Probiotic-Mediated Decrease of Elevated Serine Protease Activity in Fecal Supernatants of Diarrheic IBS is Positively Correlated With an Improvement of the Symptomatology: A New Biomarker in IBS Probiotic Treatment Efficacy

Abstract

Background Probiotics are live microorganisms that promote gut health and regulate intestinal homeostasis. How probiotics work is incompletely understood, but may involve induction of cell survival pathways (Akt) and stress/MAP kinase-dependent induction of heat shock proteins. The possibility that probiotics might induce autophagy, however, has not been previously explored. Autophagy is believed to have an important role in promoting cell survival under conditions of stress and in clearance of potentially disease-causing intracellular microorganisms. Polymorphisms in autophagy genes have recently been linked to increased risk of human IBD. Methods Intestinal epithelial cells (human colonic Caco2BBE or rat jejunal IEC18) were treated with conditioned media from Bifidobacterium breve (BB-CM) or other intestinal bacteria (Lactobacillus plantarum, Lactobacillus rhamnosus GG). Initially time dependence was determined using 1% CM and subsequently concentration dependence was determined at 30 or 120 min and 24 hours for rapid transduction events (MAP kinase, LC3 conjugation, or Akt activation) versus long lived cellular survival proteins (heat shock protein induction), all assessed by Western blot analysis. Results BB-CM induced autophagy in a timeand concentration-dependent manner. Western blot analysis demonstrated LC3II activation by conjugation to phosphatidylethanolamine by 120 min after BB-CM. BB-CM also activated p38 MAP kinase and ERK1/2, but within 30 min of addition. Both Lactobacillus plantarum and LGG-CM also stimulated LC3, thereby demonstrating stimulation of autophagy. For BB-CM, inhibition of either p38 MAP kinase with SB203580 ort ERK1/2 activation with PD98059 blocked LC3 activation. A wider panel of gram positive and gram negative bacteria were tested only on LC3 activation and while most gram positive bacteria stimulated LC3 activation, most gram negative did not. Conclusions These studies provide evidence that bioactive agents secreted by probiotic and commensal bacteria can induce autophagy in gut epithelial cells. The induction of autophagy may underlie some of the beneficial clinical effects attributed to a healthy enteric microbiota and probiotic agents.