The overall goal of this research will be to understand the fundamental mechanisms by which trichothecene mycotoxins target leukocytes and dysregulate immune function. The trichothecene mycotoxins are common food and indoor air contaminants that have been etiologically linked to human and animal disease. In experimental animals, how level trichothecene exposure evokes upregulated cytokine production with attendant autoimmune sequelae, whereas exposure to higher levels induces leukocyte apoptosis with accompanying immune suppression. Upon examining upstream events that may contribute to these divergent effects, it was observed that mitogen-activated kinases (MAPKs) JNK 1 / 2, ERK 1 / 2, and p38 are rapidly and variably phosphorylated following exposure to trichothecenes in vitro and in vivo. Based on these findings and known downstream functions of these kinases, it is hypothesized that immune stimulation and suppression by trichothecenes are mediated by differential activation of MAPK, signaling cascades. Three SPECFIC AIMS are proposed to test this hypothesis.
In Aim 1, trichothecene-mediated activation of specific MAPKs will be related to changes in transcriptional and post-transcriptional regulation of cytokine gene expression in RAW 264.7 cells, a cloned macrophage model. To define contributions of specific MAPKs, the effects of selectively impairing JNK 1 / 2, ERK 1 / 2, and p38 function/expression by pharmacologic and genetic means on regulation of cytokine expression will be measured.
In Aim 2, trichothecene-medicated activation of specific MAPKs will be related to apoptosis in RAW 264.7 cells. The contributions of p53 activation and expression of downstream apoptotic effectors Bax, Bcl-XI and caspase-3 in trichothecene-induced apoptosis will be assessed. The effects of selectively blocking JNK 1 / 2, ERK 1 / 2, and p38 function/expression on regulation of the apoptotic pathway will be determined.
In Aim 3, putative mechanisms will be further verified in mice orally exposed to trichothecenes by qualifying the effects of trichothecene dose and exposure time on MAPK phosphorylation/activation and relating these to regulation of cytokine mRNA expression and apoptosis in lymphoid tissues. The sensitivity of various tissues and leukocyte phenotypes to trichothecene-induced MAPK phosphorylation will be compared. The effects of selectively inhibiting JNK 1 / 2, ERK 1 / 2 and p38 in vivo and ex vivo on cytokine mRNA expression and apoptosis will be addressed. Over the long term, this research will help characterize hazards associated with trichothecene exposure that can be used in human risk assessment.
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