Garlic consumption in relation to colorectal cancer risk and to alterations of blood bacterial DNA.

PURPOSE: Garlic consumption has been inversely associated to intestinal adenoma (IA) and colorectal cancer (CRC) risk, although evidence is not consistent. Gut microbiota has been implied in CRC pathogenesis and is also influenced by garlic consumption. We analyzed whether dietary garlic influence CRC risk and bacterial DNA in blood. METHODS: We conducted a case-control study in Italy involving 100 incident CRC cases, 100 IA and 100 healthy controls matched by center, sex and age. We used a validated food frequency questionnaire to assess dietary habits and garlic consumption. Blood bacterial DNA profile was estimated using qPCR and16S rRNA gene profiling. We derived odds ratios (ORs) and the corresponding 95% confidence intervals (CIs) of IA and CRC according to garlic consumption from multiple conditional logistic regression. We used Mann-Whitney and chi-square tests to evaluate taxa differences in abundance and prevalence. RESULTS: The OR of CRC for medium/high versus low/null garlic consumption was 0.27 (95% CI = 0.11-0.66). Differences in garlic consumption were found for selected blood bacterial taxa. Medium/high garlic consumption was associated to an increase of Corynebacteriales order, Nocardiaceae family and Rhodococcus genus, and to a decrease of Family XI and Finegoldia genus. CONCLUSIONS: The study adds data on the protective effect of dietary garlic on CRC risk. Moreover, it supports evidence of a translocation of bacterial material to bloodstream and corroborates the hypothesis of a diet-microbiota axis as a mechanism behind the role of garlic in CRC prevention.

Reduction of Staphylococcus epidermidis in the mammary tumor microbiota induces antitumor immunity and decreases breast cancer aggressiveness.

The mammary gland hosts a microbiota, which differs between malignant versus normal tissue. We found that aerosolized antibiotics decrease murine mammary tumor growth and strongly limit lung metastasis. Oral absorbable antibiotics also reduced mammary tumors. In ampicillin-treated nodules, the immune microenvironment consisted of an M1 profile and improved T cell/macrophage infiltration. In these tumors, we noted an under-representation of microbial recognition and complement pathways, supported by TLR2/TLR7 protein and C3-fragment deposition reduction. By 16S rRNA gene profiling, we observed increased Staphylococcus levels in untreated tumors, among which we isolated Staphylococcus epidermidis, which had potent inflammatory activity and increased Tregs. Conversely, oral ampicillin lowered Staphylococcus epidermidis in mammary tumors and expanded bacteria promoting an M1 phenotype and reducing MDSCs and tumor growth. Ampicillin/paclitaxel combination improved the chemotherapeutic efficacy. Notably, an Amp-like signature, based on genes differentially expressed in murine tumors, identified breast cancer patients with better prognosis and high immune infiltration that correlated with a bacteria response signature. This study highlights the significant influence of mammary tumor microbiota on local immune status and the relevance of its treatment with antibiotics, in combination with breast cancer therapies.

Flavonoid Intake in Relation to Colorectal Cancer Risk and Blood Bacterial DNA.

Flavonoids have been inversely associated to colorectal cancer (CRC) and are plausible intermediaries for the relation among gut microbiome, intestinal permeability and CRC. We analyzed the relation of flavonoid intake with CRC and blood bacterial DNA. We conducted a case-control study in Italy involving 100 incident CRC cases and 200 controls. A valid and reproducible food-frequency questionnaire was used to assess dietary habits and to estimate six flavonoid subclass intakes. We applied qPCR and 16S rRNA gene profiling to assess blood bacterial DNA. We used multiple logistic regression to derive odds ratios (ORs) of CRC and Mann-Whitney and chi--square tests to evaluate abundance and prevalence of operational taxonomic units (OTUs) according to flavonoid intakes. Inverse associations with CRC were found for anthocyanidins (OR for the highest versus the lowest tertile = 0.24, 95% confidence interval, CI = 0.11-0.52) and flavanones (OR = 0.18, 95% CI = 0.08-0.42). We found different abundance and prevalence according to anthocyanidin and flavanone intake for OTUs referring to Oligoflexales order, Diplorickettsiaceae family, Staphylococcus, Brevundimonas, Pelomonas and Escherischia-Shigella genera, and Flavobacterium and Legionella species. The study provides evidence to a protective effect of dietary anthocyanidins and flavanones on CRC and suggests an influence of flavonoids on blood bacterial DNA, possibly through intestinal permeability changes.

The relevance of urolithins-based metabotyping for assessing the effects of a polyphenol-rich dietary intervention on intestinal permeability: A post-hoc analysis of the MaPLE trial.

A polyphenol-rich diet reduced intestinal permeability (IP) in older adults. Our aim was to evaluate if participants categorized according to urolithin metabotypes (UMs) exhibited different responses in the MaPLE trial. Fifty-one older adults (mean age: 78 years) completed an 8-week randomized-controlled-crossover trial comparing the effects of a polyphenol-rich vs. a control diet on IP, assessed through zonulin levels. Plasma and urinary metabolomics were evaluated with a semi-targeted UHPLC-MS/MS method. Gut microbiota was characterized by 16S rRNA gene profiling. UMs were determined according to urolithin excretion in 24 h urine samples. Multivariate statistics were used to characterize the differences in metabolomic and metataxonomic responses across UMs. Thirty-three participants were classified as urolithin metabotype A (UMA), 13 as urolithin metabotype B (UMB), and 5 as urolithin metabotype 0 (UM0) according to their urinary excretion of urolithins. Clinical, dietary, and biochemical characteristics at baseline were similar between UMs (all p > 0.05). After the polyphenol-rich diet, UMB vs. UMA participants showed a 2-fold higher improvement of zonulin levels (p for interaction = 0.033). Moreover, UMB vs. UMA participants were characterized for alterations in fatty acid metabolism, kynurenine pathway of tryptophan catabolism, and microbial metabolization of phenolic acids. These changes were correlated with the reduction of zonulin levels and modifications of gut microbes (increased Clostridiales, including, R. lactaris, and G. formicilis). In conclusion, urolithin-based metabotyping identified older adults with a higher improvement of IP after a polyphenol-rich diet. Our results reinforce the concept that UMs may contribute to tailor personalized nutrition interventions.

Apyrase-mediated amplification of secretory IgA promotes intestinal homeostasis.

Secretory immunoglobulin A (SIgA) interaction with commensal bacteria conditions microbiota composition and function. However, mechanisms regulating reciprocal control of microbiota and SIgA are not defined. Bacteria-derived adenosine triphosphate (ATP) limits T follicular helper (Tfh) cells in the Peyer's patches (PPs) via P2X7 receptor (P2X7R) and thereby SIgA generation. Here we show that hydrolysis of extracellular ATP (eATP) by apyrase results in amplification of the SIgA repertoire. The enhanced breadth of SIgA in mice colonized with apyrase-releasing Escherichia coli influences topographical distribution of bacteria and expression of genes involved in metabolic versus immune functions in the intestinal epithelium. SIgA-mediated conditioning of bacteria and enterocyte function is reflected by differences in nutrient absorption in mice colonized with apyrase-expressing bacteria. Apyrase-induced SIgA improves intestinal homeostasis and attenuates barrier impairment and susceptibility to infection by enteric pathogens in antibiotic-induced dysbiosis. Therefore, amplification of SIgA by apyrase can be leveraged to restore intestinal fitness in dysbiotic conditions.

A Polyphenol-Rich Diet Increases the Gut Microbiota Metabolite Indole 3-Propionic Acid in Older Adults with Preserved Kidney Function.

SCOPE: Dietary polyphenols can alter the gut microbiota (GM) and promote the production of bioactive metabolites. Several indoles result of GM metabolism of dietary tryptophan have been associated with intestinal barrier integrity. Our aim is to study the changes in GM-derived indoles during a polyphenol-rich (PR) diet intervention in older adults. METHODS AND RESULTS: Randomized, controlled, crossover trial in adults ≥ 60 years living in a residential care facility during an 8-week PR versus control diet (n = 51). Seven GM-tryptophan metabolites are measured in serum, and metataxonomic analysis of GM is performed on fecal samples. Exploratory subgroup analyses are performed based on renal function (RF). The PR-diet significantly increases serum indole 3-propionic acid (IPA) in subjects with normal RF, but not in subjects with impaired RF. Other GM-tryptophan metabolites are not affected. Comparison of baseline GM composition shows shifts in Bacteroidales order members as well as higher abundance of Clostridiales in participants with normal RF. During the trial, variations of IPA are associated with changes in C-reactive protein (ß = 0.32, p = 0.010) and GM, particularly with the Clostridiales (r = 0.35, p < 0.001) and Enterobacteriales (r = -0.15, p < 0.05) orders. CONCLUSION: A PR diet increases the serum concentration of IPA in older adults with normal RF. Our findings may be important when defining appropriate dietary interventions for older adults. TRIAL REGISTRATION NUMBER: ISRCTN10214981 (https://doi.org/10.1186/ISRCTN10214981).

Higher bacterial DNAemia can affect the impact of a polyphenol-rich dietary pattern on biomarkers of intestinal permeability and cardiovascular risk in older subjects.

PURPOSE: Aging can be characterized by increased systemic low-grade inflammation, altered gut microbiota composition, and increased intestinal permeability (IP). The intake of polyphenol-rich foods is proposed as a promising strategy to positively affect the gut microbiota-immune system-intestinal barrier (IB) axis. In this context, we tested the hypothesis that a PR-dietary intervention would affect the presence of bacterial factors in the bloodstream of older adults. METHODS: We collected blood samples within a randomized, controlled, crossover intervention trial in which older volunteers (n = 51) received a polyphenol-enriched and a control diet. We quantified the presence of bacterial DNA in blood by qPCR targeting the 16S rRNA gene (16S; bacterial DNAemia). Blood DNA was taxonomically profiled via 16S sequencing. RESULTS: Higher blood 16S levels were associated with higher BMI and markers of IP, inflammation, and dyslipidemia. PR-intervention did not significantly change bacterial DNAemia in the older population (P = 0.103). Nonetheless, the beneficial changes caused by the polyphenol-enriched diet were greatest in participants with higher bacterial DNAemia, specifically in markers related to IP, inflammation and dyslipidemia, and in fecal bacterial taxa. Finally, we found that the bacterial DNA detected in blood mostly belonged to γ-Proteobacteria, whose abundance significantly decreased after the polyphenol-rich diet in subjects with higher bacterial DNAemia at baseline. CONCLUSIONS: This study shows that older subjects with higher bacterial DNAemia experienced a beneficial effect from a polyphenol-rich diet. Bacterial DNAemia may be a further relevant marker for the identification of target populations that could benefit more from a protective dietary treatment. REGISTRATION: This trial was retrospectively registered at www.isrctn.org (ISRCTN10214981) on April 28, 2017.

Blood Bacterial DNA Load and Profiling Differ in Colorectal Cancer Patients Compared to Tumor-Free Controls.

Inflammation and immunity are linked to intestinal adenoma (IA) and colorectal cancer (CRC) development. The gut microbiota is associated with CRC risk. Epithelial barrier dysfunction can occur, possibly leading to increased intestinal permeability in CRC patients. We conducted a case-control study including 100 incident histologically confirmed CRC cases, and 100 IA and 100 healthy subjects, matched to cases by center, sex and age. We performed 16S rRNA gene analysis of blood and applied conditional logistic regression. Further analyses were based on negative binomial distribution normalization and Random Forest algorithm. We found an overrepresentation of blood 16S rRNA gene copies in colon cancer as compared to tumor-free controls. For high levels of gene copies, community diversity was higher in colon cancer cases than controls. Bacterial taxa and operational taxonomic unit abundances were different between groups and were able to predict CRC with an accuracy of 0.70. Our data support the hypothesis of a higher passage of bacteria from gastrointestinal tract to bloodstream in colon cancer. This result can be applied on non-invasive diagnostic tests for colon cancer control.

Data on cecal and fecal microbiota and predicted metagenomes profiles of female mice receiving whole flaxseed or its oil and secoisolariciresinol diglucoside components.

Dietary flaxseed (FS) and its components including FS oil (FSO), secoisolariciresinol diglucoside (SDG) and fiber, are processed by the gut microbiota. These data are in support of the article entitled "Discriminatory and cooperative effects within the mouse gut microbiota in response to flaxseed and its oil and lignan components", Journal of Nutritional Biochemistry [1]. Here we describe data generated by 16S rRNA sequencing of DNA obtained from cecum contents and feces of C57BL/6 female mice fed either a basal diet (BD, AIN93G), or isocaloric diets containing 10% FS, or 10% FS-equivalent amounts of FSO or SDG for 21 days. These include bacterial community composition and inferred KEGG pathways; the raw data are publicly available at the NCBI SRA database (BioProject ID PRJNA683934). Furthermore, this work includes detailed experimentation procedures, total bacterial counts (qPCR) in the cecum content and feces, and correlation analysis between a selected bacterial genus, Bacteroides and a predicted metabolic pathway. FS is utilized worldwide, especially for the prevention and/or treatment of diseases including cardiovascular diseases, diabetes and cancer. These data will be valuable as a reference to study different FS cultivars and SDG- or FSO- enriched products on the gut microbiota, to study gut microbial responses to FS and its components in different mouse strains and mammalian hosts to elucidate individualized effects, and to understand the importance of the gut microbiota for FS benefits.

Crosstalk among intestinal barrier, gut microbiota and serum metabolome after a polyphenol-rich diet in older subjects with "leaky gut": The MaPLE trial.

BACKGROUND &AIM: The MaPLE study was a randomized, controlled, crossover trial involving adults ≥60 y.o. (n = 51) living in a residential care facility during an 8-week polyphenol-rich (PR)-diet. Results from the MaPLE trial showed that the PR-diet reduced the intestinal permeability (IP) in older adults by inducing changes to gut microbiota (GM). The present work aimed at studying the changes in serum metabolome in the MaPLE trial, as a further necessary step to depict the complex crosstalk between dietary polyphenols, GM, and intestinal barrier. METHODS: Serum metabolome was monitored using a semi-targeted UHPLC-MS/MS analysis. Metataxonomic analysis (16S rRNA gene profiling) of GM was performed on faecal samples. Clinical characteristics and serum levels of the IP marker zonulin were linked to GM and metabolomics data in a multi-omics network. RESULTS: Compared to the control diet, the PR-diet increased serum metabolites related to polyphenols and methylxanthine intake. Theobromine and methylxanthines, derived from cocoa and/or green tea, were positively correlated with butyrate-producing bacteria (the order Clostridiales and the genera Roseburia, Butyricicoccus and Faecalibacterium) and inversely with zonulin. A direct correlation between polyphenol metabolites hydroxyphenylpropionic acid-sulfate, 2-methylpyrogallol-sulfate and catechol-sulfate with Butyricicoccus was also observed, while hydroxyphenylpropionic acid-sulfate and 2-methylpyrogallol-sulfate negatively correlated with Methanobrevibacter. The multi-omics network indicated that participant's age, baseline zonulin levels, and changes in Porphyromonadaceae abundance were the main factors driving the effects of a PR-diet on zonulin. CONCLUSION: Overall, these results reveal the complex relationships among polyphenols consumption, intestinal permeability, and GM composition in older adults, and they may be important when setting personalized dietary interventions for older adults. TRIAL REGISTRATION NUMBER: ISRCTN10214981.

Discriminatory and cooperative effects within the mouse gut microbiota in response to flaxseed and its oil and lignan components.

Gut microbial processing of dietary flaxseed (FS) contributes to its health benefits, but the relative effects of its bioactive components (lignans, omega-3 fatty acids, fiber) on the microbiota are unclear. We investigated the gut microbial compositional and functional responses to whole FS and its isolated components, FS oil (FSO) and secoisolariciresinol diglucoside (SDG) (precursor to microbial-derived enterolignans) to help understand their contribution to whole FS benefits. Cecum content and fecal samples were collected from C57BL/6 female mice fed a basal diet (AIN93G) or isocaloric diets containing 10% FS or 10% FS-equivalent amounts of FSO or SDG for 21 days. Cecal and fecal microbiota composition and predicted genomic functions, and their relationship with serum enterolignans were evaluated. Only FS modified the community structure. Shared- and diet-specific enriched taxa and functions were identified. Carbohydrate and protein processing functions were enriched in FS mice, and there was a positive correlation between select enriched taxa, encompassing fiber degraders and SDG metabolizers, and serum enterolignans. This was not observed in mice receiving isolated FSO and SDG, suggesting that FS fiber supports SDG microbial metabolism. In conclusion, the cooperative activities of a diverse microbiota are necessary to process FS components and, when administered at the amount present in FS, these components may act together to affect SDG-derived enterolignans production. This has implications for the use of FS, FSO and SDG in clinical practice.

Association between Food Intake, Clinical and Metabolic Markers and DNA Damage in Older Subjects.

The use of DNA damage as marker of oxidative stress, metabolic dysfunction and age-related diseases is debated. The present study aimed at assessing the level of DNA damage (evaluated as DNA strand-breaks, endogenous and oxidatively-induced DNA damage) in a group of older subjects with intestinal permeability enrolled within the MaPLE (Gut and Blood Microbiomics for Studying the Effect of a Polyphenol-Rich Dietary Pattern on Intestinal Permeability in the Elderly) intervention trial, to evaluate its association with clinical, metabolic and dietary markers. DNA damage in peripheral blood mononuclear cells was assessed by the comet assay in 49 older subjects participating in the study. Clinical and metabolic markers, markers of inflammation, vascular function and intestinal permeability were determined in serum. Food intake was estimated by weighted food diaries. On the whole, a trend towards higher levels of DNA damage was observed in men compared to women (p = 0.071). A positive association between DNA damage and clinical/metabolic markers (e.g., uric acid, lipid profile) and an inverse association with dietary markers (e.g., vitamin C, E, B6, folates) were found and differed based on sex. By considering the importance of DNA stability during aging, the results obtained on sex differences and the potential role of dietary and metabolic factors on DNA damage underline the need for further investigations in a larger group of older adults to confirm the associations found and to promote preventive strategies.

Gut Microbiota Condition the Therapeutic Efficacy of Trastuzumab in HER2-Positive Breast Cancer.

Emerging evidence indicates that gut microbiota affect the response to anticancer therapies by modulating the host immune system. In this study, we investigated the impact of gut microbiota on immune-mediated trastuzumab antitumor efficacy in preclinical models of HER2-positive breast cancer and in 24 patients with primary HER2-positive breast cancer undergoing trastuzumab-containing neoadjuvant treatment. In mice, the antitumor activity of trastuzumab was impaired by antibiotic administration or fecal microbiota transplantation from antibiotic-treated donors. Modulation of the intestinal microbiota was reflected in tumors by impaired recruitment of CD4+ T cells and granzyme B-positive cells after trastuzumab treatment. Antibiotics caused reductions in dendritic cell (DC) activation and the release of IL12p70 upon trastuzumab treatment, a mechanism that was necessary for trastuzumab effectiveness in our model. In patients, lower α-diversity and lower abundance of Lachnospiraceae, Turicibacteraceae, Bifidobacteriaceae, and Prevotellaceae characterized nonresponsive patients (NR) compared with those who achieved pathologic complete response (R), similar to antibiotic-treated mice. The transfer of fecal microbiota from R and NR into mice bearing HER2-positive breast cancer recapitulated the response to trastuzumab observed in patients. Fecal microbiota ß-diversity segregated patients according to response and positively correlated with immune signature related to interferon (IFN) and NO2-IL12 as well as activated CD4+ T cells and activated DCs in tumors. Overall, our data reveal the direct involvement of the gut microbiota in trastuzumab efficacy, suggesting that manipulation of the gut microbiota is an optimal future strategy to achieve a therapeutic effect or to exploit its potential as a biomarker for treatment response. SIGNIFICANCE: Evidence of gut microbiota involvement in trastuzumab efficacy represents the foundation for new therapeutic strategies aimed at manipulating commensal bacteria to improve response in trastuzumab-resistant patients.See related commentary by Sharma, p. 1937 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2195/F1.large.jpg.

A polyphenol-rich dietary pattern improves intestinal permeability, evaluated as serum zonulin levels, in older subjects: The MaPLE randomised controlled trial.

BACKGROUND & AIM: Increased intestinal permeability (IP) can occur in older people and contribute to the activation of the immune system and inflammation. Dietary interventions may represent a potential strategy to reduce IP. In this regard, specific food bioactives such as polyphenols have been proposed as potential IP modulator due to their ability to affect several critical targets and pathways that control IP. The trial aimed to test the hypothesis that a polyphenol-rich dietary pattern can decrease serum zonulin levels, an IP surrogate marker involved in tight junction modulation, and can beneficially alter the intestinal microbiota, and IP-associated biochemical and clinical markers in older subjects. METHODS: A randomised, controlled, cross-over intervention trial was performed. Sixty-six subjects (aged ≥ 60 y) with increased IP based on serum zonulin levels, were randomly allocated to one of the two arms of the intervention consisting of a control diet (C-diet) vs. a polyphenol-rich diet (PR-diet). Each intervention was 8-week long and separated by an 8-week wash out period. At the beginning and at the end of each intervention period, serum samples were collected for the quantification of zonulin and other biological markers. Faecal samples were also collected to investigate the intestinal microbial ecosystem. In addition, anthropometrical/physical/biochemical parameters and food intake were evaluated. RESULTS: Fifty-one subjects successfully completed the intervention and a high compliance to the dietary protocols was demonstrated. Overall, polyphenol intake significantly increased from a mean of 812 mg/day in the C diet to 1391 mg/day in the PR-diet. Two-way analysis of variance showed a significant effect of treatment (p = 0.008) and treatment × time interaction (p = 0.025) on serum zonulin levels, which decreased after the 8-week PR-diet. In addition, a treatment × time interaction was observed showing a reduction of diastolic blood pressure (p = 0.028) following the PR-diet, which was strongest in those not using antihypertensive drugs. A decrease in both diastolic (p = 0.043) and systolic blood pressure (p = 0.042) was observed in women. Interestingly, a significant increase in fibre-fermenting and butyrate-producing bacteria such as the family Ruminococcaceae and members of the genus Faecalibacterium was observed following the PR intervention. The efficacy of this dietary intervention was greater in subjects with higher serum zonulin at baseline, who showed more pronounced alterations in the markers under study. Furthermore, zonulin reduction was also stronger among subjects with higher body mass index and with insulin resistance at baseline, thus demonstrating the close interplay between IP and metabolic features. CONCLUSIONS: These data show, for the first time, that a PR-diet can reduce serum zonulin levels, an indirect marker of IP. In addition, PR-diet reduced blood pressure and increased fibre-fermenting and butyrate-producing bacteria. These findings may represent an initial breakthrough for further intervention studies evaluating possible dietary treatments for the management of IP, inflammation and gut function in different target populations. THIS STUDY WAS REGISTERED AT WWW.ISRCTN. ORG AS: ISRCTN10214981.

Increased Intestinal Permeability in Older Subjects Impacts the Beneficial Effects of Dietary Polyphenols by Modulating Their Bioavailability.

Polyphenols have great potential in regulating intestinal health and ameliorating pathological conditions related to increased intestinal permeability (IP). However, the efficacy of dietary interventions with these phytochemicals may significantly be influenced by interindividual variability factors affecting their bioavailability and consequent biological activity. In the present study, urine samples collected from older subjects undergoing a crossover intervention trial with polyphenol-rich foods were subjected to metabolomics analysis for investigating the impact of increased IP on the bioavailability of polyphenols. Interestingly, urinary levels of phase II and microbiota-derived metabolites were significantly different between subjects with healthier intestinal barrier integrity and those with increased IP disruption. Our results support that this IP-dependent impaired bioavailability of polyphenols could be attributed to disturbances in the gut microbial metabolism and phase II methylation processes. Furthermore, we also observed that microbiota-derived metabolites could be largely responsible for the biological activity elicited by dietary polyphenols against age-related disrupted IP.

Estimated Intakes of Nutrients and Polyphenols in Participants Completing the MaPLE Randomised Controlled Trial and Its Relevance for the Future Development of Dietary Guidelines for the Older Subjects.

The evaluation of food intake in older subjects is crucial in order to be able to verify adherence to nutritional recommendations. In this context, estimation of the intake of specific dietary bioactives, such as polyphenols, although particularly challenging, is necessary to plan possible intervention strategies to increase their intake. The aims of the present study were to: (i) evaluate the nutritional composition of dietary menus provided in a residential care setting; (ii) estimate the actual intake of nutrients and polyphenols in a group of older subjects participating in the MaPLE study; and (iii) investigate the impact of an eight-week polyphenol-rich dietary pattern, compared to an eight-week control diet, on overall nutrient and polyphenol intake in older participants. The menus served to the participants provided ~770 mg per day of total polyphenols on average with small variations between seasons. The analysis of real consumption, measured using weighed food diaries, demonstrated a lower nutrient (~20%) and polyphenol intake (~15%) compared to that provided by the menus. The feasibility of dietary patterns that enable an increase in polyphenol intake with putative health benefits for age-related conditions is discussed, with a perspective to developing dietary guidelines for this target population.

Effect of a polyphenol-rich dietary pattern on intestinal permeability and gut and blood microbiomics in older subjects: study protocol of the MaPLE randomised controlled trial.

BACKGROUND: During aging, alterations of the intestinal microbial ecosystem can occur contributing to immunosenescence, inflamm-aging and impairment of intestinal barrier function (increased intestinal permeability; IP). In the context of a diet-microbiota-IP axis in older subjects, food bioactives such as polyphenols may play a beneficial modulatory role. METHODS: MaPLE is a project centered on a randomized, controlled cross-over dietary intervention trial [polyphenol-rich diet (PR-diet) versus control diet (C-diet)] targeted to older people (≥ 60 y) living in a well-controlled setting (i.e. nursing home). The 8-week interventions are separated by an 8-week wash-out period. Three small portions per day of selected polyphenol-rich foods are consumed during intervention in substitution of other comparable products within the C-diet. Biological samples are collected before and after each treatment period to evaluate markers related to IP, inflammation, vascular function, oxidative stress, gut and blood microbiomics, metabolomics. A sample size of 50 subjects was defined based on IP as primary outcome. DISCUSSION: Evidence that increasing the consumption of polyphenol-rich food products can positively affect intestinal microbial ecosystem resulting in reduced IP and decreased translocation of inflammogenic bacterial factors into the bloodstream will be provided. The integration of data from gut and blood microbiomics, metabolomics and other IP-related markers will improve the understanding of the beneficial effect of the intervention in the context of polyphenols-microbiota-IP interactions. Finally, findings obtained will provide a proof of concept of the reliability of the dietary intervention, also contributing to future implementations of dietary guidelines directed to IP management in the older and other at risk subjects. TRIAL REGISTRATION: The trial is registered at (ISRCTN10214981); April 28, 2017.

Polyphenols and Intestinal Permeability: Rationale and Future Perspectives.

Increasing evidence links intestinal permeability (IP), a feature of the intestinal barrier, to several pathological or dysfunctional conditions. Several host and environmental factors, including dietary factors, can affect the maintenance of normal IP. In this regard, food bioactives, such as polyphenols, have been proposed as potential IP modulators, even if the mechanisms involved are not yet fully elucidated. The aim of the present paper is to provide a short overview of the main evidence from in vitro and in vivo studies supporting the role of polyphenols in modulating IP and briefly discuss future perspectives in this research area.

Surface Layer of Lactobacillus helveticus MIMLh5 Promotes Endocytosis by Dendritic Cells.

Surface layers (S-layers) are proteinaceous arrays covering the cell walls of numerous bacteria. Their suggested properties, such as interactions with the host immune system, have been only poorly described. Here, we aimed to elucidate the role of the S-layer from the probiotic bacterial strain Lactobacillus helveticus MIMLh5 in the stimulation of murine bone-marrow-derived dendritic cells (DCs). MIMLh5 induced greater production of interferon beta (IFN-ß), interleukin 10 (IL-10), and IL-12p70, compared to S-layer-depleted MIMLh5 (naked MIMLh5 [n-MIMLh5]), whereas the isolated S-layer was a poor immunostimulator. No differences in the production of tumor necrosis factor alpha (TNF-α) or IL-1ß were found. Inhibition of the mitogen-activated protein kinases JNK1/2, p38, and ERK1/2 modified IL-12p70 production similarly in MIMLh5 and n-MIMLh5, suggesting the induction of the same signaling pathways by the two bacterial preparations. Treatment of DCs with cytochalasin D to inhibit endocytosis before the addition of fluorescently labeled MIMLh5 cells led to a dramatic reduction in the proportion of fluorescence-positive DCs and decreased IL-12 production. Endocytosis and IL-12 production were only marginally affected by cytochalasin D pretreatment when fluorescently labeled n-MIMLh5 was used. Treatment of DCs with fluorescently labeled S-layer-coated polystyrene beads (Sl-beads) resulted in much greater uptake of beads, compared to noncoated beads. Prestimulation of DCs with cytochalasin D reduced the uptake of Sl-beads more than plain beads. These findings indicate that the S-layer plays a role in the endocytosis of MIMLh5 by DCs. In conclusion, this study provides evidence that the S-layer of L. helveticus MIMLh5 is involved in endocytosis of the bacterium, which is important for strong Th1-inducing cytokine production.IMPORTANCE Beneficial microbes may positively affect host physiology at various levels, e.g., by participating in immune system maturation and modulation, boosting defenses and dampening reactions, thus affecting the whole homeostasis. As a consequence, the use of probiotics is increasingly regarded as suitable for more extended applications for health maintenance, not only microbiota balancing. This implies a deep knowledge of the mechanisms and molecules involved in host-microbe interactions, for the final purpose of fine tuning the choice of a probiotic strain for a specific outcome. With this aim, studies targeted to the description of strain-related immunomodulatory effects and the identification of bacterial molecules responsible for specific responses are indispensable. This study provides new insights in the characterization of the food-origin probiotic bacterium L. helveticus MIMLh5 and its S-layer protein as a driver for the cross-talk with DCs.