable varieties), dietary modifications and
faecal microbiota transplantation (FMT) are promising
approaches in intestinal diseases, including FGIDs207–210.
Early work showed that monoassociation of germ-free
mice with B. infantis could suppress the stress response184
and now a randomized, placebo-controlled trial has
demonstrated symptom improvement and favourable
modulation of mucosal immune responses following
administration of B. infantis in patients with IBS211.
Nonabsorbable antibiotics offer the opportunity for
intestinal microbial modulation without the possibility
of systemic adverse effects. A landmark phase III trial
reporting the outcome of two separate studies with iden-
tical protocols (TARGET1 and TARGET2) has shown
the efficacy of a 2‑week course of rifaximin in diarrhoea-­
predominant IBS, including reduced abdominal pain and
reduced diarrhoea episode frequency 212. Dietary modifi­
cation is now also a mainstay of IBS treatment. A diet
low in fermentable, oligosaccharides, disaccharides,
monosaccharides and polyols (FODMAPs) substan-
tially altered the composition of the intestinal micro-
biota in patients with IBS213, and is efficacious for IBS
symptoms213,214. Evidence also exists that low FODMAP
diets modulate host metabolic and immune pathways,
including an eightfold reduction in urinary histamine
concentration in patients with IBS consuming a low
FODMAP diet215.
Looking forward, smarter selection and focused
manipulation of the composition of the microbiota with
probiotics or FMT might be required and a manufactured
‘therapeutic bacterial community’ might comprise high
levels of diversity, depletion of pro-inflammatory species
(known to adhere to the epithelium) and enrichment of
immunosuppressive bacteria. Indeed, supplementation
of bacteria favouring anti-inflammatory IL‑10 responses,
such as B. fragilis and particular clusters of Clostridia,
might hold particular promise to limit excessive mucosal
immune responses in IBD and FGIDs. From a preci-
sion medicine perspective, it is conceptually attractive
to consider that individual patients will ultimately be
stratified according to clinical phenotypes (pattern of
functional symptoms and magnitude or profile of psycho­
logical symptoms), together with biomarkers reflecting
patterns of mucosal and systemic immune activ­ation,
as well as in‑depth profiling of intestinal bacterial com-
munities. Patients could then be stratified to specific
treatments based on implicated pathways. For instance,
patients with prominent IBS symptoms and major anx-
iety symptoms that are linked to selective enrichment
of IL‑6 in serum and/or in mucosal responses might be
expected to benefit from blockade of the IL‑6 pathway.
Similarly, patients with reduced numbers of regu­latory
T cells, diminished IL‑10 responses and depletion of
clostridial clusters or B. fragilis might be better served
by administering probiotics or FMT that are selectively
supplemented with these bacteria. Clinical trials stratified
by these parameters are eagerly awaited by the scientific
and clinical communities.
Conclusions
In conclusion, lines of communication between the brain
and gut play a key part in the biology, clinical manifes-
tations and clinical outcomes of gut diseases, but most
notably in FGIDs. The central pathways through which
this dialogue is mediated are neural and immune net-
works, including crosstalk between these systems. The
bidirectional trafficking of these signals opens the pos-
sibility of some gut diseases being driven by aberrant
brain function, and conversely, disordered gut homeo-
stasis being responsible for driving brain pathology and,
particularly, mood disorders in other patients. Overall,
the mucosal immune system is potentially the key gate-
keeper of these pathways as it specifically interacts with
nerves, the luminal microbiota and is an important tar-
get of the stress response. What effect these insights will
have on clinical practice remains to be seen, although it
is conceptually appealing to envisage the emergence of
new diagnostic criteria, biomarkers and psychological
scoring tools in patients with gut diseases. With the emer-
gence of precision medicine, strategies in which patients
with FGID or IBD might be stratified beyond standard
gut symptom complexes might offer the opportunity to
­tailor therapies to individual patients. In particular, anti-­
cytokine therapy or efforts to manipulate the intestinal
microbiota could hold the key to effectively uncoupling
pathological brain–gut dialogue, with the potential to
disrupt the proximal drivers of these important diseases.

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Author contributions
N.P. and M.M.W. researched data for the article, N.P. wrote
the article and N.P., M.M.W. and N.J.T. made substantial
contributions to discussion of content and reviewed or edited
the manuscript before submission.
Competing interests statement
The authors declare no competing interests.

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