Infections with Shiga toxin (Stx)-producing bacteria Shigella dysenteriae 1 and Escherichia coli cause bloody diarrheal diseases which continue to be a major public health concern in developed and developing countries. Patients infected with Stx-producing bacteria are at increased risk for the development of life- threatening complications involving vascular damage to the kidneys and CNS. Prior studies have shown that the cytokines tumor necrosis factor-alpha (TNF) and interleukin-1 (IL-1) sensitize vascular endothelial cells to the cytotoxic action of Stxs via a mechanism involving the upregulated expression of the toxin receptor glycolipid Gb3 on target cell membranes. Thus, the innate immune response elicited by bacterial products such as Stxs or lipopolysaccharides (LPS) may contribute to vascular damage. We showed that human macrophages and macrophage-like cell lines respond to Stxs by secreting TNF and IL-1, and the response is augmented by LPS. This is a paradoxical finding as Stxs have been shown to act on ribosomes to inhibit protein synthesis. How is it that protein synthesis inhibiting toxins cause increased protein expression in macrophages? Following the burst of cytokine expression, however, Stxs induce apoptotic cell death in macrophages. We propose to continue our studies on the intracellular signaling mechanisms activated by Stxs and/or LPS, including MAPK and caspase cascades, leading to macrophage cytokine expression and apoptosis, respectively. We will determine the role of MAPKs, upstream kinases such as Akt, and the translation initiation factors elF4E and elF4E-BPs in regulating cytokine expression at transcriptional and post-transcriptional levels. Inhibitors of intracellular signaling molecules will be used to block Stx-induced cytokine expression and cell death. Given the capacity of Stxs to elicit the expression of TNF, we will examine the role, if any, of endogenously derived TNF in apoptosis induced by Stxs and/or LPS. Previous studies have suggested that TNF may activate anti-apoptotic pathways in macrophages, and we hypothesize that Stxs may inhibit the normal survival response activated in TNF producing macrophages. The timing of exposure to Stxs and TNF may also be important in determining whether apoptosis or cell survival will occur. We will determine the importance of prolonged MAPK and upstream kinase activation on apoptosis vs. cell survival. Finally, we will use a mouse model of Stx-induced renal damage to correlate our in vitro findings with in vivo responses. There currently are no effective treatments to prevent or treat diseases caused by Stx-producing bacteria. An improved understanding of how these toxins cause disease is necessary to formulate effective therapeutic agents to block disease progression.
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