Undergraduate Researcher Nationwide Children's Hospital Bridgeville, Pennsylvania, United States
Background: Urinary tract infections (UTIs) are the most common bacterial infections encountered by pediatricians, leading to over 1 million healthcare visits annually. Uropathogenic E. coli (UPEC) is the primary cause of UTI and recurrent infections place children at risk for kidney scarring, hypertension, and chronic kidney disease. Antimicrobial peptides (AMPs) play a vital role in combating UTIs by limiting UPEC colonization and invasion. These cationic peptides are produced by uroepithelial cells and are regulated by activation of transcription factors (TFs) in response to pathogens. Identifying the role of TFs on AMP expression can lead to the development of therapeutic treatments that enhance uroepithelial immunity and reduce antibiotic dependence, ultimateily improving outcomes for children susceptible to UTI. Objective: This study was designed to understand how the silencing of transcription factors affects the expression of AMPs that are involved in the prevention of UTIs. Design/Methods: The ENCODE project ChIP-seq matrix was referenced to identify TFs that bind to AMP promoters. RNA-sequencing bladder tissue was used to prioritize TFs with high urothelial expression. AMP genes of interest included those expressed in the human urinary tract: LCN2 (NGAL), RNASE4, RNASE7, SLPI, S100A7, CAMP (cathelicidin), and DMBT1. Primary human bladder epithelial cells were transfected with siRNAs targeting top-candidate TFs, including ELF3 and CEBPB. After 48 hours, cells were collected for RNA extraction to assess downstream expression of AMPs during TF silencing using real-time PCR (qRT-PCR). Results: ENCODE ChIP-seq analysis and RNA-seq identified ELF3 and CEBPB as highly expressed TFs with predicted binding to AMP promoters. siRNA-mediated ELF3 knockdown decreased mRNA expression for S100A7, CAMP, DMBT1, and RNASE7, but increased RNASE4 expression. When CEBPB was silenced, expression decreased for CAMP and increased for DMBT1, RNASE7, and LCN2.
Conclusion(s): ELF3 and CEBPB are TFs that are involved in the regulation of AMP expression that contribute to UTI defenses. Differential AMP responses following TF knockdown demonstrate that ELF3 predominantly promotes AMP expression, whereas CEBPB can act as either an activator or repressor depending on the AMP target. Because pediatric populations are highly susceptible to UTI and long-term UTI sequelae, targeting TF-driven AMP regulation may offer a novel, antibiotic-sparing strategy to strengthen innate immunity to manage UTIs.
