Fatty acids are also known to

Fatty acids are also known to activate G-protein coupled receptors (GPCRs). LCFAs activate the GPCRs for free fatty gaba receptor antagonist (FFA)1 and FFA4 (Briscoe et al., 2003; Hirasawa et al., 2005), whereas SCFAs activate FFA2 and FFA3 receptors, which are expressed in bovine neutrophils (Alarcon et al., 2018; Carretta et al., 2013). Omega-3 fatty acids are ligands for FFA4, also known as GPR120, which has the ability to regulate gut incretin glucagon-like peptide-1 secretion (Hirasawa et al., 2005) and reduce inflammasome activation in macrophages (Oh et al., 2010). FFA4 has been described in the intestine enteroendocrine cells, lung, adipose tissue, hypothalamus, and macrophages (Cintra et al., 2012; Hirasawa et al., 2005; Miyauchi et al., 2009; Oh et al., 2010); however, its expression in bovine neutrophils has not yet been established. Omega-3 fatty acids have been proposed as a strategy to improve immune function in dairy cows. Diets containing fatty acids rich in omega-3 increased phagocytic capacity of neutrophils isolated from transition dairy cows and decreased expression of pro-inflammatory markers in milk cells and peripheral blood mononuclear cells (Gandra et al., 2016; Rezamand et al., 2016). Also, DHA attenuated inflammatory response in primary bovine mammary epithelial cells (He et al., 2017). Since neutrophils play a key role in innate immunity, and omega-3 fatty acids have potential beneficial effects in dairy cattle, the goal of our study was to determine the presence of FFA4 receptor in bovine neutrophils, and the effects of both natural and synthetic FFA4 ligands, DHA (Hirasawa et al., 2005; Oh et al., 2010) and TUG-891 (Hudson et al., 2013; Shimpukade et al., 2012), respectively, on intracellular calcium mobilisation. We further investigated the effect of DHA and TUG-891 on the release of MMP-9 granules and superoxide production given the well known role of calcium in these processes (Sengelov et al., 1993; Yin and Heit, 2017).
Materials and methods
Results
Discussion Several studies have described the mechanisms by which omega-3 fatty acids have beneficial effects on the immune response, such as their effects on the inflammasome and autophagy in human or murine cells, highlighting a role of FFA4 as the main omega-3 receptor (Oh et al., 2010; Williams-Bey et al., 2014). In this study, we further demonstrate that bovine neutrophils express FFA4 receptor, which induces intracellular calcium release. In addition, FFA4 agonists could induce the release of MMP-9 granules and superoxide production in neutrophils. A previous study showed that DHA and eicosapentaenoic acid could differentially modulate certain responses in rat neutrophils such as superoxide release, phagocytic capacity, and cytokine release (Paschoal et al., 2013); however, the role of FFA4 was not assessed. The detection of mRNA and protein levels of FFA4 receptor in bovine neutrophils, activated by natural and synthetic FFA4 agonists, further suggest a role of FFA4 in the immune response. Both DHA and TUG-891 increased intracellular calcium mobilisation, a well known response of FFA4 activation. Similarly, intracellular calcium release has been observed in different cell types with the use of other FFA4 agonists such as grifolic acid, FFA4 agonist III, α-linolenic acid, along with TUG-891 (Briscoe et al., 2006; Hudson et al., 2013; Paillamanque et al., 2017). To assess the participation of FFA4 in the intracellular calcium mobilisation induced by DHA and TUG-891, we used the pharmacological antagonist AH7614, and observed a statistically significant reduction in this response; however, AH7614 did not completely block intracellular calcium mobilisation, even at higher concentrations. The -log half-maximal inhibitory concentration of AH7614 was reported to be 7.1; however, several concentrations have been tested in various studies (1–100 μM) to inhibit responses stimulated by FFA4 agonists (Houthuijzen et al., 2017; Quesada-Lopez et al., 2016; Sparks et al., 2014; Tran et al., 2016; Villegas-Comonfort et al., 2017). By contrast, one study described that AH7614 alone induced intracellular calcium in rat spermatids (Paillamanque et al., 2017). These differences in the effect of AH7614 could be due to its role as a non-competitive negative allosteric modulator of FFA4 agonists (Watterson et al., 2017). Currently, AH7614 is the only available tool to assess the role of FFA4 in neutrophils, because a knockdown assay is difficult owing to the short lifespan and high level of activation in neutrophils after typical transfection protocols (Tamassia et al., 2012).