Activation of the omega-3 fatty acid receptor GPR120 mediates anti-inflammatory actions in immortalized hypothalamic neurons

The hypothalamic cell line from the embryonic rat, rHypoE-7, was isolated and immortalized as previously described [22, 23]. Cell lines were maintained and grown to confluency in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma, St. Louis MO, USA) supplemented with 5% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin (GIBCO, Big Cabin, OK, USA) and seeded into 60 mm culture plates 24 hrs prior to experimental treatments.

Total cellular RNA was isolated as previously described using the guanidium thiocyanate phenol chloroform extraction method [24]. Contaminating DNA was removed from all RNA samples by Turbo DNAase (Ambion, Austin, TX, USA) treatment (1 hr, 37°C). For quantitative real-time reverse transcriptase-PCR (qRT-PCR) experiments, cDNA was produced using the Applied Biosystems High Capacity cDNA Reverse Transcriptase kit and 50 ng of cDNA per sample was analyzed using an Applied Biosystems Prism 7000 Real-Time PCR machine together with gene-specific primers [See Additional file 1: Table S1] and SYBR green master mix.

DNAse treated RNA (200 μg) was subjected to one-step reverse transcriptase-PCR (RT-PCR) analysis (Qiagen, Mississauga, ON, CAN) according to manufacturer’s instructions and using primers listed in Additional file 1: Table S1. Product size and nucleotide sequencing confirmed the identities of all PCR products.

For induction of the inflammatory response, rHypoE-7 cells were treated with tumor necrosis factor α (TNFα) (Sigma; 1 to 100 ng/mL) for 10 min for the assessment of protein phosphorylation levels or 2 to 6 hr for measurement of the mRNA response. For the anti-inflammatory experiments, cells were serum-starved for 1 hr and exposed to 100 μM docosahexaenoic acid (DHA) or GW9508 (4-(3-phenoxybenzylamino)-phenylpropionic acid) (Sigma) in DMSO for 1 hr prior to TNFα treatment. Unless otherwise indicated, all inhibitor studies were done by pretreating cells for 1 hr prior to DHA exposure. These inhibitors which include N-(6-Chloro-9H-pyrido[3,4-b]indol-8-yl)-3-pyridinecarbox-amide dihydrochloride (PS1145; 20 μM), staurosporine aglycone (1 μM), and Wortmannin (1 μM) were purchased from Sigma. For experiments involving DHA-bovine serum albumin (BSA) complexes, BSA and DHA were co-incubated at identical concentrations for 1 hr prior to their use.

Cells were treated with DMSO or DHA (100 μM) for 30 min prior to being lysed in radioimmunoassay (RIPA) buffer supplemented with 0.2% (v/v) SDS, 0.1% (v/v) Triton X-100 and 1 mM PMSF (Sigma). The soluble fraction was incubated with the anti-TAB1 antibody (1 μg; Abcam, Cambridge, MA, USA) overnight at 4°C and for an additional hour with equilibrated protein A/G sepharose beads (Santa Cruz). The beads were washed three times with RIPA buffer supplemented with SDS and Triton X-100 as above, and protein complexes were eluted into sample buffer (1 M Tris-Cl, 8% (w/v) SDS, 40% (v/v) glycerol, 50 mM EDTA, 4% (v/v) β-mercaptoethanol, and 1 mM bromophenol blue) by boiling.

After two washes with phosphate buffered saline (PBS), cells were scrapped on ice into lysis buffer (Cell Signaling Technology Inc., Danvers, MA, USA) supplemented with 1 mM PMSF and the soluble fraction was isolated after centrifugation (14000 rpm, 10 min, 4°C). Protein was quantified using a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) according to manufacturer’s protocol, and lysates were resolved on 8% poly-acrylamide gels and transferred onto Immobilon-P PVDF membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% milk in Tris buffered saline with 0.1% Tween-20 (TBST) for 1 hr, followed by an overnight incubation at 4°C in primary antibody (1:1000). Membranes were washed with TBST before and after exposure to goat-anti-rabbit HRP secondary antibody (1 hr; Cell Signaling) and protein were visualized using Kodak 1D Image Analysis Software 3.6 and a Kodak Image Station 2000R (Eastman Kodak Company, Rochester, NY, USA). Primary antibodies used for western blotting include phospho-p44/42 MAPK (ERK1/2), p44/42 MAPK (ERK1/2), phospho-AKT, AKT, phospho-JNK, phospho-IKK, phospho-NF-κB, phospho-elF-2α, and phospho-TAK1 were obtained from Cell Signaling, anti-GPR120 was obtained from Abcam and anti-β-actin and anti-TNFα were obtained from Sigma.

Cell viability was assessed using the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide method after TNFα treatment using a Vybrant MTT Cell Proliferation Assay Kit (Invitrogen, Eugene, OR, USA) according to the manufacturer’s protocol. Briefly, cells were plated into a 96 well plate, treated with 10 μM palmitate for 2 hr, and incubated for 4 hr at 37°C with 1 mM MTT reagent. Formazan produced was solublized in DMSO and quantified by measuring the absorbance at 570 nm. All values are normalized to solvent controls.

The rHypoE-7 cells were plated into 6 well plates and GPR120 mRNA and protein were knocked down using Dharmafect transfection reagent (Thermo Scientific) and a TriFECTa™ Kit (Integrated DNA Technology, Coralville, Iowa, USA) containing a non-targeting DsiRNA 5′-GGU AAA CCA UGG UGU GC-3′ and antisense 5′-CUU UAC AUG CAC ACC AUG-3 and three DsiRNA oligonucleotide sets specifically targeting rat GPR120: GPR120a sense 5′-GGA CCA GCA AAU UAA GGA ACG AUC G-3′ and antisense 5′-CGA UCG UUC CUU AAU UUG CUG GUC CUG-3′, GPR120b sense 5′-CCC AAC CGC AUA GGA GAA AUC UCA T-3′ and antisense 5′-AUG AGA UUU CUC CUA UGC GGU UGG GCC-3′, GPR120c sense 5′-GGU AAA CCA UGG UGU GCA UGU AAA G-3′and antisense 5′-CUU UAC AUG CAC ACC AUG GUU UAC CUG-3′. Dharmafect (1 nM) and siRNA (20 ng) complexes were added to the rHypo-7 cells 24 hr and GPR120 mRNA and protein was quantified 24 hr after transfection. The GPR120c set of DsiRNAs was the most effective in reducing the mRNA levels of GPR120 within the rHypoE-7 cell line and thus this set was used in all experiments where endogenous GPR120 levels were reduced.

Pixel intensity was quantified using the ImageJ program (U.S. National Institutes of Health, Bethesda, Maryland, USA, http://​imagej.​nih.​gov/​ij/​, 1997-2014) and all statistics were calculated using Graphpad prism 5.0 (San Diego, CA, USA). Groups were compared by a two-tailed t-test or by a one-way or two-way ANOVA with the Bonferroni test for post hoc comparisons where appropriate. Data are presented as the mean ± SEM and the significance is denoted by *P <0.05, **P <0.01, ***P <0.001.

Generation of the rHypoE-7 cell line from the embryonic rat hypothalamus was completed as previously described and semi-quantitative RT-PCR was used to characterize the gene expression of numerous markers, neuropeptides, receptors, and signal transduction components [22, 23]. Specifically, the rHypoE-7 cell line was screened for mRNA transcripts encoding the free FA sensing G protein coupled receptors (GPRs; GPR-40 and GPR-120), the neuronal marker neuron-specific enolase (NSE), hypothalamic neuropeptides (AgRP, NPY, and POMC), and inflammatory receptors and cytokines (Figure 1A). Consistent to their origin rHypoE-7 cells expressed NSE and hypothalamic neuropeptides including AgRP and NPY (Figure 1A). Furthermore mRNA transcripts encoding various cytokine receptors including the TNFα receptor 2 (TNFαR), interleukin (IL)-1β receptor (IL-1Rec), and IL-6 receptor (IL-6Rec), as well as IKK-β/NF-κB cascade components, were also identified, indicating that the rHypoE-7 neuronal model has the sufficient machinery to illicit an innate immune response against the pro-inflammatory cytokines. For this study inflammation was induced in rHypoE-7 cell model by exposure to the pro-inflammatory cytokine, TNFα.

In order to determine if the rHypoE-7 cell line is capable of eliciting an innate immune response to inflammatory cytokines, we assessed the activation of IKK-β/NF-κB cascade upon TNFα exposure using western blotting and qRT-PCR. Essentially, treatment of rHypoE-7 cells with TNFα (1 to 100 ng/mL) dissolved in H₂0 led to a rapid (10 min) increase in phosphorylation levels of IKK-β, and JNK (Figure 1B) relative to the vehicle control (H₂0). A modest increase in the phosphorylation levels of the transforming growth factor-β-activated kinase 1 (TAK1) was also observed up to 10 ng/mL TNFα. Furthermore, TNFα treatment led to an upregulation of mRNA levels of IκBα and TNFα at 2 to 6 hr (Figure 1Ci, ii). Treatment with 10 ng/mL TNFα (2 hr) was sufficient to dramatically enhance TNFα mRNA relative to control (1.66 ± 0.09 and 0.46 ± 0.10 TNFα/histone mRNA, respectively) and TNFα protein levels relative to vehicle (1.39 ± 0.07 TNFα/β-actin protein) within this cell line (Figure 1Di, ii). To ensure that the upregulation of TNFα mRNA and protein was the result of the activation of the specific IKK-β/NF-κB cascade, the rHypoE-7 cell line was treated with the IKK-β inhibitor, PS1145 prior to TNFα exposure [15]. As predicted 20 μM PS1145 was sufficient in reducing the transcriptional inflammatory response of IκBα (DMSO: 2.95 ± 0.25 and PS1145: 1.10 ± 0.08 IκBα/histone mRNA) and TNFα (DMSO: 2.84 ± 0.32 and PS1145: 0.56 ± 0.10 TNFα/histone mRNA) (Figure 1Ei, ii). Together, these results indicate that the canonical inflammatory pathway is functionally conserved in the rHypoE-7 cell model. Given that an effective transcriptional and translational inflammatory response was observed upon treatment with TNFα at 10 ng/mL for 2 hr, this incubation time and concentration was used in all subsequent studies.

As the IKK-β/NF-κB cascade has been associated with the induction of endoplasmic reticulum (ER) stress or apoptotic cascades both pathways were examined in order to fully characterize the inflammatory state of rHypoE-7 cells [25]. Relative to H₂0 treatment alone, TNFα did not increase the phosphorylation levels of the ER transcription factor, elongation factor 2-α (p-elF-2α; 0.71 ± 0.05 and 0.81 ± 0.08 p-elF-2α/β-actin, respectively) or the mRNA levels of the ER chaperone, GRP-78 (glucose responsive protein-78; 1.03 ± 0.05 and 1.17 ± 0.07 GRP78/histone mRNA, respectively) or transcription factor CHOP (C/EBP homologous protein; 0.78 ± 0.11 and 0.92 ± 0.07 CHOP/histone mRNA, respectively) (Figure 2A, B). It should be noted that a longer exposure to a higher TNFα concentration (6 hr, 100 ng/mL) elevated CHOP mRNA levels suggesting ER stress may come into play later in the inflammatory process (data not shown). Activation of apoptotic pathways was also ruled out by the MTT assay, which revealed an identical cell number/and or metabolic activity across treatments (H₂0: 0.12 ± 0.002 and TNFα: 0.13 ± 0.004 AB_570nm) (Figure 2C). Together with the findings in Figure 1, an acute exposure to modest TNFα concentrations is sufficient to produce a robust transcriptional and translational inflammatory response, without accompanying ER stress or apoptosis. This finding will enable us to specifically examine activation of the IKK-β/NF-κB pathway in the rHypoE-7 neuronal model.

To determine if long-chain omega-3 FAs exhibit an anti-inflammatory property in rHypoE-7 cells, we pretreated the cells with 100 μM FAs or DMSO vehicle 1 hr prior to exposure to TNFα (10 ng/mL, 10 min) and monitored activation of the IKK-β/NF-κB cascade by Western blotting. As DHA is the most abundant omega-3 FA in the brain, we focused on using this FA in our studies [26]. DHA pretreatment reduced phospho-TAK1 and phospho-NF-κB levels relative to DMSO control, suggesting this FA can inhibit signaling through the IKK-β/NF-κB cascade (Figure 3A). DHA exposure lowered the level of active TAK1 relative to DMSO even without an inflammatory challenge, suggesting that the rHypoE-7 cell model may exhibit a lower basal level of signaling through the IKK-β/NF-κB pathway.

Furthermore, DHA pretreatment significantly abrogated the TNFα-dependent increase in mRNA levels of both proinflammatory markers: IκBα (DMSO: 1.83 ± 0.22 and DHA: 1.07 ± 0.09 IκBα/histone mRNA) and TNFα (DMSO: 2.33 ± 0.21 and DHA: 0.44 ± 0.04 TNFα/histone mRNA) (Figure 3Bi, ii). As expected from the mRNA results, DHA pretreatment also significantly reduced TNFα protein production and thus the translational inflammatory response (Figure 3C). These findings ensure that DHA exhibits anti-inflammatory properties in the hypothalamic rHypoE-7 cell model.

To determine if the anti-inflammatory actions of DHA is mediated through the phosphoinositide 3-kinase (PI3K)/AKT (protein kinase b) or mitogen activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) pathways, rHypoE-7 cells were co-treated with DHA and their respective inhibitors prior to TNFα exposure. Pretreatment of rHypoE-7 cells with the PI3K inhibitor, Wortmannin (Wort) significantly reduced AKT phosphorylation (pAKT) levels in response to DHA (DHA: 3.60 ± 0.13 and DHA/Wort: 1.13 ± 0.03 pAKT/AKT) but failed to prevent the anti-inflammatory effects of DHA as shown from the analysis of IκBα mRNA (DHA: 0.09 ± 0.01 and Wort/DHA (W + D): 0.12 ± 0.01 IκBα/histone mRNA) and TNFα mRNA (DHA: 0.83 ± 0.14 and W + D: 0.92 ± 0.08 TNFα/histone mRNA) (Figure 4A, B). Treatment of the rHypoE-7 cells with the PKC inhibitor, Staurosporine algyone (Stauro) reduced ERK phosphorylation (DHA: 2.61 ± 0.29 and DHA/Stauro: 1.36 ± 0.28 pERK/ERK) indicating pERK formation upon DHA exposure is PKC-dependent (Figure 4C). Pretreatment with Stauro abolished or reduced the enhancement of IκBα or TNFα respectively (Figure 4Di, ii), indicating that PKC itself participates in the induction of the transcriptional inflammatory response to TNFα in the rHypoE-7 cell line (a comparison denoted by # in Figure 4D). Relative to DHA pretreatment alone, DHA and Stauro (D + S) co-pretreatment prior to TNFα exposure did not cause a further reduction in IκBα mRNA levels (DHA: 0.38 ± 0.17 and SD + S: 0.44 ± 0.08 IκBα/histone mRNA) but led to a further decrease in TNFα mRNA levels (DHA: 1.19 ± 0.07 and D + S: 0.48 ± 0.03 TNFα/histone mRNA (Figure 4D). These results indicate that the ability of DHA to activate PKC activity plays a role in its anti-inflammatory actions. These results suggest that although DHA activates PI3K and PKC, neither signaling cascade plays significant roles in mediating the anti-inflammatory actions of this omega-3 FA.

The transcriptional anti-inflammatory actions of DHA in rHypoE-7 cells was abolished by the presence of BSA as shown upon quantifying mRNA levels of IκBα (DHA + H₂0: 0.20 ± 0.003 and DHA + BSA: 0.94 ± 0.09 IκBα/histone mRNA) and TNFα, (DHA + H₂0: 0.51 ± 0.02 and DHA + BSA: 1.51 ± 0.09 TNFα/histone) after TNFα exposure (Figure 5A). As the addition of BSA and the formation of DHA-BSA complexes abolished the ability of DHA to inhibit the transcriptional inflammatory response, it is clear that DHA must exist in its free form to mediate its acute anti-inflammatory actions and may interact with free FA receptors at the cell surface.

The free FA receptor, GPR120, was recently identified to mediate the anti-inflammatory actions of omega-3 FAs in macrophages, and to determine if GPR120 may be involved in the anti-inflammatory actions in the hypothalamic rHypoE-7 cell model, we employed the GPR120 synthetic agonist GW9508 (GW) [19, 27]. It must be noted here that GW can also activate GPR40, but as GPR40 is not expressed in the rHypoE-7 cell model, this agonist is considered a GPR120-specific in our studies (Figure 1A). Pretreatment of rHypoE-7 cells with GW (100 μM, 1 hr) prior to TNFα exposure significantly reduced mRNA levels of inflammatory indicators, IκBα (DMSO: 1.61 ± 0.22 and GW: 1.28 ± 0.05 IκBα/histone mRNA) and TNFα (DMSO: 2.33 ± 0.21 and GW: 1.31 ± 0.07 TNFα/histone mRNA) (Figure 5B). This finding indicates that GPR120 activation exhibits anti-inflammatory properties in the rHypoE-7 cell line.

In addition to enhancing the levels of phospho-ERK and phospho-AKT, activated GPR120 can also associate with TAB1. This interaction prevents the subsequent activation of its partner protein TAK1 and halts signaling through the pro-inflammatory IKK-β/NFκB cascade [19]. To determine if this interaction is conserved in the hypothalamic cell model we treated rHypoE-7 cells with DMSO or DHA for 30 min and monitored the GPR120-TAB1 association by co-immunoprecipitation. DHA treatment increased the GPR120-TAB1 association relative to DMSO control indicating that a similar mechanism identified in the periphery is likely conserved in the hypothalamic cell model (DMSO: 1.44 ± 0.40 and DHA: 2.55 ± 0.28 GPR120/TAB1) (Figure 5C).

To reduce endogenous level of GPR120 in the rHypoE-7 neuronal model we used the small interfering RNA (siRNA) method and GPR120-specific oligonucleotides. Importantly, siRNA resulted in approximately 40% reduction in GPR120 mRNA (data not shown) and 75% reduction in protein levels as evident from qRT-PCR and Western blotting, respectively (Figure 6A). Reduction of endogenous GPR120 levels enabled us to directly test the role of this GPR in mediating the anti-inflammatory actions of DHA. Reduction of endogenous levels of GPR120 protein significantly impaired the anti-inflammatory actions of DHA as seen by an increase in inflammatory transcripts relative to control for IκBα (Scr: 0.63 ± 0.09 and GPR120: 1.11 ± 0.07 IκBα/histone mRNA) and TNFα (Scr: 0.82 ± 0.06 and GPR120: 1.35 ± 1.67 TNFα/histone mRNA) (Figure 6B). Despite the observation that DHA could lower TNFα protein production by roughly 70% in the Scr controls (DMSO = 0.63 ± 0.04 and DHA = 0.20 ± 0.03 TNFα/βactin), no significant difference could be observed upon a reduction in endogenous GPR120 levels (DMSO = 0.55 ± 0.05 and DHA = 0.44 ± 0.03 TNFα/βactin) (Figure 6C). Taken together, GPR120 is functionally active in the rHypoE-7 cell model, wherein its activation by DHA inhibits the transcriptional and translational inflammatory response against the pro-inflammatory cytokine TNFα.