Ethanol increases post-burn intestinal permeability: IL-6-induced MLCK activation

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6. Abstract Ethanol is a common factor in traumatic injury, including burn injury. Previous studies from our laboratory indicate that ethanol increases both pulmonary and gastrointestinal inflammation as well as susceptibility to infection in burn-injured mice; however, the mechanisms of these responses are not entirely known. Moreover, after burn injury, bacteria and their products leak out of the intestinal lumen and into the bloodstream. These bacteria can disperse throughout the body leading to pulmonary damage, acute lung injury, sepsis, and death. Ethanol in combination with burn injury has been shown to decrease intestinal barrier function greater than either insult alone. Long (210 kDa) smooth muscle myosin light chain kinase (MLCK), an enzyme important for epithelial tight junction maintenance, has been implicated in barrier alterations after burn injury or ethanol exposure alone. With this knowledge, we hypothesize that the combination of acute ethanol exposure and burn injury causes an increase in intestinal barrier dysfunction due to interleukin-6-mediated activation of myosin light chain kinase. To test this hypothesis, three aims are proposed: to determine whether 1) elevated myosin light chain kinase activation mediates the intestinal permeability alterations in mice receiving acute ethanol and burn injury treatment, 2) interleukin-6 (IL-6) activates myosin light chain kinase and, 3) IL-6 mediates MLCK activation after ethanol exposure and burn injury. Movement of a fluorescent- tagged protein out of the ileum lumen to the bloodstream along with immunofluorescent staining for tight junction protein localization will be performed to investigate intestinal barrier function and integrity. MLCK activation will be determined by Western blot analysis. Inhibition and knock out of MLCK will be employed as well to examine the role of this molecule on intestinal permeability after acute ethanol and burn injury exposure. We will investigate MLCK activation and tight junction integrity in IL-6 knock out mice and wild type mice given an IL-6 neutralizing antibody. Finally, we will examine how IL-6 affects the MLCK activation signaling by examining the activation of MLCK regulators PKC, Src kinase, and Rho kinase. Finally, activation of activation of myosin light chain phosphatase (MLCP) will be assessed to investigate whether IL- 6 mediates its activation to a greater degree after the combined injury. These studies will help gain an understanding for how even acute ethanol in combination with burn injury can result in decreased barrier function that has been clinically observed and can result in increased morbidity and mortality.