Mechanisms of Endothelial Inflammatory Memory in Post-Sepsis Pneumonia and Sepsis - Sepsis survivors remain highly vulnerable to secondary infections, particularly pneumonia, which significantly increases their long-term morbidity and mortality, which are frequently associated with sustained vascular dysfunction and impaired endothelial repair, increasing the risk of long-term organ failure and mortality. Anti- inflammatory strategies have not been successful in post-sepsis pneumonia, so uncovering new mechanisms to target inflammation better is an unmet clinical need. Endothelial cells (EC) can establish inflammatory memory and may predispose ECs to exaggerated responses during secondary infections. There is a major gap in our understanding of how epigenetic mechanisms, including transcription factor regulation and chromatin remodeling, establish inflammatory memory in ECs and contribute to organ-specific responses. We aim to investigate the molecular and epigenetic mechanisms that establish and maintain endothelial priming, focusing on the roles of transcription factors STAT3 and JunB, and the histone methyltransferase EZH2. A clinically relevant two-hit infection model of cecal ligation and puncture (CLP), followed by Streptococcus pneumoniae challenge at day 20 post-CLP, will be used to examine lung and kidney ECs at days 3 (acute), 10 (recovery), and 22 (secondary challenge). Our work showed CLP survivors clinically returned to normal by day 10, but they displayed persistent kidney dysfunction at day 13, which worsened following the SP challenge. Notably, 100% of CLP+SP died within 4 days of SP exposure (day 24), while sham+SP mice survived. Blood inflammation profiling at day 22 revealed significantly elevated IL-6, TNF-α, and MCP-1 in the CLP+SP group. Notably, our data showed that JunB is persistently elevated in ECs, correlating with amplified proinflammatory gene expression, whereas EZH2 expression is reduced, suggesting a loss of epigenetic repression that may perpetuate endothelial priming. We hypothesize that ECs undergo epigenetic reprogramming during acute inflammation, creating a primed state that persists after resolution. This persistent primed state contributes to the heightened mortality observed in sepsis survivors during secondary inflammatory events. We propose two aims: (Aim 1) to determine how STAT3 and JunB orchestrate chromatin remodeling and establish inflammatory memory, characterized by endothelial activation, barrier dysfunction, and heightened immune cell recruitment; and (Aim 2) investigate whether EZH2-mediated epigenetic modifications can amplify or resolve this priming, highlighting potential therapeutic targets to restore vascular homeostasis. Survival outcomes, vascular barrier integrity, immune profiling, and multi-omic analyses will reveal key regulators of persistent endothelial dysfunction. These aims address a fundamental gap in our understanding of the role of ECs priming in post- sepsis pneumonia or inflammatory events. Findings may also inform broader strategies for managing chronic inflammatory diseases linked to endothelial dysregulation.
