Project Grant F31AI203121

DEFINING ROLES OF GASDERMIN FAMILY AND NINJ1 CLUSTERING EVENTS IN NEUTROPHIL DEATH - THE NLRP3 INFLAMMASOME HAS BEEN IMPLICATED IN NUMEROUS COMMON DISEASES AFFECTING MULTIPLE ORGAN SYSTEMS. NLRP3 WAS FIRST IDENTIFIED AS THE CAUSE OF A RARE MONOGENIC AUTOINFLAMMATORY DISEASE TERMED CRYOPYRIN-ASSOCIATED PERIODIC SYNDROMES (CAPS), WHICH RESULTS FROM ACTIVATING MUTATIONS IN NLRP3. CAPS IN HUMANS AND MICE IS CHARACTERIZED BY NEUTROPHILIC INFLAMMATION, AND MOUSE MODELS OF CAPS PROVIDE A CLINICALLY RELEVANT SYSTEM TO STUDY INFLAMMASOME ACTIVATION IN NEUTROPHILS. ALTHOUGH MUCH PROGRESS HAS BEEN MADE IN UNDERSTANDING INFLAMMASOME FUNCTION, A KNOWLEDGE GAP REMAINS REGARDING THE BIOCHEMICAL AND BIOPHYSICAL CHANGES THAT OCCUR UPON GASDERMIN AND NINJ1 ACTIVATION, PARTICULARLY IN NEUTROPHILS. UNDERSTANDING THE CONTRIBUTIONS OF GASDERMINS AND NINJ1 TO CYTOKINE SECRETION, NET FORMATION, INFLAMMATORY CELL DEATH, AND ULTIMATELY NEUTROPHILIC INFLAMMATION WILL SUPPORT THE DEVELOPMENT OF MORE EFFECTIVE, SAFER, AND TARGETED THERAPIES. ANALYSIS OF NEUTROPHILS EXPRESSING MUTANT NLRP3 INDICATES THAT GASDERMIN D (GSDMD) AND GASDERMIN E (GSDME) ACT COOPERATIVELY-RATHER THAN REDUNDANTLY-TO DRIVE IL-1B RELEASE, PYROPTOSIS, AND SEVERE AUTOINFLAMMATION. NLRP3 ACTIVATION IN NEUTROPHILS INDUCES CLUSTERING OF GSDMD AND GSDME AND SUBSEQUENT CAVITATION OF THE PLASMA MEMBRANE. THE PROPOSED STUDY HAS THE FOLLOWING OBJECTIVES. AIM 1 WILL CHARACTERIZE THE IMPACT OF NINJ1 DEFICIENCY ON CAPS AUTOINFLAMMATION AND THE EFFECTS OF NINJ1-MEDIATED NEUTROPHIL RUPTURE ON TISSUE INFLAMMATION. AIM 2 USES COMPLEMENTARY IMAGING METHODS TO INVESTIGATE NEUTROPHIL CELL DEATH: (A) LIVE-CELL KINETIC ANALYSIS OF PYROPTOSIS; (B) SUPER-RESOLUTION MICROSCOPY TO TRACK THE SUBCELLULAR ORGANIZATION OF GASDERMINS AND NINJ1; (C) MINFLUX NANOSCOPY TO DEFINE THE STRUCTURE OF INDIVIDUAL PORES AND THE POTENTIAL FOR GSDMD-GSDME HETERODIMERIZATION IN THE PLASMA MEMBRANE; AND (D) AFM-TIRF TO CORRELATE THE LOCALIZATION OF GASDERMINS AND NINJ1 WITH CORRESPONDING CHANGES IN PLASMA MEMBRANE TOPOGRAPHY AND STIFFNESS. AIM 3 LEVERAGES A HIGH-THROUGHPUT MICROFLUIDIC SYSTEM WITH HYDRODYNAMIC TRAPPING TO ACHIEVE SINGLE-CELL IMMOBILIZATION, ENABLING REAL-TIME Z-STACK ACQUISITION OF NEUTROPHILS AT SUPER-RESOLUTION. THIS APPROACH WILL HELP ELUCIDATE THE DISTINCT FEATURES OF NUCLEAR DECONDENSATION AND NET RELEASE FROM THE PLASMA MEMBRANE OF PYROPTOTIC NEUTROPHILS, IN COMPARISON TO APOPTOTIC NEUTROPHILS THAT EXHIBIT CHROMATIN CONDENSATION AND RELY ON GSDME ALONE FOR MEMBRANE RUPTURE. OVERALL, THESE STUDIES ARE LIKELY TO PROVIDE TRANSFORMATIVE ADVANCES IN OUR UNDERSTANDING OF INFLAMMASOME SIGNALING THAT CULMINATES IN NEUTROPHIL PYROPTOSIS. THIS RESEARCH WILL HAVE BROADER IMPLICATIONS FOR REGULATED CELL DEATH THAT ARE KEY TO UNDERSTANDING MECHANISMS OF STERILE INFLAMMATION, PYROPTOTIC-TRIGGERED NET FORMATION, RESPONSES TO PATHOGENS, AND CHRONIC INFLAMMATION.