FUNCTION OF PAIRED ACTIVATING AND INHIBITORY FCR SIGNALING IN IMMUNE REGULATION - PROJECT SUMMARY RECOGNITION OF SECRETED IGG BY FC RECEPTORS (FCRS) ON INNATE IMMUNE CELLS IS CRITICAL FOR CLEARING PATHOGENS BY PHAGOCYTOSIS. HOWEVER, OVERACTIVE FCR SIGNALING CAN DAMAGE HEALTHY TISSUE. MACROPHAGES, AN INNATE IMMUNE CELL TYPE, REGULATE TISSUE HOMEOSTASIS AND PATHOGEN CLEARANCE THROUGH IMMUNE EFFECTOR FUNCTIONS INCLUDING CYTOKINE RELEASE, ANTIBODY-DEPENDENT CELLULAR TOXICITY, ANTIGEN PRESENTATION, AND PHAGOCYTOSIS. MACROPHAGES EXPRESS FIVE ITAM-BEARING ACTIVATING FCRS, AND A SINGLE ITIM-BEARING INHIBITORY FCR: FCGRIIB. MISREGULATED FCGRIIB SIGNALING IS ASSOCIATED WITH CHRONIC AUTOIMMUNE CONDITIONS. FROM A SYSTEMS-PERSPECTIVE, FCGRIIB IS PUZZLING. IF MACROPHAGES CAN SIMPLY DOWNREGULATE ACTIVATING FCR, WHY DOES FCGRIIB EXIST? GIVEN THAT FCGRIIB UNIQUELY SHARES THE LIGAND OF IGG WITH ITS COGNATE ACTIVATING FCGRS, THE DISSECTION OF ITS SPECIFIC EFFECTS FROM THE ACTIVATING FCRS IS PARTICULARLY CHALLENGING. TO SOLVE THIS, WE DESIGNED A SYNTHETIC RECEPTOR SYSTEM THAT ALLOWS SELECTIVE STIMULATION OF FCRIIB AND ACTIVATING FCRS INDEPENDENTLY USING DNA NANOTECHNOLOGY. USING THIS SYSTEM, OUR PRELIMINARY DATA DEMONSTRATES THAT FCGRIIB STIMULATION DOES NOT INHIBIT A SPECIFIC STAGE OF PHAGOCYTOSIS BUT RATHER SETS A TIME LIMIT ON THE DURATION OF THE PHAGOCYTIC ATTEMPT, THEREBY INCREASING THE FREQUENCY OF PHAGOCYTIC FAILURES. WE CONFIRMED THAT THIS EFFECT REQUIRED WITH RECRUITMENT OF SHIP1, AN INOSITOL PHOSPHATASE KNOWN TO COMPLEX WITH FCGRIIB'S ITIM AND DEPHOSPHORYLATE PI(3,4,5)P3 (PIP3), A KEY SIGNALING LIPID REQUIRED FOR PHAGOCYTOSIS. FURTHER, A COMPUTATIONAL MODEL OF PHOSPHOLIPID CONVERSION DURING CONCURRENT ACTIVATING AND INHIBITORY FCR SIGNALING GENERATES AN INCOHERENT FEEDFORWARD LOOP THAT SUPPORTS OUR IN VIVO OBSERVATIONS. THEREFORE, THE CENTRAL HYPOTHESIS OF THIS PROPOSAL IS THAT FCGRIIB INHIBITS PHAGOCYTOSIS BY ENACTING A PHOSPHOLIPID MEDIATED TIME-LIMIT ON PHAGOCYTOSIS. TO TEST THIS HYPOTHESIS, I WILL UTILIZE GENETICALLY ALTERED MACROPHAGES WITH FCGRIIB KNOCKED OUT OR OVEREXPRESSED. IN AIM 1, I WILL TEST THE HYPOTHESIS THAT THE DRAMATICALLY INHIBITED FCGRIIB PHENOTYPE IS A RESULT OF DECREASED PHAGOCYTOSIS ATTEMPT DURATIONS THROUGH HIGH RESOLUTION QUANTITATIVE IMAGING ANALYSIS OF TIMELAPSES CAPTURING PHAGOCYTOSIS. IN AIM 2, I WILL DEMONSTRATE THAT DIFFERENCES IN PHAGOCYTIC ATTEMPT DURATION IN MACROPHAGES WITH AND WITHOUT FCGRIIB SIGNALING ARE MEDIATED BY CHANGES TO PIP3 DYNAMICS AT THE PROGRESSING PHAGOCYTIC CUP. I WILL ACCOMPLISH THIS WITH THE USE OF A HIGHLY SPECIFIC FLUORESCENT PIP3 PROBE. FINALLY, AIM 3 WILL PROVIDE INSIGHT INTO THIS INHIBITORY MECHANISM'S ROLE IN CONTROLLING MACROPHAGE BEHAVIOR IN RESPONSE TO VARIOUS IGG ISOTYPES DURING DIFFERENT INFLAMMATORY CONTEXTS, BY MEASURING BOTH PHAGOCYTIC TIMING AND PIP3 DYNAMICS DURING ENGULFMENT OF TARGETS OPSONIZED WITH DIFFERENT IGG ISOTYPES. COLLECTIVELY, THIS WORK WILL ESTABLISH FCGRIIB AS A PIP3-DRIVEN MOLECULAR TIMER THAT ENFORCES STRICT TIME LIMITS ON PHAGOCYTIC PERSISTENCE, RESOLVING HOW ITS DYSREGULATION CAUSES PATHOGENIC OUTCOMES AND PROVIDING MECHANISTIC CRITERIA TO OPTIMIZE THERAPEUTIC ANTIBODY ISOTYPE SELECTION FOR TUNING EFFECTOR CELL ENGAGEMENT.