Neutrophil Extracellular Traps Are Found in Bronchoalveolar Lavage Fluids of Horses With Severe Asthma and Correlate With Asthma Severity

Asthma encompasses a spectrum of heterogenous immune-mediated respiratory
disorders sharing a similar clinical pattern characterized by cough, wheeze and exercise
intolerance. In horses, equine asthma can be subdivided into severe or moderate asthma
according to clinical symptoms and the extent of airway neutrophilic inflammation. While
severe asthmatic horses are characterized by an elevated neutrophilic inflammation of the
lower airways, cough, dyspnea at rest and high mucus secretion, horses with moderate
asthma show a milder neutrophilic inflammation, exhibit intolerance to exercise but no
labored breathing at rest. Yet, the physiopathology of different phenotypes of equine
asthma remains poorly understood and there is a need to elucidate the underlying
mechanisms tailoring those phenotypes in order to improve clinical management and
elaborate novel therapeutic strategies. In this study, we sought to quantify the presence of
neutrophil extracellular traps (NETs) in bronchoalveolar lavage fluids (BALF) of moderate or
severe asthmatic horses and healthy controls, and assessed whether NETs correlated
with disease severity. To this end, we evaluated the amounts of NETs by measuring cell-
free DNA and MPO-DNA complexes in BALF supernatants or by quantifying NETs release
by BALF cells by confocal microscopy. We were able to unequivocally identify elevated
NETs levels in BALF of severe asthmatic horses as compared to healthy controls or
moderate asthmatic horses. Moreover, we provided evidence that BALF NETs release
was a specific feature seen in severe equine asthma, as opposed to moderate asthma,
and correlated with disease severity. Finally, we showed that NETs could act as a
predictive factor for severe equine asthma. Our study thus uniquely identifies NETs in
BALF of severe asthmatic horses using three distinct methods and supports the idea that
moderate and severe equine asthma do not rely on strictly similar pathophysiological
mechanisms. Our data also suggest that NETs represent a relevant biomarker, a putative
driver and a potential therapeutic target in severe asthma disease