Human cellular models of TB and innate immunity - Tuberculosis (TB) is one of the largest causes of global infectious disease death. Current treatments of TB infection involve complex drug regimens that require months of treatment and are plagued by drug toxicities and drug resistance. Furthermore, TB has been the cause of death in a third to a half of the approximately 40 million AIDS deaths and remains a major cause of death in those HIV-infected individuals especially those who are immunosuppressed. Therefore, host-directed therapeutics (HDT) that increase innate immune responses to TB when combined with current antimicrobial therapies, could potentially enhance TB cure and clinical outcomes. However, the critical knowledge about the human innate immune response to MTb required to develop such HDTs is lacking as well as physiological in vitro lab models to study and validate such early innate host responses to TB infection. Here, we propose to overcome these barriers by establishing a new physiological in vitro lung alveolar epithelial cell organoid model from human bronchoalveolar lavage fluid (BALF) (Aim 1). Specifically, building on our studies with co-investigator Carla Kim, where we developed a new model for Mycobacterium tuberculosis (MTb) infection using pulmonary alvelolar epithelial type II (AT2) organoids derived from human lung tissue, in Aim 1, we propose to develop an AT2 organoid MTb infection model from easily obtainable human bronchalveolar lavage fluid (BALF), which contains primary AT2 cells and other cells including macrophages from the same individual. These BALF-AT2 organoids will allow us to use advanced imaging microscopy in collaboration with Dr. Tom Kirchausen to study MTb infection of AT2 and macrophage cells and their interaction, as well as the effect of nitazoxanide (NTZ), an experimental MTb HDT, which inhibits MTb growth in concert with broadly amplifying type I and III IFN signaling. Using AT2 tissue organoids and NTZ as a probe, in Aim 2 we will perform bulk and single-cell (sc)-seq analyses to identify factors that are associated with NTZ inhibition of early MTb infection and growth in collaboration with Dr. Brad Rosenberg. Critical pathways and novel factors in innate immune restriction of early MTb infection will then be functionally validated using CRISPR approaches. We anticipate will provide new model physiological platforms for study of TB infection and that these studies will elucidate critical innate immune mechanisms that may serve as HDT targets for further study and inhibition of MTb infection.
