TLR2 and TLR9 modulate enteric nervous system inflammatory responses to lipopolysaccharide

Abstract

Background: 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. The aim of this study was to characterise the expression and functionality of TLR2/4/9 in the ENS.

Methods: TLR2/4/9 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.

Results: TLR2/4/9 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.

Conclusions: Our 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.

Keywords: Chemoattraction; Enteric nervous system; Enteric neuron; Inflammation; TLR2; TLR4; TLR9.

Fig. 1

TLR2/4/9 are expressed in enteric neurons of adult rat colon tissue. TLR localisation in whole-mount preparations of the SMP and the LMMP. B-tubulin III was used instead of HuC/D as neuronal marker for colocalisation with TLR9 due to antibody detection incompatibilities. Scale bars: 25 μm

Fig. 2

Embryonic ENS culture neurons express TLR2/4/9. a Agarose gel showing specific products of real-time PCR for the assayed genes in ENS culture (PC); water was used as a no-template control (H₂O) and rat colon cDNA as positive control (+C). b TLR relative expression in ENS culture in basal conditions (n = 8). c Localisation of TLR in ENS culture ganglia. B-tubulin III was used instead of HuC/D as neuronal marker for colocalisation with TLR9 due to antibody detection incompatibilities. White arrows point to neuronal somas. Scale bars: 25 μm

Fig. 3

MAMP stimulation induces activation of the NF-kB pathway in embryonic ENS culture. a Rat embryonic ENS culture was incubated for 8 h with the indicated MAMPs or the IkB phosphorylation inhibitor Bay 11-7082, and cell protein extraction and Western blot were performed to determine phosphorylated IkB (P-IkBα). B-actin was used as a loading control. b Time-course densitometric quantification of P-IkBα bands corrected to β-actin and related to basal activation levels; representative bands are shown in Additional file 4. Statistical analysis was performed independently for each ligand, using one-way ANOVA followed by Dunnett’s test (n = 4 for each ligand and time point; *P < 0.05, **P < 0.01 and ***P < 0.001). c Representative micrographs showing NF-kB p65 subunit localisation in basal conditions (Ctrl) or 1 h after LPS challenge (LPS). White arrows point to neuronal nuclei displaying p65 positive staining. Scale bars: 25 μm

Fig. 4

ENS culture releases cytokines and chemokines in response to LPS. Rat embryonic ENS culture was stimulated for 24 h and culture supernatants were collected and centrifuged prior to measuring cytokine and chemokine secretion. a TNF-α (n = 4–8; LPS vs. control and Bay 11-7082 + LPS, ***P < 0.001; Bay 11-7082 + LPS vs. control, ***P < 0.001). b IL-6 (n = 4–8; LPS vs. control and Bay 11-7082 + LPS, ***P < 0.001). c MCP-1 (n = 4–8; LPS vs. control, ***P < 0.001; LPS vs. Bay 11-7082 + LPS, **P < 0.01). d Distribution studies show IL-6 and MCP-1 colocalisation with TLR4⁺ neurons. White arrows show TLR4^- IL-6-producing cells. Scale bars: 25 μm

Fig. 5

TLR activation in ENS culture is associated to TLR2 up-regulation. TLR mRNA levels were assessed in rat ENS culture stimulated for 8 h with the indicated MAMPs. a Pam2CSK4 (n = 5–10; TLR2 ligand vs. control, ***P < 0.001). b LPS (n = 5–10; TLR2 ligand vs. control, ***P < 0.001; TLR4 and TLR9 ligand vs. control, *P < 0.05). c ODN 1826 (n = 5–10; TLR2 ligand vs. control, **P < 0.01; TLR9 ligand vs. control, *P < 0.05)

Fig. 6

TLR2 blockade reduces ENS culture responses to LPS. Embryonic ENS culture was pre-incubated for 1 h with 10 μg/mL of TLR2 neutralising antibody before an LPS challenge for 24 additional hours. a TNF-α (n = 3; LPS vs. αTLR2 + LPS, ***P < 0.001). b IL-6 (n = 3; LPS vs. αTLR2 + LPS, ***P < 0.001). c MCP-1 (n = 3; LPS vs. αTLR2 + LPS, *P < 0.05)

Fig. 7

TLR4/9 combined stimulation elicits synergic pro-inflammatory responses in ENS culture. Embryonic ENS cultures were incubated with the specified combinations of MAMPs for 24 h. Randomised block design analysis followed by Tukey’s post hoc test was applied to minimise statistical differences due to intrinsic culture responsiveness. a TNF-α (n = 4–9; ODN 1826 + LPS vs. LPS, ***P < 0.001; ODN 1826 + LPS vs. Bay 11-7082 + ODN 1826 + LPS, **P < 0.01). b IL-6 (n = 4–9; ODN 1826 + LPS vs. LPS, ***P < 0.001; ODN 1826 + LPS vs. Bay 11-7082 + ODN 1826 + LPS, *P < 0.05). c MCP-1 (n = 4–9; LPS vs. Bay 11-7082 + LPS, ***P < 0.001)

Fig. 8

Stimulated ENS culture conditioned media induce chemoattraction of RAW 264.7 macrophages. RAW 264.7 macrophages were seeded in the upper chamber of 8-μm-pore transwell inserts and left to migrate for 4 h towards lower chambers filled with stimulated ENS culture conditioned media. Representative micrographs of the evaluated fields in the lower chamber of a transwell insert mounted upon unstimulated (Ctrl), LPS-stimulated (LPS) or ODN 1826 + LPS-stimulated (ODN 1826 + LPS) ENS culture medium. Scale bars: 200 μm. Number of migrating cells per field (n = 4, with two replicates for experiment; LPS vs. unstimulated ENS culture supernatant, #P = 0.08; Pam2CSK4 + LPS and ODN 1826 + LPS vs. unstimulated ENS culture supernatant, ***P < 0.001). Randomised block design analysis was performed to minimise the variability in migration due to RAW 264.7 passage, followed by Tukey’s post hoc test