ISGylation regulates lung endothelial inflammation

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Abstract Excessive lung microvascular endothelial inflammation is a pathological hallmark of acute respiratory distress syndrome (ARDS). Upon inflammatory stimuli, lung endothelial cells (ECs) increase chemokine and adhesion molecule expression, such as IL-6, and intercellular adhesion molecule 1, ultimately causing neutrophil accumulation at the site of inflammation, endothelial barrier dysfunction, and lung tissue damage in acute lung injury and sepsis. Nuclear transcriptional factor κB (NF-κB) plays a pivotal role in EC inflammation. Inflammatory stimuli trigger phosphorylation of NF-κB component p65, leading to increase p65 transcriptional activity. We revealed that NF-κBp65 can be ISGylated. The ISGylated p65 is inactive in the resting ECs. We also discovered that SCFFBXL19 E3 ligase catalyzes ISGylation of p65, impedes p65 phosphorylation, and mitigates lung EC inflammation. These observations led us to hypothesize that p65 ISGylation by SCFFBXL19 dampens its phosphorylation, transcriptional activation, and lung EC inflammation; and increases in FBXL19 stability mitigates lung EC inflammation through reducing NF-kB activation. To better understand the new modification of p65, in this proposal, we will determine molecular mechanisms by which SCFFBXL19 catalyzes p65 ISGylation and its role in human lung microvascular EC inflammation. And then, we will determine the molecular mechanisms by which p65 ISGylation impedes its phosphorylation and activation in human lung microvascular ECs. Lastly, we will determine if stabilization of FBXL19 alleviates lung EC inflammation in murine models of acute lung injury and sepsis. This application will be the first to characterize SCFFBXL19-mediated ISGylation of NF-κBp65 and determine its role in the regulation of p65 phosphorylation, transcriptional activation, and lung EC inflammation in acute lung injury and sepsis.