Fever is an evolutionarily conserved response, the key feature of which is a temporary, regulated increase in core temperature. We have shown that the temperature increase itself is an immune modulator, one key feature of which is attenuation of the proinflammatory cytokine, tumor necrosis factor-alpha(TNF). We showed that sub-heat shock febrile-range hyperthermia (FRH) activates the heat/stress-activated transcription factor, heat shock factor-1 (HSF1) to a distinct repressor form. We identified an HSF1 binding site in the murine TNFalpha 5'UTR that represses TNF promoter activity and found evidence of additional HSF1-dependent repressors in the TNF promoter. In rnacrophages exposed to FRH, TNF transcription begins normally after treatment with bacterial lipopolysaccharide (LPS), but ends early, thereby reducing TNF expression to a brief short pulse. This raised the question of how TNF transcription could begin at all in the presence of a trans-repressor. In this regard, we found that HSF1 is transiently inactivated after LPS treatment that HSF1 inactivation coincides with onset of TNF transcription, and that subsequent HSF1 reactivation coincides with TNF transcriptional silencing. Preliminary data indicates that the inactivation and reactivation of HSF1 is caused by its phosphorylation and subsequent dephosphorylation over a 60-minute cycle. We hypothesize that LPS activate a cycle of HSF1 phosphorylation/dephosphorylation and that this process temporally restricts TNF expression. The overall goal of this research proposal is to elucidate the molecular mechanisms through which HSF1 modifies TNF expression in macrophages. Specifically, we will use novel HSFl-null macrophage model to: (i) define the mechanisms through which HSF1 represses TNF expression focusing on its interaction with the proximal TNF promoter and the associated enhanceosome, and its effect on local chromatin structure; (ii) identify HSF1 domains required for transrepression of TNF; (iii) elucidate the LPS-activated phosphorylation event(s) that transiently inactivate HSF1 trans-repression, thus allowing transient TNF expression in FRH-treated cells; and (iv) determine whether HSF1 directly blocks TLR signaling, whether HSF1 binds directly to the inhibited signaling elements, and identify the active HSF1 domains. The proposed studies will provide new insight into how the biochemical events that regulate host defenses is modified by the temperature increase that occurs during fever.
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