Abstract
The present chapter provides a comprehensive overview of the multifaceted links connecting the immune system, the intestinal microbiota, and the diet, covering also some recent, less explored, and emerging topics such as the “trained immunity” and the immune cell metabolic activity. The main characteristics of the innate and adaptive immune system are described, as well as the gut-associated lymphoid tissue (GALT). Gut microbiota structure and function are also presented. Particular emphasis is given to the diet as a modulator of the microbiota-immune system crosstalk, focusing on the impact of the three main dietary components (carbohydrates, proteins, and fats) and the different dietary profiles on the gut microbiota, by shaping its composition and the deriving microbial metabolites that influence host health, also through interaction with the immune system. Western and Mediterranean diets are described and chosen as representative models of detrimental and beneficial dietary patterns, respectively.
Keywords
- innate immunity
- GALT
- intestinal microbiome
- Western diet
- Mediterranean diet
- metabolic inflammation
1. Introduction
The immune system is fundamental to protect the organism from pathogens and toxic exogenous agents, by discriminating between “self” and “nonself” antigens, and in normal physiological conditions it is programmed to react against “nonself.” At the intestinal level, the gut-associated lymphoid tissue (GALT) is a key component of immune defense, protecting the body from foreign antigens and pathogens, while allowing tolerance to commensal bacteria and dietary antigens [1]. The intestinal microbiota, defined as the complex microbial community residing in the host’s digestive tract, is recognized as an effective integral component of the host immune system, capable of finely tuning both the innate and adaptive immune responses during the entire lifespan. Indeed, the intimate relationship set up between microbiota and immune cells in the intestine is crucial for the maintenance of immunological homeostasis and, mostly, for the “education” of the immune system during the early stages of life [2]. Diet has a strong influence on the gut microbiota, acting both as a modulator able to select specific microbial groups and providing substrates that can be metabolized by the microbiota producing metabolites that impact host health also through interaction with the immune system [3, 4]. 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.
2. The immune system
The immune system is a complex network designed to react against harmful foreign agents as well as pathogens. The immune system is immature during fetal and neonatal life. The fetus receives passive protection from the mother
2.1 Innate immune response
The innate immune response is less specialized and generally less effective than the adaptive one. The cellular mechanisms of innate immunity are characterized by phagocytic and cytotoxic activities, while the humoral component is based on the complement system.
2.1.1 Cellular component
Neutrophil granulocytes are normally found in the bloodstream. During the acute phase of inflammation, they are among the first inflammatory cells migrating from blood vessels to the inflamed site, recruited by chemical signals such as interleukin-8 (IL-8), through a process called chemotaxis. Similarly, monocytes migrate from the bloodstream to tissues in response to chemokine release at infection sites, become activated, and differentiate into macrophages. Macrophages constitute, together with neutrophils, the largest group of cells endowed with phagocytic activity, they carry out their defense action by surrounding foreign microorganisms with pseudopodia, i.e., extroversions of the plasma membrane, forming the phagosome. The phagosome then merges with the numerous granules present in the cytoplasm, containing various compounds toxic for microorganisms, such as defensins, cathelicidins, lysozyme, and lactoferrin, forming the phagolysosome. Alternatively, the so-called respiratory or oxidative burst is activated, resulting in the formation of reactive oxygen species finally producing hypochlorite, hypobromite, and hypoiodite that kill microorganisms. In addition to the phagocytic function, macrophages are also responsible for the processing and presentation of antigens to T cells, as mentioned above [7]. Natural killer (NK) cells eliminate virus-infected and tumor cells through a cytotoxic activity, mediated by perforin-containing granules and granzymes. The former form pores in the plasma membrane and the latter, entering through these pores, induce the caspase cascade, leading to apoptosis of target cells. NK cells can also kill target cells through another mechanism, referred to as antibody-dependent cellular cytotoxicity (ADCC), in which NKs recognize target cells to which IgG has been previously bound [7, 8]. Many cells of the innate immune system are activated through receptors expressed on their membrane, namely pattern recognition receptors (PRR), with a long evolutionary history, which are able to recognize conserved structural patterns expressed by microorganisms, such as the microbe- and pathogen- associated molecular patterns (MAMP and PAMP, respectively). In particular, the type of PRR recognizing MAMP and PAMP is represented by the toll-like receptors (TLRs), a family of transmembrane proteins primarily expressed on the surface of immunocompetent cells, i.e., monocytes, macrophages, and dendritic cells, but also on intestinal epithelial cells. When a TLR recognizes a MAMP, a complex protein signal transduction cascade is triggered generating the appropriate immune response for that microorganism [9]. In most cases, the inflammatory response activated by TLRs leads to the activation of the nuclear factor-kappaB (NF-κB), which induces the transcription of numerous pro-inflammatory genes, including IL-8 [10]. TLRs can also be activated by endogenous danger signals such as the damage-associated molecular patterns (DAMP), molecules that are released in the intracellular or extracellular space following tissue injury, cellular stress, or apoptosis. Some innate responses can activate the inflammasome, a multiprotein complex resident in the cytosol as an inactive form, particularly in macrophages. A 2-hit-theory has been postulated, stating that for inflammasome activation two distinct signals are required. The first signal, triggered by PAMPs or DAMPs, activates the TLR signaling cascade, leading to the expression of some pro-inflammatory cytokines in an inactive form, such as proIL-1β and proIL-18. The second stimulus activates the inflammasome and generates caspase-1. Only thereafter proIL-1β and pro-IL-18 are cleaved by caspase-1 to mature IL-1β and IL-18, which can be secreted by macrophages and promote the inflammatory response [11]. Dendritic cells (DCs) are specialized to “sample” the entry sites of potential infectious agents, so they are found as immature cells in nonlymphoid tissues where antigens can be encountered, such as skin and other mucosal sites. The antigen recognition, through TLRs’ activation, initiates the maturation process of DCs, which are induced to secrete various pro-inflammatory cytokines. After the encountering, the antigen is internalized through phagocytosis or pinocytosis and processed by the DCs, which migrate to secondary lymphoid organs (lymph nodes, spleen), where the exposed antigens are presented to populations of T lymphocytes, both naive and memory cells [6]. Mast cells and basophil granulocytes, similarly to monocytes, circulate in the blood as immature progenitor cells, differentiating into mature cells in different tissues in response to cytokine secretion. Mast cells and basophils are particularly found in association with blood vessels and nerves, in close proximity to mucosal surfaces that interface with the external environment, where they are able to detect infectious agents through TLRs. Upon activation, mast cells and basophils immediately extrude histamine from granules and, within a few minutes, release lipid mediators (such as prostaglandins, leukotrienes, and thromboxane), promoting vascular permeability, vasodilation, and rapid recruitment of eosinophils, neutrophils, and other immune cells [7]. Eosinophils are another type of circulating granulocytes that can be recruited to sites of inflammatory reactions, where their numbers can be 100-fold higher than in the blood. When activated, eosinophils release the contents of their granules (numerous enzymes, major basic protein, eosinophilic cationic protein), which act primarily on extracellular helminthic parasites. Eosinophils also actively participate in allergic diseases [8]. Innate lymphoid cells (ILCs), although lacking antigen-specific receptors, play an important role in the inflammatory response and the maintenance of immune homeostasis, particularly in mucosal tissues. Based on their phenotypic and functional features, ILCs have been grouped into three major subsets. Among them, group 3 ILC (ILC3) are implicated in intestinal homeostasis as they produce IL-22, a key regulator of the intestinal barrier [12]. Recent studies have shown that some myeloid cells of the innate immune system, essentially macrophages and NK cells, can develop a nonspecific immunological memory, i.e., these cells, after a first stimulus, acquire the ability to respond effectively to a subsequent stimulus, different from the first. Effector stimuli for such “innate memory” are represented by various components of bacteria or fungi, such as lipopolysaccharide (LPS) or β-glucans, as well as viruses, and such innate memory is called “trained immunity” [13]. Following activation, the cells involved in this phenomenon undergo processes of chromatin unfolding, which thus becomes more accessible for gene transcription. These processes, globally referred to as “epigenetic reprogramming,” include methylations, acetylations, and phosphorylations at specific chromatin sites. The activation of gene transcription following the first stimulus is therefore accompanied by the acquisition of specific “epigenetic profiles,” which are only partially lost after the elimination of the stimulus. In this way, a kind of nonspecific “memory” is developed, which makes some innate immune cells more easily and rapidly activated, following a subsequent heterologous stimulus. Trained immunity has gained increasing scientific relevance in recent years, for the hypothesis that previous infections can induce a metabolic and epigenetic reprogramming of some cells of innate immunity, leading to an improved defense response during subsequent infections of various types, at the same time trained immunity could also be negatively involved in hyperactivation of the immune system leading to chronic inflammation, as in atherosclerosis [14].
2.1.2 Humoral component
The complement system represents a set of plasma and membrane proteins endowed with enzymatic activity that can result in direct lysis of the foreign agent. These proteins circulate in the blood as functionally inactive molecules, called components. Complement activation occurs by cascade mechanism events, leading to sequential activation of the various inactive components. There are distinct pathways of complement activation: classical (activated by antigen–antibody binding), alternative, and lectinic, which are triggered by different mechanisms, but then converge in a common pathway leading to the formation of the membrane attack complex, which, by binding to the microorganism membranes, determines their osmotic lysis, through the formation of pores on the membrane itself [7].
2.2 Adaptive immune response
The adaptive immune response has the ability to recognize specific antigens and to remember those antigens in case of a subsequent exposure: this immunological memory allows a very rapid response, as the particular antigen has been already previously encountered and recognized. T and B lymphocytes, mediators of this response, undergo clonal expansion when they encounter the specific antigen they are programmed to recognize. At that moment, lymphocytes experience a real metabolic switch, increasing their metabolic needs for glucose and aminoacids, and passing from the normal oxidative phosphorylation typical of naive cells to aerobic glycolysis, in which pyruvate produced by glycolysis is reduced to lactic acid, with the simultaneous generation of NAD+ molecules, which promote the continuous production of 2 ATP molecules for each metabolized glucose molecule. This process, which occurs in the presence of oxygen, is less efficient than oxidative phosphorylation, but much faster, and thus able to meet the high ATP demand required to rapidly increase the biosynthesis of lipids, proteins, and nucleic acids of activated lymphocytes [15]. T lymphocytes exclusively recognize antigenic peptides exposed on the membrane of APCs via the major histocompatibility complex (MHC), whereas B lymphocytes recognize soluble, circulating antigens. Lymphocytes are present in an immature form in the primary lymphoid organs (bone marrow and thymus), where they differentiate into mature lymphocytes through a particular process of nonhomologous genetic recombination in the genes coding for antigen receptors (antibodies for B lymphocytes and T-cell receptors (TCR) for T lymphocytes). This somatic gene rearrangement accounts for the vast heterogeneity of lymphocytes, allows each individual to have a large and unique immunological repertoire, able to recognize a very large number of molecular configurations present in foreign agents, and thus counteract the majority of infections encountered during life [6].
2.2.1 T lymphocytes
T lymphocytes, effectors of the cell-mediated adaptive response, are divided into two major populations: T helper (Th) lymphocytes, bearing the CD4 receptor, which recognizes antigens presented by the MHC type II molecules, expressed on so-called “professional” APCs (dendritic cells, macrophages, and B lymphocytes), and cytotoxic T lymphocytes (Tc), with the CD8 receptor, which recognizes antigens presented by the MHC type I molecule, expressed on all nucleated cells [7]. CD4 and CD8 are co-stimulatory molecules, which bind to the MHC complex together with the TCR, contributing to T lymphocyte activation, which triggers different signaling cascades acting via various molecules and second messengers [16]. Th cells are critical in coordinating the immune response of other T cells and assist B cells in antibody secretion, whereas Tc cells are involved in the direct removal of damaged, pathogen-infected, or tumor cells. Th cells can develop into T helper 1 (Th1) or T helper 2 (Th2) cells, depending on the context in which antigen presentation occurs. Indeed, the cytokine secretion profile by APCs determines the “fate” of Th lymphocyte differentiation (Figure 1). The Th1 response is established in microenvironments where APCs produce essentially IL-12. This cytokine induces T lymphocytes to secrete IL-2 and interferon (IFN)-γ, through which cell-mediated responses, such as the antiviral response, are triggered. In particular, IFN-γ activates macrophages, inducing respiratory burst. Other important cytokines activated in this cascade, classically defined as pro-inflammatory, are IL-1, tumor necrosis factor (TNF)-α, and IL-8. In contrast, the Th2 response is associated with the production of IL-4, IL-5, and IL-13 cytokines, which mainly attract eosinophils and mast cells. The Th2 response, particularly involved in parasitic infections and allergies, is also associated with the expression of cytokines, such as IL-10, defined as anti-inflammatory, as they inhibit or reduce inflammatory-type responses. The maintenance of the correct balance between Th1 and Th2 subpopulations plays an important role in inflammation resolution [6]. Other relevant Th lymphocyte subtypes are the Th17, producing IL-17 and IL-22 cytokines, that are important in the response to extracellular pathogens such as fungi and bacteria, but also in some intestinal inflammatory responses; and the regulatory T (Treg) lymphocytes, involved in immune homeostasis especially in the intestine, as they contribute to the maintenance of oral tolerance to nonself harmless antigens, derived from food, but also environmental, such as pollen (Figure 1). The correct Th17/Treg balance is now recognized as fundamental for the maintenance of health status, in fact, this equilibrium results altered in many diseases with an autoimmune component, such as inflammatory bowel disease (IBD) [17].
2.2.2 B lymphocytes
B lymphocytes, expressing the surface receptor CD19, are responsible for the production of antibodies (or immunoglobulins), mediators of the adaptive humoral response. Upon encountering antigen, B lymphocytes can differentiate into short-lived antibody-producing plasma cells or long-lived memory cells. Plasma cells produce one of the five classes of immunoglobulins: IgG, IgM, IgA, IgD, and IgE, each with a specific role. IgM, representing about 10% total Ig in serum, are the first Ig produced in response to a foreign antigen. After 5–6 days from infection normally IgM reach their peak concentration and, thanks to the specific profiles of cytokines released, the so-called “isotypic switch” to IgG occurs. IgG are the predominant and most important class of immunoglobulins, in fact, they represent about 70–75% total Ig in serum. IgG reach the peak of secretion about 14 days after infection and persist for long periods. Normally IgG are the most effective in foreign antigen removal, through the above-mentioned opsonization process, as well as through activation of the complement system [6, 8]. IgD are found in serum at low concentrations (representing less than 1% of all plasma Ig) and their biological functions, related to the regulation of peripheral tolerance to self-antigens and in the maintenance of mucosal homeostasis, involving also host-microbiota interactions, have been elucidated only recently [18]. IgA represent 15–20% serum Ig, but their concentration is higher in secretions (saliva, breast milk, tears, sweat, respiratory, and intestinal secretions) and in the mucosa, i.e., those tissues covering the hollow organs and therefore in contact with the external environment (digestive, respiratory, and genital apparatus), where IgA contribute to preventing microorganisms from adhering to and penetrating inside the body through epithelial cells. In intestinal mucosa, IgA are found in dimeric form (secretory IgA, sIgA), particularly important for the protection against bacteria and viruses from the lumen, but also for the maintenance of oral tolerance to harmless food antigens, as detailed below. IgE, present in serum only in trace amounts, play a role in the removal of extracellular parasites (such as helminths) by opsonization, but are also important mediator in allergic responses. Indeed, IgE bind to a receptor expressed on the membrane of basophils and mast cells, stimulating the degranulation and release of histamine and lipid mediators into the intercellular space, triggering the allergic reaction, as described above [8].
2.3 Soluble mediators of the immune response
Soluble mediators, called cytokines, low molecular weight “messenger proteins” secreted by many cell types, both immune and nonimmune, are involved in both innate and adaptive immune responses. Cytokines send intracellular signals by binding to specific membrane receptors present on the same cells that produced them, or on other target cells, that can be in proximity or not, thus acting in an autocrine, paracrine, or endocrine manner. In general, it is possible to distinguish four different types of cytokines, on the basis of their biological effect: 1 - cytokines produced by leukocytes, having effects on the leukocytes themselves: interleukins; 2 - cytokines with chemoattractant properties, i.e., with a positive effect on cell motility: chemokines; 3 - cytokines that induce differentiation and proliferation of stem cells: colony-stimulating factors; 4 - cytokines that interfere with viral replication: interferons. Cytokines can exert a pro- or anti-inflammatory action, but often the outcome depends on the context of the microenvironment where they are secreted, and on the cells involved [7].
3. The intestinal immune system
The intestinal immune system is the most extensive lymphoid tissue, given the enormous surface area of the intestinal mucosa with which it is associated. It is called gut-associated lymphoid tissue (GALT) and is mainly composed of: organized lymphatic follicles, called Peyer’s patches (PPs); mesenteric lymph nodes (MLN);
3.1 Inductive sites
PPs represent the main sites where antigenic presentation occurs, called inductive sites, where the intestinal immune response is triggered. The PPs are covered by an epithelial layer, containing specialized membranous cells, the M cells, responsible for the transport of antigens, bacteria, and macromolecules from the intestinal lumen into the patches. These specific characteristics on the one hand make M cells designated for the transepithelial transport of antigens, and on the other hand make them more easily accessible by pathogens. In fact, many pathogens use M cells as a “gateway” to cross the intestinal barrier. M cells do not have brush border nor glycocalyx, but they have an extensive system of endocytic vesicles and a large intraepithelial pocket, where vesicles, containing antigens from the lumen, are released. In the pocket are APCs, which acquire the material carried by M cells and present the antigens to naive lymphocytes, present in the underlying subepithelial layer, organized in lymphatic follicles. In such follicles, B lymphocytes are located in the germinal centers, whereas T lymphocytes preferentially occupy the periphery and interfollicular spaces. DCs are also able to expose luminal antigens through various mechanisms: in the
3.2 Effector sites
In the intestine, activated B and T lymphocytes essentially target two different lymphoid compartments: the
4. The gut microbiota
4.1 Structure and function
Body surfaces facing the external environment, namely the skin and all mucosal surfaces (nasal, oral, gastrointestinal, etc.) are colonized by a huge number of microorganisms, collectively called microbiota. Most of them reside in the gut, in a
4.2 Influence of gut microbiota on the immune system
The intestinal microbiota is recognized as an effective integral component of the host immune system, capable of finely tuning the immune responses, innate and adaptive, in the different phases of life. Indeed, the close relationship established between bacteria and immune cells in the gut is crucial for the maintenance of immunological homeostasis and, mostly, for the “education” of the immune system during the early stages of life [2]. In fact, according to the most recent theories, the interaction between microbiota and the immune system is necessary to “train,” first, and “keep trained,” then, the various functions of the latter. Thanks to the continuous contact with the gut microorganisms, with the molecules they synthesize, with those they produce from undigested food components, the immune system satisfies two apparently conflicting needs: to defend the organism from real threats, and to tolerate microbes and molecules not harmful to the organism. Indeed, the large variety of microorganisms constituting the microbiota can be functionally distinguished into symbionts and pathobionts, also referred to as opportunistic pathogens, both fundamental, as the former educate the immune system to tolerance, while the latter train it to pathogen recognition and attack [31]. In the physiological condition of eubiosis, symbionts and pathobionts are present in equilibrium. If this balance is altered, for example, due to an excessive antibiotic treatment, one of the two groups becomes predominant, leading to the onset of one of two possible extreme conditions: hyperstimulation of the immune system (inflammation) or hypostimulation (immunosuppression) [32] (Figure 3). It is worth noting that pathobionts, that are not harmful and are even necessary to educate the immune system in physiological conditions, become dangerous when the equilibrium is altered, as in dysbiosis. The immunological surveillance of the intestinal microorganisms involves the above-mentioned TLRs, which recognize MAMPs and PAMPs [9, 33]. These receptors differently act in distinct cellular compartments. Indeed, recognition of these receptors on the apical surface of the epithelium (i.e., the one in contact with the intestinal lumen) generally promotes tolerance towards commensal bacteria and foodborne antigens, and low (basal) inflammatory tone; conversely, activation of these same receptors on the basolateral side, in contact with the underlying mucosa, promotes strong inflammatory responses. Numerous microbial stimuli activate inflammatory cascades through signal transduction pathways that essentially involve the nuclear transcription factor NF-κB, with consequent production of pro-inflammatory cytokines, such as IL-1 and IL-6, or anti-inflammatory factors more directly related to the extinguishing of inflammation/immune response, such as IL-10, thus playing a crucial role in maintaining intestinal homeostasis [10]. The different communities of the intestinal microbiota, characterized by metabolic specialization, complementarity, and cooperation, constitute a very complex network of microbe-microbe and microbe-host interaction, in the form of a symbiotic or mutualistic relationship, resulting in a continuous cross-talk. The host derives substantial immunological and metabolic benefits from the physical proximity of microbial populations in the gut and underlying tissues, but at the same time, this proximity poses an ongoing threat to health. In fact, although the immune system is designed to establish the proper balance between tolerance to the gut microbiota, maintaining a low level of basal inflammation and surveillance against infectious agents and opportunistic pathogens, the disruption of this balance, for example, due to inflammatory diseases or following the excessive use of antibiotics, induces a malfunction of the intestinal barrier with consequent opening of the junctions between enterocytes. The assembly and maintenance of tight junctions are regulated by several signaling pathways, that can be altered by pro-inflammatory cytokines, in particular TNF-α, IFN-γ, and IL-1β. Thus, an increase of these cytokines due to an inflammatory status can induce a decrease in the expression of tight junction proteins, or alter their phosphorylation status, causing a “loosening” of tight junctions [34]. This condition, referred to as a leaky gut syndrome, facilitates the translocation of pathogenic bacteria or harmful antigens from the intestinal lumen to the underlying mucosa (Figure 3). This process determines the establishment of endotoxemia, i.e., the presence of LPS in the circulation. The LPS, present on the cell wall of Gram-negative bacteria, is one of the microbial components able to act as an immune activator, therefore, representing a MAMP that binds to TLR4 and triggers an inflammatory response, which from local becomes systemic. The polysaccharide A of
5. Diet as a modulator of the microbiota-immune system cross-talk
An adequate and appropriate nutritional status and composition of the diet, in terms of foods, nutrients, and bioactive substances, are critical for the proper functioning of the immune system, which in turn is a fine sensor of the nutritional status of the individual [39]. When the immune system is activated to respond to nonself antigens, the demand for energy and nutrients increases and cells undergo the metabolic switch [40], as previously mentioned in the 2.2 paragraph. The dependence of the immune response on energy, and therefore the onset of immune deficits as a result of undernutrition, is known for a long time, but recently it has been observed that also the excessive consumption of food and excessive intake of calories alter the immune system. In fact, if on the one hand a serious caloric restriction impairs immune system functionality and increases the risk of infections (as observed in childhood malnutrition, still widely spread in developing countries), on the other hand, an unbalanced diet rich in high-calorie foods leads to negative consequences, inducing an inflammatory state and metabolic disorders. Many metabolic diseases are in fact characterized by a chronic low-grade systemic inflammation, called metaflammation (metabolic inflammation). Obesity and overnutrition are both associated with this inflammatory state leading to an increased risk of cardiovascular disease, heart attack, type 2 diabetes, and nonalcoholic steatohepatitis [41].
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 (
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 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
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
5.3 Western diet and Mediterranean diet: examples of detrimental and beneficial dietary profiles
The scientific literature describes the diet as the most characterized factor capable of shaping gut microbiota and immune system. Indeed, the nutritional status of an individual and the composition of the diet, in terms of foods, nutrients, and bioactive substances, influence immunity. A recent analysis by Rinninella and colleagues [58] highlighted the effects of different dietary habits on gut microbiota composition, by comparing vegetarian/vegan, gluten-free, ketogenic, low FODMAP (i.e., low in highly fermentable but poorly absorbable carbohydrates and polyols), Western and Mediterranean diets. Overall, restrictive diets (gluten-free, ketogenic, low FODMAP) have been shown to exert negative effects on the intestinal microbiota, in terms of reduction of biodiversity and alteration of eubiosis, impacting also on the integrity of the intestinal epithelium (especially in the case of ketogenic diet), and on inflammatory status. Among the different dietary profiles, the most consistent evidence concerns the Western Diet and the Mediterranean Diet, indeed Western Diet was shown to negatively impact gut microbiota composition and diversity, and to reduce the intestinal mucus layer, thus favoring bacterial translocation and endotoxemia, while Mediterranean Diet was associated to increased bacterial diversity and improved gut barrier function [58]. The Western Diet is typically described as a diet high in calories and rich in ultra-processed foods with high levels of sugars, saturated and trans fats, salt and food additives, while complex carbohydrates, fiber, vitamins and minerals, and other bioactive molecules (such as polyphenols and omega-3 fatty acids) are scarcely present. The main effects of this diet concern the elevation of plasma glucose and insulin levels, with a consequent increase in the accumulation of lipids in adipose tissue, which induces a rapid weight gain compared to more balanced diets. Furthermore, recent rodent and human studies have established that the Western dietary pattern is associated with elevated levels of inflammatory biomarkers, suggesting a direct or indirect action on the immune system [59]. It is noteworthy that macronutrients in food are part of a complex microstructure from which the physical, sensorial and nutritional properties, and health implications derive. “The complex assembly of nutrients and non-nutrients interacting physically and chemically, that influences the release, mass transfer, accessibility, digestibility, and stability of many food compounds” has been described as food matrix [60]. Therefore, diverse food matrices can differently affect the digestion and absorption processes of food compounds and play a role in the microbial fermentation of unabsorbed components. Ultra-processed foods and beverages are considered an important hallmark of the Western Diet, and high consumption of these foods appears to be correlated with an increased risk of morbidity. Food processing involves applying controlled procedures in order to preserve, destroy, transform, and create edible structures, whose aim is to prolong the shelf -life of foods. Ultra-processed foods are microbiologically safe, highly palatable, ready-to-eat, and highly profitable products composed primarily of ingredients not routinely found in “real foods” (e.g., hydrogenated/de-esterified oils or additives designed to provide the previously mentioned characteristics). The poor and uncomplex matrix of these foods, together with their low fiber content, generates an unfavorable environment in the gut and microbiota, thus leading to dysbiosis and immune alterations. Therefore, the Western Diet, intended also as an incorrect lifestyle, would induce low-grade inflammatory processes, which are a risk factor for the development of various chronic inflammatory diseases, predisposing the individual to metabolic inflammation, through various mechanisms, acting at both levels of microbiota and intestinal permeability [61]. The action on the microbiota leads to the onset of dysbiosis, intended both as taxonomic (shifts in microbial groups composition), but especially as metabolic (changes in microbial function). Moreover, the decreased bacterial diversity makes the microbial ecosystem less resilient and more susceptible to external stressors. The increase in pathogenic bacteria also causes an increase in pro-inflammatory metabolites, which can influence the response at the level of GALT, with which they are intimately linked. When abnormalities occur in these interactions, intestinal permeability can increase and the leaky gut phenomenon occurs, leading to metabolic endotoxemia, as described previously. But metabolic inflammation arises primarily at the level of white adipose tissue, where adipocytes, cells almost entirely formed by a single large lipid droplet, release numerous adipokines, cytokine-like molecules, in response to changes in lipid accumulation and in local and systemic inflammation. Adipokines can be either pro- or anti-inflammatory and play a key role in linking metabolism with immune function [62]. In individuals with normal metabolic status, pro- and anti-inflammatory adipokines are correctly balanced, and Th2 lymphocytes, Treg cells, and macrophages with an anti-inflammatory phenotype predominate in adipose tissue. Treg cells secrete IL-10 and also stimulate IL-10 secretion by macrophages. Eosinophils secrete IL-4 and IL-13, further contributing to the anti-inflammatory and insulin-sensitive phenotype. A long-term hypercaloric diet causes an increase in the number and size of adipocytes, which become hypertrophic and dysfunctional, starting to secrete pro-inflammatory adipokines, especially TNF-α. In addition, circulating immune cells, mainly monocytes, are recruited from the bloodstream in response to chemotactic signals (particularly monocyte chemoattractant protein 1, MCP-1) produced in adipose tissue, transmigrate there, and differentiate into macrophages secreting high amounts of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6. These cytokines act in a paracrine manner, inducing changes in T lymphocyte populations, with a decrease in Treg and an increase in Th1 cells, which in turn secrete pro-inflammatory cytokines, thus generating a vicious circle, where the inflammatory state becomes systemic. Indeed, cytokines and chemokines from adipose tissue can act in an endocrine way and promote inflammation in other tissues, also causing the onset of insulin resistance and other metabolic disorders associated with obesity [63]. Adipocytes in visceral adipose tissue are metabolically very active and very sensitive to lipolysis, so following a prolonged positive caloric balance, very high amounts of free fatty acids (FFAs) are generated and released into the portal system. Insulin resistance results from an excess of circulating FFAs and excess TNF-α in adipose tissue, as both molecules result in functional blockade of the insulin receptor and its associated signal transduction. In particular, FFAs and TNF-α block insulin receptor signaling by activating phosphorylation of the insulin receptor substrate (IRS)-1 at a serine residue. Serine phosphorylation of IRS-1 causes it to detach from the insulin receptor, resulting in functional blockade of the receptor and of insulin signal transmission itself. In addition, TNF-α, secreted by adipocytes and adipose tissue macrophages, also acts by another mechanism, namely by inducing dephosphorylation of IRS-1 at tyrosine residues. Tyrosine-dephosphorylation has the same effect as serine-phosphorylation, thus IRS-1 is inactivated and detached from the insulin receptor [64]. It is known that several components characterizing the Western Diet determine an inflammatory state through the activation of the innate immune response, for example, excess cholesterol is considered the main cause of inflammation in the atherosclerotic process. In addition, an excess of free cholesterol crystals causes damage to lysosomes, with subsequent release of the pro-inflammatory cytokines IL-1β and IL-18 through activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome, resulting in a systemic response characterized by a chronic low-grade inflammatory state, associated to insulin resistance and the onset of some related diseases, including colorectal cancer [65]. Saturated fatty acids carried through excessive consumption of animal-derived foods also have cytotoxic effects and can activate endoplasmic reticulum stress as well as the NLRP3 inflammasome. More recent theories suggest that saturated fatty acids induce dysbiosis and subsequent release of metabolites that alter intestinal permeability, inducing metabolic endotoxemia [59]. From a taxonomic point of view, an excess of fat causes an increase in the Firmicutes/Bacteroidetes ratio, while some unrefined oils, such as palm oil, may cause a decrease in
Acknowledgments
This work was funded by the Italian Ministry of Agriculture, Food, and Forestry Policies (MiPAAF), within the trans-national project INTIMIC–Knowledge Platform on food, diet, intestinal microbiomics, and human health of the Joint Programming Initiative Healthy Diet for a Healthy Life (JPI-HDHL), Expression of Interest n. 795 (MICROFLUX Project, MiPAAF DM 36954/7303/18).
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