[en] 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.
Disciplines :
Veterinary medicine & animal health
Author, co-author :
Waldschmidt, Ingrid; CIRALE-National Veterinary School of Alfort, Goustranville, France
Pirottin, Dimitri ; Université de Liège - ULiège > Département de sciences fonctionnelles > Département de sciences fonctionnelles
Art, Tatiana ; Université de Liège - ULiège > Département de sciences fonctionnelles > Phys. neuro-muscul., de l'effort - Méd. sport. des animaux
Audigie, Fabrice; CIRALE-National Veterinary School of Alfort, Goustranville, France
Bureau, Fabrice ; Université de Liège - ULiège > Département de sciences fonctionnelles > GIGA-R : Biochimie et biologie moléculaire
Tosi, Irène ; Université de Liège - ULiège > Département de sciences fonctionnelles > Phys. neuro-muscul., de l'effort - Méd. sport. des animaux
El Abbas, Sophie ; Université de Liège - ULiège > Département de sciences fonctionnelles > Physiologie
Farnir, Frédéric ; Université de Liège - ULiège > Département de productions animales > Biostatistiques et bioinformatique appliquées aux sc. vétér.
Richard, Eric; Frank Duncombe Laboratory, Caen, France
Dupuis, Marie-Capucine; CIRALE-National Veterinary School of Alfort, Goustranville, France
Language :
English
Title :
Experimental model of equine alveolar macrophage stimulation with TLR ligands.
Akira S. Mammalian toll-like receptors. Curr. Opin. Immunol. 2003, 15:5-11.
Akira S., Uematsu S., Takeuchi O. Pathogen recognition and innate immunity. Cell 2006, 124:783-801.
Bedoret D., Wallemacq H., Marichal T., Desmet C., Quesada Calvo F., Henry E., Closset R., Dewals B., Thielen C., Gustin P., de Leval L., Van Rooijen N., Le Moine A., Vanderplasschen A., Cataldo D., Drion P.V., Moser M., Lekeux P., Bureau F. Lung interstitial macrophages alter dendritic cell functions to prevent airway allergy in mice. J. Clin. Invest. 2009, 119:3723-3738.
Beekman L., Tohver T., Leguillette R. Comparison of cytokine mRNA expression in the bronchoalveolar lavage fluid of horses with inflammatory airway disease and bronchoalveolar lavage mastocytosis or neutrophilia using REST software analysis. J. Vet. Intern. Med. 2012, 26:153-161.
Berndt A., Derksen F.J., Venta P.J., Ewart S., Yuzbasiyan-Gurkan V., Robinson N.E. Elevated amount of toll-like receptor 4 mRNA in bronchial epithelial cells is associated with airway inflammation in horses with recurrent airway obstruction. Am. J. Physiol. Lung. Cell Mol. Physiol. 2007, 292:L936-L943.
Couetil L.L., Hoffman A.M., Hodgson J., Buechner-Maxwell V., Viel L., Wood J.L., Lavoie J.P. Inflammatory airway disease of horses. J. Vet. Intern. Med. 2007, 21:356-361.
Couetil L.L., Rosenthal F.S., DeNicola D.B., Chilcoat C.D. Clinical signs, evaluation of bronchoalveolar lavage fluid, and assessment of pulmonary function in horses with inflammatory respiratory disease. Am. J. Vet. Res. 2001, 62:538-546.
Figueiredo M.D., Vandenplas M.L., Hurley D.J., Moore J.N. Differential induction of MyD88- and TRIF-dependent pathways in equine monocytes by Toll-like receptor agonists. Vet. Immunol. Immunopathol. 2009, 127:125-134.
Fortier G., van Erck E., Fortier C., Richard E., Pottier D., Pronost S., Miszczak F., Thiry E., Lekeux P. Herpesviruses in respiratory liquids of horses: putative implication in airway inflammation and association with cytological features. Vet. Microbiol. 2009, 139:34-41.
Gornik K., Moore P., Figueiredo M., Vandenplas M. Expression of Toll-like receptors 2, 3, 4, 6, 9, and MD-2 in the normal equine cornea, limbus, and conjunctiva. Vet. Ophthalmol. 2011, 14:80-85.
Hughes K.J., Nicolson L., Da Costa N., Franklin S.H., Allen K.J., Dunham S.P. Evaluation of cytokine mRNA expression in bronchoalveolar lavage cells from horses with inflammatory airway disease. Vet. Immunol. Immunopathol. 2011, 140:82-89.
Joubert P., Cordeau M.E., Lavoie J.P. Cytokine mRNA expression of pulmonary macrophages varies with challenge but not with disease state in horses with heaves or in controls. Vet. Immunol. Immunopathol. 2011, 142:236-242.
Juarez E., Nunez C., Sada E., Ellner J.J., Schwander S.K., Torres M. Differential expression of toll-like receptors on human alveolar macrophages and autologous peripheral monocytes. Respir. Res. 2010, 11:2.
Kumar H., Kawai T., Akira S. Toll-like receptors and innate immunity. Biochem. Biophys. Res. Commun. 2009, 388:621-625.
Kwon S., Vandenplas M.L., Figueiredo M.D., Salter C.E., Andrietti A.L., Robertson T.P., Moore J.N., Hurley D.J. Differential induction of toll-like receptor gene expression in equine monocytes activated by toll-like receptor ligands or TNF-alpha. Vet. Immunol. Immunopathol. 2010, 138:213-217.
Laan T.T., Bull S., Pirie R.S., Fink-Gremmels J. Evaluation of cytokine production by equine alveolar macrophages exposed to lipopolysaccharide. Aspergillus fumigatus, and a suspension of hay dust. Am. J. Vet. Res. 2005, 66:1584-1589.
Lavoie J.P., Cesarini C., Lavoie-Lamoureux A., Moran K., Lutz S., Picandet V., Jean D., Marcoux M. Bronchoalveolar lavage fluid cytology and cytokine messenger ribonucleic acid expression of racehorses with exercise intolerance and lower airway inflammation. J. Vet. Intern. Med. 2011, 25:322-329.
Liu Y., Song M., Che T.M., Bravo D., Pettigrew J.E. Anti-inflammatory effects of several plant extracts on porcine alveolar macrophages in vitro. J. Anim. Sci. 2012, 90:2774-2783.
Mercier E., Bolognin M., Hoffmann A.C., Tual C., Day M.J., Clercx C. Influence of age on bronchoscopic findings in healthy beagle dogs. Vet. J. 2011, 187:225-228.
Mignot C.C., Pirottin D., Farnir F., de Moffarts B., Molitor C., Lekeux P., Art T. Effect of strenuous exercise and ex vivo TLR3 and TLR4 stimulation on inflammatory gene expression in equine pulmonary leukocytes. Vet. Immunol. Immunopathol. 2012, 147:127-135.
Muehlmann L.A., Michelotto P.V., Nunes E.A., Grando F.C., da Silva F.T., Nishiyama A. PAF increases phagocytic capacity and superoxide anion production in equine alveolar macrophages and blood neutrophils. Res. Vet. Sci. 2012, 93:393-397.
Naessens T., Vander Beken S., Bogaert P., Van Rooijen N., Lienenklaus S., Weiss S., De Koker S., Grooten J. Innate imprinting of murine resident alveolar macrophages by allergic bronchial inflammation causes a switch from hypoinflammatory to hyperinflammatory reactivity. Am. J. Pathol. 2012, 181:174-184.
Richard E.A., Fortier G.D., Lekeux P.M., Van Erck E. Laboratory findings in respiratory fluids of the poorly-performing horse. Vet. J. 2010, 185:115-122.
Richard E.A., Fortier G.D., Pitel P.H., Dupuis M.C., Valette J.P., Art T., Denoix J.M., Lekeux P.M., Erck E.V. Sub-clinical diseases affecting performance in standardbred trotters: diagnostic methods and predictive parameters. Vet. J. 2010, 184:282-289.
Quintana A.M., Landolt G.A., Annis K.M., Hussey G.S. Immunological characterization of the equine airway epithelium and of a primary equine airway epithelial cell culture model. Vet. Immunol. Immunopathol. 2011, 140:226-236.
Schneberger D., Caldwell S., Suri S.S., Singh B. Expression of toll-like receptor 9 in horse lungs. Anat. Rec. (Hoboken) 2009, 292:1068-1077.
Singh Suri S., Janardhan K.S., Parbhakar O., Caldwell S., Appleyard G., Singh B. Expression of toll-like receptor 4 and 2 in horse lungs. Vet. Res. 2006, 37:541-551.
Takeda K., Akira S. TLR signaling pathways. Sem. Immunol. 2004, 16:3-9.
Wattrang E., Palm A.K., Wagner B. Cytokine production and proliferation upon in vitro oligodeoxyribonucleotide stimulation of equine peripheral blood mononuclear cells. Vet. Immunol. Immunopathol. 2012, 146:113-124.
Wilsher S., Allen W.R., Wood J.L. Factors associated with failure of thoroughbred horses to train and race. Equine Vet. J. 2006, 38:113-118.
Wood J.L., Newton J.R., Chanter N., Mumford J.A. Association between respiratory disease and bacterial and viral infections in British racehorses. J. Clin. Microbiol. 2005, 43:120-126.
Yang M., Kumar R.K., Hansbro P.M., Foster P.S. Emerging roles of pulmonary macrophages in driving the development of severe asthma. J. Leuk. Biol. 2012, 91:557-569.
Zarember K.A., Godowski P.J. Tissue expression of human toll-like receptors and differential regulation of toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J. Immunol. 2002, 168:554-561.
