HYPOXIC STRESS, HEPATOCYTES AND CACO-2 VIABILITY AND SUSCEPTIBILITY TO Shigella flexneri INVASION

Abstract

SUMMARYInflammation due to Shigella flexneri can cause damage to the colonic mucosa and cell death by necrosis and apoptosis. This bacteria can reach the bloodstream in this way, and the liver through portal veins. Hypoxia is a condition present in many human diseases, and it may induce bacterial translocation from intestinal lumen. We studied the ability of S. flexneri to invade rat hepatocytes and Caco-2 cells both in normoxic and hypoxic microenvironments, as well as morphological and physiological alterations in these cells after infection under hypoxia. We used the primary culture of rat hepatocytes as a model of study. We analyzed the following parameters in normoxic and hypoxic conditions: morphology, cell viability, bacterial recovery and lactate dehydrogenase (LDH) released. The results showed that there were fewer bacteria within the Caco-2 cells than in hepatocytes in normoxic and hypoxic conditions. We observed that the higher the multiplicity of infection (MOI) the greater the bacterial recovery in hepatocytes. The hypoxic condition decreased the bacterial recovery in hepatocytes. The cytotoxicity evaluated by LDH released by cells was significantly higher in cells submitted to hypoxia than normoxia. Caco-2 cells in normoxia released 63% more LDH than hepatocytes. LDH increased 164% when hepatocytes were submitted to hypoxia and just 21% when Caco-2 cells were in the same condition. The apoptosis evaluated by Tunel was significantly higher in cells submitted to hypoxia than normoxia. When comparing hypoxic cells, we obtained more apoptotic hepatocytes than apoptotic Caco-2 cells. Concluding our results contribute to a better knowledge of interactions between studied cells and Shigella flexneri. These data may be useful in the future to define strategies to combat this virulent pathogen.

The impact of hypoxia on intestinal epithelial cell functions: consequences for invasion by bacterial pathogens

“…P. aeruginosa internalizes into host epithelial cells by the trigger mechanism, using one of its injected effector proteins, ExoS that binds to the mammalian factor FXYD3 [ 29 ]. Interestingly, these abovementioned three intestinal pathogenic bacteria exhibit decreased levels of internalization into intestinal epithelial cells (for Y. enterocolitica and S. flexneri ) or pulmonary epithelial cells (for P. aeruginosa) pre-incubated under 1 % O 2 for 24 h [ 30 , 31 ]. In light of these results, understanding the cellular effects of hypoxia on epithelial cells, e.g., membrane alterations, cytoskeletal rearrangements, or endoplasmatic reticulum (ER) stress during infections, offers a new potential to find targets for pharmacological interference.…”

Hypoxia Decreases Invasin-Mediated Yersinia enterocolitica Internalization into Caco-2 Cells

“…Interestingly, a decrease of bacterial internalization under hypoxia was shown for Shigella flexneri into host epithelial cells in a predicted HIF-1α -dependent manner [ 90 ]. Moreover, Pseudomonas aeruginosa entry into alveolar cells was decreased under hypoxia and after DMOG treatment, confirming the role of HIF-1α in an in vivo pneumonia model [ 91 ].…”

Developing models of the small intestine

“…Inflammation raises local vasculitis thus reduced O 2 availability to the inflamed region, furthermore, transmigration of neutrophils can consume regional O 2 (Karhausen et al 2004). In addition, hypoxia can alter gene expression, intra-and extra-cellular pH and membrane receptors (Lima et al 2013). In the current study, the influence of hypoxia on the cellular behaviour in monocultures and co-cultures was examined.…”

“…Apoptotic cells were less frequent in 90% Caco-2 / 10% HT29-MTX than 169 seen in 75% Caco-2 / 25% HT29-MTX co-culture. Caco-2 cells consume less oxygen under hypoxia and thus may be better adapted to hypoxic conditions (Lima et al 2013).…”

“…Caco-2 cells have also been used to study the intestinal epithelial response to hypoxia (Zeitouni et al 2016b). Lima et al (2013) studied the effect of Shigella flexneri on co-cultured Caco-2 cells and rat hepatocytes in normoxia and hypoxia (<1% O 2 ) conditions, resulted in apoptosis. Similary, Caco-2 cells cultured on polystyrene and grown under hypoxia at 1% O 2 showed a significant decrease in brush border membrane expression of β1 integrins, which resulted in decreased Y. enterocolitica entry into Caco-2 cells (Zeitouni et al 2016b).…”