Project Grant R16AI184345

ROLE OF REACTIVE OXYGEN SPECIES IN CONTROLLING T. GONDII ACUTE INFECTION - PROJECT SUMMARY / ABSTRACT TOXOPLASMA GONDII IS A PROTOZOAN PARASITE THAT INFECTS APPROXIMATELY ONE-THIRD OF HUMANS WORLDWIDE AND CAUSES TOXOPLASMOSIS, A NEGLECTED INFECTIOUS DISEASE IN THE UNITED STATES. ALTHOUGH MOST HUMAN INFECTIONS ARE MILD OR ASYMPTOMATIC, T. GONDII INFECTION CAN RESULT IN LIFE-THREATENING DISEASE IN IMMUNOCOMPROMISED INDIVIDUALS AND IN THE DEVELOPING FETUS DUE TO CONGENITAL INFECTION, UNDERSCORING THE ROLE OF THE HOST IMMUNE SYSTEM IN CONTROLLING THE PARASITE. ALTHOUGH IT IS WELL DOCUMENTED THAT NEUTROPHILS INFILTRATE THE SITE OF ACUTE T. GONDII INFECTION, THERE IS LIMITED UNDERSTANDING OF HOW HUMAN NEUTROPHILS RESPOND TO T. GONDII. AS THERAPEUTIC STRATEGIES ARE STILL LIMITED, AND NO VACCINE IS AVAILABLE, THERE IS A CRITICAL NEED TO UNCOVER THE PERTINENT INNATE IMMUNE RESPONSES TO T. GONDII INFECTION. NEUTROPHILS CONTROL INFECTIOUS PATHOGENS BY A VARIETY OF MECHANISMS, INCLUDING THE RELEASE OF REACTIVE OXYGEN SPECIES (ROS). THE PRODUCTION OF ROS HAS BEEN DEMONSTRATED IN T. GONDII-INFECTED NEUTROPHILS; HOWEVER, THE CELLULAR AND MOLECULAR MECHANISMS INVOLVED IN THESE PROCESSES AND THEIR CONTRIBUTION TO PARASITE KILLING ARE UNKNOWN. IN THE CURRENT PROPOSAL, WE WILL: I) DETERMINE THE CELLULAR EVENTS REQUIRED TO INDUCE ROS IN T. GONDII-INFECTED NEUTROPHILS, II) DETERMINE WHICH RECEPTORS ARE ASSOCIATED WITH T. GONDII RECOGNITION AND ROS PRODUCTION, III) DETERMINE WHETHER T. GONDII-INDUCED ROS IS MEDIATED BY THE NOX2- DEPENDENT OR NOX2-INDEPENDENT (MITOCHONDRIAL) PATHWAY IN NEUTROPHILS, AND IV) DETERMINE WHETHER ROS AND PHAGOSOME MATURATION CONTRIBUTE TO NEUTROPHIL-MEDIATED T. GONDII KILLING. WE WILL USE PRIMARILY THE CELL LINE HL- 60, WHICH IS A DEVELOPMENTAL PRECURSOR OF NEUTROPHILS AND CAN BE DIFFERENTIATED INTO NEUTROPHIL-LIKE CELLS (NLCS) FOR FUNCTIONAL TESTS. THIS IS A SUITABLE MODEL THAT ALLOWS US TO DECIPHER SIGNALING PATHWAYS BY USING GENETIC LOSS- OF-FUNCTION EXPERIMENTS. ADDITIONALLY, WE WILL CONFIRM OUR CENTRAL FINDINGS IN PRIMARY HUMAN NEUTROPHILS ISOLATED FROM HEALTHY BLOOD DONORS. OUR PRELIMINARY DATA ON NLCS AND PRIMARY HUMAN NEUTROPHILS HAVE INDICATED THAT THESE CELLS PRODUCE ROS AND KILL PHAGOCYTOSED PARASITES. THE RATIONALE FOR THIS PROJECT IS THE RELEVANCE OF UNDERSTANDING THE VERY EARLY IMMUNE EVENTS THAT AFFECT THE PROGRESSION OF THE HOST RESPONSE AND THE DISEASE. COMPLETION OF THESE STUDIES IS THEREFORE EXPECTED TO HAVE A SIGNIFICANT IMPACT BY PROVIDING FOUNDATIONAL KNOWLEDGE THAT CAN AFFECT OUR ABILITY TO DEVELOP THERAPEUTICS, THUS CONTROLLING T. GONDII SPREAD TO SITES SUCH AS THE HEART AND THE BRAIN AND ATTENUATING DISEASE PATHOGENESIS.