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The Laboratory of Fang Yan, M.D., Ph.D.
A probiotic-derived protein, p40, regulates intestinal inflammation
Probiotic bacteria may have beneficial effects on the clinical course of several intestinal disorders, but the understanding of the mechanisms underlying the effects of probiotics is limited. Dr. Yan's lab isolated a protein secreted by the probiotic bacterium, Lactobacillus rhamnosus GG (LGG), named p40 by Dr. Yan. Her group has analyzed the mechanisms by which p40 regulates cellular responses in intestinal epithelial cells. They have demonstrated how the p40 protein transactivates epidermal growth factor (EGF) receptor, and its downstream target, PI3K-dependent Akt, through stimulation of a disintegrin and metalloproteinase 17 (ADAM17)-mediated release of an EGFR ligand, HB-EGF, in intestinal epithelial cells. In further studies, a functional analysis of p40 has revealed that p40 preserves barrier function, suppresses cytokine-induced apoptosis, promotes mucin production in intestinal epithelial cells, and enhances IgA production through up-regulation of the expression of a proliferation-inducing ligand (APRIL) in intestinal epithelial cells for promoting B cell class switching and IgA production. Activation of an EGF receptor is required for these cellular responses in intestinal cells regulated by p40.
Dr. Yan's lab has also developed a pectin/zein hydrogel bead system to specifically deliver p40 to the mouse colon, activating an EGF receptor in mouse colon epithelial cells. Administration of the p40-containing beads reduces intestinal epithelial apoptosis and inflammation disruption of barrier function in the colon epithelium in an EGF receptor-dependent manner, thereby preventing and treating intestinal inflammation in mouse models of colitis. These, and ongoing, studies define a previously unrecognized mechanism of probiotic-derived soluble proteins in protecting the intestine from injury and inflammation.
Supplementation of p40 in early life promotes epidermal growth factor receptor-dependent intestinal development and long-term health outcomes
The beneficial effects of the gut microbiota on growth in early life are well known. However, knowledge about the mechanisms underlying regulating intestinal development by the microbiota is limited. Dr Yan’s lab has revealed that treatment with p40-containing hydrogels from postnatal day 2 to 21 significantly enhanced bodyweight gain prior to weaning and functional maturation of the intestine, including intestinal epithelial cell proliferation, differentiation, and tight junction formation, and IgA production in early life in wild-type mice. These p40-induced effects were abolished in mice with specific deletion of EGF receptor in intestinal epithelial cells, suggesting that transactivation of EGF receptor in intestinal epithelial cells may mediate p40-regulated intestinal development. Furthermore, neonatal p40 treatment reduced the susceptibility to intestinal injury and colitis and promoted protective immune responses, including IgA production and differentiation of regulatory T cells, in adult mice. These findings reveal novel roles of neonatal supplementation of probiotic-derived factors in promoting EGF receptor-mediated maturation of intestinal functions and innate immunity, which likely promote long-term beneficial outcomes.
Intestinal epithelial cells contribute to the function of the gut microbiota
Currently, discovering solutions to promote the host and microbiota interaction is an urgent need. It is known that the composition of the gut microbiota is shaped by host genetics, diets, and immune responses. However, knowledge about the impact of the host on functions of the gut microbial community is limited. The intestinal epithelial cells serve as the first line of host responses toward the gut microbiota. Thus, Dr. Yan’s lab is focused on exploring whether intestinal epithelial cells play roles in regulation of the function of the microbiota. By utilizing LGG as a model bacterium, intestinal epithelial cell-released extracellular vesicles have been found to participate in communication between intestinal epithelial cells and LGG. Colonic epithelial cell-derived constitutes contained in extracellular vesicles promote production of p40 by LGG, which indicates the specific regulatory effects of intestinal epithelial cells on LGG’s function. Furthermore, treatment of LGG with epithelial cell-derived-constitutes increases the ability of LGG for maintenance of intestinal integrity and the protective effect of LGG against colitis. This work is significant due to its focus on providing a proof-of-concept model to reveal a previously unrecognized contribution of intestinal epithelial cells to reinforcement of function of microbiota. Application of the knowledge gained from this work will be helpful for development of microbiota-targeted therapies to enhance the beneficial efficacy of probiotics for prevention and treatment of intestinal inflammatory disorders.
Regulation of gastrointestinal inflammation and cancer development by epidermal growth factor receptor signaling in macrophages
Macrophages display protective and detrimental functions in patients with inflammatory bowel disease and in animal models of colitis. It has been reported that EGF receptor and its family of ligands are present in macrophages, and EGF promotes the migration and proliferation of macrophages. However, the impact of EGF receptor activation on regulating immune responses in macrophages remains poorly understood. Results from Dr. Yan’s lab reveal a concept of cell-type specific (epithelial cell vs macrophage) function of the EGF receptor signaling in intestinal inflammation and tumor development.
Activation of the epidermal growth facotr receptor in macrophages accelerates colitis
Dr. Yan’s lab aims to determine potential roles and mechanisms of EGF receptor activation in macrophages for regulating cytokine production and experimental colitis. EGF receptor is activated in colonic macrophages in mice with colitis and in patients with ulcerative colitis. By using DSS-induced intestinal injury and inflammation model, Dr. Yan’s lab provides evidence that selective deletion of EGF receptor in macrophages leads to early increases in both pro- and anti-inflammatory cytokine production in response to inflammatory stimuli, and the sustained increase of the IL-10 level ultimately suppresses pro-inflammatory cytokine production, resulting in protection of mice from intestinal inflammation. Furthermore, LPS transactivates EGF receptor in macrophages and inhibition of EGF receptor kinase activity increases LPS plus IFN-g-stimulated TNF and IL-10 production. These results reveal an integrated response of macrophages regulated by EGF receptor activation in intestinal inflammatory disorders.
Activation of epidermal growth factor receptor in macrophages mediates feedback inhibition of alternative polarization and gastrointestinal tumor cell growth
Tumor-associated macrophages (TAMs) have characteristics of an M2 phenotype, which exhibits pro-tumorigenic activities. However, TAMs have been reported to be associated with diverse clinical outcomes in human cancers. Currently, the molecular mechanisms underlying the diverse functions of TAMs in different tumor microenvironment remains unclear. Clinical assessment of human cancer samples by Dr. Yan’s group has revealed that EGF receptor expression in macrophages is significantly decreased in advanced stages of gastric cancer, but not in colorectal cancer. Since it has been found that TAMs are associated with poor prognosis in intestinal type of gastric adenocarcinoma, but positive prognosis in colorectal adenocarcinoma, these results further suggest an association between EGF receptor signaling in TAMs and cancer development.
Further studies from Dr. Yan’s lab have demonstrated that IL-4, a known cytokine for inducing M2 polarization, stimulates HB-EGF release and transactivates EGF receptor activation, resulting in EGF receptor down-regulation in macrophages. Transactivation of EGF receptor in macrophages has a negative feedback effect on HB-EGF production and M2 polarization in in vitro and in vivo experiments. Accordingly, IL-4-conditioned medium from macrophages with blocking EGF receptor kinase activity and EGF receptor knock out enhances cell growth, colony formation, and epithelial-to-mesenchymal transition in gastric epithelial cells and colonic tumor cells, and promotes tumor growth in the colonic xenograft model. Thus, EGF receptor activation in macrophages could serve as a negative feedback mechanism involved in alternative polarization, thereby inhibiting gastrointestinal tumor growth. These findings provide pivotal information for understanding the cell-type specific function of EGF receptor in tumor development.