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Modern Diseases and the Gut Microbiota

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The microbiota plays a major role in health and disease in humans; as well as harvesting energy and performing a variety of metabolic functions – the gut microbiota perhaps more importantly, interacts with the immune system by providing signals to ensure the development of immune functions.

Mounting evidence points to consequences of disrupting the microbiota we have coevolved with over millennia. The colonization of the microbiota in our gut usually takes place over the first 10 years of our lives and continues throughout the course of our lives unless disrupted through the use of antibiotics. For example nearly all adults in developing countries harbor H. pylori, and this was likely true for humans in general for most of our evolutionary history. Interestingly, there is strong evidence linking the decreased prevalence of H. pylori in the Western world to increased antibiotic use and to the elevated rates of asthma and allergic disorders in this same population (; ). Moreover, increased prevalence of gastresophageal reflux disease (GERD), adenocarcinoma of the esophagus, and Barrett’s esophagus has been linked to decreased prevalence of cagA+ strains of H. pylori (reviewed in ). Overall, the strong link between H. pylori and modern diseases illustrates how the disruption of the human microbiota can have dire health consequences.

Chronic Diseases Linked with Our Gut Microbiota

Obesity-Related Diseases

Starting around 2004, the hallmark studies of Gordon et al demonstrated a potential relationship between the gut microbiome and development of an obese phenotype. An increase in relative abundance of Firmicutes and a proportional decrease in Bacteroidetes were associated with the microbiota of obese mice,23 which was confirmed in a human dietary intervention study demonstrating that weight loss of obese individuals (body mass index, BMI>30) was accompanied by an increase in the relative abundance of Bacteroidetes.24 Nevertheless, based on most human studies, the obesity-associated decrease in the ratio of Bacteroidetes to Firmicutes (B:F) remains controversial.24,25 This is likely due to heterogeneity among human subjects with respect to genotype and lifestyle. Recent studies have identified diet, especially fat, as a strong modulator of the microbiota, particularly in inbred and age-standardised laboratory animals. The sources of variation in the microbiota are mainly limited to the experimental diets used, and there is growing evidence that the high fat intake rather than obesity per se had a direct effect on the microbiota and linked clinical parameters.26 However, in humans the microbiome is exposed to fundamentally different ‘environmental’ factors in obese and lean individuals that go beyond BMI alone, including diet26 and host hormonal factors.27 In addition, the aetiology of obesity and its metabolic complications, including low grade inflammation, hyperlipidaemia, hypertension, glucose intolerance and diabetes, reflect the complex interactions of these multiple genetic, behavioural and environmental factors.28 Lastly, the accuracy of BMI as an indicator for obesity is limited; 25% of obese people could in fact be regarded as metabolically ‘healthy’ (ie, with normal lipid and glucose metabolism).29 Therefore, linking GI tract microbial composition directly and exclusively to obesity in humans will remain challenging due to the various confounding factors within the heterogeneous population.

This complexity has led to a shift from treating obesity as a single phenotype, to attempts at correlating microbial signatures to distinct or multiple features associated with (the development of) metabolic syndromes such as T2D. Recently, two (meta)genome-wide association studies were performed, with 345 Chinese individuals30 and 145 European women.31 In both studies, de novo generated metagenomic species-level gene clusters were employed as discriminant markers which, via mathematical modelling, could better differentiate between patients and controls with higher specificity than a similar analysis based on either human genome variation or other known risk factors such as BMI and waist circumference. At the functional level, membrane transporters and genes related to oxidative stress were enriched in the microbiota of patients,31 while butyrate biosynthesis was decreased.30 Although both studies observed high similarities in microbial gene-encoded functions, the most discriminant metagenomic species-level gene clusters differed between the cohorts (Akkermansia did not contribute to the classification in the European cohort whereas Lactobacillus showed no contribution in the Chinese study population), indicating that diagnostic biomarkers could be specific to the population studied.

In another metagenomic study, a bimodal distribution of microbial gene richness in obese individuals was observed, stratifying individuals as High Gene Count or Low Gene Count (HGC and LGC).32 HGC individuals were characterised by higher prevalence of presumed anti-inflammatory species such as F. prausnitzii, and an increased production potential of organic acids (including butyrate). In contrast, LGC individuals showed higher relative abundance of potentially proinflammatory Bacteroides spp and genes involved in oxidative stress response. Remarkably, only biochemical obesity-associated variables, such as insulin resistance, significantly correlated with gene count while weight and BMI did not, underscoring the inadequacy of BMI as an indicator for ‘Obesity and its Associated Metabolic Disorders’ (OAMD).33 An accompanying paper demonstrated that a diet-induced weight-loss intervention significantly increased gene richness in the LGC individuals which was associated with improved metabolic status.34 Although gene richness was not fully restored, these findings support the reported link between long-term dietary habits and the structure of the gut microbiota.31 It also suggests permanent adjustment of the microbiota may be achieved through diet.

Most studies involving the microbiome have been solely correlative but recently a causal relationship was established between host glucose homoeostasis and gut microbial composition. FMT from lean donors to individuals with metabolic syndrome significantly increased their insulin sensitivity.33 The transplant produced an increase in faecal butyrate concentrations, microbial diversity and the relative abundance of bacteria related to the butyrate-producing Roseburia intestinalis.

Together, these studies produce a body of evidence that the microbiome plays a role in host energy homoeostasis and the establishment and development of OAMD, although the exact mechanisms remain obscure. Previous contradictory findings might be attributed to miscellaneous approaches,35 and also heterogeneity in genotype, lifestyle and diet of humans combined with the complex aetiology of OAMD. Nonetheless, a clearer picture is emerging. The gut of individuals with OAMD is believed to harbour an inflammation-associated microbiome, with a lower potential for butyrate production and reduced bacterial diversity and/or gene richness. Although the main cause of OAMD is excess caloric intake compared with expenditure, differences in gut microbial ecology might be an important mediator and a new therapeutic target or a biomarker to predict metabolic dysfunction/obesity in later life.

Liver Disease

The liver receives 70% of its blood supply from the intestine via the portal vein, thus it is continually exposed to gut-derived factors including bacterial components, endotoxins (lipopolysaccharide, flagellin and lipoteichoic acid) and peptidoglycans. Multiple hepatic cells, including Kupffer cells, sinusoidal cells, biliary epithelial cells and hepatocytes, express innate immune receptors known as pathogen-recognition-receptors that respond to the constant influx of these microbial-derived products from the gut.36 It is now recognised that the gut microbiota and chronic liver diseases are closely linked. Characterising the nature of gut dysbiosis, the integrity of the gut barrier and mechanisms of hepatic immune response to gut-derived factors is potentially relevant to development of new therapies to treat chronic liver diseases.37 Furthermore the field of bile acid signalling has thrown open the concept of the gut:liver axis as being active and highly regulated.38

Non-Alcoholic Fatty Liver Disease

The pathophysiology of NAFLD is multifactorial with strong genetic and environmental contributions. Recent evidence demonstrates that gut microbiota dysbiosis can result in the development of obesity-related non-alcoholic fatty liver disease (NAFLD), and patients with NAFLD have small intestinal bacterial overgrowth and increased intestinal permeability.39 In the 1980s, development of non-alcoholic steatohepatitis (NASH) and small intestinal bacterial overgrowth was observed in humans after intestinal bypass and, interestingly, regression of hepatic steatosis after metronidazole treatment, suggesting a possible role for the gut bacteria in NAFLD.40 Disruption of the murine inflammasomes (see box 1) is associated with an increase in Bacteroidetes and reduction in Firmicutes and results in severe hepatic steatosis and inflammation.41 Faecal microbiota analysis of patients with NAFLD and NASH has produced variable results due to significant variation of patient demographics, severity of liver disease and methodology. A lower proportion of Ruminococcaceae was noted in patients with NASH compared with healthy subjects42 and a study which characterised gut microbiota of children with NASH, obesity and healthy controls showed that patients with NASH had a higher proportion of Escherichia compared with other groups.43 Patients with NAFLD also have increased gut permeability suggesting that translocation of bacteria or microbe derived products into the portal circulation contributes to the pathogenesis.39

Alcoholic Liver Disease

Since not all alcoholics develop liver injury, it appears that chronic alcohol abuse is necessary but not sufficient to cause liver dysfunction. Numerous animal model and human observational studies indicate that gut bacterial products like endotoxin may mediate inflammation and function as cofactors for the development of alcohol-related liver injury.44 Serum endotoxin levels are elevated in humans and rats with alcoholic liver disease, and monocytes from alcoholics are primed to produce cytokines after endotoxin exposure. Alcohol causes intestinal bacterial overgrowth in humans and bacterial numbers were significantly higher in jejunal aspirates from patients with chronic alcohol abuse compared with controls, with similar findings in patients with alcohol-induced cirrhosis.45 The degree of overgrowth correlates with the severity of cirrhosis. Tsukamoto-French model mice fed intragastrically with alcohol for 3 weeks showed increased relative abundance of Bacteroidetes and Akkermansia spp and a reduction in Lactobacillus, Leuconostoc, Lactococcus and Pediococcus while control mice showed a relative predominance of Firmicutes.46 Patients with alcoholic liver disease also show increased gut permeability, allowing translocation of bacteria and bacterial products to the liver.47

Autoimmune Liver Diseases

These consist of primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC) and autoimmune hepatitis and represent at least 5% of all chronic liver diseases. They are presumed autoimmune conditions but the expectation is that the gut microbiota is relevant to pathogenesis, particularly because (A) PSC is associated with IBD and aberrant lymphocyte tracking, and (B) significant gut:liver axes exist through bile acid signalling. Patients with PSC develop a distinct form of IBD thus understanding the relationship between PSC and IBD is essential in uncovering the pathogenesis of PSC, which remains largely undetermined. However, it is likely that in genetically susceptible individuals, intestinal bacteria could trigger an abnormal or inadequate immune response that eventually leads to liver damage and fibrosis. Recently it was shown that patients with PSC have distinct gut microbiota. Analysis of colon biopsy microbiota revealed that patients with PSC-IBD and IBD showed reduced abundance of Prevotella and Roseburia (a butyrate-producer) compared with controls.48 ,49 Patients with PSC-IBD had a near-absence of Bacteroides compared with patients with IBD and control patients, and significant increases in Escherichia, Lachnospiraceae and Megasphaera. Randomised controllled trials (RCTs) investigating antibiotic therapy in PSC have shown these to be superior in improving biochemical surrogate markers and histological parameters of disease activity compared with ursodeoxycholic acid alone.50 In a recent prospective paediatric case series, oral vancomycin was shown to normalise or significantly improve liver function tests.51 There is evidence that mucosal integrity is compromised in patients with PSC, supporting the traditional leaky gut hypothesis of microbe-derived products translocating to the liver and biliary system to trigger an inflammatory reaction.52 It was also demonstrated that tight junctions of hepatocytes were impaired in patients with PSC and infusion of non-pathogenic E. coli into portal circulation caused portal fibrosis in animal models.53 These findings collectively suggest that bacterial antigens translocate across a leaky and possibly inflamed gut wall into the portal and biliary system to induce an abnormal immune response and contribute to PSC pathogenesis.

PBC is a chronic cholestatic liver disease with an uncertain aetiology. It is generally believed to be an autoimmune disease triggered by environmental factors in individuals with genetic susceptibility. As yet, there have been no studies directly characterising the gut microbiota in patients but molecular mimicry has been suggested as a proposed mechanism for the development of autoimmunity in PBC, with serum antibodies of patients cross-reacting with conserved bacterial pyruvate dehydrogenase complex component E2 (PDC-E2) homologues of E. coli, Novosphingobium aromaticivorans, Mycobacterium and Lactobacillus species. Hence it has been speculated that these bacteria (of possible GI origin) may initiate molecular mimicry and development of PBC in genetically susceptible hosts.54

Treating Liver Disease With Probiotics

Probiotics have shown promise in ameliorating liver injury by reducing bacterial translocation and hepatic inflammation.55 A recent meta-analysis concluded that probiotics can reduce liver aminotransferases, total cholesterol, tumour necrosis factor α and improve insulin resistance in patients with NAFLD.56 A recent study in patients with cirrhosis with ascites showed that the probiotic VSL#3 significantly reduced portal hypertension.57 A further study evaluated the role of FMT in modulating liver disease by transferring the NAFLD phenotype from mice with liver steatosis to germ-free mice.58 There remains a need for detailed descriptive and interventional studies focused on bacterial diversity and mechanisms linking gut dysbiosis with inflammatory, metabolic and autoimmune/biliary liver injury.

Inflammatory Bowel Disease (IBD)

Early studies implicating bacteria in IBD pathogenesis focused on identifying a potential culprit that could initiate the inflammatory cascade typical of IBD. Many organisms have been proposed: Mycobacterium avium subsp paratuberculosis and a number of Proteobacteria including enterohepatic Helicobacter, non-jejuni/coli Campylobacter and adherent and invasive E. coli. The focus has recently shifted with the realisation that the gut microbiota as a whole is altered in IBD. The concept of an altered gut microbiota or dysbiosis is possibly the most significant development in IBD research in the past decade. A definitive change of the normal gut microbiota with a breakdown of host-microbial mutualism is probably the defining event in IBD development.59

Changes in the gut microbiota have been repeatedly reported in patients with IBD, with certain changes clearly linked to either Crohn’s disease (CD) or UC: the most consistent change is a reduction in Firmicutes.60 This has been balanced by reports of increased levels of Bacteroidetes phylum members,61 although a reduction in Bacteroidetes has also been reported.62 There is a suggestion that there may be spatial reorganisation of the Bacteroides species in patients with IBD, with Bacteroides fragilis being responsible for a greater proportion of the bacterial mass in patients with IBD compared with controls.63

Reduction in the Firmicutes species F. prausnitzii has been well documented in patients with CD, particularly those with ileal CD, although an increase in F. prausnitzii has been shown in a paediatric cohort, suggesting a more dynamic role for the species that merits further study.64 Other studies have also demonstrated a decrease in Firmicutes diversity, with fewer constituent species detected in patients with IBD compared with controls.65 Changes in the two dominant phyla, Firmicutes and Bacteroidetes, are coupled with an increase in abundance of members of the Proteobacteria phylum, which have been increasingly found to have a key role in IBD.66 Studies have shown a shift towards an increase in species belonging to this phylum, suggesting an aggressor role in the initiation of chronic inflammation in patients with IBD.67 More specifically, increased numbers of E. coli, including pathogenic variants, have been documented in ileal CD.68 The IBD metagenome contains 25% fewer genes than the healthy gut with metaproteomic studies showing a correlative decrease in proteins and functional pathways.69 Specifically, ileal CD has been shown to be associated with alterations in bacterial carbohydrate metabolism and bacterial-host interactions, as well as human host-secreted enzymes.69 A detailed investigation of functional dysbiosis during IBD built on this by including inferred microbial gene content from 231 subjects and an additional 11 metagenomes.70 This study identified enrichment in microbial pathways for oxidative stress tolerance, immune evasion and host metabolite uptake, with corresponding depletions in SCFA biosynthesis and typical gut carbohydrate metabolism and amino acid biosynthetic processes. Intriguingly, similar microbial metabolic shifts have been observed in other inflammatory conditions such as T2D,30 suggesting a common core gut microbial response to chronic inflammation and immune activation. In addition, recent work suggests a role for viruses in IBD, with a significant expansion of Caudovirales bacteriophage in patients.71

Several clinical trials have examined the approach of modulating the microbiota in patients with IBD, many of which predate the ‘omics’ era. Such trials provide a ‘proof of concept’ for the importance of the role of the gut microbiota in IBD, but marrying up individual approaches with the complex multifactorial nature of IBD remains a challenge, particularly in addressing the different phenotypes and genotypes of disease and the different ‘phases’ of the disease process: for example, prophylaxis, maintenance of remission, treatment of relapses.

Ulcerative Colitis

In terms of probiotic research, one of the largest clinical trials in IBD was the use of E. coli Nissle 1917 in the setting of remission maintenance in UC. Patients (n=327) were assigned to a double-blind, double-dummy trial to receive either the probiotic or mesalazine.72 Both treatments were deemed equivalent with regards to relapse. E. coli Nissle is now considered an effective alternative to 5-aminosalicylate for remission maintenance in UC.73 There are two published clinical trials of the multistrain probiotic VSL#3 in the setting of mild to moderate flares of UC.74 Both demonstrate that high doses improve disease activity scores but whether such improvements in scores are clinically meaningful for patients, particularly compared with other treatment options, remains to be clarified. An alternative approach is transplantation of the whole gut microbiota from a healthy donor: FMT. In IBD, a recent systematic review and meta-analysis has shown that of nine cohort studies, eight case studies and one randomised controlled trial, overall 45% (54/119) achieved clinical remission. When only cohort studies were analysed 36% achieved clinical remission.75 Since that meta-analysis, two randomised controlled trials in UC show discrepant results. One trial, in which two faecal transplants were given via the upper GI route, showed no difference in clinical or endoscopic remission between the faecal transplant group and the control group (given autologous stool).76 A second trial, in which patients with UC were randomised to weekly faecal enemas from healthy donors or placebo enemas for 6 weeks, demonstrated remission in a greater percentage of patients given FMT compared with the control group (given water enema).77 There are unanswered questions regarding mode of delivery, frequency of delivery and optimal donor/host characteristics.

Crohn’s Disease

Antibiotics demonstrate efficacy in particular groups of patients with CD but some antibiotics may be detrimental, showing a complex interplay between host and microbiota. Patients who have had a resection for CD have a decreased rate of endoscopic and clinical recurrence when metronidazole or ornidazole are used as prophylactic therapies.78 Several studies have assessed the specific role of antimycobacterial therapies in CD treatment but overall results are disappointing. There is no clinically relevant evidence base for the use of probiotics in CD and in terms of prebiotics, although an open label trial of fructo-oligosaccharide in CD showed promise,79 and a subsequent randomised placebo-controlled trial of fructo-oligosaccharide did not support any clinical benefit.80

Pouchitis

Restorative proctocolectomy with ileal-pouch anal anastomosis is the operation of choice for patients with UC requiring surgery. Pouchitis has an incidence of up to 50% of patients although it is a significant clinical problem for only about 10%. Antibiotics are used as primary therapy; if single antibiotics fail, dual antibiotics used for longer periods of time or antibiotics tailored to the microbiota in an individual patient can be used. VSL#3 reduced the risk of disease onset and maintained an antibiotic-induced disease remission in pouchitis.81 A meta-analysis has shown that VSL#3 significantly reduced the clinical relapse rates for maintaining remission in patients with pouchitis.82

CRC

Many microbiome studies have focused on colitis-associated cancers83 or rodent preclinical models.84 Despite this, there is increasing evidence that the colonic microbiota plays an important role in the cause of sporadic CRC.85 Reduced temporal stability and increased diversity has been shown for the faecal microbiota of subjects with established CRC and polyposis,86 and now metagenomic and metatranscriptomic studies have identified an individualised oncogenic microbiome and specific bacterial species that selectively colonise the on-tumour and off-tumour sites.87

Several competing theories of the microbial regulation of CRC have emerged (figure 1) to explain these observations. The keystone-pathogen hypothesis88 and the α-bug hypothesis both state that certain, low abundance microbiota members (such as enterotoxigenic B. fragilis) possess unique virulence traits, which are pro-oncogenic and remodel the microbiome and in turn promote mucosal immune responses and colonic epithelial cell changes.89 Tjalsma et al90 have also proposed the ‘driver-passenger’ model for CRC: a first hit by indigenous intestinal bacteria (‘bacterial drivers’), which drive the DNA damage that contributes to CRC initiation. Second, tumorigenesis induces intestinal niche alterations that favour the proliferation of opportunistic bacteria (‘bacterial passengers’). For example, CRCs have an increased enrichment of opportunistic pathogens and polymicrobial Gram-negative anaerobic bacteria91 but it is not yet clear whether these opportunistic pathogens merely benefit from the CRC microenvironment or influence disease progression. However, colonic polyps demonstrate higher bacterial diversity and richness when compared with control patients, with higher abundance of mucosal Proteobacteria and lower abundance of Bacteroidetes.92 This may in part be explained by the mucosal defensive strategies designed to manage the commensal microbiota. For example, α-defensin expression is significantly increased in adenomas resulting in an increased antibacterial activity compared with normal mucosa.93

Figure 1

Figure 1: Proposed mechanisms of the gut microbiome in colon cancer aetiology.

At present, human studies have involved small patient numbers, with evidence of sampling heterogeneity, limited tumour phenotyping and oncological data. Despite this, a small number of specific pathobionts have now been linked with adenomas and CRC including Streptococcus gallolyticus,94Enterococcus faecalis95 and B. fragilis.84E. coli is also overexpressed on CRC mucosa; it expresses genes that confer properties relevant to oncological transformation including M cell translocation, angiogenesis and genotoxicity.96 Enrichment of Fusobacterium nucleatum has also been identified in adenoma versus adjacent normal tissue and is more abundant in stools from CRC and adenoma cases than in healthy controls. F. nucleatum‘s fadA, a unique adhesin, allows it to adhere to and invade human epithelial cells, eliciting an inflammatory response97 and stimulating cell proliferation.98 Novel mechanisms from previously unassociated bacteria are also being described to explain how bacterial proteins target proliferating stem-progenitor cells. For example, AvrA, a pathogenic product of Salmonella, has been shown to activate β-catenin signals and enhance colonic tumorigenesis.99

Work has also focused on the metabolic function of the gut microbiome and dietary microbiome interactions in the aetiology of CRC. It is likely that the metabolism of fibre is critical to this. Metagenomic analyses have consistently identified a reduction of butyrate-producers in patients with CRC,100 a finding replicated in animals.101 The microbiome also plays an important role in the metabolism of sulfate, through assimilatory sulfate-reduction to produce cysteine and methionine, and dissimilatory sulfate-reduction to produce hydrogen sulfide (H2S). H2S is likely to contribute to CRC development, as colonic detoxification of H2S is also reduced in patients with CRC; it also induces colonic mucosal hyperproliferation.102 There is also evidence that differences in host genotype, which affect the carbohydrate landscape of the distal gut, interact with diet to alter the composition and function of resident microbes in a diet-dependent manner.103 Therefore it is possible that patients genetically predisposed to CRC have a modified metabolically active microbiome, which is determined by their genes and by their family environment and dietary habits. There is other evidence from global studies of cancer risk, that the microbiome is important in cancer risk.

African Americans possess a colon dominated by Bacteroides, while in Africans Prevotella are more abundant.104 African Americans, who are at high risk of CRC, may have evolved a CRC-microbiota moulded by dietary habits and environmental exposures. Critically, mucosal Ki67 expression (a biomarker for cancer risk) may decrease or increase within 2 weeks of either a high fibre (>50 g/day) dietary intervention in African Americans or a high fat, high protein low fibre Westernised diet in African subjects. This short-term intervention leads to reciprocal changes in luminal microbiome co-occurrence network structures that overwhelm interindividual differences in microbial gene expression. Specifically, an animal-based diet increases the abundance of bile-tolerant microorganisms (Alistipes, Bilophila and Bacteroides) and decreases the levels of Firmicutes that metabolise dietary plant polysaccharides (Roseburia, Eubacterium rectale and Ruminococcus bromii).105,106

Chronic Diseases Linked with Gut Bacteria

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