Alterations in the gut microbiota, referred to as dysbiosis, are strongly associated with chronic inflammatory bowel diseases. Neutrophils are one of the earliest cells to respond to an inflammatory stimulus and, in the inflamed intestine, constitute a significant proportion of cells that are recruited. Whereas probiotics have been shown to exert beneficial effects on gut homeostasis, their effect on neutrophil extracellular trap (NET) formation remains undefined. Therefore, the aim of this study was to determine the effects of the probiotic Lactobacillus rhamnosus GG (LGG) on the dynamics of NET formation. Bone marrow‐derived neutrophils (BMDN) were isolated from C57BL6 mice and co‐incubated with LGG (1 h, 37°C, 5% CO2) prior to stimulation with phorbol 12‐myristate 13‐acetate (PMA, 100 nM), which is an inducer of NET formation. Measurements of extracellular DNA (Sytox Green, 5 nM) and immunofluorescence microscopy staining for DNA, elastase and histones demonstrated that LGG mediated a concentration‐dependent reduction in NET‐osis. Given the proximity of recruited neutrophils to the lumen of the inflamed gut and the resident intestinal microbiota, it is important to define how neutrophil‐microbe interactions affect the local microenvironment. This work was funded by CIHR (operating grant MOP‐89894).

Intestinal epithelia regulate barrier integrity when challenged by inflammation, oxidative stress, and microbes. Transforming growth factor-β1 (TGF-β1) is a cytokine with known beneficial effects on intestinal epithelia, including barrier enhancement, after exposure to proinflammatory cytokines and infectious agents. The aim of this study was to determine whether TGF-β1 directly protects intestinal epithelia during hypoxia-reoxygenation (HR). Intestinal epithelial monolayers (T84, Caco-2) were exposed to either hypoxia (1% O2, 1 h) or oxidative stress (hydrogen peroxide, 1 mM), followed by normoxic atmosphere for different time points in the absence and presence of varying concentrations of TGF-β1. Transepithelial electrical resistance (TER) assessed barrier function, with RNA extracted for reverse transcription polymerase chain reaction analysis of GPx-1, HIF-1, heme-oxygenase-1 (HO-1), and NOX-1. In some experiments, intestinal epithelia were exposed to enterohemorrhagic Escherichia coli (EHEC) O157:H7 during the reoxygenation period and TER recorded 7 h after the infectious challenge. Hypoxia-reoxygenation significantly decreased TER in intestinal epithelia compared with normoxic controls. Transforming growth factor-β1 pretreatment ameliorated HR-induced epithelial barrier dysfunction in T84 (at 1 - 3 h) and Caco-2 (1 h) monolayers. Transforming growth factor-β1 preserved barrier integrity for up to 16 h after challenge with hydrogen peroxide. In TGF-β1-treated epithelial monolayers, only HO-1 mRNA significantly increased after HR (P < 0.05 vs. normoxic controls). The EHEC-induced epithelial barrier dysfunction was significantly worsened by intestinal HR (P < 0.05 vs. normoxia-EHEC-infected cells), but this was not protected by TGF-β1 pretreatment. Transforming growth factor-β1 preserves loss of epithelial barrier integrity caused by the stress of HR via a mechanism that may involve the upregulation of HO-1 transcription. Targeted treatment with TGF-β could lead to novel therapies in enteric diseases characterized by HR injury.

Alterations in the gut microbiota, referred to as dysbiosis, are strongly associated with chronic inflammatory bowel diseases. Neutrophils are one of the earliest cells to respond to an inflammatory stimulus and, in the inflamed intestine, constitute a significant proportion of cells that are recruited. Whereas probiotics have been shown to exert beneficial effects on gut homeostasis, their effect on neutrophil extracellular trap (NET) formation remains undefined. Therefore, the aim of this study was to determine the effects of the probiotic Lactobacillus rhamnosus GG (LGG) on the dynamics of NET formation. Bone marrow‐derived neutrophils (BMDN) were isolated from C57BL6 mice and co‐incubated with LGG (1 h, 37°C, 5% CO2) prior to stimulation with phorbol 12‐myristate 13‐acetate (PMA, 100 nM), which is an inducer of NET formation. Measurements of extracellular DNA (Sytox Green, 5 nM) and immunofluorescence microscopy staining for DNA, elastase and histones demonstrated that LGG mediated a concentration‐dependent reduction in NET‐osis. Given the proximity of recruited neutrophils to the lumen of the inflamed gut and the resident intestinal microbiota, it is important to define how neutrophil‐microbe interactions affect the local microenvironment. This work was funded by CIHR (operating grant MOP‐89894).