Tuft cells mediate commensal remodeling of the small intestinal antimicrobial landscape.

Succinate produced by the commensal protist Tritrichomonas musculis (T. mu) stimulates chemosensory tuft cells, resulting in intestinal type 2 immunity. Tuft cells express the succinate receptor SUCNR1, yet this receptor does not mediate antihelminth immunity nor alter protist colonization. Here, we report that microbial-derived succinate increases Paneth cell numbers and profoundly alters the antimicrobial peptide (AMP) landscape in the small intestine. Succinate was sufficient to drive this epithelial remodeling, but not in mice lacking tuft cell chemosensory components required to detect this metabolite. Tuft cells respond to succinate by stimulating type 2 immunity, leading to interleukin-13-mediated epithelial and AMP expression changes. Moreover, type 2 immunity decreases the total number of mucosa-associated bacteria and alters the small intestinal microbiota composition. Finally, tuft cells can detect short-term bacterial dysbiosis that leads to a spike in luminal succinate levels and modulate AMP production in response. These findings demonstrate that a single metabolite produced by commensals can markedly shift the intestinal AMP profile and suggest that tuft cells utilize SUCNR1 and succinate sensing to modulate bacterial homeostasis.

CCR2-dependent CX3CR1+ colonic macrophages promote Enterococcus faecalis dissemination.

Enterococci are common commensal bacteria that colonize the gastrointestinal tracts of most mammals, including humans. Importantly, these bacteria are one of the leading causes of nosocomial infections. This study examined the role of colonic macrophages in facilitating Enterococcus faecalis infections in mice. We determined that depletion of colonic phagocytes resulted in the reduction of E. faecalis dissemination to the gut-draining mesenteric lymph nodes. Furthermore, we established that trafficking of monocyte-derived CX3CR1-expressing macrophages contributed to E. faecalis dissemination in a manner that was not reliant on CCR7, the conventional receptor involved in lymphatic migration. Finally, we showed that E. faecalis mutants with impaired intracellular survival exhibited reduced dissemination, suggesting that E. faecalis can exploit host immune cell migration to disseminate systemically and cause disease. Our findings indicate that modulation of macrophage trafficking in the context of antibiotic therapy could serve as a novel approach for preventing or treating opportunistic infections by disseminating enteric pathobionts like E. faecalis.

A serum-induced gene signature in hepatocytes is associated with pediatric nonalcoholic fatty liver disease.

OBJECTIVE: Pediatric nonalcoholic fatty liver disease (NAFLD) is a growing problem, but its underlying mechanisms are poorly understood. We used transcriptomic reporter cell assays to investigate differences in transcriptional signatures induced in hepatocyte reporter cells by the sera of children with and without NAFLD. METHODS: We studied serum samples from 45 children with NAFLD and 28 children without NAFLD. The sera were used to induce gene expression in cultured HepaRG cells and RNA-sequencing was used to determine gene expression. Computational techniques were used to compare gene expression patterns. RESULTS: Sera from children with NAFLD induced the expression of 195 genes that were significantly differentially expressed in hepatocytes compared to controls with obesity. NAFLD was associated with increased expression of genes promoting inflammation, collagen synthesis, and extracellular matrix remodeling. Additionally, there was lower expression of genes involved in endobiotic and xenobiotic metabolism, and downregulation of peroxisome function, oxidative phosphorylation, and xenobiotic, bile acid, and fatty acid metabolism. A 13-gene signature, including upregulation of TREM1 and MMP1 and downregulation of CYP2C9, was consistently associated with all diagnostic categories of pediatric NAFLD. CONCLUSION: The extracellular milieu of sera from children with NAFLD induced specific gene profiles distinguishable by a hepatocyte reporter system. Circulating factors may contribute to inflammation and extracellular matrix remodeling and impair xenobiotic and endobiotic metabolism in pediatric NAFLD.

Enteric defensins are essential regulators of intestinal microbial ecology.

Antimicrobial peptides are important effectors of innate immunity throughout the plant and animal kingdoms. In the mammalian small intestine, Paneth cell alpha-defensins are antimicrobial peptides that contribute to host defense against enteric pathogens. To determine if alpha-defensins also govern intestinal microbial ecology, we analyzed the intestinal microbiota of mice expressing a human alpha-defensin gene (DEFA5) and in mice lacking an enzyme required for the processing of mouse alpha-defensins. In these complementary models, we detected significant alpha-defensin-dependent changes in microbiota composition, but not in total bacterial numbers. Furthermore, DEFA5-expressing mice had striking losses of segmented filamentous bacteria and fewer interleukin 17 (IL-17)-producing lamina propria T cells. Our data ascribe a new homeostatic role to alpha-defensins in regulating the makeup of the commensal microbiota.

Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract.

Enterococcus faecalis is both a common commensal of the human gastrointestinal tract and a leading cause of hospital-acquired infections. Systemic infections with multidrug-resistant enterococci occur subsequent to gastrointestinal colonization. Preventing colonization by multidrug-resistant E. faecalis could therefore be a valuable approach towards limiting infection. However, little is known about the mechanisms E. faecalis uses to colonize and compete for stable gastrointestinal niches. Pheromone-responsive conjugative plasmids encoding bacteriocins are common among enterococcal strains and could modulate niche competition among enterococci or between enterococci and the intestinal microbiota. We developed a model of colonization of the mouse gut with E. faecalis, without disrupting the microbiota, to evaluate the role of the conjugative plasmid pPD1 expressing bacteriocin 21 (ref. 4) in enterococcal colonization. Here we show that E. faecalis harbouring pPD1 replaces indigenous enterococci and outcompetes E. faecalis lacking pPD1. Furthermore, in the intestine, pPD1 is transferred to other E. faecalis strains by conjugation, enhancing their survival. Colonization with an E. faecalis strain carrying a conjugation-defective pPD1 mutant subsequently resulted in clearance of vancomycin-resistant enterococci, without plasmid transfer. Therefore, bacteriocin expression by commensal bacteria can influence niche competition in the gastrointestinal tract, and bacteriocins, delivered by commensals that occupy a precise intestinal bacterial niche, may be an effective therapeutic approach to specifically eliminate intestinal colonization by multidrug-resistant bacteria, without profound disruption of the indigenous microbiota.

Microbiome Signatures Associated With Steatohepatitis and Moderate to Severe Fibrosis in Children With Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: The intestinal microbiome might affect the development and severity of nonalcoholic fatty liver disease (NAFLD). We analyzed microbiomes of children with and without NAFLD. METHODS: We performed a prospective, observational, cross-sectional study of 87 children (age range, 8-17 years) with biopsy-proven NAFLD and 37 children with obesity without NAFLD (controls). Fecal samples were collected and microbiome composition and functions were assessed using 16S ribosomal RNA amplicon sequencing and metagenomic shotgun sequencing. Microbial taxa were identified using zero-inflated negative binomial modeling. Genes contributing to bacterial pathways were identified using gene set enrichment analysis. RESULTS: Fecal microbiomes of children with NAFLD had lower α-diversity than those of control children (3.32 vs 3.52, P = .016). Fecal microbiomes from children with nonalcoholic steatohepatitis (NASH) had the lowest α-diversity (control, 3.52; NAFLD, 3.36; borderline NASH, 3.37; NASH, 2.97; P = .001). High abundance of Prevotella copri was associated with more severe fibrosis (P = .036). Genes for lipopolysaccharide biosynthesis were enriched in microbiomes from children with NASH (P < .001). Classification and regression tree model with level of alanine aminotransferase and relative abundance of the lipopolysaccharide pathway gene encoding 3-deoxy-d-manno-octulosonate 8-phosphate-phosphatase identified patients with NASH with an area under the receiver operating characteristic curve value of 0.92. Genes involved in flagellar assembly were enriched in the fecal microbiomes of patients with moderate to severe fibrosis (P < .001). Classification and regression tree models based on level of alanine aminotransferase and abundance of genes encoding flagellar biosynthesis protein had good accuracy for identifying case children with moderate to severe fibrosis (area under the receiver operating characteristic curve, 0.87). CONCLUSIONS: In an analysis of fecal microbiomes of children with NAFLD, we associated NAFLD and NASH with intestinal dysbiosis. NAFLD and its severity were associated with greater abundance of genes encoding inflammatory bacterial products. Alterations to the intestinal microbiome might contribute to the pathogenesis of NAFLD and be used as markers of disease or severity.

Fecal Microbial Transplant Capsules Are Safe in Hepatic Encephalopathy: A Phase 1, Randomized, Placebo-Controlled Trial.

Hepatic encephalopathy (HE) can cause major morbidity despite standard of care (SOC; rifaximin/lactulose). Fecal microbial transplant (FMT) enemas postantibiotics are safe, but the effect of FMT without antibiotics using the capsular route requires investigation. The aim of this work was to determine the safety, tolerability, and impact on mucosal/stool microbiota and brain function in HE after capsular FMT in a randomized, single-blind, placebo-controlled clinical trial in Virginia. Patients with cirrhosis with recurrent HE with MELD (Model for End-Stage Liver Disease) <17 on SOC were randomized 1:1 into receiving 15 FMT capsules versus placebo from a single donor enriched in Lachnospiraceae and Ruminococcaceae. Endoscopies with duodenal and sigmoid biopsies, stool analysis, cognition, serum lipopolysaccharide-binding protein (LBP), and duodenal antimicrobial peptide (AMP) expression at baseline were used. Clinical follow-up with SOC maintenance was performed until 5 months. FMT-assigned patients underwent repeat endoscopies 4 weeks postenrollment. Twenty subjects on lactulose/rifaximin were randomized 1:1. MELD score was similar at baseline (9.6 vs. 10.2) and study end (10.2 vs. 10.5). Six patients in the placebo group required hospitalizations compared to 1 in FMT, which was deemed unrelated to FMT. Infection/HE episodes were similar between groups. Baseline microbial diversity was similar in all tissues between groups. Post-FMT, duodenal mucosal diversity (P = 0.01) increased with higher Ruminococcaceae and Bifidobacteriaceae and lower Streptococcaceae and Veillonellaceae. Reduction in Veillonellaceae were noted post-FMT in sigmoid (P = 0.04) and stool (P = 0.05). Duodenal E-cadherin (P = 0.03) and defensin alpha 5 (P = 0.03) increased whereas interleukin-6 (P = 0.02) and serum LBP (P = 0.009) reduced post-FMT. EncephalApp performance improved post-FMT only (P = 0.02). Conclusion: In this phase 1 study, oral FMT capsules are safe and well tolerated in patients with cirrhosis and recurrent HE. FMT was associated with improved duodenal mucosal diversity, dysbiosis, and AMP expression, reduced LBP, and improved EncephalApp performance. Further studies are needed to prove efficacy.

A requisite role for induced regulatory T cells in tolerance based on expanding antigen receptor diversity.

Although both natural and induced regulatory T (nTreg and iTreg) cells can enforce tolerance, the mechanisms underlying their synergistic actions have not been established. We examined the functions of nTreg and iTreg cells by adoptive transfer immunotherapy of newborn Foxp3-deficient mice. As monotherapy, only nTreg cells prevented disease lethality, but did not suppress chronic inflammation and autoimmunity. Provision of Foxp3-sufficient conventional T cells with nTreg cells reconstituted the iTreg pool and established tolerance. In turn, acute depletion of iTreg cells in rescued mice resulted in weight loss and inflammation. Whereas the transcriptional signatures of nTreg and in vivo-derived iTreg cells were closely matched, there was minimal overlap in their T cell receptor (TCR) repertoires. Thus, iTreg cells are an essential nonredundant regulatory subset that supplements nTreg cells, in part by expanding TCR diversity within regulatory responses.

Lactobacillus plantarum 299v Supplementation Improves Vascular Endothelial Function and Reduces Inflammatory Biomarkers in Men With Stable Coronary Artery Disease.

RATIONALE: A strong association has emerged between the gut microbiome and atherosclerotic disease. Our recent data suggest Lactobacillus plantarum 299v (Lp299v) supplementation reduces infarct size in male rats. Limited human data are available on the impact of Lp299v on the vasculature. OBJECTIVE: To determine whether oral Lp299v supplementation improves vascular endothelial function and reduces systemic inflammation in humans with stable coronary artery disease (CAD). METHODS AND RESULTS: Twenty men with stable CAD consumed a drink containing Lp299v (20 billion CFU) once daily for 6 weeks. After a 4-week washout, subjects were given an option of additionally participating in a 10-day study of oral liquid vancomycin (250 mg QID). Vascular endothelial function was measured by brachial artery flow-mediated dilation. Before and after Lp299v, plasma short-chain fatty acids, trimethylamine oxide, and adipokine levels were measured. Additional plasma samples underwent unbiased metabolomic analyses using liquid chromatography/mass spectroscopy. 16S rRNA sequencing was used to determine changes of the stool microbiome. Arterioles from patients with CAD were obtained, and endothelium-dependent vasodilation was measured by video microscopy after intraluminal incubation with plasma from Lp299v study subjects. Lp299v supplementation improved brachial flow-mediated dilation ( P=0.008) without significant changes in plasma cholesterol profiles, fasting glucose, or body mass index. Vancomycin did not impact flow-mediated dilation. Lp299v supplementation decreased circulating levels of IL (interleukin)-8 ( P=0.01), IL-12 ( P=0.02), and leptin ( P=0.0007) but did not significantly change plasma trimethylamine oxide concentrations ( P=0.27). Plasma propionate ( P=0.004) increased, whereas acetate levels decreased ( P=0.03). Post-Lp299v plasma improved endothelium-dependent vasodilation in resistance arteries from patients with CAD ( P=0.02).16S rRNA analysis showed the Lactobacillus genus was enriched in postprobiotic stool samples without other changes. CONCLUSIONS: Lp299v improved vascular endothelial function and decreased systemic inflammation in men with CAD, independent of changes in traditional risk factors and trimethylamine oxide. Circulating gut-derived metabolites likely account for these improvements and merit further study. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov . Unique identifier: NCT01952834.

Sortase-Dependent Proteins Promote Gastrointestinal Colonization by Enterococci.

The human gastrointestinal tract (GIT) is inhabited by a dense microbial community of symbionts. Enterococci are among the earliest members of this community and remain core members of the GIT microbiota throughout life. Enterococci have also recently emerged as opportunistic pathogens and major causes of nosocomial infections. Although recognized as a prerequisite for infection, colonization of the GIT by enterococci remains poorly understood. One way that bacteria adapt to dynamic ecosystems like the GIT is through the use of their surface proteins to sense and interact with components of their immediate environment. In Gram-positive bacteria, a subset of surface proteins relies on an enzyme called sortase for covalent attachment to the cell wall. Here, we show that the housekeeping sortase A (SrtA) enzyme promotes intestinal colonization by enterococci. Furthermore, we show that the enzymatic activity of SrtA is key to the ability of Enterococcus faecalis to bind mucin (a major component of the GIT mucus). We also report the GIT colonization phenotypes of E. faecalis mutants lacking selected sortase-dependent proteins (SDPs). Further examination of the mucin binding ability of these mutants suggests that adhesion to mucin contributes to intestinal colonization by E. faecalis.

Longitudinal changes in the gut microbiome of infants on total parenteral nutrition.

BACKGROUND: Animal studies suggest that total parenteral nutrition (TPN) may alter bacterial colonization of the intestinal tract and contribute to complications. Progressive changes in gut microbiome of infants receiving TPN are not well understood. METHODS: Infants with and without TPN/soy lipid were enrolled in a prospective, longitudinal study. Weekly fecal samples were obtained for the first 4 weeks of life. High throughput pyrosequencing of 16S rDNA was used for compositional analysis of the gut microbiome. RESULTS: 47 infants were eligible for analyses, 25 infants received TPN, and 22 infants did not (control). Although similar between TPN and control groups in the first week, fecal bacterial alpha diversity was significantly lower in the TPN group compared to controls at week 4 (Shannon index 1.0 vs 1.5, P-value = 0.03). The TPN group had significantly lower Bacteroidetes and higher Verrucomicrobia abundance compared to controls (P-values < 0.05), and these differences became more pronounced over time. At the genus level, TPN was associated with lower abundance of Bacteroides and Bifidobacterium in all weeks. CONCLUSIONS: TPN is associated with significant loss of biodiversity and alterations in the pattern of gut microbial colonization of infants over time. TPN-associated dysbiosis may predispose infants to adverse NICU outcomes.

Ceftriaxone Administration Disrupts Intestinal Homeostasis, Mediating Noninflammatory Proliferation and Dissemination of Commensal Enterococci.

Enterococci are Gram-positive commensals of the mammalian intestinal tract and harbor intrinsic resistance to broad-spectrum cephalosporins. Disruption of colonization resistance in humans by antibiotics allows enterococci to proliferate in the gut and cause disseminated infections. In this study, we used Enterococcus faecalis (EF)-colonized mice to study the dynamics of enterococci, commensal microbiota, and the host in response to systemic ceftriaxone administration. We found that the mouse model recapitulates intestinal proliferation and dissemination of enterococci seen in humans. Employing a ceftriaxone-sensitive strain of enterococci (E. faecalis JL308), we showed that increased intestinal abundance is critical for the systemic dissemination of enterococci. Investigation of the impact of ceftriaxone on the mucosal barrier defenses and integrity suggested that translocation of enterococci across the intestinal mucosa was not associated with intestinal pathology or increased permeability. Ceftriaxone-induced alteration of intestinal microbial composition was associated with transient increase in the abundance of multiple bacterial operational taxonomic units (OTUs) in addition to enterococci, for example, lactobacilli, which also disseminated to the extraintestinal organs. Collectively, these results emphasize that ceftriaxone-induced disruption of colonization resistance and alteration of mucosal homeostasis facilitate increased intestinal abundance of a limited number of commensals along with enterococci, allowing their translocation and systemic dissemination in a healthy host.

Modulators of Enterococcus faecalis Cell Envelope Integrity and Antimicrobial Resistance Influence Stable Colonization of the Mammalian Gastrointestinal Tract.

The Gram-positive bacterium Enterococcus faecalis is both a colonizer of the gastrointestinal tract (GIT) and an agent of serious nosocomial infections. Although it is typically required for pathogenesis, GIT colonization by E. faecalis is poorly understood. E. faecalis tolerates high concentrations of GIT antimicrobials, like cholate and lysozyme, leading us to hypothesize that resistance to intestinal antimicrobials is essential for long-term GIT colonization. Analyses of E. faecalis mutants exhibiting defects in antimicrobial resistance revealed that IreK, a determinant of envelope integrity and antimicrobial resistance, is required for long-term GIT colonization. IreK is a member of the PASTA kinase protein family, bacterial transmembrane signaling proteins implicated in the regulation of cell wall homeostasis. Among several determinants of cholate and lysozyme resistance in E. faecalis, IreK was the only one found to be required for intestinal colonization, emphasizing the importance of this protein to enterococcal adaptation to the GIT. By studying ΔireK suppressor mutants that recovered the ability to colonize the GIT, we identified two conserved enterococcal proteins (OG1RF_11271 and OG1RF_11272) that function antagonistically to IreK and interfere with cell envelope integrity, antimicrobial resistance, and GIT colonization. Our data suggest that IreK, through its kinase activity, inhibits the actions of these proteins. IreK, OG1RF_11271, and OG1RF_11272 are found in all enterococci, suggesting that their effect on GIT colonization is universal across enterococci. Thus, we have defined conserved genes in the enterococcal core genome that influence GIT colonization through their effect on enterococcal envelope integrity and antimicrobial resistance.

Alternatively Activated Macrophages Boost Induced Regulatory T and Th17 Cell Responses during Immunotherapy for Colitis.

Induced regulatory T (iTreg) and Th17 cells promote mucosal homeostasis. We used a T cell transfer model of colitis to compare the capacity of iTreg and Th17 cells to develop in situ following the transfer of naive CD4(+)CD45RB(hi)T cells intoRag1(-/-)C57BL/6 or BALB/c mice, the prototypical Th1/M1- and Th2/M2-prone strains. We found that the frequency and number of Foxp3(+)iTreg cells and Th17 cells were significantly reduced in C57BL/6 mice compared with the BALB/c strain. C57BL/6 mice with colitis were also resistant to natural Treg cell immunotherapy. Pretreatment of C57BL/6Rag1(-/-)mice with IL-4 plus IL-13, or with M2a but not M1 macrophages, dramatically increased the generation of iTreg and Th17 cells. Importantly, M2a transfers, either as a pretreatment or in mice with established colitis, allowed successful immunotherapy with natural Treg cells. M2a macrophages also reduced the generation of pathogenic iTreg cells that lost Foxp3 expression, suggesting that they stabilize the expression of Foxp3. Thus, polarized M2a macrophages drive a directionally concordant expansion of the iTreg-Th17 cell axis and can be exploited as a therapeutic adjuvant in cell-transfer immunotherapy to re-establish mucosal tolerance.

Black Raspberries and Their Anthocyanin and Fiber Fractions Alter the Composition and Diversity of Gut Microbiota in F-344 Rats.

Natural compounds can alter the diversity and composition of the gut microbiome, potentially benefiting our health. We previously demonstrated chemopreventive effects of black raspberries (BRBs) in colorectal cancer, which is associated with gut dysbiosis. To investigate the effects of whole BRBs and their fractions on gut microbiota, we fed F-344 rats a control diet, 5% BRBs, the BRB anthocyanin fraction, or the BRB residue fraction for 6 weeks. Feces were collected at baseline and at weeks 3 and 6, and bacterial sequence counts were analyzed. We observed distinct patterns of microbiota from different diet groups. Beta diversity analysis suggested that all diet groups exerted time-dependent changes in the bacterial diversity. Hierarchical clustering analysis revealed that post-diet fecal microbiota was segregated from baseline fecal microbiota within each diet. It is interesting to note that fractions of BRBs induced different changes in gut bacteria compared to whole BRBs. The abundance of specific microbial species known to have anti-inflammatory effects, such as Akkermansia and Desulfovibrio, was increased by whole BRBs and their residue. Further, butyrate-producing bacteria, e.g., Anaerostipes, were increased by whole BRBs. Our results suggest that whole BRBs and their fractions alter the gut microbiota in ways that could significantly influence human health.

Continued Alcohol Misuse in Human Cirrhosis is Associated with an Impaired Gut-Liver Axis.

BACKGROUND: Cirrhosis and alcohol can independently affect the gut-liver axis with systemic inflammation. However, their concurrent impact in humans is unclear. METHODS: Our aim was to determine the effect of continued alcohol misuse on the gut-liver axis in cirrhotic patients. Age- and MELD-balanced cirrhotic patients who were currently drinking (Alc) or abstinent (NAlc) and healthy controls underwent serum and stool collection. A subset underwent upper endoscopy and colonoscopy for biopsies and duodenal fluid collection. The groups were compared regarding (i) inflammation/intestinal barrier: systemic tumor necrosis factor levels, intestinal inflammatory cytokine (duodenum, ileum, sigmoid), and ileal antimicrobial peptide expression; (ii) microbiota composition: 16SrRNA sequencing of duodenal, ileal, and colonic mucosal and fecal microbiota; and (iii) microbial functionality: duodenal fluid and fecal bile acid (BA) profile (conjugation and dehydroxylation status), intestinal BA transporter (ASBT, FXR, FGF-19, SHP) expression, and stool metabolomics using gas chromatography/mass spectrometry. RESULTS: Alc patients demonstrated a significant duodenal, ileal, and colonic mucosal and fecal dysbiosis, compared to NAlc and controls with lower autochthonous bacterial taxa. BA profile skewed toward a potentially toxic profile (higher secondary and glycine-conjugated BAs) in duodenal fluid and stool in Alc patients. Duodenal fluid demonstrated conjugated secondary BAs only in the Alc group. There was a greater expression of all ileal BA transporters in Alc patients. This group also showed higher endotoxemia, systemic and ileal inflammatory expression, and lower amino acid and bioenergetic-associated metabolites, without change in antimicrobial peptide expression. CONCLUSIONS: Despite cirrhosis, continued alcohol misuse predisposes patients to widespread dysbiosis with alterations in microbial functionality such as a toxic BA profile, which can lead to intestinal and systemic inflammation.

Intestinal alkaline phosphatase deficiency leads to dysbiosis and bacterial translocation in the newborn intestine.

BACKGROUND: Intestinal alkaline phosphatase (IAP) has been shown to help maintain intestinal homeostasis. Decreased expression of IAP has been linked with pediatric intestinal diseases associated with bacterial overgrowth and subsequent inflammation. We hypothesize that the absence of IAP leads to dysbiosis, with increased inflammation and permeability of the newborn intestine. METHODS: Sprague-Dawley heterozygote IAP cross-matches were bred. Pups were dam fed ad lib and euthanized at weaning. The microbiotas of terminal ileum (TI) and colon was determined by quantitative real-time polymerase chain reaction (qRT-PCR) of subphylum-specific bacterial 16S ribosomal RNA. RT-PCR was performed on TI for inflammatory cytokines. Intestinal permeability was quantified by fluorescein isothiocyanate-dextran permeability and bacterial translocation by qRT-PCR for bacterial 16S ribosomal RNA in mesenteric lymph nodes. Statistical analysis was done by chi-square analysis. RESULTS: All three genotypes had similar concentrations of bacteria in the TI and colon. However, IAP knockout (IAP-KO) had significantly decreased diversity of bacterial species in their colonic stool compared with heterozygous and wild-type (WT). IAP-KO pups had a nonstatistically significant 3.9-fold increased inducible nitric oxide synthase messenger RNA expression compared with WT (IAP-KO, 3.92 ± 1.36; WT, 1.0 ± 0.27; P = 0.03). IAP-KO also had significantly increased bacterial translocation to mesenteric lymph nodes occurred in IAP-KO (IAP-KO, 7625 RFU/g ± 3469; WT, 4957 RFU/g ± 1552; P = 0.04). Furthermore, IAP-KO had increased permeability (IAP-KO, 0.297 mg/mL ± 0.2; WT, 0.189 mg/mL ± 0.15 P = 0.07), but was not statistically significant. CONCLUSIONS: Deficiency of IAP in the newborn intestine is associated with dysbiosis and increased inflammation, permeability, and bacterial translocation.

Gut Microbial Dysbiosis Due to Helicobacter Drives an Increase in Marginal Zone B Cells in the Absence of IL-10 Signaling in Macrophages.

It is clear that IL-10 plays an essential role in maintaining homeostasis in the gut in response to the microbiome. However, it is unknown whether IL-10 also facilitates immune homeostasis at distal sites. To address this question, we asked whether splenic immune populations were altered in IL-10-deficient (Il10(-/-)) mice in which differences in animal husbandry history were associated with susceptibility to spontaneous enterocolitis that is microbiome dependent. The susceptible mice exhibited a significant increase in splenic macrophages, neutrophils, and marginal zone (MZ) B cells that was inhibited by IL-10 signaling in myeloid, but not B cells. The increase in macrophages was due to increased proliferation that correlated with a subsequent enhancement in MZ B cell differentiation. Cohousing and antibiotic treatment studies suggested that the alteration in immune homeostasis in the spleen was microbiome dependent. The 16S rRNA sequencing revealed that susceptible mice harbored a different microbiome with a significant increase in the abundance of the bacterial genus Helicobacter. The introduction of Helicobacter hepaticus to the gut of nonsusceptible mice was sufficient to drive macrophage expansion and MZ B cell development. Given that myeloid cells and MZ B cells are part of the first line of defense against blood-borne pathogens, their increase following a breach in the gut epithelial barrier would be protective. Thus, IL-10 is an essential gatekeeper that maintains immune homeostasis at distal sites that can become functionally imbalanced upon the introduction of specific pathogenic bacteria to the intestinal track.

Dysbiosis--a consequence of Paneth cell dysfunction.

The complex community of colonizing microbes inhabiting the mucosal surfaces of mammals is vital to homeostasis and normal physiology in the host. When the composition of this microbiota is unfavorably altered, termed dysbiosis, the host is rendered more susceptible to a variety of chronic diseases. In the mammalian small intestine, specialized secretory epithelial cells, named Paneth cells, produce a variety of secreted antimicrobial peptides that fundamentally influence the composition of the microbiota. Recent investigations have identified numerous genetic and environmental factors that can disrupt normal Paneth cell function, resulting in compromised antimicrobial peptide secretion and consequent dysbiosis. These findings suggest that Paneth cell dysfunction should be considered a common cause of dysbiosis.

Selection of models for the analysis of risk-factor trees: leveraging biological knowledge to mine large sets of risk factors with application to microbiome data.

MOTIVATION: Establishment of a statistical association between microbiome features and clinical outcomes is of growing interest because of the potential for yielding insights into biological mechanisms and pathogenesis. Extracting microbiome features that are relevant for a disease is challenging and existing variable selection methods are limited due to large number of risk factor variables from microbiome sequence data and their complex biological structure. RESULTS: We propose a tree-based scanning method, Selection of Models for the Analysis of Risk factor Trees (referred to as SMART-scan), for identifying taxonomic groups that are associated with a disease or trait. SMART-scan is a model selection technique that uses a predefined taxonomy to organize the large pool of possible predictors into optimized groups, and hierarchically searches and determines variable groups for association test. We investigate the statistical properties of SMART-scan through simulations, in comparison to a regular single-variable analysis and three commonly-used variable selection methods, stepwise regression, least absolute shrinkage and selection operator (LASSO) and classification and regression tree (CART). When there are taxonomic group effects in the data, SMART-scan can significantly increase power by using bacterial taxonomic information to split large numbers of variables into groups. Through an application to microbiome data from a vervet monkey diet experiment, we demonstrate that SMART-scan can identify important phenotype-associated taxonomic features missed by single-variable analysis, stepwise regression, LASSO and CART.