Abstract BACKGROUND Chronic abdominal pain is common in patients with chronic gastrointestinal disorders, including Inflammatory Bowel Disease (IBD). Accumulating data suggests that the metabolism of bioactive lipids, such as LPC and LPA, is altered in patients with IBD. We have found that fecal microbiota transplantation from patients with chronic pain and high fecal LPC/LPA levels induces visceral hypersensitivity in the recipient mice. However, the underlying mechanisms are not fully understood. HYPOTHESIS LPC and LPA contribute to visceral hypersensitivity both through direct neuronal activation and pro-inflammatory effects on immune cells. AIMS 1) Assess whether LPC and LPA induce neuronal activation. 2) Investigate the pro-inflammatory effects of LPC and LPA on enteric glial cells and splenocytes in vitro. METHODS Dorsal root ganglia (DRG) sensory neurons from conventional C57BL/6 mice were cultured for 48 hours. Calcium mobilization was measured by a Cytation C10 imaging reader using a fluorescent probe Fluo-4 (1mM) after being stimulated with different concentrations of LPC (1 to 30 𝜇M) and LPA (1 to 100 𝜇M). Rat enteric glial cells (CRL-2690, ATCC) and splenocytes from specific pathogen-free mice were cultured and treated with either a vehicle, a positive control lipopolysaccharide (LPS; 100 ng/mL) or LPC (5 to 300 𝜇M) and/or LPA (50 to 100 𝜇M) for 24 hours. Cell health was assessed by counting the number of viable cells following incubation, evaluating cell morphology and measuring the production of the pro-inflammatory cytokine IL-6, with known neuroactive properties, by ELISA. RESULTS Both LPC and LPA induced an increase in the percentage of responding DRG neurons and the intensity of their responses, in a dose-dependent manner. Enteric glial cells incubated with LPC but not with LPA exhibited a cytotoxic effect, leading to a significant reduction in the number of viable cells as LPC concentration increased. Interestingly, no visible cell adherence was observed at LPC concentrations exceeding 50 𝜇M. IL-6 production by enteric glial cells was increased with LPA to levels similar to LPS, but not LPC, likely as a consequence of the fewer viable cells. In contrast, LPC and LPA induced cell proliferation in splenocytes. Viable cells increased approximately 21-fold and 35-fold following incubation with the highest concentrations of LPC and LPA, respectively. However, there was no increase in IL-6 production compared to the vehicle-treated cells. CONCLUSION Our data suggests that LPC and LPA induce visceral hypersensitivity through direct neuronal activation, with immunomodulatory effects also possibly being involved. Further studies are required to investigate the putative role of cytokines and other immune mediators.