Lactobacillus rhamnosus GG (LGG) is one of the best-studied probiotic bacteria in clinical trials for treating and/or preventing several intestinal disorders, includig inflammatory bowel disease (IBD). However, the clinical application of LGG and other probiotics is limited by the paucity of information regarding their mechanisms of action. We have successfully purified and cloned two novel LGG-derived soluble proteins (p40 and p75) that prevent cytokine-induced apoptosis through activating Akt in intestinal epithelial cells. We focus on p40, a secreted protein which exerts more potent effects than p75, and have found that p40 activates epidermal growth factor (EGF) receptor, an known upstream signaling pathway regulating Akt and cell survival. Since increased production of inflammatory cytokines and epithelial cell apoptosis are two major pathogenic fcators for IBD, the goal of this research proposal is to define mechanisms by which p40 regulates intestinal epithelial cell function and determine the effects of p40 on intestinal inflammation. We will test the hypothesis that p40 prevents and/or treats intestinal inflammation through activating anti-apoptotic signals to inhibit cytokine-induced intestinal epithelial cell apoptosis.
Three Specific Aims are proposed to address this hypothesis:
Aim 1. To define p40-regulated signaling pathways for Akt activation and inhibition of cytokine- induced apoptosis in intestinal epithelial cells. We will focus on determining the requirement of EGF receptor activation by p40 for Akt activation and inibition of apoptosis using intestinal epithelial cells lacking EGF receptor expression. To further invstigate the mechanism of EGF receptor activation by p40, we will identify p40-stimulated EGF receptor ligand release using ELISA assays.
Aim 2. To determine the structure-functional requirements of p40 for LGG-regulated signaling pathways and survival of intestinal epithelial cells. We will precisely define the functional domain using deletion mutagenesis. Then we will generate p40 functional domain and mutant p40 with the functional domain deletion fusion proteins and determine their in vitro and in vivo effects on signaling and intestinal inflammation. The requirement of p40 for LGG's regulatory effects will be determined by inactivating p40 from the LGG chromosome using a single-crossover insertional integration system and comparing effects of wild-type to mutated LGG on cell signaling and survival.
Aim 3. To define the in vivo effects of p40 on intestinal inflammation in animal models of colitis. We will determine the optimal conditions for delivering p40 to the colon using the specific colon delivery strategy, the pectin/zein hydrogel system. The effects of p40 on prevention and/or treatment of inflammation and intestinal epithelial apoptosis will be detected in two mouse models of colitis, interleukin-10 and kinase suppressor of Ras double deficiency- elicited colitis and dextran sodium sulfate-induced colitis. The requirement of EGF receptor for p40's effects on inflammation will be analyzed using a EGF receptor kinase inhibitor and EGF receptor defective mice. Our long-term goal is to use p40 as a novel therapeutic agent for human intestinal inflammatory disorders.

Public Health Relevance

This proposal will provide fundamental knowledge of understanding the mechanisms by which p40, a secreted protein from a probiotic bacterium, Lactobacillus GG, regulates intestinal epithelial cell function and will dedermine the in vivo effects of p40 on preventing and/or treating intestinal inflammation using animal models of colitis. Therefore, the findings from this proposal will support a scientific basis for a potential therapeutic application of p40 for preventing and/or treating human gastrointestinal inflammatory disorders.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Gastrointestinal Mucosal Pathobiology Study Section (GMPB)
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Grey, Michael J
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Vanderbilt University Medical Center
Schools of Medicine
United States
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Shen, Xi; Liu, Liping; Peek, Richard M et al. (2018) Supplementation of p40, a Lactobacillus rhamnosus GG-derived protein, in early life promotes epidermal growth factor receptor-dependent intestinal development and long-term health outcomes. Mucosal Immunol 11:1316-1328
Yan, F; Liu, L; Cao, H et al. (2017) Neonatal colonization of mice with LGG promotes intestinal development and decreases susceptibility to colitis in adulthood. Mucosal Immunol 10:117-127
Wang, Y; Liu, L; Moore, D J et al. (2017) An LGG-derived protein promotes IgA production through upregulation of APRIL expression in intestinal epithelial cells. Mucosal Immunol 10:373-384
Zhao, Gang; Liu, Liping; Peek Jr, Richard M et al. (2016) Activation of Epidermal Growth Factor Receptor in Macrophages Mediates Feedback Inhibition of M2 Polarization and Gastrointestinal Tumor Cell Growth. J Biol Chem 291:20462-72
Li, Ran; Zhang, Yufeng; Polk, D Brent et al. (2016) Preserving viability of Lactobacillus rhamnosus GG in vitro and in vivo by a new encapsulation system. J Control Release 230:79-87
Wang, Lihong; Cao, Hailong; Liu, Liping et al. (2014) Activation of epidermal growth factor receptor mediates mucin production stimulated by p40, a Lactobacillus rhamnosus GG-derived protein. J Biol Chem 289:20234-44
Lu, Ning; Wang, Lihong; Cao, Hailong et al. (2014) Activation of the epidermal growth factor receptor in macrophages regulates cytokine production and experimental colitis. J Immunol 192:1013-23
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Yu, Jinpu; Wang, Yue; Yan, Fang et al. (2014) Noncanonical NF-?B activation mediates STAT3-stimulated IDO upregulation in myeloid-derived suppressor cells in breast cancer. J Immunol 193:2574-86
Wang, Lihong; Cao, Hailong; Lu, Ning et al. (2013) Berberine inhibits proliferation and down-regulates epidermal growth factor receptor through activation of Cbl in colon tumor cells. PLoS One 8:e56666

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