A central feature of recurrent airway obstruction (RAO) is an enhanced sensitivity to hay dust (HD) compared to control horses, but the cellular and molecular mechanisms accounting for this differential airway sensitivity are unknown. The microbial components of HD can stimulate pathogen recognition receptors (PRRs), which are abundant on alveolar macrophages (AM). Thus inhaled HD would be expected to evoke an inflammatory response from AM, however, work in other species indicates that the nature of the response depends on the macrophage phenotype. Two major polarized phenotypes, classified by their distinct gene expression and functions, have been described. The classically-activated M1 exerts a robust pro-inflammatory response and the alternatively-activated M2 produces anti-inflammatory cytokines, and little in the way of pro-inflammatory cytokines. Thus, our overarching hypothesis was that the HD hypersensitivity observed in RAO may be mediated by the presence of pro-inflammatory (M1) or the absence of immune-regulatory (M2) phenotypes. There are limited data characterizing equine M1 and M2 phenotypic markers, thus to address this, equine AM were cultured in IFNγ+LPS or IL-4 generating equine M1 and M2 phenotypes respectively, and the gene expression of predicted surrogate M1/M2 genes and the effect of polarization on the response to pro-inflammatory agonists was evaluated. Equine M1s were characterized by increased expression of pro-inflammatory genes (TNFα, IL1β, IL-12p40, IL-8, CD80), regulatory IL-10 and a potentiated pro-inflammatory gene expression response when stimulated. Equine M2s were characterized by elevated scavenger receptor CD206, low expression of M1 associated genes and potently suppressed pro-inflammatory cytokines and IL-10 when stimulated. Further, this study determined that canonical murine macrophage markers iNOS and arginase were not M1 or M2 associated in the horse. Next the expression of equine M1 and M2 transcriptional markers and the cytokine response to HD components was assessed in AM from RAO-susceptible and control horses prior to (at baseline) and following exposure to hay. These results determined that different AM phenotypes exist in RAO-susceptible and control horses at baseline: RAO-susceptible horses had greater gene expression of IL-10 and enhanced responsiveness to LPS stimulation suggesting an M2-like, immune regulatory phenotype that maintains LPS responsiveness. Further, exposure to hay induced different phenotypes in both groups: AM from control horses expressed elevated IL-1β, IL-8, IL-10, CD206, and developed enhanced responsiveness to LPS indicating a mixed M1/M2 phenotype. In RAO-susceptible horses, hay exposure only increased the expression of CD206, an M2 marker. In summary, these data provide the first characterization of the transcriptional signature of equine AM M1 and M2 phenotypes that will assist in studying their role in equine pulmonary disease. Furthermore, these data demonstrate that divergent AM exist in RAO-susceptible and control horses, indicating that the AM plays a role in RAO-immunopathology.
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