Functional impacts of the intestinal microbiome in the pathogenesis of inflammatory bowel disease.

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Functional Impacts of the Intestinal Microbiome in the Pathogenesis of Inflammatory Bowel Disease Jennifer Li, BSc,* James Butcher, BSc,* David Mack, MD,† and Alain Stintzi, PhD*

Abstract: The human intestinal microbiome plays a critical role in human health and disease, including the pathogenesis of inflammatory bowel disease (IBD). Numerous studies have identified altered bacterial diversity and abundance at varying taxonomic levels through biopsies and fecal samples of patients with IBD and diseased model animals. However, inconsistent observations regarding the microbial compositions of such patients have hindered the efforts in assessing the etiological role of specific bacterial species in the pathophysiology of IBD. These observations highlight the importance of minimizing the confounding factors associated with IBD and the need for a standardized methodology to analyze well-defined microbial sampling sources in early IBD diagnosis. Furthermore, establishing the linkage between microbiota compositions with their function within the host system can provide new insights on the pathogenesis of IBD. Such research has been greatly facilitated by technological advances that include functional metagenomics coupled with proteomic and metabolomic profiling. This review provides updates on the composition of the microbiome in IBD and emphasizes microbiota dysbiosis-involved mechanisms. We highlight functional roles of specific bacterial groups in the development and management of IBD. Functional analyses of the microbiome may be the key to understanding the role of microbiota in the development and chronicity of IBD and reveal new strategies for therapeutic intervention. (Inflamm Bowel Dis 2015;21:139–153) Key Words: Crohn’s disease, ulcerative colitis, intestinal microbiota, dysbiosis

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he human intestinal microbiome consists of a complex ecosystem of microbial cells that play a critical role in human health. Under normal physiological conditions, this dynamic population acts in symbiosis with its host. Consequently, a rift within this mutualistic relationship is thought to manifest into various disorders, including inflammatory bowel disease (IBD).1–3 IBD is largely defined by 2 chronic inflammatory disorders, Crohn’s disease (CD) and ulcerative colitis (UC), which are characterized by chronic persistent inflammation of the intestinal mucosa lining the intestinal tract. The inflammation during CD extends deeper into the bowel wall than UC.4 The etiopathogenesis of IBD has been conceptualized as occurring in genetically susceptible individuals after a triggering event that Received for publication June 30, 2014; Accepted August 6, 2014. From the *Ottawa Institute of Systems Biology and Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada; and † Department of Pediatrics and CHEO Research Institute, University of Ottawa, Ottawa, ON, Canada. A. Stintzi and D. Mack are funded by the Government of Canada through Genome Canada and the Ontario Genomics Institute (OGI-067), CIHR grant number GPH-129340, CIHR grant number MOP-114872, and the Ontario Ministry of Economic Development and Innovation (REG1-4450). D. Mack and A. Stintzi also acknowledge funding from the 3C Foundation of Canada and the Crohn’s and Colitis Canada (CCC). The authors have no conflicts of interest to disclose. Reprints: David Mack, MD, Department of Pediatrics, CHEO, 401 Smyth Road, Ottawa, ON K1H 8L1, Canada (e-mail: [email protected]); or Alain Stintzi, PhD, Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology; University of Ottawa, 451 Smyth Rd., Ottawa, ON, Canada, K1H 8M5 (e-mail: [email protected]). Copyright © 2014 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1097/MIB.0000000000000215 Published online 21 September 2014.

Inflamm Bowel Dis Volume 21, Number 1, January 2015

generates an aberrant host immune response, and increasing evidence suggests that the intestinal microbiota is an important contributor to the disease.1,2,5–8 In recent years, numerous studies have been conducted to characterize the intestinal microbiota in patients with IBD. These studies have been particularly facilitated by methodological advances in high-throughput sequencing, metagenomics, proteomics, and metabolomic profiling.3,9–11 The integration of this knowledge into hypothesis-driven research using animal models can help define the etiological role or causal effect of the intestinal microbiota and/or microbial metabolic pathways in the development of intestinal inflammation. To date, a wealth of information has been generated regarding the altered microbiota composition, or dysbiosis, at different taxonomic levels that manifest in patients with IBD and animal models for IBD. Although no microbe has yet been identified as a sole causative agent for IBD, the specific changes observed within the microbiome during IBD have been proposed for use as biomarkers for IBD diagnosis, for predicting disease outcomes and for monitoring patient responses to therapeutic interventions.3,8,12–14 Lingering questions still remain on whether potential biomarker microbes are actively involved in the clinical features seen in IBD or whether they are simply bystanders that thrive in the inflamed milieu present during active IBD. Given the numerous confounding factors that affect microbial species distribution and abundance, it is not surprising that various studies have reported not only highly variable but also inconsistent observations on the compositional changes of microbiota that result from IBD. However, an emerging paradigm postulates that the metabolic functions fulfilled by the gut microbiota are more www.ibdjournal.org |

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relevant than the specific microbes that are fulfilling these roles and that identifying the dysregulation of these metabolic processes will yield greater insight into disease progression than simply identifying specific microbes that are present or absent. As a result, functional predictions from altered microbiota are required to address this knowledge gap. Researchers in the current -omics era have explored combinatorial approaches such as metagenomics, metatranscriptomics, metaproteomics, and metabolomics to assess the composition, gene expression, protein activity, and metabolic potential of the gut microbiota15–19; these interdisciplinary approaches should provide powerful tools to help define the structure, dynamics, and function of gut microbiota. This article is aimed at highlighting studies on altered microbiota with an emphasis on linking these changes in microbiota composition with functional changes that occur during IBD. These studies provide insights on the role of microbial dysbiosis and various microbial metabolic pathways that may contribute to the pathophysiology of IBD. Further unraveling the potential metabolic roles of the microbiota in IBD should lead to a better understanding of the root causes of IBD and may provide alternative therapies that target the diseased microbiota (as opposed to the diseased host) and enhance disease management.

GASTROINTESTINAL MICROBIOTA: COMPOSTION, FUNCTION, AND DISEASE The human gastrointestinal tract is colonized by a diverse population of more than 1000 bacterial species along with archaea, eukaryotes, fungi, and viruses.20–24 The combined microbial metagenome of .5 million genes is more than 100 times larger than its host, which is consistent with our ever-increasing understanding of the diverse functions that the microbiota contribute in normal human physiology and during disease pathology.23,25,26 Under normal physiological conditions, the gut microbes provide mutualistic benefits to their host with major contributions in metabolic, protective, and trophic functions.27–29 The diversity and composition of a healthy human microbiome for specific individuals is profoundly affected by diet, host genetics, early microbial exposures, and environment. Extensive sampling of the human microbiome through oral and stool samples has revealed diverse microbial communities at the family level, which are strongly influenced by the habitat in which they are found.30 However, studies indicate that the functional repertoire of the gut microbiome is relatively more stable than the compositional diversity,30 which highlights the importance of examining the human microbiome in diseased populations from a functionality point of view. There are pronounced temporal variations within a normal individual’s microbiome even in short timespans.31 However, there is general consensus that one’s microbiome does become more stable and more similar to the general population as one enters adulthood; this stability then declines in the elderly.32–35 Although the diversity of bacterial species varies greatly between individuals, recent studies have identified common core subsets within the microbiome that are relatively stable throughout

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large populations and could even last for decades.12,36 A recently published article has narrowed the 1000 bacterial species of the gastrointestinal microbiome into a long-term core of approximately 100 species.36 Many species within this subset are considered early colonizers that likely originated from immediate family members.36 The Firmicutes and Bacteroidetes phyla (which begin to colonize in infants within the first week of birth) later represent the majority of the intestinal microbiota in adults with Bacteroides as the most abundant, yet most variable, genus.12,37–40 An in vivo study characterized the chronological establishment of healthy microbiota in mice.41 Like the human microbiome, the Firmicutes and Bacteroidetes phyla also dominate the mouse gut microbial ecosystem.41 Nevertheless, at the family level, different studies showed variable results, suggesting the importance of analyzing microbiota at the lower phylogenetic levels.41 It is interesting to note that lowabundance organisms provide specialized functions that are beneficial to the host12; thus, even small shifts in microbial composition may have a measurable impact on one’s health. The impacts of the intestinal microbiome have been studied in many aspects of health and disease. A study assessing the intestinal microbiome composition in the elderly clearly showed a correlation between microbial composition, diet, and health status (including clinical measures of frailty, comorbidity, nutritional status, markers of inflammation, and with metabolites) suggesting a role for dietdriven microbiota changes.32 Interestingly, a metagenomic-wide association study characterizing the intestinal microbiome of individuals with type 2 diabetes revealed 6000 microbial markers for classifying type 2 diabetes.42 The gut microbiota also interacts intensely with the host immune system through the intestinal mucosal surface.21 Curiously, it has also been established that the gut microbiota can interact with distant organs, such as the brain.43 Altogether, these reports strongly emphasize the critical role of gut microbiota in human health through host–microorganism functional interactions.

MICROBIOME IN IBD Intestinal dysbiosis (specifically a compositional imbalance of commensal bacteria) is the central characteristic of the gut microbiota in IBD and has been considerably documented.8,13,44–46 Copious studies have compared the distinct microbial compositions of healthy individuals and patients with IBD through 16S rRNA gene analysis.47–49 Such studies have generally used biopsies (for mucosa-associated microbiota) or fecal samples to determine specific bacterial alterations in patients with IBD.47–49 Moreover, researchers have uncovered remarkable dissimilarity in the intestinal microbiota of healthy individuals and patients with IBD from fecal samples.50–52 This inconsistency makes it challenging to assess the clinical significance of species-specific variability because the composition of the gut microbiota is also affected by age, diet, smoking, geography, and condition of health (Fig. 1).53,54 Nevertheless, there are certain bacterial species or phylotypes that are found more frequently in either healthy or diseased populations. A common feature of the intestinal microbiome in IBD is the reduced abundance of several types of bacteria, particularly


Functional Impacts of the Intestinal Microbiome

FIGURE 1. The interplay between host microbiota dysbiosis, genetic status and environmental factors contribute to the pathogenesis of IBD.

members of the Firmicutes and Bacteroidetes phyla.13,45,46 In fact, species richness (a-diversity) as a whole is seen to decrease in patients with IBD.46 Losses of the core microbiome and temporal stability at differing functional levels have also been identified.46,50,55–60 Although some researchers have also reported similar microbiota alterations in biopsy and fecal samples from patients with IBD,61 others have documented differences related to sources of samples.13 A recent investigation studying the intestinal microbiome in the largest pediatric inception, treatment-naive CD cohort to date has convincingly revealed the importance of using ileal or rectal samples as compared with fecal sources, suggesting that the assessment of rectal mucosal-associated microbiome offers a unique potential for convenient and early diagnosis of CD.13 Despite the fact that species-driven dysbiosis in CD and UC can be different,51,62–65 overall species diversity is generally reduced in both CD and UC compared with healthy populations.51,66,67 However, these changes are often inconsistent and occasionally even conflicting among different studies as further assessed in the next section below. Hence, it is imperative that researchers consider the sampling time, sample source (e.g., ileum, rectum, feces, etc), and patient disease status when reporting results and drawing conclusions. Additionally, pharmacological treatment or other therapeutic interventions are also potential confounders in studying the composition of the microbiota in IBD and should also be taken into account.49 Considering the complexity of microbial compositions, an ecological analytical approach has been used to identify modules of interacting bacteria that represent quantitative reproducible features of microbial compositions.68 This study has revealed the presence of 5 microbial modules in endoscopic lavage samples from patients with IBD

and healthy controls; 2 of these modules showed a distinct metabolic functionality and were reciprocally associated with IBD.68 These 2 modules could act as potential indicators to better characterize IBD from both a compositional and functional perspective and thus aid in IBD disease management. It has long been known that the host’s genetic makeup also affects their vulnerability towards IBD.6 Although some genetic risk factors are specific to either CD or UC, there are a group of common genes between the 2 conditions as well. Although CD and UC are both associated with genomic regions that implicate products of genes involved in leukocyte trafficking, there is evidence for association patterns that are distinct between CD and UC.69 CD-predominant associations include nucleotide-binding oligomerization domain 2 (NOD2) and genes that regulate autophagy.69–72 Interestingly, Cuthbert et al72 determined that homozygotes and compound heterozygotes of the NOD2 mutation have .20-fold increased risk to CD. NOD2 recognizes bacterial peptidoglycan (muramyl dipeptide) and is required for the production of several intestinal antimicrobial peptides that provide protective immunity to the host.73 NOD2 mutations lead to a loss of function, which increases susceptibility to CD.74 NOD2 is also known to interact with autophagy-related protein 16-like 1 (ATG16L1) or immunity-related GTPase M (IRGM) for autophagic clearance of intracellular pathogens throughout the body.75 Thus, when NOD2 is mutated, ATG16L1 or IRGM alone are insufficient for the induction the autophagic response, which, in turn, can exacerbate microbial dysbiosis. Similarly, ATG16L12/2 mice also displayed an increased risk for CD.76 In contrast, extracellular matrix protein 1 is a susceptibility locus specific for UC.77 Extracellular matrix protein 1 is expressed in the intestinal tracts and activates NF-kB, an important immune response regulator.77 www.ibdjournal.org |

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Genes involved in the unfolded protein response are also associated with IBD.75 The Il102/2, Il22/2, Il10r22/2Tgfbr22/2, and Tbx212/2Rag22/2 genotypes act as risk factors that promote the development of spontaneous colitis in mice.78 For instance, many patients diagnosed with early-onset IBD have mutations affecting the interleukin-10 (IL-10) receptor. Because IL-10 is an anti-inflammatory cytokine, disruption of this pathway yields a hyperinflammatory immune response within the intestine.79

COMPOSITIONAL CHANGES OF SPECIES-SPECIFIC BACTERIA IN IBD The intestinal microbiota signatures of patients with IBD often differ from healthy populations. The compositional shifts that differ between the groups include reduced microbial diversity and beneficial commensal species, as well as increased levels of potential pathobionts.13,51,67,80 Still, these compositional changes have evidently been complex. Table 1 provides a list of speciesspecific bacterial abundance changes (increased or decreased) in patients with CD and UC; although, as discussed above, there are conflicting reports for several bacteria. The dominant gut microbe, Bacteroides, can both positively and negatively affect the host.133 In certain circumstances, individual species of Bacteroides may be reduced, whereas the overall genus is enriched in patients with IBD.14,67 A study on the spatial organization and composition of the mucosal biota of patients with IBD indicated Bacteroides fragilis biofilm as a main feature of IBD.134 Recent studies continue to reveal significant abundance changes of several bacteria including Bdellovibrio bacteriovorus, Erysipelotrichales, Pasteurellaceae, and Veillonellaceae in patients with CD (Table 1).13,106 Specifically, reduced B. bacteriovorus occurs along the intestine in patients with IBD from the duodenum to rectum with the most dramatic decrease in the ileum.106 Patients with UC have higher bacterial densities associated with biopsies than patients with CD.62 Reduced Faecalibacterium and Bifidobacterium as well as increased Bacteroides were found in patients with active CD from multiple IBD research centers.86 The reduced levels of F. prausnitzii were found to be inversely related to the severity of UC.64 Furthermore, the reduction of F. prausnitzii indicated a higher risk for postoperative recurrence of ileal CD.135 In addition, characterizing the mucosa-associated microbiota of twin pairs discordant or concordant for CD revealed a reduced abundance of F. prausnitzii and elevated abundance of Escherichia coli in patients with CD.53 A consistent reduction of F. prausnitzii was observed in fecal samples of patients with UC during remission, particularly those with a history of frequent relapse, whereas maintenance of remission was associated with F. prausnitzii increase.102 However, despite these multiple reports of reduced levels of F. prausnitzii, Hansen et al51 detected increased F. prausnitzii in de novo pediatric CD patients at the onset of disease but not in UC. In another study, comparing twins with CD, patients with colonic CD and ileal CD exhibited an increase and decrease in F. prausnitzii, respectively.52 The differences between these studies can likely be attributed to differences between sampling

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sources and times (e.g., colonic mucosa versus stool, de novo versus established IBD). Again, these comparisons highlight the importance of a standardized sampling methodology to minimize confounding factors and to facilitate comparability between studies. Patients with CD also showed a lower relative abundance of Bacteroides uniformis and higher relative abundances of Bacteroides ovatus and Bacteriodes vulgatus.66 Reduced bacterial populations within the Clostridium Cluster IV and XIVA groups were observed in fecal samples from CD and UC patients, respectively.98,136 However, a recent case-control study has suggested that reduced numbers of the Clostridium Cluster IV group occurs in both CD and UC.101 Another dominant species, Roseburia hominis, also appears to be reduced significantly in patients with UC and correlates inversely to UC disease activity and duration.64 The abundances of F. prausnitzii and R. hominis parallel with each other, suggesting that these microbes may potentially define UC dysbiosis and could thus be used to manage the microbial balance in patients with UC by the administration of probiotics or prebiotics.64 Interestingly, these bacterial populations do not directly correlate with the reduced levels of short-chain fatty acid (SCFA) metabolites that are known to regulate inflammation, which again suggests that compositional analysis alone may not be sufficient enough to characterize IBD.64 A study on the fecal microbiota of patients with UC identified 8 distinct clusters, 7 of which distinguish patients with UC from the single healthy individual cluster.137 Furthermore, these groups consist of various unclassified bacteria, which include Eubacterium, Fusobacterium, Ruminococcus, Gammaproteobacteria, Bacteroides, and Lactobacillus.137 Altogether, these bacteria seem to be able to distinguish patients with UC from those of healthy individuals and thus could be potential IBD biomarkers. Pathogenic bacteria such as Escherichia coli (particularly adherent invasive E. coli [AIEC]), Rhodococcus spp., Shigella spp., and Stenotrophomonas maltophilia are increasingly observed in patients with IBD.87,122,123,131,138 Analysis of fecal samples from pediatric IBD, revealed that E. coli was positively and Bacteroides, negatively, correlated with age in patients with CD, whereas patients with UC showed the inverse.65 Additionally, patients with IBD showed an increased urogenital biofilm of Gardnerella vaginalis, a key pathogen in bacterial vaginosis.139 It should be noted that patients with IBD can suffer from various infections associated with opportunistic pathogens; and thus, the increased levels of these pathogenic bacteria may be due to the secondary infections unrelated to IBD pathogenesis.140,141 The overall incidence of IBD is rapidly increasing worldwide.142 Although IBD is the most commonly diagnosed disease in young adults (20–30 yr old), it can manifest at any age. Alarmingly, epidemiological studies have shown that the incidence of IBD onset in children is increasing.73,143 These results are quite intriguing because the microbiome is known to change immensely over the course of aging.144 The microbiome of a newborn infant is much less complex than that of an adult, with birth-route, type of feeds, and genetics as the major influences on the initial microbial abundance and diversity.3 The microbiome stabilizes as one becomes a young adult, at which life-stage, the environment has a larger influence on



TABLE 1. Abundance Changes of Gut Microbiota Identified in Biopsy and/or Fecal Samples of Patients with IBDa Bacterial Taxa Phylum/Class

Order, Family, Genus or Species

CD

UC

References

Actinobacteria

(Not specified) Bifidobacterium B. bifidum Mycobacterium avium paratuberculosis Bacteroides, B. ovatus, B. vulgatus Bacteroidale, Bacteriodes or B. uniformis Odoribacter Parabacteroides Prevotella Enterococcus Lactobacillus Leuconostocaceae Listeria Streptococcaceae Clostridiales Clostridium difficile Clostridium coccoides Clostridium leptum Eubacterium rectale Faecalibacterium prausnitzii Lachnospiraceae Phascolarctobacterium Roseburia or R. hominis Ruminococcaceae Ruminococcus gnavus/R. torques Erysipelotrichales Veillonellaceae Fusobacteriaceae Bdellovibrio bacteriovorus Desulfovibrio Campylobacter Helicobacter Enterobacteriaceae Escherichia coli Escherichia coli (AIEC) Pasteurellacaeae Shigella Stenotrophomonas maltophilia (Not specified) (Not specified) (Not specified) Akkermansia Methanosphaera stadtmanae

+ +/2 2 + + 2

+ +

Refs. 46,68,81,82 Ref. 49 Ref. 49 Refs. 83–85 Refs. 66,86–88 Refs. 13,66,67,81,82,89,90 Refs. 17 Refs. 91 Refs. 47,92,93 Refs. 89,94 Ref. 49 Ref. 17 Ref. 89 Ref. 61 Refs. 47,87 Refs. 82,89,95–97 Refs. 98–100 Refs. 98,100,101 Refs. 94,99 Refs. 45,46,49,52,53,64,81,86,89,94,100,102–104 Refs. 46,61,91 Ref. 17 Refs. 17,52,64,100,104 Refs. 46,89,91 Refs. 52,92,105 Ref. 13 Ref. 13 Refs. 13,68,91 Ref. 106 Refs. 107–110 Refs. 80,97,111–114 Refs. 114–119 Refs. 13,47,52,61,88,114,120,121 Refs. 49,53,87,89,94,121 Refs. 92,114,122–130 Ref. 13 Refs. 87,89 Ref. 131 Refs. 46,47,68,81,82,94,103,114 Ref. 68 Ref. 68 Refs. 14,90,105 Ref. 132

Bacteroidetes

Firmicutes/Bacilli

Firmicutes/Clostridia

Firmicutes/Erysiplotrichi Firmicutes/Negativicutes Fusobacteria Proteobacteria/Deltaproteobacteria Proteobacteria/Epsiloproteobacteria Proteobacteria/Gammaproteobacteria

Proteobacteria Tenericutes Verrucomicrobia Euryarchaeota (Archaea)

2 + + +

2 2 +

2 + 2 +/2 +/¼ 2 2 +/2 2 2 2 + 2 + + 2 +/¼ +/¼ + + + + + + + + + 2 +

2 + +/¼ 2 2 2/¼ 2 2 2 +

+ + +/¼ +/¼ + + +/¼

+ + + 2 +

a The plus (+) and minus (2) signs denote a reported increase or decrease in abundance for the relevant microbiota respectively. The equal sign (¼) represents that no difference in abundance was found in that relevant study.

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its composition.3 Age-specific taxonomic changes have been identified in the intestinal microbiome.54 A meta-analysis of human microbiota from multiple studies has shown an age-related fecal bacterial gradient with a transition from communities enriched in Bifidobacteriaceae, Clostridiaceae, Enterococcaceae, Enterobacteriaceae, Lactobacillaceae, and Streptococcaceae in early age to communities enriched in Bacteroidaceae Lachnospiraceae, Prevotellaceae, and Ruminococcaceae in adults.145 Among all of the compositional changes of the microbiome during IBD, it is particularly important to highlight the dysbiosis caused by pathobionts. Pathobionts are distinguishable from pathogens as they only become harmful to predisposed individuals when an environmental stimulus is triggered.146,147 Helicobacter hepaticus is such a pathobiont causing colitis in immunocompromised mice.148,149 Yet, the type VI secretion system of H. hepaticus has been found to induce anti-inflammatory gene expression within intestinal epithelial cells, thus promoting a balance between host colonization and intestinal inflammation.72 Escherichia coli, such as AIEC, is another important pathobiont that may play a role in IBD development150 (see details below in section Antiinflammatory Effects of Microbiota: Function and Mechanisms). The expression of the mannose-binding FimH adhesin is critical for the role of AIEC in IBD.151,152 AIEC strains contain point mutations in FimH that cause a pathoadaptive alteration which enhances intestinal inflammatory response.153 AIEC also alters the microbiota composition in susceptible mice by elevating the production of bioactive lipopolysaccharide (LPS) and flagellin.154 Other relevant pathobionts include Clostridium difficile, Klebsiella pneumoniae, Proteus mirabilis, and Prevotellaceae.155 Nevertheless, given that the microbiota exhibits important control over pathobionts and pathogens,156 interventions can be applied to prevent the etiological effect of pathobionts. For example, the microbial molecule polysaccharide A (which is produced by Bacteroides fragilis) has been demonstrated to prevent H. hepaticus-induced experimental IBD in animal models, likely by inducing IL-10 production and subsequent suppression of colitis.149

ANTI-INFLAMMATORY EFFECTS OF MICROBIOTA: FUNCTION AND MECHANISMS The anti-inflammatory properties of the intestinal microbiota involve host–microbiota interactions that use various metabolic pathways and the immune system. An early study showed that the development of spontaneous colitis and activation of the immune system requires the presence of enteric bacteria.157 Metagenomic computational assessment of the intestinal microbiome identified an overall shift associated with IBD that was seen at both the microbial gene-level and metabolic network-level.3 Such studies demonstrate the importance of evaluating changes in microbial functionality to understand their effects on IBD pathogenesis. Functional signatures of the CD microbiota from stool samples include alterations in bacterial carbohydrate metabolism, bacteria–host interactions, and host-secreted enzymes compared with healthy controls.158 These signatures could be used as potential targets for biomarkers

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and therapeutic interventions. We have an increased appreciation for the potential role of specific bacteria in the etiopathogenesis of IBD. This section highlights some key microbial functions and mechanisms that may influence IBD disease progression.

Metabolite-producing Microbiota The metabolic functions of the intestinal microbiota greatly influence the intestinal environment and thus, the host’s health.2 Changes in microbial composition can lead to an imbalance in metabolites production which in turn might be linked to IBD pathogenesis. Various bacteria, including Faecalibacterium, produce SCFAs, such as acetate, propionate, and butyrate.159,160 Acetates are a substrate for butyrate production, whereas butyrate is a major energy source for colonic epithelial cells; thus, butyrate-producing bacteria play an important role in epithelial barrier integrity.159,160 Indeed, Machiels et al64 found a decrease in butyrate-producing bacteria and a reduction of acetate and proprionate levels in fecal samples from patients with UC. Additionally, a study using an in vitro simulator of the human intestinal microbial ecosystem found lowered SCFA and butyrate production by the luminal communities present in patients with UC.99 The abundance of known SCFA producers (Clostridium Clusters IV and XIVA, F. prausnitzii, and Roseburia spp.) and genes for butyryl-CoA:acetate CoA transferase (responsible for butyrate production) were also reduced.99 Diet greatly influences the composition of the microbiome and the metabolites that are produced by the bacterial species. Both long- and short-term dietary styles have been linked to microbial enterotypes. For example, Bacteroides species increase with protein and animal-fat rich diets and Prevotella increases with carbohydrate rich diets.161 Increases in animal-based diets result in an increased abundance of bile-tolerant bacteria such as Bacteroidetes (Alistipes and Bacteroides) and Proteobacteria (Bilophila) and a decreased abundance of Firmicutes (Roseburia, Eubacterium rectale and Ruminococcus bromii).162 Bile acid (e.g., taurocholic acid) production increases as well in animal-based diets, affecting microbial composition by promoting the expansion of pathogens such as sulphite-reducing Bilophila wadsworthia, which increase microbiota dysbiosis perturbing immune homeostasis.163 Tryptophan may be also involved in the etiopathogenesis of IBD. Interleukin-27 (IL-27) is a mediator of gut epithelial barrier protection and is elevated during IBD.164 The increase of IL-27 impacts the expression of hundreds of genes including indoleamine 2,3dioxygenase. Indoleamine 2,3-dioxygenase is an anti-inflammatory and antibacterial protein that is involved in initiating tryptophan catabolism.165 IL-27–induced indoleamine 2,3-dioxygenase leads to microbiome dysbiosis by inhibiting the growth of the intestinal bacteria through local tryptophan depletion.164 Interestingly, tryptophan reduction is involved in inflammation throughout the human body; deficiencies in tryptophan hydroxylase-1 (which converts tryptophan to serotonin) are known to intensify neuroinflammation.166

Microbiota Disruption of the Intestinal Barrier The intestinal barrier is composed of a single layer of epithelial cells linked through tight junctions and a mucus shield


with strong surface hydrophobicity.167 This cell layer is composed of various cell types, including goblet and Paneth cells, that produce mucin and antimicrobial peptides (e.g., defensins, cathelicidins, and lysozymes), respectively. Additionally, goblet and Paneth cells provide strong barrier functions that control the equilibrium between tolerance to microbes and non-self-antigens.168,169 Consequently, disruption of epithelial barrier integrity has been implicated in IBD pathogenesis through a wide range of specific mechanisms encompassing structural, metabolic, and innate immune pathways.170 Barrier defects include increased permeability of the mucus to water and various small or large molecules that can elicit immune response either as a cause or a consequence of IBD. Various bacteria produce hemolysins, which damage host cell membranes. Hemolysin secreted by Escherichia coli increases the initial lesions in the colon mucosa (dubbed as “focal leaks”), which in turn reduces transepithelial electrical resistance and increased macromolecular uptake.171,172 Mucosal samples between patients with IBD and healthy controls show similar levels of a-hemolysin-producing E. coli. However, patients with active UC display 10-fold higher levels of a-hemolysin, suggesting a role of a-hemolysin in disruption of the epithelial barrier, again highlighting the importance of measuring the metabolic capacity of the microbiota rather than simply its composition.172 Bacterial proteases are also known to affect intestinal integrity. Gelatinase, a metalloproteinase produced by Enterococcus faecalis, disrupts the epithelial barrier and increases inflammation in mice that are genetically susceptible to IBD.173 Enterococcal isolates from IBD show higher frequencies of virulence genes including gelE that encodes for gelatinase.174 Similar to other pathogenic species such as Campylobacter, Escherichia coli, and Shigella species, certain Helicobacter species produce cytolethal distending toxins,175 which cause cell cycle arrest, chromatin fragmentation, and apoptosis and are speculated to adversely influence IBD development.176 The gut microbiota composition of IL-10–deficient mice of different research facilities has been found to differ, and this variation was associated with differing susceptibility to colitis induced by Helicobacter hepaticus.177 Indeed, increased prevalence of enterohepatic Helicobacter species in patients with CD and UC have been observed,115 although there are conflicting reports whether Helicobacters (specifically Helicobacter pylori) contribute to IBD progression.178 As discussed above, such discrepancies can likely be attributed to different sampling methodologies. Adherent Lactobacillus spp. can induce upregulation of extracellular mucin secretion and thus protect the gut mucosal surface against the adherence of enteropathogens.179 Intriguingly, frequent use of nonantibiotic non-steroidal anti-inflammatory agents was also found to be a potential risk factor for IBD180 because non-steroidal anti-inflammatory agents are well known to disrupt mucosal integrity along the intestinal tract30 and might impact the microbiota composition.

Adherent Invasive E. coli The colonization of AIEC is frequently observed in patients with IBD and, its role in IBD seems to be multifactorial (Table 1). Mucosa-associated AIEC strains with a 54 kb polyketide synthase


genotoxic island were identified at a significantly higher rate in IBD and colorectal cancer patients.181 The polyketide synthase island encodes the multienzymatic machinery involved in the synthesis of colibactin (a peptide–polyketide hybrid genotoxin), and colibactin was involved in inducing tumorigenesis.181 In addition, defects in Paneth cell function or an autophagy deficiency of the host can affect one’s ability to control AIEC infection. Patients with IBD exhibit abnormal expression of carcinoembryonic antigen-related cell adhesion molecule 6 in the ileum, which can also promote AIEC colonization.182,183 IBD-derived AIEC produces pili variants that enhance their adhesion to ileal epithelial cells.124,153 AIEC are known to induce the expression of claudin-2, a pore-forming tight junction protein, and the production of proinflammatory cytokines by infected macrophages.124,125,184 AIEC also induces inflammation by upregulating gut epithelial cell microRNAs that reduce autophagy and whose perturbation is known to be associated with autoimmune diseases like IBD.146,185 Together with genetic factors, IBD-associated AIEC also modulates the ubiquitin proteasome system by decreasing the accumulation of polyubiquitin conjugates, increasing 26S proteasome activities and reducing protein levels of the NF-kB de-ubiquitinase CYLD in the intestine, resulting in aberrant NF-kB activation present during IBD pathogenesis.126 A study on transient AIEC colonization in mice revealed that mice lacking Toll-like receptor 5, which recognizes the presence of bacterial flagellin, are prone to inflammation development.186 This colonization is correlated with microbiota compositional alterations and elevated levels of bioactive LPS and flagellin, implicating an additional role for AIEC in IBD by altering the microbiota composition through a flagellin-dependant manner.186 Furthermore, a higher abundance of the Clostridia class (notably Lachnospiraceae family) was observed, which produces flagellins that are immunodominant antigens in several T-cell–mediated colitis models and CD.186,187 Thus, microbiota alterations are likely involved in promoting the chronic inflammatory process. A recent study also found that a high-fat highsugar diet resulted in microbiota dysbiosis in mice with altered host barrier function; this dysbiosis favored AIEC colonization.92 In agreement with these results, dietary supplementation with the polysaccharide maltodextrin-induced type 1 pili expression in Escherichia coli enhanced AIEC adhesion through type 1 pili-dependent mechanism and enhanced biofilm formation.188

Microbiota in Immune Response Although microbiota–host immune system interactions have been clearly shown to play a major role in IBD, these interactions will be only briefly discussed in this review. Readers are encouraged to read the following reviews for a more detailed discussion of these interactions.40,44,69 Instead, several key reports that demonstrate the direct link between IBD immune response and the intestinal microbiota are highlighted. Reduced levels of F. prausnitzii in IBD can be explained by the anti-inflammatory properties of this species that function by partly blocking NF-kB activation and IL-8 production in IBD animal models and thus improving the dysbiosis seen in www.ibdjournal.org |

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chemically induced mouse models of colitis.135 Bacteriodes fragilis, a major commensal, produces antigens, such as polysaccharide A, that induce the conversion of CD4+ T cells into FoxP3 regulatory T cells, which in turn produce the IL-10 cytokines that dampen the inflammatory response.149,189,190 In addition, Lactobacillus casei/paracasei species produce a bacterial protease, lactocepin, which selectively degrades the lymphocyte-recruiting chemokine IP-10.191 IP-10 is a proinflammatory chemokine that has been implicated in IBD; thus, its degradation by lactocepin can partially explain the beneficial effects from Lactobacillus spp.191,192 Patients with CD also show a dysregulated response to microbial DNA that is mediated through upregulation of IL-17A, a proinflammatory cytokine.193 The microbiota can also produce bacterial peptidoglycans that can activate the host’s nucleotide-binding oligomerization domain-containing protein 2 (NOD2), a major player implicated in IBD pathogenesis and stimulate the release of proinflammatory cytokines, autophagy, and antigen presentation.6 LPS is a key outer membrane component of Gram-negative bacteria such as Enterobacteriaceae, and its endotoxicity contributes to T-cell mediated colitis development in mice.194 Yet, mutated LPS with low endotoxicity is associated with mucosal immune homeostasis and prevents colitis with fecal samples displaying higher Bacteroidetes and lower Enterobacteriaceae abundances.194

a role for oxygen in intestinal dysbiosis.4 In support of this, there seems to be a shift from anaerobiosis in the healthy state to dysanaerobiosis in IBD with an elevated oxygen level in the gut.199 However, the potential use of antioxidants or probiotics to decrease oxygen levels for the management/treatment of IBD remains to be fully tested. Interestingly, in contrast to this hypothesis, another potentially microbiota-related metabolic disease, type 2 diabetes, has been recently hypothesized to be a redox disease, where the lack of oxygen could be a trigger for type 2 diabetes.200 These findings also support further study into the contribution of oxygen to IBD. Indeed, Walujkar et al201 recently observed both microbial dysbiosis and dysanaerobiosis in patients with UC.

MICROBIOTA-ASSOCIATED INTERVENTIONS FOR IBD Because there is no identified cure for IBD, disease control is a primary focus for therapeutics. Microbiota-involved pathogenesis in IBD has prompted the investigation on the effect of various interventions that specifically target the microbiome in IBD disease progression. The impacts from the administration of antibiotics, probiotics, prebiotics, or dietary supplementation are among the best studied interventions that are currently in use clinically.

Antibiotics Role of the Inflammasome Colonic microbiota can be regulated by host cytoplasmic multiprotein complexes, known as the inflammasome, which constitutes one of the nucleotide-binding oligomerization domain protein-like receptors and provides a regulatory sensing system within the colon.195 NLRP6 suppresses colitogenic microbiota and increases the abundance of Prevotella through IL-18; genetic deletion of NLRP6 has an adverse impact on microbial composition by causing a shift towards a proinflammatory environment that may contribute to IBD initiation and maintenance.93 Treatment with metronidazole and ciprofloxacin led to a reduction of Prevotella species and an ameliorated, anticolitogenic microbiota.93 These results provide evidence showing a perturbation in inflammasome pathway, which could potentially be considered a risk factor for IBD. Prolonged IBD increases the risk in developing colorectal cancer.196 Hence, impact of NLRP6 deficiency on microbiota composition through IL-18 was also found to lead to an enhanced tendency for inflammation-induced colorectal tumorigenesis.93 Additionally, NLRP6 also negatively regulates innate immunity and host defense against pathogenic bacteria, such as Listeria monocytogenes, Salmonella typhimurium, and Escherichia coli.197 Microbiota-induced inflammation driven by chemokine production promotes epithelial cell proliferation through local activation of the IL-6 pathway.198

Role of Oxygen and Reactive Oxygen Species (Hypothesis) The decrease of obligate anaerobes of the Firmicutes phylum and increase in facultative anaerobes in colitic models suggests

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A meta-analysis of placebo-controlled trials supports the long-term (a median of 6 mo) treatment of CD with nitroimidazole or clofazimine.202 This can be explained by the spectrum activity of nitroimidazole against anaerobes and the immunosuppressive effects displayed by clofazimine. However, a 2-year combination therapy with clarithromycin, rifabutin, and clofazimine for CD did not show sustainable benefits; there was only short-term improvement that was attributed to the nonspecific antibacterial activity of these drugs.203 Another pilot clinical trial conducted in patients with CD suggested that remission and beneficial response were more frequently seen in patients treated with ciprofloxacin for 10 weeks as compared with metronidazol treatment and placebo.204 However, another meta-analysis of randomized controlled trials did not reveal evidence of the benefit of antibiotics in patients with CD.205 The conflicting results seen in various studies could be readily illustrated by the fact that therapeutic doses of antibiotics can alter the overall gut microbiota composition and function because antibiotics are generally nondiscriminant in their targets.206 Although older patients with IBD seem to receive limited benefits from antibiotics, antibiotic treatment at early stages in life (especially in newborns) seems to be a risk factor for the development of IBD. Various retrospective studies have showed an association between pediatric IBD development and antibiotic use,207–209 with one study suggesting a strong association of 3 cephalosporin purchases with CD (but not UC).209 Additionally, Gevers et al13 were able to assess a small subset of the patients with CD that were on antibiotics during sample collection, allowing a comparison between the microbiome in patients with CD



TABLE 2. Microbiota Dysbiosis-involved Functional Alterations of Gut Microbiome: Examples of Plausible, Causal or Contributory Mechanisms in the Pathogenesis of IBD Bacteria Taxa Firmicutes (including species of Eubacterium, Faecalibacterium, Roseburia, and Phascolarctobacterium) Bifidobacterium, Faecalibacterium prausnitzii Bifidobacterium, Bacteroides, Clostridium, Enterobacter, Lactobacillus Faecalibacterium prausnitzii Clostridia, Bacteroides Firmicutes/Clostridia (Ruminococcus torques) Firmicutes (Lactobacillus acidophilus) Firmicutes (Lactobacillus or L. casei/ paracasei) Firmicute (Lactobacillus plantarum) Pathogens (e.g., Enterobacteriaceae)

AIEC

Enterococci Pathogens (e.g., Helicobacter) Pathogens (e.g., E. coli, Campylobacter and Helicobacter) Proteobacteria (e.g., Bilophila, Desulfovibrio) Proteobacteria (e.g., Bdellovibrio bacteriovorus)

Functional Considerations Maintain metabolic symbiosis associated with production of SCFAs (e.g., acetate, propionate, and butyrate) that affect colonic pH, the growth of pathogens, and energy supply to the colonic epithelial cells Decrease glycan-degrading enzymes Promote the production of host mucosal glycans Produce choline metabolites that modulate lipid metabolism and glucose homeostasis Produce bile acid metabolites to facilitate lipid absorption, barrier function, and energy homeostasis Block NF-kB activation and IL-8 production Provide antigens that induce regulatory T cells to generate antiinflammatory cytokines to protect commensal gut microbiota Degrade mucin Reduce platelet-activating factor-induced NF-kB activation and IL-8 production in gut epithelial cells Promote mucin secretion Produce lactocepin protease that selectively degrades proinflammatory chemokines Produce an extracellular immunomodulatory peptide mediating microbiota/host interaction Produce mucinases to hydrolyze mucus Produce toxins Produce hemolysin Produce lipopolysaccharide Produce flagellin, a dominant antigen in IBD Manipulate ubiquitination and deubiquitination pathways Epithelial barrier disruption with microbial invasion, aberrant immune response with increased T-helper 17 cells, and cytokine production (e.g., IL-27) Alter microbiota composition Produce hemolysin Disrupt gut barrier Survive inside macrophages and induce production of proinflammatory cytokines by infected macrophages Modulate ubiquitin proteasome system Upregulate microRNAs in gut epithelial cells to reduce autophagy Produce bacterial proteases (e.g., gelatinase) Affect metabolite pathways including tryptophan metabolism Produce cytolethal distending toxins Produce hydrogen sulfide Prey other Gram-negative bacteria for survival and act as a population balancer Reduce pathogen colonization

References Refs. 2,119,159, 218,219

Refs. 2,159

Ref. 2 Ref. 135 Refs. 189,220 Ref. 92 Ref. 221 Refs. 179,191,192

Ref. 222 Refs. 126,164,186, 187,194,222–226

Refs. 124– 126,153,185,186

Refs. 173,174,227 Refs. 119,228 Refs. 175,176, 229–231 Refs. 163,177 Refs. 106,232


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with and without antibiotic exposure. A strong effect was observed on the microbial composition, and exposure to antibiotics generally amplified the dysbiosis described in their treatment-naive inception cohort of pediatric patients.13 These results indicate that the effects of antibiotics on the microbiota composition during the period of commensal gut microbiota establishment in newborns may result in the establishment of microbiota’s that are susceptible to developing IBD later in life.

Probiotics Probiotics comprises a number of strains of microbes and offer a method to potentially alter the intestinal microbiome.210,211 Probiotic usage by patients with IBD is common (e.g., in 40%– 50% of patients with IBD in 2 separate survey-based studies conducted in the United Kingdom).212,213 Various randomized trials have been conducted to understand the capacity of probiotics as induction, maintenance, and postoperative prophylaxis in IBD.210 Current probiotics have shown no benefit in any aspect of CD therapy, whereas there remains some advocacy for their use in mild UC.210 One highly concentrated probiotic mixture, VSL#3, contains 8 strains of Bifidobacterium, Lactobacillus, or Streptococcus spp. and has received attention for its beneficial role in the management of chronic recurrent pouchitis192,210,214–216 that follows in patients undergoing ileoanal anastomosis and J-Pouch creation for patients with UC who failed conventional medical therapies. Another study showed that giving daily doses of Lactobacillus spp. (2.5 · 109 cfu/g) to patients with UC with pouchitis for 2 months could restore the mucosal barrier against Escherichia coli although there were no significant differences in the mucosal microbiota compositions.217 The beneficial effects of Lactobacillus spp. on the amelioration of IBD are indeed mechanistically supported as their metabolites or products can reduce proinflammatory cytokines (Table 2).179,191,192,221 Nevertheless, because of the increased abundance of Bidfidobacterium and Lactobacillus species observed in patients with active CD, cautions against probiotic use have also been suggested.49 Altogether, it is still unclear whether the increase of apparently beneficial microbes is a secondary phenomenon in these patients with CD.

Prebiotics Prebiotics, by definition, are compounds that change the composition and/or activity of the intestinal microbiota in a manner that is beneficial to the host.233 Inulin and oligofructose are 2 prebiotics, which promote the growth of indigenous Bifidobacterium and Lactobacillus spp.234 Patients with CD receiving oligofructoseenriched inulin showed elevated levels of acetaldehyde and butyrate among the analyzed fecal metabolites.235 In chemically induced colitic rats, difructose dianhydride-enriched caramels exhibit effects, such as promoting beneficial colon microbiota, increasing the levels of SCFAs and inhibiting the production of proinflammatory cytokines.236 Similarly, other prebiotics, such as cellobiose and rice fiber also possess anti-inflammatory effects by modulating the colonic environment and reducing proinflammatory cytokines in experimental colitis.237,238 Although prebiotics seem to be promising as

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a potential treatments, their effects on modifying gut microbiota may vary between healthy individuals and patients with IBD.239 This concern is consistent with the current caution in not supporting the use of prebiotics in the dietary management of CD adults.240

Diet Both long- and short-term diet modifications are known to profoundly influence the structure and activity of the intestinal microbiome.161,162 Observational studies of high-fat and sugar (“Western”) diets show a remarkable increase of Escherichia coli populations (including AIEC as discussed above)92 and a decreased abundance of sulphite-reducing pathogens that exacerbate dysbiosis.163 Additionally, experimental studies have demonstrated the effect of dietary supplementation on animal colitis. Dietary fiber (cellulose) supplementation in the early lives of mice stimulated noteworthy shifts in the mice’s gut microbiota with decreased abundance of Coribacteriaceae and increased levels of Peptostreptococcaceae and Clostridiaceae. This was associated with reduced gut inflammation in adult mice.241 Oral administration of the anti-inflammatory peptide pyroglutamyl leucine (an enzymatic hydrolysate of wheat gluten) in mice improved dextran sulfate sodium–induced colitis with increased and decreased abundance of Bacteroidetes and Firmicutes, respectively.242 Aberrant DNA methylation is a purported risk factor for IBD.243,244 In keeping with this hypothesis, animal studies have shown that transient prenatal or maternal exposure to diets supplemented with methyl donor micronutrients induced a colitogenic microbiome and augmented colitis in both the maternal mice and their offspring.243,244 In another study assessing functional behavior of microbes and dietary interventions, a set of mouse experiments were performed inoculating intestinal microbes isolated from children suffering from kwashiorkor (a condition characterized by severe protein–calorie deprivation). Germ-free mice transplanted with the intestinal microbiota of infants with kwashiorkor gained weight with an interventional diet but returned to losing weight when fed a typical diet.245 Thus, recurrence of disease inducing functionality by the microbiota with dietary resumption speaks to the resiliency of the functions provided by these microbes. Persistence will need to be considered because we address intestinal microbiome interventions as treatment for IBD. Taken together, these studies do align with the dietary management of IBD as currently advocated.240,246

CONCLUSIONS IBD is a multifactorial disorder that is suspected to be highly influenced by the functional characteristics of one’s intestinal microbiota. Extensive and expansive analyses through large cohort studies have been aided by recent technological advances to expand our knowledge of altered gut microbiota composition and function in IBD. Nevertheless, many studies have only targeted the higher taxonomic levels of bacteria; and thus, a shift to identify specific families, genera, or species can broaden our understanding of their functional role(s) in the pathogenesis of IBD. Functional dissection of the microbiome and thus


elucidation of relevant mechanisms pose a significant challenge because of the complex relationship within and among the microbiota, host, and environment. Elucidating the functional parameters that govern these relationships will likely prove as far more important than phenotypic characterization of the intestinal microbiota. New technologies coupled with both compositional and functional strategies may contribute in defining the role of specific bacterial species and their relevant metabolic influences. Such studies could reveal whether the microbiome plays an etiological or contributing role in the onset or progression of this complex disease and thus, revolutionize our understanding of IBD.

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153

Inflammatory bowel disease: pathogenesis.

Irritable bowel syndrome, inflammatory bowel disease and the microbiome.

Dynamics of the human gut microbiome in inflammatory bowel disease.

The microbiome in inflammatory bowel disease and beyond.

Microbiome, Metabolome and Inflammatory Bowel Disease.

Genetics and Pathogenesis of Inflammatory Bowel Disease.

Inflammatory bowel disease as a model for translating the microbiome.

The genetics and pathogenesis of inflammatory bowel disease.

Intestinal Permeability in Inflammatory Bowel Disease: Pathogenesis, Clinical Evaluation, and Therapy of Leaky Gut.

Intestinal barrier homeostasis in inflammatory bowel disease.

Intestinal obstruction following operation for inflammatory disease of the bowel.

Inflammatory Bowel Disease: Genetics, Epigenetics, and Pathogenesis.

Inflammatory bowel disease pathogenesis: where are we?

Host gene-microbiome interactions: molecular mechanisms in inflammatory bowel disease.

Intestinal microbiota pathogenesis and fecal microbiota transplantation for inflammatory bowel disease.

16S sequencing and functional analysis of the fecal microbiome during treatment of newly diagnosed pediatric inflammatory bowel disease.

Intestinal expression of the anti-inflammatory interleukin-1 homologue IL-37 in pediatric inflammatory bowel disease.

Recent Advances: The Imbalance of Cytokines in the Pathogenesis of Inflammatory Bowel Disease.

The role of innate immunity receptors in the pathogenesis of inflammatory bowel disease.

Colorectal Cancer in Inflammatory Bowel Disease: Epidemiology, Pathogenesis and Surveillance.

The microbiome in inflammatory bowel disease: current status and the future ahead.

Finger clubbing in inflammatory bowel disease: its prevalence and pathogenesis.

Mimicry between intestinal Behçet's disease and inflammatory bowel disease.

Diet in the pathogenesis and treatment of inflammatory bowel diseases.