Abstract
BackgroundAccumulating evidence suggest that the enteric nervous system (ENS) plays important roles in gastrointestinal inflammatory responses, which could be in part mediated by Toll-like receptor (TLR) activation. The aim of this study was to characterise the expression and functionality of TLR2/4/9 in the ENS.MethodsTLR2/4/9 expression was assessed in the plexuses of adult rats and embryonic ENS cultures by immunofluorescence and quantitative PCR. Following stimulation with TLR2/4/9 ligands or their combinations, activation of NF-kB, production of TNF-α, IL-6 and MCP-1 and chemoattraction of RAW264.7 macrophages were evaluated by means of Western blot, ELISA, immunofluorescence and migration assays in transwell inserts.ResultsTLR2/4/9 staining colocalised with enteric neuronal markers, whereas their presence in enteroglial processes was low to inexistent. Stimulation of ENS cultures with selective ligands induced NF-kB activation and release of cytokines and chemokines by neurons and resident immunocytes. TLR2 neutralisation before lipopolysaccharide (LPS) challenge reduced production of inflammatory mediators, whereas combination of TLR2/4 ligands promoted macrophage migration. Combined stimulation of cultures with LPS and the CpG oligonucleotide 1826 (TLR4/9 ligands) caused a synergic increase in chemoattraction and cytokine production.ConclusionsOur results suggest that the ENS, and particularly enteric neurons, can integrate a variety of microbial signals and respond in a relatively selective fashion, depending on the particular TLRs stimulated. These findings additionally suggest that the ENS is capable of initiating a defensive response against pathogens and expanding inflammation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0653-0) contains supplementary material, which is available to authorized users.
Highlights
Accumulating evidence suggest that the enteric nervous system (ENS) plays important roles in gastrointestinal inflammatory responses, which could be in part mediated by Toll-like receptor (TLR) activation
TLR2 staining was found in all neurons of the submucosal plexus (SMP) and the longitudinal muscle myenteric plexus (LMMP) (Fig. 1, upper panels), whereas TLR4 was only observed in discrete neuronal somas and fibres (Fig. 1, middle panels, and Additional file 1)
TLR2 immunoreactivity was observed in some enteroglial processes in the LMMP (Additional file 3), indicating that this receptor is expressed in both neurons and enteric glial cells (EGC)
Summary
Accumulating evidence suggest that the enteric nervous system (ENS) plays important roles in gastrointestinal inflammatory responses, which could be in part mediated by Toll-like receptor (TLR) activation. Expression of some TLRs in the ENS has been described [18, 19], and their roles in intestinal motility, apoptosis and normal development have progressively been unravelled [19, 20] Their immune functions have been addressed by some authors [7, 21], but there are still several questions to be answered, such as whether differential recognition of MAMPs by the ENS translates into microbial-selective responses, or whether TLRmediated signalling is involved in expansion of the inflammatory response. Our results show that neurons are the main TLR-expressing cells in enteric plexuses and integrate TLR signals promoting diverse responses depending on the stimulus received, bringing some new evidence regarding their roles in defence against pathogens and inflammation enhancement
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