Direct bacteria-neuron interaction via Toll-Like Receptors (TLRs): consequences for orofacial pain.

Objectives: Expression of TLRs on high-threshold sensory neurons (nociceptors) permits direct interaction between neurons and pathogens. Previously we have shown that TLR4 is preferentially expressed by nociceptors within the trigeminal ganglion (TG). Here we test the hypothesis that sensory neurons directly detect and respond to challenge by the TLR4 agonists E. coli- and P. gingivalis-derived lipopolysaccharide (LPS) and the TLR2 agonist Pam3CSK4.
Methods: Indirect, dual-labelled immunohistochemistry was performed on fixed, frozen rat TG to detail the expression of TLR2 within specific neurochemically-identified sensory neuron sub-populations. Primary TG neuron cell cultures were prepared from mechanically and enzymatically dissociated TG. Cultures were maintained for 48 hours prior to exposure to either E. coli LPS (O111:B4, 1μg/mL), P. gingivalis LPS (1690/1435 isoforms, 1μg/mL) or Pam3CSK4 (500ng/mL) for 2 hours. Induction of TNFα, IL-1β, IL-6 and IFNβ gene expression was measured by qPCR using GAPDH, β-actin and 18s as endogenous controls. TLR2 (CU CPT 22, 8μM) and TLR4 (CLI-095, 3μM) specific inhibitors were used to demonstrate receptor specificity of the various ligands.
Results: TLR2 is expressed by 27.4±3.1% of total neurons, 62.7±7.9% of TRPV1-positive and 61.5±6.8% of P2X3-positive neurons within the TG (n=3). Following exposure to E. coli LPS both TNFα (8.1±0.5 fold, p<0.0001) and IL-1β (4.1±0.5 fold, p<0.001) gene expression are significantly induced relative to endogenous controls. Pam3CSK4 exposure significantly induced TNFα (18.2±1.5 fold, p<0.001) and IL-1β (4.6±0.3 fold, p<0.001) gene expression. Investigations into the response to P. gingivalis LPS isoforms are currently underway.
Conclusions: Expression of TLR4 and TLR2 within TG sensory neurons is nociceptor specific. Acute activation of these TLRs induces TNFα and IL-1β gene expression, both of which contribute toward the onset of inflammatory pain. Thus, TG nociceptors may directly respond to pathogenic challenge by synthesis of pro-inflammatory cytokines impacting on acute nociception and the local inflammatory response.