Activation of the innate immune response commonly triggers the acute pathophysiology characteristic of critical illnesses. This conserved response is necessary for host survival in the face of infections;however, if left unregulated, this proinflammation may impact organ function and host survival. A paradigm focused on proinflammation has driven recent therapeutic approaches, but with little success in ameliorating critical illnesses. Thus, a paradigm shift that places greater emphasis on understanding the counter-regulation of this biology may be necessary. It is likely that substantial immunologic dissonance exists in critically ill patients so that therapeutically targeting one end of this spectrum is unlikely to be successful. Therefore, our focus is on elucidating regulatory mechanisms that limit proinflammation, acknowledging that these can also become dysregulated resulting in the functional immunodeficiency often reported late in sepsis and multiple organ dysfunction syndrome. A canonical pathway examined in both states of dysregulation is LPS induction of tumor necrosis factor (TNF)-1 which is dependent on transcriptional and post-transcriptional regulation by MAP kinase pathways. Preliminary data shows that inhibiting PP2A activity results in augmented TNF-1 expression after LPS stimulation in a manner dependent on p38 activation. Furthermore, stimulation of cells is rapidly followed by changes in the subcellular localization, post-translational modification, and activitation of PP2A suggesting these mechanisms are crucial for targeting kinase regulation. These findings prompt the question as to whether similar mechanisms are at play in states of immune deactivation in vivo. Therefore, the central hypothesis of this proposal is that PP2A negatively regulates MAPK signaling pathways triggered by inflammatory stimuli and that this is modulated by sub-cellular localization and post-translational modifications of PP2A and regulated by the ceramide. Furthermore, we hypothesize that induction of the hyporesponsive monocyte/macrophage in vivo is modulated by increased PP2A activity. As a result, we aim to: 1) determine the molecular mechanisms by which PP2A regulates endotoxin-induced TNF-1 biosynthesis in macrophages;2) determine the composition of, post-translational modifications, and subcellular localization of PP2A following cellular activation;3) define the contribution of TNF-1, ceramide and PP2A activation in mediating an LPS hyporesponsive macrophage phenotype;4) define the in vivo role of PP2A in mediating a deactivated state of immune reactive cells in children subjected to a predictable inflammatory challenge. The results from these studies will provide novel insight into the functional regulatory mechanisms mediated by PP2A involving key inflammatory signaling pathways. Results should define a broader model of phosphatase regulation of inflammatory-triggered gene expression and aid in the identification of innovative strategies for modifying the timing and degree of proinflammatory gene expression.
The human immune system has evolved so that patients who are afflicted with infections can mount a strong immune response, termed proinflammation, to kill and clear invading pathogens from the body. Often, this proinflammatory response is so substantial that it affects the normal physiologic functions of the individual thereby creating the need for intensive medical care because of organ failure. Unfortunately, targeting our therapies to block this response has not been very successful. We have learned that the body also mounts a response to counter proinflammation and therefore avoid this acute damage;however, this process can also become too intense causing a state of immune dysfunction through cellular deactivation. The goals of the proposed studies are to understand the molecular mechanisms by which the protein phosphatase, PP2A, regulates a typical proinflammatory pathway and to determine whether PP2A activity is responsible for the state of cellular deactivation. A combination of cellular and human studies will be used with the aim of defining a broader model by which phosphatases regulate inflammation. We also aim to identify innovative strategies for modifying the timing and degree of cellular activation and/or deactivation in states of critical illness.
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