Hypothermia enhances inflammatory cytokine expression via NF-kappa B

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DESCRIPTION (provided by applicant): The goal of the proposed research is for Dr. Fairchild to transition into an independent investigator elucidating the cellular, molecular, and in vivo effects of hypothermia on NF kappa B-dependent gene expression. Therapeutic hypothermia (HT) is under investigation in children with traumatic brain injury and neonates with hypoxic ischemic encephalopathy, and there are many other potential uses of this therapy in pediatric and neonatal critical care. While HT appears to be neuroprotective in some patients, concerns remain over potential detrimental effects. Dr. Fairchild has shown that HT in the range used in clinical trials increases activation of the transcription factor nuclear factor kappa B (NF-/kB) and expression of the pro- inflammatory cytokines TNFa, IL-1/3, and IL-8 in LPS-stimulated human macrophages. When overexpressed, these cytokines can be cytotoxic. Preliminary experiments in juvenile mice show that in vivo hypothermia also significantly alters cytokine production. Our overall goal is to understand the mechanisms of hypothermic enhancement of NF-/kB-dependent gene expression in order to better anticipate and perhaps prevent adverse effects of therapeutic HT. Our preliminary data show that HT alters key phosphorylation and acetylation events which favor association of the p65 transactivation component of NF-/kB with cytokine gene promoters on chromatin. In Aim 1 we will elucidate the role of altered NF-/kB p65 transactivation in the hypothermic enhancement of cytokine production, using Gal4-p65 reporter gene studies and lentiviral mediated RNA interference. Experiments in Aim 2 will determine how alterations in critical phosphorylation and acetylation events contribute to the HT effect on pro-inflammatory cytokines, through immunoblotting, in vitro phosphatase and deacetylase assays, and RNA interference. Using chromatin immunoprecipitation, we will elucidate effects of HT on loading of phosphorylated and acetylated p65 and its coactivators and compressors on specific cytokine gene promoters. Finally, in Aim 3, we will develop a system for studying in vivo effects of HT on NF-/kB activation and cytokine expression in juvenile transgenic NF-/kB reporter mice. HT is under investigation as a neuroprotective therapy for children and neonates with brain injury, and many other clinical uses of HT have been proposed. Ultimately, our studies may provide insight into appropriate indications, protocols, and adjunct strategies to optimize outcome of patients undergoing therapeutic HT.