Summary/Abstract This “test of concept” R21 is to explore a novel concept on the role of circular RNAs in aging-related sepsis and organ damage. Circular RNA (circRNA) is a type of single-stranded RNA that forms a covalently closed continuous loop. The biological function of most circRNAs is unclear. CircRNA does not have 5' or 3' ends. The unique structure of circRNAs provides them with a longer half-life and more resistance to RNase R than linear RNAs, which makes them potential candidates for diagnostic biomarkers and therapeutic strategies. People who are aging are more susceptible to pneumonia than any other group and are also more likely to develop infections from gram-negative (G-) organisms than younger adults. Moreover, the mortality of acute respiratory distress syndrome (ARDS) increases significantly in older patients. Macrophages (MΦs) form the first line of host defense against microbial pathogens via protective inflammatory responses. The high mortality and morbidity after bacterial infections often result from an imbalance in host defense between bactericidal and an excessive inflammatory response that leads to tissue damage. Currently, the initiation, propagation, and regulation of inflammatory responses in the presence of G-bacterial infection remain incompletely explored, particularly in aging populations, impeding the development of novel and specific therapeutics for the elderly. We initially screened circRNAs in mΦs from both young and old mice with and without LPS. We found that circular RNA-20033 (circ20033) is highly upregulated by LPS in the BALF EVs from older mice (78 weeks, 78w) compared to the younger ones (12w). We previously reported that BALF EVs are mostly derived from MΦs after sepsis and infections.17 Therefore, we will focus on MΦs in this proposal. Circ20033 is derived from the Slc39a10 gene via back-splicing. Modulating circ20033 does not have an impact on its host gene suggesting that circ20033 carries a distinct function. Computational modeling analysis suggests that circ20033 interacts with several ‘anti- inflammatory’ miRNAs, including but not limited to the miR-182/183, miR-330, miR-326, and miR-192. Elevated Circ20033 in the elderly but not in the young ones, along with its prolonged half-life, potentially inhibits the ‘anti- inflammatory’ miRNAs via sponging effects, subsequently promoting a runaway inflammation too. Our central hypothesis is that aging-associated circ20033 un-regulation facilitates G-bacteria/LPS-induced, dysregulated lung inflammation and lung injury via promoting NLRP3 inflammasome activation in macrophages. We propose two specific aims: Aim I: To determine the function and mechanisms of aging-induced circ20033 in macrophage pro-inflammatory activation after bacterial infections. Aim II: To determine the role of aging-induced circ20033 in lung inflammation in vivo after bacterial infections.