ABSTRACT This is a request for an Administrative Supplement to expand our current research on the regulation of NLRP3 inflammasome activation, carried out under parent award AI043477, with the goal of developing a novel anti- inflammatory therapy for COVID-19 related acute respiratory distress syndrome (ARDS). Like its predecessor, the Severe Acute Respiratory Syndrome-related coronavirus (SARS-CoV-1), the novel SARS-CoV-2 virus, the cause of the COVID-19 pandemic, can establish lower airway infections that cause viral pneumonia that may progress to ARDS. ARDS is a potentially fatal, severe medical condition that has been estimated to cause 200,000 yearly cases in the U.S., prior to the COVID-19 pandemic and many more now. Several innate immune cell types including platelets, neutrophils, macrophages and dendritic cells partake in mounting uncontrolled inflammation and tissue injury in ARDS, regardless of its initial trigger. These cells produce numerous inflammatory mediators and cytokines in response to the initial insult, which in the case of COVID-19 is viral replication within lung epithelial cells and subsequent cell death. Dying epithelial cells release damage associated molecular patterns (DAMPs), of which IL-1α and ATP are of primary importance. Together these molecules lead to priming (IL-1a) of alveolar macrophages and activation (ATP) of the NLRP3 inflammasome, which mediates production of mature IL-1β and IL-18, which amplify and propagate the inflammatory response that culminates in ARDS. Inhibition of this response should reduce much of the mortality and morbidity associated with COVID-19. However, since total IL-1 blockade with currently available drugs increases the risk of bacterial infections, the only suitable strategies for inhibition of SARS-CoV-2 elicited ARDS are either selective IL-1α blockade or inhibition of the NLRP3 inflammasome, which is not involved in anti-microbial defenses. So far, targeting of the downstream cytokine IL-6 had produced mixed results and IL-1a specific antibodies are still under clinical development. Moreover, anti-cytokine drugs are quite costly. We recently found the widely prescribed anti-diabetic drug metformin to be an effective inhibitor of NLRP3 inflammasome activation and IL-1β production by activated macrophages in vitro and in vivo. Accordingly, we now ask for additional funding to test and improve the ability of metformin to block the onset of ARDS, first in LPS-challenged Bl6 mice and then in SARS-CoV-2 infected hACE2-transgenic mice. As metformin has a short half-life and macrophages do not express the metformin transporters expressed by hepatocytes, we will examine whether metformin-loaded nanoparticles or exosomes given by inhalation allow for more effective inhibition of SARS-CoV-2 elicited ARDS. Importantly, metformin is a very safe and inexpensive drug with strong anti-aging properties that may be of further value in attenuating the well documented age-related increases in ARDS and COVID-19 risk, attributed to inflamma-aging.

PROJECT NARRATIVE Having found that the safe anti-diabetic drug metformin is an effective inhibitor of NLRP3 inflammasome activation and IL-1β secretion by macrophage, we plan to repurpose metformin for the prevention and treatment of acute respiratory distress syndrome (ARDS). In particular, we will evaluate metformin for its ability to inhibit and ameliorate SARS-CoV-2 induced ARDS, the major cause of death in COVID-19 patients. To improve the stability of metformin and increase its delivery to alveolar macrophages, we will evaluate the efficacy of metformin-loaded nanoparticles and exosomes given by inhalation to hACE2-transgenic mice infected with SARS-CoV-2.