Indole Metabolites as Xenobiotic Danger signals in Acute Lung Injury

ABSTRACT This application is for an NHLBI R01 entitled “Indole Metabolites as Xenobiotic Danger Signals in Acute Lung Injury.” I am a physician in Pulmonary and Critical Care Medicine at the University of Pittsburgh, and I am building a research program focused on the studying the intersection of danger signals and host responses in epithelial

cells in the pathogenesis of Acute Respiratory Distress Syndrome (ARDS). ARDS is an urgent public health problem characterized by severe inflammatory lung injury with impaired gas exchange, a high mortality risk (up to 40%), and long-term morbidity in survivors. Beyond supportive clinical care bundles that define the standard-of-care for ARDS,

there are few effective therapeutic strategies. Recent work has convincingly demonstrated that mortality in ARDS and a larger group of patients at risk for ARDS is linked to excessive inflammation. However, the underlying biological pathways driving these processes are only partially understood. Danger signals are proximal activators

of the innate immune system, and recognition of danger signals by the host initiate regulated cell death programs and inflammatory pathways in lung epithelial cells. Danger signal activation of regulated cell death and inflammation evolved to promote pathogen clearance, but these processes are deleterious and even fatal to the

host when activated in excess. In this proposal we describe novel danger signals to lung epithelial cells derived from metabolites that function as Metabolism-Associated Molecular Patterns (MAMPs). Using high throughput screens of metabolite libraries, we identified microbe-derived indole metabolites as novel danger signals that

activate regulated cell death pathways in lung epithelial cells. We show indole metabolites exacerbate LPS- induced lung injury in vivo in mice, and in humans with ARDS we measured indole metabolites and found several to be highly elevated compared to mechanically-ventilated patients without lung injury. In this proposal we will

investigate the following Aims: Aim 1: Determine the mechanism of indole metabolite-mediated cell death. We will investigate indole metabolite toxicity to lung epithelial cells through CYP2F1/2-dependent biotransformation and activation of inflammatory cell death programs. Aim 2: Determine the role of cAMP signaling in protecting from

indole metabolite cellular toxicity. We will validate key candidate genes identified through an RNAi screen and repurposed drugs that augment cAMP signaling for protective effects against indole metabolite toxicity. Aim 3: Determine ex-vivo toxicity of indole metabolites to human lungs and associations of indole metabolite levels with

hyperinflammatory respiratory failure and clinical outcomes. We will test indole metabolites in a human ex-vivo lung perfusion platform and enroll patients in our Acute Lung Injury Registry to determine if indole metabolite levels are associated with the hyperinflammatory respiratory failure subphenotype. This work will advance understanding

of the molecular inputs contributing to regulated cell death and innate immune activation in the lungs and provide a new paradigm in lung injury – that metabolites are danger signals.