Insights into mechanisms of antifungal activity by innate immune cells

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen that causes over 180,000 annual deaths in HIV/AIDS patients. Innate phagocytes such as dendritic cells (DCs) and macrophages are the first cells to interact with the pathogen, with destruction inside a mature lysosome through degradation with acid hydrolases and reactive oxygen/nitrogen species. This work investigates the mechanisms of antifungal activity by innate immune cells. Previous studies have shown that innate immune cells will either allow for intracellular growth or kill the fungus by recognizing, phagocytosing, and finally destroying the fungus within a mature lysosome using both oxidative and nonoxidative mechanisms. We hypothesize that there are certain subsets that allow for differential interactions of the fungus and that killing by nonoxidative means is made possible by many different lysosomal proteins. Results showed several lysosomal proteins affected the growth of C. neoformans. Proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, protein concentrations needed for cryptococcal inhibition were found to be non-cytotoxic to mammalian cells. Results also showed all macrophage and dendritic cell subsets interacted with the fungus, and both living and killed morphologies were discernable within the subsets using imaging flow cytometry. Single-cell RNA-seq identified several different clusters of cells which more closely related to interactions with C. neoformans and its protective capacity against the pathogen rather than discrete cellular subsets. Differential gene expression analyses identified several changes in the innate immune cell as it kills the fungus, including increases of TNF-α and a switch to fatty acid metabolism by upregulation of FABP4. Also, increases of TNF-α correlated to cryptococcal interactions. These data show signaling networks that regulate expression of many different genes—both metabolic and immune—as certain clusters of cells mount a protective response. This protective response is facilitated by proteins that have antifungal activity and are well tolerated by mammalian cells. Future studies will examine these proteins, genes, and networks to understand the exact mechanism(s) these subsets use to kill C. neoformans in order to develop immunotherapeutic strategies to combat this deadly disease.