TLR4 mediates upregulation and sensitization of TRPV1 in primary afferent neurons in 2,4,6-trinitrobenzene sulfate-induced colitis

Abstract

Elevated excitability of primary afferent neurons underlies chronic pain in patients with functional or inflammatory bowel diseases. Recent studies have established an essential role for an enhanced transient receptor potential vanilloid subtype 1 (TRPV1) signaling in mediating peripheral hyperalgesia in inflammatory conditions. Since colocalization of Toll-like receptor 4 (TLR4) and TRPV1 has been observed in primary afferents including the trigeminal sensory neurons and the dorsal root ganglion neurons, we test the hypothesis that TLR4 might regulate the expression and function of TRPV1 in primary afferent neurons in 2,4,6-trinitrobenzene sulfate (TNBS)-induced colitis using the TLR4-deficient and the wild-type C57 mice. Despite having a higher disease activity index following administration of 2,4,6-trinitrobenzene sulfate, the TLR4-deficient mice showed less inflammatory infiltration in the colon than the wild-type mice. Increased expression of TLR4 and TRPV1 as well as increased density of capsaicin-induced TRPV1 current was observed in L4-S2 dorsal root ganglion neurons of the wild-type colitis mice till two weeks post 2,4,6-trinitrobenzene sulfate treatment. In comparison, the TLR4-deficient colitis mice had lower TRPV1 expression and TRPV1 current density in dorsal root ganglion neurons with lower abdominal withdrawal response scores during noxious colonic distensions. In the wild type but not in the TLR4-deficient dorsal root ganglion neurons, acute administration of the TLR4 agonist lipopolysaccharide increased the capsaicin-evoked TRPV1 current. In addition, we found that the canonical signaling downstream of TLR4 was activated in 2,4,6-trinitrobenzene sulfate-induced colitis in the wild type but not in the TLR4-deficient mice. These results indicate that TLR4 may play a major role in regulation of TRPV1 signaling and peripheral hyperalgesia in inflammatory conditions.

Keywords: TLR4; TRPV1; colitis; pain; primary afferent neurons.

Figure 1.

Characterization of TNBS-induced colitis in WT and TLR4-deficient mice. (a) Photomicrographs of colonic sections from WT-TNBS, TLR4^−/−-TNBS, TLR4^−/−, and WT littermate controls as indicated. Scale bar = 100 μm. (b) DAI score for four subgroups during two-week period post TNBS or vehicle treatment (n = 5 per group, *p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA followed by Bonferroni post hoc test). (c) Index of inflammatory infiltration for four subgroups. (d) Index of crypt epithelial damage for four subgroups (n = 5 per group, *p < 0.05; **p < 0.01; ***p < 0.001; one-way ANOVA followed by post hoc test). WT: wild type; TLR4: Toll-like receptor 4; KO: knockout; TNBS: 2,4,6-trinitrobenzene sulfate.

Figure 2.

TLR4 deficiency causes reduction of TRPV1 expression in DRG and alleviation of visceral hypersensitivity in TNBS-induced colitis. (a) TLR4 and TRPV1 transcription in DRG of WT and TLR4-deficient group post TNBS treatment. (b and c) TLR4 and TRPV1 protein level in DRG of WT and TLR4-deficient mice post TNBS treatment. Differences in the expression level at different time points within group were analyzed using repeated measures one-way ANOVA followed by Bonferroni’s multiple comparisons test (*p < 0.05; **p < 0.01; ***p < 0.001). Differences in expression level at a given time points between two groups were compared with student’s t test (^#p < 0.05; ^##p < 0.01; ^###p < 0.001). (d) TLR4 deficiency resulted in significant alleviation of visceral hypersensitivity in TNBS-induced colitis mice compared to control mice (*p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA followed by Bonferroni post hoc test). WT: wild type; TLR4: Toll-like receptor 4; KO: knockout; TNBS: 2,4,6-trinitrobenzene sulfate; TRPV1: transient receptor potential vanilloid subtype 1; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; mRNA: messenger RNA.

Figure 3.

Colocalization of TRPV1 (red) and TLR4 (green) in DRG neurons. An increasing number of TRPV1-positive neurons were seen in TNBS-treated WT mice and further statistical comparisons of the percentage of TRPV1-positive neurons in different groups shown in Figure 4. Scale bar = 100 μm. WT: wild type; TLR4: Toll-like receptor 4; KO: knockout; TNBS: 2,4,6-trinitrobenzene sulfate; TRPV1: transient receptor potential vanilloid subtype 1; DAPI: 4′,6-diamidino-2-phenylindole.

Figure 4.

Percentage of TRPV1-positive neurons in L5–L6 DRG of four subgroups of mice. *p < 0.05; **p < 0.01, vehicle-treated WT versus TNBS-treated WT mice; ^###p < 0.001, TNBS-treated TLR4-KO versus TNBS-treated WT mice (with Bonferroni’s post hoc test). WT: wild type; TLR4: Toll-like receptor 4; KO: knockout; TNBS: 2,4,6-trinitrobenzene sulfate; TRPV1: transient receptor potential vanilloid subtype 1.

Figure 5.

LPS sensitizes TRPV1 through TLR4 pathway in DRG neurons. (a) Representative traces of capsaicin (0.1 µM)-evoked currents in acutely dissociated DRG neurons from four groups of mice (WT, TNBS-treated WT, TLR4-KO, and TNBS-treated TLR4-KO). Cells were treated with the vehicle for LPS for 5 min. (b) Acute treatment with LPS for 5 min increased amplitude of the capsaicin-evoked currents in WT DRG neurons. Note the scale bar is different from that of panel (a). (c) The average TRPV1 current density in the DRG neurons with vehicle or LPS treatment. *p < 0.05; **p < 0.01; ***p < 0.001(one-way ANOVA followed by Bonferroni’s multiple comparison test); ^#p < 0.05; ^##p < 0.01; ^###p < 0.001(student’s t test). WT: wild type; TLR4: Toll-like receptor 4; KO: knockout; TNBS: 2,4,6-trinitrobenzene sulfate; LPS: lipopolysaccharide.

Figure 6.

TNBS-induced colitis activates the canonical TLR4 signaling pathway in DRG neurons. (a) Representative western blot gel bands for adaptor proteins MyD88 and TRIF, IL-1β, IL-6, and NF-κB in the DRG from vehicle or TNBS-treated WT and TLR4-KO mice. (b) Statistic graphs showing activation of the canonical TLR4 signaling pathway in TNBS-induced colitis (n = 6 per group). *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA followed by Bonferroni’s multiple comparisons test), ^#p < 0.05; ^##p < 0.01; ^###p < 0.001 (Student’s t test). WT: wild type; TLR4: Toll-like receptor 4; KO: knockout; TNBS: 2,4,6-trinitrobenzene sulfate; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; IL: interleukin; NF-κB: nuclear factor kappa B; TRIF: Toll/IL-1 receptor domain-containing adapter inducing interferon-beta; RU: response units.