Introduction
Gastro-oesophageal reflux disease (GORD) is characterised by visceral pain hypersensitivity, manifesting as heartburn, and distal oesophageal inflammation. In the oesophageal mucosa of GORD patients, even without visible inflammation (non-erosive reflux disease; NERD) there is increased innervation of sensory nerve fibres containing Substance P (SP) (Xu et al., 2013). SP may be released in response to luminal refluxate or inflammatory mediators to potentiate neuro-immune pathways relevant to GORD pathogenesis. We aimed to investigate the contribution of SP and its receptor NK1R to GORD symptoms.
Aims & Methods
To assess expression of NK1R on native tissue epithelium, biopsies were collected from healthy controls (HC), functional heartburn (FH), NERD and erosive reflux disease (ERD) patients following informed consent (Royal London Hospital ethics number: 19/LO/1506) and prepared for immunohistochemistry. NK1R was co-stained with epithelial cell marker E-Cadherin, pan-leukocyte marker CD45, and general T lymphocyte marker CD3. NK1R protein level was determined by mean fluorescence intensity of 3 FOVs. Cells of the NE-1 oesophageal epithelial cell line were incubated with media containing 100 nM and 300 nM SP for 30 minutes. Cells were lysed and intracellular inflammatory pathway activation was determined by p-NF-κB/NF-κB ratio through Western blot. mRNA levels of the inflammatory cytokine CXCL8 were determined via qPCR with 18S rRNA as a housekeeping gene.
Results
Quantification of mucosal tissue samples showed NK1R mean fluorescence intensity was significantly higher in NERD (fold-change 1.52; p<0.05) and ERD (fold change 1.70; p<0.01) oesophageal mucosa compared to healthy control (n=6-8). Interestingly, NK1R was also found expressed in a sub-group of CD3+ T lymphocytes specifically in the papillae and epithelium of GORD patients. in NE-1 oesophageal epithelial cells, 30 minutes exposure to 100 nM (fold change 1.41; p<0.05) and 300 nM (fold change 1.92; p<0.0001) SP induced phosphorylation of NF-κB in a dose dependent manner (n=7-8). CXCL8 expression in NE-1 cells exposed to 30 nM, 100 nM, or 300 nM SP for 30 minutes resulted in significantly higher mRNA expression at the highest concentration (fold change 6.29; p<0.05), although a dose-dependent trend was observed at lower concentrations (n=3).
Conclusion
The significant increase in NK1R expression in NERD and ERD patients compared to healthy volunteers, combined with expression on T lymphocytes provides evidence, for the first time, that SP may have a critical role in potentiating pain sensation in the oesophagus. Indeed, SP directly induces NF-κB phosphorylation and increases transcription of inflammatory cytokine IL-8 in oesophageal epithelial cells. This demonstrates a novel neuroimmune pathway regulated by SP and NK1R that may be critical in GORD symptom generation.
References
Xu, X., Li, Z., Zou, D., Yang, M., Liu, Z., & Wang, X. (2013). High expression of calcitonin gene-related peptide and substance P in esophageal mucosa of patients with non-erosive reflux disease. Dig Dis Sci, 58(1), 53-60. doi:10.1007/s10620-012-2308-z
