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γ-Conglutin (γ-C) from lupin seeds has been identified as a potent allergen with cross reactivity to peanuts. Here, we investigated how γ-C affected the response in bone marrow-derived dendritic cells (DCs) to bacterial stimuli. γ-C enhanced L. acidophilus NCFM (LaNCFM)-induced IL-12, IL-10, and IL-23 dose-dependently. In contrast, together with E. coli Nissle or LPS, γ-C reduced the production of IL-12 but not of IL-23 and IL-10. Enzyme-hydrolyzed γ-C also enhanced LaNCFM-induced IL-12 and IL-23 production. All preparations induced ROS production in the DCs. The mannose receptor ligands mannan and dextran and the clathrin inhibitor monodansylcadaverine partly inhibited the endocytosis of γ-C. Kunitz trypsin inhibitor and the scavenger receptor ligand polyG also enhanced LaNCFM-induced IL-12, indicating the involvement of receptors other than C-type lectin receptors. The endocytosis of labeled γ-C increased dose-dependently by addition of unlabeled γ-C, which coincided with γ-C's tendency to aggregate. Taken together, γ-C aggregation affects endocytosis and affects the cytokine production induced by gram-positive and gram-negative bacteria differently. We suggest that γ-C is taken up by the same mechanism as other food proteins but due to aggregation is present in higher concentration in the DCs. This could influence the resulting T-cell response in a microbial stimuli-dependent way.

Background: Conditions in utero influence intrauterine and postnatal infant growth and a few studies indicate that maternal inflammation and insulin resistance might affect birth and breastfeeding outcomes. Furthermore, hormones in human milk (HM) may influence infant appetite-regulation and thereby milk intake, but the associations are less understood., Objective: (1) To investigate associations between maternal inflammatory, lipid and metabolic markers and birth and breastfeeding outcomes, and (2) to assess predictors of maternal inflammatory, lipid and metabolic markers in pregnancy., Methods: Seventy-one mother-infant dyads participating in the Mothers, Infants and Lactation Quality (MILQ) study were included in the present study. Fasting blood samples were collected around 28th gestational week, and HM samples at three time points from 1.0 to 8.5 months, where milk intake was assessed using 24-h test weighing. Maternal plasma inflammatory, lipid and metabolic markers included high-sensitive C-reactive protein (hs-CRP), tumor-necrosis factor-α (TNFα), interferon-γ (IFNγ), Interleukin (IL)-6, IL-8, high-, low-, and very-low-density lipoprotein (HDL, LDL, VLDL), total-cholesterol, triglycerides, leptin, adiponectin, insulin, C-peptide, the homeostasis model assessment of insulin resistance (HOMA-IR) and glucose concentration at t = 120 min following an oral glucose tolerance test. Of these, TNFα, IFNγ, IL-6, IL-8, leptin, adiponectin and insulin were also measured in HM samples., Results: HDL in pregnancy was inversely associated with gestational age (GA) at birth and GA-adjusted birthweight z-score, whereas triglycerides and glucose ( t = 120) were positively associated with GA-adjusted birthweight z-score. Higher hs-CRP, VLDL and triglycerides were associated with a higher placental weight. Furthermore, higher HDL, insulin, leptin and HOMA-IR were associated with longer duration of exclusive breastfeeding (EBF). Higher pre-pregnancy BMI was the main predictor of higher levels of hs-CRP, log-TNFα, leptin, insulin, C-peptide, and HOMA-IR., Conclusion: Maternal lipid and metabolic markers influenced birthweight z-score and placental weight as well as duration of EBF. Furthermore, pre-pregnancy BMI and maternal age predicted levels of several inflammatory and metabolic markers during pregnancy. Our findings indicate that maternal lipid and metabolic profiles in pregnancy may influence fetal growth and breastfeeding, possibly explained by overweight and/or higher placental weight., Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT03254329., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Christensen, Rom, Greve, Lewis, Frøkiær, Allen, Mølgaard, Renault and Michaelsen.)

Background: The mechanisms underlying the protective effect of older siblings on allergic disease remain unclear but may relate to the infant gut microbiota., Objective: We sought to investigate whether having older siblings decreases the risk of IgE-mediated food allergy by accelerating the maturation of the infant gut microbiota., Methods: In a birth cohort assembled using an unselected antenatal sampling frame (n = 1074), fecal samples were collected at 1 month, 6 months, and 1 year, and food allergy status at 1 year was determined by skin prick test and in-hospital food challenge. We used 16S rRNA gene amplicon sequencing to derive amplicon sequence variants. Among a random subcohort (n = 323), microbiota-by-age z scores at each time point were calculated using fecal amplicon sequence variants to represent the gut microbiota maturation over the first year of life., Results: A greater number of siblings was associated with a higher microbiota-by-age z score at age 1 year (β  = 0.15 per an additional sibling; 95% CI, 0.05-0.24; P = .003), which was in turn associated with decreased odds of food allergy (odds ratio, 0.45; 95% CI, 0.33-0.61; P < .001). Microbiota-by-age z scores mediated 63% of the protective effect of siblings. Analogous associations were not observed at younger ages., Conclusions: The protective effect of older siblings on the risk of developing IgE-mediated food allergy during infancy is substantially mediated by advanced maturation of the gut microbiota at age 1 year., (Copyright © 2023 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Stilbenoids are anti-inflammatory and antioxidant compounds, with resveratrol being the most investigated molecule in this class. However, the actions of most other stilbenoids are much less studied. This study compares five monomeric (resveratrol, piceatannol, pterostilbene, pinostilbene, and trimethoxy-resveratrol) and two dimeric (dehydro-δ-viniferin and trans -δ-viniferin) stilbenoids for their capability to modulate the production of bacteria-induced cytokines (IL-12, IL-10, and TNF-α), as well as lipopolysaccharide (LPS)-induced reactive oxygen species (ROS), in murine bone marrow-derived dendritic cells. All monomeric species showed dose-dependent inhibition of E. coli -induced IL-12 and TNF-α, whereas only resveratrol and piceatannol inhibited IL-10 production. All monomers, except trimethoxy-resveratrol, inhibited L. acidophilus -induced IL-12, IL-10, and TNF-α production. The dimer dehydro-δ-viniferin remarkably enhanced L. acidophilus -induced IL-12 production. The contrasting effect of resveratrol and dehydro-δ-viniferin on IL-12 production was due, at least in part, to a divergent inactivation of the mitogen-activated protein kinases by the two stilbenoids. Despite having moderate to high total antioxidant activity, dehydro-δ-viniferin was a weak inhibitor of LPS-induced ROS formation. Conversely, resveratrol and piceatannol potently inhibited LPS-induced ROS formation. Methylated monomers showed a decreased antioxidant capacity compared to resveratrol, also depending on the methylation site. In summary, the immune-modulating effect of the stilbenoids depends on both specific structural features of tested compounds and the stimulating bacteria.

Background: Appetite-regulating hormones (ARH) in human milk (HM) are suggested to affect infants' milk intake and possibly infant growth. Maternal adiposity might contribute to higher levels of ARH in HM, either from the mammary gland or from raised circulating levels due to higher adiposity. Counterfactual-based mediation analysis can define indirect and direct effects between HM ARH and maternal and infant factors, and might be an important tool when investigating the mother-milk-infant triad., Objective: We aim to investigate whether potential associations between (1) maternal adiposity and HM ARH and (2) HM ARH and infant milk intake and growth are mediated through maternal and infant plasma ARH, respectively., Materials and Methods: Maternal and infant anthropometry and body composition, HM and blood samples were collected from 223 mother-infant dyads participating in the Mother, Infant and Lactation Quality study at three postpartum visits from 1 to 8.49 months. Leptin, insulin and adiponectin were analyzed using immunoassays. Mediation analyses using linear mixed-effect models were applied to investigate the direct and indirect effects through maternal and infant plasma hormone concentrations., Results: A positive association between maternal body-mass-index (BMI) and HM leptin was mediated by maternal plasma leptin by 29% when fixing BMI to < 25 kg/m 2 , and through 51% when fixing BMI to ≥ 25 kg/m 2 ( p interaction < 0.01). There was no mediated effect through plasma insulin in the association between BMI and HM insulin ( p = 0.068). We found negative and positive associations between HM insulin and total milk intake and infant weight, respectively, however, these diminished in mediation analyses with reduced sample sizes., Conclusion: Our main results suggest that the association between maternal adiposity and HM leptin was mediated through circulating leptin to a stronger degree for mothers with overweight compared to mothers with normal-weight. This indicates that excess maternal adiposity, and the resulting rise of circulating leptin and possible concomitant low-grade inflammation, may be reflected in HM composition., Clinical Trials Registry Number: NCT03254329., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Christensen, Lewis, Larnkjær, Frøkiær, Allen, Mølgaard and Michaelsen.)

Background: Preclinical studies have shown that maternal gut microbiota during pregnancy play a key role in prenatal immune development but the relevance of these findings to humans is unknown. The aim of this prebirth cohort study was to investigate the association between the maternal gut microbiota in pregnancy and the composition of the infant's cord and peripheral blood immune cells over the first year of life., Methods: The Barwon Infant Study cohort ( n =1074 infants) was recruited using an unselected sampling frame. Maternal fecal samples were collected at 36 weeks of pregnancy and flow cytometry was conducted on cord/peripheral blood collected at birth, 6 and 12 months of age. Among a randomly selected sub-cohort with available samples ( n =293), maternal gut microbiota was characterized by sequencing the 16S rRNA V4 region. Operational taxonomic units (OTUs) were clustered based on their abundance. Associations between maternal fecal microbiota clusters and infant granulocyte, monocyte and lymphocyte subsets were explored using compositional data analysis. Partial least squares (PLS) and regression models were used to investigate the relationships/associations between environmental, maternal and infant factors, and OTU clusters., Results: We identified six clusters of co-occurring OTUs. The first two components in the PLS regression explained 39% and 33% of the covariance between the maternal prenatal OTU clusters and immune cell populations in offspring at birth. A cluster in which Dialister, Escherichia , and Ruminococcus were predominant was associated with a lower proportion of granulocytes ( p =0.002), and higher proportions of both central naïve CD4 + T cells (CD4 + /CD45RA + /CD31 - ) ( p <0.001) and naïve regulatory T cells (Treg) (CD4 + /CD45RA + /FoxP3 low ) ( p =0.02) in cord blood. The association with central naïve CD4 + T cells persisted to 12 months of age., Conclusion: This birth cohort study provides evidence consistent with past preclinical models that the maternal gut microbiota during pregnancy plays a role in shaping the composition of innate and adaptive elements of the infant's immune system following birth., Competing Interests: The findings described in this paper are the subject of a provisional patent, licensed to Prevatex Pty Ltd, in which the following authors have a financial interest: PV, MOH, SR, ALP. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gao, O’Hely, Quinn, Ponsonby, Harrison, Frøkiær, Tang, Brix, Kristiansen, Burgner, Saffery, Ranganathan, Collier and Vuillermin.)

Methicillin resistant Staphylococcus aureus (MRSA) has developed resistance to most β-lactam antibiotics leaving few treatment options against infections with MRSA. Through mannose receptors, mannan potentiates IL-12 production induced by Gram-positive bacteria, a cytokine crucial in the clearance of S. aureus infection. We investigated the IL-12 potentiating effect of mannan pre-treatment of bone marrow-derived dendritic cells prior to stimulation with clinical MRSA strains. Mannan almost doubled IL-12 as well as IFN-β production in response to USA300, also when USA300 was treated with the β-lactam cefoxitin. The MRSA-induced IL-12 production was dependent on bacterial uptake and reactive oxygen species (ROS). Mannan alone induced ROS production, and in combination with USA300, the ROS produced corresponded to the sum induced by mannan and USA300. Addition of a monoclonal antibody against the mannose receptor likewise enhanced USA300-induced IL-12 and induced ROS production. Mannan addition further increased the endocytosis as well as the rate of endosomal killing of bacteria. Pre-treatment with soluble β-glucans also induced ROS and potentiated the USA300-induced IL-12 indicating that other C-type receptors may play a similar role. In the presence of the pro-inflammatory mediators, GM-CSF or IFN-γ, the mannan-enhanced IL-12 production increased further. The USA300-induced and the mannan-facilitated enhanced IFN-β and IL-12 showed same dependency on MAPK c-Jun N-terminal kinase signaling, suggesting that mannan enhances the signals already induced by the bacteria, rather than changing them. We suggest that the C-type lectin-induced ROS production is a key factor in the IFN-β and IL-12 potentiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Eld, Johnsen, Nielsen, Jørgensen, Lindstrøm-Svendsen, Baldry, Ingmer and Frøkiær.)

Methicillin resistant Staphylococcus aureus (MRSA) constitute a serious health care problem worldwide. This study addresses the effect of β-lactam treatment on the ability of clinically relevant MRSA strains to induce IL-12 and IL-23. MRSA strains induced a dose-dependent IL-12 response in murine bone-marrow-derived dendritic cells that was dependent on endocytosis and acidic degradation. Facilitated induction of IL-12 (but not of IL-23) called for activation of the MAP kinase JNK, and was suppressed by p38. Compromised peptidoglycan structure in cefoxitin-treated bacteria - as denoted by increased sensitivity to mutanolysin -caused a shift from IL-12 towards IL-23. Moreover, cefoxitin treatment of MRSA led to a p38 MAPK-dependent early up-regulation of Dual Specificity Phosphatase (DUSP)-1. Compared to common MRSA, characteristics associated with a persister phenotype increased intracellular survival and upon cefoxitin treatment, the peptidoglycan was not equally compromised and the cytokine induction still required phagosomal acidification. Together, these data demonstrate that β-lactam treatment changes the MRSA-induced IL-12/IL-23 pattern determined by the activation of JNK and p38. We suggest that accelerated endosomal degradation of the peptidoglycan in cefoxitin-treated MRSA leads to an early expression of DUSP-1 and accordingly, a reduction in the IL-12/IL-23 ratio in dendritic cells. This may influence the clearance of S. aureus., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Some oligosaccharides induce growth of anti-inflammatory bacterial species and induce regulatory immunity in humans as well as animals. We have shown that the equine gut microbiota and the immune-microbial homeostasis largely stabilize within the first 50 days of life. Furthermore, we have previously established that certain bacterial species in the equine gut correlated with regulatory immunity. Accordingly, we hypothesized that an oligosaccharide rich diet fed to foals during the first 50 days would increase the abundance of bacterial species associated with regulatory immunity, and that this would influence immune responses in the foals. Eight pregnant mares and their foals were fed an oligosaccharide rich diet from 4 weeks before expected parturition until 49 days post-partum. Six mares and foals served as control. Fecal microbiota from mares and foals was characterized using 16S rRNA gene amplicon high throughput sequencing. On Day 49 the test foals had significantly higher abundances of Akkermansia spp. Blood sampled from the foals in the test group on Day 7, 28, and 49 showed non-significant increases in IgA, and decreases in IgG on Day 49. In BALB/cBomTac mice inoculated with gut microbiota from test and control foals we found increased species richness, increased relative abundance of several species identified as potentially anti-inflammatory in horses, which were unclassified Clostridiales, Ruminococcaceae, Ruminococcus , Oscilospira , and Coprococcus . We also found increased il10 expression in the ileum if inoculated with test foal microbiota. We conclude that an oligosaccharide diet fed to foals in the "window of opportunity," the first 50 days of life, increases the abundance of anti-inflammatory species in the microbiota with potentially anti-inflammatory effects on regulatory immunity., Competing Interests: FL works at Brogaarden Diets. AKH declares that he has several industrial collaborations as further described on https://ivh.ku.dk/english/employees/?pure=en/persons/107126. The study received funding from Innovation Fund Denmark, who was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. The study received funding from Brogaarden which is the employer of FL, and was therefore involved in the study design and execution., (Copyright © 2021 Lindenberg, Lützhøft, Krych, Fielden, Kot, Frøkiær, van Galen, Nielsen and Hansen.)

A dietary shift from resource-demanding animal protein to sustainable food sources, such as protein-rich beans, lowers the climate footprint of food production. In this study, we examined the nutrients and antinutrients in 15 fava bean varieties cultivated in Sweden to select varieties with high nutritional value. On a dry weight basis, the fava beans were analyzed for their content of protein (range 26-33%), amino acids (leucine range: 50.8-72.1 mg/g protein, lysine range: 44.8-74.8 mg/g protein), dietary fiber (soluble fraction range: 0.55-1.06%, insoluble fraction range: 10.7-16.0%), and iron (1.8-21.3 mg/100 g) and zinc contents (0.9-5.2 mg/100 g), as well as for the following antinutrients: lectin (0.8-3.2 HU/mg); trypsin inhibitor (1.2-23.1 TIU/mg) and saponin (18-109 µg/g); phytate (112-1,281 mg/100 g); total phenolic content (1.4-5 mg GAE/g); and vicine(403 µg/g - 7,014 µg/g), convicine (35.5 µg/g - 3,121 µg/g) and the oligosaccharides raffinose (1.1-3.9 g/kg), stachyose (4.4-13.7 g/kg) and verbascose (8-15 g/kg). The results indicate substantial differences between cultivars in relation to their contents of nutrients and antinutrients. Only one of the cultivars studied (Sunrise) have adequate estimated bioavailability of iron, which is of major concern for a diet in which legumes and grains serve as important sources of iron. The nutritional gain from consuming fava beans is significantly affected by the cultivar chosen as the food source., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Diet is an important component in weight management strategies, but heterogeneous responses to the same diet make it difficult to foresee individual weight-loss outcomes. Omics-based technologies now allow for analysis of multiple factors for weight loss prediction at the individual level. Here, we classify weight loss responders (N = 106) and non-responders (N = 97) of overweight non-diabetic middle-aged Danes to two earlier reported dietary trials over 8 weeks. Random forest models integrated gut microbiome, host genetics, urine metabolome, measures of physiology and anthropometrics measured prior to any dietary intervention to identify individual predisposing features of weight loss in combination with diet. The most predictive models for weight loss included features of diet, gut bacterial species and urine metabolites (ROC-AUC: 0.84-0.88) compared to a diet-only model (ROC-AUC: 0.62). A model ensemble integrating multi-omics identified 64% of the non-responders with 80% confidence. Such models will be useful to assist in selecting appropriate weight management strategies, as individual predisposition to diet response varies.

Elevated postprandial plasma glucose is a risk factor for development of type 2 diabetes and cardiovascular disease. We hypothesized that the inter-individual postprandial plasma glucose response varies partly depending on the intestinal microbiome composition and function. We analyzed data from Danish adults (n = 106), who were self-reported healthy and attended the baseline visit of two previously reported randomized controlled cross-over trials within the Gut, Grain and Greens project. Plasma glucose concentrations at five time points were measured before and during three hours after a standardized breakfast. Based on these data, we devised machine learning algorithms integrating bio-clinical, as well as shotgun-sequencing-derived taxa and functional potentials of the intestinal microbiome to predict individual postprandial glucose excursions. In this post hoc study, we found microbial and clinical features, which predicted up to 48% of the inter-individual variance of postprandial plasma glucose responses (Pearson correlation coefficient of measured vs. predicted values, R = 0.69, 95% CI: 0.45 to 0.84, p<0.001). The features were age, fasting serum triglycerides, systolic blood pressure, BMI, fasting total serum cholesterol, abundance of Bifidobacterium genus, richness of metagenomics species and abundance of a metagenomic species annotated to Clostridiales at order level. A model based only on microbial features predicted up to 14% of the variance in postprandial plasma glucose excursions (R = 0.37, 95% CI: 0.02 to 0.64, p = 0.04). Adding fasting glycaemic measures to the model including microbial and bio-clinical features increased the predictive power to R = 0.78 (95% CI: 0.59 to 0.89, p<0.001), explaining more than 60% of the inter-individual variance of postprandial plasma glucose concentrations. The outcome of the study points to a potential role of the taxa and functional potentials of the intestinal microbiome. If validated in larger studies our findings may be included in future algorithms attempting to develop personalized nutrition, especially for prediction of individual blood glucose excursions in dys-glycaemic individuals., Competing Interests: The authors have declared that no competing interests exist.

Aim: The aim was to examine associations between thymus size and anthropometric measurements, sex, age, breastfeeding status, presence of siblings, household pets, and infections and allergies since birth in 8- to 13-month-old healthy Danish infants., Methods: Data collected from 256 healthy infants enrolled in the ProbiComp study were used. Thymus size was assessed using sonographic measures, and thymic index (TI) and thymus weight index (TWI) was used as an absolute and a relative volume estimate, respectively., Results: In terms of TI and TWI, boys had approximately 15% and 5% larger thymus than girls (P < .001 and P < .02, respectively). TWI was larger in girls who were still breastfed than girls who were no longer breastfed (β: 0.16 cm 3 /kg; 95% CI: 0.004, 0.29; P = .01), but no difference was observed for boys. Having household pets was associated with a larger TI (P = .02), which seemed to be driven by associations for boys (β: 1.38 cm 3 ; 95% CI: 0.02, 2.74). No other factors associated with thymus size were identified., Conclusion: Thymus size was associated with current breastfeeding in girls and with having household pets in boys. Sex-specific associations should be further explored in future studies on factors associated with thymus size., (© 2019 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.)

This study examines different factors influencing the 13-valent pneumococcal conjugate vaccine (PCV13) specific antibody response in 8-13 months old Danish children starting in day care. We present secondary findings to the ProbiComp study, which included nose swabs, buccal swabs and blood samples from the children before entering day care (baseline) and again after 6 months. Pneumococci isolated from nose swabs were identified by latex agglutination kit and Quellung reaction. Luminex-based assay was used for antibody measurements against specific anti-pneumococcal capsular IgG. Buccal gene expression was analyzed by qPCR. Statistical analyses were performed in R and included Pearson's Chi-squared test, Welch two sample t-test and linear regression models. The PCV13 antibody response was unaffected by whether the children were carriers or non-carriers of any pneumococcal serotype. Having siblings increased the risk of carrying serotype 21 before day care (p = 0.020), and having siblings increased the PCV13 antibody response at the end of study (p = 0.0135). Hepatitis B-vaccination increased the PCV13 antibody response before day care attendance (p = 0.005). The expression of IL8 and IL1B was higher in children carrying any pneumococcal serotype at baseline compared to non-carriers (p = 0.0125 and p = 0.0268 respectively).

Staphylococcus aureus has evolved different strategies to evade the immune response, which play an important role in its pathogenesis. The bacteria express and shed various cell wall components and toxins during different stages of growth that may affect the protective T cell responses to extracellular and intracellular S. aureus However, if and how the dendritic cell (DC)-mediated T cell response against S. aureus changes during growth of the bacterium remain elusive. In this study, we show that exponential-phase (EP) S. aureus bacteria were endocytosed very efficiently by human DCs, and these DCs strongly promoted production of the T cell polarizing factor interleukin-12 (IL-12). In contrast, stationary-phase (SP) S. aureus bacteria were endocytosed less efficiently by DCs, and these DCs produced small amounts of IL-12. The high level of IL-12 production induced by EP S. aureus led to the development of a T helper 1 (Th1) cell response, which was inhibited after neutralization of IL-12. Furthermore, preincubation with the staphylococcal cell wall component peptidoglycan (PGN), characteristically shed during the exponential growth phase, modulated the DC response to EP S. aureus PGN preincubation appeared to inhibit IL-12p35 expression, leading to downregulation of IL-12 and an increase of IL-23 production by DCs, enhancing Th17 cell development. Taken together, our data indicate that exponential-phase S. aureus bacteria induce a stronger IL-12-dependent Th1 cell response than stationary-phase S. aureus and that this Th1 cell response shifted toward a Th17 cell response in the presence of PGN., (Copyright © 2019 American Society for Microbiology.)

The mannose receptor (MR) is a C-type lectin involved in endocytosis and with a poorly defined ability to modulate cellular activation. We investigated the effect of mannan treatment prior to stimulation of murine bone marrow-derived dendritic cells with the Gram-positive bacteria Lactobacillus acidophilus NCFM (L. acidophilus ) on the induction of Interleukin (IL)-12. Mannan enhanced the IL-12 production induced by L. acidophilus in a dose dependent manner (up to 230% enhancement). Additionally, mannan-enhanced IL-12 induction was also demonstrated with another Gram-positive bacteria, Staphylococcus aureus (S. aureus) , while an IL-12 reducing effect was seen on Escherichia coli stimulated cells. Furthermore, the expression of Interferon β ( Ifnb ) was increased in cells treated with mannan prior to stimulation with L. acidophilus . The addition of mannan but not of bacteria led to endocytosis of MR, while addition of mannan prior to L. acidophilus or S. aureus resulted in increased endocytosis of bacteria, a faster killing of endocytosed bacteria, and increased reactive oxygen species production. Expression of signaling lymphocytic activation molecule (SLAMF)1 shown previously to be involved in the facilitation of endosomal degradation was upregulated by mannan but not by L. acidophilus and S. aureus . The IL-12 enhancement by mannan but not the IL-12 induced by the bacteria was abrogated by addition of inhibitors of clathrin coated pits (chlorpromazine and monodansylcadaverine). Furthermore, the addition of acid sphingomyelinase, a facilitator of ceramide raft formation, prior to addition of L. acidophilus enhanced the IL-12 production and the endocytosis of bacteria. In summary, our results show that mannan increases the IL-12 production induced by some Gram-positive bacteria through MR-endocytosis, which increases bacterial endocytosis and endosomal killing. The differential effect of MR activation on the IL-12 production induced by Gram-positive and Gram-negative bacteria may influence the immune response toward allergens and other glycoproteins., (Copyright © 2019 Mathiesen, Eld, Sørensen, Fuglsang, Lund, Taverniti and Frøkiær.)

Shortly after birth the mammalian gut is colonized, by a transient microbiota, highly susceptible to environment and diet, that eventually stabilizes and becomes the resident gut microbiota. In a window of opportunity during the colonization, oral tolerance is established towards resident bacteria. In this study, the development of the equine gut microbiota was investigated in ten foals from parturition until post weaning. We found great differences in the core species of the gut microbiota composition between time-matched samples on Day 7 and 20 post-partum. Between day 20 and Day 50 post-partum, we saw the gut microbiota became increasingly dominated by fiber fermenting species. After Day 50, no significant changes in species abundance were observed. Gene expression analysis of pro- and anti-inflammatory cytokines in the blood revealed no significant changes before and after weaning. In summary, relative stability of the gut microbiota was reached within 50 days post-partum and, weaning did not have a major impact on the microbial composition.

Billions of bacteria inhabit the gastrointestinal tract. Immune-microbial cross talk is responsible for immunological homeostasis, and symbiotic microbial species induce regulatory immunity, which helps to control the inflammation levels. In this study we aimed to identify species within the equine intestinal microbiota with the potential to induce regulatory immunity. These could be future targets for preventing or treating low-grade chronic inflammation occurring as a result of intestinal microbial changes and disruption of the homeostasis. 16S rRNA gene amplicon sequencing was performed on samples of intestinal microbial content from ileum, cecum, and colon of 24 healthy horses obtained from an abattoir. Expression of genes coding for IL-6, IL-10, IL-12, IL-17, 18 s, TNFα, TGFβ, and Foxp3 in the ileum and mesenteric lymph nodes was measured by qPCR. Intestinal microbiota composition was significantly different in the cecum and colon compared to the ileum, which contains large abundances of Proteobacteria. Especially members of the Clostridiales order correlated positively with the regulatory T-cell transcription factor Foxp3 and so did the phylum Verrucomicrobia. We conclude that Clostridiales and Verrucomicrobia have the potential to induce regulatory immunity and are possible targets for intestinal microbial interventions aiming at regulatory immunity improvement.

Objective: Children with severe acute malnutrition (SAM) may have impaired intestinal function, which can result in malabsorption, diarrhoea, and poor growth. This study evaluated the gut function of children with SAM using fecal and blood biomarkers and assessed their correlates., Methods: A cross-sectional study, nested in a randomized trial (www.isrctn.com, ISRCTN 16454889), was conducted at Mulago hospital, Uganda among subgroups of 400 children with complicated SAM and 30 community controls. Gut function was evaluated by 5 biomarkers: plasma citrulline, fecal myeloperoxidase and fecal neopterin, bacterially derived 16S rRNA gene and internal transcribed Spacer region (ITS) specific for Candida spp. in blood., Results: Compared with controls, children with SAM had lower median plasma citrulline (5.14 vs 27.4 μmol/L, P < 0.001), higher median fecal myeloperoxidase (18083 vs 7482 ng/mL, P = 0.001), and fecal neopterin (541 vs 210 nmol/L, P < 0.001). A higher blood concentration of 16S rRNA gene copy numbers was observed among children with SAM (95 vs 28 copies/μl, P = 0.05), whereas there was no difference in the blood concentration of Candida-specific ITS fragment.Among those with SAM, plasma citrulline was lower in children with edema, diarrhoea, dermatosis, and plasma C-reactive protein (CRP) >10 mg/L. Fecal neopterin was positively correlated with symptoms of fever and cough whereas it was negatively correlated with mid-upper arm circumference (MUAC), weight-for-height z score (WHZ), edema, and dermatosis., Conclusions: Children with complicated SAM seem to have impaired gut function characterized by reduced enterocyte mass, intestinal inflammation, and increased bacterial translocation.

Purpose: We explored the effect of winter cholecalciferol (vitamin D 3 ) supplementation on innate immune markers in healthy Danish children (55°N)., Methods: In the double-blind, placebo-controlled trial, ODIN Junior, 119 healthy, white, 4-8 year-olds were randomized to 0 (placebo), 10 or 20 µg/day of vitamin D 3 for 20 weeks (October-March). Cheek mucosal swabs, blood samples, and questionnaires on acute respiratory infections the previous month were collected at baseline and endpoint. Innate immune markers were measured as secondary outcomes including in vivo oral mucosal gene expression of calprotectin (S100A9), lipocalin-2 (LCN2), beta-defensin-4 (DEFB4), interleukin-8 (IL-8), viperin (RSAD2), and the cathelicidin-antimicrobial-peptide (CAMP); ex vivo whole-blood lipopolysaccharide (LPS)-induced cathelicidin, IL-8, and IL-6; and plasma cathelicidin, together with serum 25-hydroxyvitamin D [25(OH)D]., Results: Serum 25(OH)D was 56.7 ± 12.3 nmol/L at baseline and 31.1 ± 7.5, 61.8 ± 10.6, and 75.8 ± 11.5 nmol/L at endpoint after placebo, 10 and 20 µg/day of vitamin D 3 (P < 0.0001), respectively. A decreased oral mucosal S100A9 expression with placebo [- 18 (95% CI - 1; - 32)%] was marginally avoided with 20 µg/day [6 (- 13; 28)%] (P = 0.06). Likewise, a decreased LPS-induced IL-8 with placebo [- 438 (95% CI - 693; - 184) ng/L] was marginally avoided with 20 µg/day [- 109 (- 374; 157) ng/L] (P = 0.07). All other immune markers and respiratory infection episodes were unaffected by vitamin D 3 supplementation (all P > 0.11)., Conclusions: Winter vitamin D 3 supplementation of 10 µg/day did not affect innate immune markers, whereas 20 µg/day tended to maintain the capacity to produce a few markers in healthy children.

We compared 6 frequently used mouse blood-sampling methods (lateral tail incision; tail-tip amputation; sublingual, submandibular, and saphenous vein puncture; and retrobulbar sinus puncture during isoflurane anesthesia) with regard to induction of local and systemic inflammation, stomach contents, weight changes, and corticosterone levels at 6 h to 12 d after sampling. Local inflammation was assessed through histopathology and assessment of the expression of inflammation and tissue damage-related genes ( S1008/9A , Cxcl2 , Il1b , Nlrp3 , Il6 , and Il33 ) in sampled tissue. Systemic inflammation was assessed through quantification of plasma haptoglobin levels, measurement of blood Il1b expression, and evaluation of histopathologic changes in lung, kidney, liver, and spleen. Apart from slight, transient increases in plasma haptoglobin levels after lateral tail incision, retrobulbar sinus puncture, and saphenous vein puncture, no other signs of systemic inflammation were found. Mice subjected to retrobulbar sinus puncture, sublingual puncture, or isoflurane anesthesia only showed the highest plasma corticosterone concentrations. Retrobulbar sinus puncture had the largest effect on body weight loss. Retrobulbar sinus puncture, sublingual puncture, and submandibular puncture only showed minor and in, most cases, fastresolving inflammation. By contrast, blood sampling by lateral tail incision, tail-tip amputation, or saphenous vein puncture caused tissue damage and inflammation locally at the sampling site, which resolved more slowly compared with head-region sampling techniques, according to results from pathologic and gene expression assessments. Expression of S1008/9A , Cxcl2 , Il1b, and Nlrp3 increased 10- to 1000-fold and did not return to baseline until day 6 after sampling or later and did not resolve after tail-tip amputation within the 12-d observation period. Increased expression of genes involved in inflammation and tissue repair correlated with histopathologic changes and may thus represent a quantitative supplement to histopathology. In conclusion, none of the tested methods for obtaining blood samples from mice is superior, according to simultaneous immunologic, histopathologic, and animal welfare-related parameters.

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-β), 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-1β 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., (Copyright © 2019 American Society for Microbiology.)

Objectives: To examine the effect of a combination of probiotics on the antibody response to pneumococcal and pertussis vaccination in healthy Danish children, aged 8-14 months, at the time of starting day care. Moreover, the cytokine response to lipopolysaccharide of whole blood was assessed., Methods: A total of 290 children were randomly allocated to receive a combination of Bifidobacterium animalis ssp. lactis and Lactobacillus rhamnosus GG daily for a 6-month intervention period, and blood samples were drawn at the start and end of the study. Specific antibody response towards Streptococcus pneumoniae serotypes and Bordetella pertussis toxin, as well as endotoxin-induced interleukin-6 (IL-6) and interferon-γ (IFN-γ) production in blood were analysed by Luminex and ELISA., Results: There was no significant difference between the average individual changes from baseline to end of study in antibody concentrations for S. pneumoniae for both the probiotics (340.4% ± 11.2%) and the placebo group (382.9% ± 10.4%) (p 0.525), nor for B. pertussis toxin in the two groups (probiotics 190.1% ± 12.6% versus placebo 238.8% ± 1.1%, p 0.340). The average individual change in IL-6 concentration was significantly lower in the probiotics versus the placebo group (2.9% ± 10.3% versus 33.7% ± 9.0%, p 0.024), whereas there was no difference in IFN-γ concentration (0.0% ± 0.2% versus -0.2% ± 0.1%, p 0.279)., Conclusions: The probiotic intervention did not affect the antibody response against S. pneumoniae and B. pertussis toxin in healthy Danish children., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: To investigate whether a whole grain diet alters the gut microbiome and insulin sensitivity, as well as biomarkers of metabolic health and gut functionality., Design: 60 Danish adults at risk of developing metabolic syndrome were included in a randomised cross-over trial with two 8-week dietary intervention periods comprising whole grain diet and refined grain diet, separated by a washout period of ≥6 weeks. The response to the interventions on the gut microbiome composition and insulin sensitivity as well on measures of glucose and lipid metabolism, gut functionality, inflammatory markers, anthropometry and urine metabolomics were assessed., Results: 50 participants completed both periods with a whole grain intake of 179±50 g/day and 13±10 g/day in the whole grain and refined grain period, respectively. Compliance was confirmed by a difference in plasma alkylresorcinols (p<0.0001). Compared with refined grain, whole grain did not significantly alter glucose homeostasis and did not induce major changes in the faecal microbiome. Also, breath hydrogen levels, plasma short-chain fatty acids, intestinal integrity and intestinal transit time were not affected. The whole grain diet did, however, compared with the refined grain diet, decrease body weight (p<0.0001), serum inflammatory markers, interleukin (IL)-6 (p=0.009) and C-reactive protein (p=0.003). The reduction in body weight was consistent with a reduction in energy intake, and IL-6 reduction was associated with the amount of whole grain consumed, in particular with intake of rye., Conclusion: Compared with refined grain diet, whole grain diet did not alter insulin sensitivity and gut microbiome but reduced body weight and systemic low-grade inflammation., Trial Registration Number: NCT01731366; Results., Competing Interests: Competing interests: MV Lind was partly supported by an unrestricted grant from Cereal Partners Worldwide, a joint venture between Nestlé SA and General Mills Ltd. There were no conflict of interest to declare for the other authors. Intervention products were sponsored by Kohberg, Lantmännen, AXA, Wasa, Urtekram, Finax and Doves Farm. Sponsors of grants and products played no role in the design, methods, data management and analysis nor in the decision to publish., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Adherence to a low-gluten diet has become increasingly common in parts of the general population. However, the effects of reducing gluten-rich food items including wheat, barley and rye cereals in healthy adults are unclear. Here, we undertook a randomised, controlled, cross-over trial involving 60 middle-aged Danish adults without known disorders with two 8-week interventions comparing a low-gluten diet (2 g gluten per day) and a high-gluten diet (18 g gluten per day), separated by a washout period of at least six weeks with habitual diet (12 g gluten per day). We find that, in comparison with a high-gluten diet, a low-gluten diet induces moderate changes in the intestinal microbiome, reduces fasting and postprandial hydrogen exhalation, and leads to improvements in self-reported bloating. These observations suggest that most of the effects of a low-gluten diet in non-coeliac adults may be driven by qualitative changes in dietary fibres.

Scope: This study addresses whether administration of Lactobacillus rhamnosus HN001 could mitigate the effects of a compromised gut microbiota on the composition of mature leukocytes and granulocyte-macrophage progenitor cells (GMPs) in newborn mice., Methods and Results: Pregnant dams receive oral broad-spectrum antibiotics, which dramatically decrease the gut microbial composition analyzed by 16S rRNA sequencing. Perinatal antibiotic treatment decreases the proportions of bone marrow (BM) GMPs (postnatal day (PND2): 0.5% vs 0.8%, PND4: 0.2% to 0.6%) and mature granulocytes (33% vs 24% at PND2), and spleen granulocytes (7% vs 17% at PND2) and B cells (PND2:18% vs 28%, PND4:11% vs 22%). At PND35, T helper (Th) cells (20% vs 14%) and cytotoxic T (Tc) cells (10% vs 8%) decrease in the spleen. Oral administration of L. rhamnosus HN001 to neonatal pups (PND1-7) restores the antibiotic-induced changes of GMPs and granulocytes in BM and spleen, and further increases splenic granulocytes in control pups. At PND35, splenic proportions of B and Th but not Tc cells are restored., Conclusion: Postnatal administration of a single bacterial strain efficiently restores granulopoiesis and most T cell activation in neonatal mice that suffer from a perinatal antibiotic-induced compromised gut microbiota at birth., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intergenerational transmission of bacteria during birth initiates the natural successional development of the intestinal microbiota in mammals. This process can be disrupted by antibiotic exposure, potentially affecting early-life microbiota-dependent metabolic programming. In the present study, we specifically investigate the metabolic consequences of exposing neonate Wistar rats to an antibiotic-perturbed low-diversity microbiota from birth until weaning, without exposing the pups directly to antibiotics. Here, we show that pups born from both amoxicillin and vancomycin-treated dams gain less weight than controls. This was concordant with lower feed intake as well as increased colonic expression of the PYY satiety hormone gene at weaning. The weight difference persists into adulthood even though the initial differences in gut microbiota subsided. Our results demonstrate that early-life exposure to an antibiotic-perturbed low-diversity microbiota is sufficient to cause changes in body weight persisting into adulthood., Competing Interests: The authors declare no competing interests.

Previous studies demonstrated that oral antibiotic (ABX) treatment prior to and during influenza A virus (IAV) infection of adult mice profoundly altered gut microbiota (GM) and was associated with increased susceptibility and impaired immunity to IAV. We examined the impact of ABX during critical times relevant to the establishment of GM in early life (using perinatal treatment of neonates and direct treatment of juvenile mice) and asked whether cessation of ABX treatment in early life had lasting effects on GM composition and anti-IAV immunity. ABX treatment significantly changed GM composition in juvenile mice and in ABX-treated dams. However, if ABX treatment ceased at the time of infection, neither neonates nor juvenile mice showed enhanced susceptibility to IAV, nor were major differences detected in cellular and humoral adaptive antiviral immunity. Thus, while ABX treatment alters GM diversity in early life, cessation and subsequent re-colonization correlates with effective immunity against IAV.

Background: Low serum 25-hydroxyvitamin D [25(OH)D] has been associated with unfavorable cardiometabolic risk profiles in many observational studies in children, but very few randomized controlled trials have investigated this., Objective: We explored the effect of winter-time cholecalciferol (vitamin D3) supplementation on cardiometabolic risk markers in young, white, 4- to 8-y-old healthy Danish children (55°N) as part of the pan-European ODIN project., Methods: In the ODIN Junior double-blind, placebo-controlled, dose-response trial, 119 children (mean ± SD age: 6.7 ± 1.5 y; 36% male; 82% normal weight) were randomly allocated to 0, 10 or 20 µg/d of vitamin D3 for 20 wk (October-March). Cardiometabolic risk markers including BMI-for-age z score (BMIz), waist circumference, systolic and diastolic blood pressure, serum triglycerides and cholesterol (total, LDL, HDL, and total:HDL), plasma glucose and insulin, and whole-blood glycated hemoglobin were measured at baseline and endpoint as secondary outcomes together with serum 25(OH)D. Intervention effects were evaluated in linear regression models as between-group differences at endpoint adjusted for baseline value of the outcome, and additionally for age, sex, baseline serum 25(OH)D, BMIz, time since breakfast, and breakfast content., Results: Mean ± SD serum 25(OH)D was 56.7 ± 12.3 nmol/L at baseline and differed between groups at endpoint with concentrations of 31.1 ± 7.5, 61.8 ± 10.6, and 75.8 ± 11.5 nmol/L in the 0-, 10-, and 20 µg/d groups, respectively (P < 0.0001). Vitamin D3 supplementation had no effect on any of the cardiometabolic risk markers in analyses adjusted for baseline value of the outcome (all P ≥ 0.05), and additional covariate adjustment did not change the results notably., Conclusions: Preventing the winter decline in serum 25(OH)D with daily vitamin D3 supplementation of 10 or 20 µg had no cardiometabolic effects in healthy 4- to 8-y-old Danish children. This trial was registered at www.clinicaltrials.gov as NCT02145195.

Background: Low-grade systemic inflammation (LGSI) is often characterized by elevated levels of interleukin (IL)6, tumor necrosis factor (TNF)α, and C-reactive protein (CRP). Other serum proteins, ex vivo-stimulated cytokine production, and leukocyte count have, however, also been suggested LGSI-markers, but their associations with the metabolic syndrome (MS) are less clear. We aimed to evaluate mutual relationships between in vivo and ex vivo inflammatory markers and their association with MS and its subcomponents., Methods: A cross-sectional study of 118 overweight adults with one or several features of MS. Inflammatory markers included fasting serum levels of IL6, TNFα, CRP, and pentraxin-3 (PTX3), IL1-receptors, leukocytes, and whole-blood ex vivo-produced IL1β, IL6, TNFα, and IL8 after lipopolysaccharide stimulation., Results: All classical serum LGSI-markers correlated with each other, and IL6 and CRP were also correlated with leukocyte count. Ex vivo-produced cytokines were intercorrelated and correlated with leukocyte count, but did not correlate with the serum immune markers. MS score, body mass index, and glycated hemoglobin (HbA1c) were associated with 8%-16% higher inflammatory score per standard deviation increment (P = 0.030, 0.001, and 0.034, respectively), primarily driven by higher serum IL6. Serum PTX3 was only significantly associated with high-density lipoprotein cholesterol (1.19[1.04; 1.37], P = 0.013). HbA1c was inversely associated with surface expression of IL1R1 on monocytes and IL1R2 on granulocytes (P < 0.01) and with a 3%-9% lower ex vivo production of cytokines when adjusting for leukocyte count, as were plasma triacylglycerol (9%-10% lower IL1β and IL6). Leukocyte count was most consistently associated with MS and its subcomponents, although not with HbA1., Conclusions: The classical fasting serum markers of LGSI and leukocyte counts associated best with measures of MS-associated LGSI, whereas ex vivo cytokine production was only associated with prevailing glycemia and dyslipidemia. Taken together, this indicates that the relationship between in vivo and ex vivo inflammatory markers is complex and may depend on the MS phenotype.

Faecalibacterium prausnitzii is a highly abundant human gut microbe in healthy individuals, but it is present at reduced levels in individuals with gastrointestinal inflammatory diseases. It has therefore been suggested to constitute a marker of a healthy gut and is associated with anti-inflammatory properties. However, factors affecting the colonization of F. prausnitzii in the human gut during early life are very poorly understood. By analysis of 16S rRNA amplicon sequencing data from three separate infant study populations, we determined the colonization dynamics of Faecalibacterium and factors affecting its establishment in the gut. We found that in particular, the presence of older siblings was consistently associated with Faecalibacterium gut colonization during late infancy and conclude that acquisition of Faecalibacterium is very likely to be accelerated through transfer between siblings. IMPORTANCE Faecalibacterium prausnitzii has been suggested to constitute a key marker of a healthy gut, yet the factors shaping the colonization of this highly oxygen-sensitive, non-spore-forming species in the intestinal environment remain poorly understood. Here, we provide evidence from three separate infant study populations that F. prausnitzii colonization in the gut happens during late infancy and is affected by the number of older siblings in the family. We conclude that Faecalibacterium acquisition is highly likely to be accelerated by contact between siblings. Bearing in mind the immunoregulatory properties of F. prausnitzii and the well-established protective effects against allergic disorders related to the presence of older siblings, early colonization of this species may have profound consequences for child health.

Group A streptococci (GAS) cause 20-30% of pediatric pharyngitis episodes, which are a major cause of ambulatory care visits. Therefore, a strategy to prevent GAS dissemination in children could significantly benefit public healthcare. Contextually, we assessed the possibility of employing alternative food-grade strategies to be used with the oral probiotic L. helveticus MIMLh5 for the prevention of pharyngeal infections. First, we demonstrated through an antagonism-by-exclusion assay that guaran may potentially prevent S. pyogenes adhesion on pharyngeal cells. Subsequently, we showed that an anthocyanin-rich fraction extracted from wild blueberry (BbE) exerts anti-inflammatory effects on the human macrophage cell line U937. Finally, we showed that BbE reduces interferon-β expression in MIMLh5-stimulated murine dendritic cells, resulting in a reduction in the pro-inflammatory cytokines IL-12 and TNF-α. In conclusion, this proof-of-concept study indicates that different food-grade strategies may be concomitantly adopted to potentially prevent GAS colonization and modulate local immune defences.

Staphylococcus aureus is a serious human pathogen and antibiotic resistant, community-associated strains, such as the methicillin resistant S. aureus (MRSA) strain USA300, continue to spread. To avoid resistance, anti-virulence therapy has been proposed where toxicity is targeted rather than viability. Previously we have shown that norlichexanthone, a small non-reduced tricyclic polyketide produced by fungi and lichens, reduces expression of hla encoding α-hemolysin as well as the regulatory RNAIII of the agr quorum sensing system in S. aureus 8325-4. The aim of the present study was to further characterise the mode of action of norlichexanthone and its effect on biofilm formation. We find that norlichexanthone reduces expression of both hla and RNAIII also in strain USA300. Structurally, norlichexanthone resembles ω-hydroxyemodin that recently was shown to bind the agr two component response regulator, AgrA, which controls expression of RNAIII and the phenol soluble modulins responsible for human neutrophil killing. We show that norlichexanthone reduces S. aureus toxicity towards human neutrophils and interferes directly with AgrA binding to its DNA target. In contrast to ω-hydroxyemodin however, norlichexanthone reduces staphylococcal biofilm formation. Transcriptomic analysis revealed that genes regulated by the SaeRS two-component system are repressed by norlichexanthone when compared to untreated cells, an effect that was mitigated in strain Newman carrying a partially constitutive SaeRS system. Our data show that norlichexanthone treatment reduces expression of key virulence factors in CA-MRSA strain USA300 via AgrA binding and represses biofilm formation., Competing Interests: The authors have declared that no competing interests exist.

Interactions between members of the intestinal microbiota and the mucosal immune system can significantly impact human health, and in this context, fungi and food-related yeasts are known to influence intestinal inflammation through direct interactions with specialized immune cells in vivo. The aim of the present study was to characterize the immune modulating properties of the food-related yeast Kluyveromyces marxianus in terms of adaptive immune responses indicating inflammation versus tolerance and to explore the mechanisms behind the observed responses. Benchmarking against a Saccharomyces boulardii strain with probiotic effects documented in clinical trials, we evaluated the ability of K. marxianus to modulate human dendritic cell (DC) function in vitro. Further, we assessed yeast induced DC modulation of naive T cells toward effector responses dominated by secretion of IFNγ and IL-17 versus induction of a Treg response characterized by robust IL-10 secretion. In addition, we blocked relevant DC surface receptors and investigated the stimulating properties of β-glucan containing yeast cell wall extracts. K. marxianus and S. boulardii induced distinct levels of DC cytokine secretion, primarily driven by Dectin-1 recognition of β-glucan components in their cell walls. Upon co-incubation of yeast exposed DCs and naive T cells, S. boulardii induced a potent IFNγ response indicating TH1 mobilization. In contrast, K. marxianus induced a response dominated by Foxp3+ Treg cells, a characteristic that may benefit human health in conditions characterized by excessive inflammation and positions K. marxianus as a strong candidate for further development as a novel yeast probiotic., Competing Interests: I have read the journal’s policy and have the following conflicts to declare. IMS and AB are employees of Chr. Hansen A/S, a manufacturer of probiotic products, and the described work was carried out at Chr. Hansen A/S facilities in Hørsholm, Denmark. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Preterm neonates are highly sensitive to systemic infections in early life but little is known about systemic immune development following preterm birth. We hypothesized that preterm neonates have immature systemic immunity with distinct developmental trajectory for the first several weeks of life, relative to those born at near-term or term. Using pigs as a model, we characterized blood leukocyte subsets, antimicrobial activities and TLR-mediated cytokine production during the first weeks after preterm birth. Relative to near-term and term pigs, newborn preterm pigs had low blood leukocyte counts, poor neutrophil phagocytic rate, and limited cytokine responses to TLR1/2/5/7/9 and NOD1/2 agonists. The preterm systemic responses remained immature during the first postnatal week, but thereafter showed increased blood leukocyte numbers, NK cell proportion, neutrophil phagocytic rate and TLR2-mediated IL-6 and TNF-α production. These immune parameters remained different between preterm and near-term pigs at 2-3 weeks, even when adjusted for post-conceptional age. Our data suggest that systemic immunity follows a distinct developmental trajectory following preterm birth that may be influenced by postnatal age, complications of prematurity and environmental factors. Consequently, the immediate postnatal period may represent a window of opportunity to improve innate immunity in preterm neonates by medical, antimicrobial or dietary interventions.

Lactobacillus acidophilus induces a potent interferon-β (IFN-β) response in dendritic cells (DCs) by a Toll-like receptor 2 (TLR2) -dependent mechanism, in turn leading to strong interleukin-12 (IL-12) production. In the present study, we investigated the involvement of different types of endocytosis in the L. acidophilus-induced IFN-β and IL-12 responses and how TLR2 or TLR4 ligation by lipopolysaccharide and Pam3/4CSK4 influenced endocytosis of L. acidophilus and the induced IFN-β and IL-12 production. Lactobacillus acidophilus was endocytosed by constitutive macropinocytosis taking place in the immature cells as well as by spleen tyrosine kinase (Syk) -dependent phagocytosis but without involvement of plasma membrane TLR2. Stimulation with TLR2 or TLR4 ligands increased macropinocytosis in a Syk-independent manner. As a consequence, incubation of DCs with TLR ligands before incubation with L. acidophilus enhanced the uptake of the bacteria. However, in these experimental conditions, induction of IFN-β and IL-12 was strongly inhibited. As L. acidophilus-induced IFN-β depends on endocytosis and endosomal degradation before signalling and as TLR stimulation from the plasma membrane leading to increased macropinocytosis abrogates IFN-β induction we conclude that plasma membrane TLR stimulation leading to increased macropinocytosis decreases endosomal induction of IFN-β and speculate that this is due to competition between compartments for molecules involved in the signal pathways. In summary, endosomal signalling by L. acidophilus that leads to IFN-β and IL-12 production is inhibited by TLR stimulation from the plasma membrane., (© 2016 John Wiley & Sons Ltd.)

Staphylococcus aureus is a major human pathogen that has evolved very efficient immune evading strategies leading to persistent colonization. During different stages of growth, S. aureus express various surface molecules, which may affect the immune stimulating properties, but very little is known about their role in immune stimulation and evasion. Depending on the growth phase, S. aureus may affect antigen presenting cells differently. Here, the impact of growth phases and the surface molecules lipoteichoic acid, peptidoglycan and poly-N-acetyl glucosamine on the induction of IL-12 imperative for an efficient clearance of S. aureus was studied in dendritic cells (DCs). Exponential phase (EP) S. aureus was superior to stationary phase (SP) bacteria in induction of IL-12, which required actin-mediated endocytosis and endosomal acidification. Moreover, addition of staphylococcal cell wall derived peptidoglycan to EP S. aureus stimulated cells increased bacterial uptake but abrogated IL-12 induction, while addition of lipoteichoic acid increased IL-12 production but had no effect on the bacterial uptake. Depletion of the capability to produce poly-N-acetyl glucosamine increased the IL-12 inducing activity of EP bacteria. Furthermore, the mutant dltA unable to produce D-alanylated teichoic acids failed to induce IL-12 but like peptidoglycan and the toll-like receptor (TLR) ligands LPS and Pam3CSK4 the mutant stimulated increased macropinocytosis. In conclusion, the IL-12 response by DCs against S. aureus is highly growth phase dependent, relies on cell wall D-alanylation, endocytosis and subsequent endosomal degradation, and is abrogated by receptor induced macropinocytosis.

Staphylococcus aureus infections are becoming increasingly difficult to treat due to antibiotic resistance with the community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 being of particular concern. The inhibition of bacterial virulence has been proposed as an alternative approach to treat multi-drug resistant pathogens. One interesting anti-virulence target is the agr quorum-sensing system, which regulates virulence of CA-MRSA in response to agr-encoded autoinducing peptides. Agr regulation confines exotoxin production to the stationary growth phase with concomitant repression of surface-expressed adhesins. Solonamide B, a non-ribosomal depsipeptide of marine bacterial origin, was recently identified as a putative anti-virulence compound that markedly reduced expression of α-hemolysin and phenol-soluble modulins. To further strengthen solonamide anti-virulence candidacy, we report the chemical synthesis of solonamide analogues, investigation of structure-function relationships, and assessment of their potential to modulate immune cell functions. We found that structural differences between solonamide analogues confer significant differences in interference with agr, while immune cell activity and integrity is generally not affected. Furthermore, treatment of S. aureus with selected solonamides was found to only marginally influence the interaction with fibronectin and biofilm formation, thus addressing the concern that application of compounds inducing an agr-negative state may have adverse interactions with host factors in favor of host colonization.

Neonatal studies in different mouse strains reveal that early life colonization affects the development of adaptive immunity in mice. The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes, but neonatal studies of NOD mice are lacking. We hypothesized that NOD mice deviate from another much used mouse strain, C57BL/6, with respect to postnatal microbiota and/or hematopoiesis and compared this in newborn mice of dams housed under the same conditions. A distinct bacteria profile rich in staphylococci was found at postnatal days (PND) 1-4 in NOD mice. Furthermore, a distinct splenic cell profile high in a granulocytic phenotype was evident in the neonatal NOD mice whereas neonatal C57BL/6 mice showed a profile rich in monocytes. Neonatal expression of Reg3g and Muc2 in the gut was deviating in NOD mice and coincided with fewer bacteria attaching to the Mucosal surface in NOD compared to C57BL/6 mice.

Immature immunity may predispose preterm neonates to infections and necrotizing enterocolitis (NEC). Intravenous antibiotics are frequently given to prevent and treat sepsis, while oral antibiotics are seldom used. We hypothesized that oral antibiotics promote maturation of systemic immunity and delay gut bacterial colonization and thereby protect preterm neonates against both NEC and bacteremia in the immediate postnatal period. Preterm pigs were given formula and administered saline (CON) or broad-spectrum antibiotics orally (ORA) or systemically (SYS) for 5 d after birth. Temporal changes in blood parameters and bacterial composition in the intestine, blood and immune organs were analyzed. Newborn preterm pigs had few blood neutrophils and a high frequency of progenitor cells. Neutrophils gradually matured after preterm birth with increasing CD14 and decreasing CD172a expressions. Preterm neutrophil and monocyte TLR2 expression and TLR2-mediated blood cytokine responses were low relative to adults. ORA pigs showed enhanced blood neutrophil maturation with reduced cell size and CD172a expression. Only ORA pigs, but not SYS pigs, were protected from a high density of gut Gram-positive bacteria, high gut permeability, Gram-positive bacteremia and NEC. Neonatal oral antibiotics may benefit mucosal and systemic immunity via delayed gut colonization and enhanced blood neutrophil maturation just after preterm birth., (© The Author(s) 2015.)