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Modulation of lipopolysaccharide-induced neuronal response by activation of the enteric nervous system.

Coquenlorge S, Duchalais E, Chevalier J, Cossais F, Rolli-Derkinderen M, Neunlist M - J Neuroinflammation (2014)

Bottom Line: Activation of extracellular signal-regulated kinase (ERK) and 5'-adenosine monophosphate-activated protein kinase (AMPK) pathways was analyzed by immunocytochemistry and Western blot analysis.Signaling analyses showed that LPS induced activation of ERK but not AMPK, which was constitutively activated in rENSpc neurons.In the presence of LPS, EFS inhibited the ERK and AMPK pathways.

View Article: PubMed Central - PubMed

Affiliation: Neuropathies of the enteric nervous system and digestive diseases, INSERM UMR913, School of Medicine, University of Nantes, 1, rue Gaston Veil, Nantes, F-44035, France. sabrina.coquenlorge@univ-nantes.fr.

ABSTRACT

Background: Evidence continues to mount concerning the importance of the enteric nervous system (ENS) in controlling numerous intestinal functions in addition to motility and epithelial functions. Nevertheless, little is known concerning the direct participation of the ENS in the inflammatory response of the gut during infectious or inflammatory insults. In the present study we analyzed the ENS response to bacterial lipopolysaccharide, in particular the production of a major proinflammatory cytokine, tumor necrosis factor-alpha (TNF-α).

Methods: TNF-α expression (measured by qPCR, quantitative Polymerase Chain Reaction) and production (measured by ELISA) were measured in human longitudinal muscle-myenteric plexus (LMMP) and rat ENS primary cultures (rENSpc). They were either treated or not treated with lipopolysaccharide (LPS) in the presence or not of electrical field stimulation (EFS). Activation of extracellular signal-regulated kinase (ERK) and 5'-adenosine monophosphate-activated protein kinase (AMPK) pathways was analyzed by immunocytochemistry and Western blot analysis. Their implications were studied using specific inhibitors (U0126, mitogen-activated protein kinase kinase, MEK, inhibitor and C compound, AMPK inhibitor). We also analyzed toll-like receptor 2 (TLR2) expression and interleukin-6 (IL-6) production after LPS treatment simultaneously with EFS or TNF-α-neutralizing antibody.

Results: Treatment of human LMMP or rENSpc with LPS induced an increase in TNF-α production. Activation of the ENS by EFS significantly inhibited TNF-α production. This regulation occurred at the transcriptional level. Signaling analyses showed that LPS induced activation of ERK but not AMPK, which was constitutively activated in rENSpc neurons. Both U0126 and C compound almost completely prevented LPS-induced TNF-α production. In the presence of LPS, EFS inhibited the ERK and AMPK pathways. In addition, we demonstrated using TNF-α-neutralizing antibody that LPS-induced TNF-α production increased TLR2 expression and reduced IL-6 production.

Conclusions: Our results show that LPS induced TNF-α production by enteric neurons through activation of the canonical ERK pathway and also in an AMPK-dependent manner. ENS activation through the inhibition of these pathways decreased TNF-α production, thereby modulating the inflammatory response induced by endotoxin.

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ENS activation in rat primary cultures inhibits the LPS-induced increase in TNF-α transcript and protein levels.(A) Evaluation of the impact of electrical field stimulation (EFS) on TNF-α secretion was performed by ELISA in rENSpc treated (+) or not (−) with LPS (0.1 μg/ml) (twelve independent experiments). (B)TNF-α mRNA expression was measured in rENSpc by qPCR (twelve independent experiments). (C) The effect of ATP (100 μM) and involvement of P2X7 receptor on TNF-α secretion were measured by ELISA in ENS cultures pretreated or not with BzATP (P2X7 agonist; 100 μM) or A439079 (P2X7 antagonist; 30 μM) (five independent experiments). Values represent the mean ± SEM (Mann-Whitney U test; *P <0.05 as compared with control without LPS; #P <0.05 LPS with EFS, agonist or antagonist versus LPS alone; §P <0.05 LPS with ATP compared with LPS with ATP in presence of A438079). ATP, adenosine-5'-triphosphate; BzATP, 2’(3’)-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate triethylammonium salt; CT, control; EFS, electrical field stimulation; LPS, lipopolysaccharide; mRNA, messenger ribonucleic acid; rENSpc, rat enteric nervous system primary culture; SEM, standard error of the mean; TNF-α, tumor necrosis factor alpha.
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Fig2: ENS activation in rat primary cultures inhibits the LPS-induced increase in TNF-α transcript and protein levels.(A) Evaluation of the impact of electrical field stimulation (EFS) on TNF-α secretion was performed by ELISA in rENSpc treated (+) or not (−) with LPS (0.1 μg/ml) (twelve independent experiments). (B)TNF-α mRNA expression was measured in rENSpc by qPCR (twelve independent experiments). (C) The effect of ATP (100 μM) and involvement of P2X7 receptor on TNF-α secretion were measured by ELISA in ENS cultures pretreated or not with BzATP (P2X7 agonist; 100 μM) or A439079 (P2X7 antagonist; 30 μM) (five independent experiments). Values represent the mean ± SEM (Mann-Whitney U test; *P <0.05 as compared with control without LPS; #P <0.05 LPS with EFS, agonist or antagonist versus LPS alone; §P <0.05 LPS with ATP compared with LPS with ATP in presence of A438079). ATP, adenosine-5'-triphosphate; BzATP, 2’(3’)-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate triethylammonium salt; CT, control; EFS, electrical field stimulation; LPS, lipopolysaccharide; mRNA, messenger ribonucleic acid; rENSpc, rat enteric nervous system primary culture; SEM, standard error of the mean; TNF-α, tumor necrosis factor alpha.

Mentions: To assess if ENS activity could regulate TNF-α production, we measured LPS-induced TNF-α production in the supernatants of rENSpc after EFS and in those not treated with EFS. EFS had no significant effect on basal TNF-α production. However, EFS significantly inhibited TNF-α production induced by LPS in rENSpc (Figure 2A). qPCR analysis indicated that EFS inhibited the increase in TNF-α mRNA level observed after LPS (Figure 2B). These results show that LPS induce an increase in TNF-α transcript and protein levels, and that ENS activation reduces TNF-α production.Figure 2


Modulation of lipopolysaccharide-induced neuronal response by activation of the enteric nervous system.

Coquenlorge S, Duchalais E, Chevalier J, Cossais F, Rolli-Derkinderen M, Neunlist M - J Neuroinflammation (2014)

ENS activation in rat primary cultures inhibits the LPS-induced increase in TNF-α transcript and protein levels.(A) Evaluation of the impact of electrical field stimulation (EFS) on TNF-α secretion was performed by ELISA in rENSpc treated (+) or not (−) with LPS (0.1 μg/ml) (twelve independent experiments). (B)TNF-α mRNA expression was measured in rENSpc by qPCR (twelve independent experiments). (C) The effect of ATP (100 μM) and involvement of P2X7 receptor on TNF-α secretion were measured by ELISA in ENS cultures pretreated or not with BzATP (P2X7 agonist; 100 μM) or A439079 (P2X7 antagonist; 30 μM) (five independent experiments). Values represent the mean ± SEM (Mann-Whitney U test; *P <0.05 as compared with control without LPS; #P <0.05 LPS with EFS, agonist or antagonist versus LPS alone; §P <0.05 LPS with ATP compared with LPS with ATP in presence of A438079). ATP, adenosine-5'-triphosphate; BzATP, 2’(3’)-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate triethylammonium salt; CT, control; EFS, electrical field stimulation; LPS, lipopolysaccharide; mRNA, messenger ribonucleic acid; rENSpc, rat enteric nervous system primary culture; SEM, standard error of the mean; TNF-α, tumor necrosis factor alpha.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4279994&req=5

Fig2: ENS activation in rat primary cultures inhibits the LPS-induced increase in TNF-α transcript and protein levels.(A) Evaluation of the impact of electrical field stimulation (EFS) on TNF-α secretion was performed by ELISA in rENSpc treated (+) or not (−) with LPS (0.1 μg/ml) (twelve independent experiments). (B)TNF-α mRNA expression was measured in rENSpc by qPCR (twelve independent experiments). (C) The effect of ATP (100 μM) and involvement of P2X7 receptor on TNF-α secretion were measured by ELISA in ENS cultures pretreated or not with BzATP (P2X7 agonist; 100 μM) or A439079 (P2X7 antagonist; 30 μM) (five independent experiments). Values represent the mean ± SEM (Mann-Whitney U test; *P <0.05 as compared with control without LPS; #P <0.05 LPS with EFS, agonist or antagonist versus LPS alone; §P <0.05 LPS with ATP compared with LPS with ATP in presence of A438079). ATP, adenosine-5'-triphosphate; BzATP, 2’(3’)-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate triethylammonium salt; CT, control; EFS, electrical field stimulation; LPS, lipopolysaccharide; mRNA, messenger ribonucleic acid; rENSpc, rat enteric nervous system primary culture; SEM, standard error of the mean; TNF-α, tumor necrosis factor alpha.
Mentions: To assess if ENS activity could regulate TNF-α production, we measured LPS-induced TNF-α production in the supernatants of rENSpc after EFS and in those not treated with EFS. EFS had no significant effect on basal TNF-α production. However, EFS significantly inhibited TNF-α production induced by LPS in rENSpc (Figure 2A). qPCR analysis indicated that EFS inhibited the increase in TNF-α mRNA level observed after LPS (Figure 2B). These results show that LPS induce an increase in TNF-α transcript and protein levels, and that ENS activation reduces TNF-α production.Figure 2

Bottom Line: Activation of extracellular signal-regulated kinase (ERK) and 5'-adenosine monophosphate-activated protein kinase (AMPK) pathways was analyzed by immunocytochemistry and Western blot analysis.Signaling analyses showed that LPS induced activation of ERK but not AMPK, which was constitutively activated in rENSpc neurons.In the presence of LPS, EFS inhibited the ERK and AMPK pathways.

View Article: PubMed Central - PubMed

Affiliation: Neuropathies of the enteric nervous system and digestive diseases, INSERM UMR913, School of Medicine, University of Nantes, 1, rue Gaston Veil, Nantes, F-44035, France. sabrina.coquenlorge@univ-nantes.fr.

ABSTRACT

Background: Evidence continues to mount concerning the importance of the enteric nervous system (ENS) in controlling numerous intestinal functions in addition to motility and epithelial functions. Nevertheless, little is known concerning the direct participation of the ENS in the inflammatory response of the gut during infectious or inflammatory insults. In the present study we analyzed the ENS response to bacterial lipopolysaccharide, in particular the production of a major proinflammatory cytokine, tumor necrosis factor-alpha (TNF-α).

Methods: TNF-α expression (measured by qPCR, quantitative Polymerase Chain Reaction) and production (measured by ELISA) were measured in human longitudinal muscle-myenteric plexus (LMMP) and rat ENS primary cultures (rENSpc). They were either treated or not treated with lipopolysaccharide (LPS) in the presence or not of electrical field stimulation (EFS). Activation of extracellular signal-regulated kinase (ERK) and 5'-adenosine monophosphate-activated protein kinase (AMPK) pathways was analyzed by immunocytochemistry and Western blot analysis. Their implications were studied using specific inhibitors (U0126, mitogen-activated protein kinase kinase, MEK, inhibitor and C compound, AMPK inhibitor). We also analyzed toll-like receptor 2 (TLR2) expression and interleukin-6 (IL-6) production after LPS treatment simultaneously with EFS or TNF-α-neutralizing antibody.

Results: Treatment of human LMMP or rENSpc with LPS induced an increase in TNF-α production. Activation of the ENS by EFS significantly inhibited TNF-α production. This regulation occurred at the transcriptional level. Signaling analyses showed that LPS induced activation of ERK but not AMPK, which was constitutively activated in rENSpc neurons. Both U0126 and C compound almost completely prevented LPS-induced TNF-α production. In the presence of LPS, EFS inhibited the ERK and AMPK pathways. In addition, we demonstrated using TNF-α-neutralizing antibody that LPS-induced TNF-α production increased TLR2 expression and reduced IL-6 production.

Conclusions: Our results show that LPS induced TNF-α production by enteric neurons through activation of the canonical ERK pathway and also in an AMPK-dependent manner. ENS activation through the inhibition of these pathways decreased TNF-α production, thereby modulating the inflammatory response induced by endotoxin.

Show MeSH
Related in: MedlinePlus