Endothelial responses to anthrax lethal toxin and pathogenic implications
Warfel, Jason Michael.
Thesis (Ph.D.)--Georgetown University, 2010.; Includes bibliographical references.; Text (Electronic thesis) in PDF format. Anthrax lethal toxin (LT) is a primary virulence factor of Bacillus anthracis, the microorganism that causes anthrax. Recent evidence indicates that LT has direct effects on endothelial cells supporting our central hypothesis that LT plays a causative role in the vascular pathologies associated with anthrax, including vascular leakage, vasculitis, and faulty innate immune response. In order to further understand anthrax pathogenesis, it is necessary to determine the precise role of LT on these vascular pathologies. Here, I use a primary human endothelial cell culture model to investigate the effects of LT on two critical functions of the endothelium: 1) the immune response, which is essential to coordinate leukocyte transmigration, and 2) the barrier function, which regulates passage of fluid and proteins out of the bloodstream. With respect to the immune response, I show that LT differentially regulates the cytokine-induced expression of pro-inflammatory genes. For example, LT enhances TNF-induced expression of the adhesion molecule VCAM-1, but suppresses expression of numerous chemokines, including MCP-1. The underlying mechanisms for these contrasting actions involve modulation of pro-inflammatory transcription factor activation by LT. Specifically, LT is shown to enhance or prolong activation of IRF-1, NF-κB, and STAT1, while significantly inhibiting activation of the AP-1 family. In addition, the prolonged NF-κB activation by LT is shown to involve enhanced activity of the upstream IκB kinase (IKK), which leads to delayed reaccumulation of NF-κB-inhibitory IκB proteins. Another novel finding in this thesis is that LT-induced endothelial barrier dysfunction occurs by two independent mechanisms. LT-induced morphological changes, including actin stress fiber formation and cell elongation, are shown to be dependent upon Rho kinase (ROCK) phosphorylation of myosin light chain. However, LT-induced barrier dysfunction is only partially ROCK-dependent. Characterizing a second mechanism, I show that LT-treated endothelial cells display decreased expression of the tight junction adhesion molecule claudin-5. This suppression involves inhibition of the kinase Akt, and subsequent increased activity of FoxO1, a claudin-5 transcriptional repressor. Together, the novel findings presented in this thesis provide a mechanistic basis for understanding the role of endothelial dysfunction in anthrax pathogenesis.
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