RESUMO
Pulmonary diseases are common in horses and have a major economic impact on the equine industry. Some of them could be associated with an inadequate immune response in the lung, but methods to evaluate this response in horses are lacking. The aim of this study was to develop and validate an experimental model that could be applied in several physiological and pathological conditions to assess the innate immune response of equine pulmonary cells. Equine alveolar macrophages (AMs) obtained from bronchoalveolar lavages were isolated from other cells by adhesion. TLR2, 3, and 4 expression in AMs was studied and their responses to commercial ligands (respectively FSL-1, Poly(I:C), and LPS) were evaluated after determination of the appropriate dose and time of incubation. TLR responses were assessed by measuring cytokine production using (1) gene expression of TNFα, IFNß, Il-1ß, and IFNα by qPCR (indirect method); and (2) cytokine production for TNFα and IFNß by ELISA (direct method). TLR 2, 3, and 4 were expressed by AMs. TLR 2 stimulation with 10 ng/mL of FSL-1 during 3h significantly increased IL-1ß and TNFα gene expression. TLR 3 stimulation with 1000 ng/mL of Poly(I:C) during 1h increased IFNß, IFNα, Il-1ß and TNFα expression. TLR 4 stimulation with 100 ng/mL of LPS during 3h increased TNFα, IFNß, and Il-1ß expression. Results obtained by ELISA quantification of TNFα and IFNß produced by AMs following stimulation during 6h were similar: FSL-1 increased TNFα production but not IFNß, Poly(I:C) and LPS increased production of IFNß and TNFα. In conclusion, pulmonary innate immunity of horses can be assessed ex vivo by measuring cytokine production following stimulation of AMs with TLR agonists. This experimental model could be applied under several conditions especially to improve the understanding of equine respiratory disease pathogenesis, and to suggest novel therapeutic opportunities.