Title: The Cell Gene Expression and Function in Response to Low Dose and Acute Radiation PI name: Daila S. Gridley, Ph.D.

Abstract:
The goal of this project, submitted in response to DOE Office of Science Notice DE-FG02-06ER06-10, is to determine if low dose, low linear-energy transfer (LET) radiation-induced changes In T lymphocytes influence the response to subsequent acute irradiation. The focus is on the T helper subsets (Th1, Th2 and Th3) that regulate both innate and adaptive immune defenses by virtue of the potent cytokines they secrete. Persons in certain occupations (nuclear power plant workers, astronauts and medical personnel), as well as many patients, are exposed to low levels of radiation. These same individuals may be subject to acute irradiation due to accidents, terrorist acts, solar particle events, or radiotherapy. The impact that these radiation conditions have on immunoregulatory mechanisms that are highly important to human health, have not yet been firmly established. Our pilot studies with whole-body irradiated mice show strikingly different gene expression patterns based on Th1/Th2/Th3 microarray technology depending upon dose rate and properties of the acute radiation regimen. Especially intriguing was the finding that the gene for interferon-γ (IFN-γ), a Th1-derived cytokine with radioprotective properties, was up-regulated in mice exposed to low dose γ-rays plus simulated solar flare protons. These and other data have led to the following overall hypothesis: Whole-body exposure to low dose, low-LET radiation will significantly ameliorate the effects of acute radiation. More specifically, we propose that: a) Th cell hypo-responsiveness following acute exposure will be due to defective TCR/CD3 signaling; b) The protective effect of low dose exposure will include up-regulation of IFN-γ; and c) The efficacy of low dose-induced protection will be dependent upon the quality of acute radiation. These hypotheses will be addressed in three Specific Aims: 1) Identify Th cell cytokine genes affected by low dose photons and determine if the genetic changes correlate with secreted cytokines; 2) Identify Th cell cytokine genes affected by acute radiation (photons, protons and simulated solar flare protons) and determine if the genetic changes correlate with secreted cytokines; and 3) Determine if the genetic changes induced by low dose photons modify the cytokine profile of Th cells when exposed to the three acute radiation regimens. Whole-body irradiated C57BL/6 mice will be used throughout. In aim 1, low dose photons (57Co γ-rays) will be delivered to total doses of 0, 0.01, 0.05, and 0.10 gray (Gy) at a dose rate of 0.03 cGy/hr. In the second aim, 2 Gy acute irradiation will include photons (60Co), high-energy protons and simulated solar flare protons. In aim 3, one low dose of priming radiation will be selected to combine with each of the acute radiation protocols. In all three aims, analyses will be performed immediately after the end of irradiation (day 0) and again on day 21 post-exposure. Gene expression patterns will be obtained for negatively isolated splenic CD4+ Th cells using cytokine cDNA microarrays. Integrity of signal transduction pathways initiated via TCR/CD3 and CD28 activation (normally involved in Th cell activation during antigen presentation in vivo) will be assessed using anti-CD3 +/- anti-CD28 antibodies; phorbol myristate acetate (PMA) and calcium ionophore will be used to bypass these early events in Th cell activation. Secreted cytokines characteristic for each of the Th subsets will be quantified using Luminex technology; secreted and plasma TGF-ß1 will be quantified by ELISA to determine levels of this highly immunosuppressive cytokine. Specific leukocyte types in the spleen will be quantified using an automated hematology analyzer and rapid 2-tube flow cytometry to characterize the cellular milieu in which the Th cells reside in vivo. Collectively, the data will provide valuable new information on the underlying mechanisms by which whole-body exposure to low dose/low-LET radiation, alone and in concert with three different acute irradiation events, influences Th cell gene expression and function.

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