Vasoactive intestinal peptide prevents PKCε-induced intestinal epithelial barrier disruption during EPEC infection

We previously showed that vasoactive intestinal peptide (VIP) protects against bacterial pathogen-induced epithelial barrier disruption and colitis, although the mechanisms remain poorly defined. The aim of the current study was to identify cellular pathways of VIP-mediated protection with use of pharmacological inhibitors during enteropathogenic Escherichia coli (EPEC) infection of Caco-2 cell monolayers and during Citrobacter rodentium-induced colitis. EPEC-induced epithelial barrier disruption involved the PKC pathway but was independent of functional cAMP, Rho, and NF-kappa B pathways. VIP mediated its protective effects by inhibiting EPEC-induced PKC activity and increasing expression of the junctional protein claudin-4. Short-term treatment with TPA, which is known to activate PKC, was inhibited by VIP pretreatment, while PKC degradation via long-term treatment with TPA mimicked the protective actions of VIP. Immunostaining for specific PKC isotypes showed upregulated expression of PKC theta and PKC epsilon during EPEC infection. Treatment with specific inhibitors revealed a critical role for PKC epsilon in EPEC-induced barrier disruption. Furthermore, activation of PKC epsilon and loss of barrier integrity correlated with claudin-4 degradation. In contrast, inhibition of PKC epsilon by VIP pretreatment or the PKC epsilon inhibitor maintained membrane-bound claudin-4 levels, along with barrier function. Finally, in vivo treatment with the PKCe inhibitor protected mice from C. rodentium-induced colitis. In conclusion, EPEC infection increases intracellular PKC epsilon levels, leading to decreased claudin-4 levels and compromising epithelial barrier integrity. VIP inhibits PKC epsilon activation, thereby attenuating EPEC-induced barrier disruption.