Cloning, Identification and Functional Characterization of Bovine Free Fatty Acid Receptor-1 (FFAR1/GPR40) in Neutrophils

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.


Introduction
Neutrophils are the first line of host defense, and they are one of the first cells to migrate from the blood into injured or infected tissues. Neutrophils exert their defensive role through FFAR3, analysis and functional studies have not been performed to demonstrate whether this sequence is a receptor for short chain fatty acids or long chain fatty acids.
In the present study, we cloned the putative bovine cDNA FFAR1 receptor obtained from bovine neutrophils and analyzed its similarity to human FFAR1. Then, we evaluated the effects of activating the bFFAR1 receptor using synthetic or natural ligands of long and short chain fatty acid receptors in both CHO-K1 cells expressing the bFFAR1 receptor and bovine neutrophils. Finally, we examined the intracellular mechanisms involved in MMP-9 release and ROS production after FFAR1 receptor activation.

Neutrophil isolation
Blood was collected by jugular venipuncture of five healthy Holstein heifers from a Universidad Austral de Chile herd, and samples were collected in ACD Blood Collection Tubes (Becton Dickinson, USA). All experiments were conducted in strict accordance with protocols approved by the Ethical Committee of the Universidad Austral de Chile (Permit Number: 06/10). Neutrophils were isolated according to a previously described method [18]. Viability was determined by trypan blue exclusion assays, and it was at least 97% for all experiments. Neutrophil purity was at least 94% ( Fig. 1), as assessed by flow cytometry (BD FACSCanto II, USA) using a forward-scatter versus side-scatter dot plot to determine the relative size and granularity of cells [19].

Cloning of bovine FFAR1 receptor
Total RNA was extracted from bovine neutrophils using the Total RNA Kit I (Omega Bio-Tek Inc, Norcross, GA, USA), according to the manufacturer's instructions. RNA was Putative bovine FFAR1 (bFFAR1) receptor cDNA was obtained by PCR amplification using the following oligonucleotides synthesized based on the genomic DNA sequence of the putative bFFAR1 receptor (GenBank Accession No. XM_870502): 5 0 -CTCGAGGGTACCATGGACCTGCCCCCGCAGCTC-3 0 (forward) and 5 0 -GCGGCCGCGGATCCCTATTTCTGGGATGCCCCTGC-3 0 (reverse). PCR was performed using Green Master Mix DNA polymerase and Pfu DNA Polymerase (1:3) under the following conditions: 1 min at 95°C; 35 cycles of 20 s at 95°C, 20 s at 60°C and 1 min at 72°C; and 3 min at 72°C. A fraction of the PCR product was resolved on a 1.2% agarose gel, and the band was excised from the gel, purified and subcloned into the pGEM T-Easy vector (Promega, Madison, WI, USA). Plasmids from the resulting bacterial clones were isolated, and the full insert was verified by sequencing. Next, pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) was digested with NotI and BamHI (New England Biolabs Inc., Ipswich, MA, USA) and bFFAR1 cDNA was ligated at both ends. bFFAR1 in pcDNA3.1 (pcDNA3.1-bFFAR1) was successfully transfected into CHO-K1 cells using FuGene 6 reagent (Promega, Madison, WI, USA), according to the manufacturer's instructions.

Sequence analysis and homology modeling
The bFFAR1 cDNA and predicted protein sequence were analyzed using the BLAST search [20] and Clustal Omega [21]. The predicted protein sequence was obtained using EMBOSS Transeq (http://www.ebi.ac.uk/Tools/st/emboss_transeq/). A non-hydrogen atom homology model of bovine FFAR1 was obtained using the automated methodology implemented in MODELLER [22] (Fig. 2A). The crystal structure of human FFAR1 was used as template. A sequence alignment produced from Clustal Omega and refined manually was used as the input for model construction (Fig. 2B). The C-terminal bovine sequence, G280-K300, was omitted from the model due to the absence of these residues in the crystal structure. The stereochemical quality of the bovine FFAR1 homology model was evaluated using WHAT_CHECK [23].

Determination of MMP-9 activity
One million neutrophils in 500 μl of HBSS were incubated with vehicle or either 10 μM GW1100, 2 μM U73122 or 1 μM staurosporine (Selleckchem, Houston, TX, USA) for 15, 3 or 15 min, respectively, at 37°C. Then, neutrophils were stimulated with 300 μM oleic acid, 100 μM linoleic acid, 10 μM GW9508 or vehicle for 5 min at 37°C. As positive control, 100 nM FFAR3 was performed using Clustal Omega. Colored sequence annotations based on 4PHU identify the approximate extent of transmembrane helices. Residues highlighted in grey indicate a mismatch between the aligned sequences. platelet activating factor (PAF) for 5 min at 37°C was used. After incubation, the cells were centrifuged at 600 × g for 6 min, and equal amount of supernatants were assayed for gelatinase activity by zymography [29,30]. Ten microliters of supernatant was loaded into 10% polyacrylamide gels (0.75 mm thick) containing 0.28% gelatin. The gels were run at 200 V for 1 h in a Bio-Rad Mini Protean II apparatus (Bio-Rad Laboratories, Richmond, CA) and soaked twice in 2.5% Triton X-100 in distilled water on a shaker at room temperature for 30 min. Then, the gels were soaked in reaction buffer consisting of 100 mM Tris (pH 7.5) and 10 mM CaCl 2 at 37°C overnight. The gels were stained in 0.5% Coomassie Brilliant Blue R-250 in acetic acid: methanol:water (1:3:6). Enzymatic activity was determined by measuring non-staining areas where the gelatin was degraded. Gelatinolytic bands were compared with a recombinant MMP-9 standard (Sigma-Aldrich, Saint Louis, MO, USA). To measure activity, the gels were digitalized, and the band intensity was determined using ImageJ 1.35s software.

Statistical analysis
All experimental protocols were performed in quintuplicate. Experiments in neutrophils were done from 5 different animals. Bar graphs present the arithmetic mean ± SEM. For statistical analyses, a one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test, and t-tests were performed. All analyses were performed using Graph Pad Prism v5.0 software, assuming that a p-value less than 0.05 represents a significant result.

Sequence analysis of the bovine FFAR1 receptor
To analyze the sequence of the bovine FFAR1 receptor, we cloned the bovine FFAR1 from mRNA obtained from bovine neutrophils into the pGEM T-Easy vector and sequenced the obtained 902 bp fragment. A nucleotide BLAST search of the cDNA sequence showed 100% identity to a predicted Bos taurus FFAR3 transcription variant X2 [GenBank Accession No. XR_238380.1], 84% identity to human FFAR1 [GenBank Accession No. EU432113.1] and no significant similarity to human FFAR3 [GenBank Accession No. EU432115.1]. Next, we obtained the predicted protein sequence (300 amino acids) of the bovine cDNA FFAR1 receptor and performed a protein BLAST search [20] of putative bovine FFAR1. This search indicated that the bovine FFAR1 sequence shares more similarity with human FFAR1 compared to human FFAR3. Furthermore, sequence alignment performed using Clustal Omega [21] revealed that bovine FFAR1 shares 84% identity with human FFAR1 (GenBank Accession No. AAH95536.1) and only 31% identity with human FFAR3 (GenBank Accession No. NP_005295.1) (Fig. 2C). Using the predicted protein sequence of the bovine FFAR1 receptor, we constructed a homology model of bovine FFAR1 using the recently published human FFAR1 crystal structure (PDB: 4PHU) [31] at 2.3 Å resolution as template. In summary, sequence analysis and homology modeling of the bovine FFAR1 receptor strongly suggests that its sequence is more related to the FFAR1 receptor than the FFAR3 receptor.

LCFAs increase intracellular calcium in CHO-K1/bFFAR1 cells
The human FFAR1 receptor (hGPR40) is activated by long and medium chain free fatty acids, and its activation increases intracellular calcium in CHO cells overexpressing this receptor (CHO-hGPR40) [25]. We cloned the putative bovine FFAR1 cDNA into the pcDNA3.1 expression plasmid and transiently transfected CHO-K1 cells with pcDNA3.1-FFAR1 (CHO-K1/bFFAR1 cells) or empty pcDNA3.1 vector (CHO-K1/pcDNA3.1 cells). Next, we used FACS to evaluate the expression of bFFAR1 and the ability of the transfected cells to increase intracellular calcium levels after stimulation with different concentrations of propionic acid, oleic acid, linoleic acid or the synthetic FFAR1 agonist, GW9508. FACS analysis revealed that fluorescence increased in CHO-K1/bFFAR1 cells, but not in CHO-K1 cells transfected with the empty vector, indicating that CHO-K1/bFFAR1 cells expressed the bFFAR1 receptor (Fig. 3A). Bovine neutrophils and MCF-7 cells served as controls because these cells express the FFAR1 receptor [4,14]. As a negative control, all cell types were incubated without the FFAR1 antibody, producing a lower signal compared with cells incubated with the primary antibody.
Next, we analyzed the intracellular calcium mobilization in Fura 2-AM-loaded CHO-K1/ bFFAR1. We observed that different concentrations of the LCFA, oleic (100-500 μM) and linoleic (50-200 μM), and the synthetic FFAR1 receptor agonist, GW9508 (10-100 μM), increased intracellular calcium (Fig. 3 B-D, upper graphs). The curve representing the oleic and linoleic acid-induced increase in intracellular calcium was biphasic, displaying a rapid increase, followed by a slight decrease, and a second phase of increase. In contrast, the GW9508-induced intracellular calcium response displayed a rapid and sustained increase. The analysis of the area under the curve (AUC) between 60-150 s (AUC (60-150 s) ) revealed a significant increase in intracellular calcium induced by oleic acid at 300 and 500 μM, linoleic acid at 100 and 200 μM and GW9508 at 10, 50 and 100 μM (Fig. 3B-D, middle graphs). Incubation of CHO-K1/ bFFAR1 cells with different concentrations of propionic acid, a short chain fatty acid, did not increase intracellular calcium (Fig. 3E). Ionomycin (2 μM) and thapsigargin (2 μM), positive controls, increased the intracellular calcium in CHO-K1/bFFAR1 cells, being the response stimulated by ionomycin higher compared with thapsigargin (Fig. 3F). Additionally, intracellular calcium levels did not increase in cells transfected with empty pcDNA3.1 vector after stimulation with each stimulus (Fig. 3B-E, bottom graphs). Thus, these results indicate that intracellular calcium is only induced by LCFAs and the synthetic agonist of the FFAR1 receptor, but not by a short chain fatty acid, suggesting that the sequence cloned corresponds to a LCFA receptor.

bFFAR1 activation increases intracellular calcium through PLC in CHO-K1/bFFAR1 cells
The human FFAR1 receptor is coupled to the Gαq protein, and it stimulates PLC activity [25,32]. To confirm the participation of the bFFAR1 receptor and investigate the mechanism by which it increases intracellular calcium in response to ligands of bFFAR1, we used the synthetic antagonist of the FFAR1 receptor, GW1100, and the PLC inhibitor, U73122.
To inhibit PLC activity, CHO-K1/bFFAR1 cells were incubated with 2 μM U73122 for 3 min and then stimulated with each stimulus. U73122 significantly decreased the induction of intracellular calcium in response to 300 μM oleic acid (AUC (60-150 s) (p < 0. 05 Fig. 4B), 100 μM linoleic acid (AUC (60-150s) (p< 0.05, Fig. 4D) and 10 μM GW9508 (AUC (60-150 s) (Fig. 4F)). These results demonstrate that bGPR40 activation increases intracellular calcium via PLC activation.  Oleic and linoleic acid increase intracellular calcium through bFFAR1 and PLC in bovine neutrophils Hidalgo et al., [4] demonstrated that bovine neutrophils express the FFAR1 receptor at the protein and mRNA levels, and they demonstrated that intracellular calcium increased in response to oleic and linoleic acid [4,9]. To demonstrate that oleic and linoleic acid increase intracellular calcium through FFAR1 activation in bovine neutrophils, we used the FFAR1 antagonist GW1100 to assess the role of FFAR1 in neutrophils. Once isolated from blood, these cells have a short life, making it difficult to silence gene expression. Fluo-4AM-loaded neutrophils were incubated with 10 μM GW1100 for 15 min, followed by the addition of ligand. GW1100 decreased the increase in intracellular calcium induced by 300 μM of oleic acid (AUC (60-150 s) (p < 0.05, Fig. 5A) and 100 μM linoleic acid (AUC (60-150 s) (p < 0.05, Fig. 5C). To assess whether the increase in intracellular calcium induced by LCFAs is mediated by PLC activation in bovine neutrophils, similar to CHO-K1/bFFAR1 cells, we incubated neutrophils with 2 μM U73122 for 3 min, followed by vehicle, oleic or linoleic acid. U73122 significantly decreased the increase in intracellular calcium levels induced by 300 μM oleic acid (AUC (60-150 s) (p < 0.05, Fig. 5B) and 100 μM linoleic acid (AUC (60-150 s) (p < 0.05, Fig. 5D). As controls, ionomycin and thapsigargin increased the intracellular calcium in bovine neutrophils (Fig. 5E). Thus, the increase in intracellular calcium levels in response to oleic or linoleic acid in bovine neutrophils is mediated by FFAR1 and PLC activation.
Taken together, these results demonstrate that the PLC and PKC pathways mediate the FFAR1-ligand-induced release of MMP-9 granules.
Finally, we incubated neutrophils with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) (10 μM) for 30 min, followed by vehicle, oleic acid, linoleic acid or GW9508 for 5 min. DPI significantly reduced ROS production in bovine neutrophils stimulated with the FFAR1 agonists (Fig. 8), suggesting the participation of NADPH oxidase in this response.

Discussion
Due to the significant increase in plasma of free LCFAs in cows during peripartum, identifying the exact receptors for these fatty acids is crucial for understanding the physiological processes related to innate immunity in bovines. In this study, we demonstrated that the sequence initially identified as the bovine FFAR1 receptor, which was recently named a transcription variant of FFAR3, corresponds to a receptor for LCFAs that is highly homologous to human FFAR1. Analysis of cDNA and predicted amino acid sequences revealed that this protein has high identity to human FFAR1 (84%) compared with human FFAR3 (31%). Therefore, based on sequence analysis, we identified the presence of a FFAR1-like receptor in bovine. Next, we assessed whether this receptor could be activated by long or short chain fatty acids. One method that is widely used to assess FFAR1 receptor activation is the analysis of intracellular calcium levels in heterologous expression systems [11,25]. We observed that only LCFAs, but not the short chain fatty acid propionic acid, increased intracellular calcium in CHO-K1 cells expressing bovine FFAR1. Additionally, the synthetic agonist of FFAR1, GW9508, also increased intracellular calcium levels. Reports by Itoh and Briscoe demonstrated that LCFAs are ligands of FFAR1, and they can increase intracellular calcium levels in CHO cells expressing human FFAR1 [11,25]. In bovine cells, oleic and linoleic acids increased intracellular calcium in neutrophils [4,9] and mammary epithelial cells [15]. In contrast, short chain fatty acids are ligands of FFAR3 and FFAR2 [26], receptors that are expressed in bovine neutrophils [37]. Furthermore, the short chain fatty acid, propionic acid, increases intracellular calcium in bovine neutrophils [37]. To confirm the existence of FFAR1 in bovine, we conducted experiments using the synthetic antagonist of the FFAR1 receptor, GW1100. In CHO-K1/bFFAR1 cells and bovine neutrophils, 10 μM GW1100 reduced the intracellular calcium mobilization induced by oleic and linoleic acid and GW9508. GW1100 is a selective antagonist of the FFAR1 receptor that dose-dependently inhibited the FFAR1-mediated increase in calcium stimulated by GW9508 and linoleic acid (pIC50 values of 5.99+/-0.03 and 5.99+/-0.06, respectively) in HEK cells expressing FFAR1, but GW1100 did not alter the GPR120-mediated stimulation of intracellular calcium release produced by both stimulus in HEK cells expressing GPR120 [13].
The ability of U73122 to reduce FFAR1 ligand-stimulated intracellular calcium mobilization in CHO-K1/bFFAR1 cells and bovine neutrophils suggests that PLC actively participates in this response. Activation of the human FFAR1 receptor activates G q protein in pancreatic cells [16] and G i/o in breast cancer cells [17], which leads to the activation of PLC and the generation of inositol triphosphate and diacylglycerol, the latter of which can activate PKC [16].
After identifying the existence of the FFAR1 receptor in bovine neutrophils, we assessed the functional role of this receptor in neutrophils. The release of MMP-9 granules induced by ligands of FFAR1 was reduced by GW1100, indicating that the FFAR1 receptor participates in this process. According to the classical cascade of G-protein coupled receptor activation, we demonstrated the participation of PLC and PKC in the FFAR1-ligand-mediated release of MMP-9. PLC was first reported to participate in MMP-9 release in human neutrophils activated with the neuropeptide, pituitary adenylate cyclase-activating protein, which acts through a specific G-protein coupled receptor [38]. PLC inhibition suggests the participation of a Gα q protein in MMP-9 release after FFAR1 receptor activation in bovine neutrophils; however, we cannot discard the participation of another G protein, such as Gα i protein, because different PLC isoforms can also be directly activated by Gβγ subunits [33,39,40,41,42]. In addition, we previously reported a partial decrease in intracellular calcium mobilization induced by oleic acid in bovine neutrophils treated with pertussis toxin, suggesting a partial role for the Gα i protein [4]. FFAR1 receptor-activated MMP-9 release was PKC-dependent. Activation of the PLC/PKC pathway after FFAR1 activation in HepG2 cells was previously reported [43]. In human neutrophils, PKC and intracellular calcium participate in IL-8-mediated MMP-9 release [34]. Moreover, PKC has been described to participate in the MMP-9 release induced by TNF-α [44] or the agonist of G-protein coupled receptor B(1)R in human neutrophils [45].
Our results revealed that the FFAR1-ligand-mediated increase in ROS production was dependent of FFAR1, PLC, PKC and NADPH oxidase. A role for FFAR1 in superoxide production was previously suggested in pancreatic islets. Other reports demonstrated that the activation of rat pancreatic islets with palmitic acid, another LCFA FFAR1 agonist, increased NADPH oxidasemediated ROS production and up-regulated FFAR1 protein [46], and the superoxide production induced by palmitic acid is dependent on FFAR1 and NADPH oxidase [47]. The participation of PLC and PKC in superoxide production in neutrophils is well documented. PLC isoforms (PLCβ2 and 3) are required for GPCR-induced superoxide release [33]. Additionally, PKC is required for the assembly of NADPH oxidase and activation of the respiratory burst in neutrophils [48].
In conclusion, we confirmed the presence and identity of a FFAR1 receptor in bovine neutrophils that is activated by LCFAs, such as oleic and linoleic acid, but not by short chain fatty acids, such as propionic acid. Furthermore, PLC-PKC signaling controls the release of MMP-9 granules and ROS production after FFAR1 receptor activation in bovine neutrophils. The results of this study highlight the link between metabolism and innate immunity, showing the importance of FFAR1 receptor on bovine neutrophils activation, which could contribute to the risk developing infectious diseases at calving, by mechanisms which have been involved in tissue damage. However, because FFAR1 receptor not only bind oleic or linoleic acids [11,13], future studies are necessary to assess the contribution of other LCFAs ligands for FFAR1 in the pathogenesis of inflammatory diseases, in order to establish their useful as potential biomarkers of disease risk, or to promote the presence of fatty acids beneficial for innate immunity through dietary or pharmacological approaches. It is interesting consider the known anti-inflammatory effect of omega-3 fatty acids via GPR120 receptor, which are also ligands for FFAR1 receptor [11,49], and a recent study suggested a potential anti-inflammatory role of FFAR1 receptor in bone tissue [50], however this finding could be tissue-specific and should be studied in detail.