Abstract: PD12-04
Sources of Funding: NIH

Introduction

Leukotriene B4 (LTB4), a dihydroxy fatty acid derived from arachidonic acid, acts as a chemoattractant in non-urothelial tissues and plays a role in innate host defenses. However, virtually nothing is known about the role or, the lack thereof, of LTB4 in the urinary tract, which is the focus of the present study.

Methods

Adult wild-type female mice (7-9 weeks) were inoculated via the transurethral route with type 1 piliated cystitis strain UTI89 or pyelonephritis strain CFT073 (10⁷ cfu in 20 μl PBS). Urothelial production and urinary secretion of LTB4 was measured by ELISA, and its localization determined by confocal immunofluorescence microscopy. The effects of LTB4 deficiency on UPEC infections were examined by administering the wild-type mice with zileuton, an inhibitor of LTB4 production and by using knockout mice lacking arachidonate 5-lipoxygenase (ALOX5), a key enzyme in LTB4 biosynthesis. The resultant neutrophil infiltration, intracellular bacterial community (IBC) formation and clearance, neutrophil myeloperoxidase (MPO) activity, bacterial colony forming units were assessed under specific conditions and controls.

Results

In the wild-type mice, both UPEC strains UTI89 and CFT073 triggered an 8-fold increase of LTB4 in urothelium over PBS controls 2 hours after inoculation. The urothelial level of LTB4 continued to rise, peaking at 12 hours and subsiding considerably at 24 hours, the two time points coinciding with the accumulation and clearance of IBC, respectively. Urine levels of LTB4 lagged slightly behind that of urothelium, probably reflecting time required for protein secretion. Inhibition of LTB4 by zileuton included in the UPEC inocula significantly reduced MPO activity and neutrophil infiltration in the urothelium. In ALOX5 knockout mice, UTI89 and CFT073 inoculation caused four times more IBC than in wild-type mice. Consistent with this, neutrophils were extremely sparse in ALOX5 mice in comparison to the wild-type controls. Additionally, CFT073 were recovered in large numbers from the kidneys of ALOX5 knockout mice, but not from those of wild-type mice.

Conclusions

Our study provides the first experimental evidence indicating that LTB4 signaling initially in the urothelium and later through the neutrophils plays a crucial role in eliciting innate urothelial defenses and in clearing UPECs from the lower and upper urinary tract. Deficiency in LTB4 signaling may contribute significantly to the increased susceptibility of the urinary system to uropathogenic E. coli-caused infections.

Funding

NIH