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Probiotic Bifidobacterium species stimulate human SLC26A3 gene function and expression in intestinal epithelial cells.

ABSTRACT: SLC26A3, or downregulated in adenoma (DRA), plays a major role in mediating Cl(-) absorption in the mammalian intestine. Disturbances in DRA function and expression have been implicated in intestinal disorders such as congenital Cl(-) diarrhea and gut inflammation. We previously showed that an increase in DRA function and expression by Lactobacillus acidophilus and its culture supernatant (CS) might underlie antidiarrheal effects of this probiotic strain. However, the effects of Bifidobacterium species, important inhabitants of the human colon, on intestinal Cl(-)/HCO3 (-) exchange activity are not known. Our current results demonstrate that CS derived from Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum increased anion exchange activity in Caco-2 cells (?1.8- to 2.4-fold). Consistent with the increase in DRA function, CS also increased the protein, as well as the mRNA, level of DRA (but not putative anion transporter 1). CS of all three Bifidobacterium sp. increased DRA promoter activity (-1,183/+114 bp) in Caco-2 cells (1.5- to 1.8-fold). Furthermore, the increase in DRA mRNA expression by CS of B. breve and B. infantis was blocked in the presence of the transcription inhibitor actinomycin D (5 ?M) and the ERK1/2 MAPK pathway inhibitor U0126 (10 ?M). Administration of live B. breve, B. infantis, and B. bifidum by oral gavage to mice for 24 h increased DRA mRNA and protein levels in the colon. These data demonstrate an upregulation of DRA via activation of the ERK1/2 pathway that may underlie potential antidiarrheal effects of Bifidobacterium sp.

SUBMITTER: Kumar A

PROVIDER: S-EPMC4269665 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Probiotic Bifidobacterium species stimulate human SLC26A3 gene function and expression in intestinal epithelial cells.

Kumar Anoop A Hecht Cameron C Priyamvada Shubha S Anbazhagan Arivarasu N AN Alakkam Anas A Borthakur Alip A Alrefai Waddah A WA Gill Ravinder K RK Dudeja Pradeep K PK

American journal of physiology. Cell physiology 20140820 12

SLC26A3, or downregulated in adenoma (DRA), plays a major role in mediating Cl(-) absorption in the mammalian intestine. Disturbances in DRA function and expression have been implicated in intestinal disorders such as congenital Cl(-) diarrhea and gut inflammation. We previously showed that an increase in DRA function and expression by Lactobacillus acidophilus and its culture supernatant (CS) might underlie antidiarrheal effects of this probiotic strain. However, the effects of Bifidobacterium ...[more]

PMID: 25143346

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Project description:BACKGROUND & AIMS:Diarrhea associated with inflammatory bowel diseases has been associated with increased levels of inflammatory cytokines, including tumor necrosis factor (TNF). The intestinal mucosa of patients with inflammatory bowel diseases has reduced expression of solute carrier family 26 member 3 (SLC26A3, also called DRA). We investigated whether TNF directly affects expression of DRA in human intestinal epithelial cells (IECs) and in the intestines of mice, and studied the mechanisms of these effects. METHODS:We performed quantitative reverse transcription polymerase chain reaction, immunofluorescence, and immunoblot analyses in Caco-2, HT-29, and T-84 cells human IECs cultured in 2 or 3 dimensions with or without TNF (50 ng/mL for 6-24 hours). We purified nuclear extracts and quantified nuclear factor-?B (NF-?B) activation and DNA binding. We isolated intestinal crypts from C57BL/6 mice, cultured enteroids, incubated these with TNF (50 ng/mL, 24 hours), and quantified messenger RNAs. DRA-mediated exchange of Cl- for HCO3- was measured by uptake of 125I. Expression of the NF-?B inhibitor ? (IkBa) was knocked down in Caco-2 cells with small interfering RNAs. Activation of NF-?B in response to TNF was measured by luciferase reporter assays; binding of the NF-?B subunit p65 in cells was analyzed in chromatin immunoprecipitation assays. DRA promoter activity was measured in a luciferase reporter assay. C57BL/6 mice were injected with TNF (5 ?g/mouse for 3-6 hours) or vehicle (control); intestines were collected and analyzed by immunofluorescence, or RNA and protein were collected from the mucosa. RESULTS:Incubation of IECs with TNF reduced expression of DRA. Knockdown of NF-?B inhibitor ? in IECs led to nuclear translocation of the NF-?B subunit p65 and reduced levels of DRA messenger RNA and protein. Expression of a transgene encoding p65 or p50 in IECs led to significant reductions in the promoter activity of DRA and its expression. In chromatin immunoprecipitation assays, p65 bound directly to the promoter of DRA, at the regions of -935 to -629 and -375 to -84. Injection of mice with TNF or incubation of crypt-derived enteroids with TNF reduced their expression of DRA messenger RNA and protein. CONCLUSIONS:In human IECs and intestinal tissues from mice, we found TNF to activate NF-?B, which reduced expression of the Cl- / HCO3- exchanger DRA (SLC26A3), via direct binding to the promoter of DRA. This pathway is an important therapeutic target for inflammatory bowel disease-associated diarrhea.

Project description:BackgroundOne way to improve both the ecological performance and functionality of probiotic bacteria is by combining them with a prebiotic in the form of a synbiotic. However, the degree to which such synbiotic formulations improve probiotic strain functionality in humans has not been tested systematically. Our goal was to use a randomized, double-blind, placebo-controlled, parallel-arm clinical trial in obese humans to compare the ecological and physiological impact of the prebiotic galactooligosaccharides (GOS) and the probiotic strains Bifidobacterium adolescentis IVS-1 (autochthonous and selected via in vivo selection) and Bifidobacterium lactis BB-12 (commercial probiotic allochthonous to the human gut) when used on their own or as synbiotic combinations. After 3 weeks of consumption, strain-specific quantitative real-time PCR and 16S rRNA gene sequencing were performed on fecal samples to assess changes in the microbiota. Intestinal permeability was determined by measuring sugar recovery in urine by GC after consumption of a sugar mixture. Serum-based endotoxin exposure was also assessed.ResultsIVS-1 reached significantly higher cell numbers in fecal samples than BB-12 (P < 0.01) and, remarkably, its administration induced an increase in total bifidobacteria that was comparable to that of GOS. Although GOS showed a clear bifidogenic effect on the resident gut microbiota, both probiotic strains showed only a non-significant trend of higher fecal cell numbers when administered with GOS. Post-aspirin sucralose:lactulose ratios were reduced in groups IVS-1 (P = 0.050), IVS-1 + GOS (P = 0.022), and GOS (P = 0.010), while sucralose excretion was reduced with BB-12 (P = 0.002) and GOS (P = 0.020), indicating improvements in colonic permeability but no synergistic effects. No changes in markers of endotoxemia were observed.ConclusionThis study demonstrated that "autochthony" of the probiotic strain has a larger effect on ecological performance than the provision of a prebiotic substrate, likely due to competitive interactions with members of the resident microbiota. Although the synbiotic combinations tested in this study did not demonstrate functional synergism, our findings clearly showed that the pro- and prebiotic components by themselves improved markers of colonic permeability, providing a rational for their use in pathologies with an underlying leakiness of the gut.

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Probiotic Bifidobacterium species stimulate human SLC26A3 gene function and expression in intestinal epithelial cells.

Publications

Probiotic Bifidobacterium species stimulate human SLC26A3 gene function and expression in intestinal epithelial cells.

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