97 - NADPH Oxidase Complex 2 is Required to Control Experimental Urinary Tract Infection
Friday, April 22, 2022
6:15 PM – 8:45 PM US MT
Poster Number: 97 Publication Number: 97.138
Rachel Han, Nationwide Children's Hospital, Columbus, OH, United States; Hanna Cortado, Nationwide Children's Hospital, columbus, OH, United States; Birong Li, Nationwide Children's Hospital, Columbus, OH, United States; Ashley R. Jackson, Nationwide Children's Hospital, Columbus, OH, United States; Brian Becknell, Ohio State University College of Medicine, Columbus, OH, United States; Juan de Dios Ruiz-Rosado, Nationwide Children's Hospital, Columbus, OH, United States
Neonatal-Perinatal Medicine Fellow Nationwide Children's Hospital Nationwide Children's Hospital Columbus, Ohio, United States
Background: Urinary tract infections (UTIs) rank among the most serious bacterial illnesses in early childhood with a high risk for urosepsis, acute pyelonephritis, and renal scarring as a long-term morbidity. Systemic antibiotic treatment is the only currently available therapy for the management of UTI, which are most often caused by Uropathogenic Escherichia coli (UPEC). Thus, the development of alternative non-antibiotic strategies for the treatment of UTI is necessary. NADPH oxidase complex 2 (NOX2) is the phagocyte oxidase with an essential role in host antimicrobial defense during bacterial and fungal infections, but its role during UTI remains unknown.
Objective: To study the role of NOX2 in bacterial killing and reactive oxygen species (ROS) production in experimental cystitis and pyelonephritis.
Design/Methods: We tested the role of NOX2 during cystitis and pyelonephritis through the use of NOX2 (Cybb) knockout (KO) mice and the NOX2-specific inhibitor, GSK2795039. We measured NOX2 protein expression during experimental UTI by Western blotting and determined its cellular source by immunofluorescence microscopy. UPEC burden was assessed by enumerating colony forming units (CFU). Neutrophils were isolated from murine bone marrow or peritoneal lavage following thioglycolate administration. The roles of NOX2 in neutrophil ROS production and antimicrobial activity were determined by co-culturing neutrophils with UPEC in the setting of NOX2 deficiency or chemical inhibition, followed by the measurement of intracellular and extracellular ROS, along with intracellular CFU.
Results: NOX2 protein levels increased during experimental cystitis and pyelonephritis in a manner that parallels neutrophil recruitment. Immunofluorescence microscopy localized NOX2 primarily in infiltrating neutrophils. NOX2 KO mice were more susceptible to experimental cystitis with increased bladder burden 6 and 48 hours post infection (hpi). Western blotting revealed increase in NOX2 expression at 6 and 48 hpi in the bladder, which appeared proportional to the CFUs/bladder. Likewise, chemical inhibition of NOX2 during experimental pyelonephritis resulted in attenuated intracellular ROS production in neutrophils and augmented renal UPEC burden. Neutrophils from NOX2 KO mice exhibit impaired extracellular ROS production and increased intracellular bacteria compared to wild-type controls, after challenged with UPEC in vitro. Similar effects in wild-type murine neutrophils following chemical inhibition of NOX2 in vitro.Conclusion(s): Neutrophil derived NOX2 serves a critical antimicrobial role against UPEC in vivo and in vitro.