Decoding the Interferome by Mapping Genetic Interactions in Human Tissue

PROJECT SUMMARY Non-immune cells greatly outnumber professional immune cells in the body, providing intracellular pathogens with many opportunities to shelter and take refuge. How does the immune system protect this huge landscape? My previous work described the ability of the immune cytokine interferon-g (IFN-g), classically considered a macrophage activating protein, to broadly activate non-immune cells and confer the ability to mount sterilizing cell-intrinsic responses through effectors encoded by Interferon-Stimulated Genes (ISGs), collectively termed the ‘interferome’. However, our understanding of how ISGs execute tissue localized responses is in its infancy, with remarkably few of these effectors being well characterized and virtually nothing known about how multiple ISGs functionally interact to achieve host defense. The principal barrier to understanding ISGs is that they rarely work in isolation; rather they are part of complex genetic networks that are buffered from the phenotypic effects of perturbation. I propose a radical new strategy that exploits this genetic complexity by mapping functional relationships between ISGs using combinatorial forward genetic screens. My central hypothesis is that systematically mapping genetic interactions will uncover the effectors of localized IFN-g signalling and enable hierarchical organization of ISG products into functional complexes and pathways (network) that execute specific protective and pathological responses. On a small scale with a single query gene in my postdoctoral work, this approach unveiled a pair of synergistic ISGs that execute unexpectedly potent bactericidal defense of the cytosol. Further development of this technology for use at a larger scale in my own lab will provide the framework to fully deconvolve the interferome into distinct host resistance pathways. I will develop an experimental pipeline that enlists a pooled CRISPR-based screening system for multi-locus gene perturbation paired with a novel single cell imaging and expression analysis platform. Focusing on the human airway epithelium, this innovative approach will be applied to dissect the ISG-encoded effectors that mediate cell-intrinsic control of three diverse pulmonary pathogens (C. pneumoniae, B. pertussis, and RSV), and those that mediate lung tissue damage downstream of cytokine storm. Newly identified ISGs and their connections will be validated for their protective or pathological activities using tissue explant systems from donated human lungs and models of human lung organoids. The ensuing genetic interaction network will provide unprecedented insight into the effectors that dictate infection outcome in the human lung during type 1 immune responses. These findings will reveal new local therapeutic targets and establish a paradigm for appreciating the full spectrum of immunity. The approaches pioneered here will also extend to the downstream effectors of other non-immune cells and tissues whose integrated study will define a new biological landscape of critical importance to human health.