CRF Induces Intestinal Epithelial Barrier Injury via the Release of Mast Cell Proteases and TNF-a Elizabeth L. Overman1, Jean E. Rivier2, Adam J. Moeser1* 1 College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America, 2 The Salk Institute, La Jolla, California, United States of America Abstract Background and Aims: Psychological stress is a predisposing factor in the onset and exacerbation of important gastrointestinal diseases including irritable bowel syndrome (IBS) and the inflammatory bowel diseases (IBD). The pathophysiology of stress-induced intestinal disturbances is known to be mediated by corticotropin releasing factor (CRF) but the precise signaling pathways remain poorly understood. Utilizing a porcine ex vivo intestinal model, the aim of this study was to investigate the mechanisms by which CRF mediates intestinal epithelial barrier disturbances. Methodology: Ileum was harvested from 6–8 week-old pigs, mounted on Ussing Chambers, and exposed to CRF in the presence or absence of various pharmacologic inhibitors of CRF-mediated signaling pathways. Mucosal-to-serosal flux of 4 kDa-FITC dextran (FD4) and transepithelial electrical resistance (TER) were recorded as indices of intestinal epithelial barrier function. Results: Exposure of porcine ileum to 0.05–0.5 mM CRF increased (p,0.05) paracellular flux compared with vehicle controls. CRF treatment had no deleterious effects on ileal TER. The effects of CRF on FD4 flux were inhibited with pre-treatment of tissue with the non-selective CRF1/2 receptor antagonist Astressin B and the mast cell stabilizer sodium cromolyn (1024 M). Furthermore, anti-TNF-a neutralizing antibody (p,0.01), protease inhibitors (p,0.01) and the neural blocker tetrodotoxin (TTX) inhibited CRF-mediated intestinal barrier dysfunction. Conclusion: These data demonstrate that CRF triggers increases in intestinal paracellular permeability via mast cell dependent release of TNF-a and proteases. Furthermore, CRF-mast cell signaling pathways and increases in intestinal permeability require critical input from the enteric nervous system. Therefore, blocking the deleterious effects of CRF may address the enteric signaling of mast cell degranulation, TNFa release, and protease secretion, hallmarks of IBS and IBD. Citation: Overman EL, Rivier JE, Moeser AJ (2012) CRF Induces Intestinal Epithelial Barrier Injury via the Release of Mast Cell Proteases and TNF-a. PLoS ONE 7(6): e39935. doi:10.1371/journal.pone.0039935 Editor: Jo¨ rn Karhausen, Duke University Medical Center, United States of America Received May 1, 2012; Accepted May 29, 2012; Published June 29, 2012 Copyright: 2012 Overman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by National Institutes of Health grant NIH P30 DK34987 (UNC Chapel Hill Center for Gastrointestinal Biology and Disease) and K08 DK084313 (AJM) and DKPO1026741 (JER). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ajmoeser@ncsu.edu Introduction The gastrointestinal barrier, consisting primarily of intestinal epithelial cells, mucus layer, and sub-epithelial immune cells, selectively controls the access of the immense luminal load of antigens and resident microorganism to the underlying lamina propria immune tissues [1]. It is well-known that intestinal barrier function can be adversely affected by acute or chronic psycholog- ical stress, resulting in increased intestinal permeability [2,3,4,5,6], a critical event in the onset of clinical symptoms of GI disorders including irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) [7,8,9,10]. Disturbances in intestinal barrier facilitates bacterial movement from the lumen into the lamina propria while also critically impairing other vital functions including absorption of nutrients, transport of ions, secretion [11,12], motility, and visceral hypersensitivity [13,14]. Although it is known that stress compromises intestinal barrier function, the precise mechanisms remain poorly understood. CRF is a 41 amino acid peptide, produced in the central nervous system and peripheral tissues [15,16] in response to stress and has been shown to play a central role in stress-induced intestinal pathophysiology. CRF activity is mediated by activa- tion of specific seven transmembrane G-protein coupled recep- tors (GPCRs) known as CRF1 and CRF2. CRF has been shown to induce intestinal barrier disturbances in multiple animals and human tissues models. Several studies have demonstrated that CRF mediates its effects via mast cell activation [6,11,17]. Upon activation, mast cells are capable of releasing a variety of pro- inflammatory mediators, including de novo synthesized media- tors such as prostaglandins, leukotrienes, and cytokines or preformed granule-housed mediators including histamine, serine proteases, tryptase, chymase, and cytokines [18], which pro- foundly influence intestinal epithelial barrier function; however, the mast cell mediators and signaling pathways that are responsible for CRF-mediated intestinal barrier dysfunction have not been fully elucidated. Here, utilizing a porcine model, we PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e39935