The live biotherapeutic Blautia stercoris MRx0006 attenuates social deficits, repetitive behaviour, and anxiety-like behaviour in a mouse model relevant to autism.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterised by deficits in social behaviour, increased repetitive behaviour, anxiety and gastrointestinal symptoms. The aetiology of ASD is complex and involves an interplay of genetic and environmental factors. Emerging pre-clinical and clinical studies have documented a potential role for the gut microbiome in ASD, and consequently, the microbiota represents a potential target in the development of novel therapeutics for this neurodevelopmental disorder. In this study, we investigate the efficacy of the live biotherapeutic strain, Blautia stercoris MRx0006, in attenuating some of the behavioural deficits in the autism-relevant, genetic mouse model, BTBR T+ Itpr3tf/J (BTBR). We demonstrate that daily oral administration with MRx0006 attenuates social deficits while also decreasing repetitive and anxiety-like behaviour. MRx0006 administration increases the gene expression of oxytocin and its receptor in hypothalamic cells in vitro and increases the expression of hypothalamic arginine vasopressin and oxytocin mRNA in BTBR mice. Additionally at the microbiome level, we observed that MRx0006 administration decreases the abundance of Alistipes putredinis, and modulates the faecal microbial metabolite profile. This alteration in the metabolite profile possibly underlies the observed increase in expression of oxytocin, arginine vasopressin and its receptors, and the consequent improvements in behavioural outcomes. Taken together, these findings suggest that the live biotherapeutic MRx0006 may represent a viable and efficacious treatment option for the management of physiological and behavioural deficits associated with ASD.

Microbial short-chain fatty acids modulate CD8+ T cell responses and improve adoptive immunotherapy for cancer.

Emerging data demonstrate that the activity of immune cells can be modulated by microbial molecules. Here, we show that the short-chain fatty acids (SCFAs) pentanoate and butyrate enhance the anti-tumor activity of cytotoxic T lymphocytes (CTLs) and chimeric antigen receptor (CAR) T cells through metabolic and epigenetic reprograming. We show that in vitro treatment of CTLs and CAR T cells with pentanoate and butyrate increases the function of mTOR as a central cellular metabolic sensor, and inhibits class I histone deacetylase activity. This reprogramming results in elevated production of effector molecules such as CD25, IFN-γ and TNF-α, and significantly enhances the anti-tumor activity of antigen-specific CTLs and ROR1-targeting CAR T cells in syngeneic murine melanoma and pancreatic cancer models. Our data shed light onto microbial molecules that may be used for enhancing cellular anti-tumor immunity. Collectively, we identify pentanoate and butyrate as two SCFAs with therapeutic utility in the context of cellular cancer immunotherapy.

In vitro Characterization of Gut Microbiota-Derived Bacterial Strains With Neuroprotective Properties.

Neurodegenerative diseases are disabling, incurable, and progressive conditions characterized by neuronal loss and decreased cognitive function. Changes in gut microbiome composition have been linked to a number of neurodegenerative diseases, indicating a role for the gut-brain axis. Here, we show how specific gut-derived bacterial strains can modulate neuroinflammatory and neurodegenerative processes in vitro through the production of specific metabolites and discuss the potential therapeutic implications for neurodegenerative disorders. A panel of fifty gut bacterial strains was screened for their ability to reduce pro-inflammatory IL-6 secretion in U373 glioblastoma astrocytoma cells. Parabacteroides distasonis MRx0005 and Megasphaera massiliensis MRx0029 had the strongest capacity to reduce IL-6 secretion in vitro. Oxidative stress plays a crucial role in neuroinflammation and neurodegeneration, and both bacterial strains displayed intrinsic antioxidant capacity. While MRx0005 showed a general antioxidant activity on different brain cell lines, MRx0029 only protected differentiated SH-SY5Y neuroblastoma cells from chemically induced oxidative stress. MRx0029 also induced a mature phenotype in undifferentiated neuroblastoma cells through upregulation of microtubule-associated protein 2. Interestingly, short-chain fatty acid analysis revealed that MRx0005 mainly produced C1-C3 fatty acids, while MRx0029 produced C4-C6 fatty acids, specifically butyric, valeric and hexanoic acid. None of the short-chain fatty acids tested protected neuroblastoma cells from chemically induced oxidative stress. However, butyrate was able to reduce neuroinflammation in vitro, and the combination of butyrate and valerate induced neuronal maturation, albeit not to the same degree as the complex cell-free supernatant of MRx0029. This observation was confirmed by solvent extraction of cell-free supernatants, where only MRx0029 methanolic fractions containing butyrate and valerate showed an anti-inflammatory activity in U373 cells and retained the ability to differentiate neuroblastoma cells. In summary, our results suggest that the pleiotropic nature of live biotherapeutics, as opposed to isolated metabolites, could be a promising novel drug class in drug discovery for neurodegenerative disorders.

Identification performance of MALDI-ToF-MS upon mono- and bi-microbial cultures is cell number and culture proportion dependent.

Biotyping using matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectroscopy (MS) has revolutionized microbiology by allowing clinicians and scientists to rapidly identify microbes at genus and species levels. The present study extensively assesses the suitability and reliability of MALDI-ToF biotyping of 14 different aerobic and anaerobic bacterial species as pure and mixed cultures. Reliable identification at species level was possible from biomaterial of older colonies and even frozen biomaterial, although this was species dependent. Using standard instrument settings and direct application of biomaterial onto the MALDI-ToF target plates, it was determined that the cell densities necessary for completely reliable identification of pure cultures varied between 2.40 × 108 and 1.10 × 1010 viable cell counts (VCCs) per mL, depending on the species. Evaluation of the mixed culture algorithm of the Bruker Biotyper® software showed that the performance of the algorithm depends greatly on the targeted species, on their phylogenetic distance, and on their ratio of VCC per mL in the mixed culture. Hence, the use of MALDI-ToF-MS with incorporation of the mixed culture algorithm of the software is a useful pre-screening tool for early identification of contaminants, but due to the great variability in performance between different species and the usually unknown percentage of the possible contaminant in the mixture, it is advisable to combine this method with other microbiology methods.

The flagellin of candidate live biotherapeutic Enterococcus gallinarum MRx0518 is a potent immunostimulant.

Many links between gut microbiota and disease development have been established in recent years, with particular bacterial strains emerging as potential therapeutics rather than causative agents. In this study we describe the immunostimulatory properties of Enterococcus gallinarum MRx0518, a candidate live biotherapeutic with proven anti-tumorigenic efficacy. Here we demonstrate that strain MRx0518 elicits a strong pro-inflammatory response in key components of the innate immune system but also in intestinal epithelial cells. Using a flagellin knock-out derivative and purified recombinant protein, MRx0518 flagellin was shown to be a TLR5 and NF-κB activator in reporter cells and an inducer of IL-8 production by HT29-MTX cells. E. gallinarum flagellin proteins display a high level of sequence diversity and the flagellin produced by MRx0518 was shown to be more potent than flagellin from E. gallinarum DSM100110. Collectively, these data infer that flagellin may play a role in the therapeutic properties of E. gallinarum MRx0518.

Bacteroides thetaiotaomicron Ameliorates Colon Inflammation in Preclinical Models of Crohn's Disease.

Background: Alterations in the gut microbiota are strongly associated with the development of inflammatory bowel disease (IBD), particularly with Crohn's disease, which is characterized by reduced abundance of commensal anaerobic bacteria including members of the Bacteroides genus. Our aim was to investigate the protective effects of Bacteroides thetaiotaomicron, an abundant member of this genus, in different rodent models of IBD. Methods: We assessed the effect of B. thetaiotaomicron administration on primary readouts of colitis (weight loss, histopathology, and immune parameters) in dextran sodium sulphate (DSS) and interleukin-10 knockout (IL10KO) models of IBD. Efficacy of a freeze-dried bacterial formulation and a purified recombinant protein of B. thetaiotaomicron was also investigated. Results: B. thetaiotaomicron showed protective effects in both DSS and IL10KO rodent models, as demonstrated by significant amelioration of weight loss, colon shortening, histopathological damage and immune activation. This efficacy was not exclusive to actively growing bacterial preparations but was retained by freeze-dried cells of B. thetaiotaomicron. A pirin-like protein (PLP) of B. thetaiotaomicron, identified by microarray analysis during coculture of the bacterial strain with Caco-2 cells, reduced pro-inflammatory NF-κB signalling in these intestinal epithelial cells. Recombinant PLP partially recapitulated the effect of the whole strain in a rat DSS model. Conclusions: B. thetaiotaomicron displays strong efficacy in preclinical models of IBD and protects against weight loss, histopathological changes in the colon and inflammatory markers. These data indicate that the live strain or its products may be a novel alternative to current treatment options for Crohn's disease.

Bifidobacterium breve MRx0004 protects against airway inflammation in a severe asthma model by suppressing both neutrophil and eosinophil lung infiltration.

Asthma is a phenotypically heterogeneous disease. In severe asthma, airway inflammation can be predominantly eosinophilic, neutrophilic, or mixed. Only a limited number of drug candidates are in development to address this unmet clinical need. Live biotherapeutics derived from the gut microbiota are a promising new therapeutic area. MRx0004 is a commensal Bifidobacterium breve strain isolated from the microbiota of a healthy human. The strain was tested prophylactically and therapeutically by oral gavage in a house dust mite mouse model of severe asthma. A strong reduction of neutrophil and eosinophil infiltration was observed in lung bronchoalveolar lavage fluid following MRx0004 treatment. Peribronchiolar and perivascular immunopathology was also reduced. MRx0004 increased lung CD4+CD44+ cells and CD4+FoxP3+ cells and decreased activated CD11b+ dendritic cells. Cytokine analysis of lung tissue revealed reductions of pro-inflammatory cytokines and chemokines involved in neutrophil migration. In comparison, anti-IL-17 antibody treatment effectively reduced neutrophilic infiltration and increased CD4+FoxP3+ cells, but it induced lung eosinophilia and did not decrease histopathology scores. We have demonstrated that MRx0004, a microbiota-derived bacterial strain, can reduce both neutrophilic and eosinophilic infiltration in a mouse model of severe asthma. This novel therapeutic is a promising next-generation drug for management of severe asthma.

Human gut bacteria as potent class I histone deacetylase inhibitors in vitro through production of butyric acid and valeric acid.

Overexpression of histone deacetylase (HDAC) isoforms has been implicated in a variety of disease pathologies, from cancer and colitis to cardiovascular disease and neurodegeneration, thus HDAC inhibitors have a long history as therapeutic targets. The gut microbiota can influence HDAC activity via microbial-derived metabolites. While HDAC inhibition (HDI) by gut commensals has long been attributed to the short chain fatty acid (SCFA) butyrate, the potent metabolic reservoir provided by the gut microbiota and its role in host physiology warrants further investigation in a variety of diseases. Cell-free supernatants (CFS) of 79 phylogenetically diverse gut commensals isolated from healthy human donors were screened for their SCFA profile and their total HDAC inhibitory properties. The three most potent HDAC inhibiting strains were further evaluated and subjected to additional analysis of specific class I and class II HDAC inhibition. All three HDAC inhibitors are butyrate producing strains, and one of these also produced substantial levels of valeric acid and hexanoic acid. Valeric acid was identified as a potential contributor to the HDAC inhibitory effects. This bacterial strain, Megasphaera massiliensis MRx0029, was added to a model microbial consortium to assess its metabolic activity in interaction with a complex community. M. massiliensis MRx0029 successfully established in the consortium and enhanced the total and specific HDAC inhibitory function by increasing the capacity of the community to produce butyrate and valeric acid. We here show that single bacterial strains from the human gut microbiota have potential as novel HDI therapeutics for disease areas involving host epigenetic aberrations.

Development of Immune Cells in the Intestinal Mucosa Can Be Affected by Intensive and Extensive Farm Environments, and Antibiotic Use.

Epidemiological studies have demonstrated that exposure to farm environments during childhood can be linked to reductions in the incidence of immune disorders, but generating an appropriate model is difficult. 108 half-sibling piglets were born on either extensive (outdoor) or intensive (indoor) farms: at 1 day old, a subset of piglets from each litter were transferred to a high-hygiene isolator facility to create differences in rearing environment either during birth/first day or during the subsequent 56 days of life. Interactions between CD14, CD16, MHCIIDR, and capillary endothelium were assessed using four-color quantitative fluorescence immunohistology. Effects of birth and rearing environment on the antigen-presenting microenvironment of the proximal and distal jejunum (professional and stromal) were apparent at 5, 28, and 56 days after birth However, effects on CD4+CD25+Foxp3+ regulatory T-cells (Tregs) in the intestinal mucosa were apparent around weaning at 28 days but had disappeared by 56 days. These Tregs were reduced in the isolator piglets compared to their farm-reared siblings, but this effect was less marked in piglets born on the extensive farm and required administration of antibiotics. Our results suggest that there may be at least two windows of opportunity in which different farm environments were influencing immune development: one during the perinatal period (up to the first day of life), and one during later infancy. Furthermore, the differences on Tregs suggest that the effects of early life influences may be particularly critical around weaning.

Human Gut Symbiont Roseburia hominis Promotes and Regulates Innate Immunity.

OBJECTIVE: Roseburia hominis is a flagellated gut anaerobic bacterium belonging to the Lachnospiraceae family within the Firmicutes phylum. A significant decrease of R. hominis colonization in the gut of ulcerative colitis patients has recently been demonstrated. In this work, we have investigated the mechanisms of R. hominis-host cross talk using both murine and in vitro models. DESIGN: The complete genome sequence of R. hominis A2-183 was determined. C3H/HeN germ-free mice were mono-colonized with R. hominis, and the host-microbe interaction was studied using histology, transcriptome analyses and FACS. Further investigations were performed in vitro and using the TLR5KO and DSS-colitis murine models. RESULTS: In the bacterium, R. hominis, host gut colonization upregulated genes involved in conjugation/mobilization, metabolism, motility, and chemotaxis. In the host cells, bacterial colonization upregulated genes related to antimicrobial peptides, gut barrier function, toll-like receptors (TLR) signaling, and T cell biology. CD4+CD25+FoxP3+ T cell numbers increased in the lamina propria of both mono-associated and conventional mice treated with R. hominis. Treatment with the R. hominis bacterium provided protection against DSS-induced colitis. The role of flagellin in host-bacterium interaction was also investigated. CONCLUSION: Mono-association of mice with R. hominis bacteria results in specific bidirectional gene expression patterns. A set of genes thought to be important for host colonization are induced in R. hominis, while the host cells respond by strengthening gut barrier function and enhancing Treg population expansion, possibly via TLR5-flagellin signaling. Our data reveal the immunomodulatory properties of R. hominis that could be useful for the control and treatment of gut inflammation.

16S sequencing and functional analysis of the fecal microbiome during treatment of newly diagnosed pediatric inflammatory bowel disease.

The human microbiome is of considerable interest to pediatric inflammatory bowel disease (PIBD) researchers with 1 potential mechanism for disease development being aberrant immune handling of the intestinal bacteria. This study analyses the fecal microbiome through treatment in newly diagnosed PIBD patients and compares to cohabiting siblings where possible. Patients were recruited on clinical suspicion of PIBD before diagnosis. Treatment-naïve fecal samples were collected, with further samples at 2 and 6 weeks into treatment. Samples underwent 16S ribosomal ribonucleic acid (RNA) gene sequencing and short-chain fatty acids (SCFAs) analysis, results were analyzed using quantitative-insights-into-microbial-ecology. Six PIBD patients were included in the cohort: 4 Crohn disease (CD), 1 ulcerative colitis (UC), 1 inflammatory bowel disease (IBD) unclassified, and median age 12.6 (range 10-15.1 years); 3 patients had an unaffected healthy sibling recruited. Microbial diversity (observed species/Chao1/Shannon diversity) was reduced in treatment-naïve patients compared to siblings and patients in remission. Principal coordinate analysis using Bray-Curtis dissimilarity and UniFrac revealed microbial shifts in CD over the treatment course. In treatment-naïve PIBD, there was reduction in functional ability for amino acid metabolism and carbohydrate handling compared to controls (P = .038) and patients in remission (P = .027). Metabolic function returned to normal after remission was achieved. SCFA revealed consistent detection of lactate in treatment-naïve samples. This study adds in-depth 16S rRNA sequencing analysis on a small longitudinal cohort to the literature and includes sibling controls and patients with UC/IBD unclassified. It highlights the initial dysbiosis, reduced diversity, altered functional potential, and subsequent shifts in bacteria from diagnosis over time to remission.

Antibiotics modulate intestinal immunity and prevent necrotizing enterocolitis in preterm neonatal piglets.

Preterm birth, bacterial colonization, and formula feeding predispose to necrotizing enterocolitis (NEC). Antibiotics are commonly administered to prevent sepsis in preterm infants, but it is not known whether this affects intestinal immunity and NEC resistance. We hypothesized that broad-spectrum antibiotic treatment improves NEC resistance and intestinal structure, function, and immunity in neonates. Caesarean-delivered preterm pigs were fed 3 days of parenteral nutrition followed by 2 days of enteral formula. Immediately after birth, they were assigned to receive either antibiotics (oral and parenteral doses of gentamycin, ampicillin, and metronidazole, ANTI, n = 11) or saline in the control group (CON, n = 13), given twice daily. NEC lesions and intestinal structure, function, microbiology, and immunity markers were recorded. None of the ANTI but 85% of the CON pigs developed NEC lesions by day 5 (0/11 vs. 11/13, P < 0.05). ANTI pigs had higher intestinal villi (+60%), digestive enzyme activities (+53-73%), and goblet cell densities (+110%) and lower myeloperoxidase (-51%) and colonic microbial density (10(5) vs. 10(10) colony-forming units, all P < 0.05). Microarray transcriptomics showed strong downregulation of genes related to inflammation and innate immune response to microbiota and marked upregulation of genes related to amino acid metabolism, in particular threonine, glucose transport systems, and cell cycle in 5-day-old ANTI pigs. In a follow-up experiment, 5 days of antibiotics prevented NEC at least until day 10. Neonatal prophylactic antibiotics effectively reduced gut bacterial load, prevented NEC, intestinal atrophy, dysfunction, and inflammation and enhanced expression of genes related to gut metabolism and immunity in preterm pigs.

The NF-κB binding site located in the proximal region of the TSLP promoter is critical for TSLP modulation in human intestinal epithelial cells.

Thymic stromal lymphopoietin (TSLP) is constitutively secreted by intestinal epithelial cells. It regulates gut DCs, therefore, contributing to the maintenance of immune tolerance. In the present report, we describe the regulation of TSLP expression in intestinal epithelial cells and characterize the role of several NF-κB binding sites present on the TSLP promoter. TSLP expression can be stimulated by different compounds through activation of p38, protein kinase A, and finally the NF-κB pathway. We describe a new NF-κB binding element located at position -0.37 kb of the promoter that is crucial for the NF-κB-dependent regulation of TSLP. We showed that mutation of this proximal NF-κB site abrogates the IL-1ß-mediated transcriptional activation of human TSLP in several epithelial cell lines. We also demonstrated that both p65 and p50 subunits are able to bind this new NF-κB binding site. The present work provides new insight into epithelial cell-specific TSLP regulation.

Microbiome and immunological interactions.

The healthy human gut supports a complex and diverse microbiota, dominated by bacterial phylotypes belonging to Bacteroidetes and Firmicutes. In the inflamed gut, overall diversity decreases, coincident with a greater representation of Proteobacteria. There is growing evidence supporting an important role for human gut bacteria in mucosal immunity; interactions at the level of both intestinal and colonic epithelial cells, dendritic cells, and T and B immune cells have been documented. These interactions influence gut barrier and defense mechanisms that include antimicrobial peptide and secretory IgA synthesis. The functional effects of commensal bacteria on T helper cell differentiation have led to the emerging concept that microbiota composition determines T effector- and T regulatory-cell balance, immune responsiveness, and homeostasis. The importance of this biology in relation to immune homeostasis, inflammatory bowel disease, and the rising incidence of autoimmune diseases will be discussed. The detailed description of the human gut microbiota, integrated with evidence-based mechanisms of immune modulation, provides an exciting platform for the identification of next-generation probiotics and related pharmaceutical products.

Direct experimental evidence that early-life farm environment influences regulation of immune responses.

BACKGROUND: In mammals, early-life environmental variations appear to affect microbial colonization and therefore competent immune development, and exposure to farm environments in infants has been inversely correlated with allergy development. Modelling these effects using manipulation of neonatal rodents is difficult due to their dependency on the mother, but the relatively independent piglet is increasingly identified as a valuable translational model for humans. This study was designed to correlate immune regulation in piglets with early-life environment. METHODS: Piglets were nursed by their mother on a commercial farm, while isolator-reared siblings were formula fed. Fluorescence immunohistology was used to quantify T-reg and effector T-cell populations in the intestinal lamina propria and the systemic response to food proteins was quantified by capture ELISA. RESULTS: There was more CD4(+) and CD4(+) CD25(+) effector T-cell staining in the intestinal mucosa of the isolator-reared piglets compared with their farm-reared counterparts. In contrast, these isolator-reared piglets had a significantly reduced CD4(+) CD25(+) Foxp3(+) regulatory T-cell population compared to farm-reared littermates, resulting in a significantly higher T-reg-to-effector ratio in the farm animals. Consistent with these findings, isolator-reared piglets had an increased serum IgG anti-soya response to novel dietary soya protein relative to farm-reared piglets. CONCLUSION: Here, we provide the first direct evidence, derived from intervention, that components of the early-life environment present on farms profoundly affects both local development of regulatory components of the mucosal immune system and immune responses to food proteins at weaning. We propose that neonatal piglets provide a tractable model which allows maternal and treatment effects to be statistically separated.

Microbes and microbial effector molecules in treatment of inflammatory disorders.

The healthy gut tolerates very large numbers of diverse bacterial species belonging mainly to the Bacteroidetes and Firmicutes phyla. These bacteria normally coexist peacefully with the gut and help maintain immune homeostasis and tolerance. The mechanisms promoting tolerance affect various cell populations, including the epithelial cells lining the gut, resident dendritic cells (DCs), and gut-homing T cells. Gut bacteria also influence multiple signaling pathways from Toll-like receptors to nuclear factor κB and regulate the functionality of DCs and T cells. Several bacterial species have been identified that promote T-cell differentiation, in particular T-helper 17 and T-regulatory cells. Insight into the molecular mechanisms by which bacteria mediate these effects will be very important in identifying new ways of treating intestinal and extra-intestinal immune-mediated diseases. These diseases are increasing dramatically in the human population and require new treatments. It may be possible in the future to identify specific bacterial species or strains that can correct for T-cell imbalances in the gut and promote immune homeostasis, both locally and systemically. In addition, new information describing microbial genomes affords the opportunity to mine for functional genes that may lead to new generation drugs relevant to a range of inflammatory disease conditions.

Establishment of normal gut microbiota is compromised under excessive hygiene conditions.

BACKGROUND: Early gut colonization events are purported to have a major impact on the incidence of infectious, inflammatory and autoimmune diseases in later life. Hence, factors which influence this process may have important implications for both human and animal health. Previously, we demonstrated strong influences of early-life environment on gut microbiota composition in adult pigs. Here, we sought to further investigate the impact of limiting microbial exposure during early life on the development of the pig gut microbiota. METHODOLOGY/PRINCIPAL FINDINGS: Outdoor- and indoor-reared animals, exposed to the microbiota in their natural rearing environment for the first two days of life, were transferred to an isolator facility and adult gut microbial diversity was analyzed by 16S rRNA gene sequencing. From a total of 2,196 high-quality 16S rRNA gene sequences, 440 phylotypes were identified in the outdoor group and 431 phylotypes in the indoor group. The majority of clones were assigned to the four phyla Firmicutes (67.5% of all sequences), Proteobacteria (17.7%), Bacteroidetes (13.5%) and to a lesser extent, Actinobacteria (0.1%). Although the initial maternal and environmental microbial inoculum of isolator-reared animals was identical to that of their naturally-reared littermates, the microbial succession and stabilization events reported previously in naturally-reared outdoor animals did not occur. In contrast, the gut microbiota of isolator-reared animals remained highly diverse containing a large number of distinct phylotypes. CONCLUSIONS/SIGNIFICANCE: The results documented here indicate that establishment and development of the normal gut microbiota requires continuous microbial exposure during the early stages of life and this process is compromised under conditions of excessive hygiene.

Restricting microbial exposure in early life negates the immune benefits associated with gut colonization in environments of high microbial diversity.

BACKGROUND: Acquisition of the intestinal microbiota in early life corresponds with the development of the mucosal immune system. Recent work on caesarean-delivered infants revealed that early microbial composition is influenced by birthing method and environment. Furthermore, we have confirmed that early-life environment strongly influences both the adult gut microbiota and development of the gut immune system. Here, we address the impact of limiting microbial exposure after initial colonization on the development of adult gut immunity. METHODOLOGY/PRINCIPAL FINDINGS: Piglets were born in indoor or outdoor rearing units, allowing natural colonization in the immediate period after birth, prior to transfer to high-health status isolators. Strikingly, gut closure and morphological development were strongly affected by isolator-rearing, independent of indoor or outdoor origins of piglets. Isolator-reared animals showed extensive vacuolation and disorganization of the gut epithelium, inferring that normal gut closure requires maturation factors present in maternal milk. Although morphological maturation and gut closure were delayed in isolator-reared animals, these hard-wired events occurred later in development. Type I IFN, IL-22, IL-23 and Th17 pathways were increased in indoor-isolator compared to outdoor-isolator animals during early life, indicating greater immune activation in pigs originating from indoor environments reflecting differences in the early microbiota. This difference was less apparent later in development due to enhanced immune activation and convergence of the microbiota in all isolator-reared animals. This correlated with elevation of Type I IFN pathways in both groups, although T cell pathways were still more affected in indoor-reared animals. CONCLUSIONS/SIGNIFICANCE: Environmental factors, in particular microbial exposure, influence expression of a large number of immune-related genes. However, the homeostatic effects of microbial colonization in outdoor environments require sustained microbial exposure throughout development. Gut development in high-hygiene environments negatively impacts on normal succession of the gut microbiota and promotes innate immune activation which may impair immune homeostasis.

Genome analysis of Bifidobacterium bifidum PRL2010 reveals metabolic pathways for host-derived glycan foraging.

The human intestine is densely populated by a microbial consortium whose metabolic activities are influenced by, among others, bifidobacteria. However, the genetic basis of adaptation of bifidobacteria to the human gut is poorly understood. Analysis of the 2,214,650-bp genome of Bifidobacterium bifidum PRL2010, a strain isolated from infant stool, revealed a nutrient-acquisition strategy that targets host-derived glycans, such as those present in mucin. Proteome and transcriptome profiling revealed a set of chromosomal loci responsible for mucin metabolism that appear to be under common transcriptional control and with predicted functions that allow degradation of various O-linked glycans in mucin. Conservation of the latter gene clusters in various B. bifidum strains supports the notion that host-derived glycan catabolism is an important colonization factor for B. bifidum with concomitant impact on intestinal microbiota ecology.