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See detailExperimental model of equine alveolar macrophage stimulation with TLR ligands.
Waldschmidt, Ingrid; Pirottin, Dimitri ULg; Art, Tatiana ULg et al

in Veterinary Immunology and Immunopathology (2013), 155(1-2), 30-37

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 ... [more ▼]

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 TNFalpha, IFNbeta, Il-1beta, and IFNalpha by qPCR (indirect method); and (2) cytokine production for TNFalpha and IFNbeta by ELISA (direct method). TLR 2, 3, and 4 were expressed by AMs. TLR 2 stimulation with 10ng/mL of FSL-1 during 3h significantly increased IL-1beta and TNFalpha gene expression. TLR 3 stimulation with 1000ng/mL of Poly(I:C) during 1h increased IFNbeta, IFNalpha, Il-1beta and TNFalpha expression. TLR 4 stimulation with 100ng/mL of LPS during 3h increased TNFalpha, IFNbeta, and Il-1beta expression. Results obtained by ELISA quantification of TNFalpha and IFNbeta produced by AMs following stimulation during 6h were similar: FSL-1 increased TNFalpha production but not IFNbeta, Poly(I:C) and LPS increased production of IFNbeta and TNFalpha. 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. [less ▲]

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