Only the highly S100-positive EGC also expressed the TLR2 (Figure5B), but no changes were observed after LPS stimulation. ENS primary cultures (rENSpc). They were either treated or not treated with lipopolysaccharide (LPS) in the presence or not of electrical field activation (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–neutralizing antibody. == Results == Treatment of human LMMP or rENSpc with LPS induced an increase in TNF- production. Activation of the ENS by EFS significantly inhibited TNF- 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- production. In the presence of LPS, EFS inhibited the ERK and AMPK pathways. In addition, we exhibited using TNF–neutralizing antibody that LPS-induced TNF- production increased TLR2 expression and reduced IL-6 production. == Conclusions == Our results show that LPS induced TNF- production by enteric neurons through activation of the canonical ERK pathway and also in an AMPK-dependent manner. ENS activation LX 1606 Hippurate through the inhibition of these pathways decreased TNF- production, thereby modulating the inflammatory response induced by endotoxin. == Electronic supplementary material == The online version LX 1606 Hippurate of this article (doi:10.1186/s12974-014-0202-7) contains supplementary material, which is available to authorized users. Keywords:Enteric nervous system, LPS, TNF-, AMPK, ERK == Background == The enteric nervous system (ENS), composed of neurons and enteric glial cells (EGC), is usually a central regulator of gastrointestinal functions encompassing gut motility, electrolyte transport and intestinal epithelial barrier (IEB) functions [1]. Recently, the ENS has been recognized as a major player in gut protection in response to pathogen or inflammatory insult [2]. Conversely, the ENS is also affected in disease, in particular in inflammatory bowel diseases (IBD). Alterations in ENS functions and phenotype (altered excitability and neuroplastic changes) occur in IBD [3,4]. These changes are associated with gastrointestinal (GI) dysfunctions such as altered motility, diarrhea and even pain. The ENS could also be directly LX 1606 Hippurate involved in the inflammatory response to infectious or inflammatory difficulties. Indeed, CD22 in animal models of colitis, enteric neuronal hyper- and hypoplasia was associated with increased and reduced production of TNF-, respectively [5]. In addition, hypertrophy and hyperplasia of enteric neurons has been reported in IBD [6]. However, the mechanism responsible for neuronal modulation of the severity of inflammation remains unknown. Being putatively due to the modulation of neuroimmune interactions, it is tempting to speculate that enteric neurons could directly produce and regulate important cytokines involved in IBD. During inflammation, the ENS responds to a wide range of mediators, such as cytokines [7]. The ENS can also respond to bacterial difficulties such as lipopolysaccharide, as it expresses a wide array of toll-like receptors (TLRs). Enteric neurons express TLR4 [8,9]. TLR3 and TLR7 are expressed in the myenteric and submucosal plexi. TLR3, TLR4 and TLR7 are also expressed in EGC [10-13]. The functional effects of the activation of TLRs by their ligands in enteric neurons remain largely unknown. TLR2 was nevertheless shown to be necessary for maintaining ENS integrity and protection from colitis [14]. Activation of TLR4 in EGC induced the release of nitric oxide [12]. However, the ability of enteric neurons to respond to lipopolysaccharides (LPS) and to synthesize cytokines such as TNF-, as well as the LX 1606 Hippurate signaling pathways involved, remain unknown. The overall aim of this study was to determine whether the ENS can directly respond to LPS by generating the major proinflammatory cytokine TNF-, and whether ENS activity can modulate this production. == Methods == == Generation of enteric nervous system cultures == Rat ENS main cultures (rENSpc) were performed as previously explained using the small intestines of E15 Sprague-Dawley rat embryos (Janvier Laboratories SA, Le Genest-St-Isle, France) [15]. These procedures were approved by the local Animal Care and Use Committee (agreement E. 44011; INSERM, Nantes, France). Briefly, the small intestines of rat.
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