RESUMO
Long chain fatty acids (LCFAs), which are ligands for the G-protein coupled receptor FFAR1 (GPR40), are increased in cow plasma after parturition, a period in which they are highly susceptible to infectious diseases. This study identified and analyzed the functional role of the FFAR1 receptor in bovine neutrophils, the first line of host defense against infectious agents. We cloned the putative FFAR1 receptor from bovine neutrophils and analyzed the sequence to construct a homology model. Our results revealed that the sequence of bovine FFAR1 shares 84% identity with human FFAR1 and 31% with human FFAR3/GPR41. Therefore, we constructed a homology model of bovine FFAR1 using human as the template. Expression of the bovine FFAR1 receptor in Chinese hamster ovary (CHO)-K1 cells increased the levels of intracellular calcium induced by the LCFAs, oleic acid (OA) and linoleic acid (LA); no increase in calcium mobilization was observed in the presence of the short chain fatty acid propionic acid. Additionally, the synthetic agonist GW9508 increased intracellular calcium in CHO-K1/bFFAR1 cells. OA and LA increased intracellular calcium in bovine neutrophils. Furthermore, GW1100 (antagonist of FFAR1) and U73122 (phospholipase C (PLC) inhibitor) reduced FFAR1 ligand-induced intracellular calcium in CHO-K1/bFFAR1 cells and neutrophils. Additionally, inhibition of FFAR1, PLC and PKC reduced the FFAR1 ligand-induced release of matrix metalloproteinase (MMP)-9 granules and reactive oxygen species (ROS) production. Thus, we identified the bovine FFAR1 receptor and demonstrate a functional role for this receptor in neutrophils activated with oleic or linoleic acid.
Assuntos
Clonagem Molecular , Neutrófilos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cálcio/análise , Cálcio/metabolismo , Bovinos , Cricetinae , Cricetulus , Estrenos/farmacologia , Humanos , Transporte de Íons/efeitos dos fármacos , Ácido Linoleico/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Metilaminas/farmacologia , Dados de Sequência Molecular , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Ácido Oleico/metabolismo , Propionatos/farmacologia , Pirrolidinonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
Neutrophils are critical to the innate immune response; therefore, the proper function of neutrophils is critical to avoid the development of certain diseases. Linoleic acid, a polyunsaturated long-chain fatty acid, is one of the most abundant long-chain fatty acids found in the plasma of cows after giving birth. In this study, we evaluated the effects of linoleic acid treatment on bovine neutrophil adhesion, chemotaxis, metalloproteinase (MMP)-9 release, CD11b expression, intracellular calcium mobilisation, mitogen-activating protein kinase (MAPK) phosphorylation and COX-2 and IL-8 expression. Bovine neutrophils isolated from healthy heifers were incubated with different concentrations of linoleic acid, and then neutrophil responses were evaluated. Our results show that the treatment of neutrophils with 100 µM linoleic acid increased their adhesion to the bovine endothelial cell line CPA47. The results of a transwell migration assay revealed that linoleic acid could also promote the chemotaxis of bovine neutrophils. Furthermore, linoleic acid treatment increased MMP-9 activity and CD11b cell surface expression in neutrophils. Fifty and 100 µM linoleic acid also increased intracellular calcium mobilisation in neutrophils loaded with Fluo-4 AM dye. Linoleic acid also rapidly (2-5 min) stimulated the phosphorylation of ERK1/2 and p38 MAPK as evaluated by immunoblot. Finally, COX-2 and IL-8 mRNA expression increased after 2h of linoleic acid treatment. In conclusion, linoleic acid stimulates adhesion, chemotaxis, granule release and intracellular responses in bovine neutrophils.