Abstract: PD12-02
Sources of Funding: SPU 2016 research grant; K08 grant DK102594 from NIDDK

Introduction

Urinary tract infections (UTI) are a significant source of morbidity. Due to rising antibiotic resistance patterns, alternate treatments are needed. Prior work has demonstrated induction of urine and serum IL-6 in pyelonephritis and renal scarring. IL-6 has been linked to antimicrobial peptide (AMP) production in other organs via Stat3 phosphorylation on Tyr705. As a result, we sought to evaluate the potential role of IL-6/Stat3 induced signaling on urothelial AMP expression and UTI susceptibility.

Methods

We infected 6 week old female C57BL/6J or C3H/HeOuJ mice transurethrally with UPEC and measured IL-6, phosphorylated (p-)Stat3, and AMP expression by ELISA, qRT-PCR, Western blotting, and immunofluorescence microscopy. The requirement for Lipopolysaccharide (Lps) / Toll-like receptor 4 (Tlr4) signaling was established using Lps-hyporesponsive C3H/HeJ mice, compared to C3H/HeOuJ Lps-sensitive controls. We evaluated the roles of IL-6 in p-Stat3, AMP production, and UPEC clearance using IL-6 neutralizing antibody and IL-6 knock out (KO) mice. We determined the contribution of Stat3 to AMP production via tamoxifen-inducible Stat3 conditional KO mice. We measured bacterial burden by serial plating on LB agar. Results were evaluated by Mann-Whitney U test with p <0.05 being significant.

Results

Upon UPEC infection, we observed a time dependent induction of urinary IL-6 secretion and bladder p-Stat3. Sustained expression of IL-6 and p-Stat3 required intact Tlr4 signaling. P-Stat3 localized to the nuclei of infected bladder urothelium. Bladder AMP mRNA levels increased following peak IL-6 secretion and p-Stat3. IL-6 KO mice exhibited absent urothelial p-Stat3 and severely blunted AMP mRNA induction following experimental UTI, leading to impaired UPEC clearance. Conversely, intraperitoneal administration of recombinant IL-6 induced bladder p-Stat3 and AMP expression in the absence of infection. Conditional Stat3 deletion reduced bladder AMP mRNA levels at baseline and following experimental UTI. IL-6 neutralizing antibody reduced p-Stat3 levels in C3H/HeOuJ mice, with increased renal UPEC colonization and pyelonephritis severity.

Conclusions

IL-6 is both necessary and sufficient for urothelial p-Stat3 and AMP transcription, and genetic or acquired IL-6 deficiency leads to increased UTI susceptibility. This implicates IL-6 and Stat3 as master regulators of urothelial AMP transcription in states of sterility and infection. Therapeutic manipulation of the IL-6/Stat3 axis provides a mechanism to alter AMP expression to treat and even prevent UTI.

Funding

SPU 2016 research grant; K08 grant DK102594 from NIDDK