Colon-specific delivery of a probiotic-derived soluble protein ameliorates intestinal inflammation in mice through an EGFR-dependent mechanism
Fang Yan, … , Keith T. Wilson, D. Brent Polk
Fang Yan, … , Keith T. Wilson, D. Brent Polk
Published May 23, 2011
Citation Information: J Clin Invest. 2011;121(6):2242-2253. https://doi.org/10.1172/JCI44031.
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Research Article

Colon-specific delivery of a probiotic-derived soluble protein ameliorates intestinal inflammation in mice through an EGFR-dependent mechanism

Abstract

Probiotic bacteria can potentially have beneficial effects on the clinical course of several intestinal disorders, but our understanding of probiotic action is limited. We have identified a probiotic bacteria–derived soluble protein, p40, from Lactobacillus rhamnosus GG (LGG), which prevents cytokine-induced apoptosis in intestinal epithelial cells. In the current study, we analyzed the mechanisms by which p40 regulates cellular responses in intestinal epithelial cells and p40’s effects on experimental colitis using mouse models. We show that the recombinant p40 protein activated EGFR, leading to Akt activation. Activation of EGFR by p40 was required for inhibition of cytokine-induced apoptosis in intestinal epithelial cells in vitro and ex vivo. Furthermore, we developed a pectin/zein hydrogel bead system to specifically deliver p40 to the mouse colon, which activated EGFR in colon epithelial cells. Administration of p40-containing beads reduced intestinal epithelial apoptosis and disruption of barrier function in the colon epithelium in an EGFR-dependent manner, thereby preventing and treating DSS-induced intestinal injury and acute colitis. Furthermore, p40 activation of EGFR was required for ameliorating colon epithelial cell apoptosis and chronic inflammation in oxazolone-induced colitis. These data define what we believe to be a previously unrecognized mechanism of probiotic-derived soluble proteins in protecting the intestine from injury and inflammation.

Authors

Fang Yan, Hanwei Cao, Timothy L. Cover, M. Kay Washington, Yan Shi, LinShu Liu, Rupesh Chaturvedi, Richard M. Peek Jr., Keith T. Wilson, D. Brent Polk

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Figure 2

Activation of EGFR is required for p40-stimulated prevention of cytokine-induced apoptosis and disruption of tight junctions in colon epithelial cells.

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Activation of EGFR is required for p40-stimulated prevention of cytokine...
HT-29 cells transfected with EGFR siRNA or nontargeting siRNA (A and B) for 24 hours were treated with the cytokine cocktail combination of TNF (100 ng/ml), IL-1α (10 ng/ml), and IFN-γ (100 ng/ml) for 16 hours (B). HT-29 cells were dissociated, stained with annexin V–FITC and propidium iodide, and analyzed using flow cytometry. Percentage of apoptosis is shown in B. *P < 0.01 compared with control in non-target or EGFR siRNA–transfected cells; #P < 0.01 compared with TNF/IL-1α/IFN-γ treatment in non-target siRNA–transfected cells. HT-29 cells were treated with the cytokine cocktail as indicated in B for 8 hours, with or without 1-hour pretreatment using AG1478 (150 nM) or wortmannin (100 nM). (C) Caspase activity in living cells was detected using the Sulforhodamine Multi-Caspase Activity Kit, with caspase-active cells staining red. The percentage of cells with active caspase is shown in D.*P < 0.01 compared with control; #P < 0.05 and §P < 0.01 compared with TNF/IL-1α/IFN-γ treatment. Colon explants derived from 6- to 8-week-old mice were cultured in DMEM containing 0.5% FBS and treated with TNF (100 ng/ml) for 8 hours in the presence or absence of p40 (10 ng/ml). (E) Paraffin-embedded tissue sections were prepared for detecting apoptosis using ISOL staining. Apoptotic nuclei (brown nuclei) labeled with peroxidase were visualized using differential interference contrast (DIC) microcopy. The percentage of crypts with apoptotic cells is shown. n = 3 mice for each group. Three colon explants were cultured from each mouse for each treatment condition. Original magnification, ×20 (C); ×40 (E).