Tumor Necrosis Factor Alpha Effects on the Porcine Intestinal Epithelial Barrier Include Enhanced Expression of TNF Receptor 1

Tumor necrosis factor alpha (TNF alpha) has been shown to impair the intestinal barrier, inducing and maintaining inflammatory states of the intestine. The aim of the current study was to analyze functional, molecular and regulatory effects of TNF alpha in a newly established non-transformed jejunal enterocyte model, namely IPEC-J2 monolayers. Incubation with 1000 U/mL TNF alpha induced a marked decrease in transepithelial electrical resistance (TEER), and an increase in permeability for the paracellular flux marker [H-3]-D-mannitol compared to controls. Immunoblots revealed a significant decrease in tight junction (TJ) proteins occludin, claudin-1 and claudin-3. Moreover, a dose-dependent increase in the TNF receptor (TNFR)-1 was detected, explaining the exponential nature of pro-inflammatory effects, while TNFR-2 remained unchanged. Recovery experiments revealed reversible effects after the removal of the cytokine, excluding apoptosis as a reason for the observed changes. Furthermore, TNF alpha signaling could be inhibited by the specific myosin light chain kinase (MLCK) blocker ML-7. Results of confocal laser scanning immunofluorescence microscopy were in accordance with all quantitative changes. This study explains the self-enhancing effects of TNF alpha mediated by MLCK, leading to a differential regulation of TJ proteins resulting in barrier impairment in the intestinal epithelium.