Interferon γ induces translocation of commensal Escherichia coli across gut epithelial cells via a lipid raft-mediated process

Background & Aims: The "leaky gut" hypothesis proposes that leakage of enteric bacteria into the body resulting from disruption of the epithelial barrier is a critical step in the pathophysiology of various disorders such as inflammatory bowel disease and sepsis. However, the pathways and underlying mechanisms by which commensal bacteria cross the epithelial barrier in inflammatory conditions remain unclear. This study investigated the mechanisms of interferon γ-;mediated bacterial translocation across human colonic epithelial monolayers. Methods: Caco-2 and T84 monolayers were exposed to interferon γ. Barrier function was assessed by transepithelial electrical resistance and lucifer yellow permeability. Internalization and translocation of Escherichia coli strain C25 were measured by quantitative bacterial culture. Expression and distribution of junctional proteins were assessed by immunoblotting and confocal imaging. Results: Minimal apical to basolateral translocation of C25 was observed in untreated T84 and Caco-2 monolayers. Interferon γ caused a dramatic, dosedependent increase in C25 translocation, which was uncoupled from cytokine-induced increases in paracellular permeability and disruption of tight junction proteins at low interferon γ concentrations. These effects were associated with increased internalization of viable bacteria into, but not adherence to, Caco-2 cells. Interferon γ-mediated bacterial translocation was abolished by pretreatment with the cholesterol-disrupting drugs filipin and methyl-β-cyclodextrin, whereas these agents had no effect on infection of Caco-2 by the enteric pathogen Shigella sonnei. Conclusions: Normally poorly invasive enteric bacteria may, in situations of inflammatory stress, exploit lipid raft-mediated transcytotic pathways to cross the intestinal epithelium, and these effects may precede cytokine-induced disruption of tight junctions. © 2005 by the American Gastroenterological Association.