Metabolic Influences of Gut Microbiota Dysbiosis on Inflammatory Bowel Disease

1Inflammatory Bowel Disease (IBD) is a chronic and remitting disorder characterized by relapsing episodes of gastrointestinal inflammation. IBD is comprised of two major classes; ulcerative colitis (UC) and Crohn’s disease (CD). The inflammation in UC is restricted to the mucosal layer, leading to superficial damage of the bowel wall, while CD is associated with inflammation of all layers of the bowel wall. Epidemiological studies showed that IBD is spreading widely throughout the world leading to a public health challenge worldwide. It is widely accepted that the IBD pathogenesis results from an interplay between gut microbiota and the host immune system with the predisposition of genetic susceptibility and environmental factors. The microbiome-immune system interaction leads to improper immune activation responsible for the clinical and endoscopic observations in IBD patients.

2The human gastrointestinal tract comprises very rich and diverse microbial community that includes more than 100 trillion microorganisms. The gut microbiome plays an important role in human health and in the development of several chronic diseases. The altered composition of the gut microbiota (dysbiosis), in addition to the disturbance of the metabolic harmony of such microbial community plays a crucial role in the pathogenesis of IBD. Indeed, recent advances in molecular biology techniques together with improving the usability of microbial databases led to comprehensive characterization of microbial communities and revealing the association between gut microbiota dysbiosis and IBD. However, the actual contributions of this dysbiosis to the inflammation and the cause/effect relationship between gut microbe and IBD are still unclear.

3In this review, we outline the recent findings that correlate gut microbiota dysbiosis and metabolic dysfunctionality to IBD. We highlight the metabolic pathways by which microbial dysbiosis could contribute to the inflammation seen in IBD. We also consider the connection among different metabolic pathways in relation to disease progression. In addition, we clarify the strategies of manipulating gut microbiota to promote gut health in IBD.

4Our gut is populated by a complex and dynamic microbial ensemble, which is considered an additional organ of the human body and collectively known as the gut microbiota. This cohort consists mainly of bacteria with low percentage of archeae, eukaryotic and viral members. Using fecal samples from 124 Europeans and Illumina-based metagenomic sequencing, the gut microbial gene catalog was estimated to be 100–150-fold that of human genes with 99.1% of bacterial origin. The bacterial species that compose the entire community were calculated to range from 1,000 to 1,150 prevalent species with at least 160 species per individual.

5Studying mucosal and luminal microbiota structure via sequencing 16S rDNA clones has revealed that approximately 90% of gut microbes are related to main two phyla; Firmicutes (51%) and Bacteroidetes (48%), with the remaining 10% distributed among Proteobacteria, Actinobacteria, Fusobacteria, and Verrucomicrobia. At lower levels, the majority of the detected Firmicutes were related to the Clostridia class, with the vast majority classified under clostridial clusters IV, XIVa, and XVI. Bacteroidetes, on the other hand, has showed less diversity than Firmicutes with 67% of its sequences classified under 3 main phylotypesB. vulgatus, Prevotellaceae, and B. thetaiotamicron. B. thetaiotamicron was the only common Bacteroidetes among all tested individuals. Other low abundant species related to other phyla included Proteobacteria species (Sutterellawadsworthensis, Desulfomonaspigra, and Bilophilawadsworthia), Actinobacteria genera (e.g., Actinomyces, Collinsella, and Bifidobacterium) and the mucin-degrading Verrucomicrobia species, Akkermansiamuciniphila. Along with bacteria, the most common coexisting fungal genera in the gut are Saccharomyces, Candida, Galactomyces, Pleospora, Bullera, Aspergillus, Trametes, Sclerotinia, Penicillium, and Rhodotorula, while virome mainly exists in the form of bacteriophages.

6The diversity and the composition of gut bacteria vary along the length of the gastrointestinal tract (GIT) with a gradual increase in bacterial load and diversity from the stomach and duodenum (102 CFU/gm content) to the colon (1011–1012 CFU/gm content). The composition also varies at different intestinal locations starting with aerobic Streptococcus and Lactobacillus species in the duodenum and ending with strict anaerobes such as Bacteroides, Bifidobacterium and clostridial clusters in the colon. This difference in composition and diversity may be attributed to nutritional substrate availability, oxygen content, luminal acidity, and other physiological and immunological conditions at different parts of the GIT.

7In addition to the stable differences in the composition at different intestinal locations, the gut bacteria are known to, significantly, vary among individuals. Furthermore, gut bacteria have been shown to be in continuous temporal variation within the same individual as revealed by monitoring the gut microbiota structure of two subjects for 15 months. These temporal variations could be attributed to small perturbations in some environmental factor, such as a short-term change in diet, a gastrointestinal infection, or due to a reversible change in the immunological state of the host. However, the gut microbiota tolerates these temporal changes and returns to its original structure once all physical and physiological conditions of the gut return to the normal state, which is known as resilience of the gut microbial ecosystem. Contrary to this, gut microbiota can lose this resilience by exposure to certain permanent factors, such as broad spectrum antibiotics, chronic diseases [e.g., Diabetes, IBD], obesity, or with age. With regards to age, gut microbiota begins to colonize the intestine immediately after birth with a few aerobes (Enterobacteria, Staphylococcus, and Streptococcus), which gradually are replaced by anaerobic bacteria to reach the same complexity as mentioned above for adults by the first year of life. However, differences between children and adults are still significant even after the first year of life. For example, children 1–7 years of age have lower fecal microbiota diversity with higher abundance of Enterobacteria than adults. Also, a more recent comprehensive study has indicated a substantial difference in elderly people when compared to the established adult pattern, with Bacteroidetes as the predominant phyla and lower abundance of Firmicutes. This age-related pattern has been confirmed previously by comparative assessment of Firmicutes/Bacteroidetes ratio at different ages by quantitative PCR. Also, individual bacterial species such as Escherichia coli, Enterococcispp., Bacteroidesspp., Bifidobacterium spp., and lactobacilli have been demonstrated to exhibit specific age-related profiles in adults and elderly subjects. linked the microbial diversity to both age and geographical location. They surveyed the gut microbiome structure in the stool of 314 Americans, 114 Malawians, and 100 Amerindians at different ages. They reported that the inter-subject variability is higher in the early stages of life compared to the adult microbiome with a progressive increase in microbial diversity with age. The authors also illustrated that the infant microbiome is dominated by Bifidobacteria and reaches an adult-like composition by the age of three. Moreover, the composition of the gut microbiota was more similar between Malawians and Amerindians and its diversity was higher than the American’s reflecting the association between geography and gut microbiota diversity.

8Many reports have described humans as a superorganism that live in symbiosis with different microbes within various parts of the body. This ecosystem offers many benefits to the human host that are essential for good health. The gut harbors the greatest human microbial assembly. The first role of the gut bacteria involves its metabolic capacity to process undigestible food particles such as complex carbohydrates, plant glycans, choline and bile acids. The microbial processing of indigestible polysaccharides generates beneficial short chain fatty acids (SCFAs) such as butyrate, acetate and propionate, which represent around 90% of SCFAs produced in the human gut. Butyrate is considered the major source of energy to the colonocytes. For example, the colonocytes of germ-free mice develop impaired mitochondrial respiration and increased autophagy in comparison with conventionally raised mice, and these findings have been reversed by adding butyrate to germ free colonocytes. On the other hand, acetate and propionate exert extra intestinal roles, where they act as metabolic substrates for lipogenesis and gluconeogenesis. The major producers of SCFAs include the genus Bacteroides, Clostridium clusters IV and XIVa and Bifidobacterium. While Eubacterium rectale, Roseburiafaecis, Eubacterium hallii, and Faecalibacterium prausnitzii are the major butyrate producers in the gut as revealed by investigating the butyryl-CoA:acetate CoA-transferase gene, B. thetaiotamicron and B. ovatus showed a high genomic content of carbohydrate active enzymes (CAZymes) that enable them to metabolize indigestible plant and host glycans. This might explain the predominance of Bacteroidetes in the gut of rural Africans who have a mainly plant-based diet. The highest percentage of SCFAs in the large bowel is seen in the cecum and proximal colon, and decreases gradually toward the distal colon. This gradient may be explained by the higher prevalence of substrates in the proximal colon, which decreases progressively toward the rectum. The majority of the SCFAs produced are absorbed by the gut or delivered to peripheral tissues such as the liver and muscles. Hence using feces to measure the gut metabolites may be inappropriate.

9Gut microbiota, via various mechanisms, contributes to intestinal epithelial integrity. The first of these factors is the production of SCFAs, notably butyrate. Butyrate is a main modulator of mucin release, which acts as the first barrier against gut microbial invasion. In addition, butyrate controls gene expression of the colonocytes either via inhibition of histone deacetylase (HDAC) or through binding to G-protein coupled receptors (GPR41 or GPR43). For example, sodium butyrate up-regulates the expression of the tight junction proteins and their mRNA via the inhibition of HDAC. Furthermore, intra-rectal delivery of C. tyrobutyricum to immunocompetent and immunodeficient pathogen free mice showed a protective effect against dextran sulfate sodium (DSS)-induced colitis. This is mediated by inducing the expression of zonula occludens (ZO)-1 tight junction proteins, as well as MUC-2 mucin both of which are directly related to the luminal level of butyrate. In addition to SCFAs, other microbial components contribute to the epithelial integrity. Germ free mice showed a thinner mucus layer relative to microbiota colonized mice, and this was corrected following exposure to LPS or peptidoglycan. Recently, it has been shown that gut microbiota, also, induces intestinal mucosal endothelial and mesenchymal cells via stimulation of toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) like receptor pathways. Together, this emphasizes the critical role of gut microbiota or their metabolites in maintaining the integrity of the intestinal barrier.

10In addition to the development of tissues and cells, the gut microbiota is responsible for the shaping and maturation of the immune system. Germ free mice showed defective gut associated lymphoid tissues (GALT), fewer and smaller Peyer’s patches, less cellular mesenteric lymph nodes, less cellular lamina propria, lower expression of TLRs and class II major histocompatibility complex (MHC II) molecules, and finally reduced intraepithelial lymphocytes and CD4+ T cells in comparison to conventional mice. Additionally, gut colonization with microbes corrected some of these deficiencies. In relation to specific pathogen free mice, germ free mice also showed accumulation of invariant natural killer T cells (iNKT) in the lamina propria of the colon and lung, which resulted in higher mortality rates. However, the exposure of neonatal germ free mice, but not adults, to commensal bacteria was protective against iNKT-accumulation and its undesirable consequences. Likewise, mice who received CD4+CD62L+ lymphocytes from germ free mice developed colitis faster than mice who received the same regulatory T cells (Tregs) from conventionally housed mice. This suggests a critical role of the gut microbiota in the development of the intestinal immune system. This gut immune maturation is dependent on the host-specific microbiota. Colonization of germ free mice with either human or rat microbiota resulted in fewer intestinal T cells and dendritic cells, and lower antimicrobial peptide expression relative to germ free mice colonized with a murine microbiota, and humanized mice were more susceptible to salmonella infection. Both humanized mice and the mice colonized with a murine microbiota showed similar bacterial diversity at higher taxonomic levels, but they harbored different species. This indicates that each host selects a specific microbial consortium that shapes its immune system and maintains intestinal health.

11As mentioned above, our gut is populated with a complex and dynamic ecosystem, which under normal circumstances is characterized by a stable structure at various intestinal segments in everyone. Any alteration of this consortium may disrupt its functionality and eventually, a diseased state will appear. The dysbiosis of the intestinal microbiota is well reported in different diseases such as irritable bowel syndrome (IBS), obesity, diabetes, and IBD. The relationship between IBD and gut microbiota dysbiosis was first established by studying animal models of colitis. Germ free IL 10–/– mice do not develop colitis unless they are colonized by enteric bacteria. were able to clearly demonstrate that the alteration of the gut microbiota composition could induce colitis in immunocompetent mice. They reported that deficiency of T-bet, a transcriptional factor that is important for gut homeostasis, resulted in microbial population shifts into a colitogenic community. This colitogenic microbiota was able to drive the intestinal inflammation in genetically intact mice. Many molecular studies have illustrated the changes in gut microbial composition of IBD patients in comparison with non-IBD controls. The gut microbiota of individuals with IBD is characterized by low microbial diversity, a reduced abundance of Bifidobacterium spp., Lactobacillus spp., and F. prausnitzii and a higher abundance of pathobionts such as adherent/invasive E. coli and C. difficile, resulting in lower SCFA concentrations compared with healthy individuals.

12Studies using 16S rDNA sequencing have shown a decrease in the diversity of gut biota in IBD mucosal specimens. Using single strand confirmation polymorphism (SSCP) fingerprint, based on 16S rRNA showed that the diversity decreased by 50 and 30% in CD and UC, respectively. illustrated this imbalance by sequencing SS-rRNA clones from 190 biopsies. In their study, the Lachnospiraceae family of Firmicutes and Bacteroidetes were depleted in IBD subjects, with a relative increase of Proteobacteria, Actinobacteria, and Bacillus subgroups of Firmicutes. FISH analysis, on the other hand, has illustrated an increase in the relative abundance of Bacteroidetes and a low abundance of some butyrate producing bacteria such as F. prausnitziiin mucosal IBD specimens. Using 454 pyrosequencing of the 16S rRNA V5 and V6 regions extracted from the fecal materials of concordant and discordant twins, ileal CD, colonic CD and healthy individuals were differentiated from each other according to their microbial profile. However, the authors were not able to discriminate between UC and healthy subjects by following the same approach. Colonic CD was characterized by higher Firmicutes (mainly Faecalibacterium, and Ruminococsulus), Bifidobacteriaceae (Bifidobacterium), Coriobacteriaceae (Collinsella), and Aneroplasmataceae. Conversely, ileal CD showed depletion of Ruminococaceae family, especially Faecalibacterium, and Collinsella with higher abundance of Proteobacteria due to the increase of the Enterobacteriaceae family. For UC, were able to identify few differences such as depletion of Prevotella, Streptococcus, and Asteroleplasma. In contrast, other study was able to discriminate between UC and healthy individuals by calculating the mean of 16S rDNA-clone libraries taken from sigmoid colon biopsies of 62 individuals. The microbiota dysbiosis of UC was characterized by less diversity, fewer Lachnospiraceae and Ruminococcacea families with higher abundance of Proteobacteria and Actinobacteria. These two studies confirm the association between IBD and gut microbiota dysbiosis. However, the discrepancy between them, regarding the UC microbiota structure, underlines the importance of the sampling approach in these types of studies.

13For pediatric IBD, some studies have monitored the gut microbiota in IBD compared to healthy controls. described the alteration in microbiota composition along the length of the intestine in pediatric IBD patients in comparison to control subjects. They used the conventional culture-based techniques and 16S rRNA-based real time PCR for quantifying the mucosa associated bacteria at the ileum, caecum, and rectum of 42 subjects. One important observation of this study is that, in contrast to adult IBD, B. vulgatus was found at lower abundance in patients with IBD compared to controls. This supports the idea that pediatric IBD is a unique form of IBD. Moreover, a study investigating the relative abundance of 9 bacterial groups using real-time PCR showed higher number of E. coli and lower number of F. prausnitzii in children with CD in comparison to control subjects. Furthermore, microbiota diversity in pediatric IBD patients was shown to differ from that of adults. However, these studies solely examined the dominant bacterial groups by applying conventional culture-based techniques and simple molecular methodology. Since it is widely accepted that 80% of gut microbiota are unculturable, a comprehensive molecular survey of the gut microbiota in pediatric IBD is necessary. Two studies have applied a high throughput molecular approach to characterize gut microbiota in pediatric IBD. The first study applied synthetic learning in microbial ecology (SLiME) analytic approach of 454 pyrosequencing data obtained from fecal samples of 91 individuals and other published datasets. They were able to differentiate between children with IBD and healthy individuals or those with other diseases based on their microbiota composition. The drawback of this study is that they relayed upon stool samples for their analysis, and it is known that fecal materials contain different microbiota when compared to mucosa associated bacteria. Moreover, reported that the microbiota diversity is lower in CD but not in UC relative to control subjects. Pairwise comparison among the 3 groups identified only 7 differentially abundant taxa. One important result is that F. prausnitzii was highly abundant in CD compared to controls, which is the reverse of what has been previously documented in adults with IBD, indicating once again the unique microbiota composition of pediatric IBD. Using intestinal mucosal colonoscopic washes, have reported dysbiosis of gut microbiota in new onset pediatric IBD with enrichment of H2S producing bacteria. Overall, these studies support the association between intestinal microbiota imbalance and pediatric IBD at different ages. Nevertheless, the cause/effect relationship between these conditions and gut microbiota is still unclear.

14Generally, microbiota dysbiosis is significantly greater in patients with CD than with UC, where the microbial community stability and diversity is significantly lower in case of CD than in UC. In case of CD a specific microbial signature that is comprised of eight groups is well reported. Eight groups of microorganisms involving Anaerostipes, Methanobrevibacter, Faecalibacterium, an unknown Peptostreptococcaceae, Collinsella, an unknown Christensenellaceae, and Escherichia, Fusobacterium could be utilized to differentiate between CD from non-CD, where, the first six groups are relatively low and the latest two groups are relatively high in case of CD. Moreover, it is noteworthy fecal (stool) and mucosal sampling is critical during the determination of the microbial dysbiosis in IBD. The IBD stool samples are characterized by reduced diversity of microbiota in comparison to IBS and healthy population. IBD cases are characterized by decreased Verrucomicrobia and Bacteroidetes than healthy population. On the other hand, in case of IBS Bacteroidetes are increased in comparison to healthy population. Moreover, IBD are reported to harbor less population of Bacteroidetes and Verrucomicrobia, and higher abundance of Actinobacteria in comparison to IBS. Lactobacillus, Ruminococcus, and Streptococcus are significantly higher in IBD than healthy population, while Oscillospira, Lachnospiraceae, Ruminococcaceae, and Rikenellaceaeas well as Akkermansia muciniphila are diminished in IBD. In case of IBS, Pseudomonas and Lactococcus are decreased in comparison to healthy population, while Parabacteroides distasonisis relatively increased. By comparing IBS and IBD, Rikenellaceae, Bacteroides, Butyricimonas, Oscillospira, Mogibacteriaceae, Anaerostipes, Barnesiellaceae, Roseburia, Parabacteroides, P. distasonis, and A. muciniphila were more abundant in IBS than in IBD, while Granulicatella was relatively decreased. Similarly, to fecal samples, mucosal samples exhibited reduced microbiota diversity passing from healthy population to IBS to IBD. Generally, there is no significant difference in microbiota population between inflamed and not-inflamed tissue samples of IBD. On contrary, the microbiota of inflamed mucosa of IBD patients exhibited low abundance of Firmicutes and Bacteroidetes and higher abundance of Proteobacteria in comparison to healthy population. The abundance level of Enterobacteriaceae was significantly increased and the abundance levels of Lachnospiraceae, Ruminococcaceae, Rikenellaceae, Bacteroides, Coprococcus, F. prausnitzii, and P. distasonis were diminished in IBD inflamed mucosa compared to healthy population.

15In addition to dysbiosis of bacteria, the dramatic changes that occur in the fungal community named as “mycobiome” is relatively important during IBD. Mechanistically, it appears that fungi may paly crucial role in the progression of IBD through either affecting the gut microbiota composition or increasing the production of pro-inflammatory cytokines. In addition, mycobiome dysbiosis is a well-reported case in IBD. It is reported that fungal diversity is higher in CD patients than in healthy controls.

16An increased ratio of Basidiomycota/Ascomycota is a characteristic feature of IBD. reported higher abundances of Aspergillus, Wickerhamomyces, Candida, and Sterigmatomyces and lower abundances of Alternaria, Penicillium, Exophiala, Emericella, Acremonium, Epicoccum, and Trametes in patients suffering from UC in comparison to healthy controls. However, there was no significant association between Basidiomycota/Ascomycota ratio and the increased levels of pro-inflammatory cytokines. Moreover, Candida spp., particularly C. albicans, is significantly increased in patients suffering from CD or general IBD. It is reported that specific-pathogen-free (SPF) Clec7a–/– mice exhibit more severe colitis symptoms when colonized with the pathogenic fungus, C. tropicalis compared to uncolonized Clec7a–/– mice or colonized wild type mice. These studies confirm the crucial link between mycobiome dysbiosis, especially Candida spp. and IBD. illustrated higher levels of Candida spp. in the inflamed mucosa of IBD patients than in healthy population. In addition, illustrated higher colonization of familial CD patients by C. albicans. Moreover, other reports reported high levels of both C. albicans and C. glabrata in CD patients. In addition, documented the high abundance of Candida in CD patients, which was decreased by therapeutic intervention, especially with anti-TNF-α treatment.

17Besides Candida spp., Malasseziarestricta, which is a skin normal fungus, significantly increases in patients suffering from CD. This fungus was found to aggravate the colitis in mouse models via mechanisms demanding on a protein included in antifungal immunity; CARD9. Moreover, depletion of Saccharomyces cerevisiae is reported in IBD patients. depicted the protective effects of S. cerevisiae UFMG A-905 in mice suffering from UC. In addition, reported that adherent-invasive E. coli (AIEC)-induced ileal colitis can be reduced by S. cerevisiae CNCM I-3856 in a murine model. However, on the other hand, demonstrated that S. cerevisiae worsens the disease condition in a murine model of colitis.

18The crosstalk between fungi and bacteria may be a pivotal concern in IBD patients. However, in pediatric IBD patients there was no significant correlation between leading fungal species with particular bacterial taxa and it has been hypothesized that fungal dysbiosis may be a result of or a cause for the gut bacterial dysbiosis. On the other hand, previous report showed a positive correlation between C. tropicalis and both E. coli and Serratia marcescens. It is noteworthy that the correlation between mycobiota-bacterial dysbiosis with the disease severity in IBD patients may afford evidence for the crucial role for gut mycobiome during IBD.

19In addition to microbiota and mycobiota, gut virome, which is comprised of viruses infecting both prokaryotes and eukaryotes, constitute a large portion of gut microbiome. Bacteriophages are the major components of the enteric virome. Generally, in patients suffering from IBD, alteration of the virome reflects microbiota dysbiosis. One of the first studies, which reported virome dysbiosis in IBD patients revealed higher abundance of bacteriophages infecting Alteromonadales, Clostridiales, and Clostridium acetobutylicum as well as Retroviridae family in IBD patients in comparison to healthy population. It is well reported that Caudovirales phage families, including Siphoviridae, Myoviridae, and Podoviridae, are significantly enriched in IBD subjects. A previous study illustrated the high abundance of Caudovirales phage in children suffering from IBD. On the other hand, another study reported a decreased diversity and consistency of Caudovirales that is directly connected to the inflammation degree of the intestine in UC patients. Moreover, phages infecting Enterobacteria and Escherichia were reported to be abundant in UC patients. A recent study established in germ-free mice showed that certain phages including Escherichia, Lactobacillus, and Bacteroides infecting phages as well as phage DNA exacerbate gut inflammation and contribute to IBD pathogenesis through increased production of IFN-γ via a TLR9-dependent pathway.

20In addition to bacteriophages, phages infecting and incorporating into eukaryotic cells are relatively important and have been related to IBD pathogenesis due to their ability of integration in human genome and affecting physiological state of the intestinal cells. Previous reports conducted on patients suffering from UC revealed higher abundance of Pneumoviridae and lower abundance of Anelloviridae compared to healthy population. On the other hand, a study that was established on a small cohort of patients suffering from UC and CD depicted the higher abundance of Herpesviridae compared to healthy control. Other studies demonstrated that infection with Norovirus contributes to intestinal inflammation and can increase the rate of colitis incidence. Moreover, recent studies depicted the high abundance of Hepeviridae and Hepadnaviridae in the intestinal mucosa of patients suffering from CD and UC, respectively. Despite all these studies, the actual role of virome dysbiosis during IBD has not been fulfilled yet, even with some reports revealing that intestinal inflammation may be initiated by eukaryotic viruses. By detecting and identifying viruses that infect IBD patients during the early stages of intestinal inflammation, it will be promising to establish a complete correlation between virome and disease progression.

21Major dysbiosis of gut microbiota, mycobiome, and virome during IBD from different studies is summarized in Table 1. Moreover, the contribution of this dysbiosis to worsening the condition and progression of the disease is illustrated in Figure 1.

22Two metagenomic studies tried to link the dysfunctionality of the gut microbiome and IBD via two different approaches. First, treated the gut microbiota as one single organism and constructed the metabolic network of the entire community and compared the odd ratios, of the topology of different metabolic pathways, genes and biological processes of obese and IBD cases against healthy individuals. This approach yielded an association between IBD and genes involved in NO2 production and metabolism of choline and p-cresol. In contrast to the metagenomic analysis of the community as a single supra-organism, identified the microbiota composition of each disease phenotype. Then, they extracted the genome of the microbiota composing each community and constructed a gene catalog for each phenotype, followed by genes and pathway assessment using sparse multivariate analysis. They also confirmed their findings by shotgun metagenomic analysis of 4 CD and 7 healthy fecal microbiota. They found an IBD-associated enrichment of genes involved in glutathione transport and metabolism, sulfur amino acid metabolism, redox homeostasis, mucin degradation, secretion systems, adhesion, and invasion as well as a depletion of genes involved in the biosynthesis of SCFAs, some nucleotides and amino acids. Recently, an interesting study identified a network of bacterial metabolite interactions including sulfur metabolism as an important player correlated to CD activity. To better understand the dysfunctionality of gut microbiota in IBD, we will discuss the IBD-associated metabolic shift in more detail.

23Gut microbiota generate hydrogen sulfide (H2S) via 2 different biochemical pathways. First, sulfate reducing bacteria (SRB) couple the reduction of sulfate as a terminal electron acceptor to the oxidation of H2 or other organic compounds such as lactate as an electron donor during their anaerobic respiration. The second pathway is followed by bacteria that ferment sulfur containing amino acids. These bacteria might depend on desulfhydrases such as cysteine desulfhydrase or other enzymes, which have the ability to metabolize cysteine. Sulfate reducing bacteria mainly include members of Deltaproteobacteria in addition to Desulfotomaculum, Desulfosporosinus, Thermodesulfobacterium, and Thermodesulfovibrio genera. Using multiplex PCR to identify fecal-SRB isolates, luminal SRB are dominated by Desulfovibriopiger, D. fairfieldensis, and D. desulfuricans. A diversity analysis of Desulfovibrio using the dissimilatory sulfite reductase (dsrAB) gene confirmed the predominance of D. piger with the detection of a new unclassified SRB. Another study identified 8 sulfate and sulphite reducing bacteria in addition to the sulphite reducing bacterium, Bilophilawadsworthia via 454 pyrosequencing of dsrAB gene fragment. The identified bacteria include four known species; D. piger, D. vulgaris, Desulfovibrio sp. NY682, and D. desulfuricans F28-1, and four new species highly similar to D. desulfuricans F28-1 (93% dsrABsequence similarity); D. oxamicus (84% identity), Desulfotomaculum sp. Lac2 (80% identity), and D. simplex (88% identity). H2S producing microbiota through fermentation of amino acids include, for example, Fusobacterium nucleatum, Atopobium spp., Gemella sanguinis, Micromonas micros, Streptococcus spp., Actinomyces spp., Eubacterium spp., Veillonella spp., Bulleidiamoorei., Prevotella spp., Campylobacter spp., and Selenomonas spp.. have reported the enrichment of H2S producers in children with CD with one H2S generating organism; A. parvulum, has proinflammatory characteristics in IL10–/– mice. Recently, an interesting study identified a network of bacterial metabolite interactions including sulfur metabolism as an important player correlated to CD activity. The authors showed that CD patients with active disease are enriched in members belonging to Enterococcus, Fusobacterium, Haemophilus, Megasphaera, and Campylobacter, while Roseburia, Christensenellaceae, Oscillibacter, and Odoribacter are enriched in CD patients with inactive disease. Another report detected two dominating sulfate-reducing bacteria morphotypes that differ in colonial size and quantitate in the feces of healthy and patients with colitis. In the feces of healthy individuals, 93% of sulfate-reducing bacteria of morphotype I prevailed (Desulfovibrio) while morphotype II included only 7% (Desulfomicrobium); in the feces of patients with colitis, the ratio of these morphotypes was 99:1, respectively. In addition to microbiota-released H2S, it can also be synthesized endogenously by intestinal colonocytes from L-cysteine via two main enzymes; cystathionine β-synthase and cystathionine γ-lyase. Two additional pathways (3-mercaptopyruvate sulfurtransferase in combination with cysteine aminotransferase and 3-mercaptopyruvate sulfurtransferase coupled with D-amino acid oxidase) were identified in H2S production, peripherally. Normally, the luminal H2S concentration of the human large intestine is 1.0–2.4 mmol/L, while the concentration in the fecal contents ranges from 0.17 to 3.38 mmol/kg. Taking into consideration the lipid solubility and the passive diffusion of H2S through the intestinal mucosa, these concentrations are underestimated.

24At lower concentrations (<1 mM), H2S is considered a cytoprotective metabolite that induces some cellular anti-inflammatory responses. These cellular responses include, for example, prevention of caspase activation and apoptotic cell death, inhibition of leukocyte adhesion to vascular endothelium, which decreases infiltration of neutrophils and lymphocytes, induction of cyclooxygenase-2 (COX-2) expression, and promotion of neutrophil apoptosis. The later was contradicted by who concluded that H2S accelerates the resolution of the inflammation process via inhibition of polymorphonucelar (PMN) apoptosis. In another in vitro study, H2S at normal colonic concentration lowered the proliferation of different colonic cancerous and normal cells and induced autophagy through the AMP-activated protein kinase (AMPK) pathway. At higher concentrations, H2S acts as a genotoxic and/or cytotoxic transmitter to the colonocytes by affecting genes responsible for cell cycle progression, DNA repair and inflammatory responses. The main cytotoxic effect of H2S is the inhibition of cytochrome c oxidase activity, which is the terminal oxidase of mitochondrial respiration. This leads to the prevention of the oxidation of essential metabolites such as n-butyrate, L-glutamine and acetate, which eventually decreases the bioenergetic performance of the cell. Sulfide has also been shown to inhibit butyrate oxidation in rat colonocytes through inhibition of short chain acyl dehydrogenation of activated fatty acids. The antagonistic effect of H2S on butyrate may induce hyperproliferation of the colonic mucosa. An indirect cytotoxicity of H2S was reported, where increased sulfide production induces the conversion of nitrite to nitric oxide, which had a damaging effect on colonocytes.

25To keep the local concentration of H2S at a harmless level, the colonic mucosa expresses a special H2S oxidation system that degrades H2S to sulfate and thiosulfate. This oxidation system consists of sulfide quinone reductase (SQR), dioxygenase ethylmalonic encephalopathy protein 1 (ETHE1), and thiosulfate sulfur transferase (TST that is also known as rhodanese). This mitochondrial oxidation of H2S is not essential for cell respiration, but instead, its main purpose is the detoxification of excess H2S. In contrast, the respiratory capacity of the cell is an important parameter that affects the efficiency of H2S detoxification independent of the mitochondrial oxidation system.

26A higher abundance of H2S generated by gut microbiota is considered one of the strongest models associated with the pathogenesis of IBD. Higher luminal H2S concentration in IBD could arise from either increased abundance of H2S-producing bacteria or a deficient H2S-detoxification pathway. The association between H2S and IBD was first reported in UC. In reviewed the role of H2S in the pathogenesis of UC. They stated that the level of colonic sulfide and the relative abundance of sulfate reducing bacteria was higher in UC patients compared to healthy subjects. In addition, the bacteria isolated from UC patients showed higher generation of H2S than those separated from control cases. Treatment of those patients with 5-aminosalicylic acid containing drugs lowered the production of H2S as indicated by the stool sulfide level and this was proposed to contribute to the therapeutic activities of these drugs. Sulfate reducing bacteria were exclusive to patients with UC, where they were isolated from 80% of UC pouches but not form patients with familial adenomatous polyposis. Other indirect evidences for the role of H2S in the pathogenesis of UC are also available. The first comes from studies of diet consumption, where high protein intake is associated with a higher risk of IBD. Higher protein means increased sulfur containing amino acids and subsequently, elevated H2S levels. The second is the higher activity of fecal mucin sulphatase in UC patients. Mucin sulphatase releases sulfate from the mucosal sulfomucin and this endogenous sulfate provides the source for H2S biosynthesis by SRB. Regarding CD, few reports have linked H2S production with disease activity. have reported no difference in the general abundance of SRB between CD and healthy subjects. have illustrated the increased abundance of H2S producing bacteria along with downregulation of mitochondrial proteins implicated in H2S detoxification in children with CD. Some indirect links are also available. For example, increased metabolism of sulfur containing amino acids such as methionine and cysteine associated with a decrease in the metabolism of sulfur lacking amino acids such as lysine and glutamine are characteristics of ileal CD. Also, the same study reported an over representation of sulfate transport genes in CD patients. Both sulfate and sulfur containing amino acids are the main precursors for H2S biosynthesis as mentioned above. The second factor that contributes to higher colonic sulfide concentration is the dysfunctionality of H2S detoxification. The dysfunctionality of H2S detoxification genes in IBD is still up for debate. showed that the activity of thiol-methyl transferase (TMT) is higher in the peripheral blood of UC patients. This was then confirmed in 2007, when the activity of TMT and rhodanese were found to be higher in the erythrocytes of UC patients than controls. However, no difference in TMT and rhodanese activity was detected in the rectal biopsies of the same individuals. In patients with CD neither the erythrocytes nor the rectal biopsies of patients showed a change in that enzymatic activity. In 2009, the role of H2S detoxification in IBD emerged again when the activity and the expression of rhodanese were shown to decrease in parallel to the development of dextran sodium sulfate-induced colitis in mice. In concordance with formerly mentioned studies, this loss of activity is followed by an increase in its activity in red blood cells. Impaired detoxification of H2S has been confirmed for UC patients via the assessment of TST expression level and activity in colonic mucosal biopsies. As well, it has been confirmed for CD where metaproteomic and expression analyses reported the decreased abundance of H2S detoxification proteins and transcripts in children with new onset CD and UC. These includes the sulfur dioxygenase (ETHE1), the thiosulfate sulfurtransferase (TST) and the components of complexes III and IV of the mitochondrial respiratory chain, and tst, cytochrome c oxidase subunit IV (hcox41) and the sulfide dehydrogenase genes (SQRDL) transcripts. All in all, the association between higher H2S generation and IBD is well established. Additionally, the impaired intestinal H2S detoxification in IBD is evident.

27Butyrate is known as the salt or ester of butanoic (butyric) acid, which is a weak acid with a pKa of 4.8. By considering the pH of the intestine, which is approximately neutral, most of the butyrate in the intestine will be in the anionic form rather than the free acid form. Butyrate biosynthesis by the gut microbiota starts by condensation of two molecules of acetyl coA to generate one molecule of butyrylcoA. Next, butyrate are generated from butyrylcoA via two main pathways; the enzymes butyrate kinase and phosphotransbutyrylase or butyryl-CoA: acetate-CoA-transferase. The second pathway has been shown to be predominant among butyrate producing microbiota in human colon. The major butyrate producing bacteria that inhabit the human gut are related to Clostridium clusters XIVa and IV with few percentage of Clostridium clusters I, XV, and XVI, while acetate and propionate are mainly produced by Bacteroides. It was indicated that the terminal ileum and proximal colon are the main sites of butyrate production, while acetate and propionate are generated in the distal colon, where Bacteroidesis the predominant bacteria. Particularly, the role of butyrate in preventing IBD is illustrated in several studies as mentioned below.

28A large body of evidence has revealed the association between IBD, perturbations of butyrate metabolism and/or depleted butyrate producing bacteria in the gut. Initially, a raised luminal butyrate level, as a result of impaired oxidation by colonocytes, was correlated with the severity of mucosal inflammation and was considered as a biomarker in UC patients. On the other hand, butyrate intake was shown to have a protective effect against development of colitis independent of restored butyrate oxidation in UC patients. For example, administration of butyrate either orally or via local enema to mice or rats with chemically induced colitis alleviated the mucosal inflammation. Indeed, butyrate exhibits several anti-inflammatory activities. First, butyrate inhibits NFκB activation, which results in suppression of proinflammatory cytokines in UC patients and rats with trinitrobenzene sulphonic acid (TNBS) induced colitis. This inhibitory action is impaired in IBD individuals as revealed by assessment of butyrate effect on cytokines production by peripheral blood mononuclear cells (PBMC) isolated from IBD and healthy subjects in response to TLR-2 activation. Secondly, butyrate was reported to induce Fas-mediated apoptosis of T cells via inhibition of HDAC-1 in mice. This in turn inhibits IFN-γ-induced STAT1 activation, which results in reduced colonocyte expression of inflammatory mediators such as nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). Finally, Butyrate is known to reduce the inflammation through contributions to intestinal barrier integrity. For example, intra-rectal inoculation of C. tyrobutyricum, the potent butyrate producer, has restored MUC-2 secretion, upregulated expression of (ZO)-1 tight junction protein and reduced cytokine release in DSS-treated mice. Recently, compared impacts of butyrate on the intestinal immune profile of UC patients with active disease and non-inflamed controls. They found that butyrate exhibits different impact on gene regulation and more strongly leads to down-regulation of +expressed genes of inflammatory pathways in non-inflamed controls than in inflamed tissue of UC patients. According to the authors, such discrepancies can partly elucidate why expected anti-inflammatory impacts of local butyrate stimulation or supplementation are not always obtained.

29Regarding butyrate producing bacteria, screening of fecal microbiota of 6 healthy and 6 CD individuals via 16S rDNA clone sequencing and FISH analysis revealed a depletion of C. leptum cluster in CD cases compared to the healthy group. In agreement with this, illustrated that F. prausnitzii was depleted in CD patients mucosal microbiota and this reduction in abundance was associated with a higher risk of recurrence of ileal CD. Though, they have shown that the anti-inflammatory activity of this bacterium is independent of butyrate production using in vitro cellular models and in vivo colitis model of mice. Recently, depletion of F. prausnitziiand other butyrate producers was confirmed in the fecal microbiota of adults with CD compared to their unaffected relatives and other healthy controls via denaturing gradient gel electrophoresis and real time PCR. This is not the case in pediatric CD, where some studies reported that F. prausnitzii had higher abundance in CD in comparison to controls. An important difference in the pediatric studies is that the samples were collected at the time of diagnosis by colonoscopy, in contrast to where specimens were taken at surgical resection. This indicates the importance of sample collection time for these kinds of microbiota studies. The age groups screened by different studies were also different confirming the different phenotypes of the disease at different ages.

30reported the therapeutic effect of butyrate-producing bacteria in CD patients in addition to its role in enhanced intestinal epithelial barrier integrity. The four-strain probiotic supplement (containing Lactobacillus acidophilus NCIMB 30175, Lactobacillus plantarum NCIMB 30173, Lactobacillus rhamnosus NCIMB 30174, and Enterococcus faecium NCIMB 30176) was found to positively influence the immune response via colonic butyrate production in vitro and facilitating modulation of the gut microbiota composition and metabolic enhancing.

31Bile acids are self signaling molecules that regulate their own biosynthesis through a negative feedback mechanism. Primary bile acids are the product of cholesterol breakdown in the liver, while secondary bile acids are the products of gut microbiota metabolism. primary and secondary bile acids have been found to act as signaling players on a group of cell membrane and nuclear receptors together named “bile acid-activated receptors.” These receptors are highly expressed all over the gastrointestinal tract and control the bilateral communications of the intestinal microbiota with the host immune system. Bile acid absorption is affected in models of inflammatory bowel disease. The expression of bile acid transporter apical sodium dependent bile acid transporter was inhibited in rats with colitis in addition to murine, canine and rabbit models of intestinal inflammation.

32Bile acids mainly act as digestive aids to facilitate digestion of cholesterol, fat-soluble vitamins and triglycerides into water soluble products so they can be absorbed from the small intestine. They are also nutrient regulatory molecules that mediate some endocrine functions. They activate some nuclear receptors and cell signaling pathways to regulate lipid and glucose metabolism, energy expenditure and triglyceride homeostasis. In addition, they have been shown to exert antimicrobial activity either directly through their detergent characteristics on bacterial membranes or via indirect physiological function. Their indirect antimicrobial activity mainly occurs in the distal small intestine, where they promote the activation of nuclear farnesoid X receptor (FXR). Activation of FXR induces the expression of several host genes involved in mucosal defense including genes involved in oxidative stress, and antibacterial peptide biosynthesis. The expression and function of bile acid-activated receptors including FXR in addition to other receptors; G-Protein bile acid-activated receptor, pregnane-X-receptor, vitamin D receptor, and related orphan receptor gamma are strongly linked to the composition of the intestinal microbiota and negatively regulated by intestinal inflammation.

33The enterohepatic circulation of bile acids starts at the liver, where 14 enzymes are required to synthesize the two primary forms of bile acids namely cholic acid and chenodeoxycholic acid from cholesterol. Thereafter, they are conjugated to glycine or taurine to form bile salts (also termed conjugated bile acids), which are stored in the gallbladder during interdigestive periods. After meals, bile salts are released into the duodenum via the biliary duct to facilitate lipid digestion and absorption. In the gut lumen, the microbiota induce biotransformation of the primary forms of bile acids into secondary bile acids products via deconjugation, oxidation of hydroxyl groups and dehydoroxylation. This increases the hydrophobicity of bile acids so that they can passively diffuse through the small intestine and proximal colon in addition to their active transport from the small intestine to the blood stream and back to the liver. Approximately 95% of bile acids are recycled and transported through the circulation to return to the liver while 5% (400–600 mg) enter the colon, where they are dehydroxylated by colonic bacteria into secondary bile acid products [Deoxycholic acid (DCA) and Lithocholic acid (LCA); ].

34Gut microbiota start the metabolism of bile salts via the deconjugation step through bile salt hydrolases (BSH) that generate free primary bile acids and amino acids. The distribution of BSH activity among gut microbiota was estimated via metagenomic analysis of 89,856 fecal clones. Firmicutes were the most predominant BSH-expressing bacteria (30%) followed by Bacteroidetes (14.4%) and Actinobacteria (8.9%). Firmicutes with BSH activity include the genera Eubacterium, Coprococcus, Clostridium, Ruminococcus, Dorea, Lactobacillus, Enterococcus, Listeria, and Lactococcus while Bifidobacterium and Collinsella are the two Actinobacteria genera that express BSH. The same study revealed that Firmicutes and Actinobacteria are capable of deconjugating both glyco- and tauro-conjugated bile acids while Bacteroidetes are only able to hydrolyse tauro-conjugated bile acids. Also 9 BSH open reading frames have been identified among the gut microbiota community, one of them shares 56% homology with a protein encoded by the gut, archeon Methanobrevibacter smithii, which has been confirmed to have BSH activity against both glyco- and tauro-conjugated bile acids. The second step of bile acid metabolism by intestinal bacteria is the oxidation of the hydroxyl group at carbon 3, 7, or 12 of deconjugated bile acids via hydroxysteroid dehydrogenases to generate the oxo-derivative of bile acids. Gut microbiota expressing hydroxysteroid dehydrogenases include, but are not limited to Clostridium spp., Eggerthella lentum, Ruminococcus spp., Bacteroides spp., and E. coli. Simultaneously, some microbes promote 7α-/β-dehydroxylation of free cholic acid and chenodeoxycholic acid to generate deoxycholic acid (DCA) and lithocholic acid (LCA), respectively. This dehydroxylation activity is common among the order Clostridiales of gut bacteria including Ruminococcaceae, Lachnospiraceae, and Blautia. performed an interesting study that included a systematic workflow to computationally model bile acid metabolism by gut microbes and microbial communities. The authors found that, each microbe could produce maximally 6 secondary bile acids in silico, while microbial pairs could produce up to 12 bile acids, suggesting bile acid biotransformation being a microbial community task. performed a shotgun metagenomic analysis of the bile salt biotransformation genes and their distribution at the phyla level. They reported that IBD patients harbored a significantly lower abundance of these genes in comparison with healthy individuals; many of these genes originated from Firmicutes.

35Bile acids metabolism by gut microbes has a controversial contribution to both the host and the microbe. The deconjugation step has been suggested to benefit gut microbiota colonization by increasing their resistance to bile salts, where the antimicrobial effect of bile acids benefit the host via alleviating the bacterial overgrowth. Also, bile acid modification results in reducing cholesterol level and controls lipid metabolism, which is considered a protective factor against metabolic disorders such as obesity, cardiac diseases and diabetes. Another link between bile acids and metabolic diseases arises from its effect on gut microbiota composition. Using 16S rDNA clones sequencing of rats’ cecal microbiota has revealed that cholic acid is a host modifier of the gut microbiota structure that is equivalent to high fat diet. One more benefit to the host from bile acid metabolism is the protection from pathogen colonization such as vegetative C. difficile. However, high levels of secondary bile acids are associated with some diseases such as gastrointestinal cancer and gallstones.

36Four decades of research have established the association between IBD and bile acid dysmetabolism. Different studies have linked the dysmetabolism of bile acids to gut microbiota dysbiosis in IBD. It is clear now that cholic acid or a high fat diet act as a regulatory host factor that selects for gut microbes that have the machinery to detoxify bile acid metabolites or utilize their conjugates, glycine and taurine as metabolic substrates. reported that diets high in saturated fat induce the expansion of the H2S producing pathobiont, Bilophila wadsworthia, which in turn promotes colitis in IL10–/– mice. This proinflammatory effect of high saturated fat diet was attributed to taurocholic acid. Taurine, the conjugate in taurocholic acid, is known to have a sulfonic acid moiety that could be dissimilated by gut microbiota into H2S as a microbial by-product. Diets high in fat or meat lead to more conjugation of taurine to bile acids and consequently more H2S production, which is considered as a potential risk factor for IBD. Alternatively, microbial dysbiosis associated with IBD may result in bile acid dysmetabolism and this in turn may affect the anti-inflammatory characteristics of bile acids. has reported the dysfunctionality of bacterial metabolism of bile acids because of microbial dysbiosis. Moreover, they illustrated that this intestinal dysmetabolism disturbed the intestinal bile acid pool, which in turn impacted the anti-inflammatory characteristics of bile acids. Indeed, bile acids are known as anti-inflammatory mediators that inhibit NFκB activation and consequently reduce cytokine production by macrophages. This anti-inflammatory effect is characteristic of secondary bile acids but not the conjugated forms, which stresses the importance of deconjugation by gut microbiota. Another anti-inflammatory effect of bile acids may be attributed to the activation of FXR, the bile acid receptors, which has been revealed as a protective factor against chemically induced colitis in mice. Mice lacking FXR receptors have developed compromised intestinal barrier and antimicrobial defense in small intestine. The role of bile acids in protection against inflammation has been confirmed recently after identifying the association between a genetic variation of NR1H4, the gene encoding FXR receptor, and IBD. A pilot study of fecal bile acid and microbiota profiles in inflammatory bowel disease demonstrated that bile acid profiles were in general alike among patients with IBD and healthy controls. Oral administration of secondary bile acids in mice was reported to reduce the severity of colitis and ameliorate colitis-associated fecal dysbiosis at the phylum level. In accordance with the last study, dysbiosis of gut microbiota was found to induce deficiency in secondary bile acids in inflammatory-prone UC patients, which in turn leads to pro-inflammatory status in the intestine that may be treated via restoring secondary bile acids. Like bile acid dysmetabolism, bile acid malabsorption was reported to be common reason of diarrhea in CD and colitis patients.

37Oxidative stress is thought to be one of the key players in the tissue damage associated with IBD. Oxidative stress is defined as an imbalance between reactive oxygen species and intracellular antioxidants. Hence, oxidative stress arises from either a higher production of oxidative free radicals and/or deficient antioxidant machinery. The inflammatory cascade starts by the infiltration of proinflammatory cells to the intestinal mucosa, which release reactive oxygen species (ROS) and/or reactive nitrogen metabolites. For example, chemiluminescence analysis of ROS has elucidated a higher release of these free radicals by monocytes and polymorphonuclear cells extracted from both CD and UC biopsies. These free radicals induce more infiltration of proinflammatory cells and so this cycle is sustained and eventually causes tissue damage. The disruption of the intestinal epithelium exposes the immune system to the gut microbiota or other antigenic luminal components, which exacerbate the inflammation resulting in the active phenotype of the disease. For the cells to protect themselves against oxidative stress, they have to induce the production of antioxidant metabolites, which is not the case in IBD. It has been reported that IBD is associated with a depleted total antioxidant capacity of the cells or individual antioxidants. For example, IBD patients are characterized by a depletion of copper/zinc containing protein (superoxide dismutase and metallothionein), lower glutathione transferase activity with higher glutathione peroxidase in UC, and lower colonic ascorbate. Indeed, it is well reported that increased oxidative stress level in IBD patients and the detection of oxidative stress index rate could be used as predictors for the pathogenesis of IBD. showed that plasma free thiols are reduced in patients with CD, reflection of systemic oxidative stress, in clinical remission. The authors recommended that systemic oxidative stress and plasma free thiols may be a relevant therapeutic target and biomarker to monitor disease activity in CD. Oral administration of probiotics in IBD patients were reported to effectiveness via the decrease of oxidative stress values.

38For the gut microbiota to maintain their homeostasis at this oxidative stress, they must develop oxidative stress resistance machinery. demonstrated a shift of IBD microbiota toward microbes that possess the glutathione generation and reduction capability, which enables them to compensate for the oxidative stress. This machinery includes higher cysteine biosynthesis, which is a precursor of glutathione, riboflavin and NADPH, which are cofactors of glutathione reduction reaction and glutathione transfer gene. Glutathione is a tripeptide (γ-glutamylcysteinylglycine) thiol that is produced by the majority of living cells. The reduced form of glutathione protects the cells from toxic oxygen metabolites and other electrophiles via keeping the cell in a reduced state. It also has other protective functions via regulation of gene expression, cell apoptosis and transport of organic solutes. With regards to gut microbiota, glutathione has been reported to be biosynthesized by Proteobacteria members and a limited number of gram positive bacteria such as some Streptococcus spp. and Staphylococcus aureus, but not by Clostridium, Bacillus or Micrococcus members. Also, glutathione sulfur transferase encoding genes that possess peroxidase activity are expressed by Proteobacteria members. It is well documented now that IBD microbiota is dominated by Proteobacteria, which is known to produce some proinflammatory metabolites such as enterotoxins or LPS. This might generate a testable hypothesis that the inflammation associated stresses in the gut, such as oxidative stress, might constitute a selective pressure that induces a microbial shift toward the stress-resistant microbes. Furthermore, this shift might develop a colitogenic microbiota that could maintain the active chronic phenotype of the inflammation.

39Medications for IBD can be classified into five classes: aminosalicylates (sulfasalazine, olsalazine, and mesalamine), Corticosteroids (cortisone, prednisone, prednisolone, hydrocortisone, methylprednisolone, beclometasone, and budesonide), immunosuppressive agents (6-Mercaptopurine, azathioprine, methotrexate, and tacrolimus), antibody agents (Anti-TNF agents (infliximab, adalimumab, certolizumab pegol), and antibiotics (metronidazole, ciprofloxacin and rifaximin). The choice of the treatment strategy depends on the severity of the disease and the response to previous therapy. The effect of some of the above-mentioned drugs on gut microbiota are summarized in Table 2. Interestingly some drugs that are used for treatment of IBD requires metabolic activation via the gut microbiota e.g., sulfasalazine, balsalazide (mesalamine prodrug), olsalazine, and methotrexates.

40It should be mentioned that some IBD medications are reported to affect either the metabolism of gut microbiota or the metabolic status of intestinal cells by altering the intestinal biota. Sulfasalazine was reported to enhance carbohydrate metabolism, citrate cycle and decrease the oxidative stress (riboflavin, sulfur, cysteine). showed that, mesalamine was able to decrease polyphosphate levels in bacteria, including members of the human gut microbiota. This reduction leads to bacterial sensitization to oxidative stress and decreases bacterial colonization. performed an in-silico metabolic prediction analysis by including azathioprine or anti-TNF antibodies-treated IBD groups and assessed the effect of gut microbiota function on remission status. They found that the predicted butyrate synthesis was significantly enriched in patients achieving clinical remission. The use of oral steroids in IBD patients was demonstrated to affect two biosynthetic pathways of methanogenesis and one pathway in the biosynthesis of vitamin B2 and nucleosides. The oral administration of metronidazole was found to reduce basal oxidative stress in colonic tissue of healthy rat and increase the thickness of colonic mucosal layer by about twofolds. Moreover, the metronidazole-treated microbiota in murine fecal donors retained its ability to control inflammation co-occurring with enrichment of Lactobacillus and innate immune responses including invariant natural killer T cells in experimental colitis.

41Probiotics are living organisms that when are given in appropriate amount to the host result in health benefit. Prebiotics are substances, which are utilized by probiotics leading to enhancing the health. Postbiotics are non-living microorganisms with or without their cell components and metabolites that confer health benefits. Although probiotics have the ability to modulate microbiome composition resulting in enhancing the growth of good species and inhibiting the growth of pathogenic ones, their use in IBD treatment is recommended only in the context of clinical trials in adults and children. Probiotics have anti-inflammatory impact and enhance intestinal barrier functions. In adults and children with pouchitis, the use of eight-strain combination of Lactobacillus plantarum, Lactobacillus paracasei subspparacasei, Lactobacillus delbrueckii subsp bulgaricus, Lactobacillus acidophilus, Bifidobacterium breve, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, and Streptococcus salivarius subsp. thermophilus is recommended. It is well documented that the probiotic cocktail VSL#3, which is composed of 3 Bifidobacteria strains, 4 lactobacilli strains, and 1 Streptococcus strain, is promising for treatment of patients suffering from IBD. Also, the probiotic Lactobacillus reuteri ATCC 55730 was speculated to be helpful during the treatment of IBD cases. In addition, the probiotic cocktail composed of Lactobacillus acidophilus NCIMB 30175, Lactobacillus plantarum NCIMB 30173, Lactobacillus rhamnosus NCIMB 30174, and Enterococcus faecium NCIMB 30176 has a well-documented effect in the treatment of IBD patients especially UC. More recently, next generation probiotics such as A. muciniphila and F. prausnitzii and their supernatants (postbiotics) are reported to exhibit beneficial effects during IBD treatment. Fermented foods are good sources of probiotics including miso, tempeh, kefir, kimchi, pickled vegetables, yogurt with live active cultures, kombucha tea and sauerkraut.

42Also, prebiotics such as inulin, resistant starch, gums, pectins, and fructo-oligosaccharides are reported to be beneficial in the treatment of IBD patients through enhancing functions of the intestinal barrier and protecting vs. invasion and translocation of pathogens. Although there are many commercial products of these prebiotic fibers, healthy diets are considered their main source such as bananas, beans, onions, raw version of leeks, oats, dandelion greens, wheat, garlic, asparagus, artichokes, barley, seaweed, and other fruits and vegetables that are rich in fibers and indigestible carbohydrates. Such prebiotics aid the growth of normal gut microbiota and the production of SCFAs, which results in enhancing the activity of immune cells, maintaining the levels of glucose and cholesterol as well as decreasing the pH of the colon, which results in enhancing the condition.

43Phages are the most ubiquitous organisms worldwide and they are characterized by their selectivity and specificity to bind their target host. Phages can bind and lyse specific bacterial strains within certain species. This capability gives the phage the advantage to be safer during the treatment of bacterial infections than commonly used antibiotics and to have limited effect on microbiota of the host.

44The use of Russian coliphage or oral T4-like coliphages in children with bacterial infection-induced diarrhea does not induce any side effect, however, it was unsuccessful to enhance the conditions. Recently, it is recommended to use phages against AIEC in patients suffering from IBD. AIEC is an abnormal pathogen, which is commonly found in the ileal mucosa of IBD patients. The use of bacteriophages to lyse AIEC significantly reduced the symptoms of DSS-induced colitis in transgenic mice expressing the human receptor for AIEC named CEACAM6. Therefore, phages targeting AIEC may be a promising therapeutic approach for the treatment of IBD patients.

45FMT is defined as the transplantation of healthy donor fecal microbiota into the gut of a patient as a trial to reverse the dysbiosis, restore homeostasis of the gut microbiota and improve the condition. This approach was reported to be successful during the treatment of recurrent C. difficile infection (CDI). The successful treatment of IBD patients using FMT depends on three main factors including; early treatment, content of fecal matter of the donor and usage of multiple FMTs. Treatment of IBD patients using FMT is based on the idea that regaining the gut microbiota homeostasis will alter the growth of pathogens like C. difficile that are responsible for the disease. This is achieved through competition between the regained microbiota and pathogens for nutrients and colonization, production of antimicrobials by regained microbiota that directly affect pathogens, and vegetative growth and spore germination inhibition through bile acid mediated mechanism. However, it is noteworthy that the fungal and viral content of the stool of donors may affect the outcome of IBD treatment using FMT. It is reported that using stool rich in C. albicans significantly decreases the treatment efficacy. Moreover, it was depicted that following FMT, different patients who received fecal matter from the same donor exhibited extremely individualized virus colonization patterns.

46showed that 1 week treatment with anaerobically prepared fecal material, in mild to moderate UC patients, led to a higher probability of remission after 8 weeks. Another interesting work followed a randomized controlled study to investigate the role of FMT to maintain remission in CD patients. The authors found a low similarity index between donor and recipient microbiota in some patients suggesting that a single FMT might not be enough to stimulate significant changes in these patients. An interesting study aimed to investigate the optimum timing of FMT for maintaining the long-term clinical benefits in UC. The authors demonstrated that UC patients could take the 2nd course of FMT within 4 months after the initial course of FMT, which will allow clinicians to consider sequential FMTs as a long-term treatment strategy for UC. Moreover, they showed that the relative abundance of Eubacterium, Ruminococcus, Eggerthella, and Lactobacillus in UC patients can be used as predictors for the long-term efficacy of FMT. Similarly, the species Eubacterium hallii, Ruminococcus bromii, and Roseburia inulivorans were recommended to predict the success of FMT therapy in UC patients. However, the application of FMT has many disadvantages. First, different samples represent different bacterial ensembles which means variable efficacy and the outcome will rely on the sample. Additionally, safety risks may arise from the presence of undesirable strain/functionality within the donated stool sample such as transferable antibiotic resistance elements, virulence factors or pathogenic strains enteropathogenic E. coli.

47There is a tight bond between diet, gut microbiota, colonocytes, and immune cells during both the intestinal healthy conditions (homeostasis) and inflammation. Healthy diet patterns including diets rich in fruits, vegetables, probiotics, fermentable food, fibers, prebiotics, and adequate concentrations of vitamin D are good for intestinal health and homeostasis. Healthy diets generally result in more microbial diversity and an increase of good bacteria including F. prausnitzii, Bifidobacterium spp., and Lactobacillus spp. that result in increased production of SCFAs, especially butyrate. Butyrate has strong anti-inflammatory characters as it improves the function of intestinal barrier and endorses the proliferation of regulatory T cells (Treg). Moreover, butyrate promotes the dendritic cells (DC) within the lamina propria and commensal microbiota antigens within the intestinal lumen. Dendritic cells mainly trigger Treg cells to produce transforming growth factor-β (TGF-β) and interleukin-10 (IL-10) through the production of TGF-β resulting in tolerant immune response and intestinal homeostasis. These modulations result in more thick mucus layer, which is an important property of intestinal homeostasis, giving a protective shield between epithelial cells and luminal bacteria. Moreover, healthy diets represent a good source of aryl hydrocarbon receptor (AhR) which induce the production of IL-22, via the innate lymphoid cell 3 (ILC3) that maintains intestinal barrier function.

48Healthy diets harboring adequate concentrations of vitamin D as well as vitamin D supplements are reported to be helpful during IBD cases. It is reported that vitamin D is crucial for maintaining the composition of gut normal microbiota, where vitamin D receptor (VDR) deficient mice exhibited increased abundance of Bacteroidetes and Proteobacteria as well as diminished Lactobacillus spp. resembling the dysbiotic cases of IBD patients. Vitamin D enhances the epithelial barrier function and promotes the production of antimicrobial peptides. It also promotes the proliferation of dendritic cells, which in turn promotes the production of the immunosuppressive IL-10 through provoking Treg cells. Vitamin D also decreases the production of pro-inflammatory cytokines including TNF-γ, IL-17, and IL-21 through its inhibitory effect on IL-12 and IL-23 that are responsible for the responses of Th-1 and Th-17, respectively.

49On the other hand, unhealthy diets such as Western dietary patterns those are low in fruits, vegetables, probiotics, fermentable food, fibers, prebiotics, and vitamin D are directly related to dysbiosis of the microbiota and diminished microbial diversity, decreasing SCFAs production, reducing good bacteria and increasing the pathobionts such as E. coli and Clostridium difficile. This results in more patchy and thinner mucus layer, which as a result provides less protection between colonocytes and luminal bacteria. In addition, the compromised intestinal barrier function, due to decreased expression of cellular tight junctions, leads to escape of harmful bacterial products including lipopolysaccharides (LPS) from the intestinal lumen to the lamina propria. LPS trigger the macrophages via binding to toll-like receptors (TLR) leading to the production of TNF-α. TNF-α endorses the proliferation of T helper cell type 1 (Th1) that produce the proinflammatory cytokines including TNF-α and TNF-γ resulting in inflammation and dysfunctionality of the intestinal barrier. Moreover, the condition may worsen due to decreasing Treg cells responsible for the production of IL-10, which mitigates the intestinal inflammation.

50From what has been discussed above, gut microbiota exerts many beneficial roles to human health, where any permanent disturbance of this ensemble could metabolically lead to a disease state. Gut microbiota of IBD patients is characterized by depletion of Firmicutes and Bacteroidetes with increased abundance of Proteobacteria and Actinobacteria. These changes in microbial composition result in perturbations of microbial functions that could be summarized in less short chain fatty acid production, less bile acid hydrolysis, higher redox potential and increased H2S production (Figure 2). This pattern of microbial metabolic perturbations is associated with defectiveness of some human cellular pathways such as defective H2S detoxification, SCFAs transport and oxidation, and high luminal redox potential. The interaction between both sides of dysfunctionality is considered pathogenic to the host and might modulate the chronicity of the disease. However, the knowledge of the cause/effect relationship between these metabolic perturbations and the inflammation has so far been limited. In addition, the actual molecular mechanisms that regulate the interaction between both sides of the equation still need to be identified. Still, different strategies are being developed to manipulate gut microbiota to reduce the intestinal inflammation and improve the disease outcomes. These include live biotherapeutics such as FMT and probiotics. Taking in account the disadvantages of FMT and enrichment-based approaches in general, new approaches may rely on bottom-up rational to design identified bacterial consortia that are metabolically interdependent and exert a variety of protective functions to the host and the gut microbiota. These functions include generation of beneficial metabolites such as SCFAs and indole, deconjugation and conversion of bile acids, competition for critical nutrients and synthesis of antimicrobial molecules against opportunistic pathogens.

51All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

52The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

53All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.