**3. Role of the microbiota in the development of the mucosa-associated immune system**

From an immunological perspective, the mucous membranes which cover a total area of about 400 m2 , represent both an anatomical and functional entity, because they are populated by a large number of immune cells.

The intestinal microbiota, epithelium and digestive, respiratory, genital, urinary mucosa-associated immune system form a functional triad whose components influence each other close interactions, with a rapid dynamic of change, induced by population changes of the microbiota, due to diet variation and/or administration of antibiotics. The modification of the functional parameters of a component of the triad has major influences on the physiology of the whole organism. The microbiota interacts directly with the epithelium of the adjacent mucosa and influences its permeability, and both local and systemic inflammatory responses [10] The interaction of the microbiota with the mucosal immune system (gut-associated lymphoid tissue—GALT) induces the synthesis of a wide set of cytokines, with local regulatory action of intestinal physiology [20, 21].

The microbiota has an essential role in the functional modulation (education), first of all of the GALT structures. Germ-free and gnotobiotic animal studies have made a decisive contribution to understanding the functional relationships of the microbiota-epithelium-immune system triad and provided new evidence for the role of the intestinal microbiota as a whole, but also of different groups of bacteria in the functional development and maturation of the systemic immune system, especially GALT. Germ-free mice have structural and functional defects of the immune system—decreased TCD4 lymphocyte count and Th-2 predominance in the spleen, altered Th-17 and T-reg differentiation in the lamina propria, and restoration of deficiencies after colonization with *Bacteroides* and segmented filamentous bacteria (SFB). The balance of effector T lymphocytes is disturbed in intestinal dysbiosis and accelerates or suppresses the autoimmune reactions [22, 23]. The constant interaction of the microbiota with the cells of adaptive immunity prevents bacterial invasion and pathogenesis, but also the systemic immune response with detrimental effects against the microbial antigens [24]. The structural but especially functional peculiarities of GALT tend to delimit it more and more from the systemic immune system.

M cells that cover the subepithelial immune structures engulf the luminal antigens, through the mechanism of pinocytosis and transfer them unaffected to the immune structures in the underlying follicles (i.e., macrophages, dendritic cells, T and B lymphocytes). Macrophages and dendritic cells respond to microbiota antigens in a nonspecific manner by TLR recognition followed by cytokines release (i.e., IFNα, IL-18, and IL-22), which stimulate the epithelial cells to synthesize antimicrobial peptides.

The microbiota, through the composition of bacterial phyla, has a major influence on the development of T lymphocyte subpopulations and in maintaining the numerical balance of Th-2/Th-1 lymphocyte populations in lymphoid organs. The differentiation of T lymphocyte sets is influenced by the antigenic specificity of the dominant bacterial population and its metabolic properties—(i) some bacteria stimulate the predominant differentiation of proinflammatory TCD4 lymphocytes that synthesize IFNγ and IL-17A [25]; and (ii) others stimulate the differentiation of regulatory CD25+ and Foxp3+ TCD4 lymphocytes (T-reg), the essential mediator of immune tolerance by decreasing Th-17 lymphocytes [26, 27]. The direct relationship between the concentration of butyric acid and the number of T-reg lymphocytes is well known. SCFA, particularly butyric acid harbor important roles, that is, stimulate gene transcription for mucin synthesis, strengthen the intestinal barrier and render it impermeable to toxins and bacterial cell translocation, thus preventing chronic systemic inflammation, inhibiting the synthesis of pro-inflammatory interleukins (IL) (TNFα and IL-6) induced by LPS and regulate the innate and adaptive immunity [13, 14]. Th-17 lymphocytes play an essential role in anti-bacterial and anti-fungal defense, but at the same time have an important role in the initiation of inflammatory diseases, through the synthesis of pro-inflammatory IL-17 and IL-22 and the recruitment of neutrophils. In germ-free animals, the lamina propria is populated by a very small number of Th-17 lymphocytes [9]. Th-17 lymphocytes also decrease after antibiotic treatment [27]. The group of *Clostridium* SFB, following the colonization of the epithelium, induces an increase in the number of Th-17 lymphocytes, whose proinflammatory IL can promote the onset of rheumatoid arthritis and multiple sclerosis in gnotobiotic animals. In patients suffering from inflammatory bowel disease, which manifests clinically similar to Crohn's disease and ulcerative colitis, the number of T-reg lymphocytes with immunosuppressive function decreases in the lamina propria and the population of lymphocytes that have TCR for the bacterial microbiota antigens increases abnormally. The density of T-reg lymphocytes increases in gnotobiotic animals colonized by *Clostridium* SFB group, while the polysaccharide A of *Bacteroides fragilis* (which is attributed to symbiotic factor status) induces the differentiation of TCD4 lymphocytes to T-reg lymphocytes [9, 28]. TCD4 Foxp3 + lymphocytes secrete IL-10, the main anti-inflammatory cytokine, thus being involved in tolerance to microbial antigens. In germ-free animals, the dominance of Th-2 subpopulation in the spleen that favors allergic manifestations is restored by the polysaccharide A of *Bacteroides fragilis*.

The microbiota has also a profound influence on the development of B lymphocytes—it stimulates the synthesis of antibodies, especially of IgA type, targeted against thymus-dependent (Td), and thymus-independent (Ti) antigens. The *Clostridium* SFB and *Alcaligenes* group of bacteria are potential inducers of IgA synthesis specific for the intestinal microbiota antigens. In the absence of IgA, the *Clostridium* group is enriched, whereas the *Alcaligenes* group is diminished [9].

In germ-free animals, GALT structures play a key role in inducing immune tolerance against auto-antigens from the intestinal mucosa, are less developed and indicators of immune response activation are lacking. In these animals, the number of TCD4 lymphocytes and IgA-secreting plasma cells decreases in Peyer's patches, while in the spleen and lymph nodes, the number of B lymphocytes and germinal centers decreases.

In conclusion, the development, maturation, and function of the immune system are closely associated with the level of exposure to microbial antigens during early life, and as an opposite, insufficient exposure to various antigens increases the risk of autoimmune disorders occurrence [29].
