Inna Grosheva1, Danping Zheng2, Maayan Levy3, Omer Polansky4, Alexandra Lichtenstein4, Ofra Golani5, Mally Dori-Bachash6, Claudia Moresi6, Hagit Shapiro6, Sara Del Mare-Roumani7, Rafael Valdes-Mas6, Yiming He2, Hodaya Karbi6, Minhu Chen8, Alon Harmelin9, Ravid Straussman4, Nissan Yissachar7, Eran Elinav10, Benjamin Geiger11. 1. Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel; Immunology Department, Weizmann Institute of Science, Rehovot, Israel. 2. Immunology Department, Weizmann Institute of Science, Rehovot, Israel; Department of Gastroenterology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. 3. Immunology Department, Weizmann Institute of Science, Rehovot, Israel; Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania; Institute for Immunology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania. 4. Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. 5. Department of Life Sciences Core Facilities, Weizmann Institute of Science, Rehovot, Israel. 6. Immunology Department, Weizmann Institute of Science, Rehovot, Israel. 7. The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan, Israel. 8. Department of Gastroenterology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. 9. Department of Veterinary Resources, Weizmann Institute of Science, Rehovot, Israel. 10. Immunology Department, Weizmann Institute of Science, Rehovot, Israel; Cancer-Microbiome Research Division, DKFZ, Heidelberg, Germany. Electronic address: eran.elinav@weizmann.ac.il. 11. Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel; Immunology Department, Weizmann Institute of Science, Rehovot, Israel. Electronic address: benny.geiger@weizmann.ac.il.
Abstract
BACKGROUND & AIMS: The intestinal barrier protects intestinal cells from microbes and antigens in the lumen-breaches can alter the composition of the intestinal microbiota, the enteric immune system, and metabolism. We performed a screen to identify molecules that disrupt and support the intestinal epithelial barrier and tested their effects in mice. METHODS: We performed an imaging-based, quantitative, high-throughput screen (using CaCo-2 and T84 cells incubated with lipopolysaccharide; tumor necrosis factor; histamine; receptor antagonists; and libraries of secreted proteins, microbial metabolites, and drugs) to identify molecules that altered epithelial tight junction (TJ) and focal adhesion morphology. We then tested the effects of TJ stabilizers on these changes. Molecules we found to disrupt or stabilize TJs were administered mice with dextran sodium sulfate-induced colitis or Citrobacter rodentium-induced intestinal inflammation. Colon tissues were collected and analyzed by histology, fluorescence microscopy, and RNA sequencing. RESULTS: The screen identified numerous compounds that disrupted or stabilized (after disruption) TJs and monolayers of epithelial cells. We associated distinct morphologic alterations with changes in barrier function, and identified a variety of cytokines, metabolites, and drugs (including inhibitors of actomyosin contractility) that prevent disruption of TJs and restore TJ integrity. One of these disruptors (putrescine) disrupted TJ integrity in ex vivo mouse colon tissues; administration to mice exacerbated colon inflammation, increased gut permeability, reduced colon transepithelial electrical resistance, increased pattern recognition receptor ligands in mesenteric lymph nodes, and decreased colon length and survival times. Putrescine also increased intestine levels and fecal shedding of viable C rodentium, increased bacterial attachment to the colonic epithelium, and increased levels of inflammatory cytokines in colon tissues. Colonic epithelial cells from mice given putrescine increased expression of genes that regulate metal binding, oxidative stress, and cytoskeletal organization and contractility. Co-administration of taurine with putrescine blocked disruption of TJs and the exacerbated inflammation. CONCLUSIONS: We identified molecules that disrupt and stabilize intestinal epithelial TJs and barrier function and affect development of colon inflammation in mice. These agents might be developed for treatment of barrier intestinal impairment-associated and inflammatory disorders in patients, or avoided to prevent inflammation.
BACKGROUND & AIMS: The intestinal barrier protects intestinal cells from microbes and antigens in the lumen-breaches can alter the composition of the intestinal microbiota, the enteric immune system, and metabolism. We performed a screen to identify molecules that disrupt and support the intestinal epithelial barrier and tested their effects in mice. METHODS: We performed an imaging-based, quantitative, high-throughput screen (using CaCo-2 and T84 cells incubated with lipopolysaccharide; tumor necrosis factor; histamine; receptor antagonists; and libraries of secreted proteins, microbial metabolites, and drugs) to identify molecules that altered epithelial tight junction (TJ) and focal adhesion morphology. We then tested the effects of TJ stabilizers on these changes. Molecules we found to disrupt or stabilize TJs were administered mice with dextran sodium sulfate-induced colitis or Citrobacter rodentium-induced intestinal inflammation. Colon tissues were collected and analyzed by histology, fluorescence microscopy, and RNA sequencing. RESULTS: The screen identified numerous compounds that disrupted or stabilized (after disruption) TJs and monolayers of epithelial cells. We associated distinct morphologic alterations with changes in barrier function, and identified a variety of cytokines, metabolites, and drugs (including inhibitors of actomyosin contractility) that prevent disruption of TJs and restore TJ integrity. One of these disruptors (putrescine) disrupted TJ integrity in ex vivo mouse colon tissues; administration to mice exacerbated colon inflammation, increased gut permeability, reduced colon transepithelial electrical resistance, increased pattern recognition receptor ligands in mesenteric lymph nodes, and decreased colon length and survival times. Putrescine also increased intestine levels and fecal shedding of viable C rodentium, increased bacterial attachment to the colonic epithelium, and increased levels of inflammatory cytokines in colon tissues. Colonic epithelial cells from mice given putrescine increased expression of genes that regulate metal binding, oxidative stress, and cytoskeletal organization and contractility. Co-administration of taurine with putrescine blocked disruption of TJs and the exacerbated inflammation. CONCLUSIONS: We identified molecules that disrupt and stabilize intestinal epithelial TJs and barrier function and affect development of colon inflammation in mice. These agents might be developed for treatment of barrier intestinal impairment-associated and inflammatory disorders in patients, or avoided to prevent inflammation.