Abstract: ARDS accounts for one in ten intensive care unit (ICU) admissions. Mortality in ARDS is estimated at 30 – 50%. Decades of clinical trials have failed to identify targeted pharmacotherapeutics that significantly impact disease course. New insights are needed to broaden our understanding of the underlying mechanisms that lead to ARDS to promote the development of novel therapeutics. Transfer RNA (tRNA) constitutes 10 – 20% of the total cellular RNA content. Although classically thought of passive translational machinery, more recent studies show that tRNA can be dynamically regulated in response to the environment. The post-translation modification of tRNA by methylation is one mechanism by which tRNAs respond to cellular stress. Methylation of tRNAs can alter translation, change the distinction between ‘self’ and ‘non-self’, and protect tRNAs from fragmentation. Despite this, the role of tRNAs in infection and critical illness is unknown. We identified a methyltransferase enzyme, termed TRMT1 that is upregulated in the human lung in response to infection. We also find that the methylation of tRNA by TRMT1 is altered in response to inflammatory stimuli. Our preliminary data suggest that the TRMT1 is vital for macrophage viability and host defense in infection, and for maintaining cytokine responses. This application focuses on the role of the TRMT1 in acute lung injury and the investigation of tRNA biology, unexplored territory in ARDS and critical care illnesses. During the execution of these studies, we will examine how myeloid TRMT1 contributes to immunopathology in experimental lung injury. We will determine how TRMT1 alters inflammatory signaling and regulates cell death in vitro and in vivo. To examine the mechanistic role of TRMT1, we will measure the methylation of tRNA in response to inflammatory stimuli and the production of tRNA fragments. To further explore the regulation of TRMT1, we will examine how subcellular trafficking of TRMT1 differentially regulates cytoplasmic and mitochondrial tRNA methylation and how this alters the inflammatory response. Finally, because cellular concentrations of TRMT1 protein may be crucial in regulating macrophage behavior and viability we will examine the E3 ligase mediated ubiquitination and degradation of TRMT1. These studies will be the first to elucidate the role of TRMT1 in acute lung injury. In addition, these studies will provide new insight into the role tRNA methylation in lung injury. The execution of these studies will provide significant mechanistic and biological advances in the field of acute lung injury and ARDS and lead to novel interventional targets for therapeutics.