Intestinal barrier function is compromised in inflammatory, infectious, ischemic, and immune-mediated intestinal disease. In the previous cycle we studied mechanisms and impact of tumor necrosis factor- (TNF-) induced barrier loss using in vitro and in vivo models to: i) define TNF-induced, myosin light chain kinase- (MLCK-) dependent tight junction regulation;ii) show that MLCK is required for TNF-induced diarrhea;iii) identify and characterize the human MLCK promoter;iv) document MLCK activation in human inflammatory bowel disease (IBD);and v) show that chronic epithelial MLCK activation stimulates mucosal immune cells and sensitizes mice to experimental IBD. We have now begun to define the mechanisms by which TNF specifically activates MLCK1, a long MLCK splice variant, to trigger endocytosis of the tight junction protein occludin. This creates tight junction 'leaks'that allow paracellular flux of large macromolecules without affecting ion selectivity. Our preliminary data show that expression of constitutively-active MLCK in vivo increases flux across the 'leak'pathway. Moreover, unlike in vitro MLCK activation, chronic in vivo MLCK activation alters ion selectivity of the paracellular barrier. This is due to increased mucosal IL-13 production, which induces epithelial claudin-2 expression to create small, cation-selective 'pores'that allow Na+, but not large macromolecules, to traverse the paracellular pathway. This in vivo result emphasizes the presence of, as well as interactions between, two distinct pathways across the tight junction;a high capacity 'pore'pathway that allows small uncharged solutes and specific ions to pass and a low capacity 'leak'pathway that is permeable to larger, uncharged macromolecules but is not ion selective. The fact that 'pore'and 'leak'pathways interact through the mucosal immune system, i.e. IL-13 production, suggests that these pathways may make distinct contributions to disease pathogenesis. These and other data have led to the central hypothesis that at least two mechanisms of regulation modulate unique paracellular pathways, i.e. 'pore'and 'leak,'to differentially impact mucosal homeostasis and disease pathogenesis.
The aims of this application are to i) define the mechanisms by which TNF triggers MLCK1 perijunctional trafficking and enzymatic activation to enhance flux across the tight junction 'leak'pathway;ii) identify the mechanisms that regulate claudin-2 expression and 'pore'pathway flux;and iii) determine the impact of MLCK, occludin, and claudin-2 expression on 'pore'and 'leak'pathway function as well as initiation and progression of chronic intestinal disease. As a whole, these studies will define the relative roles of specific epithelial regulatory processes and immune signaling in modulation of 'pore'and 'leak'pathway paracellular flux and disease pathogenesis. The data, which will provide new understanding of the mechanisms by which intestinal barrier function contributes to human health and disease, will lay the foundation necessary for development of novel therapeutic strategies to correct barrier dysfunction.

Public Health Relevance

The intestinal lining (epithelium) must maintain a barrier that keeps the intestinal contents separate from the remainder of the body. This function is frequently compromised in intestinal disease and has been implicated as an early step in disease development. The proposed studies will advance mechanistic understanding of barrier regulation and dysregulation and, therefore, lead to development of novel therapeutic approaches to improve human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK068271-08
Application #
8290500
Study Section
Special Emphasis Panel (ZRG1-DKUS-C (03))
Program Officer
Grey, Michael J
Project Start
2004-07-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
8
Fiscal Year
2012
Total Cost
$472,473
Indirect Cost
$168,742
Name
University of Chicago
Department
Pathology
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Ronaghan, Natalie J; Shang, Judie; Iablokov, Vadim et al. (2016) The serine protease-mediated increase in intestinal epithelial barrier function is dependent on occludin and requires an intact tight junction. Am J Physiol Gastrointest Liver Physiol 311:G466-79
Choi, Vivian M; Herrou, Julien; Hecht, Aaron L et al. (2016) Activation of Bacteroides fragilis toxin by a novel bacterial protease contributes to anaerobic sepsis in mice. Nat Med 22:563-7
Herrmann, Jeremy R; Turner, Jerrold R (2016) Beyond Ussing's chambers: contemporary thoughts on integration of transepithelial transport. Am J Physiol Cell Physiol 310:C423-31
Setty, Mala; Discepolo, Valentina; Abadie, Valérie et al. (2015) Distinct and Synergistic Contributions of Epithelial Stress and Adaptive Immunity to Functions of Intraepithelial Killer Cells and Active Celiac Disease. Gastroenterology 149:681-91.e10
Luther, Jay; Garber, John J; Khalili, Hamed et al. (2015) Hepatic Injury in Nonalcoholic Steatohepatitis Contributes to Altered Intestinal Permeability. Cell Mol Gastroenterol Hepatol 1:222-232
Pan, Jieyan; Zhang, Lili; Odenwald, Matthew A et al. (2015) Expression of human decay-accelerating factor on intestinal epithelium of transgenic mice does not facilitate infection by the enteral route. J Virol 89:4311-8
Sideri, Aristea; Stavrakis, Dimitris; Bowe, Collin et al. (2015) Effects of obesity on severity of colitis and cytokine expression in mouse mesenteric fat. Potential role of adiponectin receptor 1. Am J Physiol Gastrointest Liver Physiol 308:G591-604
Lingaraju, Amulya; Long, Tiha M; Wang, Yitang et al. (2015) Conceptual barriers to understanding physical barriers. Semin Cell Dev Biol 42:13-21
Edelblum, Karen L; Singh, Gurminder; Odenwald, Matthew A et al. (2015) γδ Intraepithelial Lymphocyte Migration Limits Transepithelial Pathogen Invasion and Systemic Disease in Mice. Gastroenterology 148:1417-26
Weber, Christopher R; Liang, Guo Hua; Wang, Yitang et al. (2015) Claudin-2-dependent paracellular channels are dynamically gated. Elife 4:e09906

Showing the most recent 10 out of 81 publications