Project Grant R21AI184357

TRAINING HUMAN IPSC-DERIVED NEUTROPHILS FOR ANTIMICROBIAL FUNCTION - PROJECT SUMMARY NEUTROPHILS ARE TERMINALLY DIFFERENTIATED CELLS OF THE INNATE IMMUNE SYSTEM THAT ARE NECESSARY FOR HOST DEFENSE. EMERGING EVIDENCE SUGGESTS THAT NEUTROPHILS HAVE MORE HETEROGENEITY AND PLASTICITY THAN PREVIOUSLY THOUGHT. HOWEVER, THERE IS A GAP IN UNDERSTANDING NEUTROPHIL DEVELOPMENTAL HETEROGENEITY AND FUNCTION BECAUSE THEY ARE SHORT-LIVED EX VIVO AND ARE NOT GENETICALLY TRACTABLE. INDUCED PLURIPOTENT STEM CELLS (IPSC)-DERIVED HUMAN NEUTROPHILS (INEUTROPHLS) OFFER THE OPPORTUNITY TO GENETICALLY AND DEVELOPMENTALLY PROGRAM NEUTROPHILS FOR DESIGNED PROPERTIES THAT MAY BE USED BOTH TO UNDERSTAND NEUTROPHIL BIOLOGY AND PROVIDE AN AVENUE FOR ENGINEERING NEUTROPHILS FOR HUMAN TREATMENT. WE HAVE ENGINEERED GMP-COMPATIBLE HUMAN INEUTROPHILS THAT SHOW ANTIMICROBIAL FUNCTION IN VITRO BUT DISPLAY SIGNIFICANT HETEROGENEITY WITH DISTINCT SUBTYPES BASED ON PRELIMINARY SINGLE CELL ANALYSIS. OUR PRELIMINARY DATA ALSO SUGGEST THAT LPS TREATMENT OF PROGENITOR CELLS "TRAINS" THE INEUTROPHILS FOR AN INCREASED RESPONSIVENESS TO SECONDARY STIMULI. HERE WE PROPOSE TO USE SINGLE-CELL MULTI-OMICS TO UNDERSTAND THE HETEROGENEITY AND FUNCTION OF INEUTROPHILS AND TO TEST THE HYPOTHESIS THAT INEUTROPHILS CAN BE USED TO UNDERSTAND THE METABOLIC AND GENOMIC MECHANISMS OF TRAINED IMMUNITY OF HUMAN NEUTROPHILS. SPECIFICALLY, WILL USE SINGLE-CELL MULTI-OMICS AND OPTICAL METABOLIC IMAGING TO IDENTIFY THE TRANSCRIPTIONAL, EPIGENETIC AND METABOLIC HETEROGENEITY THAT COULD INFORM FUNCTIONS OF DISTINCT INEUTROPHIL SUBSETS AND THE EFFECTS OF IMMUNE TRAINING. WE WILL ALSO DEVELOP ZEBRAFISH LARVAE AS AN IN VIVO MODEL TO SCREEN FOR INEUTROPHIL ANTIMICROBIAL FUNCTIONS IN MODELS OF BACTERIAL AND FUNGAL INFECTION. THE PROPOSED WORK WILL PROVIDE A GENETICALLY TRACTABLE SYSTEM TO UNDERSTAND THE HETEROGENEITY OF HUMAN NEUTROPHILS, AND THE EFFECT OF IMMUNE TRAINING ON NEUTROPHIL FUNCTION THAT MAY OPTIMIZE FOR ANTIMICROBIAL EFFECTS.