NIH R01 HL 075557-01 (Principal Investigator - J. Christman, 2003-2007)
This grant,"Macrophage Gene Expression in Acute Lung Injury", is designed to investigate the basic molecular mechanism by which the ets-family transcription factor, PU.1 is involved in the response of macrophages to endotoxin with the hope that this leads to novel treatments for ARDS.
Abstract: The mechanisms that regulate inflammatory response in humans with severe sepsis need to be better defined in order to design specific therapies that can be used to protect the lungs and prevent death from multiple organ failure. Pulmonary macrophages are immune-effector cells that mediate the molecular pathobiology of neutrophilic lung inflammation in response to endotoxin. The overall goal of this proposal is to investigate whether PU.1 gene expression is critical determinant of pulmonary macrophage involvement in ARDS. We hypothesize that PU.1 regulates the response of pulmonary macrophages to endotoxin through two integrated mechanisms: a) induction of Toll-like receptors that can trigger the NF-kB activation pathway and b) through enhancement of inflammatory gene production, such as COX-2, that contributes to the development of cytokine and chemokine mediated lung inflammation. We propose three specific aims: 1) To define changes in PU.1 gene expression or activation state in response to treatment with endotoxin, 2) To determine the mechanisms by which PU.1 increases production of inflammatory mediators in response to treatment with endotoxin, and 3)To identify the relative contribution of PU.1 to the development of neutrophilic lung inflammation in a murine model of peritoneal sepsis. In the setting of severe sepsis, signaled recruitment of differentiation of macrophages may represent a renewable pool of endotoxin responsive pulmonary macrophages that contribute to the initiation,intensity, and duration of neutrophilic lung inflammation. Our studies are designed to investigate the basic molecular mechanism by which PU.1 is involved in the response of macrophages to endotoxin with the hope that this leads to novel treatments for ARDS.
Department of Veterans Affairs Merit Review Grant (Principal Investigator - J. Christman, 2004-2009)
This grant is in its 15-20 year and is entitled "Regulation of Neutrophilic Inflammation" and addressed the interaction between NADPH oxidase, toll-like 4 receptors, PU.1 and nuclear factor kappa B in the pathogenesis of neutrophilic lung infalmmation.
Abstract: The acute respiratory distress syndrome (ARDS) associated with Gram-negative sepsis has a substantial morbidity and high mortality. Accumulating clinical data indicate that macrophages have a central role in the pathogenesis of neutrophilic lung inflammation that leads to ARDS but specific molecular pathways in macrophages have not been defined. We have reasoned that detailed knowledge of regulatory pathways in macrophages could lead to new treatments for ARDS. Our overall goal is to define interactions among critical regulatory pathways in macrophages that contribute to the initiation and progression of neutrophilic lung inflammation. Four integrated hypotheses will be addressed in the context of endotoxin-induced neutrophilic lung inflammation. 1) toll-like receptor 4 (TLR4) and accessory proteins on macrophages are essential for generation of neutrophilic lung inflammation, 2) reactive oxygen species via the membrane bound NADPH oxidase enzyme complex regulates macrophage activation by modulating activation of the NF-?B pathway in response to endotoxin, 3) activation of NF-?B in macrophages is fundamentally necessary for generation of neutrophilic lung inflammation, and 4) PU.1 is a co-regulatory partner, along with NF-?B that augments macrophage production of key inflammatory proteins that contribute to the initiation and progression of neutrophilic lung inflammation. These hypotheses will be addresses with two specific aims: SA 1: To determine the necessity of macrophage toll-like 4 receptors and NADPH oxidase for the development of neutrophilic lung inflammation in response to treatment with endotoxin. Our approach will be to use fetal liver transplantation (FLT) to reconstitute TLR4 and NADPH deficient macrophages in wild-type mice in order to examine the impact of macrophage TLR4 and NADPH oxidase on the response to endotoxin in mice. Recently, TLR4 receptors and components of NADPH oxidase system have been identified in cells other than macrophages, including endothelial cells but the relative contribution of these protein complexes in individual cell types to lung inflammation is not clear. We will address the importance of macrophage TLR4 gene expression and NADPH oxidase by generating reciprocal bone marrow chimerics with TLR4-/- null donor and wild-type recipient mice and NADPH oxidase (p47phox) -/- null donor and wild-type recipient mice, respectively. The relative importance of macrophage TLR4 and NADPH oxidase will be identified by determining the magnitude of differences in the endotoxin response between these knockout and wild-type bone marrow chimeric mice. We think that isolating the relative role of macrophage TLR4 and NADPH oxidase to macrophage will lead to cell directed interventions in the pathogenesis of ARDS. SA-2: To determine whether PU.1 contributes to the endotoxin response by regulating macrophage phenotype and acting as a co-regulatory partner with NF-?B to augments production of pro-inflammatory mediators. We have observed that PU.1 regulates TLR4 and COX-2 gene expression and therefore influences the sensitivity of macrophages to endotoxin and could modulate the inflammatory response, respectively. We propose to address the critical role of PU.1 to the endotoxin response by using a duel approach. First, we will employ a chromatin immunoprecipitation (ChIP) assay to determine the timing and intensity of binding of PU.1 to the promoters of inflammatory genes (including TLR-4 and COX-2) in lung macrophages from endotoxin-treated mice. This approach has the unique advantage of detecting PU.1 interactions with endogenous TLR4 and COX-2 genes in the context of whole animal model of ARDS. For these studies, we will use a ChIP assay on whole lung tissue and lung macrophages from endotoxin-treated mice. Second, we will examine the evolution of the endotoxin response in conditional PU.1 knock-in mice. Macrophages from these mice lack PU.1 but normal PU.1 levels can restored by treating the mice with Tamoxifan, allowing us to identify the full impact of PU.1 on inflammatory gene expression. These studies, together, should provide important and unique insights into the molecular regulation of neutrophilic lung inflammation, and could provide evidence for novel therapeutic approaches to limit lung injury and restore health in patients suffering from ARDS and other inflammation diseases.
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