Using meta-analysis to understand the impacts of dietary protein and fat content on the composition of fecal microbiota of domestic dogs (Canis lupus familiaris): A pilot study.

The interplay between diet and fecal microbiota composition is garnering increased interest across various host species, including domestic dogs. While the influence of dietary macronutrients and their associated microbial communities have been extensively reviewed, these reviews are descriptive and do not account for differences in microbial community analysis, nor do they standardize macronutrient content across studies. To address this, a meta-analysis was performed to assess the impact of dietary crude protein ("protein") and dietary crude fat ("fat") on the fecal microbiota composition in healthy dogs. Sixteen publications met the eligibility criteria for the meta-analysis, yielding a final data set of 314 dogs. Diets were classed as low, moderate, high, or supra in terms of protein or fat content. Sequence data from each publication were retrieved from public databases and reanalyzed using consistent bioinformatic pipelines. Analysis of community diversity indices and unsupervised clustering of the data with principal coordinate analysis revealed a small effect size and complete overlap between protein and fat levels at the overall community level. Supervised clustering through random forest analysis and partial least squares-discriminant analysis indicated alterations in the fecal microbiota composition at a more individual taxonomic level, corresponding to the levels of protein or fat. The Prevotellaceae Ga6A1 group and Enterococcus were associated with increasing levels of protein, while Allobaculum and Clostridium sensu stricto 13 were associated with increasing levels of fat. Interestingly, the random forest analyses revealed that Sharpea, despite its low relative abundance in the dog's fecal microbiome, was primarily responsible for the separation of the microbiome for both protein and fat. Future research should focus on validating and understanding the functional roles of these relatively low-abundant genera.

A Diet Enriched with Lacticaseibacillus rhamnosus HN001 and Milk Fat Globule Membrane Alters the Gut Microbiota and Decreases Amygdala GABA a Receptor Expression in Stress-Sensitive Rats.

Brain signalling pathways involved in subclinical anxiety and depressed mood can be modulated via the gut brain axis (GBA), providing the potential for diet and dietary components to affect mood. We investigated behavioural, physiological and gut microbiome responses to the Lacticaseibacillus rhamnosus strain HN001 (LactoB HN001™), which has been shown to reduce postpartum anxiety and depression, and a milk fat globule membrane-enriched product, Lipid 70 (SurestartTM MFGM Lipid 70), which has been implicated in memory in stress-susceptible Wistar Kyoto rats. We examined behaviour in the open field, elevated plus maze and novel object recognition tests in conjunction with the expression of host genes in neuro-signalling pathways, and we also assessed brain lipidomics. Treatment-induced alterations in the caecal microbiome and short-chain fatty acid (SCFA) profiles were also assessed. Neither ingredient induced behavioural changes or altered the brain lipidome (separately or when combined). However, with regard to brain gene expression, the L. rhamnosus HN001 + Lipid 70 combination produced a synergistic effect, reducing GABAA subunit expression in the amygdala (Gabre, Gat3, Gabrg1) and hippocampus (Gabrd). Treatment with L. rhamnosus HN001 alone altered expression of the metabotropic glutamate receptor (Grm4) in the amygdala but produced only minor changes in gut microbiota composition. In contrast, Lipid 70 alone did not alter brain gene expression but produced a significant shift in the gut microbiota profile. Under the conditions used, there was no observed effect on rat behaviour for the ingredient combination. However, the enhancement of brain gene expression by L. rhamnosus HN001 + Lipid 70 implicates synergistic actions on region-specific neural pathways associated with fear, anxiety, depression and memory. A significant shift in the gut microbiota profile also occurred that was mainly attributable to Lipid 70.

A bioactive bovine whey protein extract improves intestinal barrier function in vitro.

The human intestine plays an important role as a barrier against the ingress of pathogens and other harmful antigens. Accordingly, proper regulation of the intestinal barrier is essential for optimal health. Intestinal barrier function is regulated in part by the interactions between dietary compounds and the intestinal immune system. Bioactive whey proteins from bovine milk (such as lactoferrin, lactoperoxidase, and immunoglobulins) are known to exert a range of physiological functions, including modulation of the immune system, and thus have the potential to regulate intestinal barrier function. While the effects of individual whey proteins on intestinal barrier function have been studied to some extent, less is known about the potentially synergistic properties of whey protein mixtures. Here we investigated the effects of a bioactive bovine whey protein (BWP) extract containing all whey proteins with an isoelectric point >6.8 on intestinal barrier function in vitro. Intestinal epithelial cell (Caco-2) monolayers were treated with BWP before measuring the barrier integrity over 48 h by means of trans-epithelial electrical resistance (TEER). Treatment of epithelial monolayers with 1 mg/mL BWP resulted in an increase in TEER compared with untreated epithelial monolayers. To determine whether BWP could mitigate immune-mediated intestinal barrier dysfunction, we challenged differentiated Caco-2 cell monolayers with tumor necrosis factor α (TNFα) to obtain an in vitro model of a "leaky" intestinal epithelium. The TNFα challenge led to a decrease in TEER over time across untreated control monolayers, indicating a loss of barrier function. This loss of barrier function was mitigated in monolayers treated with 1 mg/mL BWP, but not monolayers treated with the equivalent amount of lactoferrin present in 1 mg/mL BWP. These data suggest that naturally co-occurring bioactive proteins together may enhance intestinal barrier integrity and protect against inflammation-induced barrier dysfunction to a greater extent than lactoferrin alone. Further work is required to determine the key proteins and protein combinations within BWP, and the mechanisms through which BWP modulates intestinal barrier function.

The Role of Segmented Filamentous Bacteria in Immune Barrier Maturation of the Small Intestine at Weaning.

The microbiological, physical, chemical, and immunological barriers of the gastrointestinal tract (GIT) begin developing in utero and finish maturing postnatally. Maturation of these barriers is essential for the proper functioning of the GIT. Maturation, particularly of the immunological barrier, involves stimulation by bacteria. Segmented filamentous bacteria (SFB) which are anaerobic, spore-forming commensals have been linked to immune activation. The presence and changes in SFB abundance have been positively correlated to immune markers (cytokines and immunoglobulins) in the rat ileum and stool samples, pre- and post-weaning. The abundance of SFB in infant stool increases from 6 months, peaks around 12 months and plateaus 25 months post-weaning. Changes in SFB abundance at these times correlate positively and negatively with the production of interleukin 17 (IL 17) and immunoglobulin A (IgA), respectively, indicating involvement in immune function and maturation. Additionally, the peak in SFB abundance when a human milk diet was complemented by solid foods hints at a diet effect. SFB genome analysis revealed enzymes involved in metabolic pathways for survival, growth and development, host mucosal attachment and substrate acquisition. This narrative review discusses the current knowledge of SFB and their suggested effects on the small intestine immune system. Referencing the published genomes of rat and mouse SFB, the use of food substrates to modulate SFB abundance is proposed while considering their effects on other microbes. Changes in the immune response caused by the interaction of food substrate with SFB may provide insight into their role in infant immunological barrier maturation.

Ruminant Milk-Derived Extracellular Vesicles: A Nutritional and Therapeutic Opportunity?

Milk has been shown to contain a specific fraction of extracellular particles that are reported to resist digestion and are purposefully packaged with lipids, proteins, and nucleic acids to exert specific biological effects. These findings suggest that these particles may have a role in the quality of infant nutrition, particularly in the early phase of life when many of the foundations of an infant's potential for health and overall wellness are established. However, much of the current research focuses on human or cow milk only, and there is a knowledge gap in how milk from other species, which may be more commonly consumed in different regions, could also have these reported biological effects. Our review provides a summary of the studies into the extracellular particle fraction of milk from a wider range of ruminants and pseudo-ruminants, focusing on how this fraction is isolated and characterised, the stability and uptake of the fraction, and the reported biological effects of these fractions in a range of model systems. As the individual composition of milk from different species is known to differ, we propose that the extracellular particle fraction of milk from non-traditional and minority species may also have important and distinct biological properties that warrant further study.

Whole tissue homogenization preferable to mucosal scraping in determining the temporal profile of segmented filamentous bacteria in the ileum of weanling rats.

Segmented filamentous bacteria (SFB) are thought to play a role in small intestine immunological maturation. Studies in weanling mice have shown a positive correlation between ileal SFB abundance and plasma and faecal interleukin 17 (IL-17) and immunoglobulin A (IgA) concentrations. Although the first observation of SFB presence was reported in rats, most studies use mice. The size of the mouse ileum is a limitation whereas the rat could be a suitable alternative for sufficient samples. Changes in SFB abundance over time in rats were hypothesized to follow the pattern reported in mice and infants. We characterized the profile of SFB colonization in the ileum tissue and contents and its correlation with two immune markers of gastrointestinal tract (GIT) maturation. We also compared two published ileum collection techniques to determine which yields data on SFB abundance with least variability. Whole ileal tissue and ileal mucosal scrapings were collected from 20- to 32-day-old Sprague-Dawley rats. SFB abundance was quantified from proximal, middle and distal ileal tissues, contents and faeces by quantitative PCR using SFB-specific primers. Antibody-specific ELISAs were used to determine IL-17 and IgA concentrations. Significant differences in SFB abundance were observed from whole and scraped tissues peaking at day 22. Variability in whole ileum data was less, favouring it as a better collection technique. A similar pattern of SFB abundance was observed in ileum contents and faeces peaking at day 24, suggesting faeces can be a proxy for ileal SFB abundance. SFB abundance at day 26 was higher in females than males across all samples. There were significant differences in IgA concentration between days 20, 30 and 32 and none in IL-17 concentration, which was different from reports in mice and infants.

Short communication: Processed bovine colostrum milk protein concentrate increases epithelial barrier integrity of Caco-2 cell layers.

Colostrum plays an important role in initiating the development of the intestinal barrier in newborn mammals. Given its bioactivity, there is much interest in the potential use of bovine colostrum to improve human gastrointestinal health throughout the life span. There is evidence that bovine colostrum is effective at improving small intestinal barrier integrity and some indication that it may alter colonic motility. However, for colostrum to be used as a product to improve intestinal health, it needs to be bioactive after processing. The aim of this study was to determine whether industrial processing of bovine colostrum affects its ability to improve small intestinal barrier integrity or alter distal colon motility. Three colostrum sample types were compared; raw whole colostrum powder (WCP), raw skim colostrum powder (SCP), and industrially produced colostrum milk protein concentrate (CMPC). To determine whether these colostrum powders had different effects on small intestinal barrier integrity, their effects on the transepithelial electrical resistance across an in vitro intestinal epithelial layer (Caco-2 cells) were measured, both with and without a challenge from the proinflammatory cytokine tumor necrosis factor-α. These results showed that CMPC enhanced transepithelial electrical resistance across unchallenged epithelial cell layers, whereas the raw colostrum samples, WCP and SCP, did not have an effect. The colostrum samples were also compared to determine how they affect contractility in the distal colon isolated from the rat. Skim colostrum powder was the only sample to act directly on colonic tissue to modulate motility, increasing the amplitude of contractions. The results show that bovine colostrum is able to improve small intestinal barrier integrity and alter colon motility, and they implicate different components. The barrier integrity enhancement was apparent only in the industrial CMPC, which may have been due to the increase in protein concentration or the release of small peptides as a result of processing. The ability to alter colon motility was present in SCP but absent in WCP, again implying that an increase in protein concentration is responsible for the effect. However, this effect was not apparent for the industrially processed CMPC, suggesting denaturation or degradation of the active component. The beneficial effect of colostrum on small intestinal barrier integrity was present after processing, confirming that it is feasible to industrially produce an active product for gut health.

Bovine dairy complex lipids improve in vitro measures of small intestinal epithelial barrier integrity.

Appropriate intestinal barrier maturation is essential for absorbing nutrients and preventing pathogens and toxins from entering the body. Compared to breast-fed infants, formula-fed infants are more susceptible to barrier dysfunction-associated illnesses. In infant formula dairy lipids are usually replaced with plant lipids. We hypothesised that dairy complex lipids improve in vitro intestinal epithelial barrier integrity. We tested milkfat high in conjugated linoleic acid, beta serum (SureStart™Lipid100), beta serum concentrate (BSC) and a ganglioside-rich fraction (G600). Using Caco-2 cells as a model of the human small intestinal epithelium, we analysed the effects of the ingredients on trans-epithelial electrical resistance (TEER), mannitol flux, and tight junction protein co-localisation. BSC induced a dose-dependent improvement in TEER across unchallenged cell layers, maintained the co-localisation of tight junction proteins in TNFα-challenged cells with increased permeability, and mitigated the TEER-reducing effects of lipopolysaccharide (LPS). G600 also increased TEER across healthy and LPS-challenged cells, but it did not alter the co-location of tight junction proteins in TNFα-challenged cells. SureStart™Lipid100 had similar TEER-increasing effects to BSC when added at twice the concentration (similar lipid concentration). Ultimately, this research aims to contribute to the development of infant formulas supplemented with dairy complex lipids that support infant intestinal barrier maturation.

Live Faecalibacterium prausnitzii induces greater TLR2 and TLR2/6 activation than the dead bacterium in an apical anaerobic co-culture system.

Inappropriate activation of intestinal innate immune receptors, such as toll-like receptors (TLRs), by pathogenic bacteria is linked to chronic inflammation. In contrast, a "tonic" level of TLR activation by commensal bacteria is required for intestinal homeostasis. A technical challenge when studying this activation in vitro is the co-culturing of oxygen-requiring mammalian cells with obligate anaerobic commensal bacteria. To overcome this, we used a novel apical anaerobic co-culture system to successfully adapt a TLR activation assay to be conducted in conditions optimised for both cell types. Live Faecalibacterium prausnitzii, an abundant obligate anaerobe of the colonic microbiota, induced higher TLR2 and TLR2/6 activation than the dead bacterium. This enhanced TLR induction by live F. prausnitzii, which until now has not previously been described, may contribute to maintenance of gastrointestinal homeostasis. This highlights the importance of using physiologically relevant co-culture systems to decipher the mechanisms of action of live obligate anaerobes.

Live Faecalibacterium prausnitzii Does Not Enhance Epithelial Barrier Integrity in an Apical Anaerobic Co-Culture Model of the Large Intestine.

Appropriate intestinal barrier maturation during infancy largely depends on colonization with commensal bacteria. Faecalibacterium prausnitzii is an abundant obligate anaerobe that colonizes during weaning and is thought to maintain colonic health throughout life. We previously showed that F. prausnitzii induced Toll-like receptor 2 (TLR2) activation, which is linked to enhanced tight junction formation. Therefore, we hypothesized that F. prausnitzii enhances barrier integrity, an important factor in appropriate intestinal barrier maturation. In order to test metabolically active bacteria, we used a novel apical anaerobic co-culture system that allows the survival of both obligate anaerobic bacteria and oxygen-requiring intestinal epithelial cells (Caco-2). The first aim was to optimize the culture medium to enable growth and active metabolism of F. prausnitzii while maintaining the viability and barrier integrity, as measured by trans-epithelial electrical resistance (TEER), of the Caco-2 cells. This was achieved by supplementing the apical cell culture medium with bacterial culture medium. The second aim was to test the effect of F. prausnitzii on TEER across Caco-2 cell layers. Live F. prausnitzii did not improve TEER, which indicates that its benefits are not via altering tight junction integrity. The optimization of the novel dual-environment co-culturing system performed in this research will enable the investigation of new probiotics originating from indigenous beneficial bacteria.

Influence of the Fruit Juice Carriers on the Ability of Lactobacillus plantarum DSM20205 to Improve in Vitro Intestinal Barrier Integrity and Its Probiotic Properties.

This study investigates the influence of tomato and feijoa juices as fermentable carriers of Lactobacillus plantarum (LP DSM20205) on the ability of the bacterium to improve intestinal barrier function using the trans-epithelial electrical resistance (TEER) assay in an apical anaerobic model. The survival of LP DSM20205 in different fruit juices during in vitro digestion, its adhesion capacity, and potential cytotoxic effect on Caco-2 cells were also studied. The results showed that carrier fruit juices have a significant influence on LP DSM20205 growth, survival during in vitro digestion, adhesion capacity, and TEER. All fermented samples were not cytotoxic to Caco-2 cells. The fermented tomato juice showed the largest improvement to intestinal barrier integrity. The digested fermented juices did not increase TEER, although the LP DSM20205 in these samples adhered well. Therefore, LP DSM20205 has the potential to be used as a probiotic in the production of fermented tomato and feijoa juices.

Promotility Action of the Probiotic Bifidobacterium lactis HN019 Extract Compared with Prucalopride in Isolated Rat Large Intestine.

Attention is increasingly being focussed on probiotics as potential agents to restore or improve gastrointestinal (GI) transit. Determining mechanism of action would support robust health claims. The probiotic bacterium Bifidobacterium lactis HN019 reduces transit time, but its mechanisms of action and effects on motility patterns are poorly understood. The aim of this study was to investigate changes in GI motility induced by an extract of HN019 on distinct patterns of colonic motility in isolated rat large intestine, compared with a known promotility modulator, prucalopride. The large intestines from male Sprague Dawley rats (3-6 months) were perfused with Kreb's buffer at 37°C in an oxygenated tissue bath. Isometric force transducers recorded changes in circular muscle activity at four independent locations assessing contractile propagation between the proximal colon and the rectum. HN019 extract was perfused through the tissue bath and differences in tension and frequency quantified relative to pre-treatment controls. Prucalopride (1 µM) increased the frequency of propagating contractions (by 75 ± 26%) in the majority of preparations studied (10/12), concurrently decreasing the frequency of non-propagating contractions (by 50 ± 11%). HN019 extract had no effect on contractile activity during exposure (n = 8). However, following wash out, contraction amplitude of propagating contractions increased (by 55 ± 18%) in the distal colon, while the frequency of non-propagating proximal contractions decreased by 57 ± 7%. The prokinetic action of prucalopride increased the frequency of synchronous contractions along the length of colon, likely explaining increased colonic rate of transit in vivo. HN019 extract modified motility patterns in a different manner by promoting propagating contractile amplitude and inhibiting non-propagations, also demonstrating prokinetic activity consistent with the reduction of constipation by B. lactis HN019 in humans.

Comparative innate immune interactions of human and bovine secretory IgA with pathogenic and non-pathogenic bacteria.

Secretory IgA (SIgA) from milk contributes to early colonization and maintenance of commensal/symbiotic bacteria in the gut, as well as providing defence against pathogens. SIgA binds bacteria using specific antigenic sites or non-specifically via its glycans attached to α-heavy-chain and secretory component. In our study, we tested the hypothesis that human and bovine SIgA have similar innate-binding activity for bacteria. SIgAs, isolated from human and bovine milk, were incubated with a selection of commensal, pathogenic and probiotic bacteria. Using flow cytometry, we measured numbers of bacteria binding SIgA and their level of SIgA binding. The percentage of bacteria bound by human and bovine SIgA varied from 30 to 90% depending on bacterial species and strains, but was remarkably consistent between human and bovine SIgA. The level of SIgA binding per bacterial cell was lower for those bacteria that had a higher percentage of SIgA-bound bacteria, and higher for those bacteria that had lower percentage of SIgA-bound bacteria. Overall, human and bovine SIgA interacted with bacteria in a comparable way. This contributes to longer term research about the potential benefits of bovine SIgA for human consumers.

Short communication: Early-lactation, but not mid-lactation, bovine lactoferrin preparation increases epithelial barrier integrity of Caco-2 cell layers.

Bovine lactoferrin is an important milk protein with many health-promoting properties, including improving intestinal barrier integrity. Dysfunction of this barrier, commonly referred to as "leaky gut," has been linked to inflammatory and autoimmune diseases. With some processing techniques, lactoferrin isolated from milk collected at the start of the milking season (early lactation) may have lower purity than that isolated from milk collected during the rest of the milking season (mid-lactation) and could result in differences in bioactivity based on the stage of lactation. We compared reversed-phase HPLC chromatographs of early-lactation and mid-lactation preparations and found that both had large chromatograph peaks at the time predicted for lactoferrin. The notable difference between the 2 chromatographs was a much larger peak in the early-lactation lactoferrin sample that was determined to be angiogenin. Angiogenin was first identified due to its ability to induce new blood vessel formation, but is now known to be involved in numerous physiological processes. Then, we compared the effects of early-lactation and mid-lactation lactoferrin preparations in 2 bioassays: trans-epithelial electrical resistance (TEER), a measure of intestinal barrier integrity, and peripheral blood mononuclear cell cytokine secretion, a measure of immune-stimulatory properties. We found that early-lactation lactoferrin increased TEER across Caco-2 cell layers compared with control from 10 to 48 h, mid-lactation lactoferrin did not alter TEER. We also found that early-lactation lactoferrin reduced the amount of IL-8 produced by peripheral blood mononuclear cells (compared with those treated with control medium) to a greater extent than mid-lactation lactoferrin. A pro-inflammatory chemokine, IL-8 is also known to decrease barrier function. These results suggest that the decrease in IL-8 production in the presence of early-lactation lactoferrin may be the mechanism by which it increases TEER. The anti-inflammatory effect of early-lactation lactoferrin may be related to the presence of angiogenin, which is known to suppress inflammatory responses. This work indicates that products rich in angiogenin may have intestinal health benefits, and further work to investigate this is warranted.

Influence of Bovine Whey Protein Concentrate and Hydrolysate Preparation Methods on Motility in the Isolated Rat Distal Colon.

Whey protein concentrate (WPC) and hydrolysate (WPH) are protein ingredients used in sports, medical and pediatric formulations. Concentration and hydrolysis methods vary for whey sourced from cheese and casein co-products. The purpose of this research was to investigate the influence of whey processing methods on in vitro gastrointestinal (GI) health indicators for colonic motility, epithelial barrier integrity and immune modulation. WPCs from casein or cheese processing and WPH (11% or 19% degree of hydrolysis, DH) were compared for their effects on motility in a 1 cm section of isolated rat distal colon in an oxygenated tissue bath. Results showed that WPC decreased motility irrespective of whether it was a by-product of lactic acid or mineral acid casein production, or from cheese production. This indicated that regardless of the preparation methodology, the whey protein contained components that modulate aspects of motility within the distal colon. WPH (11% DH) increased contractile frequency by 27% in a delayed manner and WPH (19% DH) had an immediate effect on contractile properties, increasing tension by 65% and frequency by 131%. Increased motility was associated with increased hydrolysis that may be attributed to the abundance of bioactive peptides. Increased frequency of contractions by WPH (19% DH) was inhibited (by 44%) by naloxone, implicating a potential involvement of opioid receptors in modulation of motility. Trans-epithelial electrical resistance and cytokine expression assays revealed that the WPC proteins studied did not alter intestinal barrier integrity or elicit any discernible immune response.

Human oral isolate Lactobacillus fermentum AGR1487 induces a pro-inflammatory response in germ-free rat colons.

Lactobacilli are thought to be beneficial for human health, with lactobacilli-associated infections being confined to immune-compromised individuals. However, Lactobacillus fermentum AGR1487 negatively affects barrier integrity in vitro so we hypothesized that it caused a pro-inflammatory response in the host. We compared germ-free rats inoculated with AGR1487 to those inoculated with another L. fermentum strain, AGR1485, which does not affect in vitro barrier integrity. We showed that rats inoculated with AGR1487 had more inflammatory cells in their colon, higher levels of inflammatory biomarkers, and increased colonic gene expression of pro-inflammatory pathways. In addition, our in vitro studies showed that AGR1487 had a greater capacity to activate TLR signaling and induce pro-inflammatory cytokines in immune cells. This study indicates the potential of strains of the same species to differentially elicit inflammatory responses in the host and highlights the importance of strain characterization in probiotic approaches to treat inflammatory disorders.

Lactobacillus fermentum AGR1487 cell surface structures and supernatant increase paracellular permeability through different pathways.

Lactobacillus fermentum is commonly found in food products, and some strains are known to have beneficial effects on human health. However, our previous research indicated that L. fermentum AGR1487 decreases in vitro intestinal barrier integrity. The hypothesis was that cell surface structures of AGR1487 are responsible for the observed in vitro effect. AGR1487 was compared to another human oral L. fermentum strain, AGR1485, which does not cause the same effect. The examination of phenotypic traits associated with the composition of cell surface structures showed that compared to AGR1485, AGR1487 had a smaller genome, utilized different sugars, and had greater tolerance to acid and bile. The effect of the two strains on intestinal barrier integrity was determined using two independent measures of paracellular permeability of the intestinal epithelial Caco-2 cell line. The transepithelial electrical resistance (TEER) assay specifically measures ion permeability, whereas the mannitol flux assay measures the passage of uncharged molecules. Both live and UV-inactivated AGR1487 decreased TEER across Caco-2 cells implicating the cell surfaces structures in the effect. However, only live AGR1487, and not UV-inactivated AGR1487, increased the rate of passage of mannitol, implying that a secreted component(s) is responsible for this effect. These differences in barrier integrity results are likely due to the TEER and mannitol flux assays measuring different characteristics of the epithelial barrier, and therefore imply that there are multiple mechanisms involved in the effect of AGR1487 on barrier integrity.

Live Faecalibacterium prausnitzii in an apical anaerobic model of the intestinal epithelial barrier.

Faecalibacterium prausnitzii, an abundant member of the human commensal microbiota, has been proposed to have a protective role in the intestine. However, it is an obligate anaerobe, difficult to co-culture in viable form with oxygen-requiring intestinal cells. To overcome this limitation, a unique apical anaerobic model of the intestinal barrier, which enabled co-culture of live obligate anaerobes with the human intestinal cell line Caco-2, was developed. Caco-2 cells remained viable and maintained an intact barrier for at least 12 h, consistent with gene expression data, which suggested Caco-2 cells had adapted to survive in an oxygen-reduced atmosphere. Live F. prausnitzii cells, but not ultraviolet (UV)-killed F. prausnitzii, increased the permeability of mannitol across the epithelial barrier. Gene expression analysis showed inflammatory mediators to be expressed at lower amounts in Caco-2 cells exposed to live F. prausnitzii than UV-killed F. prausnitzii, This, consistent with previous reports, implies that live F. prausnitzii produces an anti-inflammatory compound in the culture supernatant, demonstrating the value of a physiologically relevant co-culture system that allows obligate anaerobic bacteria to remain viable.

Understanding how commensal obligate anaerobic bacteria regulate immune functions in the large intestine.

The human gastrointestinal tract is colonised by trillions of commensal bacteria, most of which are obligate anaerobes residing in the large intestine. Appropriate bacterial colonisation is generally known to be critical for human health. In particular, the development and function of the immune system depends on microbial colonisation, and a regulated cross-talk between commensal bacteria, intestinal epithelial cells and immune cells is required to maintain mucosal immune homeostasis. This homeostasis is disturbed in various inflammatory disorders, such as inflammatory bowel diseases. Several in vitro and in vivo studies indicate a role for Faecalibacterium prausnitzii, Bacteroides thetaiotaomicron, Bacteroides fragilis, Akkermansia muciniphila and segmented filamentous bacteria in maintaining intestinal immune homeostasis. These obligate anaerobes are abundant in the healthy intestine but reduced in several inflammatory diseases, suggesting an association with protective effects on human health. However, knowledge of the mechanisms underlying the effects of obligate anaerobic intestinal bacteria remains limited, in part due to the difficulty of co-culturing obligate anaerobes together with oxygen-requiring human epithelial cells. By using novel dual-environment co-culture models, it will be possible to investigate the effects of the unstudied majority of intestinal microorganisms on the human epithelia. This knowledge will provide opportunities for improving human health and reducing the risk of inflammatory diseases.

Human oral isolate Lactobacillus fermentum AGR1487 reduces intestinal barrier integrity by increasing the turnover of microtubules in Caco-2 cells.

Lactobacillus fermentum is found in fermented foods and thought to be harmless. In vivo and clinical studies indicate that some L. fermentum strains have beneficial properties, particularly for gastrointestinal health. However, L. fermentum AGR1487 decreases trans-epithelial electrical resistance (TEER), a measure of intestinal barrier integrity. The hypothesis was that L. fermentum AGR1487 decreases the expression of intestinal cell tight junction genes and proteins, thereby reducing barrier integrity. Transcriptomic and proteomic analyses of Caco-2 cells (model of human intestinal epithelial cells) treated with L. fermentum AGR1487 were used to obtain a global view of the effect of the bacterium on intestinal epithelial cells. Specific functional characteristics by which L. fermentum AGR1487 reduces intestinal barrier integrity were examined using confocal microscopy, cell cycle progression and adherence bioassays. The effects of TEER-enhancing L. fermentum AGR1485 were investigated for comparison. L. fermentum AGR1487 did not alter the expression of Caco-2 cell tight junction genes (compared to L. fermentum AGR1485) and tight junction proteins were not able to be detected. However, L. fermentum AGR1487 increased the expression levels of seven tubulin genes and the abundance of three microtubule-associated proteins, which have been linked to tight junction disassembly. Additionally, Caco-2 cells treated with L. fermentum AGR1487 did not have defined and uniform borders of zona occludens 2 around each cell, unlike control or AGR1485 treated cells. L. fermentum AGR1487 cells were required for the negative effect on barrier integrity (bacterial supernatant did not cause a decrease in TEER), suggesting that a physical interaction may be necessary. Increased adherence of L. fermentum AGR1487 to Caco-2 cells (compared to L. fermentum AGR1485) was likely to facilitate this cell-to-cell interaction. These findings illustrate that bacterial strains of the same species can cause contrasting host responses and suggest that food-safe status should be given to individual strains not species.