Transcriptional Profiling of Stat3-Overexpressing Mice During Urinary Tract Infection (UTI)

Background: Chronic UTI is a bothersome clinical scenario of prolonged bacterial infection of the bladder. We previously demonstrated Signal Transducer and Activator of Transcription 3 (STAT3) limits chronic UTI. Although STAT3 regulates several cellular processes including the immune response, cellular proliferation, and cellular barrier function in various pathologies, it has not yet been investigated in chronic UTI. We hypothesize that STAT3 regulates cellular pathways maintaining epithelial barrier, increasing cellular regeneration (K14, Ki67, Reg3), enhancing antimicrobial peptide production (AMP), and regulating the innate immune response to minimize tissue damage and confer protection during UTI. Methods: 6-8 week old FVB/N wild type (WT) and constitutively active Stat3 (Stat3C) mice underwent experimental UTI. Serial dilution and plating were used to determine urine and bladder burden up to 7 days post infection (dpi). FVB/N vs STAT3C bladder RNA was extracted using Qiagen RNeasy mini kit. Bulk RNAseq was performed on bladders at baseline, 24 hours post infection and 7dpi. Pairwise comparisons were performed to identify differentially expressed genes across relevant groups (adj P ≤ 0.05, absolute fold change ≥ 1.5). Functional enrichment analyses were conducted using the cluster Profiler R package, and upstream transcriptional regulators were predicted using Ingenuity Pathway Analysis (IPA). Immunofluorescence (IF) against K14 and Ki67 was performed on bladders. Results: Stat3C mice demonstrated reduced urine and then bladder burden by 7dpi (p=0.0123). RNAseq analysis identified specific genes upregulated at baseline in STAT3C mice, with further significant increases observed at 24hpi and 7dpi. IPA and gene ontology enrichments revealed upregulation of genes involved in cellular regeneration, innate immunity, cytokine response, AMP production and metabolic pathways. Confirmation of Krt14 and Ki67 by IF showed increased expression in Stat3C. Conclusion: Stat3 overexpression in the urothelium results in upregulation of genes in cellular pathways involved in antimicrobial targeting (i.e. AMPs) and epithelial barrier function and urothelial integrity. This would indicate that STAT3 regulates transcription in urothelial cells to promote urothelial fitness in protecting against chronic UTI. Funding: NIDDK Support