Equine Asthma Is Characterised by Severity-Dependent Correlations Between Blood Neutrophil Cholesterol Content and NET Formation

Equine asthma (EA) is the most prevalent chronic lung disease in horses. Neutrophils are the main effector cells in severe EA. Neutrophil extracellular traps (NETs) have been described as contributors to severity in human asthma and chronic obstructive pulmonary disease. Thus, we aimed to investigate if NET-related factors in equine neutrophils, blood and bronchoalveolar lavage fluid (BALF) allow us to differentiate EA severities and to identify NET-related mechanistic insights in EA. We quantified NETs and NET-related factors in the blood and BALF of eight healthy horses and 18 horses with differing EA severities. The proportion of activated cells in BALF increased with EA severity, accompanied by dysregulation of local NET regulators in severe EA. Furthermore, circulating anti-neutrophil cytoplasmic antibodies (ANCAs = NET-autoantibodies) were found elevated in severely diseased horses. In line with these findings, NET formation by circulating neutrophils was found to depend on the severity of EA. Finally, we analysed the cholesterol content of circulating neutrophils and identified an asthma-severity-dependent decrease in cellular cholesterol, which correlated with increased NET formation and hypoxia. Local and systemic modifications—particularly in neutrophil cellular cholesterol content—provide further insight into the partially understood pathogenesis of EA and point to a systemic cholesterol-associated inflammatory component fuelling disease progression.