199 TNF-α Induced Expression of Enteric Neuronal Neuropeptide Y (NPY) Influences Intestinal Epithelial Barrier Functions

Abstract

Introduction: The integrity of the intestinal epithelium is crucial to maintain boundaries between luminal contents and mucosal immune system. Increased epithelial permeability has been noted in 10-20% of pre-symptomatic Crohn's disease patients. We investigated if enteric neurons producing neuropeptide Y (NPY) could influence epithelial permeability and modulate TNF-α signaling. Methods: Caco2BBE epithelial cells were grown on transwell plates and NPY was added to the basolateral medium (0.1 μM). The epithelial permeability was measured by transepithelial resistance (TER) and flux of FITC-Dextran (4 Kd) at 2 and 24 h. The changes in the expression of tight junction proteins were compared in control and NPY-treated cells by real-time polymerase chain reaction (qRT-PCR) and Western blotting. The enteric neurons transfected with NPY promoter constructs (-1078 (full length), -952, -836, -769, -728, -597, -448, -278) were treated with TNF-α, and NPY promoter activity was assessed by luciferase assay. NPY expression in enteric neurons treated with TNF-α was also determined by qRT-PCR. Results: The addition of NPY increased the TER and FITC-Dextran flux across the CaCo2-BBE monolayer (p<0.05), thus increasing colonic epithelial permeability. NPY also induced a two-fold increase in the claudin-2 expression at mRNA (p< 0.01) and protein (p< 0.05) levels. NPY mRNA was up regulated by two-fold in TNF-α treated enteric neurons (p< 0.05) as seen by qRT-PCR. Luciferase assay demonstrated a significant increase in the NPY promoter activity, specifically in the regions between -728 and -836 of the NPY promoter (p< 0.05. Transcription factor (TRANSFAC) analysis showed Activator Protein-1 (AP-1) binding sites in this region of the NPY promoter, supporting the responsiveness to TNF-α. Conclusions: TNF-α upregulates NPY expression in enteric neurons; NPY in turn alters barrier functions of the colonic epithelium via modulation of leaky tight junction proteins like Claudin-2. Taken together, enteric neurons producing NPY aggravate pro-inflammatory signaling and increases epithelial permeability. Our studies demonstrate a role of NPY regulation of epithelial permeability and participation of enteric neuronal signaling in propagating inflammation during inflammatory bowel disease.