**5.1 Impact of diet on gut microbiota**

Diet has a profound influence also on the gut microbiota, acting both as a modulator able to select specific microbial groups, and as a provider of substrates that can be metabolized by the microbiota producing metabolites that impact on host health status, also through interaction with the immune system. Therefore, there is a close

connection between diet, gut microbiota, and immune system, orchestrated by a fine tuning of the complex mechanisms underlying this cross-talk. The influence of diet in modulating gut microbiota composition is related to the concept of "enterotype." Indeed, although a wide inter-individual variability is observed among the bacterial groups present in the gut, the microbiota of most individuals can be classified into one of three variants or enterotypes, based on the dominant genera (*Bacteroides*, *Prevotella*, or *Ruminococcus*), which constitutes a relatively stable "core" [3, 26]. These enterotypes are associated with long-term dietary regimens [42]. In particular, enterotype 1, characterized by a predominance of the genus *Bacteroides*, able to extract the maximum energy from the fermentation of carbohydrates and proteins and to produce high amounts of vitamins B2 (riboflavin), B7 (biotin), and ascorbic acid (vitamin C), is associated with a diet rich in animal proteins and fats and low in fiber and vegetables, typical of the "Western Diet" profile. This enterotype may be related to increased intestinal inflammation and consequently to an increased state of general inflammation. Enterotype 2, dominated by the *Prevotella* genus, able to degrade complex polysaccharides and to produce high levels of vitamin B1 (thiamine) and vitamin B9 (folic acid), is instead correlated with a diet profile rich in fiber and carbohydrates. Finally, enterotype 3 is characterized by a predominance of bacteria of the genus *Ruminococcus* and is associated with a dietary profile rich in simple sugars [43]. Although most published papers demonstrate how long-term dietary regimen affects the structure and activity of the gut microbiota, there is still evidence suggesting the ability of the microbiota to respond to short-term dietary change in terms of macronutrients. For example, short-term consumption of diets composed entirely of animal or plant products has been shown to alter the structure of the gut bacterial community, minimizing inter-individual differences. Specifically, the most pronounced effect has been found for diets based on animal products, resulting in increased levels of bile-tolerant microorganisms (*Alistipes*, *Bilophila*, and *Bacteroides*), and decreased levels of Firmicutes capable of metabolizing plant polysaccharides (*Roseburia*, *Eubacterium rectale,* and *Ruminococcus bromii*) [44].

### *5.1.1 Effect of macronutrients*

Among macronutrients, the effect of carbohydrates on the microbiota is the most described, while for proteins and lipids the mechanisms are less defined. Micronutrient intake is also critical for gut well-being; in fact, vitamin deficiencies have been associated with alterations in barrier function and GALT immune response. However, it is important to emphasize that modifications to the immune system and microbiota are primarily associated with the composition of the diet as a whole, and not with specific foods or nutrients [4]. Many complex carbohydrates are known to act as prebiotics, selectively stimulating, in the intestine, the growth of microorganisms beneficial to human health, such as bifidobacteria. Dietary fiber is a heterogeneous and complex mixture of different combinations of monosaccharides, with a minimum of 10 monomeric units or oligosaccharides containing from 3 to 9 monomeric units. A further classification of dietary fiber is related to its water solubility, viscosity, and fermentability. Polysaccharides are further categorized in non-starch polysaccharides and resistant starch, while oligosaccharides include resistant oligosaccharides. Soluble fiber is typically fermented to SCFA by the intestinal microbiota. A growing body of literature shows that dietary fiber has the potential to change the gut microbiota and alter metabolic regulation in humans. Most findings supporting the fiber hypothesis are based on short-term dietary interventions, while only sparse data

### *Immune System, Gut Microbiota and Diet: An Interesting and Emerging Trialogue DOI: http://dx.doi.org/10.5772/intechopen.104121*

evaluating the impact of long-term dietary fiber on the gut microbiome exist. Specific sources of dietary fiber were differentially associated with the gut microbiome. Fiber from fruit and vegetable intake was related to the gut microbiome composition, characterized by an increased abundance of Clostridia, an important class of dietary fiber fermenters producing SCFA. Other evidence showed an association between legume fiber intake and Actinobacteria abundance, particularly Bifidobacteriales [45]. A recent systematic review demonstrated that the most consistent results can be related to an increased abundance of SCFA-producers, alterations in microbiota diversity, and in the *Prevotella*/*Bacteroides* ratio. However, to what extent a dietary intervention with fiber may affect the human gut microbiota and hence metabolic regulation is currently not well described, due to differences in methodologies and lack of standardization that hamper the interpretation of the results [46]. It is known that also proteins can shape gut microbiome, and that different protein sources differently impact its profile. As an example, a diet rich in pea protein has been shown to increase *Bifidobacterium* and *Lactobacillu*s levels [47]. Approximately 12–18 g of dietary protein reaches the human colon daily. Several gut microbiota species such as *Clostridum* spp., *Bacteroides* spp., and *Lactobacillus* spp. can metabolize proteins through different proteases [48]. Microbial metabolites deriving from dietary protein fermentation by gut microbiota include short branched chain fatty acids, sulfur-containing products, aromatic compounds, polyamines, and ammonia. Interestingly, several neuroactive compounds including neurotransmitters such as GABA, norepinephrine, dopamine, serotonin, and histamine are produced from amino acids by gut microbiota, and this is one of the most attractive topics to understand the role of microbiota in gut-brain axis [48]. On the other hand, the pro-atherogenic metabolite trimethylamine-Noxide (TMAO) is produced by the combined activity of microbial and host enzymes after consumption of animal proteins, with a negative impact on health. Most of the ingested fatty acids are absorbed in the human small intestine, but a small fraction (about 7%) reaches the colon. With respect to carbohydrates and protein, the impact of dietary fats on gut microbiota profile is less reported. The most characterized effect of a high-fat diet is related to a decreased Bacteroidetes/Firmicutes ratio [49].

### **5.2 Effect of microbial metabolites on the host immune system**

Most of the physiological effects of the microbiota are mediated by metabolites produced by the bacteria themselves or derived from the microbial transformation of host molecules. In fact, the gut microbiota has a high potential to synthesize bioactive compounds by acting on molecules of endogenous origin or derived from the diet. As previously mentioned, SCFAs are the principal metabolites derived from the microbial fermentation of complex polysaccharides. Acetate and propionate are mostly produced by Bacteroidetes, while Firmicutes are the principal butyrate-producing microorganisms [50]. While propionate and acetate reach the liver through the portal vein, where they contribute to gluconeogenesis and lipogenesis, respectively, butyrate, mainly produced by Firmicutes, plays a fundamental role in the intestine and represents the major fuel for enterocytes. SCFAs, especially butyrate, are molecules fully capable of transducing signals, as they are ligands of G-Protein Coupled Receptors (GPCRs). This interaction activates various molecular signaling pathways in the different intestinal cells, resulting in strengthening the intestinal barrier and exerting an anti-inflammatory action. In particular, Paneth cells are stimulated to release antimicrobial substances; intestinal endocrine L cells release satiety peptides, glucagon-like-1 (GLP-1) and peptide YY (PYY); goblet cells are stimulated to produce mucin, while in epithelial cells butyrate exerts a trophic effect, promoting the expression of junction proteins and cell regeneration. SCFAs also have important actions on both innate and adaptive immune cells present in the intestine, increasing IL-10 expression levels and promoting Treg cell differentiation. SCFAs are also epigenetic modulators, as they act as inhibitors of histone deacetylase enzymes, resulting in transcriptional activation of several genes, including a Treg cell-specific transcription factor, Foxp3, that leads to an anti-inflammatory phenotype, through inhibition of NF-κB. In other contexts, however, it has been observed that SCFAs may have opposite, pro-inflammatory effects, especially in the presence of LPS or TNF-α. This observation demonstrates how the same molecule can have beneficial or detrimental effects, depending on the concurrent conditions of eubiosis or dysbiosis [4, 51]. The microbiota plays an essential role also in the metabolism of bile acids, influencing their profile with over 20 different secondary bile acids produced. Such diversity of bile acids composition differently affects the physiology and metabolism of the entire body*.* Cholesterol-derived primary bile acids, essentially cholic and chenodeoxycholic acid, are first conjugated with taurine and glycine in the liver to form the corresponding conjugated bile salts which are stored in gallbladder. Released into the duodenum after an abundant meal, most bile salts (95%) are reabsorbed from the terminal ileum and colon and delivered back to the liver via the portal vein in a process known as enterohepatic circulation [52]. A small percentage of bile salts, estimated at around 5%, reaches the colon, where they are deconjugated in a reaction catalyzed by bile salt hydrolase (BSH), and mediated by a broad spectrum of aerobic and anaerobic bacteria (Gram-positive *Bifidobacterium*, *Lactobacillus*, *Clostridium*, and *Enterococcus*, and Gram-negative *Bacteroides*). Then bacterial dehydrogenase enzymes convert primary bile acids into the secondary bile acids deoxycholic and lithocholic acids. This reaction is mediated by a limited number of bacteria belonging to B*acteroides, Clostridium, Eubacterium, Lactobacillus*, and *Escherichia* genera [53]. Thus, gut microbiota composition determines the profile of secondary bile acids that are produced. The secondary bile acids are absorbed into the colon, return to the liver and after being conjugated enter the enterohepatic circulation. Secondary bile acids can undergo epimerization, sulfation, glucuronidation, and conjugation with N-acetylglucosamine in the liver, kidneys, and gut to form tertiary bile acids [52]. Bile acids exert multiple physiological functions, which are: 1 - intestinal detergent activity that solubilizes dietary lipids and fat-soluble vitamins promoting their absorption; 2 - hormone- like properties by acting as signaling molecules via two independent pathways, farnesoid X receptor (FXR) and G protein-coupled bile acid receptor (TGR5) signaling. Binding FXR, bile acids can regulate their homeostasis, as well as lipogenesis, gluconeogenesis, tumor suppression, and intestinal barrier function; while through TGR5, they regulate glucose homeostasis, energy expenditure, and anti-inflammatory response. Different bile acids have different affinities towards these receptors, with secondary bile acids preferentially activating TGR5; 3- antibacterial properties providing protection against invasive microorganisms, and acting as mediators of gut innate defense. However, it is important to note that bile acids can become cytotoxic at high concentrations, and excessive accumulation can lead to oxidative stress, apoptosis, and liver damage [54]. In this context, any dietary component, which could influence gut microbiota composition, may also modulate bile acid homeostasis and the ability to impact host health. High dietary fat intake is known to increase primary bile acids release into the small intestine and stimulate secondary bile acid synthesis mediated by various bacteria, including *Lactobacillus*, *Bifidobacterium,* and *Bacteroides* [55]. Milk fat has been shown to induce shifts in hepatic conjugation of bile acids in mice,
