‘SUPPLEMENT ‘ay Mechanisms of Action of Probiotics Julio Plaza-Diaz,"* Francisco Javier Ru Ojeda,* Mercedes Gil-Campos/* and Angel Gil ® "+ Department of Biochemistry and Molecular Biology I, School of Pharmacy, Univesity of Granade, and Istiut of Nurtin and Food Tecoogy ose Mara” Biomedical Research Center, University of Granade, Armle, Granada, Span Institute de Investigacion Biosanitaria IBS GRANADA, Complejo ‘Hospital Universitario de Granada, Granada Spain; *CREROBN Physiopathology of Obesity and Nuttin CB12/03/30038) Institut de Salud Carlos I Madd, Spon: and "Pediat Research ond Metabolism Uni, Reina Sofe University Hospital, Maimonides Institute for Biomedical Research, Cordoba, Spain Cos Probiotics ate Ining microorganisms that confer heath benefits to the host when administered in adequate amounts; however, dead bacteria. and thelr components can also exhibit probiotic properties ifdobacteium and sans of lactic acid bacteria are the most widely used bactetla ‘that exhibit probiotic properties an are included in many functional foods and dietary supplements, Probictics have been shown to prevent and ameliorate the course of gestive dsorders suchas acute, nosocomial and antblti-asoclated larheallergc sores suchas atopic dermatts (eczema) and allergic hits in infants: and Clostictum cifcie-associated dares and some irlammatory bowel disorders in adults. In ation, robotics may be of interest as coadjuvats inthe tteatment of metabolic disorders, Including obesity, metabolc syndrome, nonalcoholic ety Iver dlsease and type 2 diabetes. However, the mechanisms of action of probiotics which are diverse, heterogeneous, and stan specific have recelvd Ite attention, Thus, the alm ofthe present work was to review the main mechanisms of action of probotcs, Including cololzation and normalization of perturbed intestinal microbial communitiesin children and adults compertve exclusion ofpathogens and bacterocn production; ‘adulation of fecal enzymatic actives associated with the metabolizaton of bilary salts and inactivation of carcinogens and other xenablatcs, production of short-chain and branched-chain faty acids, whic in tun, have wide effects not only inthe Intestine but also in peripheral tissues va interactions with short-chain fatty aid receptors madulating main tissue nsulln senstvty cell adhesion and mucin production;modulation ‘ofthe immunesyster, which resus mainly inthe dferentlaton of -reguatory cel and upregulation of antinlammatory cytokines and growth factors, Le, Intereukn-10 and transforming growth factor and interaction withthe braln-qut ais by regulation of endacrine and neurologic ‘unetons Further esearch to elucidate the precse molecular mechanisms of action of probate s warranted. Ad Nutr 2018:10S49-56, Keywords: biidobacteria lactic acid bacteria lactobacll, mechanism of action probiotics, volatile fatty acids, brain-gut avs, immune system Introduction ‘The term “probiotics” refers to microorganisms that confer health benefits to hosts when administered in adequate amounts (1-3). A true probiotic should preferably be of Aieiedina memento ani ition Peart te sal sen aon sdeners DNS 23tsoral Congres Nin CC en Bes es, ‘ren, Osta 15-2027. 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Moreover, a probiotic should exhibit antagonism against pathogens and stimulation of the immune system and, ultimately, must have demonstrable beneficial effects on the host. Finally, maintenance of the activity, viability, and growth efficacy of the probiotic upon technologic treatment should be demonstrated (4, 5). ‘eseratoe va AD at oem dre ECA bareee many a8 ELE sats om dense CA con cee cA Euepen od Sake Aer ACE. eens eos case forse? Foes BoxP3: OR (peen- cued eps; HD, array Bove asa, ate be 9nd Us aa 304-rtichy tcr NAFLD, nok a as NE, ea maces Wr, race tancten curt AMY patagenasocted ne ter PR paterson ese ACT. a TT eb TR a Mean Hg pty eT ype 2 dans. S49Iya Ilyich Mechnikov (6) performed the first investi gations on lactic acid-producing bacteria (LAB) and their health effects in humans, and results from these first inves tigations suggested that LAB ingestion improved host health. LAB are a heterogeneous group of microorganisms that are often present in the human gut, being introduced via the ingestion of fermented foods, such as yogurt and other fer ‘mented milk products, various cheeses, and fermented cured, meat by-products. Strains of Bifidobacterium, Enterococcus, Lactobacillus, Saccharomyces boulardii, and Escherichia coli Nissle 1917 are the most widely used probiotic bacteria However, other strains such as Lactococcus, Leuconastoc, Pediococcus, and Streptococcus are also used as probiotics (7~ 9%), ‘In 2014, the International Scientific Association for Probi otics and Prebiotics stated that the development of metabolic by-products, dead microorganisms, or other microbe-based, nonviable products has potentiak however, these do not fall under the probiotic construct (3). Nevertheless, several stud jes have shown that dead bacteria and bacterial molecular components display probiotic properties 4,5, 10). Currently, the term “postbiotic” refers to soluble components with biological activity that, could therefore be a safer alternative to the use of whole bacteria (11). The effets of probiotics on host health have been reported in many articles, reviews, and systematic reviews (12, 13). These studies have documented the role of probioties in the prevention of health problems, including digestive dis orders such as diarthea caused by infections (4), antibiotic: associated diarrhea (14), irritable bowel syndrome (IBS) (15), Clostridium dificile-associated diarrhea in adults and. children (16), inflammatory bowel disease (IBD), only in ulcerative colitis (17), and allergic disorders such as atopic dermatitis (eczema) (18) and allergic rhinitis (19). Even though many probiotic strains are well documented as safe or denoted “generally recognized as safe,” the Euro: pean Food Safety Authority (EFSA) and the US FDA do not attribute the ability to prevent or treat diseases to probiotic administration, Probiotis are purchased as dietary supple ‘ments in many countries and follow current market policies. The EFSA has not approved any product with health claims associated with probiotic administration, More than 300 approval requests have been submitted for 200 probiotic strains or combinations of strains, claiming > 60 beneficial effects (20), The principal reasons for these approval requests being denied were as follows: insufficient characterization, undefined claims, nonbeneficial claims, lack of relevant hhuman studies, lack of measurable outcomes that reflect direct benefit for humans, and finally, the quality of the presented studies (20) In addition, the FDA might regulate probiotic strains asa dietary supplement, food ingredient, or drug (21). Similarly to the EFSA, the FDA has not approved any probiotics to prevent or treat health problems (22). Both food agencies have emphasized the following notions: each health claim is unique for each probiotic strain; scientific requirements have to be considered in the context of each application; guidelines and past evaluations are valuable sources of information; it is important to understand the $50, PlzeDazetal rationale behind the principles being applied; no recipe for success can be provided: and finaly, researchers and ‘companies need to try, fai, learn, and try again (20, 22), Moreover, many studies of probiotics lack insight into the potential mechanism of action. However, Health Canada approves a multistrain probi otic [Streptococcus sativarius subsp. thermophilus (SD5207), Bifidobacterium breve (SD5206), Lactobacillus plantarum ($5209), Lactobacillus paracasei(SD5218), Bifidobacterium ‘animalis subsp. lactis ($D5220, D219), Lactobacilus ac dophilus ($5212), and Lactobacillus heveticus (SD5210)] and a single strain of B. animalis spp. lactis LAFTI B94 as natural health products for relief of IBS symptoms, such as abdominal discomfort, gas, and bloating (23) Currently, it is accepted that gut dysbiosis refers to changes in the quantitative and qualitative composition ‘of microbiota, that these changes may lead to altered host microbial interaction that can contribute to a disease state often with inflammation, and that this is associated with the development of many noncommunicable human diseases, but the mechanisms via which homeostasis is maintained are not yet completely understood (4, 24). Re- cent investigations have proposed that, during homeostasis, epithelial hypoxia limits oxygen availability in the colon, leading to the maintenance of a balanced microbiota that functions as a microbial organ, producing metabolites that contribute to host nutrition, immune training, and niche security (24,28) Probiotics are a current strategy to treat dysbiosis, restoring microbial diversity and altering the perturbed intestinal microbiota with specific mechanisms of action that have not been completely elucidated (26, 27). For this reason, we performed a literature review of the varied mechanisms of action of probiotics to understand the role of various strains in host homeostasis. A comprehensive search of the relevant literature was performed with the use of electronic databases, including MEDLINE (PubMed), EMBASE, and the Cochrane Library. MEDLINE through PubMed was searched for scientific articles in English through the use of the terms “probiotics” combined with ‘mechanism of action,” “competitive exclusion,” “volatile fatty acids,” “mucin,” “immune system,” and “brain-gut axis.” The following mechanisms have been reviewed: 1) colonization and normalization of perturbed intestinal mi- 1 organ systems, mainly the gastrointestinal tract and/or skin (58). Many infants develop symptoms in >2 organ systems. Typical IgE: mediated symptoms include urticaria, angioedema, vom. iting, diarrhea, and anaphylaxis. Dermatitis and rhinitis can be IgE and non-IgE mediated. Vomiting, constipation, hemosiderosis, malabsorption, villous atrophy, eosinophilic proctocolitis, enterocolitis, and eosinophilic esophagitis are non-IgE-mediated reactions (58). Samples from subjects with CMA showed that Firmicutes and Clostridia were enriched in the infant gut microbiome of subjects whose milk allergy resolved by age 8 y, whereas Bacteroidetes and Enterobacter were characteristic of subjects whose mill: allergy did not resolve by age 8 y (60). Specifically, there seems to be a link between dysbiosis in the composition of the intestinal microbiota and the pathogenesis of CMA. The administration of probiotics such as L. rhamnosus GG in an extensively hydrolyzed formula led to increased tolerance in infants with CMA compared with those treated with hydrolyzed formula alone, which was due in part to changes in the structure of the bacterial community in the intestines ofthe infants (61). $52. Plz»Dazetal ‘The use of probiotics in adult diseases After the administration or consumption of probiotic strains, the process of colonization begins. Few studies have assessed this step, and most have only evaluated major outcomes and drawn associations between those results and microbial administration. In healthy adults, probiotic administration increases the production of SCFAs (see below), fecal moisture, frequency of defecation, and volume of stools (62). Recorded gas- trointestinal symptoms, defecation frequency, and stool consistency were not influenced by L. rhamnosus PRSP- L477, indicating that this bacterium was well tolerated. ‘The detection of L. rhamnosus in the feces of subjects in the probiotic-treated group was an important issue (63). ‘Tolerance to Lactobacillus salivarius CECTS713 was assessed, in healthy adults; the strain was tolerated, and no adverse effects were detected, but no attempt was made to evaluate Intestinal colonization by this strain (64). However, intestinal persistence was observed in volunteers who received L, rhamnosus CNCM 1-4036, as detected through the use of a specific primer for qRT-PCR (65). The effects of probiotics ‘go beyond health status; subjects living with overweight and, obesity are good candidates to receive probiotic strains, as Individual treatments or as multistrain preparations. De Si- ‘mone formulation is a multistrain probiotic preparation that hasbeen tested in subjects living with overweight. De Simone formulation administration reduced the concentrations of lipids and indlammatory markers such as high-sensitivity C- reactive protein, enhanced insulin sensitivity, and produced changes in the composition of the gut microbiota (66). In patients living with obesity and hypertension, L. plantarum ‘TENSIA decreased BMI and blood pressure (67). IBD Is a term used to describe a group of systemic pathologies that affect the gastrointestinal tract. In these conditions, the function of the epithelial barrier is affected and is a main factor in the onset of the disease and in further complications (4). Alterations in the gut microbiota might be associated with the initiation and progression of IBD. Probiotic treatment trials in patients living with Crohn disease (CD) showed no remission effect; in contrast, probiotic consumption by patients with ulcerative colitis ‘seems to be more effective in the remission of the pathology, ‘especially upon treatment with De Simone formulation and a combination of Lactobacillus and prebiotics (68). Butyrate-producing bacterial strains (Butyricicoccus pul- licaecorum 25-37, Butyricicaccus pullicaecorum 1.20, Fae- calibacterium prausniteli, and a mix of Butyricicaccus pul licaecorum 25-3T, Faecalibacterium prausnitzii, Roscburia hominis, Eubacterium hall, and Anaerostipes caccae) were tested in patients with CD to evaluate mucus stimula- tion. All the assayed strains exhibited increased butyrate production and improved the integrity of the epithelial barrier (69) With regard to C.difiile-assoclated diarrhea, moderate- ‘quality evidence suggests that probiotic administration re. sults inefficient alleviation of this condition (70).Competitive Exclusion of Pathogens and Bacteriocin Production Competitive exclusion refers t0 the situation in which 1 species of bacteria competes for receptor sites in the intestinal tract more vigorously than other species (71). The specific pathways and key regulatory mechanisms underlying these effects of probiotics are largely unknown, Reduction in luminal pH. competition for nutritional sources, and production of bacteriocin or bacteriocin-like substances are among the main proposed mechanisms for competitive exclusion of pathogens (72). Most studies have focused on the reduction of human pathogens such as Salmonella typhi and E. col (73). Hence, some probiotic metabolites appear to play a role in the modulation of diverse signaling and metabolic pathways in cells, Indeed, components of the probiotic metabolome (organic acids, bacteriocins, hydrogen peroxide, amines, et.) have been reported to interact with multiple targets in some metabolic pathways that regulate cellular proliferation, Jitferentiation, apoptosis, inflammation, angiogenesis, and metastasis (74). Some lactobacilli and bifidobacteria can produce an- timicrobial peptides known as bacteriocins, which prevent the proliferation of selected pathogens. The term “eol- ‘nization resistance’ refers to the use of probiotics to prevent or treat enteric pathogens (71). Bacteriocins are small cationic molecules composed of ~30-60 amino acids ‘These moleculesact at bacterial cytoplasmic membranes and target energized membrane vesicles to disrupt the proton- motive force (75). Bacteriocins are classified into 4 main types based on their primary structures, molecular weights, post-translational modifications, and genetic characteristics (76), In particular, some of these compounds produced by L. plantarum and L. acidaphitus have been shown to inhibit the growth of Helicobacter, C. dificle,rotaviruses, and multicrug-resistant Shigella spp. and E. coli in some gastrointestinal conditions (77) and have activity against a number of uropathogens (76). Fatty Acids Enzymatic activities ‘The enzymatic activities of probiotics in the gut lumen can play a role in the biological effects of these probiotics Lactobacilli and bifidobacteria exhibit >20 different enzy- matic activities, with 6-galactosidase activity being the most typical Intestinal bacterial B-glucuronidase hydrolyzes glucuronidated metabolites to thelr toxic forms in intestines, resulting in intestinal damage. In addition, low f-glucuronidase activity in fecal material has been linked to an increase in the amounts of substances such as ‘carcinogens in the colonic lumen (78). B, longun, when added to the diet, contributes to changes In the intestinal microbiota, lowering the activity of f-glucuronidase, which Is associated with the inhibition of aberrant crypt formation and is an early preneoplastic marker of malignant potential in the process of colon carcinogenesis (79). Moreover, in a systematic review of randomized clinical trials (RCTS) testing probiotics, prebiotics, or both (synbiotics) for the treatment of nonalcoholic fatty liver disease (NAFLD) in adult patients, a reduction in liver aminotransferase activity was documented (80). In an RCT involving 30 healthy adults to evaluate the effects of a fermented product containing 2 probiotic strains (Lactobacillus gasseri CECTS714 and Lactobacillus coryniformis CECTS711), compared with standard yogurt, ‘on host intestinal function, 19 enzymatic activities were detected in the feces of volunteers. The pattern of enzymatic activity exhibited by the control and the probiotic-treated groups was very stable throughout the study. However, the naphthol-AS-Bi-phosphohydrolase activity a typical feature of lactobacilli, was augmented in the feces ofthe probiotic treated group. In addition, the leucine arylamidase activity, which is characteristic of probiotic strains, also increased, ‘whereas the f-glucuronidase activity exhibited a decreasing trend (62), Probiotics interact with bile acids in the gut lumen, ‘modifying bile acid metabolism and in turn influencing cholesterol absorption. Bile salt hydrolase (BSH) is an enzyme produced by bacterial species of several genera associated with the gastrointestinal tract and by most of the known probiotics; this enzyme may participate in the first reaction ofthe deconjugation of biliary salts (1). Con sidering these beneficial effects of BSH-containing bacteria, BSH activity has been included in FAO/WHO guidelines for the evaluation of probiotics for food use (1). Enzymatic deconjugation of bile acids by BSH from probiotics has been considered to be one of the main mechanisms of the hypocholesterolemic effect attributed to probiotics (81, 82). Volatile fatty acids In an RCT with adult volunteers the treatment group that received L. gasseri CECTS714and L. coryniformis CECTS71L exhibited increased production of fecal butyrate compared ‘with a group who received yogurt. Similarly, production of propionic and acetic acid was higher in the probiotic-treated group after 2 wk of treatment. At the end of the washout period, the production of butyrate in the probiotic-treated group vas still higher than that in the control group (62, 83. ‘Another RCT study, conducted to determine the impact alter 4 wk of daily consumption of a capsule containing 24 x 10? viable L. paracasei DG on the intestinal microbial ecology of healthy volunteers, reported that participants with a butyrate concentration of >100 mmol of wet feces, had a butyrate reduction of 49% 21% (mean + SD) and a concomitant decrease in the total abundance of 6 genera of Clostridiales, namely, Faccalibacterium, Blautia, Anacrostipes, Pseudobutyrvibrio, Clostridium, and Butyrivibrio, ater the probiotic intervention. However, in subjects with initial butyrate concentrations of <25 mmol/kg of wet feces, the probiotic contebuted to a very high increase in butyrate concentrations concomitantly with a ~55% decrease in Rumsinococcus lbundance and a 150% increase in an abundantly represented unclassified Bacteroidales genus Mechanisms of action of probiotics $53Therefore, the authors concluded that the impact of the Intake of L. paracasei DG on the microbiota and on SCPAs seems to depend on the initial characteristics ofthe intestinal microbial ecosystem, and specifically, fecal butyrate content right represent an important biomarker for identifying subjects who may benefit from probiotic treatment (84). ‘Another RCT study recruited 33 healthy subjects, in cluding young (mean age of 26 y), middle-aged (mean age of 51 y), and elderly (mean age of 76 y) volunteers, wo were given a single daily oral dose of L. plantarum Lp-8 The concentrations of both acetate and propionate, but not butyrate, increased significantly and peaked at week 5 in all 3 age groups. After Lp-8 consumption was terminated, the concentrations of both acetate and propionate gradually decreased but remained higher than the baseline concen trations (85). Hence, the production of fecal butyrate by diferent probiotics appears to strictly depend on the specific bacteria used. Worthy etal. (86) carried out a 4-wk crossover RCT of resistant stach and Bifidbacterion lactis, either alone or as a combined synbiotic preparation, in 20 human volunteers. This synbiotic supplementation at the doses used induced unique changes inthe fecal microbiota but did not signif cantly alter any other fecal, serum, or epithelial variables: for example, fecal SCEA concentrations were unchanged from the baseline. In contrast, 4-wk crossover RCT carried out in 43 older volunteers that used a synbiotic comprising the probiotic B, longum and an inulin-based prebiotic reported Increased production of acetate, succinate, butyrate, and Isobutyrate compared with the placebo-treated group at the end of the treatment. Thus, short-term synbiotic use could be effective in improving the metabolic activity ofthe colonic bacterial microbiota in older people (83). Osmotic diarhea and antibiotic-associateddiarthea are significant problems in patients receiving total enteral nutrition, particularly elderly patients, because enteral feeding may change the intestinal microbiota and SCEA composition (87) The results of an RCT showed that short-term treatment (~64) with the probiotic yeast S, bouladit may decrease the incidence of diarehea in patients receiving total enteral nu trition. Fecal butyrate concentrations were lower in patients than in controls, and treatment with S. boulardif increased the total fecal SCFA concentrations in the patients. At the end of the treatment with 8 Boulad, the patients had higher fecal and total SCEA concentrations, which remained high 9 dafter treatment was discontinued. Thus, the increase in fecal SCFA concentrations, particularly butyrate, may contribute to explaining the preventive effects of S. boulardit on total enteral nutrition-induced diarrhea (83). L. plantarum 299v has been shown to enhance the concentrations of fecal SCFAs in patients with recurrent C, difiile-associated diarchea, contributing to reducing the adverse effects of antibiotics Infact, after consumption of ‘metronidazole, a significant decrease in total SCFA concen trations was observed in the placebo-treated group but not i the probiotic-treated group. Moreover, the concentration of fecal butyrate was higher in the Lactobacillus-treated group $54 Plaza Dizetal than in the placebo-treated group. At the end of the study and fier cessation of placebo or probiotic treatment, the total SCFA concentrations were restored to the pre-antbiotic: treatment levels in the placebo-treated group (89). Nagata et al. (90) conducted an RCT in 77 elderly people (mean age of 84 y) at a long-stay health service facility. ‘The study evaluated the effect of the intake of probiotic- fermented milk containing Lactobacillus casei strain Shirota ‘on norovirus gastroenteritis, which occurs in the winter season, during the intake period. While the duration of norovirus-gastroenteritis-related processes decreased, Bifi dobacterium and Lactobacillus were found tobe the dominant ‘genera: the abundance of Enterobacteriaceae decreased in the fecal samples ofthe probiotic-treated group; and a significant increase in fecal acetic acid concentration was observed. Ina more recent RCT carried out by Nagata etal. (91) wit elderly residents who randomly received either L. casei Shirota or 4 placebo beverage once daily for 6 mo, the counts of C. dificile were significantly lower and the fecal acetic acid concentration and total acidity were significantly higher in the L. casei Shirota-treated group, and these results were associated with significantly lower incidence of fever and improved bowel movements. Changes in fecal SCFA or branched-chain fatty acid (BCFA) concentrations can partially explain the effect of probiotics and the role of probiotics in the nutritional status ‘of, and risk of diarrhea in, children. L. paracasei Lpc-37 or B, lactis HNO19 consumption by 2- to 5-y-old children was found to reduce the risk of diarrhea and was associated with higher concentrations of selected SCEAs and BCFAs in sub- jects who had experienced diarrhea. The concentrations of SCFAs, namely, acetate, propionate, and butyrate were found to correlate with each other. Likewise, the concentrations of the BCFAs isobutyrate, 2-methylbutyrate, and isovalerate also correlated with each other, After this intervention, L. paracasei Lpe-37 abundance correlated positively with total Bifidobacterium counts and isovalerate concentrations B. lactis HNO19 counts were found to correlate positively with total bacterial counts and negatively with propionate concentrations (92). Riezzo et al, (93), in an RCT, evaluated the effects of probiotic-enriched artichokes, compared with ordinary artichokes, on SCFA patterns in constipated subjects, Each patient consumed 180 g of ordinary artichokes/d or art chokes/d enriched with L. paracasei IMPC 2.1 for 15 4. Propionic acid concentrations were higher than baseline in the probiotic-treated group, and this result was associated, with a lower constipation score (93) ‘The addition of prebiotics or probiotics to infant formula to improve the intestinal microbiota of formula-fed infants is ‘a matter of great interest for consumers and stakeholders and for the food industry. An RCT evaluated the effects of an infant formula ‘containing L.salivarius CECTS713 compared with a control standard formula over 6 mo. Consumption of the probiotic formulated to an increase inthe fecal lactobacilli content and the fecal concentration of butyric acid over 6 mo (94),Abnormal colonization of low-birth-weight infants usu ally occurs because of a number of reasons. including cesarean delivery, prolonged hospital stay, and immature intestines, and can have adverse effects on health. An RCT evaluated the oral application of a probiotic, usually called B. lactis Bb12 (true name B, arimalis),on selected indicators of health status in preterm infants. The fecal pH in the probiotic-treated group was significantly lower than that im the placebo-treated group, and the fecal concentrations of acetate and lactate were 42% and 38% higher in the probiotic-treated group than in the placebo-treated group, respectively (95). The lower fecal pH in feces of infants, as seen in breastfed infants, is associated with a lower incidence of diarrhea. Another RCT compared the effect ‘of 2 prebiotic/probiotic products on the weight gain, stool microbiota, and stool SCFA content of premature infants Even the bifidobacteria content was higher inthe infants who were fed the probiotic formulas, and significant differences in fecal SCFA content were detected between the groups (96). Based on the roles of probiotics in the modulation of the immune system (see below), variousstudieshave investigated the potential effects of probiotics in the prevention of childhood eczema and allergies. Probiotic supplementation (B. bifidum W23, B. animalis subsp. lactis W52, and Lac- fococcus lactis W58) resulted in fewer children developing eczema at the age of 3 mo compared with the controls, In addition, the probiotic-treated group exhibited higher concentrations of lactate and SCEAS (acetate, butyrate propionate, and isobutyrate) and lower concentrations of lactose and succinate than the group who had received the placebo. Moreover, lower concentrations of SCFAs, succinate, phenylalanine, and alanine were detected in fecal samples ‘of the children who later developed eczema, whereas the amounts of glucose, galactose, lactate, and lactose were higher than those in the children who did not develop ‘eczema (78). These results emphasize the roles that SCFAs and other probiotic metabolites may play inthe regulation of the immune system, Extensively hydrolyzed casein formula (eHCF) represents an elective treatment for infants diagnosed with CMA, and supplementation with probiotics, particularly L. rhamnosus GG (eHCF + LGG), seems to accelerate antigenic tolerance (61), Regardless of changesin the gut microbial communities, after treatment with HCE, most tolerant infants showed a significant increase in fecal butyrate concentrations, which was associated with enrichment of Blautia and Roseburia species (61). Thus, HCF + LGG treatment seemsto promote tolerance in infants with CMA by influencing the bacterial ‘community structure and the capacity to produce SCFAS, mainly butyrate Both human and experimental obesity is associated with changes in the intestinal microbiota, as characterized by relatively lower abundances of Firmicutes and higher abundances of Bacteroidetes. In addition, some obesity- associated comorbidities, namely type 2 diabetes (T2D) and NAFLD, also exhibit perturbation of the intestinal micro- biota, In these conditions, both probiotics and synbioties may provide beneficial health effects because these treatments can influence the intestinal microbial ecology and immunity. A recent article by our group reviewed the effects of probiotics and synbiotics on obesity, insulin resistance syndrome, T2D, and NAFLD in a human RCT (97), Selected probiotics and synbiotics exhibited beneficial effects in patients with obesity, ‘mainly affecting the BMI and fat mass. Some probiotics had beneficial effects on insulin resistance syndrome, decreasing the concentrations of some biomarkers of cardiovascular disease. Moreover, selected probiotics improved the carbo: hydrate metabolism, fasting blood glucose concentrations, insulin sensitivity, and antioxidant status and reduced the ‘metabolic stress in subjects with T2D. Some probiotics and. synbiotics also improved the liver and metabolic markers in patients with NAFLD (97) ‘An RCT has evaluated the effects of L.salivarius Ls-33 on the fecal microbiota of obese adolescents over 12 wk. The ratio of bacteria of the Bacteroides-Prevotella-Porphyromonas group to Firmicutes, including Clostridium cluster XIV, Bautia coccoides, Eubacteria rectale, and R. intestinalis, was significantly increased after Ls-33 administration. However, the abundances of the Lactobacillus group and Bifdabac- teriur were not significant altered by the intervention, and. similarly the SCEA concentrations remained unaffected (98). De Simone formulation isa high-concentration probiotic preparation of live freeze-dried bacterial species that are normal components of the human gastrointestinal micro: biota, including 4 strains of lactobacilli (L. ease, L. plan- tarwm,L. acidophilus, and L. delbrueckii subsp. bulgaricus), 3 strains of bifidobacteria (B. longum B. breve, and B. infantis), and 8. salivarius subsp. thermophilus. De Simone formulation ‘treatment induced changes in the NAFLD urinary metabolic phenotype; these changes occurred primarily atthe level of| host amino-acid metabolism (1e. valine tyrosine, 3-amino- isobutyrate, or 8-aminoisobutyric acid), nucleic acid degra dation (pseudouridine), creatinine metabolism (methyl guanidine), and also atthe level of gut microbial amino-acid metabolism (i.e. 2-hydroxyisobutyrate from valine degra dation), Furthermore, the concentrations of some of these ‘metabolites correlated with clinical primary and secondary trial endpoints after De Simone formulation treatment, particularly alanine aminotransferase and active glucagon like peptide 1 (99). Thus, in addition to the beneficial effects of some probiotics in lowering the concentrations of liver lipids (100), the induced changes in fecal metabotite concen- trations may play an important role in the pathogenesis of NAFLD, and some of these metabolites may be considered as noninvasive effective biomarkers to evaluate the response to ‘treatment (99). Consistent with in vitro and in vivo data, SCFAs have been reported to have numerous physiologic. biochemical, and molecular effets in many tissues, including intestine, liver, adipose, muscle, and brain tissues (101). It has been proposed that acetate produced by bifidobacteria can improve the intestinal defense mediated by epithelial cells and can protect the host against lethal infection, Thus, genes encoding an ATP-binding-cassete-type carbohydrate Mechanisms of action of probiotics $55transporter present in certain bifidobacteria contribute to protecting mice against death induced by £. coli O157:H7, and this effect can be attributed, atleast in par, to increased, production of acetate and to translocation of the E. coli O1S7:H7 (102). SCFAs are an important source of energy for enterocytes and are key signaling molecules for the maintenance of gut health. In addition, SCFAs can enter the systemic circulation and interact with cell receptors in peripheral tissues. Infact, SCFAs have an important role in the regulation of energy homeostasis and metabolism, Increasing evidence, mainly derived from animal and in vitro studies, has suggested a role for SCFAs in the prevention and treatment of obesity and. obesity-related disorders in glucose metabolism and insulin resistance (101).SCFAS can interact with the SCPA receptors G protein-coupled receptor (GPR) 41 and GPR43, leading to an increase in the intestinal secretion of polypeptide YY and glucagon-like peptide 1, respectively, which, in turn, can enhance satiety (101, 103). Moreover, SCFAS might reach the adipose tissue and contribute to decreasing fat accumulation by interacting with GPR43, which would result in decreased lipolysis and inflammation and increased adipogenesis and leptin release. Propionate can increase free fatty acid uptake, possibly by affecting the lipoprotein lipase inhibitor angiopoietin-like 4. Acetate and propionate right also attenuate intracellular lipolysis via decreased hormone-sensitive lipase phosphorylation by interacting with the SCEA receptor GPR43, Similarly, acetate, propi ate, and butyrate might increase peroxisome proliferator activated receptor (PPAR)-y--mediated adipogenesis, which is possibly regulated by a GPR43-related mechanism. In addition, it has been proposed that acetate, propionate, and. butyrate, especially the latter 2, could reduce the secretion of proinflammatory cytokines and chemokines, likely by reducing local macrophage infiltration (101). Furthermore, SCFAs seem to activate AMP kinase in muscle, increasing insulin sensitivity and fatty acid oxidation and decreasing lipid accumulation (101). Figure I summarizes the potential biological effects of SCFAs in humans. Other miscellaneous biological activities of SCFAs might be attributed to probiotics asa result of epigenetic alterations, which may explain the wide range of anticarcinogenic effects attributed to probiotics (104). However, further study is needed inthis area, particularly in humans, Cell Adhesion and Mucin Production ‘When a microbial strain is indicated to be a probiotic, there are some specific prerequisites that need tobe addressed. One of them is adhesion to the intestinal mucosa for colonization and further interaction between the administered probiotic strains and the host (71). This specific interaction is required for the modulation of the antagonism against pathogens and for actions in the immune system (4, 105). Intestinal epithelial cells secrete mucin to avold the adhesion of pathogenic bacteria (72). Several Lactobacillus proteins have been shown to promote this adhesion (106), $55. PlzeDizetal exhibiting surface adhesins that facilitate attachment to the mucous layer (107). Over the last 30 the Caco-2 cell line has been extensively used to determine the adhesion capacity of probiotics in vitro (108). These cells form « homogeneous monolayer that resembles that of mature enterocytes in the small intestines of humans (109) and form crypts, which are typical structures, ofthe epithelial monolayer (7, 110). L. rhamnosus ATCC 7469 was tested in the presence of an Féexpressing E, coli strain (serotype 0149: KSI, K88ac) in intestinal porcine epithelial J2 cell. The ex pression of Toll-like receptor (TLR)-4 and nucleotide binding oligomerization domain-containing protein (NOD) 2(NOD2) wasaugmented by the presence of Ecol, and these increases were attenuated by L. rhamnosus treatment (111). Pretreatment with L. rhamnosus enhanced Akt phosphoryla tion and increased zonula occludens-1 and occludin protein expression. The probiotic maintained the epithelial barrier and promoted intestinal epithelial cell activation in response to bacterial infection (111), Inanother study the effects of 3 L. plantarum stains were «evaluated on in vivo small intestinal barrier function and gut mucosal gene transcription in human subjects. L. plantarum TIENIOL modulated gene transcription pathways; notably this probiotic upregulated the matrix metalloproteinase 2, tissue inhibitors of metalloproteinase 1 and 3, and muc? genes and downregulated genes involved inthe tricarboxylic acid cycle I pathway (112). Modulation of the Immune System ‘The gut microbiota modulates the immune system via the production of molecules with immunomodulatory and anti-inflammatory functions that are capable of stimulating immune cells. These immunomodulatory effects are due to the interaction of probiotic bacteria with epithelial cells and DCs and with monocytes/macrophages and lymphocytes (113). One of the major mechanisms of action of probiotics is the regulation of host immune response. Thus, the Immune system is divided into the innate and adaptive systems. The adaptive immune response depends on B and T lymphocytes, which bind to specific antigens. In contrast, the innate system responds to common structures, called pathogen-associated molecular patterns (PAMPs), shared by ‘a majority of pathogens. The primary response to pathogens Is produced by pattern recognition receptors (PRRs), which bind to PAMPS. Consequently, PRRs comprise TLRS, which are transmembrane proteins that are expressed on various Immune and nonimmune cells, such as B-cells, natural killer cells, DCs, macrophages, fibroblast cells, epithelial cells, and ‘endothelial cells, Furthermore, PRRs comprise nucleotide- binding oligomerization domains, adhesion molecules, and lectins (114). In addition to TLRs, PRRs include NOD-like intracellular receptors (NODLRs), which guard the eyto- plasmic space (115). Other PRRs have also been described, such as C-type lectin receptors, formylated peptide recep- tors, retinoic acid inducible-like helicases, and intracellular IL-1-converting enzyme protease-activating factor (116). Inon ao Probiotics polysaccharides lcprss tort { sates AMPK. @® Aw insulin son janet pom meee se treptn anaene | inuinsensviy Changs n neuronal oe i @u | tnt accumu ‘excitability freaupate FIGURE 1 Potential biolagcal effects of SCFAS n humans. AMPK, AMP Kinase; ANGPTL, anglopoletin-ke; GLP1, glucagon Uke pepe 1 (GPR, G protein receptor-y;PYY, polypeptide V¥. general, the TT cell subset, which Is involved in regulating. the immune balance, is finely tuned by the host and the ilcrobes with which the host interacts, and disequilibrium, between the effector T-helper (Th) cells and regulatory T cells (T-regs) leads to impaired immune response (117). Probiotic help to preserve intestinal homeostasis by modu: lating the immune response and inducing the development of, ‘Teregs (118). Modulation of sIgA and cytokine production slgA. is secreted by intestinal B cells and is expressed on the basolateral surface of the intestinal epithelium as an antibody transporter. sig facilitates the translocation of IgA dimers to the luminal surfaces of epithelial cells. Several studies have reported that probiotics show potent stimulation of the production of slgA, thereby enhanc- Ing barrier function (119). Regardless, probiotics interact With intestinal and specific immune cells, which results in the production of selected cytokines. Thus, L. salivarius CECTS713 consumption augmented the percentages of NK cells and monocytes as well as the plasmatie concentrations of immunoglobulins M, A, and G and IL-10 in healthy adult volunteers (64). In addition, L. casei Shirota increased the expression of the CD69 activation marker on circulating TT cells and NK cells and induced an increase In mucosal salivary IFN-y, IgA, and IgA? concentrations in healthy adults (120). In addition, administration of B. breve CNCM pled receptor; HS, homone-senstve lipase: LPL lipoprotein lipase PPAR-y, peroxsome prlifeator-activated 1.4035 resulted ina significant increase in fecal sIgA content; the plasmaticconcentrationsofll-4and IL-10 also increased, ‘whereas the concentrations of IL-12 decreased, in the sera of volunteers treated with this strain. Similar results have been obtained with 2 other probiotic strains, L. shammosus and casei (63) Recently, a probiotic strain (E. faecium ALA) was pro: posed tobe effective against Campylobacter jejuni infection in chickens. E. faecium modulates the expression of transform. ing growth factor (TGF)-p4 but downregulates the relative expression of IL-17 and activates IgA-producing cells in the caeca of chicks infected with C. jejuni (121). In mice, treatment with L. rhamnosus RCOO7 for 10 d increased the phagocytic activity of peritoneal macrophages and the number of IgA cells in the lamina propria of the small intestine. Consequently, higher concentrations of monocyte chemoattractant protein 1 (MCP-1), I-10, and INF were observed, and the ratio of anti- to proinflammatory cytokines (IL-10/TNF-a) inthe intestinal fluid increased after L rhamnosus RCOO7 treatment (122 Another Lactobacillus strain, L. plantarum O6CC2, is capable of increasing the concentration of IL-12 in co-culture with 1774.1 cells and oral administration induced Th cytokine production, activating the Tal immune response associated ‘with intestinal immunity in normal mice (123). Aktas tal. (124) investigated diferent L. casei strains for their ability to alter the murine gut microbiota. They observed that L. casei Mechanisms of action of probiotics $57species are capable of modulating the host gut microbiota and the host immune system because there is a relation between the ability ofa strain to alter the composition of the gut microbiota, PRR regulation, and antimicrobial peptide regulation (124) In addition, bifidobacteria strains also modulate the immune sysiem, Accordingly, B. longum subsp. infantis 35624 is a probiotic with immunoregulatory effects, and. it has been described that the consumption of B. longum subsp. infantis 35624 resulted in the induction of T-regs and attenuation of nuclear transcription factor xB (NF-«B) activation, preventing the excessive inflammation induced by Salmonella infection in mice. Induction of T-regs by the strain has also been shown in humans, and reduction of systemic proinflammatory biomarkers has been seen in patients with psoriasis, IBS, chronic fatigue syndrome, or ulcerative colitis (125, 126). B. breve C30 releases soluble fac tors thatalleviate the secretion of proinflammatory cytokines by immune cells. Thus. a study carried out by Heuvelin et al. (127) elucidated that B, breve C50 and the soluble factors secreted by this bacterium contribute positively to intestinal homeostasis by attenuating chemokine production, such as CXCLS secretion by epithelial cells driven by Jun proto-oncogene, AP-1 transcription factor subunit and: IkBea and decreased phosphorylation of p38-MAPK and. IkB-a molecules (127). ‘A probiotic mixture, De Simone formulation, induces NF. ‘«Bnuclear translocation in epithelial cells, which is followed by the release of TNF-c, and this effect correlates with reduced epithelial permeability and susceptibility to CD. like iletis in SAMPL/YitFc mice that spontaneously develop the disease. It has been recently shown that TNF-a can stimulate epithelial cell proliferation, and this stimulation occurs only when TNF-a induces epithelial cll apoptosis in combination with IFN-y. Hence, it is possible that probioticscan participate in epithelial barrier regeneration by “upregulating TNF-a (118, 128). A numberof selected species of lactobacilli and bifidobacteria have been demonstrated to be prominent probiotics with ant-inflammatory properties, suppressing proinflammatory responses by increasing the concentrations of IL-10 and Thi-type cytokines. Kwon et al. (128) identified a mixture of probiotics that upregulates CD4*forkhead box P3 (FoxP3)* T-regs. Thus, administra tion of the probiotic mixture induced both T cell and B. cell hyporesponsiveness and downregulated Thi, Th2, and. ThI7 cytokines without inducing apoptosis. The probiotic mixture also induced the production of CD4*FoxP3* T- regs from the CD4*CD25— population and increased the suppressor activity of naturally occurring CD4*CD25* T- regs. Conversion of T-cells to FoxP3* T-regs is directly mediated by regulatory DCs that express high levels of IL 10, TGF-, cyclooxygenase (COX)-2, and indoleamine 2,3 dioxygenase. Administration of the probiotic mixture had. therapeutic effects in experimental treatments of IBD, atopic dermatitis, and rheumatoid arthritis. Overall,administration of probiotics that enhance the generation of regulatory DCs and T-regs represents an applicable treatment for inflammatory immune disorders (129). $58 Plz Dizetal Interaction of probiotics with TLRs and cell cascade signaling ‘TLRs are a family of evolutionarily conserved PRRs that recognize a wide range of microbial components, In mam- mals, the TLR family includes 11 proteins (TLR1~TLR1), and the activation of TLRs occurs after the binding of the ligand to extracellular leucine-rich repeats. Inhumans, TLR1, ‘TLR2, TLR4, TLRS, TLR6, and TLR1O are associated with the outer membrane and primaeily respond to bacterial surface-associated PAMPs. TLR3, TLR7, TLRS, and TLRS are found on the surfaces of endosomes, where they respond primarily to nucleic-acid-based PAMPs from viruses and bacteria, The TLR signaling pathway, with the exception of TLRS, involves the recruitment of myeloid differentia tion primary response 88, which activates the MAPK and NF-« B signaling pathways. TLR3 utilizes the adaptor protein ‘TiR-domain-containing adapter-inducing IFN-f, leading to the expression of type | IFNs. TLR-mediated signaling has been shown to control DC maturation. TLRS signaling is ‘essential for the mediation of the anti-inflammatory effect of| probiotics (71, 114). Figure 2 summarizes the interaction of probiotics with TLRs. Probiotics are capable of suppressing intestinal inflam. ‘mation via the downregulation of TLR expression, secretion ‘of metabolites that may inhibit TNF-a from entering blood mononuclear cells, and inhibition of NF-«B signaling in enterocytes (130), In this sense, signaling by cell wall com. ponents of lactobacilli can potentially occur via the binding ‘of TLR2 and TLR6, stimulating cytokine production, In addition, TLR2 recognizes peptidoglycan, which is the main ‘component of gram-positive bacteria, including those of the Lactobacilus genus. L, casei 431 interacts with epithelial cells via TLR2, and the interaction between L. casei and gut- associated immune cells induces an increase in the number ‘of CD.-206 and TLR2 receptors (131). Indeed, several strains, such as L. plantarum CCFM634, L. plantarum CCFM734, L. fermentum CCEMB81, L. acidophilus CCPMI37, and S. thermophilus CCFM218, stimulate TLR2/TLR6, and these interactions between PRRs such as TLRs are strain specific. ‘Thus, TLR2/TLR6 signaling is essential in immune regula tory processes (132). In addition, Shida etal. (133) showed that L. casei induces a high amount of IL-12 production in both wild-type and TLR2-deficient macrophages and that peptidoglycan induces low amounts of I-12 production in ‘wild-type macrophages and even lower amounts in TLR2- eficient macrophages (133). Moreover, L. rhamnosus GG and L. plantarum BFE 1685 enhance TLR2 activity in human Intestinal cells, and L. casei CRL 431 has similar effects in mice infected with Salmonella enterica serovar typhimurium (134, 133). Furthermore, L. plantarum was shown to acti- vate TLR2 signaling, and subsequently, protein kinase C-ar and -5 activation has also been implicated in tight-junction modulation and epithelial permeability (136). With regard to lactobacilli, L. casei DG and its postbiotic modulate the inflammatory/immune response in postinfec tion IBS in an ex vivo organ culture model. Thus, IL-La, IL-6, and IL-8 mRNA levels and TLR4 protein expression ‘were significantly higher, whereas IL-10 mRNA levels wereLactobacilli Lactobacilli 1. casei DG spmprte Wy J Plagettin nae i : mur B. breve L rhamnosus GG, Inflammasome ‘Viral ssRNA TUR? FIGURE 2. Main effects of probiotics on the immune system. ASC, apoptosis assoclated Speck ike protein containing a CARD: breve, Bindobacterium breve, CoGONA, Cytosine-phosphate-quanasine DNA; dsRNA, Double strand DNA, ERK, extracellular regulated kinase; IK 1 kinase: RAKA, IL-1 receptor-associated kinase 4 JNK, un N-terminal kinase. case Lactobacilus case. rhamnosus Lactobacls rhamnosus, My08, myeloid ferentiation primaty response 88; NEMO, NF-x8 essential modulator, NF nuclear transcription factor NLR nucleotie-binding olgomerzation domain-tke receptors, n short NOD Ike receptors; NLRP3,NLR family pyrin domain containing 3;P Phosphate; sRNA, TABI/2/3, TAK binding protein, TAKI, ubiquitn-dependent kinase of putative mitogen-actvated protein kinase (NRK) and IKK; TBK1, seine/threonine-protein kinase 1; TLR, Toll-like receptor, TRAFE, tumor necross factor receptor associated factor 6 ‘Tat, TF-domain-containing adapter-inducing interferor-B, Vital ssRNA: Viral single strand ONA lower, in postinfection IBS than in healthy controls in both the ileum and colon. L. casei DG and postbiotic significantly reduced the mRNA levels of the proinflammatory cytokines TL-la, IL-6, and IL-8 and TLR4, whereas these bacteria Increased the mRNA levels of IL-10, in both the ileum and colon after LPS stimulation. Therefore, there was an attenuation of inflammatory mucosal response in an ex vivo organ culture model of postinfection IBS (137). Bifidobacteria also stimulate TLR2, and specifically, B breve C50 induces maturation and IL-10 production and prolongs DC survival (138). Similarly, Zeuthen et al. (139) showed that TLR2”~ DCs produce mare IL-2 and less IL-10 Jn response to bifidobacteria, and the authors concluded that the immunoinhibitory effect of bifidobacteria is dependent on TLR2 (71, 139). B. bifidum OLB 6378 stimulates TLR2 expression in the ileal epithelium and enhances COX-2 expression, increasing the production of prostaglandin E2 in rats with NEC, However, the specific mechanism for this phenomenon has not been elucidated (140). Inanother study, in which dysbiosis was induced inthe rat intestine, treatment Mechanisms of action of problotes $58with a probiotic mixture of 4 strains, namely B, breve DM8310, L. acidophilus DM8302, L. casei DM8121, and 5. ‘thermophilus DM8309, ameliorated the injury to the mucosal barrier, reduced the concentrations of proinflammatory factors and cytokines, and reduced neutrophil infiltration ‘These results are closely associated with the re-establishment of intestinal microbial homeostasis and alteration of the TLR2 and TLR4 signaling pathways (141). TLRS is another relevant TLR that is activated by probi otics, and in vivo, TLRS exhibits anti-inflammatory effects at the epithelial surface, Hence, TLR9 activation induces intracellular signaling pathways via the apical and basolateral surfaces, and TNF-«--induced NF-« Bis expressed. Thus, the abilities of different probiotic species to stimulate TLR® are likely to be different. TLR9 triggers Ik Ba: degradation and. NE-«B pathway activation, whereas apical TLR9 induces cy- toplasmic accumulation of ubiquitinated la B and inhibition of NE-«B activation (71, 142). Different strains such as B. breve, L. rhammosus, and L. casei induce different amounts of cytokine production in human and mouse primary immune cells. Thus, B. breve induces cytokine production in a TLR9-dependent manner, and the lover inflammatory profile is due to the inhibitory effects of TLR2 (143), In addition, purified genomic DNA, from L. plantarum inhibits LPS-induced TNF production and reduces TLR2, TLR4, and TLR9 gene expression in THP- 1 calls (144), A recent study demonstrated that transplantation of the hhuman gut microbiota into pigs via different dosing regimens, of L rhamnosus GG affected intestinal bacterial communities and modulated the responses of the immune signaling pathway to an oral attenuated human rotavirus vaccine. The authors reported that pigs treated with 9 doses, but not those treated with 14 doses, of L. rhamnosus GG exhibited en: hanced IL-6, IL-10, TNF-a, and TLR9 mRNA levels and p38. MAPK and extracellular regulated kinase expression in ileal mononuclear cells. Therefore, 9 doses of L, rhamnosus GG ‘were more effective in activating the TLR9 signaling pathway than 14 doses in human-gut-microbiota-containing pigs vaccinated with attenuated human rotavirus vaccine (119) ur research group has previously reported that L paracasei CNCM_1-4034 and. the culture supernatant of L. paracaseé CNCM 1-4034 modulate Salmoneta-induced inflammation in a novel trans-well co-culture of human Intestnal-like dendriticand Caco-2 cells. paracasei CNCM 1-4034 significantly increased the IL-1, IL-6, IL-8, TGF. '22; regulated upon activation, normal T cell expressed and. secreted (RANTES); and 1P-10 levels and decreased the IL-12p40, IL-10, TGF-f1, and macrophage inflammatory protein (MIP)-1e levels in DCs through a physical barrier of Caco-2 cells. In contrast, incubation of the co-culture with cell-free culture supernatants increased IL-1, IL-6, TGF. 2, and IP-10 production only when S. typhi was present. ‘This induction was correlated with an overall decrease in the expression of all TLR genes except TLRS, which was strongly upregulated (145) With regard to intestinal diseases, rhamnosus HINOO1 hasbeen reported to have beneficial activity for the treatment $60 PlzeDizetal of inflammatory diseases such as NEC, Hence, the microbial DNA of L. rhanmosus HNOOL can activate TLRS, attenuating NEC in vitro, and no evidence of toxicity has been described (146) ‘With regard to the role of probiotics in reducing allergies, the underlying mechanisms might include shifting the Iymphocyte Th1/Th2 balance toward a Thi response and consequent decreased secretion of Th2 cytokines, such as IL-4, IL-5, and IL-13, as well as decreased IgE concentra- tions and increased production of C-reactive protein and Iga (56). Interaction with the brain-gut axis Jn social groups, individuals who interacted. physically through social grooming harbored more similar commu- nities of gut bacteria to each other (147). This degree of social interaction explained why there was variation in the gut microbiota even after controlling for diet, host ‘genetics, and shared environment. Social transmission of the microbiota may be beneficial for propagating the microbes themselves, and some evidence suggests that socially transmitted microbiota could confer beneficial effects to the ‘host communities as well (148). ‘The intestinal microbiota, the brain-gut signaling system, and the interaction ofthe microbiota with genetic receptors have been shown tobe associated with the health of children and with the development of short- and long-term behavior (148). The role ofthe gut microbiota in health and disease in the first years of life has become very relevant because fof evidence that the gut microbiota can influence many aspects of human behavior (150). Preterm infants differ from term infants in that preterm infants are particularly ‘vulnerable to the effects of stress and pain. Stress activates the hypothalamic: pitutary-adrenal axis and the sympathetic nervous system, which increases intestinal permeability and allows bacteria and bacterial antigens to cross the epithelial barrie activate the mucosal immune response, and alter the composition of the microbiome (151). In addition, oxidative stress inthe intestine modulates the process of microbiome establishment in preterm infants (152) Autism spectrum disorder (ASD) isa severe neurodevel- ‘opmental disorder that impairs a childs ability to commu- nicate and interact with others. Children with neurodevel- ‘opmental disorders, including ASD, are regulaey affected by {gastrointestinal problems and dysblos's af the gut microbiota (153) For example, Hsiao et al. (154) demonstrated that B. ‘Fragilis may playa role in the improvement in ASD -associated ‘behaviors (154). Recently published data have linked the Inckdence of ASD with maternal obesity and diabetes (153, 156). high-fat maternal diet was administered to mice with the objective of inducing impaired social behavior in the offspring, and subsequently the animals were administered L. reuteri, Administration of L. reuteri failed to mitigate ansety but was able to restore oxytocin concentrations, the mesolimbic dopamine reward system, and social be- the offspring that were fed a high-fat maternal. es. (soe BO BSe— ferire| te > a we os Bn Fate ' 1 e io Sun thereover ar FIGURE 3. Probiotic mechanisms of action (A) Colonization and normalization of perturbed intestinal microbial communes in children {and adults and competitive excision of pathogens and bacteriocin production; (8) enzymatic activity and production of volatile fatty acids (C) cell adhesion, cell antagonism, and mucin production; (0) modulation ofthe immune system, and (] interaction with the bran-gut ans. AMPK, AMP kinase; ANGFTL, anglopoietin like; DC, dendritic cell FoxP3, forkhead box P3; GLP, glucagon- ike peptide; GPR, G protein-coupled receptor HSL. hormone-sensitive lipase; IFN, Interferon; IRAKT, IL-1 receptor associated kinase LPL lipoprotein lipase; "MyD8, myeloid differentiation primary response 88; NFB, nuclear transcription factor xB; PPAR-y, peroxisome prolifeator-activated receptor-y; PY, polypeptide YY; TGF. transfering qrow factor, TLR Tolbike receptor, TRF, TR-domain-cantaining adapter inducing interferon. Dinan et al. (158) demonstrated that stress caused by physical or psychological factors might be directly associ ated with the imbalance of the microbiota-brain-gut axis. Messaoudi et al. (159) showed that the consumption of I. hrelveticus ROOS2 and B. longum RO175 reduced symptoms ‘of depression in healthy human volunteers (160). Recently, changes in brain structure were found to be associated with diet-dependent changes in gut microbiome populations through the use of a machine learning classifier to quan: titatively assess the strength of microbiome-brain region associations (161), In general, the mechanisms underlying the effects of the gut intestinal microbiota on the central nervous system ‘are multifactorial (neural, endocrine, and. immunologic), but these effects are believed to principally occur via the generation of bacterial metabolites (161). SCFAs alter neuronal excitability, and gut bacteria manufacture a wide spectrum of neuroactive compounds, including dopamine, y-aminobutyric acid, histamine, acetylcholine, and trypto- pphan, which isa precursor in the biosynthesis of serotonin, “Although aelditional research is needed to test the causal- ity and directionality of the association between the micro- biota and social behavior, these initial studies have asked whether microbiota-mediated changes in social behavior affect social transmission of the microbiota and whether these interactions have consequences for both host and microbial fitness Finally, Figure 3 summarizes the mechanisms of action considered in the present review, Conclusions Probiotics are safe microorganisms that when administered to human subjects in adequate doses and at appropriate periods confer some beneficial effects to the host. The Mechanisms of action of probiotics $61mechanisms of action of probiotics involve colonization and normalization of perturbed intestinal microbial com. ‘munities in both children and adults; competitive exclusion of pathogens and bacteriocin production; modulation of enzymatic activities related to metabolization of a number of carcinogens and other toxic substances: and production of 12 volatile fatty acids, namely, SCFAs and BCFAs, which play a role in the maintenance of energy homeostasisand regulation of functionality in peripheral tissues, In addition, probiotics Increase intestinal cell adhesion and mucin production and 4 modulate the activity of gu-associated lymphoid tissue and the immune system. Similarly, probiotic metabolites are able to interact with the brain-gut axis and play a role in behavior. All the aforementioned mechanisms of action ‘Bran, Quigley EM. The eficayof probiotics inthe treatment of should encourage investigators, companies, stakeholders, _jrishtorel vate nontonstc evens at aoluso 1 and consumers to learn about the effects of probiotics as a 16 Goldenberg 12. Yap C. Iytnm L, Lo CK, Beasley , Mertz D, ‘whole and evaluate those strains that show promising results, ston C. Probie forthe prevention of Clr die These steps toward establishing “good science” may result in__ssste arhen in adults and chlten, Cochrane Database St the approval of health claims in the near future. alee the app 17, Suer-Lara MY, Gomer-Locete C, Para-Diz J, Gil A. The role of| probit ci all bacteria nd bidbacteria the pevetion ad {weatmentof inflammatory bowel disease and other related diseases systematic review ofrandomized human clini ris Bled Rest 201505878, 1A. Rather 1, Baa VK, Kumar Li J, Pack WK, Prk YH, Probiotics an sopcdermti an overview: Front Microbiol 2167507 19. Beings Mi Kraan C AltnbulklC, Gevaert, Aki M, Bacher .AltsCA, Advances and hihlghsin allergen immunotherapy: on thewayo sustain inal nim lean Alergy Clin nnn 2017140:1250-67 20, FSA. Gaidance onthe scenic requirements fr heath eis tebe othe imine system the gastontesial tract and defence sean pathogenic mcrorganiams EESA J 2016144369. DDegnan FH. The US Food and Drug Administration and probit regulatory categorization. Clin inf Dis 06 46S133-6 22, FDA. Dietary suploments- Now Dictary Ingreietnoifsions inflammation gnes upregulated inthe intestinal mocosaofabese as, ae downregulated by tree probiotic strains. Si Rep 201771838, ‘ling K, Barbosa T, Penna G, Capri F,Sonzogni A, Vale Rescigno M.Problotc and posit activity in heath and disease comparison on a novel polarised exvivo onan culture model, Gut 2012361100715 Didar T, Solis, Moar, Nikfar 8, Abdolai ML A systematic view ofthe safety of probit, Expert Opin Deg Sif2014313227- ». 13, Szujewska H. 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