Category: Diet

Prebiotics for improved gut barrier function

Prebiotics for improved gut barrier function

Consequently, LX, an oral barriier of tryptophan hydroxylase, the key enzyme for Menstrual health apps serotonin synthesis has Prfbiotics successful for treatment of patients with non-constipating IBS [ ]. Bifidobacterium longum B. Eisenhoffer GT, Loftus PD, Yoshigi M, Otsuna H, Chien CB, Morcos PA, Rosenblatt J: Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia.

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Prebiotics for improved gut barrier function -

Error bars represent SEM. Comparisons of the survival curves were tested by logrank test after 25 a , b and 30 a weeks of age, as indicated by vertical dashed lines. This separation was mainly driven by Parabacteroides spp. However, a faecal microbiota transplant from XOS- and control-fed mice to pups of antibiotic-treated NOD mice housed in germ-free isolators was not sufficient to transfer a delay in diabetes onset Fig.

Effects on the gut microbiota. a—d PCoA plots based on unweighted and weighted UniFrac distance matrices depicting data based on 16S rRNA gene tag-encoded amplicon sequencing.

PERMANOVA was used to evaluate group differences. more than one taxon could be assigned to this cluster at the given taxonomic level. Error bars represent SD. j Total number of 16S copies quantified by qPCR of the 16S rRNA gene V3 region in untreated and antibiotic-treated 4-week-old NOD mice.

The y -axis is on a base 10 logarithmic scale. In addition to the effect XOS had on pancreatic and salivary gland inflammation when fed throughout life, a tendency was also observed for reduced insulitis scores in week-old mice fed an XOS diet only during pregnancy and lactation compared with mice on a continuous control diet Fig.

To test whether microbes were necessary for the observed effects, additional groups of pregnant dams on either the control or XOS diet were given a full antibiotic cocktail in the water to deplete the majority of the gut microbiota until all pups were weaned to a control diet.

Antibiotic-treated mice on a control diet developed insulitis fully to the same degree as untreated mice Fig. Interestingly, the slight reduction in insulitis in mice fed an XOS-supplemented diet only until weaning was even greater in mice that were also given antibiotics Fig.

Male NOD mice developed less insulitis than the female mice on antibiotics, but even less when fed an XOS-supplemented diet Fig. Microbes did not seem necessary for XOS to reduce insulitis, the effect of which, unexpectedly, was more pronounced when microbes were absent.

When the degree of sialadenitis was evaluated in the same groups of mice, we found that an XOS diet early in life was sufficient to reduce the sialadenitis score later in life Fig.

Both the control- and the XOS-fed mice treated with antibiotics presented with the same low degree of sialadenitis as the untreated XOS-fed mice Fig. Hence, no synergistic protective effect was observed for the XOS-fed antibiotic-treated mice as was evident for the development of insulitis.

The antibiotic cocktail had no effect on the weight of the mice or the amount of water they drank before weaning ESM Fig. The remaining microbiome of the antibiotic-treated mice was analysed to investigate whether resistant species might remain in the gut and act synergistically with the XOS diet to protect the pancreas.

Targeting the 16S rRNA gene V3 region with qPCR demonstrated that the overall number of 16S copies was reduced more than fold by the antibiotic cocktail Fig. Amplicon sequencing indicated that the few remaining bacteria in dietary groups consisted primarily of Streptophyta spp.

Streptophyta spp. have not previously been associated with any immune or disease function. It was furthermore shown that solely one genus at very low abundance, Flavobacterium spp. Having a different diet, one based on more long-chained carbohydrates instead of monomer sugar molecules, would naturally be expected to manifest as differences in the host metabolism.

Prebiotics have been shown to improve glucose homeostasis and insulin sensitivity by SCFA-mediated activation of intestinal gluconeogenesis [ 21 ]. We found no differences in weight gain or food intake based on the nitrogen-free extract NFE of the diets ESM Fig.

There was no effect of XOS on n -butyric acid or propionic acid ESM Fig. From these findings, we concluded that the effects of XOS on the host were not mediated through any major shifts in metabolism. A gluten-free diet has been shown to have favourable effects in NOD mice [ 22 , 23 ], but the effect of gluten seems, in contrast to the XOS diet, to be mediated by direct regulation of beta cell activity [ 24 , 25 ].

We therefore suspected that a gluten-free diet in combination with XOS could provide further protection. A FITC-dextran assay was used to evaluate the gut integrity as prebiotics have previously been shown to alter gut morphology and function [ 26 ].

The barrier was indeed less permeable in mice fed the XOS-supplemented diet compared with mice fed the control diet Fig. This was confirmed with qPCR of the 16S rRNA gene in mesenteric lymph node, which showed that fewer microbes were able to cross the gut barrier and reach the draining lymph node in XOS-fed mice compared with control-fed mice Fig.

Gene expression of mucus-related genes was also upregulated in mice on the XOS diet in both the ileum Muc2 and the colon Muc1 , with the strongest effect in mice that were not switched to a chow diet at weaning Fig.

In contrast, other barrier-related genes such as Ocln and Tjp1 encoding occludin and tight junction protein 1, respectively were significantly upregulated in both the ileum and colon of mice fed XOS early in life only Fig. Supplementation with XOS in early life seemed to regulate those parts of the barrier function in a microbiota-independent manner, as the difference persisted in antibiotic-treated mice Fig.

This was, however, only the case for the large intestine, as the FITC-dextran assay, representative of ileum barrier function Fig. The intestinal tight junction modulator zonulin is upregulated in serum of individuals with type 1 diabetes before the onset of disease [ 27 ], but the effect of XOS on intestinal permeability did not seem to be mediated by zonulin as the serum level of the protein was similar between the two dietary groups Fig.

Microbiota regulated dietary improvement of the gut barrier function. e — l Relative gene expression of Muc1 e , g , Muc2 f , h , Ocln i , k and Tjp1 j , l in colon and ileum biopsies sampled at 13 weeks of age from female NOD mice fed the standard chow diet C , the XOS-supplemented diet for their whole life X or only until weaning XC , and antibiotic-treated mice fed either chow CA or the XOS- supplemented diet until weaning XCA.

RQ, relative quantification. The gut barrier is important for homeostasis of the immune system both within and beyond the mucosal immune populations. Potential anti-inflammatory effects of the XOS diet were therefore analysed by flow cytometry both locally and systemically.

This was confirmed by qPCR of Cd8a , Gzmb encoding granzyme B and Fasl encoding Fas ligand , which showed expression of these genes was similarly downregulated in XOS-fed mice, but not in those switched to the control diet at weaning Fig. The Tgfβ gene was expressed at a higher level in mice fed the XOS diet early in life Fig.

In addition, F4. In fact, this population was decreased in the spleen Fig. It is likely that the increased abundance of macrophages was due to an increase in anti-inflammatory M2 macrophages, which are also major producers of TGF-β.

This was also suggested by gene expression of the M2 macrophage marker Arg1 encoding arginase 1 , which was expressed at higher levels in XOS-fed mice Fig. XOS reduced intestinal inflammation. c — e Relative gene expression in colon of c Cd8a , d Gzmb and e Fasl. i , j Relative gene expression of the M2 macrophage markers i Arg1 and j Tgfβ.

The transfer induced diabetes in both groups and, surprisingly, the effect was greater in the XOS group Fig. Perhaps the lower proportion of regulatory T cells evident in the spleen of XOS-fed mice Fig.

In addition, we transferred splenocytes from XOS- and control-fed NOD mice treated with antibiotics early in life, as the most prominent effect of XOS on insulitis was evident in these groups Fig.

Nonetheless, the incidence of diabetes was similar between the two groups Fig. Hence, the systemic effect of XOS on macrophages seems not to, in either antibiotic-treated Fig. The explanation for the effects of XOS on diabetes development may lie more in the local environment surrounding the pancreas.

Adoptive transfer of splenocytes failed to delay diabetes onset. Onset of diabetes was monitored by weekly blood glucose measurements for 9—11 weeks post transfer. c — f Flow cytometric analysis of the splenocytes transferred from the mice treated with antibiotics until weaning demonstrated the percentage of c F4.

Comparisons of the survival curves were tested by logrank test. Here, we demonstrate that the prebiotic compound XOS can change the gut microbiota and delay the onset of type 1 diabetes in NOD mice and reduce autoimmune cellular reactions against pancreatic beta cells and salivary glands.

This is in line with previous results demonstrating that a variety of intestinal bacteria can regulate the degree of both insulitis and sialadenitis in NOD mice [ 16 , 28 , 29 ].

The high degree of insulitis in antibiotic-treated mice resembles that observed in germ-free NOD mice [ 29 ]. However, the unexpected reduction in insulitis in antibiotic-treated mice fed XOS early in life suggested that the alleviating effect is not necessarily a product of microbiota-induced anti-diabetogenic signals.

Increasing evidence is establishing direct pathways by which dietary oligosaccharides can act on the immune system: for example, the gut barrier is permeable to milk oligosaccharides during infancy [ 31 ], and it can bind specific sugar receptors on human cells [ 32 , 33 ] and thereby modulate the immune response systemically.

Of course, the shift in microbial composition towards species with known probiotic potential cannot be excluded from also playing a protective role. There was, for example, a clear increase in Parabacteroide s spp. which have been associated with anti-inflammatory and diabetes-reducing properties [ 34 , 35 ].

Nonetheless, faecal microbiota transplantation from XOS-fed mice was not sufficient to transfer a reduction in time to diabetes onset.

It is worth considering that in such a scenario, without XOS as an available nutrient source, the microbiota may function differently and, therefore, a microbiota-mediated anti-inflammatory effect of an XOS-supplemented diet should therefore not be ruled out.

In contrast to the effect on insulitis, the development of sialadenitis was clearly regulated by gut bacteria, which is comparable with our previous study in germ-free mice [ 16 ]. These findings warrant further investigation into the regulatory role of Parabacteroides spp. in the development of sialadenitis rather than insulitis.

Functional studies in BB-DP rats demonstrate that increased permeability of the small intestine, but not the colon, appeared before the development of insulitis [ 37 ].

A later study verified that BB-DP rats had a higher urinary excretion ratio of lactulose:mannitol than Wistar rats before the onset of diabetes [ 38 ].

However, no difference was observed in survival or islet inflammation for BB-DP rats when butyrate was administered to strengthen intestinal tight junctions, bringing into question the importance of a leaky gut, at least in the small intestine.

If and how the mucosal inflammation contributes to inducing an autoimmune cascade in the nearby pancreas remain unknown, but the antibiotic-treated mice shed some light on the importance of a leaky gut.

The small intestinal barrier in these mice was no longer improved by the XOS diet, in contrast to markers of colonic barrier function. This is in accordance with a study from the 90s, demonstrating that dietary fibre increased crypt and mucin-containing goblet cell numbers in germ-free rats [ 26 , 39 ] and that colonisation of the gut actually abolished this effect.

The discrepancy between the different sections of gut is interesting as the XOS diet was also able to reduce islet inflammation in microbiota-depleted mice, suggesting that the colonic barrier function rather than the small intestinal barrier was involved in the alleviating effect.

In support of this, a study demonstrated that glucagon-like peptide-2 reduced small intestinal transepithelial resistance in NOD mice but failed to delay the onset of type 1 diabetes [ 40 ].

The latter study also showed that the small intestinal permeability was increased at 12 weeks of age, long after islet inflammation began, but not before 12 weeks.

In conclusion, the inconsistency between studies investigating the ability of the gut barrier to regulate the development of diabetes appears to highlight the need to distinguish segments of the gastrointestinal tract based on functional differences.

It is important to note that the small intestinal barrier function may play a more profound role in the systemic immune response and autoimmune reactions in the salivary glands, as the XOS diet not only failed to alleviate small intestinal permeability in antibiotic-treated mice, but also had no effect on systemic immunity and sialadenitis in the absence of a fully colonised gut.

Functional studies combining methods to alleviate and aggravate gut permeability with environmental factors associated with a modified barrier function, such as the XOS diet, in various sections of the gastrointestinal tract are necessary to address such hypotheses. Nonetheless, considering the previous findings on increased intestinal permeability in individuals with type 1 diabetes [ 42 , 43 ], eating a prebiotic supplement seems a feasible approach that, because of the effects on mucus production and tight junction proteins, may be able to limit the impact of environmental triggers that might otherwise translocate the compromised gut barrier.

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The results of our previous studies indicated that dietary supplementation with Lactobacillus plantarum ZLP isolated from healthy piglet intestinal tract Wang et al. However, its impact on intestinal barrier function and microbiota, and the interaction between barrier function and microbiota after L.

plantarum ZLP treatment remained to be investigated. In this study, the impact of L. plantarum ZLP on intestinal epithelial function was evaluated by measuring gut permeability and the expression of TJ proteins, inflammatory cytokines, and host defense peptides HDP.

Further, we evaluated the ability of this strain to regulate microbiota composition and community structure. The regulatory effects of the microbiota modulated by the probiotic strain on intestinal epithelium function were also analyzed. plantarum ZLP was isolated in our laboratory from the intestine of a healthy piglet.

It was identified by the China Center of Industrial Culture Collection Beijing, China and preserved in the China General Microbiological Culture Collection Center CGMCC No. It was grown in improved De Man, Rogosa, and Sharpe liquid medium 10 g peptone, 5 g yeast powder, 20 g glucose, 10 g beef extract, 5 g sodium acetate, 2 g ammonium citrate dibasic, 2 g dipotassium phosphate, 0.

Enteropathic E. It was grown in Luria-Bertani medium Oxoid, Basingstoke, United Kingdom at 37°C. The porcine intestinal epithelial cell line IPEC-J2 used in this study was purchased from JENNIO Biological Technology Guangzhou, China.

It was originally derived from the jejuna of neonatal piglets. Cells were separated at each passage with 0. Changes in paracellular permeability after L. plantarum ZLP treatment were determined with fluorescein isothiocyanate-dextrans FD-4; average molecular mass, 4.

Louis, MO, United States according to the method reported by Wang et al. IPEC-2 cells were seeded into 6-well Transwell insert chambers 0. The cells were pretreated or not with L. After incubation, medium μL was sampled from the basolateral chambers, and the FD-4 concentration was quantified using a fluorescence microplate reader FLx; BioTek, Winooski, VT, United States.

Calibration curves were plotted with an FD-4 gradient series. All experiments were carried out in triplicate. IPEC-J2 cells were seeded into 6-well plates Corning, Inc. Then, the cells were washed three times with PBS and the supernatant was removed. After incubation, the cells were rinsed with PBS three times and collected for subsequent assays.

Culture medium supernatants were collected simultaneously. The mRNA expression levels of TJ proteins, cytokines, and HDPs were determined by quantitative real-time PCR RT-qPCR. IPEC-J2 cells collected after incubation were lysed with RNAzol MRC, Cincinnati, OH, United States.

RNA concentrations were determined with a NanoDrop spectrophotometer Thermo Fisher Scientific, Waltham, MA, United States and purity was verified by AA and AA absorbance ratios.

The RNA was reverse transcribed with an iScript cDNA Synthesis Kit Bio-Rad Laboratories Ltd. qPCR was performed using iTaq Universal SYBR Green Supermix Bio-Rad Laboratories Ltd.

Porcine-specific primers are listed in Supplementary Table S1. The expression of each gene was normalized to that of glyceraldehydephosphate dehydrogenase GAPDH to yield a relative transcript level.

PCR conditions were 95°C for 10 min followed by 40 amplification cycles 95°C for 30 s, 60°C for 30 s, and 72°C for 20 s. The IPEC-J2 cells were collected into precooled lysis buffer and kept on ice for 30 min. The lysed samples were centrifuged at 4°C and 12, × g for 5 min to collect the supernatants.

Protein concentrations were determined with a Bicinchoninic Acid Protein Assay Kit Thermo Fisher Scientific, Waltham, MA, United States. They were then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at 20—25°C.

The antibodies used are listed in Supplementary Table S2. Immunoreactive proteins were detected on a ChemiDoc XRS imaging system Bio-Rad Laboratories Ltd. Band densities were analyzed with ImageJ National Institutes of Health, Bethesda, MD, United States.

Results were calculated and recorded as the protein abundance relative to β-actin. Proinflammatory cytokines and porcine β-defensin 2 were measured by an enzyme linked immunosorbent assay ELISA. Cell culture medium supernatant μL was centrifuged at 4, × g for 10 min and then passed through a 0.

The experimental protocol was reviewed and approved by the Ethics Committee of the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, Beijing, PRC.

Humane animal care was practiced throughout the trial. Ten post-weaning piglets siblings; Large White × Landrace; 8. plantarum ZLP treatment or placebo control groups. Each group consisted of two males and three females.

Animals were raised at the Beijing Xiqingminfeng Farm Beijing, China in a separate room decontaminated prior to the study and were housed at 25—28°C. Each piglet was kept in an individual 1.

Each pen contained a feeder and a water nipple. Free access to feed and water was provided throughout the day trial. Piglets received a complete feed specially formulated according to the NRC and the Feeding Standard of Swine Detailed information about the diet is shown in Supplementary Table S3.

The treatment group was administered the basal diet supplemented with freeze-dried L. plantarum ZLP 5. Fresh fecal samples were individually collected from piglet recta at the end of the feeding experiment.

The samples were immediately transferred to the laboratory and processed for genomic DNA extraction with an E. Shanghai, China. Raw sequences were denoised using Trimmomatic and FLASH software and filtered according to their barcodes and primer sequences with QIIME v.

Chimeras were identified and excluded using the UCHIME algorithm v. Principle coordinates analysis PCoA was conducted to visualize differences in fecal community composition.

PCoA plots were generated on the basis of Bray—Curtis indices. The linear discriminant analysis effect size LEfSe algorithm was used to identify the taxa responsible for the differences between the treatment and control groups.

The biomarkers used in the present study had an effect-size threshold of two. Fecal SCFA concentrations were determined following modified procedures of Qiu and Jin Half-gram fecal samples were homogenized in 10 mL of double-distilled water.

After centrifugation at 12, × g for 10 min, the supernatants were removed and filtered through 0. Acetic, propionic, and butyric acids were measured with an ion chromatography system Dionex Corp.

SPSS v. The FD-4 concentration and mRNA expression levels were analyzed by one-way ANOVA. The results are expressed as the mean ± standard error of the mean SEM.

FD-4 diffusion is a good indicator of paracellular permeability. Therefore, FD-4 transport was measured in this study to evaluate the protective effect of L. plantarum ZLP on epithelial integrity Figure 1. FD-4 concentrations in the L. plantarum ZLPtreated group were not significantly different from those in the untreated control.

Pretreatment with L. FIGURE 1. FD-4 diffusion in L. plantarum ZLPtreated IPEC-J2 cells. Cells were left untreated or pretreated with L. FD-4 concentrations in the basal compartments were measured.

Values are shown as the means ± SE of three independent experiments. ETEC alone. Abundances of claudin-1 Figure 2A , occludin Figure 2B , and ZO-1 Figure 2C transcripts in IPEC-J2 cells after bacterial treatments were examined by RT-qPCR Figure 2.

plantarum ZLP treatment alone had no significant influence on TJ mRNA expression as compared to the untreated control. FIGURE 2. Relative mRNA transcript and protein levels of TJ proteins in IPEC-J2 cells left untreated or pretreated with L.

mRNA levels of claudin-1 A , occludin B , and ZO-1 C were standardized to that of GAPDH. TJ protein levels were assessed by immunoblotting. Data are western blotting results and gradation analysis of claudin-1 D , occludin E , ZO-1 F. Differences in TJ protein expression after the bacterial treatments were examined by western blotting.

These results were consistent with those for mRNA expression. plantarum ZLP treatment alone did not significantly affect protein expression relative to the untreated control. plantarum ZLP pretreatment negated the reduction in claudin-1 Figure 2D and ZO-1 Figure 2F abundance caused by ETEC treatment.

Proinflammatory cytokines in IPEC-J2 cells were quantified after the treatments Figures 3A—C. Incubation with ETEC alone significantly upregulated IL-6 , IL-8 , and TNFα transcripts. Treatment with L. plantarum ZLP alone had no significant effect on cytokine expression.

However, pretreatment with L. Therefore, L. plantarum ZLP reduced the ETEC-induced upregulation of proinflammatory cytokines. FIGURE 3. Relative mRNA transcript levels and concentrations of proinflammatory cytokines in the culture supernatant of IPEC-J2 cells left untreated or pretreated with L.

mRNA levels of IL-6 A , IL-8 B , and TNF-α C were standardized to that of GAPDH. Protein expression of IL-6 D , IL-8 E , and TNF-α F was assessed by ELISA. ELISA was used to verify the protective effect of L. plantarum ZLP on epithelial immunological function at the protein level after ETEC challenge Figures 3D—F.

The results confirmed that, while ETEC did not significantly induced IL-6, L. plantarum ZLP pretreatment suppressed the increases in IL-6, IL-8, and TNFα secretion in IPEC-J2 cells challenged with ETEC relative to the levels observed in cells incubated with ETEC alone.

The modulatory effect of L. plantarum ZLP on the innate immune response was evaluated by measuring porcine HDP mRNA expression Figures 4A,B. Cathelicidins and β-defensins are the two main mammalian HDP families.

We selected pBD2 a β-defensin, Figure 4A and PG a cathelicidin, Figure 4B as target genes in this study. The results showed that treatment with L.

ETEC exposure had no significant effect on pBD2 expression, but significantly induced PG expression. Challenge with ETEC 3 h after L. plantarum ZLP pretreatment had no significant effect on the HDP expression levels observed after incubation with L. plantarum ZLP alone.

FIGURE 4. Relative mRNA transcript levels of pBD2 A and PG1 - 5 B and concentrations of pBD2 C in the culture supernatant of IPEC-J2 cells left untreated or pretreated with L. mRNA expression levels were standardized to that of GAPDH. The pBD2 concentration was assessed by ELISA.

Effects of bacterial treatment on pBD2 secretion were evaluated by ELISA Figure 4C. The result was consistent with pBD2 mRNA expression. Exposure to L. plantarum ZLP significantly induced pBD2 secretion in IPEC-J2 cells.

In contrast, ETEC treatment had no significant effect on pBD2 secretion. plantarum ZLP pretreatment had no significant influence on pBD2 secretion by L. plantarum ZLP treatment alone. Sequencing of the amplified 16S rRNA genes produced , reads after quality checks.

An average of 37, ± 4, reads were obtained for each sample. The average read length for each sample was bp. We obtained — OTUs per sample. Based on rarefaction analysis, the sequencing depth adequately reflected species richness, suggested that the Illumina MiSeq sequencing system detected most of the fecal bacterial diversity in our study.

We used the Chao1, ACE, Shannon, and Simpson indices to estimate fecal microbiome taxon abundance and diversity Table 1. plantarum ZLPtreated groups exhibited higher diversity than the control group according to the Shannon and Simpson indices.

plantarum ZLP treatment had no significant effect on fecal microbiota abundance according to the Chao1 and ACE indices. Thus, the dominant bacterial phylotypes present in the feces were captured by this analysis. TABLE 1. Effects of L. plantarum ZLP treatment on average richness and diversity of bacterial community in piglet feces.

Taxon-dependent analysis was used to compare microbiota compositions of the feces from piglets treated with L. plantarum ZLP and those receiving the placebo Figure 5. Firmicutes was the dominant phylum and constituted Bacteroidetes accounted for Other phyla were present at lower frequencies.

Figure 5B shows a hierarchically clustered heatmap of the fecal microbiota composition at the genus level. Prevotella was the most abundant; it accounted for an average of Clostridium sensu stricto 1 Lactobacillus plantarum ZLPtreated piglets.

LEfSe analysis indicated no significant differences between the placebo- and L. plantarum -treated piglets at the phylum level in terms of relative OTU abundance. Significant differences were observed between groups at several other taxa Figure 6.

The probiotic-treated group was enriched in Bacilli at the class level, Lactobacillales at the order level, Lactobacillaceae and Ruminococcaceae at the family level, and Alloprevotella , Anaerotruncus , Faecalibacterium , Lactobacillus , Subdoligranulum , unclassified Lachnospiraceae , and no-rank Ruminococcaceae at the genus level.

Fecal microbiota composition PCoA revealed that L. plantarum ZLP treatment significantly affected overall fecal microbiota composition. The microbiota communities in the piglets treated with L. plantarum ZLP were clustered together and were distinctly separated from those of the control pigs Supplementary Figure S1.

FIGURE 5. Phylum-level microbiota profile of L. plantarum ZLPtreated piglets as compared to that of placebo-treated control piglets A.

Stacked column chart showing the relative phylum-level bacterial abundance per fecal sample. Genus-level microbiota profile of L. plantarum ZLPtreated piglets as compared to that of placebo-treated control piglets B.

Relative abundance is indicated by a color gradient from green to red, with green representing low abundance and red representing high abundance.

C and P represent the control and the probiotic-treated group, respectively. Numbers represent individual animals.

FIGURE 6. Differences between bacterial taxa in L. plantarum ZLPtreated piglets and placebo-treated control piglets. The histogram shows differentially abundant bacteria in the treatment and control groups ranked by linear discriminant analysis LDA scores.

Red bars negative LDA scores represent bacteria that are more abundant in probiotic-treated fecal samples than in controls. Green bars positive LDA scores represent bacteria that are more abundant in placebo-treated fecal samples than in probiotic-treated fecal samples.

Table 2 shows the SCFA concentrations in piglet feces. Acetic and propionic acid concentrations did not significantly differ between the placebo and L. TABLE 2. Correlations between SCFA concentration and fecal bacterial abundance relative abundance of the top 30 genera are shown in Figure 7.

FIGURE 7. Correlations between relative generic abundance and SCFA concentrations in feces obtained from post-weaning piglets. Only the relative abundances of the top 30 bacterial genera are shown.

AA, acetic acid; PA, propionic acid; BA, butyric acid. Using porcine IPEC-2 cells as a model, we demonstrated that L. plantarum ZLP plays multiple protective roles in epithelial barrier regulation. The present study showed that ETEC treatment significantly increased gut permeability to FD-4, whereas treatment with probiotic L.

plantarum ZLP alone had no significant effect on gut permeability. These findings corroborate those of a previous study, in which probiotic L. reuteri did not significantly change FD-4 fluorescence intensity in IPEC-J1 cells Wang et al.

On the other hand, L. plantarum ZLP pretreatment significantly suppressed the increase in gut permeability caused by ETEC infection, suggesting that L. plantarum ZLP can alleviate epithelial damage caused by ETEC. This result was consistent with that of a previous study in which permeability to FD-4 indicated that L.

reuteri treatment maintains the barrier integrity of IPEC-J1 cells exposed to ETEC Wang et al. TJ proteins play crucial roles in maintaining barrier integrity and function. They include transmembrane proteins such as claudins and occludins, and cytoplasmic scaffolding proteins, such as the ZO family, which have linking and sealing effects Suzuki, In this study, relative TJ transcript and protein abundances were significantly reduced after ETEC infection.

However, these reductions were abrogated by pretreatment with L. plantarum ZLP Previous studies using various probiotic strains reported similar results in vivo and in vitro Yang et al. Therefore, probiotic L. plantarum ZLP may fortify intestinal epithelial resistance to pathogens by maintaining TJ protein abundance.

Cytokines play significant regulatory roles in the intestinal inflammatory response. Several studies have demonstrated the effects of probiotics on cytokine expression. Nevertheless, this regulatory action varies with strain. reuteri ACTT shows immunosuppressive action by inhibiting TNFα overexpression in LPS-activated human monocytoid THP1 cells Lin et al.

However, L. reuteri ACTT significantly stimulates TNF-α production as an immunostimulatory action Jones and Versalovic, In the present study, L. plantarum ZLP per se did not influence the expression of proinflammatory cytokines, but inhibited their ETEC-induced overexpression, thus exerting an immunosuppressive action.

Certain pro-inflammatory cytokines reportedly are associated with pathogen-induced TJ protein changes Otte and Podolsky, ETEC K88 substantially increases IL-8 and disrupts the membrane barrier; however, this disruption can be alleviated by L.

plantarum pretreatment Wu et al. We obtained similar results in the present study. ETEC-induced increases in IL, IL, and TNF-α expression were effectively counteracted by L. plantarum ZLP pretreatment. This observation was consistent with the FD-4 assay results and TJ protein expression levels.

Similar findings indicated that L. reuteri inhibits TNF-α expression and may protect TJ proteins Yang et al. High-throughput sequencing analysis of post-weaning piglet feces after L. plantarum ZLP or control treatment revealed relatively low abundances of certain bacterial genera in the probiotic-treated group.

Some of these are associated with various pathological conditions, e. Wang et al. In the present study, Clostridium sensu stricto 1 was significantly less abundant in the probiotic-treated than in the control group.

Certain Clostridium spp. are harmful to host health. Epithelial inflammation observed in weaned piglets may be correlated with Clostridium sensu stricto 1 enrichment in their intestinal mucosa Wang et al. The protective effect of probiotics in terms of epithelial immunity may be partially explained by microbiota modulation.

One limitation of the present study was that we did not evaluate proinflammatory cytokines in the piglet intestinal tissue and thus, we could not analyze the correlation with microbiota abundance.

Such relationships merit further investigation. The secretion of HDPs, which exert both antimicrobial and immunomodulatory activities, is an epithelial innate immunity mechanism Bevins et al.

Enhancing endogenous HDP synthesis improves the early response to bacterial infection and inflammation Veldhuizen et al. Nutrients, such as VD 3 , butyrate, and zinc induce HDP secretion Talukder et al.

Probiotics can also stimulate HDP expression Schlee et al. In the present study, increased pBD2 and PG expression and pBD2 secretion were observed after L. plantarum ZLP treatment, suggesting that this strain can induce HDPs, to protect against bacterial infection.

Similar results have been reported for the probiotic strain L. reuteri I Liu et al. The administration of synthetic HDPs reportedly can improve weaned piglet growth performance, nutrient digestion and assimilation, intestinal health, and antioxidant capacity Xiao et al. Therefore, the induction of HDP expression by L.

plantarum ZLP may be correlated with the improved growth and reduced risk of diarrhea reported in our previous studies.

SCFAs, especially butyrate, induce HDPs Zeng et al. Butyrate production by enteric microbiota is the only microbial stimulus capable of inducing HDP expression Schauber et al. Most butyrate-producing bacteria belong to Clostridium clusters IV and XIVa.

Butyrate metabolism has been observed in species of Faecalibacterium and Anaerotruncus , e. colihominis has been shown to specifically colonize the lumen whereas F. prausnitzii is enriched in the mucus Van den Abbeele et al. In the present study, Faecalibacterium spp. and Anaerotruncus spp.

were significantly abundant in L. Therefore, increasing the abundances of these genera may elevate butyrate levels and epithelial HDP expression. The present study confirmed a positive correlation between fecal butyric acid and Anaerotruncus spp.

abundance after L. plantarum ZLP treatment. In addition, Faecalibacterium spp. abundance showed a positive correlation with acetic acid concentration. Acetic acid can be converted to butyrate by Eubacterium rectale Duncan and Flint, , which may enhance HDP expression. The modulation of butyrate producers with probiotics to generate butyrate to stimulate HDP levels in the epithelium may thus be a meaningful approach for future interventions that aim to improve intestinal balance.

Few studies have focused on the association between HDP production and probiotic function. Future studies involving probiotical and the non-pathogenic enteric bacterial regulation of antimicrobial peptides may elucidate the beneficial effects of probiotics against pathogen infection.

The piglet intestinal microbiota undergoes substantial dynamic changes after weaning, and this alteration can be associated with severe disorders and bowel disease.

In the present study, fecal bacterial communities were dominated by Firmicutes and Bacteroidetes, regardless of treatment. This result was expected because the colon is a strictly anaerobic environment and most of the species within these phyla are anaerobic.

Similar results were reported in previous pig studies Konstantinov et al. Prevotella , which is associated with hemicelluloses degradation, reportedly is the predominant genus in piglets at nursery stage Konstantinov et al. A high Prevotella spp. abundance may be essential for post-weaning piglets to be able to digest plant-based diets.

Post-weaning increases in the proportions of Lactobacillus spp. are desirable and beneficial. Previous studies have shown that oral administration of lactic acid bacteria enhances the relative abundance of intestinal Lactobacillus spp.

in weaned piglets Hu et al. In the present study, dietary L. plantarum ZLP supplementation significantly increased Lactobacillus spp. abundance in the post-weaning piglet intestine. plantarum ZLP may produce molecules that stimulate Lactobacillus spp.

growth in the piglet intestine Ohashi et al. Alternatively, the observed increase in Lactobacillus abundance may have resulted from the proliferation of the administered probiotic strain Takahashi et al.

Healthy snack options Gastroenterology volume 14Article Prebiotic Cite this article. Metrics details. Data are accumulating that emphasize the important role of barrifr intestinal barrier and intestinal permeability Prebiotixs health and disease. Prebiotics for improved gut barrier function, these terms are poorly defined, their assessment is a matter of debate, and their clinical significance is not clearly established. In the present review, current knowledge on mucosal barrier and its role in disease prevention and therapy is summarized. Secondly, the key element of the intestinal barrier affecting permeability are described. This barrier represents a huge mucosal surface, where billions of bacteria face the largest immune system of our body. Prebiotics for improved gut barrier function The intestine is the first Prebiotics for improved gut barrier function functioh pathogens. Its correct functionality will Fjnction decisive gur prevent the infection of pathogenic bacteria, keeping git animals healthy Nutritional needs athletes robust more functiln in: " Elements involved Sport-specific mental training gut health ". Baarrier probiotic microorganisms have been described to have the ability to interact vut the intestinal epithelium, improve improvef barrier function by Prebiootics Fat loss before and after transformations expression of proteins that form improveed intraepithelial junctions Putaala et al. On the other barroer, it is also interesting to note the role of the amino acids threonine a major component of mucinglutamine fuel for intestinal cells, it promotes the repair of intestinal morphology and arginine involved in important metabolic cycles. All of them play an essential role in the maintenance of intestinal integrity and have been attributed the ability to improve intestinal morphology and exercise protection against pathogens Pérez and Nofrarias, ; Ewaschuk et al. The immune response will sometimes have to be stimulated for example in weaning or infections and in other cases, it will have to be restricted for example against certain allergenic agents. This modulation is very important to obtain a good gut health, prevent the animals from getting sick, and it is also directly related to other vital functions such as the promotion of a favorable microbiota, absorption of water or nutrients, energy metabolism and eventually productive efficiency.

Author: Arashikinos

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