PROJECT SUMMARY A successful pregnancy is associated with an intricate immunological balance that is crucial in supporting the presence and development of a "foreign" fetus. This balance is marked by physiological changes that can alter the lung compartment, including the need for increased oxygen consumption, which can influence pregnant women's response to respiratory infections. For example, influenza A virus (IAV) infection during pregnancy is more likely to result in severe disease, including hospitalization and even death. During the 2009 pandemic, 29% of influenza-associated hospital admissions and up to 16% of deaths were in pregnant women. Maternal IAV infection can also negatively impact the offspring, including increases in spontaneous abortions, fetal death, small gestational age (SGA), and cognitive dysfunction. Despite these detrimental outcomes, insight into the underlying mechanisms is still sparse. A fundamental gap in our knowledge is understanding how remodeling of the lung microenvironment during distinct gestational stages affects the response to IAV infection. A significant barrier to further these studies has been the difficulty to reliably define gestational dates. To fill this gap in knowledge, I have employed a robust and reproducible pregnant mouse model whereby I use ultrasound analysis to more accurately determine the gestational date allowing me to infect pregnant dams at early gestation (embryonic day (E)8.5), mid-gestation (E10.5) and at the commonly used E12.5 timepoint, which represents late gestation. My in vivo studies have shown that maternal IAV infection during early gestation is associated with increased mortality due to lung damage compared to non-pregnant infected mice and mice infected later in pregnancy, highlighting stage-dependent susceptibility and disease. In pregnant mice infected early in gestation, I found that there is increased septal thickening and denuded bronchioles suggesting damage to the lung’s stroma. In collaboration with Dr. Paul Thomas's laboratory, our group recently demonstrated the critical role for stromal cells in regulating and remodeling the lung microenvironment during response and repair to influenza (Boyd et al., Nature 2020). Collectively, this leads me to hypothesize that the gestational stage modulates IAV pathogenesis by altering the lung microenvironment, including marked changes in the lung's stromal cell populations. I will utilize integrative approaches including histology, flow-cytometry-based assays, single-cell gene-expression profiling (scEX), and bioinformatics. To address this hypothesis, in Aim 1, I will further assess, quantify and functionally determine how gestational stage can mediate increased IAV pathogenesis in the pregnant mother. Aim 2 will further define the impact of gestational influenza infection on maternal lung stromal cells, a severely understudied area. Upon completion, these studies will have major implications on the critical gestational win...