Gut Microbiota The Conductor in the Orchestra of Immune ... · PDF filemodifies many of the microbial activities, which ... diseases, and the host immune system should recog-nize

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Gut Microbiota: The conductor in the Orchestra of immune-neuroendocrine communicationEl Aidy, Sahar Farouk Abdelsalam; Dinan, Timothy G; Cryan, John F

Published in:Clinical Therapeutics

DOI:10.1016/j.clinthera.2015.03.002

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Publication date:2015

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Citation for published version (APA):El Aidy, S., Dinan, T. G., & Cryan, J. F. (2015). Gut Microbiota: The conductor in the Orchestra of immune-neuroendocrine communication. Clinical Therapeutics, 37(5), 954-67. DOI: 10.1016/j.clinthera.2015.03.002

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Clinical Therapeutics/Volume 37, Number 5, 2015

Review Article

Gut Microbiota: The Conductor in the Orchestra ofImmune–Neuroendocrine Communication

Sahar El Aidy, PhD1,2; Timothy G. Dinan, PhD1,3; and John F. Cryan, PhD1,4

1Laboratory of Neurogastroenterology, Alimentary Pharmabiotic Centre, University College Cork, Cork,Ireland; 2Department of Industrial Biotechnology, Genetic Engineering and Biotechnology ResearchInstitute, Sadat City University, Sadat City, Egypt; 3Department of Psychiatry, University College Cork,Cork, Ireland; and 4Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland

ABSTRACT

Purpose: It is well established that mammals are so-called super-organisms that coexist with a complexmicrobiota. Growing evidence points to the delicacyof this host–microbe interplay and how disruptiveinterventions could have lifelong consequences. Thegoal of this article was to provide insights into thepotential role of the gut microbiota in coordinatingthe immune–neuroendocrine cross-talk.

Methods: Literature from a range of sources,including PubMed, Google Scholar, and MEDLINE,was searched to identify recent reports regarding theimpact of the gut microbiota on the host immune andneuroendocrine systems in health and disease.

Findings: The immune system and nervous systemare in continuous communication to maintain a state ofhomeostasis. Significant gaps in knowledge remainregarding the effect of the gut microbiota in coordinat-ing the immune–nervous systems dialogue. Recentevidence from experimental animal models found thatstimulation of subsets of immune cells by the gutmicrobiota, and the subsequent cross-talk between theimmune cells and enteric neurons, may have a majorimpact on the host in health and disease.

Implications: Data from rodent models, as well asfrom a few human studies, suggest that the gutmicrobiota may have a major role in coordinatingthe communication between the immune and

Accepted for publication March 4, 2015.http://dx.doi.org/10.1016/j.clinthera.2015.03.0020149-2918/$ - see front matter

& 2015 Elsevier HS Journals, Inc. All rights reserved.

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neuroendocrine systems to develop and maintainhomeostasis. However, the underlying mechanismsremain unclear. The challenge now is to fully decipherthe molecular mechanisms that link the gut micro-biota, the immune system, and the neuroendocrinesystem in a network of communication to eventuallytranslate these findings to the human situation, bothin health and disease. (Clin Ther. 2015;37:954–967)& 2015 Elsevier HS Journals, Inc. All rights reserved.

Key words: brain, enteric nervous system, gutmicrobiota, immune system.

INTRODUCTIONOur knowledge of the host–microbe interrelationshipis accelerating because of the availability of rapidlyexpanding molecular techniques, especially in combi-nation with the use of reductionist in vivo hostmodels. A growing body of research continues toshow that the normal mammalian structure andfunction are significantly dependent on their constantengagement in complex interactions with microbes.1

For example, the intestinal microbiota with itscomponents and metabolites affects the hostphysiology in various ways to control energyhomeostasis, gut barrier function, mucosal inflam-mation, and behavior.2–4 Subsequently, the hostmodifies many of the microbial activities, which

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suggests a feedback mechanism that could influencethe microbiota and drive a further cycle of biologicalchanges to host physiology.5 This multidirectionalinteractive dialogue seems to strongly influence anexpanding repertoire of human disorders, includingobesity, depression, and irritable bowel syndrome; thegoal is to decipher the tête-à-tête between the host andits commensal organisms.6,7

The present review highlights emerging evidence,which provides a framework for appreciating theimpact of the host–microbiota interplay on healththat is undoubtedly much broader than previouslythought. This review outlines the impact of the gutmicrobiota on the immune system as well as theneuroendocrine system, both of which point to apotential role of the microbiota as crucial coordina-tors in the cross-talk between these systems.

MATERIALS AND METHODSLiterature from a range of sources, including PubMed,Google Scholar, and MEDLINE, was searched toidentify recent reports on the impact of the gutmicrobiota on the host immune and neuroendocrinesystems in health and disease. All data were gatheredfor the latest available years.

RESULTSThe Host–Microbiota Dialogue

Over the past decade, remarkable advances towarda better understanding of how the host–microbeinteractome is linked with most pathways that affecthealth, disease, and aging were made possible withnovel technologies; these include high-throughputDNA sequencing, bioinformatics, and gnotobioticanimal models.1 The intimate interactions betweenthe host and its microbes that outnumber the host’scells by 10 to 1 are required to stimulate the completematuration of an efficient intestinal barrier that canpromote niche colonization by commensals andopposes colonization by pathogens.4,8 Only microbialpopulations that are capable of establishing a mutual-istic relation with the host can be maintained in thegut ecosystem,3 creating a habitat that exertsrestrictive selection on its microbial inhabitants. Thisrestrictive selection of specific microbial groups isillustrated by the relatively low phylum-level diversityobserved in the microbiota of the gastrointestinal (GI)tract of many mammalian organisms, including miceand humans; this is dominated by the phyla of the

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Bacteroidetes and the Firmicutes, with Proteobacteria,Actinobacteria, Fusobacteria, and Verrucomicrobiaphyla present in relatively low abundance.9 Incontrast, the diversity at the genus and species levelsis enormous. Advances in metagenomic approacheshelped to illustrate that despite the variation in speciescomposition, the microbial communities encompass arelatively similar set of metabolic functions in healthyindividuals, which are referred to as the “coremicrobiome.”10 Furthermore, diet and its nutritionalvalue are partly shaped by (and they in turn canshape) the gut microbial community, supporting thenotion that “we are what we eat,” a process that startsearly in life.11,12 Indeed, this scenario is now beingexploited for medical innovations along the diet–hostinterface in line with Hippocrates’ dictum that “Letfood be thy medicine and medicine be thy food.”

The primary individual microbiota colonize atbirth. However, there is growing evidence that the inutero environment may not be sterile, as originallythought: bacteria such as Enterococcus faecalis,Staphylococcus epidermidis, and Escherichia coli havebeen isolated from the meconium of healthy neo-nates.13 The infant gut microbiota is more variable inits composition and less stable over time. In the firstyear of life, the infant intestinal tract progresses fromsterility to extremely dense colonization, ending with amixture of microbes that is relatively stable and largelysimilar to that found in the adult intestine.14,15 Thecomposition of the gut microbiota is shaped by avariety of factors, including prenatal and postnatalvariables as well as birth factors.16 Altering theintestinal microbiota, particularly during early life,has been shown to have lasting consequences on thehost.17 For example, the variation in early-life environ-mental exposure that includes the method and place ofdelivery and a nourishing neonate regimen influencesthe microbiota of infants.18,19 The microbiota ofinfants delivered vaginally is dominated by microbialgroups that colonize the vagina, whereas infants bornby cesarean delivery have microbiota that more closelyresembles those of the maternal skin community.16,20

However, the duration of the influence of these factorson the host remains obscure, with contradicting find-ings available to date. As a final age-related microbialshift, the elderly have a core microbiome different fromthat of younger adults, and the composition is directlycorrelated with health outcomes and the decline in theimmune system.21

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The gut is home to a diverse array of trillions ofmicrobes that influence almost all aspects of humanbiology through their interactions with their host.1

Although the host provides the microbiota with theecological niche and nutrients required of its survival,the indigenous microbiota, in turn, provides the hostwith full maturation of the innate and adaptive armsof the immune system, modulation of the nervoussystem function, nutrient absorption and fatdistribution, contribution to digestion (eg, the abilityof microbes to break down host nondigestiblepolysaccharides), the generation of short-chain fattyacids (SCFAs), metabolism of xenobiotics (which aidsin protection from environmental toxins), and regu-lation of the gut motility.2,22–25 Colonization resist-ance is another protective function that the microbesprovide to their host. This protective function resultsfrom a combination of various functions of commen-sals, which include their metabolic competition withthe pathogen for the available nutrient resources. Themicrobiota provides colonization resistance; in addi-tion, several of the host’s anatomical and physiolog-ical factors, including protective mucosal barrier,secretion of secretory immunoglobulin A (sIgA) andnormal gastric motility, are also involved.26 Thisprocedure promotes the establishment of anintestinal environment that prevents colonizationwith disease-inducing bacteria, which are abundantin unhealthy subjects.27

The Gut Microbiota Is Engaged in a DynamicInteraction with the Host Immune System

Initially, all microorganisms were considered aspathogens that are harmful and cause infectiousdiseases, and the host immune system should recog-nize and eliminate these invaders (non-self) whiletolerating self-molecules to maintain homeostasis.However, the persistent association of host specieswith commensal organisms illustrates the major con-tribution of the gut microbiota in the education andregulation of the immune system.28 It is now wellestablished that the immune system depends oncolonization with a microbiota for its maturation.29

The initial microbial colonization is associated withprominent changes in mucosal and systemic immu-nity. For example, the physiologic phenomenon ofmaturation of the immune system, initiated within thefetal period, is dynamic in its character and expands intime through the first months (and even years) of a

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child’s life; within the neonatal period, infancy, andearly childhood, dysfunction of numerous comp-onents of the immune system is observed.30 Neonateshave a decreased expression of costimulatorymolecules, diminished dendritic cell (DC) differen-tiation, and impaired phagocytosis as well as adefective interaction between DCs, T lymphocytes,and regulatory T cells, impaired cytotoxic activity ofT cells.31,32 Moreover, the suppressive activity of thetransplacentally transmitted maternal IgG antibodiescontributes to the deficiency of specific humoral re-sponse, including the low production of IgA in humanneonates.33 Immune immaturities have also beenreported in animal studies, in which germ-free (GF)mice and those in the first days of colonization sharemany traits, characterized by smaller Peyer’s patchesand reduced cellularity of the lamina propria withlower Cd4þ and Cd8þ T cells.34,35 Plasma cells andintraepithelial lymphocytes are rare in the small in-testinal mucosa, and sIgA levels are significantly lowerwith reduced expression of genes and activationmarkers of intestinal macrophages.35,36 sIgA, for in-stance, represents an intriguing example of how themicrobiota mediates the host physiology through im-mune modulation. sIgA binds to luminal bacteria toprevent microbial translocation across the epithelialbarrier,37 but it also influences the balance of immuneand metabolic pathways in intestinal epithelial cellsthrough a microbiota-dependent mechanism.38 In theabsence of IgA, there is a shift toward the expression ofgenes involved in host defense, excessive production ofantimicrobial proteins, and inflammatory-like re-sponses to compensate for the deficiency in microbialcompartmentalization via the sIgA. Instead, in thepresence of IgA, the intestinal microbiota alters theexpression of genes involved in lipid metabolism andstorage, thereby shifting toward a homeostatic micro-biota–immune–metabolic response.38,39

The initial engagement of the microbiota in theeducation of the host immune system appears toinvolve specific strains of the microbial community(pathobionts) of potential inflammatory allies, whichsurvive and flourish in an environment enriched by theinflammatory process.25,40 To date, the few reportsavailable suggest that these specific bacteria presum-ably activate mucosal immune priming for the bacte-rial sampling process to minimize their exposure tothe systemic immune system by transient breaching ofthe epithelial barrier; they also stimulate the

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production of antimicrobial proteins and a variety ofimmune pro-inflammatory and regulatory immunecomponents.40–42 Elimination of the penetrant bacte-ria would thus lead to the induction of antigenpresentation and processing molecules, innate andadaptive constituents that reside in the lamina propria.Hence, this initial close but regulated contact couldbenefit the host by strengthening its gut barrier, and itconfers protection against true invading pathogens.Segmented filamentous bacteria represent an interest-ing example of such microbial–host initial immuneengagement. In mice, these bacteria adhere transientlyto the surface epithelium of the ileum and Peyer’spatches shortly after weaning43 to induce Th17 in theintestinal lamina propria via a mechanism thatinvolves host production of serum amyloid A.44

These segmented filamentous bacteria induce Th17locally in the GI tract; this effect was also shown toexpand to the central nervous system (CNS),45

indicating that improving our understanding of theintestinal microbiota has therapeutic implications, notonly for intestinal immunopathologies but also forsystemic immune diseases. Combined transcriptionaland immunohistochemical analyses of GF mice andex-GF mice helped in detailing how the microbiotaresults in reprogramming of the intestinal mucosalimmune responses, with the goal of initiating andmaintaining a balanced immune response that leads tomajor molecular immune responses starting only fewdays after colonization.35,46

Cumulatively, the early life stage marks the mostextensive changes in the host biology in response tothe colonization of the microbial entities. At thatpoint, the host encounters challenges, which, at themolecular level, elicit responses that involve genesassociated with several illnesses (inflammatory-like response, metabolic disorder, and behavioralchanges). Thus, abrupt shifts during the infant’sunique developmental path through this early unstablephase may have longer term health implications.47

Indeed, emerging evidence supports the concept of acritical window of development during early life thatallows for the full-scale establishment of an adequatehost–microbial homeostasis.19

After stabilization of the gut microbiota commun-ity, the immune system is continuously stimulated byseveral structural components of the microbial cellsand responds by producing a wide range of lympho-cytes and cytokines. In a state of homeostasis, the gut

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microbiota stimulates a chronic state of low-levelactivation of the (innate) immune system in the host,in which bacterial particles stimulate intestinal macro-phages and T cells to produce pro-inflammatorycytokines (eg, interleukin [IL]-1β, tumor necrosisfactor-α, IL-18).48,49 The produced cytokines createa basal state of immune activation that starts at theintestinal mucosal surface and eventually affects theentire body. The adult human gut is believed tocontain up to 1 g of lipopolysaccharides (LPS), andthe low level exposure of immune cells to thesebacterial cell wall components is essential in theestablishment and maintenance of mucosal homeo-stasis.50 The specific mechanisms underlying hostimmune–microbe cross-talk remains unclear; nonethe-less, a number of reports have been fundamental inrevealing the mechanistic interaction that orchestratesthis interaction between the host immune system andits microbiota. The regulation of the chemokineCXCL16, which is important for the invariant naturalkiller T-cell migration and homeostasis by the gutmicrobiota, represents an elegant example of thistightly regulated host–microbe dialogue. This chemo-kine regulation occurs via epigenetic changes (inparticular, the reduction of methylation status of theCXCl16 gene), thereby reducing the number of in-variant natural killer T cells in the lung and coloniclamina propria.51

Bacteroides fragilis is another well-studied examplefound to be critical for the normal function of themammalian immune system via the production of aglycosphingolipid that inhibits natural killer T-cellproliferation in the colonic lamina propria.52

B fragilis polysaccharide A, conversely, interactswith the Toll-like receptor (TLR)-2 on DCs to inducecolonic regulatory T cells (Tregs).53 Colonic Tregshave been demonstrated to also be induced by a groupof Clostridium species, presumably via bacterialproduction of SCFAs.54 Strikingly, the differencebetween Treg stimulation with B fragilis andClostridium species is that the latter can induceTregs in both healthy and inflamed tissues; however,B fragilis induces Tregs only in inflamed tissues.28

These results indicate that commensals, once theyescape their normal habitats and interact with thehost in a different context, become pathogenic.Collectively, reports to date confirm the hygienehypothesis, which has recently been reformulated asthe “Old Friends” hypothesis; it postulates that

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colonization with a “healthy” microbiota during thevulnerable developmental period exerts effects thatmay decrease susceptibility to diseases, whereas itsabsence or dysbiosis, as in antibiotic treatment inchildhood, may have reverse effects.55 This concept ishighlighted in the expanding repertoire of humandiseases that are associated with changes in theintestinal microbiota, such as inflammatory boweldisease, allergic disorders, autoimmunity, and majordepression.56

Gut Microbiota Talk to the Brain: Language Is anEnigma

For 4150 years, it has been clear that there is abidirectional gut–brain interaction, which regulateshomeostasis. However, the concept of a microbiome–gut–brain axis is a recent phenomenon, and themechanisms that underlie this axis are only slowlybeing resolved. Communication between the gut micro-biota and the gut–brain axis can occur via multipleconduits that include gut-secreted neuropeptides, vagalnerve, sensory nerves, and immune mediators.57 Thebrain can influence the commensals directly via thereceptor-mediated signaling and signaling moleculesreleased in the gut lumen from immune cells orepithelial cells (in particular, enteroendocrine cells) orindirectly via changes in the intestinal motility andsecretion.58 Similarly, the enteric bacteria have beenshown to profoundly affect brain function andbehavior.59 On the molecular level, GF mice exhibitan altered expression of genes involved in neuropep-tide production,39 which are involved in braindevelopment and behavior.60,61 On the behaviorallevel, GF mice exhibit increased spontaneous motoractivity compared with that of conventional mice60 andbecome more exploratory (more like their donors) afterfecal transfer from mice with anxiety-like behavior.62

Lactobacillus rhamnosus (JB-1), when fed to mice,reduces stress-induced corticosterone hormone levels.Moreover, the same strain makes the mice less anxious,an effect which seems to be dependent on the vagusnerve.63

Emerging data point to the involvement of bacterialcomponents and by-products in the dialogue with thegut–brain axis. The mammalian behavior and neuro-endocrine responses are dramatically affected by themetabolites circulating in the blood, many of which aredependent on the microbiota for their synthesis.64 Evencerebral metabolites are influenced by the commensals

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through the microbiota–gut–brain axis, demonstratinghow closely connected the gut microbiota is with brainhealth and disease, development, attenuation, learning,memory, and behavior.65

The gut microbiota is capable of altering the levelsof metabolites that serve as precursors for the synthesisof the signaling molecules; for example, neuropeptidessuch as glutamine and tyrosine, as revealed fromexperiments performed in gnotobiotic animals.40,66,67

Although these metabolites can communicate the mes-sage around the entire body, our knowledge of whichbacterial species produce which metabolites or displaywhich components (and which of these synthesizedproducts can bring about the host–microbial equili-brium) is limited (apart from the capsular polysacchar-ide A and glycophospholipid in B fragilis that exertpotent immunoregulatory and neurologic effects42,68).For instance, we do not know which bacteria areinvolved in the synthesis of the bacterial metabolitehippurate, which is known to be modulated by diet,stress, and disease and is widely detected in severalmammalian species.69 Bacterial LPS and peptidoglycancomponents are possible candidates in communicatingthe host–microbe interplay. For example, LPS can becarried directly to the brain via the blood circulation toact on the TLRs in the brain to give rise toneuroinflammatory processes, thereby modify thebrain function.70 These outcomes show that thephysiologic effect of the commensals is not limited tothe GI tract but extends to the systemic immune systemand the brain.57 SCFAs such as acetate, propionate,and n-butyrate, produced during fermentation ofnondigestible polysaccharides, also represent probablecandidates to be involved in setting up the balance.71

N-butyrate especially has been the focus of manystudies aiming to unravel its overall impact on thehuman physiology. It has been shown, in in vitroexperiments and in mice, to regulate energy homeo-stasis, stimulate leptin production in adipocytes, andinduce the secretion of several neuropeptides, such asglucagon-like peptide-1. This action is proposed tomodulate insulin secretion, lipid and glucose metabo-lism, and food intake, and it also exhibits anti-inflammatory effects.72,73 On the level of the CNS,n-butyrate reportedly has profound effects on moodand behavior.74 Through their effect on gastric motilityand intestinal transit stimulation, SCFAs result in anelevation in serotonin release, as reported in an in vitrocolonic mucosal system.75 Therefore, SCFAs provide a

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perfect example to illustrate the connectivity betweenthe microbiota and the gut–brain axis.76–78 For exam-ple, Wang et al79 highlighted changes in the fecalconcentrations of the SCFAs in children with autismspectrum disorders, suggesting that altered productionof these microbial metabolites, which were shown toexhibit neuroactivity, may be a mechanism by whichbacteria can alter brain function. Together, thesefindings suggest that the modification of the amountof dietary fiber across the microbe–host food networkwould influence the composition of the microbiota,which in turn modulates the luminal concentrations ofintestinal SCFAs, a potential target for restoring thehost overall physiology. In fact, an unbalanced diet thatis poor in dietary fiber content (and thus does notrequire complex metabolic interactions between themicrobial members) would result in lower microbialdiversity and in the imbalanced host–microbialrelationship that is observed in the emerging Westernlifestyle–associated human diseases.80 In a recentattempt to unravel which bacterial metabolites canbring about the host–microbial balance, Williamset al81 used a combination of genetics and bio-chemistry to identify a key microbial enzyme thatconverts dietary tryptophan to the neuropeptidetryptamine (ie, tryptophan decarboxylase enzyme,which was found to be present in several bacteriathat colonize �10% of the human population). Thisfinding suggests that the gut microbiota can sequestertryptophan from the diet and alter its metabolites in thehost, eventually resulting in altered brain levels of theneuropeptide serotonin, which ultimately affects brainfunction.82 Similarly, although the gut microbiota canalter the metabolism of several other neuropeptides, thelimited knowledge available regarding the microbialmechanisms hinders efforts to close this gap inresearch. The assumption by Williams et al issupported by the elevated levels of tryptophan in GFanimals compared with their conventional andconventionalized counterparts.66,67 Serotonin releasefrom enterochromaffin epithelial cells is stimulated bytryptamine83 and is a key regulator of the gut motilityand secretion.84 Recently, it has been shown that gutmicrobiota, acting through SCFAs, is an importantdeterminant of enteric serotonin production andhomeostasis. The gut microbiota from ex-GF micecolonized with human gut microbiota and convention-ally raised mice the colonic rate limiting for mucosalserotonin synthesis; tryptophan hydroxylase 1 mRNAs

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as well as the neuroendocrine secretion gene; chromog-ranin A with no effect on the serotonin transporter orserotonin catabolic gene; monoamine oxidase A.85

Interestingly, the reported effects did not result froman increase in the number of the cells producingserotonin but rather through the action of SCFAs.Changes in the levels of serotonin were also linked tothe pathology of GI disorders, in which modulation ofserotonin secretion is proposed as a treatment given itssignificant role in the enteric nervous system (ENS) andgut motility.86

Bacterial by-products that come in contact with thegut epithelium stimulate enteroendocrine cells (EECs)to produce several neuropeptides such as peptide YY,neuropeptide Y, cholecystokinin, glucagon-like peptide-1and -2, and substance P.87 As an example, the gut sensesthe bacterial by-product SCFAs through G protein–coupled receptors 41 and 43 expressed on EECs.88

Upon their secretion by EECs, the neuropeptidespresumably diffuse throughout the lamina propria,which is occupied with a variety of immune cells, untilthey reach the bloodstream or act on the vagal nerve orintrinsic sensory neurons.89,90 However, the exact mech-anisms and whether the neuropeptides have a directcontribution in the bidirectional communication be-tween the microbiota and CNS are still unknown.Recently, Bohorquez et al91 demonstrated a directcommunication between EECs and neurons innervatingthe small intestine and colon that the paracrinetransmission. The authors discovered a neuroepithelialcircuit that can act as both sensory channels for foodand as gut microbiota to communicate the message fromthe lumen to the ENS and CNS and vice versa. Thisphysical innervation of sensory EECs suggests anaccurate temporal transfer of the sensory signalsoriginating in the gut lumen with a real-time modulatoryfeedback onto the EECs. Nonetheless, neuropeptidesremain an intriguing way by which the gut microbiotainitiates an effective dialogue with the host.

Several reports have shown the changes in neuro-peptides and neurotransmitters in response to changesin the gut microbiota composition. For example,Lactobacillus acidophilus stimulates the expressionof cannabinoid and opioid receptors in rodents andin in vitro human epithelial cell cultures, therebyreducing experimentally evoked visceral pain.92

Antibiotic-induced microbial dysbiosis is associatedwith elevated levels of substance P in the colon.93 Notonly does the host respond to the gut microbes by

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producing neuropeptides, a variety of different com-ponents of intestinal microbiota can also produce aplethora of neurochemicals, which act to reduceinflammation, dampen the stress response, and poten-tially improve mood.94,95 For example, the metabo-lism of commensals has been shown to producehistamines and catecholamines, both of which can sti-mulate the ENS and the CNS via the primary afferentfibers of the vagal nerve.77,96 Dopamine is alsoproduced in large quantities by a variety of bacteriaincluding E coli, Bacillus cereus, Bacillus subtilis,and Staphylococcus aureus.97 However, the hostproduces enzymes that degrade these bacterialneurochemicals. For example, dopamine is silencedby the host-produced enzyme monoamine oxidase.57

It is tempting to speculate that the microbiota hasevolved gene analogues to the host signaling moleculessuch as neuropeptides to manipulate its host to createa mutualistic relationship; for the host to prevent thatmanipulation in which it conflicts with the host’sfitness interests, however, corresponding host geneshave evolved to eradicate the microbial attempts toinvade. In fact, the same strategy is emphasized in thedetoxification of enzymes evolved by mammals toeliminate gut-derived toxins and xenobiotics by ren-dering them more polar, enabling them to be easilysecreted in the urine and thereby modifying bothgut microbial products and drugs.23 For example,the compound 4-cresyl, which is formed from thepurification of tyrosine (the precursor of the neuro-peptide dopamine) by colonic microbiota, is convertedinto a more polar form (4-cresyl sulfate, an abundanturinary microbial metabolite) by host sulfotrans-ferases.98 This microbial influence on the process ofdrug detoxification may also explain individualvariations in certain drug responses, owing to theenormous interpersonal variations in the gutmicrobiota species.6,99 In fact, when rats were admin-istered the antipsychotic olanzapine in the presence ofa cocktail of broad-spectrum antibiotics, the Firmi-cutes phylum was suppressed and the relative abun-dance of Bacteroidetes was increased while at thesame time ameliorating the olanzapine-induced weightgain, uterine fat deposition, plasma free fatty acid levels,and macrophage infiltration of adipose tissue.100 Thesefindings of a microbiota-dependent, olanzapine-inducedweight gain have recently been confirmed in GF mice,and in in vitro studies point to a direct interactionbetween olanzapine and microbes.101

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Gut Microbiota Coordinates the Immune–Neuroendocrine Cross-Talk

It has been an evolving subject since the beginningof modern medicine that the immune system and thebrain, the 2 major adaptive systems of the body, talkto each other through a network of direct- andindirect-signaling pathways that involve the hypo-thalamic–pituitary–adrenal axis and the sympatheticnervous system. It is crucial for the nervous system tocommunicate with the immune system to maintain thehost homeostasis because any challenge to the im-mune system would affect body temperature, visceralpain, and reflex withdrawal, all of which lead to painsensation and behavioral changes but also negativelyimpact the immune system.102 However, assessing thehost responses toward its commensal inhabitants,which are reflected in the complex interrelationshipsbetween nutrient metabolism, the immune system, andthe neuro-endocrine system that occur at many levels(ranging from endocrine signaling to direct sensingof bacteria and their products), strongly suggests thatthe microbiota is an instrumental component ofthe psychoneuroimmunology network (Figure).103

Neuropeptides may be 1 of the signals by which themicrobiota coordinates this communication networkbecause neurons, epithelial cells, microbial cells, andimmune cells can all produce and respond to them byexpressing the appropriate receptors. For example, atthe start of a local immune response (eg, during theinitial microbial colonization), migration of immatureDCs to lymph nodes was found to be mediated viaα1-adrenergic receptors, emphasizing the early effectsof the sympathetic nervous system.104 Moreover,various immune cells travel through the blood, andwhen they come within sensing distance of a givenneuropeptide, they begin to chemotactically orienttoward it; they then communicate with otherimmune cells in the adaptive arm (eg, B and Tlymphocytes) to ensure a well-coordinated immuneresponse.

Neuropeptides are produced by the host secondbrain: the ENS (as well as the CNS), which functionsto regulate blood flow and GI motility, partly throughits interaction with the immune system and gut micro-biota.105 Any changes in the luminal chemistry aresensed by the ENS, which transfers this information tothe intrinsic neurons to cause the release of excitatoryor inhibitory neuropeptides from a subset of cellsnamed the intestinal cells of Cajal in the myenteric

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ChA

T

B cell

T cellChAT

Dendritic cell

Enteric neurons

Macrophage

Smooth muscle

Gut motility

Microbiota

Epithelium

Mucosa

Submucosa

Muscularis

GCPRs

Microbiotasignaling;

Immune–neuroendocrinesignaling

Mucosal immune signaling,Endocrine signalingVagal nerve activation

Microbial signalingEnteroendocrinesignaling

Microbiotasignaling

-microbial products,-microbial compounds, -neuropeptide analogues

Neuropeptides

Excitatory/inhibitorysignals

Excitatory/inhibitorysignals

Immune regulationMood and behaviourIntestinal motility and secretionBarrier functionNeuropeptide release

Figure. The multidirectional dialogue between the gut microbiota, the (mucosal) immune system, and theneuroendocrine system. Within the gastrointestinal tract, the intestinal microbiota (via microbialsignals) stimulates the immune system and the enteric nervous system, which in turn modulate thefunctionality of the central nervous system by various means of communication, including vagus nerveactivation and cytokine release. In response, the brain modulates these multiple signaling pathwaysvia the hypothalamic–pituitary–adrenal axis and sympathetic and vagal efferents. GCPRs ¼ G protein-coupled receptors.

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plexus.106 A recent study showed that in a state ofhomeostasis, the GI motility is regulated via theinteractions between the ENS, microbiota, and aclass of macrophages that reside in close proximityto the myenteric plexus and intestinal cells of Cajal,named muscularis macrophages (MMs). Mulleret al107 unraveled the mechanisms regulating thecross-talk between the gut microbiota, the MM

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immune cells, and eventually the ENS, in which thegut microbiota (particularly its component LPS) stim-ulates the production of the cytokine colony-stimulating factor 1 in MMs, followed by theproduction of bone morphogenetic protein (BMP) 2;this in turn stimulates the ENS via activation of thereceptors of BMP2 and downstream signaling mole-cules, resulting in modulation of the gut motility.

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Subsequent to illustrating that MMs are sensitive tochanges in the luminal environment after antibiotictreatment, the question remains whether the under-lying mechanisms use a combination of downstreamimmune and neuronal signaling in the mucosa, in-cluding signals provided by EECs.

Another plausible mechanism by which the gutmicrobiota with its components and accumulatingmetabolites can play a critical role in shuttlinginformation with the host nervous and immunesystems is through signaling pathways. These path-ways include warning signals similar to those pro-duced during injury or inflammation. In this case, thebrain may receive the sentinel signals from theperipheral afferent nerves and elicit neural reflexes,which in turn regulate the immune responses to avoidany unwarranted inflammatory response. This sce-nario resembles what happens in endotoxemia, inwhich the signaling is conducted to the brain by thevagus nerve, and it responds by activating the efferentcholinergic signaling, which prevents the immune cellsfrom producing inflammatory cytokines via the stim-ulation of their cholinergic receptors.108 Immune cellssuch as macrophages, neutrophils, DCs, and B and Tlymphocytes not only produce neuropeptides but alsoexpress receptors to the neuropeptides. For example,acetylcholine, the principle vagal neuropeptide thatattenuates the release of pro-inflammatory cytokinestumor necrosis factor-α, IL-1β, IL-6, and IL-18 (but notIL-10), is released from a subset of CD4þ T cells thattransfer the signal to other immune cells through theactivation of α7-nicotinic acetylcholine receptors onmacrophages.109 Acetylcholine is also produced by aspecific type of T cells known as ChATþ T cells, whoseactivation by norepinephrine, via β2-adrenergic recep-tors, results in the production of proinflammatory andanti-inflammatory cytokines.110 The location ofChATþ T cells is interesting: they are present in alow percentage in the spleen but are much moreabundant in the Peyer’s patches,111 which aresurrounded by enteric ganglia and are directlyinnervated by enteric and parasympathetic neuronswhose fibers transverse in close proximity to immunecells. The high abundance of ChATþ T cells in Peyer’spatches suggests that their stimulation by microbialcomponents exhibits both defensive and regulatoryroles at the gut mucosal surface, in which trillions ofmicrobiota reside. Acetylcholine receptor is alsoexpressed on B cells (named ChATþB cells); similar

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to ChATþT cells (but only in mucosal-associatedlymphoid tissues and not in other peripheral lymphoidorgans), they release acetylcholine when stimulatedby another neuropeptide (ie, cholecystokinin).112

Intriguingly, it has been shown that the expression ofChATþB cells begins after birth, following themicrobial colonization via MyD88-dependent TLRpathways in a transit manner. The assumption wasevident by the reduction in Ach receptor expressionafter antibiotic treatment. The specific function of theChATþB cells to control local recruitment of neutro-phils during endotoxemia as shown in this study seemscrucial to prevent severe tissue damage that is associ-ated with the neutrophil recruitment or dysregulationof their proteolytic enzymes, as observed in cases of GIinflammation.113 Together, these evolving data supportthe key role of the gut microbes in orchestrating theexisting immune–neuroendocrine dialogue.

When Intimate Friends Turn into FoesThe gut microbiota is normally thought of as

including the “friendly” bacteria that perform manybeneficial functions, until dysbiosis occurs. In thissituation, the imbalanced gut microbiota representsan environmental alteration and may become harmfulto the host. Microbial dysbiosis is characterized byreduced diversity and the outgrowth of pathobionts,which can lead to inadequate immune functioning andinflammatory responses by an imbalance betweenregulatory and pro-inflammatory lymphocytes, GImotility and permeability, and subsequent changes inmetabolism and extra-intestinal milieus, including thebehavior and CNS functions. Therefore, the host mayencounter major challenges, which are linked at themolecular level to diverse, complex diseases (eg,inflammatory bowel disease and metabolic, neuro-logic, and cardiovascular disorders).114

Altered exposure to the microbiota and its componentshas been associated with separately modified immune,endocrine, and CNS functions, and it may thereforedisrupt the normal immune–neuroendocrine network.For example, elevated levels of inflammatory responseduring microbial dysbiosis appear to have direct effectson the tryptophan metabolic pathway. Dysregulation ofthe kynurenine arm in the tryptophan metabolicpathway, which is observed in many disorders ofthe brain and the GI tract,66 involves the catalyticenzyme indoleamine-2,3-dioxygenase,40 the activity ofwhich is induced by inflammatory mediators and by

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corticosteroids.115 These findings suggest that alterationsin the production of neuropeptides by dysbiosis would inturn alter the neuroendocrine–immune signaling or actdirectly on the CNS. The gut microbiota also impactsbrain health in several ways.116 Exposure of GF adultmice to the gut microbiota decreased permeability of theblood–brain barrier.117 Bacteria and their componentssuch as LPS cause leakage of the gut barrier and thesubsequent stimulation of the innate immune responseand inflammation. Excessive production of cytokines isassociated with systemic inflammation and high plasmalevels of proinflammatory and anti-inflammatory cyto-kines, along with salivary and plasma cortisol andnorepinephrine levels. Elevated levels of cytokines andneuropeptides were shown to be associated with dis-rupted sleep, depressed mood, increased anxiety levels,impaired long-term memory for emotional stimuli, andelevated visceral pain sensitivity.116,118 Particularly inelderly patients, elevated levels of the gut microbiota–delivered LPS, and the resulting endotoxemia, aremore frequent as a result of small intestinal bacterialovergrowth that is caused by disturbed gut motilityand increased intestinal permeability.119 Indeed, gutmicrobiota alterations have been described in severaldiseases with altered GI motility, raising the urgencythat increased attention be given to the role of the gutmicrobiome in modulating GI function. Patients withschizophrenia are also known to have high gut bacterialtranslocation, and the presence of CD14 as a translocatormarker (which was found to be associated with thebacterial component C-reactive protein) tripled the risk ofschizophrenia.120

The host responds to specific microbes with anti-bodies or antigen-specific cellular immune responses.Thereby, when microbial dysbiosis occurs, CNS dys-function could result from the autoimmune reactioncaused by molecular mimicry between bacteria and host-self proteins, as in case of multiple sclerosis.121 Multiplesclerosis, a devastating autoimmune disease that leads toprogressive deterioration of neurologic function, hasbeen shown to be profoundly influenced by the gutmicrobiota via its ability to stimulate proinflammatoryT-cell responses during experimental-associated ence-phalomyelitis.45 Moreover, the commensal microbeswere shown to be essential in triggering immuneprocesses that led to a relapsing-remitting autoimmunedisease driven by myelin-specific CD4þT cells.122 Thesame study demonstrated that the recruitment andactivation of autoantibody-producing B cells from the

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endogenous immune repertoire depend on the availabil-ity of the target auto-antigen, myelin oligodendrocyteglycoprotein, and commensal microbiota. This findingagain highlights the crucial role of the gut microbiota incoordinating the neuro–endocrine–immune cross-talk.

CONCLUSIONSOver the past decade, preclinical trials, as well as afew clinical studies, have highlighted the crucial role ofthe gut microbiota in maintaining host homeostasis.Analogously, microbiota dysbiosis has been associatedwith a wide range of host disorders, including inflam-matory bowel disorders, neuroendocrine disorders,and behavioral disorders. This growing body ofresearch illustrates the significant direct effect thatmicrobes have on the host immune and neuroendo-crine systems. Nonetheless, the underlying molecularmechanisms that orchestrate this dialogue are far lesscertain than what is required to open new avenues oftherapeutic intervention. A better understanding of thecausative mechanisms that govern the microbiota–host interactions is urgently needed to provide novelavenues to rationally intervene in disease situations,using either microbial or dietary interventions, withthe goal of correcting situations of microbial dysbiosisand thereby restoring homeostasis.

Despite the availability of an enormous array ofdata from preclinical studies, limited information isavailable on how these findings may translate to thehuman situation in health and disease. The hugeinterindividual variation among human subjects, notonly in terms of microbiota composition and itsgenome but also diet, sex-related differences, genetics,and environmental factors, adds to the complexity ofdeciphering the host–microbe interplay in clinicalstudies. Another major challenge in clinical studies isthat the food industry does not have as strong of aclinical trials culture as there has been traditionally inthe pharma sector, and the cost of large-scale trials isoften prohibitive.

ACKNOWLEDGMENTSDrs. Dinan and Cryan are supported by ScienceFoundation Ireland (grant no. 07/CE/B1368 and 12/RC/2273) and by the Irish Health Research Board,Health Research Awards (HRA_POR/2011/23)and (HRA_POR/2012/32). Sahar El Aidy contribu-tion: literature search, figure creation, writing.Timothy G. Dinan and John Cryan: critical reviewing.

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CONFLICTS OF INTERESTThe authors’ research at the Alimentary PharmabioticCentre is funded by Science Foundation Ireland,through the Irish Government’s National DevelopmentPlan in collaboration with a variety of pharmaceuticaland food industries. The authors have indicated thatthey have no other conflicts of interest regarding thecontent of this article.

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Address correspondence to: John F. Cryan, PhD, Department of Anatomyand Neuroscience, University College Cork, Cork, Ireland. E-mail: [email protected]

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