Interferon γ Induces Translocation of Commensal Escherichia coli Across Gut Epithelial Cells via a Lipid Raft--Mediated Process

doi:10.1053/j.gastro.2005.01.046

Gastroenterology

Volume 128, Issue 5, May 2005, Pages 1258-1267

https://doi.org/10.1053/j.gastro.2005.01.046 Get rights and content

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.

Section snippets

Materials

Recombinant human IFN-γ (10⁴ IU/μg) was purchased from R&D Systems (Abingdon, UK). Monoclonal anti-occludin (OC-3F10) and polyclonal anti-claudin 1 antibodies were obtained from Zymed Laboratories Inc (San Francisco, CA). Goat anti-mouse and anti-rabbit horseradish peroxidase (HRP)-conjugated antibodies were purchased from Bio-Rad (Hemel Hempstead, UK). Fluorescein isothiocyanate-labeled goat anti-mouse was obtained from Sigma-Aldrich (Poole, UK). [¹⁴C]Mannitol (56 mCi/mmol) was purchased from

Results

Initial studies investigated the correlation between translocation of E coli C25 and loss of paracellular integrity in T84 colonic cells in response to the proinflammatory cytokine IFN-γ. Exposure to basolateral IFN-γ for 48 hours caused an increase in paracellular permeability in T84 monolayers, as judged by a loss of TEER and increased permeability to the paracellular probe LY (Figure 1A). These effects were dose dependent, and both variables showed significant changes at 10 IU/mL IFN-γ, with

Discussion

There is strong evidence that a loss of intestinal barrier function mediated by cytokines and other cellular factors is an early event in the pathogenesis of several disorders ranging from sepsis and multiple organ failure to inflammatory bowel disease.¹⁰ The hypothesis is that increased paracellular permeability mediated by the disruption of tight junctions allows increased mucosal penetration by luminal bacteria and bacterial antigens that then initiate or prolong an inflammatory response.

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: Supported by the Medical Research Council, United Kingdom, and the Digestive Disorders Foundation.

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Basic-alimentary tractInterferon γ Induces Translocation of Commensal Escherichia coli Across Gut Epithelial Cells via a Lipid Raft--Mediated Process

Section snippets

Materials

Results

Discussion

Gastroenterology

Gastroenterology

Gastroenterology

Lab Invest

J Pediatr Surg

Differentiation

Microbes Infect

Methods Enzymol

J Biol Chem

Am J Pathol

Gastroenterology

Gastroenterology

J Biol Chem

Microb Pathog

J Surg Res

Biochem Biophys Res Commun

Biochim Biophys Acta

Intestinal barrieran interface between health and disease

J Gastroenterol Hepatol

The role of the gut flora in health and disease, and its modification as therapy

Aliment Pharmacol Ther

Multiple organ failurepathophysiology and potential future therapy

Ann Surg

Intestinal epithelial hyperpermeabilityupdate on the pathogenesis of gut mucosal barrier dysfunction in critical illness

Curr Opin Crit Care

Hemorrhagic shock increases gut macromolecular permeability in the rat

Shock

IL-6 is essential for development of gut barrier dysfunction after hemorrhagic shock and resuscitation in mice

Am J Physiol

Basic-alimentary tract
Interferon γ Induces Translocation of Commensal Escherichia coli Across Gut Epithelial Cells via a Lipid Raft--Mediated Process