Mechanisms of CRF-Induced Intestinal Dysfunction Figure 5. Histologic analysis of CRF-mediated intestinal mast cell degranulation. Treatment of porcine ileal tissues mounted on Ussing chambers with CRF caused and increase in mast cell degranulation as determined by toluidine blue staining. Arrows indicate the release of mast cell granules into surrounding tissues. Sodium cromolyn, a mast cell stabilizing agent, prevented CRF-induced mast cell degranulation. Figures are representative of ileal tissue sections from n = 4 pigs. doi:10.1371/journal.pone.0039935.g005 barrier dysfunction is not completely understood. Several groups have shown that activation of intestinal mast cells is a critical component in stress-induced intestinal barrier disturbances [11,26,27]. It remains unclear how CRF triggers mast cell activation and which mast cell mediators are responsible for triggering CRF mediated barrier changes. We show here that in porcine ileum, CRF induces release of mast cell proteases and TNF-a and that each of these mediators contribute significantly to CRF-induced intestinal barrier dysfunction as pharmacologic inhibition of protease activity and anti-TNF-a reduced CRF- induced elevations in intestinal permeability. The mechanisms by which mast cell proteases and TNF-a are working to induce epithelial barrier dysfunctions are currently unclear but appear to be mediating their effects on the paracellular tight junction as CRF treatment disrupted the localization of the tight junction protein occludin. It is well-known that TNF-a and proteases can trigger epithelial barrier disruption although via different mechanisms. TNF-a can potently enhance epithelial permeability through increased myosin light-chain kinase (MLCK) expression and activity [28] which alters size selectivity of the tight junction to increase paracellular flux of uncharged macromolecules [29]. In addition, increased TNF-a levels in vivo has recently been shown to regulate intestinal epithelial tight junctions independent of the actomyosin ring [30] by triggering caveolin-1 dependent endocy- tosis of occludin that could cause loss of barrier function. Additionally, TNF-a induces an increase of tight junction permeability in vitro through the activation of NF-kB and the subsequent down-regulation and alteration of ZO-1 protein expression [28]. MC proteases such as tryptase induce barrier dysfunction via alternate signaling mechanisms [31]. In an in vitro cell co-culture model using the HMC-1 human mast cell line and T84 intestinal epithelial cells, it was shown that MC derived tryptase impaired barrier function via activation proteinase- activated-receptor-2 (PAR-2) triggering tight junction breakdown and increased paracellular permeability [31]. This pathway was shown to involve B-arrestin-dependent activation of ERK 1/2, then, regulates the reorganization of perijunctional F-actin in the tight junctions of colonocytes, causing an increase in intestinal permeability [31], [32]. Mice deficient in mast cell protease 4, a homologue for human chymase, were shown to have decreased intestinal permeability and reduced expression of claudin-3 indicating that mast cell chymase also plays an important role in regulating intestinal permeability [33]. Given that the PI cocktail used in the current study contained both tryptase and chymase inhibitors, we were not able to discern which specific mast cell proteases (tryptase and/or chymase) were responsible for CRF- mediated intestinal barrier dysfunction. In the present study, CRF induced changes in paracellular permeability as indicated by FD4 flux; but had no effect on TER. The conflicting data between paracellular flux and TER has been observed by others [6] and may be due to activation of separate epithelial permeability pathways by CRF. For example, TNF-a has been shown to initiate MLCK dependent changes in size selectivity of the tight junction to enhance paracellular flux of uncharged macromolecules without affecting charge, while IL-13 dependent claudin-2 expression was shown to increase paracel- lular permeability to cation flux in vitro and in vivo without altering tight junction size selectivity [29,30]. These ‘leak’ and ‘pore’ pathways, respectively, are quickly gaining evidence for differential regulation of paracellular permeability pathways [29,34], and PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e39935