The amount of hyaluronic acid and airway remodelling increase with the severity of inflammation in neutrophilic equine asthma.

BACKGROUND: Equine asthma (EA) is a chronic lower airway inflammation that leads to structural and functional changes. Hyaluronic acid (HA) has crucial functions in the extracellular matrix homeostasis and inflammatory mediator activity. HA concentration in the lungs increases in several human airway diseases. However, its associations with naturally occurring EA and airway remodelling have not been previously studied. Our aim was to investigate the association of equine neutrophilic airway inflammation (NAI) severity, airway remodelling, and HA concentration in horses with naturally occurring EA. We hypothesised that HA concentration and airway remodelling would increase with the severity of NAI. HA concentrations of bronchoalveolar lavage fluid supernatant (SUP) and plasma of 27 neutrophilic EA horses, and 28 control horses were measured. Additionally, remodelling and HA staining intensity were assessed from endobronchial biopsies from 10 moderate NAI horses, 5 severe NAI horses, and 15 control horses. RESULTS: The HA concentration in SUP was higher in EA horses compared to controls (p = 0.007). Plasma HA concentrations were not different between the groups. In the endobronchial biopsies, moderate NAI horses showed epithelial hyperplasia and inflammatory cell infiltrate, while severe NAI horses also showed fibrosis and desquamation of the epithelium. The degree of remodelling was higher in severe NAI compared to moderate NAI (p = 0.048) and controls (p = 0.016). Intense HA staining was observed in bronchial cell membranes, basement membranes, and connective tissue without significant differences between the groups. CONCLUSION: The release of HA to the airway lumen increases in naturally occurring neutrophilic EA without clear changes in its tissue distribution, and significant airway remodelling only develops in severe NAI.

Fatty acid fingerprints in bronchoalveolar lavage fluid and its extracellular vesicles reflect equine asthma severity.

Equine asthma (EA) is an inflammatory disease of the lower airways driven by mediators released from cells. Extracellular vesicles (EVs) are vehicles for lipid mediators, which possess either pro-inflammatory or dual anti-inflammatory and pro-resolving functions. In this study, we investigated how the respiratory fatty acid (FA) profile reflects airway inflammatory status. The FA composition of bronchoalveolar lavage fluid (BALF), BALF supernatant, and bronchoalveolar EVs of healthy horses (n = 15) and horses with mild/moderate EA (n = 10) or severe EA (SEA, n = 5) was determined with gas chromatography and mass spectrometry. The FA profiles distinguished samples with different diagnoses in all sample types, yet they were insufficient to predict the health status of uncategorized samples. Different individual FAs were responsible for the discrimination of the diagnoses in different sample types. Particularly, in the EVs of SEA horses the proportions of palmitic acid (16:0) decreased and those of eicosapentaenoic acid (20:5n-3) increased, and all sample types of asthmatic horses had elevated dihomo-γ-linolenic acid (20:3n-6) proportions. The results suggest simultaneous pro-inflammatory and resolving actions of FAs and a potential role for EVs as vehicles for lipid mediators in asthma pathogenesis. EV lipid manifestations of EA can offer translational targets to study asthma pathophysiology and treatment options.

Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses.

Extracellular vesicles (EVs) are membrane-bound particles that engage in inflammatory reactions by mediating cell-cell interactions. Previously, EVs have been isolated from the bronchoalveolar lavage fluid (BALF) of humans and rodents. The aim of this study was to investigate the number and size distribution of EVs in the BALF of asthmatic horses (EA, n = 35) and healthy horses (n = 19). Saline was injected during bronchoscopy to the right lung followed by manual aspiration. The retrieved BALF was centrifuged twice to remove cells and biological debris. The supernatant was concentrated and EVs were isolated using size-exclusion chromatography. Sample fractions were measured with nanoparticle tracking analysis (NTA) for particle number and size, and transmission electron microscopy and confocal laser scanning microscopy were used to visualize EVs. The described method was able to isolate and preserve EVs. The mean EV size was 247 ± 35 nm (SD) in the EA horses and 261 ± 47 nm in the controls by NTA. The mean concentration of EVs was 1.38 × 1012 ± 1.42 × 1012 particles/mL in the EA horses and 1.33 × 1012 ± 1.07 × 1012 particles/mL in the controls with no statistically significant differences between the groups. With Western blotting and microscopy, these particles were documented to associate with EV protein markers (CD63, TSG101, HSP70, EMMPRIN, and actin) and hyaluronan. Equine BALF is rich in EVs of various sizes, and the described protocol is usable for isolating EVs. In the future, the role of EVs can be studied in horses with airway inflammation.