# Presence of neutrophil extracellular traps (NETs) in different types of human urinary tract infections (UTI). A pilot study

## TL;DR

This pilot study found that neutrophil extracellular traps (NETs) are present in urine samples from various types of urinary tract infections, suggesting a potential role in infection pathology.

## Contribution

The study is one of the first to systematically detect and characterize NETs in different human urinary tract infection types.

## Key findings

- NETs were significantly more prevalent in UTI patients compared to healthy controls.
- Three distinct NET phenotypes were identified in UTI samples.
- NET-associated proteins were present in UTI groups but absent in controls.

## Abstract

Activated polymorphonuclear neutrophils (PMN) release neutrophil extracellular traps (NETs) composed of a web-like DNA core, concomitant with nuclear histones, granular peptides and enzymes. NETs in human urine and their potential role in human urinary tract infections (UTI) pathogenesis is still understudied. This pilot study aimed to analyze presence of NETs in urine samples of patients with different types of UTI.

Urine and blood samples were collected from three cohorts: group (A) included females (n = 24) with cystitis (n = 10), pyelonephritis (n = 6), and asymptomatic bacteriuria (n = 8); group (B) composed of males with catheter-associated UTI (n = 20) and a control group (C) consisting of healthy patients of mixed gender (n = 20). NETs in urine samples were confirmed by immunofluorescence-based detection of neutrophil elastase and citrullinated histone. The presence of granular enzymes (myeloperoxidase, cathelicidin), calprotectin (subunits S100A8, S100A9) and CD15+ PMN were detected by ELISA, western blot and flow cytometry, respectively. To study potential associations of NETs with the respective UTI microbiome, bacterial spectrum of each urine sample was estimated by 16S rRNA gene analysis.

On average, 23.29% ± 16.89% of PMN forming NETs were detected in group A [subgroups cystitis (27.72% ± 17.88%), pyelonephritis (22.75% ± 12.91%), asymptomatic bacteriuria (18.17% ± 17.14%)] and 30.63% ± 17.88% in group B, with no differences observed between UTI groups, including patients with asymptomatic bacteriuria. For the control group (group C), a low incidence of NET-releasing cells was observed (0.32% ± 1.42%), resulting in a significant difference (p < 0.05) when compared to all UTI groups studied. Furthermore, different NET-phenotypes [i. e. spread NETs (sprNETs), diffuse NETs (diffNETs) and aggregated NETs (aggNETs)] were detected in both UTI groups. The presence of NET-associated proteins was confirmed in all UTI groups, but absent in the control samples. Microbiome analyses revealed a reduced microbial variability within UTI samples with the predominance of the bacterial family Enterobacteriaceae. Overall, PMN-derived NETs were consistently found in all UTI samples, suggesting a role of NETs in diverse UTI pathologies. Future studies should investigate its utility as an inflammatory biomarker in clinical human UTI.