Analysis of the heterogeneity of cell death responses in the influenza virus-infected cells

Summary Efficient treatment of severe influenza remains a significant unmet medical need, as this disease causes hundreds of thousands of hospitalizations and tens of thousands of deaths every year. Severe flu cases are associated with extensive cell death of the lung epithelial cells, causing the loss of lung oxygen exchange function, which can be fatal even if the virus is efficiently cleared. On the other hand, cell death is an essential component of the innate immune response, helping eliminate infected cells and prime the adaptive immune response. Critical anti-viral transcriptional programs, such as activation of the interferon responses and synthesis of a variety of additional cytokines and chemokines, also intertwine with different forms of cell death to shape host defense. Furthermore, recent evidence suggests that not all flu-infected cells are eliminated through cell death; a subset can survive and clear the virus through non-lethal means. Depending on the context, these “persister” populations may either contribute to lung repair or drive long-term inflammatory pathology (i.e., long flu). Given these complexities, to target cell death therapeutically, it is critical to understand the interplay of the transcriptional and cell death responses to elucidate which cell death events can be stopped safely without compromising viral clearance. However, the challenge for accurately interpreting the transcriptional dynamics of a population of cells asynchronously undergoing cell death is the lack of RNA-seq-compatible readouts that measure the cell death status of each cell and, thereby, allow differentiation of the persisters from the cells that have initiated or executed cell death. We propose to address this challenge using a new scAnnexinV-seq assay that we recently developed, which allows the linking of each cell's transcriptome to its cell death status. This is accomplished by conversion of a frequently used Annexin V/PI live/dead assay into a format that can be read out during scRNA-seq analysis. In Aim 1, we will optimize scAnnexinV-seq using a simple in vitro system of primary macrophages induced to undergo a particularly pathogenic form of cell death - necroptosis. Aim 2 will further adapt scAnnexinV-seq to analyze the more complex and biologically relevant model system – transcriptional and cell death dynamics in the IAV-infected primary Type I alveolar epithelial cells at a single cell level. For these experiments, our assay will be expanded to include additional readouts monitoring the viral infection status of each cell. The proposed studies will provide a valuable new approach to understanding cell fates consequential to viral infections.