Luminal Microbiota Research Articles

Potent Sodium/Glucose Cotransporter SGLT1/2 Dual Inhibition Improves Glycemic Control Without Marked Gastrointestinal Adaptation or Colonic Microbiota Changes in Rodents

The sodium/glucose cotransporters (SGLT1 and SGLT2) transport glucose across the intestinal brush border and kidney tubule. Dual SGLT1/2 inhibition could reduce hyperglycemia more than SGLT2-selective inhibition in patients with type 2 diabetes. However, questions remain about altered gastrointestinal (GI) luminal glucose and tolerability, and this was evaluated in slc5a1-/- mice or with a potent dual inhibitor (compound 8; SGLT1 Ki = 1.5 ± 0.5 nM 100-fold greater potency than phlorizin; SGLT2 Ki = 0.4 ± 0.2 nM). 13C6-glucose uptake was quantified in slc5a1-/- mice and in isolated rat jejunum. Urinary glucose excretion (UGE), blood glucose (Sprague-Dawley rats), glucagon-like peptide 1 (GLP-1), and hemoglobin A1c (HbA1c) levels (Zucker diabetic fatty rats) were measured. Intestinal adaptation and rRNA gene sequencing was analyzed in C57Bl/6 mice. The blood 13C6-glucose area under the curve (AUC) was reduced in the absence of SGLT1 by 75% (245 ± 6 vs. 64 ± 6 mg/dl⋅h in wild-type vs. slc5a1-/- mice) and compound 8 inhibited its transport up to 50% in isolated rat jejunum. Compound 8 reduced glucose excursion more than SGLT2-selective inhibition (e.g., AUC = 129 ± 3 vs. 249 ± 5 mg/dl⋅h for 1 mg/kg compound 8 vs. dapagliflozin) with similar UGE but a lower renal glucose excretion threshold. In Zucker diabetic fatty rats, compound 8 decreased HbA1c and increased total GLP-1 without changes in jejunum SGLT1 expression, mucosal weight, or villus length. Overall, compound 8 (1 mg/kg for 6 days) did not increase cecal glucose concentrations or bacterial diversity in C57BL/6 mice. In conclusion, potent dual SGLT1/2 inhibition lowers blood glucose by reducing intestinal glucose absorption and the renal glucose threshold but minimally impacts the intestinal mucosa or luminal microbiota in chow-fed rodents.

Microbes vs. chemistry in the origin of the anaerobic gut lumen

The succession from aerobic and facultative anaerobic bacteria to obligate anaerobes in the infant gut along with the differences between the compositions of the mucosally adherent vs. luminal microbiota suggests that the gut microbes consume oxygen, which diffuses into the lumen from the intestinal tissue, maintaining the lumen in a deeply anaerobic state. Remarkably, measurements of luminal oxygen levels show nearly identical pO2 (partial pressure of oxygen) profiles in conventional and germ-free mice, pointing to the existence of oxygen consumption mechanisms other than microbial respiration. In vitro experiments confirmed that the luminal contents of germ-free mice are able to chemically consume oxygen (e.g., via lipid oxidation reactions), although at rates significantly lower than those observed in the case of conventionally housed mice. For conventional mice, we also show that the taxonomic composition of the gut microbiota adherent to the gut mucosa and in the lumen throughout the length of the gut correlates with oxygen levels. At the same time, an increase in the biomass of the gut microbiota provides an explanation for the reduction of luminal oxygen in the distal vs. proximal gut. These results demonstrate how oxygen from the mammalian host is used by the gut microbiota, while both the microbes and the oxidative chemical reactions regulate luminal oxygen levels, shaping the composition of the microbial community throughout different regions of the gut.

Hypoxanthine as a Checkpoint Metabolite in Epithelial Energy Modulation and Barrier Function

A primary function of intestinal epithelial cells (IECs) is to provide a selectively permeable barrier to protect the host from luminal microbiota and antigens, and to mediate the flux of nutrients, fluid, and waste across that barrier. Loss of intestinal barrier function is a hallmark of inflammatory bowel disease (IBD). Dysregulation of IEC barrier in IBD coincides with profound shifts in metabolic energy, especially in the colon, which exists in an energetically‐vulnerable state of physiologic hypoxia. The precise mechanism(s) by which metabolic pathways contribute to and control inflammation have yet to be elucidated. To delineate barrier‐related energy flux, an HPLC‐based profiling method was developed to track changes in energy and adenylate metabolites during intestinal epithelial barrier development and restitution. Cultured epithelia demonstrated a remarkable capacity to maintain ATP and phosphocreatine during barrier development. Further epithelial cell profiling identified a significant capacity for cultured human IECs to salvage hypoxanthine (Hpx) and shift their metabolic equilibrium toward the production of ATP and phosphocreatine. In a consistent manner, we have demonstrated that Hpx‐mediated shifts in metabolism result in enhanced epithelial barrier formation, increased epithelial restitution, and improved wound healing. Modulation of cellular energetics by Hpx were recapitulated with in vivo murine models, where orally‐delivered Hpx increased colonic ATP levels. Guided by an unbiased microarray analysis of Hpx treated human IECs, we identified a number of target genes important in mucosal differentiation, energy metabolism, and reactive oxygen species mediation. As the ATP consumed by IECs under energetic stress is shuttled to and pooled as Hpx, it appears not only as a central energy metabolite, but also as a regulator of gene expression. Taken together, our ongoing studies provide strong evidence that Hpx functions as a central, checkpoint energy metabolite in IEC function.Support or Funding InformationNIH/NIDDKThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hypoxanthine is a checkpoint stress metabolite in colonic epithelial energy modulation and barrier function

Intestinal epithelial cells form a selectively permeable barrier to protect colon tissues from luminal microbiota and antigens and to mediate nutrient, fluid, and waste flux in the intestinal tract. Dysregulation of the epithelial cell barrier coincides with profound shifts in metabolic energy, especially in the colon, which exists in an energetically depleting state of physiological hypoxia. However, studies that systematically examine energy flux and adenylate metabolism during intestinal epithelial barrier development and restoration after disruption are lacking. Here, to delineate barrier-related energy flux, we developed an HPLC-based profiling method to track changes in energy flux and adenylate metabolites during barrier development and restoration. Cultured epithelia exhibited pooling of phosphocreatine and maintained ATP during barrier development. EDTA-induced epithelial barrier disruption revealed that hypoxanthine levels correlated with barrier resistance. Further studies uncovered that hypoxanthine supplementation improves barrier function and wound healing and that hypoxanthine appears to do so by increasing intracellular ATP, which improved cytoskeletal G- to F-actin polymerization. Hypoxanthine supplementation increased the adenylate energy charge in the murine colon, indicating potential to regulate adenylate energy charge-mediated metabolism in intestinal epithelial cells. Moreover, experiments in a murine colitis model disclosed that hypoxanthine loss during active inflammation correlates with markers of disease severity. In summary, our results indicate that hypoxanthine modulates energy metabolism in intestinal epithelial cells and is critical for intestinal barrier function.

Sox9‐independent Control of Intestinal Paneth Cell Differentiation and Function

The genetic and signaling mechanisms controlling the generation and function of differentiated cell types are of continued interest. The intestinal Paneth cell (PC) is a post‐mitotic and dedicated secretory cell type with reported functions in supporting intestinal stem cell (ISC) maintenance in the crypt stem cell niche, and in secreting antimicrobial peptides to protect the crypt from potentially pathogenic luminal microbiota. Previous studies have reported a near‐complete loss of PCs (as defined by morphological and marker‐based criteria) in epithelium where the transcription factor Sox9 is genetically ablated, leading to the proposition that Sox9 is required for PC differentiation. Currently, however, the mechanisms through which Sox9 controls PC differentiation are not known. We have found evidence in Sox9 knockout (KO) small intestine (SI) that cells at the crypt base frequently express PC‐markers such as Lysozyme (Lyz) and Cryptdin, exhibit apical granules, and are post‐mitotic, all of which are features indicative of PC differentiation. The numbers of Lyz+ cells are highest in the distal Sox9 KO SI but are significantly reduced relative to control, and are essentially absent in the proximal SI, suggesting that regionally patterned signals may drive PC differentiation independent of Sox9. Apical granularity and expression levels of Lyz/Cryptdin are also altered, suggesting that while Sox9 KO ISCs can initiate PC differentiation, they are unable to reach full maturity. Antibody and qPCR based detection of PC gene products (including secreted antimicrobials, growth factors, and transcriptional regulators proposed to drive PC differentiation downstream of Sox9) are maintained in Sox9 KO cultured intestinal organoids and are elevated in organoids treated with pharmacological agents that drive the PC differentiation program. These results indicate that Sox9‐deficient ‘Paneth‐like’ cells are continually generated in culture and can be pushed toward PC differentiation with factors known to stimulate the PC differentiation program. We conclude that Sox9 is not required for the activation of at least sub‐sets of gene products associated with PC differentiation and function. Ongoing experiments are designed to investigate Sox9‐ dependent and independent pathways toward PC fate, and to characterize the functionality of Sox9−/− ‘Paneth‐like’ cells compared to controls.Support or Funding InformationRO1 DK091427‐05F32 DK115016‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dietary proline supplementation alters colonic luminal microbiota and bacterial metabolite composition between days 45 and 70 of pregnancy in Huanjiang mini-pigs

BackgroundPregnancy is associated with important changes in gut microbiota composition. Dietary factors may affect the diversity, composition, and metabolic activity of the intestinal microbiota. Among amino acids, proline is known to play important roles in protein metabolism and structure, cell differentiation, conceptus growth and development, and gut microbiota re-equilibration in case of dysbiosis.ResultsDietary supplementation with 1% proline decreased (P < 0.05) the amounts of Klebsiella pneumoniae, Peptostreptococcus productus, Pseudomonas, and Veillonella spp. in distal colonic contents than that in the control group. The colonic contents of Butyrivibrio fibrisolvens, Bifidobacterium sp., Clostridium coccoides, Clostridium coccoides-Eubacterium rectale, Clostridium leptum subgroup, Escherichia coli, Faecalibacterium prausnitzii, Fusobacterium prausnitzii, and Prevotella increased (P < 0.05) on d 70 of pregnancy as compared with those on d 45 of pregnancy. The colonic concentrations of acetate, total straight-chain fatty acid, and total short-chain fatty acids (SCFA) in the proline-supplemented group were lower (P < 0.05), and butyrate level (P = 0.06) decreased as compared with the control group. Almost all of the SCFA displayed higher (P < 0.05) concentrations in proximal colonic contents on d 70 of pregnancy than those on d 45 of pregnancy. The concentrations of 1,7-heptyl diamine (P = 0.09) and phenylethylamine (P < 0.05) in proximal colonic contents were higher, while those of spermidine (P = 0.05) and total bioamine (P = 0.06) tended to be lower in the proline-supplemented group than those in the control group. The concentrations of spermidine, spermine, and total bioamine in colonic contents were higher (P < 0.05) on d 70 of pregnancy than those measured on d 45 of pregnancy. In contrast, the concentration of phenylethylamine was lower (P < 0.05) on d 70 than on d 45 of pregnancy.ConclusionThese findings indicate that L-proline supplementation modifies both the colonic microbiota composition and the luminal concentrations of several bacterial metabolites. Furthermore, our data show that both the microbiota composition and the concentrations of bacterial metabolites are evolving in the course of pregnancy. These results are discussed in terms of possible implication in terms of luminal environment and consequences for gut physiology and health.

Intestinal epithelial Toll-like receptor 4 prevents metabolic syndrome by regulating interactions between microbes and intestinal epithelial cells in mice.

Little is known about the pathogenesis of metabolic syndrome, although Toll-like receptor 4 (TLR4) has been implicated. We investigated whether TLR4 in the intestinal epithelium regulates metabolic syndrome by coordinating interactions between the luminal microbiota and host genes that regulate metabolism. Mice lacking TLR4 in the intestinal epithelium (TLR4ΔIEC), but not mice lacking TLR4 in myeloid cells nor mice lacking TLR4 globally, developed metabolic syndrome; these features were not observed in TLR4ΔIEC mice given antibiotics. Metagenomic analysis of the fecal microbiota revealed differences between TLR4ΔIEC and wild type mice, while meta-transcriptome analysis of the microbiota showed that intestinal TLR4 affected the expression of microbial genes involved in the metabolism of lipids, amino acids, and nucleotides. Genes regulated by peroxisome proliferator-activated receptors (PPARs) and the antimicrobial peptide lysozyme were significantly down-regulated in TLR4ΔIEC mice, suggesting a mechanism by which intestinal TLR4 could exert its effects on the microbiota and metabolic syndrome. Supportingly, antibiotics prevented both downregulation of PPAR genes and the development of metabolic syndrome, while PPAR agonists prevented development of metabolic syndrome in TLR4ΔIEC mice. Thus, intestinal epithelial TLR4 regulates metabolic syndrome through altered host-bacterial signaling, suggesting that microbial or PPAR based strategies might have therapeutic potential for this disease.

Alfalfa-containing diets alter luminal microbiota structure and short chain fatty acid sensing in the caecal mucosa of pigs

BackgroundPork produced by outdoor-reared pigs raised mostly on alfalfa pastures attracts increasing population of consumer from most of the world. In China, pigs were raised with alfalfa-containing diets to seek for good quality pork. However, the influence of dietary alfalfa involving high level of insoluble dietary fiber (IDF) on pig intestinal luminal microbiota composition remains unclear. The objective of this study was to investigate the effects of alfalfa on luminal microbiota and short chain fatty acids (SCFA) production, and gene expressions involved in SCFA sensing, transporting and absorbing in pig caecal mucosa.ResultsTwenty-four growing pigs were randomly allotted to four diets containing 0%, 5%, 10% and 15% alfalfa meal for a 28-d experiment. Ingestion of alfalfa meal-contained diets significantly increased the ratio of body weight gain to feed consumption. Illumina MiSeq sequencing of the V3 region of the 16S rRNA genes showed that alfalfa-containing diet significantly decreased the relative abundance of genera Turicibacter, Acidiphilium, Paracoccus, Propionibacterium, Corynebacterium, Pseudomonas, Acinetobacter, and Staphylococcus, and increased the relative abundance of genera Lachnospira, Marvinbryantia, and Desulfovibrio in the caecal digesta. Butyrate concentration was significantly increased in the hindgut by the supplementation of alfalfa meal in diets. The mRNA gene expressions of FFAR3, SMCT1, MCT1, PYY, and GCG were significantly increased in the caecal mucosa of pigs fed alfalfa meal.ConclusionsOur results suggested that alfalfa-containing diet has exerted significant impacts on caecal microbiota composition, butyrate concentration and significantly upregulated mRNA expression of host caecal mucosal genes involved in SCFA sensing and absorption as well as regulation of satiety.

P125 TLR4 ACTIVATION UPREGULATES EXPRESSION OF OXIDATIVE ENZYMES: IMPLICATIONS FOR INTESTINAL TUMORIGENESIS AND LUMINAL MICROBIOTA

Overexpression of Dual oxidase 2 (Duox2) occurs as an early event in both human inflammatory bowel disease and mouse models of colitis, and has been associated with dysbiosis. We have previously shown that mice bearing constitutive activation of toll-like receptor 4 (TLR4) in intestinal epithelial cells (IECs) (villin-TLR4) have altered microbial populations, increased cytokine expression, and increased Nox1 and Duox2 expression. Villin-TLR4 mice develop spontaneous duodenal tumors. Here, we test whether TLR4-mediated induction of Nox1 and Duox2 is dependent on luminal microbiota. villin-TLR4 mice were treated or not with a combination of antibiotics for 4 weeks to deplete their microflora. IECs from duodenum and colon segments were isolated, as well as spontaneous tumors occurring in duodenum. Duodenum and colon organoids from wild type (WT) and TLR4-/- mice were stimulated with lipopolysaccharide (LPS) and/or TNF and IFN-γ. Nox1 and Duox2 expression was evaluated in all samples by quantitative PCR. Antibiotic treatment caused a marked depletion of bacterial populations in stool. Depletion of microflora decreased expression of Nox1 and Duox2 in villin-TLR4 duodenum but not in tumors or colon tissue. Even after antibiotic treatment, villin-TLR4 mice continued to have significantly elevated Nox-1 and Duox-2 expression in the duodenum and colon when compared to their untreated littermates. To further investigate whether TLR4 signaling is responsible for induction of Nox-1 and Duox-2, WT or TLR4-/- duodenum and colon organoids were treated with ultrapure LPS. This resulted in significant upregulation of Nox-1 and Duox-2 in WT but not TLR4-/- organoids. No increased expression was seen with TNF+IFN-g. TLR4 drives expression of oxidative enzymes in the intestine. LPS and luminal microbiota increase expression of Nox-1/Duox-2. We speculate that these oxidative pathways participate in TLR4-dependent neoplasia of the intestine.

Genetic risk, dysbiosis, and treatment stratification using host genome and gut microbiome in inflammatory bowel disease.

Objectives:Inflammatory bowel diseases (IBD), comprised of Crohn’s disease (CD) and ulcerative colitis (UC), are characterized by a complex pathophysiology that is thought to result from an aberrant immune response to a dysbiotic luminal microbiota in genetically susceptible individuals. New technologies support the joint assessment of host-microbiome interaction.Methods:Using whole genome sequencing and shotgun metagenomics, we studied the clinical features, host genome, and stool microbial metagenome of 85 IBD patients, and compared the results to 146 control individuals. Genetic risk scores, computed on 159 single nucleotide variants, and human leukocyte antigen (HLA) types differentiated IBD patients from healthy controls.Results:Genetic risk was associated with the need for use of biologics in IBD and, modestly, with the composition of the gut microbiome. As compared with healthy controls, IBD patients had hallmarks of stool microbiome dysbiosis, with loss of a diversified core microbiome, enrichment and depletion of specific bacteria, and enrichment of bacterial virulence factors.Conclusions:We show that genetic risk may have a role in early risk stratification in the care of IBD patients and propose that expression of virulence factors in a dysbiotic microbiome may contribute to pathogenesis in IBD.

5-Fluorouracil and irinotecan (SN-38) have limited impact on colon microbial functionality and composition in vitro.

Gastrointestinal mucositis is a debilitating side effect of chemotherapy treatment, with currently no treatment available. As changes in microbial composition have been reported upon chemotherapy treatment in vivo, it is thought that gut microbiota dysbiosis contribute to the mucositis etiology. Yet it is not known whether chemotherapeutics directly cause microbial dysbiosis, thereby increasing mucositis risk, or whether the chemotherapeutic subjected host environment disturbs the microbiome thereby aggravating the disease. To address this question, we used the M-SHIME®, an in vitro mucosal simulator of the human intestinal microbial ecosystem, as an experimental setup that excludes the host factor. The direct impact of two chemotherapeutics, 5-fluorouracil (5-FU) and SN-38 (active metabolite of irinotecan), on the luminal and mucosal gut microbiota from several human donors was investigated through monitoring fermentation activity and next generation sequencing. At a dose of 10 µM in the mucosal environment, 5-FU impacted the functionality and composition of the colon microbiota to a minor extent. Similarly, a daily dose of 10 µM SN-38 in the luminal environment did not cause significant changes in the functionality or microbiome composition. As our mucosal model does not include a host-compartment, our findings strongly indicate that a putative microbial contribution to mucositis is initially triggered by an altered host environment upon chemotherapy.

Growth performance of piglets during the first two weeks of lactation affects the development of the intestinal microbiota

The aim of this study was to evaluate the effect of newborn piglet weight gain during the first 2weeks of lactation on the luminal and mucosal microbiota of the ileum and colon. The microbiota from high-weight-gain (HWG) and low-weight-gain (LWG) 2-week-old piglets was characterized by amplicon length heterogeneity PCR (LH-PCR) and compared using diversity indices and multivariate statistical analyses. At birth, LWG piglets weighted in average 0.26kg less than HWG piglets (p=.002). The weight difference between LWG and HWG piglets increased with time and reached 2.1kg after 16days of lactation (p<.0001). Based on these growth performance differences, estimated colostrum and milk intake was greater in HWG than in LWG piglets (p<.0001). Analysis of the LH-PCR data of the microbiota using non-metric multidimensional scaling (NMS) and blocked multiresponse permutation procedure (MRBP) revealed that the microbiota of the HWG and LWG piglets tended to differ in ileal mucosa (p=.097) and differed in colonic lumen (p=.024). The microbiota of HWG piglets had higher levels of Bacteroidetes, Bacteroides and Ruminoccocaceae, and lower proportions of Actinobacillus porcinus and Lactobacillus amylovorus when compared with those of LWG piglets. As the weight gain of nursing piglets is highly correlated with the amount of ingested colostrum and milk, the results strongly suggest that colostrum and milk intake in the first 2weeks of life influenced the development of the gut microbiota.

Interplay of GTPases and Cytoskeleton in Cellular Barrier Defects during Gut Inflammation.

An essential role of the intestine is to build and maintain a barrier preventing the luminal gut microbiota from invading the host. This involves two coordinated physical and immunological barriers formed by single layers of intestinal epithelial and endothelial cells, which avoid the activation of local immune responses or the systemic dissemination of microbial agents, and preserve tissue homeostasis. Accordingly, alterations of epithelial and endothelial barrier functions have been associated with gut inflammation, for example during inflammatory bowel disease (IBD). The discriminative control of nutriment uptake and sealing toward potentially pathological microorganisms requires a profound regulation of para- and transcellular permeability. On the subcellular level, the cytoskeleton exerts key regulatory functions in the maintenance of cellular barriers. Increased epithelial/endothelial permeability occurs primarily as a result of a reorganization of cytoskeletal–junctional complexes. Pro-inflammatory mediators such as cytokines can induce cytoskeletal rearrangements, causing inflammation-dependent defects in gut barrier function. In this context, small GTPases of the Rho family and large GTPases from the Dynamin superfamily appear as major cellular switches regulating the interaction between intercellular junctions and actomyosin complexes, and in turn cytoskeleton plasticity. Strikingly, some of these proteins, such as RhoA or guanylate-binding protein-1 (GBP-1) have been associated with gut inflammation and IBD. In this review, we will summarize the role of small and large GTPases for cytoskeleton plasticity and epithelial/endothelial barrier in the context of gut inflammation.

Faecal and mucosal microbiota in patients with functional gastrointestinal disorders: Correlation with toll-like receptor 2/toll-like receptor 4 expression.

AIMTo investigate the intestinal luminal microbiota (LM) and mucosa-associated microbiota (MAM) in Chinese patients with functional gastrointestinal disorders (FGIDs) and examine the association between these communities and the expression of toll-like receptor (TLR) 2 and TLR4.METHODSThirty-two Chinese subjects who suffered from symptoms of FGIDs, as confirmed by gastroenterologists, were enrolled in this study. Fresh faecal samples and descending colonic mucosal biopsies were collected from the subjects before (faecal) and during (mucosal) flexible colonoscopy. For analysis of the samples, we performed high-throughput sequencing of the V3-V4 region of the 16S rRNA gene and reverse transcription (RT)-PCR to detect the expression of colonic TLR2 and TLR4. Differences in the stool and mucosal microbiota were examined and a correlation network analysis was performed.RESULTSThe microbiota of faecal samples was significantly more diverse and richer than that of the mucosal samples, and the LM and MAM populations differed significantly. TLR2 expression showed a significant positive correlation with TLR4 expression. In the MAM samples, the genera Faecalibacterium and Ruminococcus, which belong to the family Ruminococcaceae, were inversely correlated with TLR4 expression (r = -0.45817, P = 0.0083 and r = -0.5306, P = 0.0018, respectively). Granulicatella, which belongs to Carnobacteriaceae, and Streptococcus, which belongs to Streptococcaceae, were inversely correlated with TLR2 expression (r = -0.5573, P = 0.0010 and r = -0.5435, P = 0.0013, respectively). In the LM samples, examination at phylum, class, or order level revealed no correlation with TLR4 expression. Faecalibacterium, which belongs to Ruminococcaceae, and Streptococcus, which belongs to Streptococcaceae, were inversely correlated with TLR2 expression (r = -0.5743, P = 0.0058 and r = -0.3905, P = 0.0271, respectively).CONCLUSIONMicrobial compositions of LM and MAM in Chinese patients with FGIDs are different. Expression of TLRs may be affected by the type of bacteria that are present in the gut.

Comparison of the Luminal and Mucosa-Associated Microbiota in the Colon of Pigs with and without Swine Dysentery

Colonic contents and mucosal scrapings from pigs inoculated with Brachyspira hyodysenteriae or Brachyspira hampsonii were collected at necropsy and classified as either positive (n = 29) or negative (n = 7) for swine dysentery (SD) based upon lesions and positive culture from the source pig. The microbiota in each sample was analyzed by bacterial census taking (16S rRNA gene sequencing). Procrustes analysis revealed similar clustering by disease classification with a relatively high M2 value (0.44) suggesting differences in the microbiota between mucosal and luminal samples from the same pig. In both sample types, differences in richness and beta diversity were observed between disease statuses (P ≤ 0.014). The relative abundance of Brachyspirales, Campylobacterales, Desulfovibrionales, and Enterobacteriales was higher in pigs with dysentery for both mucosal scrapings and luminal samples while Clostridiales, Erysipelotrichales, and Fusobacteriales were significantly more abundant in the luminal contents only. For inoculated pigs that did not develop dysentery, Burkholderiales were more abundant in both sample types, Bacteroidales and Synergistales were more abundant in mucosal scrapings, and Lactobacillales and Bifidobacteriales were more abundant in luminal contents when compared with diseased pigs. Linear discriminant analysis of effect size revealed Brachyspira, Campylobacter, Mogibacterium, and multiple Desulfovibrio spp. as differential features in mucosal scrapings from pigs with dysentery while Lactobacillus and a Bifidobacterium spp. were differential in pigs without disease. These differential features were not observed in luminal samples. In summary, microbial profiles in both sample types differ significantly between disease states; however, evaluation of the mucosal microbiome specifically may be of higher value in elucidating bacterial mechanisms underlying development of SD.

Inter-individual differences determine the outcome of wheat bran colonization by the human gut microbiome.

Gut microbiota research reveals a vital role for the luminal and mucosal gut microbiota in human health. Fewer studies, however, have characterized the microbiome associated with undigested, insoluble dietary particles in the gut. These particles can act as a food source for bacteria and offer a physical platform to which they can attach. In this study, the microbiome colonizing wheat bran particles was analyzed. In a batch experiment, wheat bran particles were separately incubated with the faecal microbiota derived from 10 donors and washed after 48 h to remove loosely attached bacteria. The response of the luminal community to wheat bran and inulin, acting as a well-characterized control, was largely donor-dependent, both functionally, and with respect to the microbiome composition. Depending on the donor, wheat bran and inulin fermentation yielded proportionally higher propionate or butyrate production. Clostridium cluster XIVa and, depending on the donor, Prevotella, Roseburia, Megamonas, Bifidobacterium and Bacteroides species were enriched on the wheat bran particles. These genera include species with the documented ability to serve as primary degraders of wheat bran components and other species depending on cross-feeding to obtain their energy. Both functional groups were present in all donors, despite the large inter-individual differences.

Long chain arabinoxylans shift the mucosa-associated microbiota in the proximal colon of the simulator of the human intestinal microbial ecosystem (M-SHIME)

Prebiotic compounds aim at improving human health by targeting beneficial gut microbes. We used a dynamic in vitro model of the human digestive tract (M-SHIME®) to study the modulatory effects of long-chain arabinoxylans (LC-AX) towards luminal and mucosal microbiota. Two doses (3 and 6g/L) of LC-AX were supplied to the proximal colon compartments of the M-SHIME, three times per day for 3days. Next generation sequencing and multivariate statistical analysis revealed the distribution of key microbial genera across M-SHIME compartments. Supplementation of 6g/L of LC-AX significantly increased the relative abundance of Bifidobacterium in both lumen (from 0.34% to 0.41%, 1.2-fold) and mucus (from 0.14% to 0.29%, 2.1-fold) compared with the control (P<0.001). Propionate (1.4-fold), as well as activity of β-xylanase, β-xylosidase and α-arabinofuranosidase (P<0.05) were also increased in the lumen. LC-AX may potentially benefit host health by stimulating Bifidobacterium abundance and its metabolic activity.

Mucosa-associated biohydrogenating microbes protect the simulated colon microbiome from stress associated with high concentrations of poly-unsaturated fat.

Polyunsaturated fatty acids (PUFAs) may affect colon microbiome homeostasis by exerting (specific) antimicrobial effects and/or interfering with mucosal biofilm formation at the gut mucosal interface. We used standardized batch incubations and the Mucosal-Simulator of the Human Microbial Intestinal Ecosystem (M-SHIME) to show the in vitro luminal and mucosal effects of the main PUFA in the Western diet, linoleic acid (LA). High concentrations of LA were found to decrease butyrate production and Faecalibacterium prausnitzii numbers dependent on LA biohydrogenation to vaccenic acid (VA) and stearic acid (SA). In faecal batch incubations, LA biohydrogenation and butyrate production were positively correlated and SA did not inhibit butyrate production. In the M-SHIME, addition of a mucosal environment stimulated biohydrogenation to SA and protected F. prausnitzii from inhibition by LA. This was probably due to the preference of two biohydrogenating genera Roseburia and Pseudobutyrivibrio for the mucosal niche. Co-culture batch incubations using Roseburia hominis and F. prausnitzii validated these observations. Correlations networks further uncovered the central role of Roseburia and Pseudobutyrivibrio in protecting luminal and mucosal SHIME microbiota from LA-induced stress. Our results confirm how cross-shielding interactions provide resilience to the microbiome and demonstrate the importance of biohydrogenating, mucosal bacteria for recovery from LA stress.

Vancomycin pre-treatment impairs tissue healing in experimental colitis: Importance of innate lymphoid cells

Background and aimsThe interplay between luminal microbes and innate immunity during colonic epithelial repair has been well noted. At the same time, antibiotic has widely been used during flare-ups of ulcerative colitis. The possible effects of luminal microbiota disruption caused by antibiotics usage on epithelial repairing have been scarcely discussed. Innate lymphoid cells (ILCs) embedded in the lamina propria can be modulated by gut microbes, resulting in altered colonic IL-22/pSTAT3 levels, which is considered a prominent molecular axis in tissue repairing after epithelium damage. This study aimed to investigate whether antibiotics could interfere with ILCs-dependent tissue repair. MethodsDextran sodium sulfate (DSS)-induced colitis was established in mice pre-treated with reagent of different antibiotic spectrum. Both morphological and molecular markers of tissue repair after DSS cessation were detected. ILCs population and function status were also recorded. Further attention was paid to the response of dendritic cells after antibiotics treatment, which were claimed to regulate colonic ILC3s in an IL-23 dependent way. ResultsUsing of vancomycin resulted in delayed tissue repairing after experimental colitis. Both colonic IL-22/pSTAT3 axis and ILC3 population were found decreased in this situation. Vancomycin treatment diminished the upstream IL-23 and producer dendritic cell population. The reduced dendritic cell number may due to inadequate chemokines and colony-stimulating factors supply. ConclusionPresence of vancomycin-sensitive microbiota is required for the maturation of ILC3-activating dendritic cells hence maintain the sufficient IL-22/pSTAT3 level in the colon during tissue healing. Manipulation of colonic microbiota may help achieve colonic mucosal healing post inflammation and injury.

Cosmc is an X-linked inflammatory bowel disease risk gene that spatially regulates gut microbiota and contributes to sex-specific risk

Inflammatory bowel disease (IBD) results from aberrant immune stimulation against a dysbiotic mucosal but relatively preserved luminal microbiota and preferentially affects males in early onset disease. However, factors contributing to sex-specific risk and the pattern of dysbiosis are largely unexplored. Core 1 β3GalT-specific molecular chaperone (Cosmc), which encodes an X-linked chaperone important for glycocalyx formation, was recently identified as an IBD risk factor by genome-wide association study. We deleted Cosmc in mouse intestinal epithelial cells (IECs) and found marked reduction of microbiota diversity in progression from the proximal to the distal gut mucosa, but not in the overlying lumen, as seen in IBD. This loss of diversity coincided with local emergence of a proinflammatory pathobiont and distal gut restricted pathology. Mechanistically, we found that Cosmc regulates host genes, bacterial ligands, and nutrient availability to control microbiota biogeography. Loss of one Cosmc allele in males (IEC-Cosmc-/y) resulted in a compromised mucus layer, spontaneous microbe-dependent inflammation, and enhanced experimental colitis; however, females with loss of one allele and mosaic deletion of Cosmc in 50% of crypts (IEC-Cosmc+/-) were protected from spontaneous inflammation and partially protected from experimental colitis, likely due to lateral migration of normal mucin glycocalyx from WT cells over KO crypts. These studies functionally validate Cosmc as an IBD risk factor and implicate it in regulating the spatial pattern of dysbiosis and sex bias in IBD.