Gastroenterology 2014;146:1489–1499

THE GUT MICROBIOME AND DISEASE

The Microbiome in Inflammatory Bowel Disease: Current Statusand the Future Ahead

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1Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge; and 2Gastrointestinal Unit,Center for the Study of Inflammatory Bowel Disease, and Center for Computational and Integrative Biology, MassachusettsGeneral Hospital, Harvard Medical School, Boston, Massachusetts

Aleksandar D. Kostic Ramnik J. Xavier Dirk Gevers

Studies of the roles of microbial communities in thedevelopment of inflammatory bowel disease (IBD) havereached an important milestone. A decade of genome-wideassociation studies and other genetic analyses have linkedIBD with loci that implicate an aberrant immune responseto the intestinal microbiota. More recently, profilingstudies of the intestinal microbiome have associated thepathogenesis of IBD with characteristic shifts in thecomposition of the intestinal microbiota, reinforcing theview that IBD results from altered interactions betweenintestinal microbes and the mucosal immune system.Enhanced technologies can increase our understanding ofthe interactions between the host and its resident micro-biota and their respective roles in IBD from both a large-scale pathway view and at the metabolic level. We reviewimportant microbiome studies of patients with IBD anddescribe what we have learned about the mechanisms ofintestinal microbiota dysfunction. We describe the recentprogress in microbiome research from exploratory 16S-based studies, reporting associations of specific organismswith a disease, to more recent studies that have taken amore nuanced view, addressing the function of the micro-biota by metagenomic and metabolomic methods. Finally,we propose study designs and methodologies for futureinvestigations of the microbiome in patients with inflam-matory gut and autoimmune diseases in general.

Abbreviations used in this paper: CD, Crohn’s disease; FMT, fecal

Keywords: Microbiota; Crohn’s Disease; Ulcerative Colitis;Metagenomics.

ver the past decade, inflammatory bowel disease

microbiota transplantation; IBD, inflammatory bowel disease; iCD, ilealCrohn’s disease; SCFA, short-chain fatty acid; T2DM, type 2 diabetesmellitus; UC, ulcerative colitis.

© 2014 by the AGA Institute0016-5085/$36.00

http://dx.doi.org/10.1053/j.gastro.2014.02.009

O(IBD) has emerged as one of the most studied hu-man conditions linked to the gut microbiota.1,2 IBD com-prises both Crohn’s disease (CD) and ulcerative colitis (UC),which together affect more than 3.6 million people.3 Large-scale studies of human genetics across a total of 75,000cases and controls have revealed 163 host susceptibility loci

to date.4 These loci are enriched for pathways that interactwith environmental factors to modulate intestinal homeo-stasis.5 The incidence of the disease has been on the riseover the past few decades, further highlighting the role ofenvironmental factors in this disease. IBD was once a veryrare disorder and only began to rise dramatically in inci-dence in the second half of the 20th century in NorthAmerica and Europe, at times doubling every decade; it hasexpanded into developing countries in the past 2 decades,although there are more cases of UC than CD in the devel-oping world.6 In addition, several twin studies have shownthat the concordance rate for IBD between monozygotictwin pairs is significantly less than 50%, with the leastconcordance in CD.7 IBD is thus a multifaceted disorder inwhich not only germline genetics and the immune systembut also several environmental factors play an importantrole.8 One such factor, the gut microbial community, isgaining increasing attention for its influence on many as-pects of health in general9 and IBD in particular (Table 1).

The gut microbiota, the largest reservoir of microbes inthe body, coexists with its host in variable concentrationsthroughout the gastrointestinal tract, reaching an upper levelin the colon of 1011 or 1012 cells/g of luminal contents.10 Thiscommunity performs a range of useful functions for the host,including digesting substrates inaccessible to host enzymes,educating the immune system, and repressing the growthof harmful microorganisms.11 The extensive use of low-resolution surveys of the microbial community structure inthe past, as well as renewed efforts using next-generation

Table 1.Changes in the Microbiome Linked to IBD

Microbial composition Decrease in a diversityDecrease in Bacteroides and FirmicutesIncrease in GammaproteobacteriaPresence of E coli, specifically adherent-

invasive E coliPresence of FusobacteriumDecrease in Clostridia, Ruminococcaceae,

Bifidobacterium, LactobacillusDecrease in F prausnitzii

Microbial function Decrease in SCFAs, butyrateDecrease in butanoate and propanoate

metabolismDecrease in amino acid biosynthesisIncrease in auxotrophyIncrease in amino acid transportIncrease in sulfate transportIncreased oxidative stressIncrease in type II secretion system,

secretion of toxins

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sequencing for a high-resolution description of composition,function, and ecology,12,13 have improved our overall un-derstanding of the role of the gut microbiota in health, aprerequisite for the study of disease-related dysbiosis.Several factors can intervene with microbial gut communitycomposition, including genetics, diet, age, drug treatment,smoking, and potentially many more (Figure 1).14 The rela-tive importance of each of these factors is still unclear, butseveral of them are directly or indirectly linked to the diseasestate.

Figure 1. Factors affecting the stability and complexity of the gumicrobiome, including stability, resilience, and complexity, are inIn the healthy gut, these characteristics contribute to important ptraining of the immune system, and digestion of food to supplfactors are indicated to affect the microbiome throughout microbgenetics, diet, and medication, among others (marked in the grintroduce perturbations affecting the complexity and stability oFeatures of an imbalanced microbiome include, for example, anoxidative stress and inflammation and metabolite production.

Environmental Factors AffectingMicrobiome CompositionDiet

One of the most important environmental factorsaffecting microbial composition is dietary preference,which has been shown to determine microbiome compo-sition throughout mammalian evolution.15 Although nospecific diet has been shown to directly cause, prevent, ortreat IBD, it is important to take interactions betweennutrients and microbiota into account when studying therole of the microbiome in disease. Thus far, only limitedinformation on this topic has been gathered in humans,undoubtedly as a result of the challenge of setting up alarge-scale controlled diet study. Wu et al have shown thatlong-term dietary patterns affect the ratios of Bacteroides,Prevotella, and Firmicutes and that short-term changesmay not have major influences.16 In addition, Zimmer et alhave studied the impact of a strict vegan or vegetariandiet on the microbiota17 and found a significant reductionin Bacteroides species, Bifidobacterium species, and theEnterobacteriaceae, whereas total bacterial load remainedunaltered. Since the Enterobacteriaceae are among thetaxa that are consistently found to be increased in pa-tients with IBD (see the following text), it would be ofvalue to include both short-term and long-term dietarypatterns in future studies of the role of the microbiome inIBD. Given the complexity of dietary effects, includingsuch information will likely only be feasible in a largecohort study.18

t microbiome in health and disease. Key characteristics of thefluenced over time from infancy to adulthood and into old age.hysiological processes such as protection against pathogens,y energy and nutrients including vitamins and SCFAs. Manyiome development and even established assembly, includingay boxes at the top of the figure). Some of these factors canf the microbiome, potentially introducing microbial dysbiosis.increase in gram-negative bacteria linked to an environment of

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AgeThere is an age-related variation in the distribution of

IBD phenotypes, with 3 distinct stages of onset. A peak ageof onset is typically 15 to 30 years of age, with late-onsetcases occurring closer to 60 years of age and early onsetoccurring at younger than 10 years of age. Noticeably, thelatter group has had a significant increase in incidence overthe past decade.19 These stages correspond to phases inwhich the gut microbiota alters its diversity and stability.20

Early life is marked by a microbiome of low complexity andlow stability, one that is more volatile, is affected by thebirth route, and fluctuates with events such as changes indiet (switch from breastfeeding to solid foods), illness, andpuberty.21 It takes until adulthood for the microbialassemblage to reach maximal stability and complexity, withimproved resilience toward perturbations.22 However,decreased stability has been observed in elderly subjects(60 years of age or older).23 Given these different charac-teristics of the microbiome at the 3 distinct stages of diseaseonset, a different role for the microbiome in disease initia-tion and progression should be considered.

IBD Genetics Point to an InterplayBetween the Immune System andMicrobiota in IBD

A potential link between genetics and themicrobiome haslong been suspected. The first identified CD susceptibilitygene was NOD2,24 which stimulates an immune reaction onrecognizing muramyl dipeptide, a cell wall peptidoglycanconstituent of gram-positive and gram-negative bacteria.NOD2 is expressed in Paneth cells, which are located pre-dominantly in the terminal ileum at the base of intestinalcrypts, and produce antimicrobial defensins.25 Therefore, itmay not be surprising that mutations in NOD2 can have sig-nificant effects on the composition of the microbial milieu.Indeed, patients with IBD carrying NOD2 mutations haveincreased numbers of mucosa-adherent bacteria2 anddecreased transcription of the anti-inflammatory cytokineinterleukin 10.26 Patients with IBD carrying NOD2 andATG16L1, an IBD susceptibility gene involved in autophagy,risk alleles have significant alterations in the structure oftheir gut microbiota, including decreased levels of Faecali-bacterium and increased levels of Escherichia.27 Subjectshomozygous for loss-of-function alleles for FUT2 are “non-secretors” who do not express ABO antigen on the gastroin-testinal mucosa and bodily secretions. Nonsecretors are atincreased risk for CD28 and exhibit substantial alterations inthe mucosa-associated microbiota.29 Host genetics may thusplay a strong role in the establishment and shaping of the gutmicrobiota; indeed, monozygotic twins share more similarmicrobiomes than nontwin siblings.30 In regard to the skin, arecent study in patients with primary immunodeficiencyshowed a bidirectional dialogue between the microbiomeand the host immune system. The skin of patients with pri-mary immunodeficiency has an altered population compo-sition compared with immunocompetent subjects, which inturn results in increased susceptibility to infection by altering

the immune response toward pathogens.31 Although thereare currently no genome-wide studies examining the in-teractions between common human genetic variation and thecomposition of the microbial ecosystem, such a study couldbe of great value.5

An Overview of Gut MicrobiomeStudies in IBD

Many IBD susceptibility loci suggest an impairedresponse to microbes in disease, but the causality of thisrelationship is unclear. The pathogenesis of IBD may resultfrom a dysregulation of the mucosal immune system drivinga pathogenic immune response against the commensal gutflora.32 Some studies have shown that the gut microbiota isan essential factor in driving inflammation in IBD1; indeed,short-term treatment with enterically coated antibioticsdramatically reduces intestinal inflammation33 and has beenshown to have some efficacy in IBD and particularly inpouchitis.34 Specifically, rifaximin has shown efficacy inrecent trials in CD.35 Additionally, patients with IBD showmucosal secretion of immunoglobulin G antibodies36 andmucosal T-cell responses against commensal microbiota.37

The dramatic improvements in DNA sequencing tech-nology and analysis over the past decade have set the stagefor investigations of the IBD microbiome. Many studies havefound structural imbalances, or dysbioses, that occur in IBDsince the initial report,38 and a broad pattern has begun toemerge that includes a reduction in biodiversity, a decreasedrepresentation of several taxa within the Firmicutes phylum,and an increase in the Gammaproteobacteria.27,39

Many studies consistently report a decrease in biodi-versity, known as a diversity or species richness inecological terms, which is a measure of the total number ofspecies in a community. There is reduced a diversity in thefecal microbiome in patients with CD compared withhealthy controls,40 which was also found in pairs of mono-zygotic twins discordant for CD.41 This decreased diversityhas been attributed to reduced diversity specifically withinthe Firmicutes phylum42 and has also been associated withtemporal instability in the dominant taxa in both UC andCD.43 There is reduced diversity in inflamed versus non-inflamed tissues even within the same patient, and patientswith CD have lower overall bacterial loads at inflamed re-gions.44 The largest IBD-related microbiome study to date ison new-onset CD in a multicenter pediatric cohort.45 Thisstudy analyzed more than 1000 treatment-naïve samples,which were collected from multiple concurrent gastroin-testinal locations, from patients representing the variety ofdisease phenotypes with respect to location, severity, andbehavior. In addition to a detailed characterization of thespecific organisms either lost or associated with disease,this study indicates that assessing the rectal mucosa–associated microbiome offers unique potential for conve-nient and early diagnosis of CD.

Other nonbacterial members of the microbiota, namelythe fungi, viruses, archaea, and phage, may have a significantrole in gastrointestinal disease46; however, the vast majorityof recent studies of the microbiota are based on 16S

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sequencing, thus largely ignoring these groups of organisms.For example, norovirus infection, in the context of an intactgut microflora and mutated Atg16l1, is required for thedevelopment of CD in a mouse model.47 A number of studiesnote a relationship between fungi and IBD,48 including anoverall increase in fungal diversity in UC and CD.49 Therelationship between these organisms and IBD will no doubtbe explored in more detail in the coming years, becausemicrobiome studies will increasingly be performed by un-biased shotgun sequencing.

Microbes Enriched in IBD MayPotentiate Disease

Specific taxonomic shifts have been reported in IBD(Table 1). The Enterobacteriaceae are increased in relativeabundance both in patients with IBD and in mousemodels.50 Escherichia coli, particularly adherent-invasiveE coli strains, have been isolated from ileal CD (iCD) bi-opsy specimens in culture-based studies51 and are enrichedin patients with UC.52 This enrichment is more pronouncedin mucosal samples than in fecal samples.53 The increase inEnterobacteriaceae may indicate the preference of this cladefor an inflammatory environment. In fact, treatment withmesalamine, an anti-inflammatory drug used in patientswith IBD, decreases intestinal inflammation and is associ-ated with a decrease in Escherichia/Shigella.39,54

In addition to trends seen in the lumen, a number ofstudies have observed a shift in microbes that are attachedto the intestinal mucus layer. The small intestine has asingle mucus layer, whereas the colon has 2 mucus layers: afirmly attached inner mucus layer that is essentially sterileand an outer mucus layer of variable thickness.55 The mucuslayer consists of mucins, trefoil peptides, and secretoryimmunoglobulin A.56 Although host-microbiota interactionsare bidirectional, direct contact with the epithelium islimited by the mucus and the production of antimicrobialfactors such as defensins and RegIII-g.57–59 As long as themucus layer is relatively healthy and intact, microbes willattach to the mucus and generally do not have direct accessto epithelial cells. There is a greater overall density ofattached bacteria on the colonic mucus layer in patientswith UC compared with healthy controls.2 The adherent-invasive E coli pathovar, in particular, is at higher abun-dance in mucosal biopsy specimens from patients with CDcompared with healthy subjects and particularly high in ilealspecimens.60 Adherent-invasive E coli invades epithelialcells and can replicate within macrophages61 and inducegranuloma formation in vitro.62 In fact, E coli has also beenfound at higher levels in granulomas from CD relative toother non-CD granulomas.63

A second group of adherent and invasive bacteria is theFusobacteria. The genus Fusobacterium is a group of gram-negative anaerobes that principally colonize the oral cavitybut can also inhabit the gut. Fusobacterium species havebeen found to be at higher abundance in the colonic mucosaof patients with UC relative to control subjects,64,65 andhuman isolates of Fusobacterium varium have been shownto induce colonic mucosal erosion in mice by rectal enema.66

The invasive ability of human Fusobacterium isolates has apositive correlation with the IBD status of the host,67 sug-gesting that invasive Fusobacterium species may influenceIBD pathology. Intriguingly, Fusobacterium species haverecently been shown to be enriched in tumor vs nonin-volved adjacent tissue in colorectal cancer,68,69 and humanFusobacterium isolates have been shown to directly pro-mote tumorigenesis in a mouse model.70,71 Because IBD isamong the highest risk factors for the development ofcolorectal cancer, Fusobacterium species may represent apotential link between these diseases.

Protective Effects of Microbes in IBDSeveral lines of evidence suggest that specific groups of

gut bacteria may have protective effects against IBD. Forexample, the colitis phenotype after treatment with dextransulfate sodium is more severe in mice that are reared germ-free compared with conventionally reared mice.72 Onemechanism by which the commensal microbiota may protectthe host is colonization resistance, in which commensalsoccupy niches within the host and prevent colonization bypathogens73 and help outcompete pathogenic bacteria.74

(Interestingly, the microbiota can sometimes take on theopposite role and facilitate viral infection.75) Commensalmicrobiota can also have direct functional effects on potentialpathogens (eg, in dampening virulence-related gene expres-sion).76 In addition, the gut microbiota plays a role in shapingthe mucosal immune system. Bacteroides and Clostridiumspecies have been shown to induce the expansion of regula-tory T cells, reducing intestinal inflammation.77 Other mem-bers of the microbiota can attenuate mucosal inflammationby regulating NF-kB activation.78

A number of bacterial species, most notably the Bifido-bacterium, Lactobacillus, and Faecalibacterium genera, mayprotect the host from mucosal inflammation by severalmechanisms, including the down-regulation of inflammatorycytokines79 or stimulation of interleukin 10,80 an anti-inflammatory cytokine. Faecalibacterium prausnitzii, onesuch proposed member of the microbiota with anti-inflammatory properties, is underrepresented in IBD.81

F prausnitzii is depleted in iCD biopsy samples concomi-tant with an increase in E coli abundance,82 and low levels ofmucosa-associated F prausnitzii are associated with a higherrisk of recurrent CD after surgery.80 Conversely, recovery ofF prausnitzii after relapse is associated with maintenance ofclinical remission of UC.83

Several constituents of the gut microbiota fermentdietary fiber, a prebiotic, to produce short-chain fatty acids(SCFAs), which include acetate, propionate, and butyrate.SCFAs are the primary energy source for colonic epithelialcells84 and have recently been shown to induce the expan-sion of colonic regulatory T cells.77,85 The Ruminococcaceae,particularly the butyrate-producing genus Faecalibacte-rium,86 is decreased in IBD, especially in iCD.38,39,42,80,82

Other SCFA-producing bacteria, including Odoribacter andthe Leuconostocaceae, are reduced in UC, and Phasco-larctobacterium and Roseburia are reduced in CD.39 Inter-estingly, the Ruminococcaceae consume hydrogen and

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produce acetate that can be used by Roseburia to producebutyrate,39 and it is therefore consistent that both groupstogether are reduced in IBD.

Functional Composition of the GutMicrobiota in IBD

At the phylogenetic level, there is generally high vari-ability in the human microbiota between and within indi-vidual subjects over time.13 However, the functionalcomposition (ie, the functional potential of the gene contentof the metagenome) of the gut microbiota is strikinglystable.13 Metagenomic approaches may therefore providegreater insight into the function of the gut microbiota indisease than taxonomic profiling87,88; indeed, one such met-agenomics study of the IBD microbiome found that 12% ofmetabolic pathways were significantly different betweenpatients with IBD and healthy controls compared with just2% of genus-level clades.39 Metagenomic andmetaproteomicstudies have confirmed a decrease in butanoate and prop-anoate metabolism genes in iCD39 and lower overall levels ofbutyrate and other SCFAs in iCD,89 consistent with the de-creases in SCFA-producing Firmicutes clades seen in taxo-nomic profiling studies. Another metagenomic trend that hasbeen identified in the IBD microbiome is an increase infunctions characteristic of auxotrophic and pathobiont bac-teria, such as a decrease in biosynthesis of amino acids, andan increase in amino acid transporter genes.39 These bacteriagenerally have a reduced ability to produce their own nu-trients but rather transport them from the environmentbecause they are readily available at sites of inflammationand tissue destruction.39

A number of studies note an increase in sulfate-reducingbacteria, such as Desulfovibrio, in IBD.90 Mesalamine, acommon treatment for patients with IBD, inhibits the pro-duction of fecal sulfide; intriguingly, stool samples frompatients not treated with mesalamine show higher levels ofsulfide.91 Genes involved in the metabolism of the sulfur-containing amino acid cysteine are increased in IBD,particularly in iCD, and there is increased sulfate transportin both UC and CD.39 Saturated fat–derived taurine conju-gates to bile acids, increasing the availability of free sulfurand causing an expansion of the sulfate-reducing pathobiontBilophila wadsworthia, driving colitis in genetically suscep-tible Il10�/� but not wild-type mice.92 The IBD metagenomehas an increased propensity for managing oxidative stress, ahallmark of an inflammatory environment, as indicated byincreased glutathione transport and riboflavin metabolismin UC.39 There is also an increase in type II secretion sys-tems,39 which are involved in the secretion of toxins, and anincrease in bacterial genes with virulence-related func-tions89 in patients with CD, indicative of a shift toward aninflammation-promoting microbiome.

The Gut Microbiota inRelated Diseases

A number of parallels can be drawn between IBD andrelated metabolic diseases such as type 2 diabetes mellitus

(T2DM) and obesity. For example, there is an overalldecrease in diversity in obesity at both the phylogeneticlevel (ie, a reduced number of distinct species)30 and themetagenomic gene count level (ie, a reduced number ofdistinct genes).93 Major shifts in clade abundances include areduction of the Firmicutes and Clostridia in T2DM94 and asignificant increase in the Firmicutes to Bacteroidetes ratioin obesity in mice95; however, it is less clear whether thisshift also holds true in human obesity.30,96 There is adecrease in Bifidobacterium species in obesity and T2DM,96

and Bifidobacterium is reduced in children who becomeoverweight,97 suggesting that it may act as a predictivefactor. As in IBD, F prausnitzii is reduced in abundance inT2DM.98 In terms of gene function, there is an enrichment ofgenes involved in membrane transport,99 sulfate reduction,and oxidative stress resistance functions and a decrease infunctions involving cofactor and vitamin metabolism andbutyrate production.100 Therefore, many of the same shiftsin function of the gut microbiota, and even specific taxa, areseen across these diseases, suggesting the existence ofgeneralized features that relate these diseases and the se-lection for an auxotrophic microbiota that can thrive in aninflammatory environment.

IBD Treatments Affecting theMicrobiome

An array of antibiotics have been shown to lead to abloom of E coli.101 Because increased Enterobacteriaceae isa distinctive feature of intestinal inflammation and oxidativestress, the relationship between microbial composition,inflammation, and antibiotic use forms an important topicfor future research. In contrast, some promising data showthat antibiotic therapy specifically in IBD does induceremission or prevent relapse, but this topic will requirefurther controlled trials.102 To better understand the con-sequences of perturbing the gut microbiota of patients andthe role of the microbiota in treatment outcome, studies thatmonitor the temporal response at the levels of microbialecology and functional composition will be required.103

Thus far, several studies of healthy humans brieflyexposed to some antibiotics show the substantial pertur-bation, and the level of resilience, of the gut microbiota.104

Repeated exposures to a single antibiotic in healthy sub-jects result in cumulative and persistent changes to the gutmicrobial composition.105 Microbial homeostasis is typicallydisrupted by the loss of species complexity, particularly ofprotective microbes and thereby potentially resulting in anincreased risk of infections,106 or dysbiosis.45 Anothermechanism by which antibiotics lead to increased gut in-fections is by causing a thinning of the mucus layer, therebyweakening its barrier function.107

Instead of perturbing the existing microbiome byremoving diversity through antibiotics, repopulating the guthabitat with a healthy community has gained popularity inthe past few years. This will be an exciting new direction forthe pharmaceutical industry, expanding the focus beyondtraditional small molecules and biologics.108 The complexity

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and composition that will be used to repopulate the gutcommunity will be very important. The success of probioticsin the management of IBD ranges from mixed results toconsiderable potential109 and is dependent on the strainsused and disease subtype targeted. In contrast, the evidencethat fecal microbiota transplantation (FMT) can be highlyeffective in replenishing our complex microbiota hasreceived considerable attention because of a convincingclinical trial of treatment of relapsing Clostridium difficileinfection.110 Related studies have shown that the use of awell-selected community subset rather than whole fecalcommunities can be sufficient for recovery.111 The highsuccess rate reported for relapsing C difficile infection haselevated FMT as an emerging treatment for several gastro-intestinal and metabolic disorders112 and is actively beingconsidered for IBD.113 So far, the sparse results reported forcases of IBD have been variable with regard to the successrate for inducing remission, and well-designed randomizedcontrolled trials are currently still lacking.114 Althoughchanges in the composition of the intestinal microbiota weresignificant, and reductions in Proteobacteria as well as anincrease in Bacteroides after FMT were observed, reachingor maintaining remission has been less frequent.115

Changing protocols toward repeated FMT procedures for

multiple days in a row seems to increase the chances ofachieving clinical remission.116

Future DirectionsStudies thus far have been able to address many aspects

of IBD, including genetics, immune responses, microbialdysbiosis, and microbial functional activity. However,because of the complexity of the human microbiome as adynamically interacting system, only limited data have beenproduced to bridge the gap between pathogenesis in a hu-man host, individual microbes, and alterations in microbialmetabolism and function. This suggests the need for a moremultifaceted approach to the microbiome in IBD (Figure 2).Because the gaps in IBD are arguably the narrowest amongdiseases in the microbiome field, as indicated by the prog-ress reviewed in the preceding text, it can be considered amodel for systems-level investigations of human-associatedmicrobial communities and their interactions with the host’simmune system. Increasing our mechanistic understandingof host-microbe interactions through such a systems-levelapproach will provide new opportunities to develop di-agnostics and treatments117 but is certainly not withoutchallenges.118 Recent technological advances, including

Figure 2. A multifacetedapproach to study the roleof the microbiome in IBD.Future microbiome studiesin the context of diseasewill shift toward multi-omics approaches tostudy host-microbe re-lations more comprehen-sively. Optimized samplecollection, detailed clinicalannotation, and sampleprocessing will be key toexpanding data generationfar beyond the typicalmarker gene and shotgunsequencing approaches. Anumber of assays on thehost side (red) and micro-bial end (blue) will gainincreasing attention goingforward.

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improvements in sequencing and computational biology, arecontributing novel methods to study human-associated mi-crobial communities,119 with an increasing focus on func-tional follow-up using cultured microbes and germ-freeanimal models.

Essential to further explorations, more longitudinal sur-veys of patients before, during, and after treatment as well aslarge-scale clinical trials that take into account bothmicrobialand genetic heterogeneity will need to be performed andshould take a multifaceted approach. One approach to in-crease sample size is to combine different cohort studies.However, sample type,120 collection,121 and extraction122 canintroduce artifactual differences in microbial composition,which unfortunately makes it more challenging to combinedata sets produced under different protocols.123 Stream-lining experimental protocols across cohorts will be essentialto preserve statistical power for identifying true biologicaleffects in microbiome data sets. This will require growingefforts for standardizing the collection of patient samples andtheir clinical information. The discussion around the value ofuniform data collection has resulted in solutions already,124

but these need further adaptation for clinical information.Pursuing a true systems biology approach will require asolution for simultaneous measurement of the host state, themicrobiome, and the multidirectional signaling betweenthem. Although solutions for sequential isolation of metabo-lites, RNA, DNA, and proteins from the same unique samplehave been described,125 engineering a solution that can easilybe deployed as a self-sampling kit for patients will requirefurther exploration. Such challenges have been addressedwith other technologies, for example, in the use of micro-arrays as a tool for biomarker detection in clinical applica-tions, which led to the establishment of a consortium with amandate to set up standards and quality measures.126 Suchefforts are now also initiated in the microbiome space withboth environmental and clinical relevance (see www.hmpdacc.org, http://www.mbqc.org, and http://www.earthmicrobiome.org) and could eventually allow us tocombine several largewell-characterized cohorts without thechallenge of study-introduced biases.123

Despite promising correlations between shifts in micro-bial composition and disease phenotypes, to date no causa-tive role for the microbiome has been established, and ourunderstanding of the dynamic role of the human microbiomein IBD remains incomplete. The biological questions of in-terest enabled by prospective longitudinal studies would beto (1) identify the potential role of the intestinal microbiomein triggering disease, (2) determine if microbial compositionpredicts subsequent risk of activity flares, and (3) examinewhether the luminal flora predicts response to therapy. Theidentification of a correlative microbial pattern in humansthat induces antimicrobial defense and ameliorates inflam-mation would have considerable promise as a novel diag-nostic and therapeutic approach for themanagement of thesecomplex diseases. Existing correlative genetic-microbialstudies have helped to motivate this area but cannot speakto causality, response to treatment, or risk prediction in theabsence of multifaceted longitudinal measurements.

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Received October 29, 2013. Accepted February 17, 2014.

Reprint requestsAddress requests for reprints to: Ramnik J. Xavier, MD, PhD, Center forComputational and Integrative Biology, Richard B. Simches ResearchCenter, Massachusetts General Hospital, 185 Cambridge Street, Boston,Massachusetts 02114. e-mail: xavier@molbio.mgh.harvard.edu; fax: (617) 7246832.

Conflicts of interestThe authors disclose no conflicts.

FundingSupported by grants from the Crohn’s and Colitis Foundation of America (toR.J.X.) and National Institutes of Health grants U54 DE023798 and R01DK092405 (to R.J.X.).