Enterocyte Cytoskeleton Changes Are Crucial for Enhanced Translocation of NonpathogenicEscherichia coliacross Metabolically Stressed Gut Epithelia

Abstract

Substantial data implicate the commensal flora as triggers for the initiation of enteric inflammation or inflammatory disease relapse. We have shown that enteric epithelia under metabolic stress respond to nonpathogenic bacteria by increases in epithelial paracellular permeability and bacterial translocation. Here we assessed the structural basis of these findings. Confluent filter-grown monolayers of the human colonic T84 epithelial cell line were treated with 0.1 mM dinitrophenol (which uncouples oxidative phosphorylation) and noninvasive, nonpathogenic Escherichia coli (strain HB101, 10 6 CFU) with or without pretreatment with various pharmacological agents. At 24 h later, apoptosis, tight-junction protein expression, transepithelial resistance (TER; a marker of paracellular permeability), and bacterial internalization and translocation were assessed. Treatment with stabilizers of microtubules (i.e., colchicine), microfilaments (i.e., jasplakinolide) and clathrin-coated pit endocytosis (i.e., phenylarsine oxide) all failed to block DNP؉E. coli HB101-induced reductions in TER but effectively prevented bacterial internalization and translocation. Neither the TER defect nor the enhanced bacterial translocations were a consequence of increased apoptosis. These data show that epithelial paracellular and transcellular (i.e., bacterial internalization) permeation pathways are controlled by different mechanisms. Thus, epithelia under metabolic stress increase their endocytotic activity that can result in a microtubule-, microfilament-dependent internalization and transcytosis of bacteria. We speculate that similar events in vivo would allow excess unprocessed antigen and bacteria into the mucosa and could evoke an inflammatory response by, for example, the activation of resident or recruited immune cells.In many respects the human interaction with bacteria is one of conflict. A voluminous literature illustrates how the pathogen affects host target cells and the host response to destroy or eradicate the pathogen (23). In contrast, the human colon is home to an immense and complex microflora of commensal bacteria. Indeed, the presence of a normal microbiota is a requirement for mucosal immune cell education and normal gut physiology, such as short-chain fatty acid synthesis as an energy source for the colonocyte (33, 35). However, evidence now indicates that commensal bacteria and their products may trigger or exacerbate chronic idiopathic intestinal inflammatory diseases, such as Crohn's disease (28). For instance, virtually every rodent model of colitis, whether spontaneous or chemically induced, is significantly less severe if the animals are raised in germfree conditions (16,38). Similarly, bowel rest via surgically diverting the fecal stream and antibiotic treatment can lead to symptomatic relief in some patients with Crohn's disease (26,37).A common feature of gut inflammation is increased epithelial permeability, both paracellular (i.e., the pathway between adjacent cells) and transcellular (i.e., material cross...

Decreased epithelial barrier function evoked by exposure to metabolic stress and nonpathogenicE. coliis enhanced by TNF-α

“…Dissecting the mechanism of control of epithelial barrier function, the present study adds to a body of data illustrating that paracellular and transcellular permeability are differentially regulated. Indeed, in our earlier investigations we showed that epithelia under metabolic stress have increased internalization of noninvasive E. coli (28) and that inhibitors of endocytosis and microtubule activity significantly reduced bacterial translocation across T84 cell monolayers (27). We did not specifically address the route of bacterial transcytosis in the present study.…”

“…Support for this hypothesis comes from analyses of kidney-derived epithelia showing that ATP depletion affects the arrangement of tight junction proteins with a consequent increase in paracellular permeability (2, 5). Our analyses revealed that uncoupling oxidative phosphorylation (i.e., inducing metabolic stress) in epithelial monolayers by treatment with dinitrophenol (DNP) in the presence of viable, noninvasive, nonpathogenic Escherichia coli (E. coli) (strain HB101) resulted in increased paracellular and transcellular permeability (27,28).With the emergence of the concept of immunophysiology, it is clear that the function of the intestinal epithelium is affected by many immune cell types (30). The macrophage produces a plethora of messengers, including tumor necrosis factor (TNF)-␣.…”

Indomethacin-induced translocation of bacteria across enteric epithelia is reactive oxygen species-dependent and reduced by vitamin C

“…In accordance with these findings, using the MTT assay as a marker of mitochondrial activity, we found a slight reduction in mitochondrial function in T84 epithelia treated with indomethacin. Previous data show that metabolic stress induced by treatment of T84 cells with DNP is associated with reduced mitochondrial activity, which correlated with reduced expression of tight junction proteins (24,30), and increased internalization and translocation of E. coli (31). Given the energy dependence of tight junction form and function and endocytotic processes (i.e., paracellular and transcellular permeability), reduced mitochondrial function would result in reduced ATP synthesis, thus contributing to the indomethacininduced epithelial barrier defect.…”

“…E. coli (strain HB101) was grown and cultured overnight in an orbital shaker in Luria-Bertani (LB) broth at 37°C and added to the apical surface of confluent monolayers at a final concentration of 10 6 colony-forming units (cfu)/ml (23,30,31,41). A putative toxic effect of all drugs on the E. coli was evaluated in 24-h bacterial growth curves, with optical density and agar plate colony growth used to evaluate bacterial cell numbers.…”

In vivo actin cross-linking induced by Vibrio cholerae type VI secretion system is associated with intestinal inflammation

“…Given the cell biologic requirements for VgrG-1 translocation, initiation of inflammation could occur after transcytosis by a variety of several cell types, including as dendritic cells, Peyer's patch M cells (39), or even by epithelial cells (40). Although it is not clear how cross-linked actin in host cells could contribute to an inflammatory response, many cellular functions could be perturbed, and intracellular surveillance pathways could be activated.…”