Role of the Microbiome in Intestinal Barrier Function and Immune Defense

Chapter 13

Role of the Microbiome in Intestinal Barrier Function and Immune Defense Aline Ignacio1, Fernanda Fernandes Terra1, Ingrid Kazue Mizuno Watanabe2, Paulo Jose´ Basso1 and Niels Olsen Saraiva Caˆmara1 1

Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil; 2Department of Medicine, Federal

University of São Paulo, São Paulo, Brazil

THE HUMAN MICROBIOTA The human body, especially the mucosae, harbors complex communities of commensal microbes, including bacteria, fungi, viruses, protozoa, and archaea. This great number of microorganisms is collectively known as indigenous microbiota, and the genes codified by them are collectively called microbiome. Recent studies indicating the presence of bacteria in the placenta and meconium suggest that our colonization begins still in the feto-placental unit [1,2]. The disruption of indigenous microbiota, also named dysbiosis, may be characterized by the reduction of its number and/or diversity. Recently, dysbiosis has been associated to a myriad of intestinal and systemic diseases, such as inflammatory bowel disease (IBD), asthma, arthritis, cardiovascular diseases, and obesity. Such diseases can be triggered by both pathogenic and symbiotic microbes that can be harmful in certain circumstances.

Intestinal Microbiota: A Functional Organ Along the gut, the bacterial density increases from the stomach toward the colon, while its diversity decreases from the lumen to the intestinal epithelia [3]. Looking for the mutualistic pattern maintenance, the intestine provides an auspicious environment for the microbes to live, with free access to nutrients, and controlled temperature. Meanwhile, the gut microbiota protects the host from potential pathogens and regulates the metabolism and nutrition, besides regulating the morphogenesis and physiology of the immune system. Many other nonconventional functions, such as adiposity control, bone mass formation and behavior are also performed by the microbiota.

Key Commensals and Pathogens Most of the resident gut microbes live as commensals or in mutualism; however, a small fraction is potentially pathogenic, often called pathobionts, and can trigger some diseases when homeostasis is disrupted. Clostridium difficile, for example, is a spore-forming bacterium which composes the indigenous microbiota; however, once commensal diversity is reduced, its germination occurs and the release of toxins in the gut can lead to severe intestinal inflammation [4]. Therefore, besides protecting the host from exogenous pathogens, the microbiota must also control pathobiont growth [5].

Colonization Resistance and Pathogen Inhibition Colonization resistance is the term used to describe the microbiota’s capacity to limit the introduction of exogenous microorganisms and pathobiont expansion. Reduction in diversity and quantity of microbes in germ-free (GF) animals or by treating mice with antibiotics reveals increased susceptibility to a variety of infections, such as C. difficile [6], Shigella flexneri [7] and Salmonella enterica serovar Typhimurium [8]. In parallel, many studies have shown that microbiota

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transplantation is capable of rendering resistant animals into susceptible to pathogens, or even mimicking some disease phenotype [9,10]. Commensals can resist colonization by competing for nutritional requirements. Several microbes sharing the same niche present similar nutritional needs; therefore, the consumption of organic acids, amino acids, sugars, and other nutrients by indigenous microbiota can restrain competing pathogens. Bacteroides thetaiotaomicron, for example, limits Citrobacter rodentium by carbohydrate competition [11]. Microbiota-derived bioproducts can also directly control pathogen dissemination. Some short-chain fatty acids (SCFAs) produced during carbohydrate fermentation in the gut, such as butyrate and acetate, can control Salmonella enterica serovar Enteritidis and Typhimurium [12]. The indigenous microbiota secretes antimicrobial peptides, termed bacteriocins, which limit microorganisms of the same species or related ones. Some strains of commensal Escherichia coli are able to produce bacteriocins, which directly inhibit pathogenic enterohemorrhagic Escherichia coli (EHEC) [13] (Fig 13.1). Reduction in oxygen tension [14] and pH alteration [15], are other known mechanisms developed by commensal bacteria to turn the environment more hostile to certain pathogens. Besides, the metabolism of bile acids by microbes plays an important role in colonization resistance to C. difficile [16].

FIGURE 13.1 Microbiotaehost homeostasis. Products of bacterial metabolism, such as short-chain fatty acids (SCFAs) and secondary bile acids act synergistically to avoid gut colonization through pH alteration, reduction in oxygen tension, virulence inhibition, and direct killing of some pathogens. The secretion of inhibitory substances such as bacteriocins by some commensals may target and inhibit pathobionts and pathogens. Indigenous microbiota facilitates host barrier maintenance and plays a central role in immune system maturation. Commensals, such as segmented filamentous bacteria (SFB) and various clostridia, can induce accumulation of Th17 and Treg, respectively. Secretion of antimicrobial peptides, such as RegIII, upregulation of mucus, and IgA release can also contribute to promote gut colonization resistance.

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Despite all the strategies mentioned above, some pathogens developed evasion mechanisms that allow their maintenance in the host gut. C. difficile and S. typhimurium, for instance, exploit the metabolization of certain sugars produced by indigenous microbes, such as B. thetaiotaomicron, as energy source for their survival [17]. Other bacteria, such as EHEC, can sense the environment and change their metabolism to avoid commensal nutrient competition [18,19]. A study evaluating the colonization resistance to EHEC demonstrated that animals previously colonized with three strains of commensal E. coli, with different nutritional requirements are resistant to EHEC colonization, but when only one of the strains is inoculated the animals become susceptible [20].

METABOLITES AND PATHWAYS Microbiota can produce a large amount of small bioactive molecules during the metabolism of diet, which are joined by endogenous metabolites, mucosal-derived macromolecules, and xenobiotics. Once in the bloodstream, these molecules link the microbiota to the rest of the human body. The metabolic axis host-microbe varies along the intestine, and the synthesized metabolites play a role in both host physiology and microbiota homeostasis, such as SCFAs, aromatic amino acids derivatives, choline, and bile acids.

Short-Chain Fatty Acids Complex carbohydrates and, less extensively, amino acid fermentation by colonic microbes may yield SCFAs. Acetate, butyrate, and propionate are the most abundant SCFAs in the gut, and are sensed by G protein-coupled receptors, such as GPR41 and GPR43 [21]. While GPR41 is expressed by many different cells, GPR43 is observed in immune cells. Apart from being used as energy source to colonocytes, SCFAs regulate host energy utilization, control gut pH, and play an important role in maintaining intestinal barrier integrity [22,23].

Primary and Secondary Bile Acids Bile acids, in turn, are mainly metabolized by Lactobacillus spp., Bifidobacteria spp., Enterobacter spp., Bacteroides spp., and Clostridium spp. [24]. Bile acids are synthesized in the liver and before being released into intestine, they are conjugated to enhance their surfactant activity. Most of these conjugated bile acids are reabsorbed in the ileum and go back to the liver; however, a small amount remains in the intestine, and is metabolized into secondary bile acids by gut microbes [25]. Deoxycholate and lithocholate are the main secondary bile acids, and they activate the nuclear farnesoid X receptor (FXR) that is expressed in intestinal and hepatic cells [26]. While modulating host lipid absorption and their own metabolism [27], bile acids can disrupt bacterial membrane [28], induce DNA damage [29], alter protein conformation [30], chelate iron [31] and regulate the expression of bacterial virulence factors [32] (Fig 13.1).

Amino Acid and Choline Metabolites The catabolism of aromatic amino acids such as tryptophan by microorganisms of the intestinal microbiota generates mainly tryptamine and indole-3-acetate [33]. In intestinal epithelial cells (IECs), these molecules are capable of regulating the production of cytokines, antimicrobial peptides, and the development of regulatory T (Treg) cells, by signaling through the aryl hydrocarbon receptor(AhR) [34]. The metabolization of choline by different microbes generates trimethylamine, which is an important modulator of lipid metabolism and glucose homeostasis [35]. Changes in the composition of the microbiota, which in turn lead to the increase in choline metabolism, have been associated with cardiovascular and liver diseases.

COMMENSALS AND MUCOSAL IMMUNITY: THE CROSSTALK AGAINST PATHOGENS The commensal microbiota is an important source of nutrients and vitamins, enables the digestion of complex polysaccharides, competes with pathogens for the same niche, and shapes the host immune system. The mechanisms by which the microbiota contributes to the development of the immune system include: (1) direct contact with host cells and (2) generation of molecules either through de novo synthesis, or through modification of food components or their metabolites [36].

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Mucosal Anatomy Among the four layers that compose the intestine (the mucosa, the submucosa, the muscularis, and the serosa), the mucosa is the nearest stratum to the lumen, consisting of a single layer of epithelial cells underlying a loose connective tissue denominated Lamina Propria (LP), which contains the majority of immune cells. Immune cells are located in organized lymphoid structures (gut-associated lymphoid tissuesdGALT), such as Peyer patches (PPs), cryptopatches (CPs), and isolated lymphoid follicles (ILFs), or diffusely distributed. In mice, the development of CPs and ILFs occurs after birth, in a process influenced by dietary factors. The absence of AhR signaling pathway reduces the ILFs [37]. AhR ligands can be provided by the diet or through tryptophan metabolism by the microbiota [38], demonstrating that the microbiota can impact immune system development. In addition, the recognition of peptidoglycan from Gram-negative bacteria is also required to induce the establishing of ILFs, through binding to NOD1 (nucleotide-binding oligomerization domain containing 1) in IECs. In the small intestine (SI), ILFs generate commensal-reactive Ig-A-producing B cells essential for determining the microbiota diversity, and to prevent bacterial invasion and systemic spread. The maturation of ILFs into B cell clusters demands bacteria detection by toll-like receptors (TLRs) [39]. That way, absence of gut microbiota impacts mucosal immune system, which in turn can reciprocate on microbiota composition.

The Gut as an Immune Organ IECs form a single cell layer that constitutes a physical barrier between the luminal content and the underlying immune cells. The expression of pattern recognition receptors (PRRs), including TLRs, nucleotide-binding domain leucine-rich repeat containing receptors (NLRs), RIG-I like receptors (RLRs), C-type lectin family, and AIM2-like receptors (ALRs) on IECs, allow the proper communication between the environment and the immune compartment. The expression and localization of TLRs and the expression of antimicrobial proteins by IECs are also influenced by bacterial colonization. It has been shown that GF mice are deficient in defensins, while Gram-negative commensal microorganism B. thetaiotaomicron induces the secretion of the antimicrobial TLR-related protein RegIIIg [40,41]. Collectively, those observations regarding tissues and cellular defects on GF mice support the idea that “normal” immune function demands the presence of the gut microbiota. GPR43, also known as free fatty acid receptor 2 (FFAR2), is a SCFA’s receptor expressed by IECs, monocytes, neutrophils, eosinophils, and lymphocytes, and it has been shown that its stimulation is necessary for the resolution of inflammatory responses. GPR43-deficient mice (Gpr43
/
) showed exacerbation and unresolved inflammation in DSSinduced colitis model due to higher production of inflammatory molecules and increased immune cell infiltration in the gut. GF mice present none or low SCFAs levels, along with increased inflammation and lower resolution of inflammation, compared to colonized mice [42].

Flagellin and Pathogenic Bacteria Pro-IL1b is the immature precursor of the proinflammatory cytokine IL-1b, produced by intestinal mononuclear phagocytes (iMPs) (macrophages and dendritic cellsdDCs). The conversion of pro-IL1b into your active form requires the activation of caspase-1 via NLCR4 inflammasome, which is induced by the bacteria protein flagellin. The enteric pathogens Pseudomonas aeruginosa and S. Typhimurium express type III secretion system, and then introduce flagellin into the host cytosol, inducing the production of IL-1b. Commensal bacteria do not express type III secretion system, and in this way, do not induce the production of IL-1b. However, it has been shown that in steady state iMPs express pro-IL1b protein [43], indicating a mechanism by which pathogenic bacteria can be discriminated from commensals.

T-Cell Subsets The microbiota also stimulates adaptive immunity, by inducing the differentiation of CD4þ T cells. The gut CD4þ T cells compartment includes four main T-cell subsets: Foxp3þ regulatory T cells (Treg), T-helper 1 (Th1), Th2, and Th17 cells, and it is believed that the distribution of the different subsets is a dynamic process, guided by microbiota composition [44]. Th17 cells play an important role in maintaining the epithelial barrier, and in host immune defense against extracellular pathogens of the mucosa surface. GF mice lack Th17 cells in the SI and colon LP but can acquire them after colonization with conventional microbiota. Although not all members of the microbiota can influence the differentiation of Th17 cells, the presence of segmented filamentous bacteria (SFB), typical of rodents and important for the immune system, increases the frequency of this subset. Moreover, the colonization with SFB along with Th17 differentiation are thought to be of

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homeostatic nature, and correlates with increased expression of genes associated with inflammation and antimicrobial defenses, resulting in enhanced resistance to the intestinal pathogen C. rodentium [45].

T Regulatory Cells (Treg) Higher proportions of Foxp3þ Treg cells are found in the small and large intestinal LP. This subset can arise from the thymus, can be natural (nTreg), or can be peripherally induced (iTreg), being further subdivided based on the expression of RORgt transcription factor [46]. Several studies have demonstrated induction of iTreg following microbial colonization. The colonization of GF mice with a consortium of eight murine bacteria species (altered Schaedler floradASF), was able to promote the differentiation of iTreg in the colon, but not in the SI [47]. Clostridia members, notably, clusters IV and XIVa, have been described as efficient commensals for the induction of iTregs [48]. However, the precise role and mechanisms by which microbial antigens and metabolites regulate the differentiation of Foxp3þ Tregs subsets need to be better elucidated.

Bacteriocins Lactic acid bacteria can produce antimicrobial peptides known as bacteriocins, with the ability to antagonize the growth of harmful microorganisms, and protect the host from infectious diseases (Fig 13.1) [49].

GUT MICROBIOTA MODULATION: A FOCUS ON THERAPIES Some therapeutic benefits after use of probiotics, prebiotics, and synbiotics are described in Table 13.1. More recently, a new approach has also been highlighted in clinical trials, the Fecal Microbiota Transplantation (FMT), questing to transfer the whole microbial and substrate content from healthy intestine to sick patients [50].

Antibiotics Antibiotics are dysbiosis inducers, commonly used to eliminate or decrease pathogenic strains in bacterial-mediated diseases. Although bactericidal and bacteriostatic antibiotics are heavily used in clinical practice, the commercially available drugs persist with low target specificity, and several beneficial strains of bacteria also decrease after their use. In addition, the overuse and misuse of antibiotics accelerate the process of resistance and treatment failure in primary care [51]. Although bacteria have resilience properties, after long periods of antibiotic treatment the intestinal microbiota becomes slightly different from that found before therapy [52]. Moreover, the use of antibiotics changes the gene expression and metabolism of the remaining bacteria, and consequently alters several biological processes of the host (e.g., immune responses, susceptibility to other infections, and gut permeability) [53]. For instance, microbiota depletion impairs the course of IBD by decreasing the availability of bacterial-derived TLR ligands in the gut, which in turn, reduce the immunological surveillance and intestinal repair [54]. Although antibiotics are highly recommended in many cases, alternative therapies should be investigated to improve the outcome of the diseases without impairing the maintenance of host homeostasis.

Probiotics Most microbial species used in probiotic formulations are Lactobacillus (e.g., reuteri, rhamnosus, casei, acidophilus, gasseri) and Bifidobacterium (e.g., bifidum, animalis, breve, lactis), but also others such as Streptococcus spp., Lactococcus spp., Escherichia coli (strain Nissle), and Clostridium ssp. The yeast Saccharomyces boulardii is also commonly used as probiotic [55,56]. Probiotic strains control pathogens through production of antimicrobial substances (e.g., SCFAs, hydroperoxides, bacteriocins, and bile acids), competition for adhesion to the gut epithelium and for nutrients, and inhibition of bacterial toxin synthesis. The soluble mediators produced by probiotic strains and the release of cellular components of pathogenic bacteria (cellular wall components, DNA) increase the immune responses by enhancing immunoglobulin production, macrophage and lymphocyte activity, and production of proinflammatory cytokines [55]. For instance, Lactobacillus strains were described as early inducers of TNF-a (tumor necrosis factor a), IL-1b, IL-6, IL-8, and IL-12p70, and they also increase the bactericidal and phagocytosis capacity of human macrophages [57]. The use of probiotics by mothers during or after pregnancy also changes the microbiota composition of fetus and newborns, respectively [58,59], expanding the potential use of probiotics to modulate the dysbiotic microbiota before birth.

TABLE 13.1 Microbiota Modulation: Prebiotics, Probiotics, and Synbiotics Composition/Dosage

Duration

Status/Disease

Outcome

Probiotic1

L. casei (108 CFU/mL)

12 months

Allergic asthma and/or rhinitis

Decreased number of rhinitis episodes

8 weeks

Ulcerative colitis, proctitis

Clinical remission

2

B. longum 536 (1e3  10

3

L. delbrueckiisubsp. Bulgaricus (NI), S. thermophiulus (NI), B. animalis subsp. Lactis (1,11  107), L. acidophilus (1,11  107)

8 weeks

Type 2 diabetes

Decreased HbA1c and TNF-a levels

Probiotic Probiotic

Probiotic4

11

CFU)

L. rhamnosus GR-1 (109) and L. reuteri RC-14 (109)

4 weeks

Vulvovaginal candidiasis

Decreased number of yeast and symptoms

5

Escherichia coli Nissle 1917 (2.5e25  10 CFU)

12 weeks

Irritable bowel syndrome

Improved clinical signs

6

Prebiotic

FOS (15 g)

3 weeks

Crohn disease

Increased Bifidobacteria ssp., IL-10þTLR2þTLR4þ DCs, decreased Crohn’s disease index score

Prebiotic7

Probiotic

9

GOS (3.5e7 g)

12 weeks

Irritable bowel syndrome

Increased Bifidobacteria and decreased symptoms

8

Inulin (20 g)

3 weeks

Hypercholesterolemia

Decreased triglycerides and cholesterol levels

9

Prebiotic

GOS/FOS (9:1 mix ratio; 0.4 g/100 mL)

12 months

Intestinal and respiratory infection

Decreased number of infectious cases

Synbiotic10

L. salivarius (2.109 CFU), FOS (10 g)

6 weeks

Healthy young individuals

Decreased E. coli, lipids, cytokines (TNF-a, IL-1b, IL6), increased lactobacilli

Synbiotic11

L. acidophilus (2  109), L. casei (7  109), L. rhamnosus (1.5  109); L. bulgaricus (2  108), B. breve (2  1010), B. longum (7  109) S. thermophilus (1.5  109), FOS (100 mg)

8 weeks

Type 2 diabetes

Decreased fasting plasma glucose levels

Synbiotic12

B. longum (2  1011), FOS/Inulin(6 g, 1:1)

4 weeks

Ulcerative colitis

Reduction of inflammation

L. acidophilus (7  10 ), B. longum (4  10 ), B. infantis (3  108), L. rhamnosus (4  108), L. plantaris (3  108), L. casei (3  108), L. bulgaricus (3  108), B. breve (3  108), FOS (100 mg)

1 week

Necrotizing enterocolitis, sepsis, mortality

Reduced necrotizing enterocolitis severity

L. paracasei ssp. paracasei (108/100 mL), GOS (540 mg), FOS (61 mg)

6 months

Respiratory tract infections

Decreased number of infections

Prebiotic

Synbiotic

13

Synbiotic14

8

8

B., Bifidobacterium; DCs, dendritic cells; E., Escherichia; FOS, fructooligosaccharides; GOS, galactooligosaccharides; HbA1c, glycated hemoglobin A1c; L., Lactobacillus; NI, not informed; S., Streptococcus; TLR, Toll-like receptors. 1 Giovannini M, et al. A randomized prospective double blind controlled trial on effects of long-term consumption of fermented milk containing Lactobacillus casei in pre-school children with allergic asthma and/or rhinitis. Pediatric Res 2007;62:215. 2 Tamaki H, et al. Efficacy of probiotic treatment with Bifidobacterium longum 536 for induction of remission in active ulcerative colitis: a randomized, double-blinded, placebo-controlled multicenter trial. Dig Endosc 2016;28(1):67e74. 3 Mazloom Z, Yousefinejad A, Dabbaghmanesh MH. Effect of probiotics on lipid profile, glycemic control, insulin action, oxidative stress, and inflammatory markers in patients with type 2 diabetes: a clinical trial. Iran J Med Sci 2013;38(1):38e43. 4 Martinez RC, et al. Improved treatment of vulvovaginal candidiasis with fluconazole plus probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14. Lett Appl Microbiol 2009;48(3):269e74. 5 Kruis W, et al. A double-blind placebo-controlled trial to study therapeutic effects of probiotic Escherichia coli Nissle 1917 in subgroups of patients with irritable bowel syndrome. Int J Colorectal Dis 2012;27(4):467e74. 6 Lindsay JO, et al. Clinical, microbiological, and immunological effects of fructooligosaccharide in patients with Crohn disease. Gut 2006;55(3):348e55. 7 Silk DB, et al. Clinical trial: the effects of a trans-galactooligosaccharide prebiotic on fecal microbiota and symptoms in irritable bowel syndrome. Aliment Pharmacol Ther 2009;29(5):508e18. 8 Causey JL, et al. Effects of dietary inulin on serum lipids, blood glucose, and the gastrointestinal environment in hypercholesterolemic men. Nutr Res 2000;20(2):191e201. 9 Bruzzese E, et al. A formula containing galacto- and fructooligosaccharides prevents intestina and extra-intestinal infections: an observational study. Clin Nutr 2009;28(2):156e61. 10 Rajkumar H, et al. Effect of Probiotic Lactobacillus salivarius UBL S22 and prebiotic fructo-oligosaccharide on serum lipids, inflammatory markers, insulin sensitivity, and gut bacteria in healthy young volunteers: a randomized controlled single-blind pilot study. J Cardiovasc Pharmacol Ther 2015;20(3):289e98. 11 Asemi Z, et al. Effect of multispecies probiotic supplements on metabolic profiles, hs-CRP, and oxidative stress in patients with type 2 diabetes. Ann Nutr Metab 2013:63(1e2):1e9. 12 Furrie E, et al. Synbiotic therapy (Bifidobacterium longum Synergy 1) initiates resolution of inflammation in patients with active ulcerative colitis: a randomized controlled pilot trial. Gut 2005;54(2):242e9. 13 Nandhini LP, et al. Synbiotics for decreasing incidence of necrotizing enterocolitis among preterm neonatesda randomized controlled trial. J Matern Fetal Neonatal Med 2016;29(5):821e5. 14 Szajewska H, et al. Effects of infant formula supplemented with prebiotics compared with synbiotics on growth up to the age of 12 mo: a randomized controlled trial. Pediatr Res 2017;81(5):752e8.

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Classification/ Ref

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Prebiotics Examples of prebiotics include the fructans (fructooligosaccharidesd FOSd and inulin) and galactans (galactooligosaccharidesdGOS). However, the carbohydrates are potential sources to be used as prebiotics. These bioactive compounds mainly act on Lactobacillus spp. and/or Bifidobacterium spp., but also on Roseburia spp., Eubacterium spp., and Faecalibacterium spp. Inulin increases the proportion of the genus Bifidobacteria, and has been shown to improve both obesity- and diabetesrelated complications [60]. On the other hand, both FOS and GOS support the growth of species of Bacteroides, Lactobacilli, Enterobacteria, Streptococci, and Bifidobacteria, while decreasing some strains of Bacteroides and Candida, and they have showed good potential to treat both Listeria monocytogenes and S. typhimurium infections, with some actions on colorectal cancer [61,62]. In addition to changing microbiota composition, prebiotics enhance digestion processing, synthesis of beneficial bioactive and antibiotic compounds, increase mineral bioavailability, and enhance the viability of epithelial cells in the colon by increasing SCFAs production [63,64]. GOS- and FOS-induced SCFAs have been shown to increase mucin production by intestinal epithelial cells, number of leukocytes in GALT and blood, IgA secretion and macrophage phagocytic activity [55,65]. Moreover, the use of FOS alone was sufficient to decrease Crohn Disease Activity Index in sick patients [66].

Synbiotics Synbiotics increase good bacterial strains (e.g., Bifidobacterium spp. and Lactobacillus spp.) and production of bioactive compounds (e.g., SCFAs) [67]. They were created to increase probiotic survival and function, since the gastrointestinal tract can be a quite hostile environment. The use of synbiotics has been promising by decreasing the number of infections in postoperative patients, improving the outcome of IBD, and presenting anticancer effects [68]. The prophylactic use of a pharmaceutical formulation containing oligofructose-enriched inulin, Lactobacillus rhamnosus, and Bifidobacterium lactis Bb12, decreased cancer pathogenicity in colon cancer and polypectomized patients [69].

OTHER REPERCUSSIONS OF MUCOSAL IMMUNITY About 10% of the host transcriptome is microbially regulated, especially genes involved in immunity, cell proliferation, and metabolism [70]. The colonization of GF mice with a single commensal bacterium resulted in increased transcription of genes involved in several intestinal functions, including nutrient absorption, mucosal barrier functionality, xenobiotic metabolism, angiogenesis, and postnatal intestinal maturation [71]. Gut microbes and their products can be recognized by a variety of receptors, including TLRs and NLRs. The pattern recognition of microbial molecules by the gut mucosal immunity regulates intestinal homeostasis and influences systemic immunity and the host metabolism. PRRs in the intestinal mucosa are not only involved in the elimination of pathogens but are also involved in host metabolic homeostasis. A variety of studies using global knockout models of TLRs, including TLR2 and TLR5, observed an increase in body weight or fat mass [72,73]. Surprisingly, the phenotypes in cell-specific knockout models are rather diverse. Loss of TLR5 from intestinal epithelial cells leads to altered gut microbiota, lowgrade inflammation, and metabolic syndrome features [74]. The lack of TLR5 would allow intestinal epithelium breaching by bacteria, compromising the efficient recruitment of immune cells. On the other hand, the deletion of the signaling adaptor molecule of TLRs in IECs, Myd88 (Myeloid differentiation primary response 88), reduces fat mass accumulation and body weight and improves glucose metabolism [75]. The gut microbiota plays a critical role in arthritis. An elegant study revealed that intestinal leukocytes migrate to and from the intestine in steady state [76]. In addition, the fraction of gut-derived Th17 cells present in the spleen correlated with serum levels of autoantibodies, in a model of rheumatoid arthritis. Other studies found high levels of Th17 cells, and decreased levels of Tregs in blood of patients with active rheumatoid arthritis [77,78]. Since Th17 differentiation in intestinal mucosa is dependent on specific intestinal microbiota, exposure to disturbed gut microbiota may be critical in rheumatoid arthritis. Increasing data also reveal that microbiotaehost interactions drive brain development, function, and behavior. GF mice exhibit profound behavioral alterations and neuropathologies including psychiatric, neurodevelopmental, and neurodegenerative disorders [79]. Maturation of nervous system appears to be highly dependent on microbial colonization, during a specific time frame of development. GF mice show an exaggerated hypothalamic pituitary adrenal (HPA) stress response, which is normalized following monocolonization at 6 weeks, but not at 14 weeks [80].

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Besides bacterial products and release of gut hormones, microbiota has access to the brain via an interconnection among primary afferent neurons, immune cells, and enteroendocrine cells. Subsets of vagal afferent nerve terminals, closely located to mucosal immune cells, are responsive to signaling molecules from these cells such as mast cells products [81]. Immune cell products can also modulate enteroendocrine cell functions, including cholecystokinin secretion [82].

BACTERIAL TRANSLOCATION: JUST A THEORETICAL POSSIBILITY OR A REAL CAUSE OF SYSTEMIC INFECTION The intestinal barrier is a complex and dynamic system composed by several lines of defense against pathogens, including physical, chemical, and functional immunological barriers. In order to have access to the systemic circulation, microbes would have to pass through the luminal mucus, IgA, and antimicrobial defensins. Secondly, the epithelial barrier represents a major obstacle against invaders. Thirdly, the GIT is a major reservoir of macrophages, highly specialized in recognition and phagocytosis [83,84]. Other structures, such as blood vessels, smooth muscle layers, and the enteric nervous system, participate in the regulation of intestinal barrier. An additional clearance can be performed by the liver, where sinusoidal endothelial cells and Kupffer cells are responsive to direct stimulation by bacterial antigens [85]. Finally, in healthy conditions, circulating factors, peripheral blood monocytes, and tissue macrophages can neutralize microbial products. The intestinal physical barrier is essentially composed by the mucus layer and the epithelial monolayer, which confers a selective permeability to the intestinal mucosa. A series of intercellular junctions seals the paracellular space, regulating the transmucosal flux [86]. Enteric pathogens can disrupt tight junctions by either altering cellular cytoskeleton, or by affecting tight junction proteins, via bacterial-derived proteases or biochemical alterations [87]. In addition, proinflammatory cytokines can also increase intestinal permeability. Preclinical studies revealed that TNF-a signaling directly regulates tight junction functions. TNF-a increases intestinal permeability by both stimulating myosin light chain phosphorylation [88] and causing occludin endocytosis [89]. In case of tight junction disruption or direct epithelial cell damage, luminal content can cross the intestinal barrier in an unrestricted pathway. Bacterial translocation (BT) is defined as translocation of bacteria and/or bacterial products (lipopolysaccharides [LPS], peptidoglycans, muramyl-dipeptides, and bacterial DNA) through the gut mucosa to normally sterile tissues such as the mesenteric lymph nodes and the internal organs [90]. BT may be a physiological phenomenon that occurs in healthy individuals without deleterious consequences [91]. It has been suggested that indigenous bacteria are continuously translocating in low number from the GIT, and they would be eliminated by the host reticuloendothelial system [92]. In nonhomeostatic conditions such as immunosuppression, bacterial overgrowth, and intestinal hypomotility, intestinal permeability may be compromised, resulting in a continuous process of BT, ultimately triggering long-term inflammatory process [93]. Dietary factors may also impair intestinal permeability, by altering tight junction proteins. High-fat diet reduced expression of claudin-1, claudin-3, occludin, and junctional adhesion molecule-1 in the intestinal mucosa [94]. Individuals with IBD have increased systemic levels of proinflammatory cytokines, which has been suggested to be due to elevated levels of LPS [95], bacterial DNA [96], endotoxin core antibodies [97] and LPS-binding protein [98]. A variety of intestinal and extraintestinal disorders have been associated with altered intestinal barrier and increased intestinal permeability, including IBS, celiac disease, allergies, arthritis, and metabolic diseases [99]. The majority of these associations were merely correlative, and there is little evidence relating barrier impairment to disease pathogenesis. Most in vivo studies involve assessment of the paracellular pathway, and when increased permeability is discovered, extrapolations to bacterial translocation are made [100]. Furthermore, animal studies have revealed that physiologic breaks in the barrier must occur, for homeostatic regulatory T-cell responses, necessary for mucosa protection from inflammation [101].

LIST OF ACRONYMS AND ABBREVIATIONS AhR Aryl hydrocarbon receptor ALR AIM2-like receptor ASF Altered Schaedler flora BT Bacterial translocation CFU Colony-forming unit CP Cryptopatch DC Dendritic cell

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DNA Deoxyribonucleic acid DSS Dextran sulfate sodium EHEC Enterohemorrhagic Escherichia coli FFAR2 Free fatty acid receptor 2 FOS Fructooligosaccharide FXR Farnesoid X receptor GALT Gut-associated lymphoid tissue GF Germ free GIT Gastrointestinal tract GOS galactooligosaccharide GPR41 G-protein-coupled receptor 41 GPR43 G-protein-coupled receptor 43 HPA Hypothalamic pituitary adrenal IBD Inflammatory Bowel Disease IBS Inflammatory intestinal syndrome IEC Intestinal epithelial cell Ig Immunoglobulin ILF Isolated lymphoid follicle iMP Intestinal mononuclear phagocyte iTreg Induced Treg LP Lamina propria LPS lipopolysaccharides MyD88 Myeloid differentiation primary response 88 NLR Nucleotide-binding domain leucine-rich repeat containing receptor NLRC4 NLR family CARD domain-containing protein 4 NOD1 Nucleotide-binding oligomerization domain containing 1 nTreg Natural Treg PP Peyer patch PRR Pattern recognition receptor RLR RIG-I like receptor RORgt RAR-related orphan receptor gamma t SCFA Short-chain fatty acid SFB Segmented filamentous bacterium SI Small intestine TGF-a Transforming growth factor-beta Th T-helper cell TLR Toll-like receptor TNF-a Tumor necrosis factor a Treg Regulatory T cell WHO World Health Organization

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