Glutamine deprivation facilitates tumour necrosis factor induced bacterial translocation in Caco-2 cells by depletion of enterocyte fuel substrate

Abstract

Background and aims: Factors that induce luminal bacteria to cross the intestinal epithelium following injury remain poorly defined. The aim of this study was to investigate the interaction between glutamine metabolism, energy supply, and inflammatory mediators in determining the translocation of non-pathogenic bacteria across cultured enterocytes. Methods: The effect of tumour necrosis factor α (TNF-α) on translocation of Escherichia coli C25 across Caco-2 epithelial monolayers was studied in the presence of products and inhibitors of glutamine metabolism. Simultaneous measurements of transepithelial electrical resistance (TEER) and flux of lucifer yellow were used to assess effects on the paracellular pathway. Lactate dehydrogenase release was used to monitor enterocyte integrity. Imaging of monolayers in these experimental conditions was undertaken with transmission electron microscopy. Results: Exposure to basolateral TNF-α (20 ng/ml) for six hours induced translocation of E coli across Caco-2 but only if accompanied by simultaneous glutamine depletion (p<0.01). Translocation was inhibited by addition of glutamine for two hours (p<0.01) but not by an isonitrogenous mixture of nonglutamine containing amino acids. Inhibition of glutamine conversion to α-ketoglutarate, but not blockade of glutathione or polyamine synthesis, also induced translocation in the presence of TNF-α. Manipulations that induced bacterial translocation were associated with a marked reduction in enterocyte ATP levels. No effect of these treatments on paracellular permeability or lactate dehydrogenase release was observed. Conditions in which translocation occurred were associated with the presence of bacteria within enterocyte vacuoles but not the paracellular space. Conclusions: In inflammatory conditions, the availability of glutamine as an enterocyte fuel substrate is essential for the preservation of a functional barrier to microorganisms. In conditions of acute glutamine depletion, cytokine mediated bacterial translocation appears to be primarily a transcellular process.

Commensal-derived OMVs elicit a mild proinflammatory response in intestinal epithelial cells

“…It is well established that activation of TLRs by their agonists significantly enhances the internalisation of bacteria in both professional immune cells, such as macrophages (Blander and Medzhitov, 2004;Doyle et al, 2004), and non-professional immune cells, such as intestinal epithelial cells (Neal et al, 2006). In addition to this, E. coli C25 has been used as a model strain for bacterial translocation through the intestinal epithelial barrier (Clark et al, 2003;Macutkiewicz et al, 2008;Suzuki and Okada, 2014); therefore, we decided to explore the regulatory effects of OMVs on this process. Consequently, we demonstrate that pretreatment with C25 OMVs was able to reduce the subsequent internalisation of the C25 bacterium in intestinal epithelial cells.…”

“…As mentioned previously, past studies have utilised E. coli C25 as a model strain for bacterial translocation across the intestinal epithelium (Clark et al, 2003;Macutkiewicz et al, 2008); therefore, we sought to investigate the regulatory ability of OMVs on this process. To explore this, we performed a bacterial internalisation assay in both HT29-19A and Caco-2 cells.…”

“…The culture was centrifuged at 10,000 x g for 10 min and the bacterial pellet was resuspended in 10 ml HBSS supplemented with 10 mM sodium bicarbonate and 180 mg/dl glucose (Trans-HBSS) in order to reduce bacterial growth during the assay (Clark et al, 2003).…”

“…Nevertheless, studies utilising live C25 have demonstrated its ability to translocate through the intestinal epithelial barrier (Clark et al, 2003;Macutkiewicz et al, 2008;Suzuki and Okada, 2014) and to initiate a proinflammatory response in intestinal epithelial cell lines (Michalsky et al, 1997;Clark et al, 2003;Zareie et al, 2005;Macutkiewicz et al, 2008). …”

Nutritional interventions in critical illness

“…Glutamine deprivation increases TNF-induced bacterial translocation in Caco-2 cells (Clark et al 2003) and regulates tightjunction proteins, claudin-1, occludin and ZO-1 . However, studies in cell lines have not examined the effects of change in glutamine concentration seen pathophysiologically in human subjects.…”