In vitro stimulation of alveolar macrophages from severe asthmatic horses

International audiencePurpose of the study: The immune response in asthmatic horses is based on innate immunity with an important role of alveolar macrophages (AM) which are the first line of defense of immune response with phagocytosis and macropinocytosis mechanisms. In fact, AMs are present on the surface of lung cells and are thus directly exposed to inhaled particles. Multiple agents may be implicated in either initiation or maintenance of equine asthma syndrome like dust stables, season, bacteria or beeding. The composition of the administered hay had a greater influence than bedding on breathable dust concentrations and on endotoxin in the inhaled air. The objectives of this study were 1/ characterize alveolar macrophages population from control (CTL) and severe equine asthmatic (sEA) horses; 2/ study the alveolar macrophages phagocytosis and macropinocytosis activities from CTL and sEA horses, in vitro stimulated by different antigens whose hay dust solution; 3/ study the cell behavior and cytokinique response of AMs from CTL and sEA horses, preliminary in vivo hay challenged (Orard et al., 2018) and in vitro stimulated.Methods used: The in vivo hay challenge was previously described in Orard et al. (2018). The AMs were isolated on D-2 (pre-challenge) and D+5 (post-challenge) from the BALF. Hay dust suspensions (HDS) were realized by shaking 1kg of dry or steamed hay with Haygain HG-600, dusts were sieved and suspended at 10 ml of saline per gram of dust. The HDS were characterised for bacteria, mould, endotoxin and β-D-glucan content, inhalable and respirable particles. After 3h of adhesion, the AMs were stimulated with GFP-conjuagted E.coli bioparticles (10 by cells), FITC-conjugated BSA (1 mg/ml), LPS (500 ng/ml), Fumagilline (4 µg/ml), dry HDS (5 µl/ml) or steamed HDS (5 µl/ml). The real-time cellular activities of AMs were analysed by live cell imaging (IncuCyte® S3, Sartorius) and tested for impedance measurement (xCELLigence®, ACEA Biosciences). Immunophenotyping, macropinocytosis and phagocytosis capacities were analysed by flow cytometry (CytoFlex S, Beckman coulter). Griess test was realised at 6h and 24h post-stimulation. Summary of results: Bacteria, mould, endotoxins and beta D glucan decrease in HDS after steaming. Moreover steam treatment decreases significantly and sustainably both inhalable and respirable dust generated by hay shaking. The immunophenotyping of AMs showed that CD14+ cell proportion was more important in sEA horses compared to CTL horse with better phagocytosis and macropinocytosis capacities in CD14+ AMs in sEA horses. No significant difference of phagocytosis and macropinocytosis capacities were observed in CD206+ cells between sEA and CTL horses. When naive AMs (D-2) were only in vitro simulated, we can observe more important cell grouping with LPS and HDS in AMs from CTL horses. We can observe that the size of AMs stimulated with D-HDS is higher compared to the other stimuli. When AMs were preliminary in vivo challenged with steamed hay and in vitro stimulated with HDS, we also observed some grouping cells. This grouping are less important when AMs were preliminary challenged with dry hay. Moreover, the AMs from sEA are smaller size after dry hay challenge and HDS stimulation. Conclusions: The expression of markers of macrophages as CD14 seems to vary according to the status of the horse with better phagocytosis and macropinocytosis capacities in CD14+ AMs frome sEA horses. No difference in phagocytosis and macropinocytosis activity was observed for CD206+ cells regardless of the status of the horse. The behavior of AMs from sEA horses seem to be more affected by a previous in vivo dry hay challenge compared to AMs from CTL horses. In addition, steamed hay challenge in vivo seems appears to have less impact on the behavior of AMs compared dry hay for sEA and CTL horses