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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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ERK and AMPK inhibition prevent LPS-induced increase in TNF-α transcript and protein levels.(A) ERK pathway activation in rENSpc treated with LPS from two minutes to seven hours was measured by Western blotting using phospho-ERK antibodies (P-ERK). The relative amount of P-ERK was determined by normalizing with β-actin (four to eight independent experiments). Values represent the mean ± SEM as fold/mean t0 (one-way ANOVA test followed by Dunn’s post-hoc test; *P <0.05 as compared with t0 without LPS). (B) Localization of activated − ERK and − AMPK in rENSpc treated for two hours with LPS was performed by immunocytochemistry using P-ERK or phospho-Acetyl-CoA Carboxylase (P-ACC), with S100β and Sox10 (glial markers) or Hu and Tuj (βIII-tubulin, neuronal markers). Scale bar: 50 μm. Inserts show higher magnification images (40x for P-ERK and 100x for P-ACC). ERK- and AMPK-dependent pathway contribution to TNFα production were assessed using MEK1/2 inhibitor (U0126; 10 μM) and AMPK inhibitor (C compound, CC; 10 μM) co-treatment on rENSpc treated (+) or not (−) with LPS for seven hours. (C) Quantification of TNF-α mRNA by qPCR (six to 11 independent samples). (D) TNF-α secretion was assayed by ELISA with or without the presence of inhibitors (six independent samples). The independence of the ERK and AMPK pathways was determined by Western blotting. (E) ERK phosphorylation was not affected by CC and (F) ACC phosphorylation was not modified by U0126 (eight and three independent experiments, respectively). (Mann-Whitney U test; *P <0.05 as compared with control without LPS; #P <0.05 as compared with LPS without inhibitor). (G) The EFS impact on ERK and AMPK activation was measured on rENSpc treated (+) or not (−) with LPS and EFS for two hours by Western blotting using P-ERK and P-AMPK antibodies. Immunoblots are representative of five independent experiments with similar results.
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Fig3: ERK and AMPK inhibition prevent LPS-induced increase in TNF-α transcript and protein levels.(A) ERK pathway activation in rENSpc treated with LPS from two minutes to seven hours was measured by Western blotting using phospho-ERK antibodies (P-ERK). The relative amount of P-ERK was determined by normalizing with β-actin (four to eight independent experiments). Values represent the mean ± SEM as fold/mean t0 (one-way ANOVA test followed by Dunn’s post-hoc test; *P <0.05 as compared with t0 without LPS). (B) Localization of activated − ERK and − AMPK in rENSpc treated for two hours with LPS was performed by immunocytochemistry using P-ERK or phospho-Acetyl-CoA Carboxylase (P-ACC), with S100β and Sox10 (glial markers) or Hu and Tuj (βIII-tubulin, neuronal markers). Scale bar: 50 μm. Inserts show higher magnification images (40x for P-ERK and 100x for P-ACC). ERK- and AMPK-dependent pathway contribution to TNFα production were assessed using MEK1/2 inhibitor (U0126; 10 μM) and AMPK inhibitor (C compound, CC; 10 μM) co-treatment on rENSpc treated (+) or not (−) with LPS for seven hours. (C) Quantification of TNF-α mRNA by qPCR (six to 11 independent samples). (D) TNF-α secretion was assayed by ELISA with or without the presence of inhibitors (six independent samples). The independence of the ERK and AMPK pathways was determined by Western blotting. (E) ERK phosphorylation was not affected by CC and (F) ACC phosphorylation was not modified by U0126 (eight and three independent experiments, respectively). (Mann-Whitney U test; *P <0.05 as compared with control without LPS; #P <0.05 as compared with LPS without inhibitor). (G) The EFS impact on ERK and AMPK activation was measured on rENSpc treated (+) or not (−) with LPS and EFS for two hours by Western blotting using P-ERK and P-AMPK antibodies. Immunoblots are representative of five independent experiments with similar results.

Mentions: To decipher the signaling pathways responsible for LPS-induced TNF-α synthesis by the ENS, and its modulation by EFS, we analyzed the possible implication of the canonical extracellular signal-regulated kinase (ERK) pathway, and also of the 5'-adenosine monophosphate-activated protein kinase (AMPK) pathway. By Western blot analysis using phospho-specific antibodies recognizing the active form of the ERKs, we showed that phosphorylation of ERKs was increased after two hours of LPS stimulation, with no change in ERK expression (Figure 3A). Activation of the AMPK pathway can be measured by the phosphorylation of the Thr172 of the AMPK itself, or by the phosphorylation of acetyl-CoA carboxylase (ACC), an AMPK target, on its Ser19. Neither AMPK nor ACC phosphorylation changed significantly over time, with or without LPS treatment (data not shown). Immunocytochemical analyses revealed that the immunoreactivity for phospho-ERK and phospho-ACC concurred with Tuj and Hu (neuronal markers), but not with S100β or Sox10 (glial markers) immunoreactivity (Figure 3B). These data show that, while the AMPK pathway was constitutively activated in neuronal cells, ERKs were activated in neurons after LPS treatment. Pretreatment of rENSpc with U0126 or compound C inhibitors of the ERK or AMPK pathways, respectively, significantly reduced TNF-α mRNA expression (Figure 3C) and TNF-α production (Figure 3D). Interestingly, ERK phosphorylation was not affected by compound C (Figure 3E), and ACC-phosphorylation was not modified by U0126 (Figure 3F), again suggesting the independence of the two pathways. These results demonstrate that the AMPK and ERK pathways participate independently in LPS induction of TNF-α production by the ENS.Figure 3


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)

ERK and AMPK inhibition prevent LPS-induced increase in TNF-α transcript and protein levels.(A) ERK pathway activation in rENSpc treated with LPS from two minutes to seven hours was measured by Western blotting using phospho-ERK antibodies (P-ERK). The relative amount of P-ERK was determined by normalizing with β-actin (four to eight independent experiments). Values represent the mean ± SEM as fold/mean t0 (one-way ANOVA test followed by Dunn’s post-hoc test; *P <0.05 as compared with t0 without LPS). (B) Localization of activated − ERK and − AMPK in rENSpc treated for two hours with LPS was performed by immunocytochemistry using P-ERK or phospho-Acetyl-CoA Carboxylase (P-ACC), with S100β and Sox10 (glial markers) or Hu and Tuj (βIII-tubulin, neuronal markers). Scale bar: 50 μm. Inserts show higher magnification images (40x for P-ERK and 100x for P-ACC). ERK- and AMPK-dependent pathway contribution to TNFα production were assessed using MEK1/2 inhibitor (U0126; 10 μM) and AMPK inhibitor (C compound, CC; 10 μM) co-treatment on rENSpc treated (+) or not (−) with LPS for seven hours. (C) Quantification of TNF-α mRNA by qPCR (six to 11 independent samples). (D) TNF-α secretion was assayed by ELISA with or without the presence of inhibitors (six independent samples). The independence of the ERK and AMPK pathways was determined by Western blotting. (E) ERK phosphorylation was not affected by CC and (F) ACC phosphorylation was not modified by U0126 (eight and three independent experiments, respectively). (Mann-Whitney U test; *P <0.05 as compared with control without LPS; #P <0.05 as compared with LPS without inhibitor). (G) The EFS impact on ERK and AMPK activation was measured on rENSpc treated (+) or not (−) with LPS and EFS for two hours by Western blotting using P-ERK and P-AMPK antibodies. Immunoblots are representative of five independent experiments with similar results.
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Related In: Results  -  Collection

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Fig3: ERK and AMPK inhibition prevent LPS-induced increase in TNF-α transcript and protein levels.(A) ERK pathway activation in rENSpc treated with LPS from two minutes to seven hours was measured by Western blotting using phospho-ERK antibodies (P-ERK). The relative amount of P-ERK was determined by normalizing with β-actin (four to eight independent experiments). Values represent the mean ± SEM as fold/mean t0 (one-way ANOVA test followed by Dunn’s post-hoc test; *P <0.05 as compared with t0 without LPS). (B) Localization of activated − ERK and − AMPK in rENSpc treated for two hours with LPS was performed by immunocytochemistry using P-ERK or phospho-Acetyl-CoA Carboxylase (P-ACC), with S100β and Sox10 (glial markers) or Hu and Tuj (βIII-tubulin, neuronal markers). Scale bar: 50 μm. Inserts show higher magnification images (40x for P-ERK and 100x for P-ACC). ERK- and AMPK-dependent pathway contribution to TNFα production were assessed using MEK1/2 inhibitor (U0126; 10 μM) and AMPK inhibitor (C compound, CC; 10 μM) co-treatment on rENSpc treated (+) or not (−) with LPS for seven hours. (C) Quantification of TNF-α mRNA by qPCR (six to 11 independent samples). (D) TNF-α secretion was assayed by ELISA with or without the presence of inhibitors (six independent samples). The independence of the ERK and AMPK pathways was determined by Western blotting. (E) ERK phosphorylation was not affected by CC and (F) ACC phosphorylation was not modified by U0126 (eight and three independent experiments, respectively). (Mann-Whitney U test; *P <0.05 as compared with control without LPS; #P <0.05 as compared with LPS without inhibitor). (G) The EFS impact on ERK and AMPK activation was measured on rENSpc treated (+) or not (−) with LPS and EFS for two hours by Western blotting using P-ERK and P-AMPK antibodies. Immunoblots are representative of five independent experiments with similar results.
Mentions: To decipher the signaling pathways responsible for LPS-induced TNF-α synthesis by the ENS, and its modulation by EFS, we analyzed the possible implication of the canonical extracellular signal-regulated kinase (ERK) pathway, and also of the 5'-adenosine monophosphate-activated protein kinase (AMPK) pathway. By Western blot analysis using phospho-specific antibodies recognizing the active form of the ERKs, we showed that phosphorylation of ERKs was increased after two hours of LPS stimulation, with no change in ERK expression (Figure 3A). Activation of the AMPK pathway can be measured by the phosphorylation of the Thr172 of the AMPK itself, or by the phosphorylation of acetyl-CoA carboxylase (ACC), an AMPK target, on its Ser19. Neither AMPK nor ACC phosphorylation changed significantly over time, with or without LPS treatment (data not shown). Immunocytochemical analyses revealed that the immunoreactivity for phospho-ERK and phospho-ACC concurred with Tuj and Hu (neuronal markers), but not with S100β or Sox10 (glial markers) immunoreactivity (Figure 3B). These data show that, while the AMPK pathway was constitutively activated in neuronal cells, ERKs were activated in neurons after LPS treatment. Pretreatment of rENSpc with U0126 or compound C inhibitors of the ERK or AMPK pathways, respectively, significantly reduced TNF-α mRNA expression (Figure 3C) and TNF-α production (Figure 3D). Interestingly, ERK phosphorylation was not affected by compound C (Figure 3E), and ACC-phosphorylation was not modified by U0126 (Figure 3F), again suggesting the independence of the two pathways. These results demonstrate that the AMPK and ERK pathways participate independently in LPS induction of TNF-α production by the ENS.Figure 3

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