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A2466 - ­Role of Microtubule-Associated Signaling in Staphylococcus Aureus-Induced Lung Injury
Author Block: P. Karki1, Y. Tian2, Y. Ke3, N. Sarich2, A. Birukova1; 1Department of Medicine, University of Maryland, Baltimore, MD, United States, 2Department of Medicine, University of Chicago, Chicago, IL, United States, 3Anesthesiology, University of Maryland Baltimore, Baltimore, MD, United States.
Staphylococcus aureus is a major etiological agent of sepsis. We have previously demonstrated that heat-killed Staphylococcus aureus (HKSA) and cell wall components of S. aureus peptidoglycan G and lipoteichoic acid induce endothelial barrier dysfunction and inflammation, two major hallmarks of acute lung injury (ALI). Microtubules (MT) are dynamic cytoskeletal structures involved in many cell processes including cell division and regulation of endothelial cell (EC) permeability. This study tested role of MT in the mechanisms of HKSA-induced human pulmonary EC barrier dysfunction and inflammation. HKSA-induced endothelial barrier disruption is accompanied by a decrease in acetylated tubulin pools of MT, an indicator of MT destabilization. EC challenged with HKSA revealed increased histone deacetylase (HDAC) activity as a result of activated reactive oxygen species (ROS) production. Molecular or pharmacological inhibition of HDAC6 using gene-specific siRNA and tubastatin A, respectively, rescued HKSA-induced EC barrier disruption monitored by measurements of transendothelial electrical resistance (TER) and macromolecular permeability with FITC-avidin tracer. HKSA-induced EC permeability was associated with impaired MT-mediated delivery of cytoplasmic linker-associated protein 2 (CLASP2) to the cell periphery limiting its interaction with adherens junction proteins p120-catenin and VE-cadherin. HKSA-induced EC barrier dysfunction was also associated with increased Rho activity via activation of MT-bound Rho-specific GEF-H1 and was abolished by downregulation of HDAC6 activity. HKSA also induced activation of the NF-κB inflammatory pathway and expression of EC adhesion molecules ICAM1 and VCAM1 which was also HDAC6-dependent and mediated, at least in part, by GEF-H1/Rho-dependent mechanism. In vivo, HDAC6 knockout mice and wild type mice treated with HDAC6 inhibitor were partially protected from vascular leak and inflammation caused by both HKSA or methicillin-resistant S. aureus (MRSA), as determined by Evans blue extravasation into lung tissue, increased total cell and protein content in bronchoalveloar lavage (BAL) fluid, expression of ICAM-1/VCAM-1 and inflammatory cytokines levels in BAL. Altogether, these results demonstrate a novel signaling axis of S. aureus-induced ROS-dependent upregulation of HDAC6 activity resulting in MT destabilization and subsequent activation of GEF-H1/Rho pathway of endothelial permeability and inflammation.