Macrophages have evolved the capacity to recognize conserved structural components of pathogens such as the LPS moiety of Gram negative bacteria to facilitate making effective anti-microbial responses. The long-term objective of this research is to understand how LPS-elicited responses of macrophages are mediated by intracellular events. The production of nitric oxide (NO) by LPS-activated macrophages is known to play a central role in the host defense against infection, but in pathological immune responses, this molecule mediates severe tissue damage. For this reason, there is considerable interest in understanding the mechanisms underlying LPS regulation of NO production. To trigger this response, LPS requires a cytokine co-factor, which is believed to be macrophage-derived interferon-beta during the early stages of an Gram-negative infection. Recently, it has been observed that phosphatidylinositol 3-kinase (PI3-K) and mTOR are constituents of a signaling pathway that regulates interferon-beta synthesis and NO production. Thus, the proposed research tests the hypothesis that PI3-K and mTOR-mediate NO production in LPS-treated macrophages by activating a downstream signaling component, p70 S6 kinase, and this kinase regulates the synthesis of interferon-beta mRNA and enhances the expression and activity of NOS2, the NO-producing enzyme.
The specific aims are designed to test this hypothesis by: (1) determining how PI3-K and mTOR regulate NOS2; (2) determining how PI3-K and mTOR regulate interferon-beta; and (3) determining whether p70 S6 kinase is a downstream effector of PI3-K and mTOR leading to interferon-beta and NO production. The effect of pharmacologically-inhibiting PI3-K or mTOR on the LPS-stimulated levels of NOS2 mRNA, protein and enzyme activity will be evaluated by Northern blotting, Western blotting and by an in vitro activity assay measuring the rate of conversion of radiolabeled L-arginine to L-citrulline (and NO), respectively. The effect of pharmacologically-inhibiting PI3-K or mTOR on LPS-modulation of interferon-beta mRNA stability will be determined by measuring the rate of mRNA degradation by ribonuclease protein assay. The activity of p70 S6 kinase will be perturbed with a transfected mutant p70 S6 kinase allele and the consequence of this manipulation on the synthesis of interferon-beta and NO will be determined.
