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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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Enteric neurons produce TNF-α in response to LPS stimulation.(A) rENSpc were treated in a time- and dose-dependent manner with LPS. Quantification of TNF-α secretion was measured by ELISA. Values represent the mean ± SEM of between three and seven independent samples per condition (two-way ANOVA test followed by a Bonferroni post-hoc test; *P <0.05 as compared with the same time point without LPS). (B) Representative images of TNF-α localization in the rENSpc (four independent samples). Immunocytochemical triple labeling of ENS cultures were performed using anti-TNF-α, anti-S100β (glial marker) and anti-Tuj (neuronal marker) antibodies. Examples of neurons expressing TNF-α are depicted with white arrowheads. Scale bar: 50 μm. (C) LPS treatment of enteric glial cell cultures did not induce TNF-α production (between four and ten independent experiments). EGC, enteric glial cells; LMMP, longitudinal muscle/myenteric plexus; LPS, lipopolysaccharides; rENSpc, rat enteric nervous system primary culture; S100β, S100 calcium binding protein beta; SEM, standard error of the mean; TNF-α, tumor necrosis factor alpha; Tuj, βIII-tubulin.
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Fig1: Enteric neurons produce TNF-α in response to LPS stimulation.(A) rENSpc were treated in a time- and dose-dependent manner with LPS. Quantification of TNF-α secretion was measured by ELISA. Values represent the mean ± SEM of between three and seven independent samples per condition (two-way ANOVA test followed by a Bonferroni post-hoc test; *P <0.05 as compared with the same time point without LPS). (B) Representative images of TNF-α localization in the rENSpc (four independent samples). Immunocytochemical triple labeling of ENS cultures were performed using anti-TNF-α, anti-S100β (glial marker) and anti-Tuj (neuronal marker) antibodies. Examples of neurons expressing TNF-α are depicted with white arrowheads. Scale bar: 50 μm. (C) LPS treatment of enteric glial cell cultures did not induce TNF-α production (between four and ten independent experiments). EGC, enteric glial cells; LMMP, longitudinal muscle/myenteric plexus; LPS, lipopolysaccharides; rENSpc, rat enteric nervous system primary culture; S100β, S100 calcium binding protein beta; SEM, standard error of the mean; TNF-α, tumor necrosis factor alpha; Tuj, βIII-tubulin.

Mentions: The rENSpc were treated with lipopolysaccharides (Escherichia coli and Salmonella typhosa; 1:1, Sigma-Aldrich) at 0.1 μg/ml for the indicated time, except for Figure 1A, where different concentrations were tested. For the purpose of establishing which pathways and receptors are implicated in TNF-α and TLR2 regulation, U0126 (mitogen-activated protein kinase kinase 1/2 or MEK1/2 inhibitor; 10 μM), compound C (5’-adenosine monophosphate-activated protein kinase (AMPK) inhibitor; 10 μM) (Calbiochem, Merk Millipore, Billerica, Massachusetts, United States) and anti-rat TNF-α (1 and 10 μg/ml; eBiosciences, San Diego, California, United States) were added 30 minutes prior to the addition of LPS or ENS stimulation. Pam3CSK4 (TLR1/2 agonist; 100 ng/ml; Invivogen, San Diego, California, United States), A438079 (selective P2X7 antagonist; 30 μM; Tocris Bioscience, Bristol, United Kingdom), adenosine-5'-triphosphate (ATP) (100 μM) and 2’(3’)-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate triethylammonium salt (BzATP) (selective P2X7 agonist; 100 μM; Sigma-Aldrich) were also used to treat ENS plus or minus LPS.Figure 1


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)

Enteric neurons produce TNF-α in response to LPS stimulation.(A) rENSpc were treated in a time- and dose-dependent manner with LPS. Quantification of TNF-α secretion was measured by ELISA. Values represent the mean ± SEM of between three and seven independent samples per condition (two-way ANOVA test followed by a Bonferroni post-hoc test; *P <0.05 as compared with the same time point without LPS). (B) Representative images of TNF-α localization in the rENSpc (four independent samples). Immunocytochemical triple labeling of ENS cultures were performed using anti-TNF-α, anti-S100β (glial marker) and anti-Tuj (neuronal marker) antibodies. Examples of neurons expressing TNF-α are depicted with white arrowheads. Scale bar: 50 μm. (C) LPS treatment of enteric glial cell cultures did not induce TNF-α production (between four and ten independent experiments). EGC, enteric glial cells; LMMP, longitudinal muscle/myenteric plexus; LPS, lipopolysaccharides; rENSpc, rat enteric nervous system primary culture; S100β, S100 calcium binding protein beta; SEM, standard error of the mean; TNF-α, tumor necrosis factor alpha; Tuj, βIII-tubulin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: Enteric neurons produce TNF-α in response to LPS stimulation.(A) rENSpc were treated in a time- and dose-dependent manner with LPS. Quantification of TNF-α secretion was measured by ELISA. Values represent the mean ± SEM of between three and seven independent samples per condition (two-way ANOVA test followed by a Bonferroni post-hoc test; *P <0.05 as compared with the same time point without LPS). (B) Representative images of TNF-α localization in the rENSpc (four independent samples). Immunocytochemical triple labeling of ENS cultures were performed using anti-TNF-α, anti-S100β (glial marker) and anti-Tuj (neuronal marker) antibodies. Examples of neurons expressing TNF-α are depicted with white arrowheads. Scale bar: 50 μm. (C) LPS treatment of enteric glial cell cultures did not induce TNF-α production (between four and ten independent experiments). EGC, enteric glial cells; LMMP, longitudinal muscle/myenteric plexus; LPS, lipopolysaccharides; rENSpc, rat enteric nervous system primary culture; S100β, S100 calcium binding protein beta; SEM, standard error of the mean; TNF-α, tumor necrosis factor alpha; Tuj, βIII-tubulin.
Mentions: The rENSpc were treated with lipopolysaccharides (Escherichia coli and Salmonella typhosa; 1:1, Sigma-Aldrich) at 0.1 μg/ml for the indicated time, except for Figure 1A, where different concentrations were tested. For the purpose of establishing which pathways and receptors are implicated in TNF-α and TLR2 regulation, U0126 (mitogen-activated protein kinase kinase 1/2 or MEK1/2 inhibitor; 10 μM), compound C (5’-adenosine monophosphate-activated protein kinase (AMPK) inhibitor; 10 μM) (Calbiochem, Merk Millipore, Billerica, Massachusetts, United States) and anti-rat TNF-α (1 and 10 μg/ml; eBiosciences, San Diego, California, United States) were added 30 minutes prior to the addition of LPS or ENS stimulation. Pam3CSK4 (TLR1/2 agonist; 100 ng/ml; Invivogen, San Diego, California, United States), A438079 (selective P2X7 antagonist; 30 μM; Tocris Bioscience, Bristol, United Kingdom), adenosine-5'-triphosphate (ATP) (100 μM) and 2’(3’)-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate triethylammonium salt (BzATP) (selective P2X7 agonist; 100 μM; Sigma-Aldrich) were also used to treat ENS plus or minus LPS.Figure 1

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