During gram-negative bacteremic sepsis, a liver-lung axis of inflammation predisposes to critical organ injury due to an imbalanced expression of inflammatory vs. anti- inflammatory gene products. Such postbacteremic organ dysfunction is thought to be augmented by secondary ischemic-hypoxic stress owing to reduction-oxidation (redox)-sensitive transcription factors and subsequent amplification of inflammatory responses mediated by expression of key cytokine and noncytokine genes. The objective of the proposed research is to test the hypothesis that postbacteremic O2 limitation within the liver and the lungs differentially modulates the activation of a defined group of redox-sensitive transcription factors, thereby altering cytokine expression in a directionally- opposite and organ-specific manner. Experiments are designed to determine the effects of secondary reductions in the hepatic vs. pulmonary O2 supply in modulating postbacteremic transactivation of nuclear factor-kB (NF-kB), activator protein (AP)-1, NFIL-6, and the cyclic AMP response element binding protein (CREB). The biologic significance of these changes will be assessed by examining the concomitant expression of inflammatory cytokines (TNF-alpha, IL-1alpha, IL-1beta), anti-inflammatory cytokines (IL-6, IL-10), prostaglandin (PG) H synthase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in these organ systems in relation to redox status during ex situ organ perfusion. Experiments are also designed to define postbacteremic protein:DNA interactions during hypoxic stress and reoxygenation by assessing the activation of these transcription factors in Kupffer cells and alveolar macrophages. Resulting data will establish if an autoregulatory loop involving enhanced COX-2 and CREB activity suppresses postbacteremic cytokine expression by a PGE2-dependent mechanism in an organ-specific manner. The role of activation of the nuclear protein hypoxia inducible factor-1 in co-modulating endotoxin-induced-cytokine gene expression during subsequent hypoxia will be assessed. These endpoints will also be analyzed in conscious rats during hypoxic stress, with and without preexisting liver dysfunction following bacteremic infection. In parallel studies, the cis-acting DNA sequences that confer hypoxic suppressibility of endotoxin-induced cytokine promoter activity in RAW 264.7 cells transfected with TNF-alpha and IL-1beta reporter gene constructs will be identified. Results from these vertically-oriented studies should provide novel insights into the transcriptional regulation of cytokine and iNOS expression during gram-negative bacteremic sepsis while identifying mechanistic approaches to ameliorate lung injury and multiple organ failure.
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