Research Project 3. Vesicant-induced Lung Injury Project Lead: Debra L. Laskin, Ph.D. Co-Investigator: Jeffrey D. Laskin, Ph.D. Co-Investigator: Vasanthi Sunil, Ph.D. Co-Investigator: Rama Malaviya, Ph.D. Co-Investigator: Raymond Rancourt, Ph.D. Project Summary/Abstract Lung injury following exposure to mustard vesicants is responsible for most morbidity and mortality of these high priority chemical threat agents. As there are currently no approved therapeutics to treat mustard poisoning, it is essential to identify mechanistic targets for drug development. Our focus is on inflammatory macrophages and mediators they release which we have demonstrated contribute to both acute lung injury and chronic disease induced by sulfur mustard and nitrogen mustard. One macrophage derived proinflammatory mediator that we discovered is key in mustard lung toxicity is tumor necrosis factor alpha (TNF). Based on our findings, Janssen Pharmaceuticals is now working to move SimponiR, a fully humanized anti-TNF antibody, into advanced development for treatment of mustard lung poisoning with funds from BARDA. In recent studies we discovered that farnesoid-X receptor (FXR), a nuclear receptor important in bile acid metabolism with anti-inflammatory activity is also important in mustard lung toxicity. Following nitrogen mustard exposure, macrophage FXR activity is suppressed. This is associated with increased activity of proinflammatory macrophages and reduced activity of anti-inflammatory/proresolution macrophages. Studies during the next grant period are aimed at elucidating mechanisms underlying nitrogen mustard induced suppression of FXR. In preliminary studies we found that microRNAs that regulate the proinflammatory transcription factor NFB in macrophages are dysregulated after nitrogen mustard exposure. As a consequence, there is protracted activation of NFB signaling resulting in increased production of TNF and cytotoxic reactive nitrogen species. We hypothesize that these mediators suppress FXR and its target, NR4A1, a central regulator of macrophage reprogramming from a proinflammatory to an anti-inflammatory phenotype. To test this hypothesis, we will (1) Analyze the impact of reduced FXR activity on the development of anti- inflammatory/proresolution macrophages and lung injury following mustard exposure; and (2) Determine if protracted activation of NFB is due to mustard-induced alterations in microRNAs regulating NFB and key downstream inflammatory gene products, TNF and inducible nitric oxide synthase. Results of these studies will lead to new mechanistic insights into mustard lung poisoning and the development of novel therapeutics for mitigating toxicity.