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Escherichia coli Nissle 1917 enhances bioavailability of serotonin in gut tissues through modulation of synthesis and clearance.

Nzakizwanayo J, Dedi C, Standen G, Macfarlane WM, Patel BA, Jones BV - Sci Rep (2015)

Bottom Line: Exposure of tissue to EcN cells, but not MG1655 cells, was found to increase levels of extra-cellular 5-HT.These effects were not observed when tissues were treated with cell-free supernatant from bacterial cultures.Measurement of 5-HT precursors and metabolites indicated EcN also increases intracellular 5-HTP and reduces 5-HIAA.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, BN2 4GJ, United Kingdom.

ABSTRACT
Accumulating evidence shows indigenous gut microbes can interact with the human host through modulation of serotonin (5-HT) signaling. Here we investigate the impact of the probiotic Escherichia coli Nissle 1917 (EcN) on 5-HT signalling in gut tissues. Ex-vivo mouse ileal tissue sections were treated with either EcN or the human gut commensal MG1655, and effects on levels of 5-HT, precursors, and metabolites, were evaluated using amperometry and high performance liquid chromatography with electrochemical detection (HPLC-EC). Exposure of tissue to EcN cells, but not MG1655 cells, was found to increase levels of extra-cellular 5-HT. These effects were not observed when tissues were treated with cell-free supernatant from bacterial cultures. In contrast, when supernatant recovered from untreated ileal tissue was pre-incubated with EcN, the derivative cell-free supernatant was able to elevate 5-HT overflow when used to treat fresh ileal tissue. Measurement of 5-HT precursors and metabolites indicated EcN also increases intracellular 5-HTP and reduces 5-HIAA. The former pointed to modulation of tryptophan hydroxylase-1 to enhance 5-HT synthesis, while the latter indicates an impact on clearance into enterocytes through SERT. Taken together, these findings show EcN is able to enhance 5-HT bioavailability in ileal tissues through interaction with compounds secreted from host tissues.

No MeSH data available.


Related in: MedlinePlus

Neurotransmission process in ileal cells treated with E. coli Nissle 1917.To determine the impact of EcN treatment on the entire neurotransmission process, disaggregated intestinal villi (mucosal scrapings) were used to measure intracellular levels of 5-HT, precursors and metabolites following EcN treatment. Samples of Ileal mucosal scrapings were co-cultured with control Krebs buffer (KB), EcN cell suspensions (EcN), E. coli MG1655 cell suspensions (MG1655); EcN supernatants (EcN- SNT) or EcN modified ileal supernatants (I-EcN SNT) for 1 h at 37°C, 5% CO2, and post co-culture levels of neurochemicals were determined by HPLC. (A) Microscopic analysis of ileal mucosal cells after co-culture with live EcN, demonstrating intact villi structures post treatment. (B) Schematic of the serotonin pathway, highlighting major intermediates and key enzymes and transporters, in 5-HT synthesis and clearance. Compounds measured in these experiments are indicated by letters (C,D,E,F), and relate directly to data displayed in parts (C,D,E,F) of this figure. Compounds: 5HTP = 5-hydroxy-tryptophan; 5-HT = serotonin; 5-HIAA = 5-hydroxy-3-indoleacetic acid. Enzymes & transporters: TpH-1 = Tryptophan hydroxylase, catalyzes conversion of tryptophan to 5-HTP; AADC = Aromatic L-amino acid decarboxylase, converts 5-HTP to 5-HT in this pathway; SERT = Serotonin transporter, transports extra-cellular 5-HT into enterocytes; MAO = Monamine oxidase, catalyzes the metabolism of 5-HT to 5-HIAA in enterocytes. Cell types: IEC - Intestinal Epithelial Cell; EC - Enterochromaffin Cell; IPANs - Intrinsic Primary Afferent Neurones. (C,D,E,F) Levels of intracellular tryptophan; Intracellular 5-HTP; total intracellular and extracellular 5-HT; and intracellular 5-HIAA; respectively. Data are means ± S.E.M. (n = 4), and measurements normalized to the tissue mass by total protein. KB - Krebs buffer only; EcN – E. coli Nissle 1917; EcN 1/10 – E. coli Nissle 1917 at 1/10 cell density of EcN; MG1655 – E. coli MG1655; EcN-SNT – Supernatants derived from E. coli Nissle 1917 cultures grown in krebs buffer; I-EcN- SNT – Supernatants from untreated ileal tissue incubated with EcN cell suspensions. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 vs KB.
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f5: Neurotransmission process in ileal cells treated with E. coli Nissle 1917.To determine the impact of EcN treatment on the entire neurotransmission process, disaggregated intestinal villi (mucosal scrapings) were used to measure intracellular levels of 5-HT, precursors and metabolites following EcN treatment. Samples of Ileal mucosal scrapings were co-cultured with control Krebs buffer (KB), EcN cell suspensions (EcN), E. coli MG1655 cell suspensions (MG1655); EcN supernatants (EcN- SNT) or EcN modified ileal supernatants (I-EcN SNT) for 1 h at 37°C, 5% CO2, and post co-culture levels of neurochemicals were determined by HPLC. (A) Microscopic analysis of ileal mucosal cells after co-culture with live EcN, demonstrating intact villi structures post treatment. (B) Schematic of the serotonin pathway, highlighting major intermediates and key enzymes and transporters, in 5-HT synthesis and clearance. Compounds measured in these experiments are indicated by letters (C,D,E,F), and relate directly to data displayed in parts (C,D,E,F) of this figure. Compounds: 5HTP = 5-hydroxy-tryptophan; 5-HT = serotonin; 5-HIAA = 5-hydroxy-3-indoleacetic acid. Enzymes & transporters: TpH-1 = Tryptophan hydroxylase, catalyzes conversion of tryptophan to 5-HTP; AADC = Aromatic L-amino acid decarboxylase, converts 5-HTP to 5-HT in this pathway; SERT = Serotonin transporter, transports extra-cellular 5-HT into enterocytes; MAO = Monamine oxidase, catalyzes the metabolism of 5-HT to 5-HIAA in enterocytes. Cell types: IEC - Intestinal Epithelial Cell; EC - Enterochromaffin Cell; IPANs - Intrinsic Primary Afferent Neurones. (C,D,E,F) Levels of intracellular tryptophan; Intracellular 5-HTP; total intracellular and extracellular 5-HT; and intracellular 5-HIAA; respectively. Data are means ± S.E.M. (n = 4), and measurements normalized to the tissue mass by total protein. KB - Krebs buffer only; EcN – E. coli Nissle 1917; EcN 1/10 – E. coli Nissle 1917 at 1/10 cell density of EcN; MG1655 – E. coli MG1655; EcN-SNT – Supernatants derived from E. coli Nissle 1917 cultures grown in krebs buffer; I-EcN- SNT – Supernatants from untreated ileal tissue incubated with EcN cell suspensions. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 vs KB.

Mentions: Because the observed effect of EcN on extra-cellular 5-HT may be due to action at various stages of the serotonergic system (synthesis, release, clearance), we next investigated the impact of EcN on the 5-HT pathway as a whole. Following EcN treatment, both intracellular and extra-cellular levels of 5-HT, precursors, and metabolites were determined in dissociated ileal mucosa by HPLC (Fig. 5a,b). Intracellular levels of the 5-HT precursor tryptophan were not significantly different across the treated samples and controls (Fig. 5c). In contrast, intracellular levels of the tryptophan derived 5-HT intermediate, 5-Hydroxytryptophan (5-HTP), were shown to be significantly higher in tissues treated with both EcN cells and I-EcN-SNT (Fig. 5d). Commensurately, measurement of total 5-HT (intra and extracellular) indicated a >5-fold increase of 5-HT content in tissues treated with either EcN cells or with I-EcN-SNT, compared to untreated controls (Fig. 5e). Moreover, the stimulatory effect of EcN on 5-HTP and 5-HT was dose-dependent, and proportionally reduced when inoculum density of EcN was reduced 10-fold. The 5-HIAA content of dissociated mucosa was also significantly altered by EcN treatment, however, in this case distinct effects were observed when tissue was treated with EcN cells compared with I-EcN-SNT (Fig. 5f). In tissues treated with EcN cells 5-HIAA was significantly decreased, however, no significant alteration in 5-HIAA levels were observed when tissues were treated with I-EcN-SNT (Fig. 5f). No significant effect was elicited by the commensal MG1655 on the levels of any compound measured.


Escherichia coli Nissle 1917 enhances bioavailability of serotonin in gut tissues through modulation of synthesis and clearance.

Nzakizwanayo J, Dedi C, Standen G, Macfarlane WM, Patel BA, Jones BV - Sci Rep (2015)

Neurotransmission process in ileal cells treated with E. coli Nissle 1917.To determine the impact of EcN treatment on the entire neurotransmission process, disaggregated intestinal villi (mucosal scrapings) were used to measure intracellular levels of 5-HT, precursors and metabolites following EcN treatment. Samples of Ileal mucosal scrapings were co-cultured with control Krebs buffer (KB), EcN cell suspensions (EcN), E. coli MG1655 cell suspensions (MG1655); EcN supernatants (EcN- SNT) or EcN modified ileal supernatants (I-EcN SNT) for 1 h at 37°C, 5% CO2, and post co-culture levels of neurochemicals were determined by HPLC. (A) Microscopic analysis of ileal mucosal cells after co-culture with live EcN, demonstrating intact villi structures post treatment. (B) Schematic of the serotonin pathway, highlighting major intermediates and key enzymes and transporters, in 5-HT synthesis and clearance. Compounds measured in these experiments are indicated by letters (C,D,E,F), and relate directly to data displayed in parts (C,D,E,F) of this figure. Compounds: 5HTP = 5-hydroxy-tryptophan; 5-HT = serotonin; 5-HIAA = 5-hydroxy-3-indoleacetic acid. Enzymes & transporters: TpH-1 = Tryptophan hydroxylase, catalyzes conversion of tryptophan to 5-HTP; AADC = Aromatic L-amino acid decarboxylase, converts 5-HTP to 5-HT in this pathway; SERT = Serotonin transporter, transports extra-cellular 5-HT into enterocytes; MAO = Monamine oxidase, catalyzes the metabolism of 5-HT to 5-HIAA in enterocytes. Cell types: IEC - Intestinal Epithelial Cell; EC - Enterochromaffin Cell; IPANs - Intrinsic Primary Afferent Neurones. (C,D,E,F) Levels of intracellular tryptophan; Intracellular 5-HTP; total intracellular and extracellular 5-HT; and intracellular 5-HIAA; respectively. Data are means ± S.E.M. (n = 4), and measurements normalized to the tissue mass by total protein. KB - Krebs buffer only; EcN – E. coli Nissle 1917; EcN 1/10 – E. coli Nissle 1917 at 1/10 cell density of EcN; MG1655 – E. coli MG1655; EcN-SNT – Supernatants derived from E. coli Nissle 1917 cultures grown in krebs buffer; I-EcN- SNT – Supernatants from untreated ileal tissue incubated with EcN cell suspensions. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 vs KB.
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Related In: Results  -  Collection

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f5: Neurotransmission process in ileal cells treated with E. coli Nissle 1917.To determine the impact of EcN treatment on the entire neurotransmission process, disaggregated intestinal villi (mucosal scrapings) were used to measure intracellular levels of 5-HT, precursors and metabolites following EcN treatment. Samples of Ileal mucosal scrapings were co-cultured with control Krebs buffer (KB), EcN cell suspensions (EcN), E. coli MG1655 cell suspensions (MG1655); EcN supernatants (EcN- SNT) or EcN modified ileal supernatants (I-EcN SNT) for 1 h at 37°C, 5% CO2, and post co-culture levels of neurochemicals were determined by HPLC. (A) Microscopic analysis of ileal mucosal cells after co-culture with live EcN, demonstrating intact villi structures post treatment. (B) Schematic of the serotonin pathway, highlighting major intermediates and key enzymes and transporters, in 5-HT synthesis and clearance. Compounds measured in these experiments are indicated by letters (C,D,E,F), and relate directly to data displayed in parts (C,D,E,F) of this figure. Compounds: 5HTP = 5-hydroxy-tryptophan; 5-HT = serotonin; 5-HIAA = 5-hydroxy-3-indoleacetic acid. Enzymes & transporters: TpH-1 = Tryptophan hydroxylase, catalyzes conversion of tryptophan to 5-HTP; AADC = Aromatic L-amino acid decarboxylase, converts 5-HTP to 5-HT in this pathway; SERT = Serotonin transporter, transports extra-cellular 5-HT into enterocytes; MAO = Monamine oxidase, catalyzes the metabolism of 5-HT to 5-HIAA in enterocytes. Cell types: IEC - Intestinal Epithelial Cell; EC - Enterochromaffin Cell; IPANs - Intrinsic Primary Afferent Neurones. (C,D,E,F) Levels of intracellular tryptophan; Intracellular 5-HTP; total intracellular and extracellular 5-HT; and intracellular 5-HIAA; respectively. Data are means ± S.E.M. (n = 4), and measurements normalized to the tissue mass by total protein. KB - Krebs buffer only; EcN – E. coli Nissle 1917; EcN 1/10 – E. coli Nissle 1917 at 1/10 cell density of EcN; MG1655 – E. coli MG1655; EcN-SNT – Supernatants derived from E. coli Nissle 1917 cultures grown in krebs buffer; I-EcN- SNT – Supernatants from untreated ileal tissue incubated with EcN cell suspensions. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 vs KB.
Mentions: Because the observed effect of EcN on extra-cellular 5-HT may be due to action at various stages of the serotonergic system (synthesis, release, clearance), we next investigated the impact of EcN on the 5-HT pathway as a whole. Following EcN treatment, both intracellular and extra-cellular levels of 5-HT, precursors, and metabolites were determined in dissociated ileal mucosa by HPLC (Fig. 5a,b). Intracellular levels of the 5-HT precursor tryptophan were not significantly different across the treated samples and controls (Fig. 5c). In contrast, intracellular levels of the tryptophan derived 5-HT intermediate, 5-Hydroxytryptophan (5-HTP), were shown to be significantly higher in tissues treated with both EcN cells and I-EcN-SNT (Fig. 5d). Commensurately, measurement of total 5-HT (intra and extracellular) indicated a >5-fold increase of 5-HT content in tissues treated with either EcN cells or with I-EcN-SNT, compared to untreated controls (Fig. 5e). Moreover, the stimulatory effect of EcN on 5-HTP and 5-HT was dose-dependent, and proportionally reduced when inoculum density of EcN was reduced 10-fold. The 5-HIAA content of dissociated mucosa was also significantly altered by EcN treatment, however, in this case distinct effects were observed when tissue was treated with EcN cells compared with I-EcN-SNT (Fig. 5f). In tissues treated with EcN cells 5-HIAA was significantly decreased, however, no significant alteration in 5-HIAA levels were observed when tissues were treated with I-EcN-SNT (Fig. 5f). No significant effect was elicited by the commensal MG1655 on the levels of any compound measured.

Bottom Line: Exposure of tissue to EcN cells, but not MG1655 cells, was found to increase levels of extra-cellular 5-HT.These effects were not observed when tissues were treated with cell-free supernatant from bacterial cultures.Measurement of 5-HT precursors and metabolites indicated EcN also increases intracellular 5-HTP and reduces 5-HIAA.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, BN2 4GJ, United Kingdom.

ABSTRACT
Accumulating evidence shows indigenous gut microbes can interact with the human host through modulation of serotonin (5-HT) signaling. Here we investigate the impact of the probiotic Escherichia coli Nissle 1917 (EcN) on 5-HT signalling in gut tissues. Ex-vivo mouse ileal tissue sections were treated with either EcN or the human gut commensal MG1655, and effects on levels of 5-HT, precursors, and metabolites, were evaluated using amperometry and high performance liquid chromatography with electrochemical detection (HPLC-EC). Exposure of tissue to EcN cells, but not MG1655 cells, was found to increase levels of extra-cellular 5-HT. These effects were not observed when tissues were treated with cell-free supernatant from bacterial cultures. In contrast, when supernatant recovered from untreated ileal tissue was pre-incubated with EcN, the derivative cell-free supernatant was able to elevate 5-HT overflow when used to treat fresh ileal tissue. Measurement of 5-HT precursors and metabolites indicated EcN also increases intracellular 5-HTP and reduces 5-HIAA. The former pointed to modulation of tryptophan hydroxylase-1 to enhance 5-HT synthesis, while the latter indicates an impact on clearance into enterocytes through SERT. Taken together, these findings show EcN is able to enhance 5-HT bioavailability in ileal tissues through interaction with compounds secreted from host tissues.

No MeSH data available.


Related in: MedlinePlus