Project Grant R01AI179037

TARGETING MEDIATORS OF OXIDATIVE STRESS TO LIMIT HYPERINFLAMMATORY CELL DEATH MODALITIES DURING MYCOBACTERIUM TUBERCULOSIS INFECTION - PROJECT SUMMARY ONE OF THE BIGGEST CHALLENGES FACING THE TUBERCULOSIS FIELD IS THE HETEROGENEITY OF TB DISEASE PRESENTATION. ONLY A SMALL PERCENTAGE OF INDIVIDUALS INFECTED WITH MYCOBACTERIUM TUBERCULOSIS GO ON TO DEVELOP AN ACUTE INFECTION. WHILE SEVERAL IMPORTANT TUBERCULOSIS CO-MORBIDITIES HAVE BEEN IDENTIFIED (I.E. HIV CO-INFECTION, SMOKING, MALNU- TRITION, DIABETES), WE ARE STILL UNABLE TO ACCURATELY PREDICT WHETHER A TUBERCULOSIS PATIENT IS AT HIGH RISK FOR DEVEL- OPING SEVERE DISEASE. FOR DECADES, WE HAVE UNDERSTOOD THAT THE WAY IN WHICH MTB-INFECTED MACROPHAGES DIE PLAYS A KEY ROLE IN DICTATING MTB OUTCOMES IN VIVO. APOPTOSIS, AN "IMMUNOLOGICALLY SILENT" FORM OF CELL DEATH IS GENERALLY CONSIDERED ANTI-BACTERIAL; APOPTOSIS LIMITS BACTERIAL SPREAD AND DOES NOT RELEASE HIGH AMOUNTS OF IM- MUNOGENIC DAMPS AND PAMPS. NECROSIS, AN INFLAMMATORY FORM OF CELL DEATH, PROMOTES MTB SURVIVAL AND SPREAD AND ELICITS HARMFUL IMMUNOPATHOLOGY. ONE WOULD PREDICT THAT A TUBERCULOSIS PATIENT WITH MACROPHAGES THAT ARE PRONE TO NECROSIS WOULD BE LIKELY TO EXPERIENCE SEVERE INFLAMMATION IN RESPONSE TO MTB INFECTION. OUR PRELIMINARY DATA DEMONSTRATE THAT A COMMON HUMAN SNP IN A MITOCHONDRIAL-ASSOCIATED GENE CALLED LRRK2 PROMOTES A NEW HYPERINFLAMMATORY TYPE OF NECROSIS IN RESPONSE TO MTB INFECTION. THIS CELL DEATH WAS DUBBED GASDERMIN D-MEDIATED NECROPTOSIS, AS IT REQUIRES THE PORE FORMING PROTEIN GASDERMIN D AND RESULTS IN ACTIVA- TION OF RIPK1/RIPK3/MLKL AND CELL DEATH VIA NECROPTOSIS. THIS MUTATION (LRRK2G2019S) PROMOTES EXCESSIVE MITO- CHONDRIAL REACTIVE OXYGEN SPECIES (MTROS) PRODUCTION AND MTROS ALONE IS SUFFICIENT TO PROMOTE GASDERMIN D- MEDIATED NECROPTOSIS. BUILDING ON THESE FINDINGS, THIS PROPOSAL WILL TEST THE HYPOTHESIS THAT MITOCHONDRIAL DYSFUNCTION AND OXIDATIVE STRESS DRIVE MTB-INFECTED MACROPHAGES TO UNDERGO INFLAMMATORY FORMS OF CELL DEATH AND CONSTITUTE A NEW, IMPORTANT "CO-MORBIDITY" FOR TUBERCULOSIS PATIENTS. THE GOAL OF THIS PROPOSAL IS TO MECHANISTICALLY DEFINE HOW OXIDATIVE STRESS PROMOTES PATHOGENIC CELL DEATH MODALITIES DURING MTB INFECTION OF MACROPHAGES AND MICE. HERE, TWO MODELS OF HIGH MTROS WILL BE LEVERAGED (EXOGENOUS MTROS VIA MENADI- ONE TREATMENT AND THE HUMAN DISEASE-ASSOCIATED SNP LRRK2G2019S) TO MECHANISTICALLY DEFINE HOW OXIDATIVE STRESS CAN REPURPOSE CELL DEATH PROTEINS IN PRIMARY MURINE MACROPHAGES. AIM 1 WILL INVESTIGATE THE ABILITY OF LRRK2 KINASE ACTIVITY TO DIRECTLY ACTIVATE NECROPTOSOME PROTEINS AND PROMOTE GASDERMIN D-MEDIATED NECROPTOSIS. AIM 2 WILL DEFINE HOW VARIOUS CELLULAR STRESSES LICENSE N-GSDMD ASSOCIATION WITH MITOCHONDRIAL PHOSPHOLIPIDS TO PROMOTE GASDERMIN D-MEDIATED NECROPTOSIS. AIM 3 WILL EXAMINE THE ROLE OF LIPID OXIDATION IN DICTATING CELL DEATH MODALITY USAGE DURING MTB INFECTION. IF SUCCESSFUL, THIS PROPOSAL WILL DEFINE THE CELLULAR TRIGGERS OF HYPERINFLAM- MATORY CELL DEATH DURING MTB INFECTION OF MACROPHAGES AND IDENTIFY NOVEL POINTS FOR THERAPEUTIC INTERVENTION THAT MAY BE MORE EFFICACIOUS TO TARGET IN TB PATIENTS WITH UNDERLYING CONDITIONS OR GENETICS LINKED TO MITOCHONDRIAL DYSFUNCTION AND OXIDATIVE STRESS.