F11 dorsal root ganglion nociceptor neurons release calcitonin gene related peptide in response to pathogen associated molecular patterns

The immune and nervous systems co‐evolved to direct and mount coordinated behaviors, reflexes and immune responses to protect the host. In the neuro‐immune paradigm sensory nociceptor neurons, located in multiple peripheral ganglia, have been shown to be sensitive to pattern associated molecular patterns (PAMPs). Upon stimulation, these sensory nociceptors release the neurotransmitter calcitonin gene related peptide (CGRP) which acts as a cytokine to assist with the coordination of the innate immune response. However, it remains unclear which PAMPs are sensed by these sensory c‐fiber nerves and what specific substances are released in response to the specific PAMPs. Additionally, how long does this stimulation by PAMPs last? These are critical knowledge gaps in our understanding of how sensory neurons shape the innate immune response. To answer these questions, we cultured immortalized F11 dorsal root ganglion (DRG) neurons and exposed them to lipopolysaccharide (LPS 500 ng/mL, Gram‐negative PAMP), lipoteichoic acid (LTA 50 μg/mL, Gram‐positive PAMP), tumor necrosis factor alpha (TNFα 500 ng/mL, inflammatory cytokine) and capsaicin (1 μM, known C‐fiber agonist, serving as a positive control). The exposures to the stimuli lasted for 0.5, 1, 2, 4 or 8 hours. The experiments were run in triplicate on three separate days where a control condition was run on each day for each time point. We measured the release of CGRP with ELISA, known to be released by DRG neurons in response to LPS. We hypothesized that CGRP would be released early on as neural excitation occurs quickly. All conditions and time points were normalized to the average of respective controls for specific time points on respective days. In contrast to our hypothesis, no condition increased CGRP concentration at 0.5 hours. However, at 1‐hour LPS, LTA, TNFα and capsaicin increased CGRP release significantly (p<0.05). At 2 hours, LPS and LTA retained their neural stimulation as CGRP was still increased in comparison to controls (P<0.01). Stimulation subsided at 4 and 8 hours as CGRP concentration returned close to control conditions at respective time points. Our data demonstrate that Gram‐positive and Gram‐negative PAMPs stimulate the release of a known neurotransmitter from sensory nociceptor cells. Additional experiments with fungal PAMPs will be run as well as identifying the complete secrotome of the neuronal culture in response to a variety of PAMPs. These data coincide with the notion that neurons are stimulated early during infection by PAMPs and inflammation directly, as indicated by TNFα stimulation, in order to quickly assist with the innate immune response.