Stretch of Septic Monolayers Figure 4. Phalloidin staining of actin in sham and 2CLP monolayers in unstretched, stretched cyclically for 60 minutes to 12% DSA, and stretched cyclically for 60 minutes to 25% DSA wells. Similar staining patterns were observed in sham unstretched monolayers and sham monolayers stretched to 12% DSA. Intensity of the actin stain in unstretched 2CLP monolayers was reduced compared to unstretched sham, and a thin cortical actin ring is apparent (arrow). In 2CLP monolayers stretched to 12% DSA, stress fibers form a circular band throughout the cytoplasm of the cell, and become more prominent compared to unstretched monolayers. In cells stretched to 25% DSA, cortical rings form (arrow), which are also more prominent in 2CLP monolayers compared to sham. Scale bar 100 mm. Representative of images from 2 isolations with 2 wells per isolation. doi:10.1371/journal.pone.0038748.g004 the combined insult would then result in an amplified signal leading to barrier dysfunction, even at sub-injurious magnitude. We showed that in a culture model of the alveolar epithelium, the threshold stretch magnitude above which permeability to a 20 A˚ tracer increases is lower in monolayers formed from cells isolated from septic animals compared to sham animals. These data confirm previous results from the ex vivo whole lung in which flux of a fluorescent tracer (FITC labeled albumin, 60 A˚) across the endothelium and epithelium increases in lungs from 2CLP animals, not sham, ventilated with a tidal volume of 20 ml/kg body weight (,25% DSA) for 30 minutes [12,18]. Therefore intact lung and isolated cell preparations demonstrated stretch-induced barrier dysfunction in the epithelium of septic animals at levels where healthy epithelia are unaffected. We previously observed both sepsis and mechanical deforma- tion activate common MAPk signaling pathways. Studies in the intact animal have identified epithelial MAPk signaling as being activated following both large tidal volume ventilation (ERK, JNK) in rats and 2CLP (ERK, JNK, p38) in mice [26,27]. Additional studies have shown activation of MAPk signaling in vitro following bacterial stimulation, administration of endotoxin, and cyclic stretch [28–32]. Based on this body of literature, we hypothesized that the MAPk pathways would modulate the synergistic response to simultaneous insults. When we probed for activation of JNK, ERK, and p38 MAPk in 2CLP and sham monolayers following stretch to 12% DSA, we observed no significant phosphorylation of any MAPk above unstretched levels. We conclude that the dual insult of stretch and sepsis is not PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e38748