Sepsis Enhances Epithelial Permeability with Stretch in an Actin Dependent Manner Taylor S. Cohen, Brian C. DiPaolo, Gladys Gray Lawrence, Susan S. Margulies* Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America Abstract Ventilation of septic patients often leads to the development of edema and impaired gas exchange. We hypothesized that septic alveolar epithelial monolayers would experience stretch-induced barrier dysfunction at a lower magnitude of stretch than healthy alveolar epithelial monolayers. Alveolar epithelial cells were isolated from rats 24 hours after cecal ligation and double puncture (2CLP) or sham surgery. Following a 5-day culture period, monolayers were cyclically stretched for 0, 10, or 60 minutes to a magnitude of 12% or 25% change in surface area (DSA). Barrier function, MAPk and myosin light chain (MLC) phosphorylation, tight junction (TJ) protein expression and actin cytoskeletal organization were examined after stretch. Significant increases in epithelial permeability were observed only in 2CLP monolayers at the 12% DSA stretch level, and in both 2CLP and sham monolayers at the 25% DSA stretch level. Increased permeability in 2CLP monolayers was not associated with MAPk signaling or alterations in expression of TJ proteins. 2CLP monolayers had fewer actin stress fibers before stretch, a more robust stretch-induced actin redistribution, and reduced phosphorylated MLCK than sham monolayers. Jasplakinolide stabilization of the actin cytoskeleton in 2CLP monolayers prevented significant increases in permeability following 60 minutes of stretch to 12% DSA. We concluded that septic alveolar epithelial monolayers are more susceptible to stretch-induced barrier dysfunction than healthy monolayers due to actin reorganization. Citation: Cohen TS, DiPaolo BC, Gray Lawrence G, Margulies SS (2012) Sepsis Enhances Epithelial Permeability with Stretch in an Actin Dependent Manner. PLoS ONE 7(6): e38748. doi:10.1371/journal.pone.0038748 Editor: Rory Edward Morty, University of Giessen Lung Center, Germany Received February 6, 2012; Accepted May 11, 2012; Published June 19, 2012 Copyright: 2012 Cohen 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: Support was provided by National Institutes of Health RO1-HL057204. 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: margulies@seas.upenn.edu Introduction Acute lung injury (ALI) is a significant clinical problem with a reported mortality rate between 25% and 40% [1,2]. It is characterized by acute onset, severe hypoxemia, and pulmonary edema, and can develop following a variety of injuries such as pneumonia, drowning, reperfusion, smoke inhalation, or pulmo- nary hemorrhage [3]. As respiratory function declines, mechanical ventilation is required to maintain blood gas levels; however, while necessary for survival, ventilation can also injure the lung [4]. Mechanical ventilation has been shown to lead to ALI without the presence of other factors, but is often exacerbated by a predispos- ing condition [5], such as sepsis [6]. Previous studies have shown a synergistic relationship between ventilation, or stretch, and the expression of inflammatory mediators [7,8]. Independent studies by Nin et. al. and O’Mahony et. al. show increases in IL-6 expression following ventilation and either 2CLP in rats or administration of endotoxin (LPS) in mice, above levels observed with either ventilation or sepsis exposure independently. A clinical study on IL-6 conducted by Damas et. al. showed a strong correlation between IL-6 expression and patient mortality [9]. Together, these data demonstrate a more injurious systemic effect of the combined insult. Data identifying a synergistic effect of inflammation and ventilation on the expression of inflammatory cytokines, the development of pulmonary edema, and alveolar epithelial cell mortality have been previously published [10]. Ke-zhong et. al. have shown that in combination, intra-tracheal administration of endotoxin (LPS) and ventilation increased pulmonary edema (bronchial alveolar lavage protein concentration and lung wet/dry ratio increased) compared to LPS or ventilation alone [11]. Our lab has demonstrated the combined insult of a cecal ligation and puncture sepsis model and mechanical stretch increases alveolar epithelial permeability in the intact lung, and stretch-induced cell death of alveolar epithelial type II cells in vitro [12,13]. Taken together, these studies, as well as clinical reports of increased edema fluid in ALI patients, demonstrate that sepsis is associated with a loss of barrier function in the tissue separating the capillary and air spaces. In the lung, the alveoli are the principal sites of gas exchange. The alveolar wall is composed of opposing cell monolayers, the epithelium and endothelium, which are separated by a thin interstitial space. Of these two cell monolayers, the epithelium is thought to supply the majority of resistance to the motion of ions and proteins into the airways [14]. Previously, we demonstrated that alveolar epithelial cells isolated from the lungs of septic animals form confluent monolayers which exhibit increased permeability, altered tight junction (TJ) protein expression, and increased phosphorylation of mitogen activated protein kinase (MAPk) signaling pathways c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) [15]. Furthermore, we have shown that increases in JNK and ERK signaling activation are partially responsible for the permeability increases observed in healthy cells following 60 minutes of stretch at high magnitude (37% change in surface area (DSA) or 100% total lung capacity PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e38748