Conjugated linoleic acid (CLA) refers to a group of PUFA that exist as a mixture of positional and stereoisomers of conjugated dienoic octadecadienoate. CLA is found in ruminant food products, such as beef and dairy products(Reference Pariza and Ha1). CLA has been shown to have many potential health benefits, such as lean body mass deposition(Reference Smedman and Vessby2), antidiabetes(Reference Ryder, Portocarrero and Song3), anti-inflammation(Reference Watkins and Seifert4), anticarcinogenesis(Reference Palombo, Ganguly and Bistrian5) and anti-atherogenesis effects(Reference Lee, Kritchevsky and Pariza6).
CLA can stimulate or inhibit immune cell function, and among CLA isomers, trans-10, cis-12 (t10c12)-CLA has shown to participate in the modulation of pro- or anti-inflammatory cytokine secretion(Reference Bhattacharya, Banu and Rahman7). t10c12-CLA has also been reported to increase TNF-α and IL-6 secretion in rat spleen lymphocytes(Reference Yamasaki, Kishihara and Mansho8), as well as the phagocytosis of canine peripheral blood polymorphonuclear cells(Reference Cho, Kang and Yang9). In contrast, CLA decreased the production of PGE2, TNF-α and the inflammatory agent NO in RAW cells treated with interferon-γ(Reference Yu, Correll and Vanden Heuvel10).
PPAR represent a subfamily of nuclear hormone receptors that are activated by a variety of dietary and endogenous fatty acids. PPARγ is one of three PPAR isoforms: α, β/δ and γ(Reference Daynes and Jones11). Some PPARγ agonists, such as 15-deoxy-δ12,14-PGJ2 and troglitazone, inhibit the phorbol myristyl acetate-induced production of IL-1β, IL-6 and TNF-α in peripheral blood monocytes(Reference Jiang, Ting and Seed12). CLA isomers enhanced PPARγ activation and attenuated the production of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) in weaned pigs challenged with lipopolysaccharide (LPS)(Reference Changhua, Jindong and Defa13). PPARγ agonists might also possess pro-inflammatory activity through the inhibition of IL-10 activity mediated by the PPARγ ligand 15-deoxy-δ12,14-PGJ2(Reference Ji, Kim and Rho14). Recently, there has been increasing evidence that the expression and activation of PPARγ may participate in the activity of NF-κB. NF-κB is a ubiquitously expressed family of transcription factors, which controls the expression of numerous genes involved in inflammatory and immune responses, and in cellular proliferation(Reference Ghosh and Karin15). The most common form of NF-κB, which is found in virtually all cell types, is composed of two subunits named p50 and p65(Reference Verma, Stevenson and Schwarz16). The NF-κB family of transcription factors exists in the cytoplasm of unstimulated cells as homo- or heterodimers complexed with inhibitory κB proteins. Various stimuli lead to the phosphorylation of inhibitory κB by inhibitory κB kinase, which triggers its degradation and the activation of NF-κB. The activation and translocation of NF-κB to the nucleus is followed by the transcription of various pro-inflammatory genes including TNF-α(Reference Meldrum17, Reference Wright, Singh and Hasday18). PPARγ ligands such as 15-deoxy-δ12,14-PGJ2 and ciglitizone have been shown to interfere with the activity of NF-κB in human colon cancer cells(Reference Chen, Lee and Wang19). Furthermore, CLA isomers have been shown to increase PPARγ DNA-binding activity and decrease the DNA-binding activity of NF-κB in vascular smooth muscle cells(Reference Ringseis, Gahler and Herter20).
The aim of the present study was to examine the effect of t10c12-CLA on PPARγ activation, NF-κB activation and TNF-α expression in LPS-naive and LPS-stimulated porcine peripheral blood mononuclear cells (PBMC). In addition, the effect of PPARγ antagonism on NF-κB activation and TNF-α expression in porcine PBMC was examined.
Materials and methods
Chemicals and reagents
t10c12-CLA (>98 % purity; Matreya, Inc., Pleasant Gap, PA, USA) was purchased commercially. t10c12-CLA stock solution was prepared by dissolving t10c12-CLA in dimethyl sulphoxide to a final concentration of 50 mm; the solution was filtered through a 0·45 μm membrane (Millipore Corporation, Bedford, MA, USA) before use. LPS from Escherichia coli 0127:B8, bovine serum albumin and GW9662, a PPARγ antagonist, were all purchased from Sigma-Aldrich Company (St Louis, MO, USA).
Isolation of porcine peripheral blood mononuclear cells
All experimental procedures and animal use were approved by the ethics committee of the Chungbuk National University. Heparinised porcine peripheral blood was drawn from the anterior vena cava, diluted with an equal volume of PBS without Ca and Mg, and overlayed 1:1 on a Percoll™ solution (GE Healthcare Bio-sciences AB, Uppsala, Sweden). After centrifugation at 400 g for 45 min at room temperature, the cells in the interface between the plasma in PBS and the Percoll solution were harvested and treated with 0·83 % NH4Cl in a tris(hydroxymethyl) aminomethane base buffer (pH 7·2) for 5 min. The resulting PBMC were washed three times with PBS. PBMC were resuspended in Roswell Park Memorial Institute 1640 medium (Sigma-Aldrich) supplemented with 5 % heat-inactivated fetal bovine serum (Gibco Company, Grand Island, NY, USA).
t10c12-CLA was added to the PBMC culture media with a minimal volume ( < 1 %) of dimethyl sulphoxide as a solvent, and the same amount of dimethyl sulphoxide was added to control cells without the t10c12-CLA treatment. The PBMC seeded at a density of 2 × 106 cells/ml in a twenty-four-multiwell plate (Nunc Company, Naperville, IL, USA) were incubated with or without LPS (1 μg/ml) and with t10c12-CLA (10 μm) or t10c12-CLA (10 μm) in combination with GW9662 (1 μm) for 24 h at 37°C under a 5 % CO2-humidified atmosphere. After a 24 h incubation, all culture supernatants were collected after centrifugation at 14 000 g for 5 min, filtered through a 0·45 μm pore size membrane filter and stored at − 80°C until used for the analysis of TNF-α. The cells were harvested and stored at − 80°C to extract nuclear protein and test PPARγ and NF-κB activation.
Nuclear protein extraction
The nuclear fraction of PBMC was isolated by using a nuclear extract kit (Active Motif, Carlsbad, CA, USA). After treatment with or without LPS (1 μg/ml) and with t10c12-CLA (10 μm) or t10c12-CLA (10 μm) in combination with GW9662 (1 μm) for 24 h, PBMC previously plated on twenty-four-multiwell plates were rinsed and scraped into ice-cold PBS containing phosphatase inhibitors. The cells were centrifuged at 14 000 g at 4°C for 30 s, suspended in hypotonic buffer and lysed with 0·5 % NP-40. The nuclear pellet was collected after centrifugation of the cell lysates at 14 000 g at 4°C for 10 min. The suspended nuclear pellet was lysed and centrifuged at 14 000 g at 4°C for 10 min. The supernatant (nuclear fraction) was collected, divided into aliquots and stored at − 80°C until they were used for PPARγ and NF-κB transcription factor assays.
PPARγ transactivation assay
PPARγ activity was assayed using an ELISA-based TransAM® PPARγ transcription factor assay kit (Active Motif) following the manufacturer's protocol. In brief, the nuclear extract was added to each well of ninety-six-well plates pre-coated with immobilised oligonucleotides containing a peroxisome proliferator response element. After 1 h of incubation with gentle agitation, wells were washed three times with wash buffer and then incubated with anti-PPARγ antibody (dilution 1:1000) for 1 h at 20°C. After three successive washes, the extracts were incubated for 1 h with diluted anti-mouse horseradish peroxidase-conjugated antibody (dilution 1:1000) followed by the addition of 100 μl of developing solution. After 5 min of incubation, the reaction was blocked by adding 100 μl of stop solution reagent. Optical density was determined using an automated microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at 450 nm with a reference wavelength of 655 nm.
NF-κB p65 transcription factor assay
NF-κB activity was determined using the TransAM® NF-κB transcription factor assay kit (Active Motif) following the manufacturer's protocol (see PPARγ transactivation assay). The NF-κB transcription factor assay kit contains a ninety-six-well plate pre-coated with NF-κB consensus binding oligonucleotides.
Measurement of TNF-α production in the culture supernatant fraction of peripheral blood mononuclear cells
The culture supernatant fraction was collected after 24 h incubation. The amount of TNF-α was determined by direct sandwich ELISA using the Quantikine® P porcine TNF-α immunoassay kit (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocol. All samples, standards and controls were assayed in triplicate. Optical density was determined using an automated microplate reader (BioTek Instruments, Inc.) at 450 nm. TNF-α was quantified from eight titration points using standard curves generated with purified porcine TNF-α, and the concentrations were expressed as pg/ml. Lower and upper detection limits were 11·7 and 1500 pg/ml, respectively.
Ribonucleic acid preparation and RT-PCR
Total RNA was extracted using the Trizol reagent (Invitrogen Company, Carlsbad, CA, USA) according to the methods outlined in the protocol, and the concentration of total RNA was determined by measuring the absorbance at 260 nm. First-strand complementary DNA was prepared by subjecting total RNA (1 μg) to reverse transcription using Moloney Murine Leukemia Virus RT (Invitrogen Company) and random primers (9-mers; Takara Bio, Inc., Otsu, Shiga, Japan). To determine the conditions for logarithmic-phase PCR amplification of PPARγ, TNF-α and cytochrome c oxidase subunit (1A), mRNA aliquots (1 μg) were amplified using different numbers of cycles. The 1A gene was PCR-amplified to rule out the possibility of RNA degradation and was used to control for variations in mRNA concentration in the RT reaction. A linear relationship between PCR product band visibility and the number of amplification cycles was observed for target mRNA. The 1A and target genes were quantified using twenty-eight and thirty cycles, respectively. Complementary DNA was amplified in 20 μl PCR mixtures containing 1 unit Taq polymerase (iNtRON Biotechnology, Inc., Sungnam, Kyungki, South Korea), 2 mm-deoxyribonucleotide triphosphate and 10 pmol specific primers. PCR mixtures were denatured at 95°C for 30 s, annealed at 58°C for 30 s and extended at 72°C for 30 s. Oligonucleotides for PPARγ were based on the complementary DNA sequence (GenBank accession no. AJ006756) 5′-CTG GCA AAG CAC TTG TAT G-3′ (sense) and 5′-GGT GTA AAT GAT CTC GTG GA-3′ (antisense). Oligonucleotides for TNF-α were based on the complementary DNA sequence (GenBank accession no. X57321) 5′-CAA GGA CTC AGA TCA TCG TC-3′ (sense) and 5′-CTT GGT CTG GTA GGA GAC G-3′ (antisense). The primer for the 1A gene (GenBank accession no. AF03253) was 5′-CAC CGT AGG AGG TCT AAC G-3′ (sense) and 5′-GTA TCG TCG AGG TAT TCC G-3′ (antisense). PCR products (8 μl) were fractionated on a 2·3 % agarose gel, stained with ethidium bromide and photographed under UV illumination. The photograph was scanned using a Gel Doc EQ system (Bio-Rad Laboratories, Hercules, CA, USA).
All statistical analyses were performed using GraphPad prism 5 software (GraphPad Software, San Diego, CA, USA). One-way ANOVA was used to determine the statistical significance of the differences between the control and treatment groups, followed by a Dunnett test. Comparisons of two groups were done using the t test. P values of less than 0·05 were considered to be statistically significant. Data are expressed as means and standard deviations.
Trans-10, cis-12 conjugated linoleic acid up-regulates PPARγ activity and mRNA expression in porcine peripheral blood mononuclear cells
To examine whether t10c12-CLA treatment activates PPARγ in porcine PBMC, cells were incubated with t10c12-CLA (10 μm) or t10c12-CLA (10 μm) in combination with the PPARγ antagonist GW9662 (1 μm) for 24 h. To assess the level of PPARγ activation, we used the binding assay as described in the Materials and methods section, although the results of this type of assay may not necessarily reflect transcriptional activation. PPARγ DNA-binding activity was significantly increased (P < 0·05) by t10c12-CLA treatment when compared with vehicle (dimethyl sulphoxide) controls. Treatment with GW9662 significantly suppressed (P < 0·05) the t10c12-CLA-mediated enhancement of PPARγ DNA-binding activity in PBMC (Fig. 1(A)). RT-PCR analysis also showed that t10c12-CLA significantly increased (P < 0·05) PPARγ mRNA expression in PBMC compared with vehicle controls. GW9662 negated (P < 0·05) the t10c12-CLA-induced enhancement of PPARγ mRNA expression (Fig. 1(B)).
Trans-10, cis-12 conjugated linoleic acid increases NF-κB activity in porcine peripheral blood mononuclear cells
To examine the effect of t10c12-CLA treatment on NF-κB activation in porcine PBMC, t10c12-CLA (10 μm) was added to the cell culture for 24 h. NF-κB p65 DNA-binding activity was significantly increased (P < 0·01) by t10c12-CLA treatment in PBMC compared with vehicle controls. GW9662 (1 μm) significantly suppressed (P < 0·05) the t10c12-CLA-mediated enhancement of NF-κB p65 DNA-binding activity (Fig. 2).
Trans-10, cis-12 conjugated linoleic acid increases TNF-α expression in porcine peripheral blood mononuclear cells
To examine whether t10c12-CLA induces the expression of TNF-α through the activation of PPARγ in porcine PBMC, these cells were incubated with t10c12-CLA (10 μm) or t10c12-CLA (10 μm) in combination with GW9662 (1 μm) for 24 h. As shown in Fig. 3, t10c12-CLA significantly increased (P < 0·01) TNF-α production in PBMC compared with vehicle-treated controls. The enhancement of TNF-α production by t10c12-CLA was significantly decreased (P < 0·01) by the addition of GW9662 (Fig. 3(A)). t10c12-CLA also significantly enhanced (P < 0·05) TNF-α mRNA expression in PBMC compared with controls, and this effect was abolished by GW9962 (P < 0·001; Fig. 3(B)).
Trans-10, cis-12 conjugated linoleic acid up-regulates PPARγ activity and mRNA expression in lipopolysaccharide-stimulated porcine peripheral blood mononuclear cells
The effect of t10c12-CLA on PPARγ activation in LPS-stimulated porcine PBMC was tested by treating PBMC with LPS (1 μg/ml) and t10c12-CLA (10 μm) in the presence or absence of GW9662 (1 μm) for 24 h. PPARγ DNA-binding activity in LPS-stimulated PBMC was significantly increased (P < 0·01) by t10c12-CLA treatment when compared with vehicle plus LPS-treated controls. GW9662 treatment significantly inhibited (P < 0·01) the t10c12-CLA stimulation of PPARγ DNA-binding activity in LPS-stimulated PBMC (Fig. 4(A)). RT-PCR analysis showed that t10c12-CLA also significantly increased (P < 0·001) PPARγ mRNA expression in LPS-stimulated PBMC compared with controls (vehicle plus LPS), and this effect was significantly inhibited (P < 0·001) by the addition of GW9662 (Fig. 4(B)).
Trans-10, cis-12 conjugated linoleic acid decreases NF-κB activity in lipopolysaccharide-stimulated porcine peripheral blood mononuclear cells
To assess NF-κB activation in response to t10c12-CLA in LPS-stimulated porcine PBMC, cells were treated with LPS (1 μg/ml) and t10c12-CLA (10 μm) in the presence or absence of the PPARγ antagonist GW9662 (1 μm) for 24 h. NF-κB p65 DNA-binding activity was significantly suppressed (P < 0·01) by t10c12-CLA treatment in LPS-stimulated PBMC compared with controls (vehicle plus LPS); NF-κB DNA-binding activity was restored (P < 0·01) by the addition of GW9662 (Fig. 5).
Trans-10, cis-12 conjugated linoleic acid decreases TNF-α expression in lipopolysaccharide-stimulated porcine peripheral blood mononuclear cells
The effect of t10c12-CLA on TNF-α expression in LPS-stimulated porcine PBMC was tested by adding LPS (1 μg/ml) and t10c12-CLA (10 μm) to the PBMC culture in the presence or absence of GW9662 (1 μm) for 24 h. As shown in Fig. 6, t10c12-CLA significantly decreased (P < 0·05) TNF-α production in LPS-stimulated PBMC compared with vehicle plus LPS-treated controls, and this effect was reversed (P < 0·05) by the addition of GW9662 (Fig. 6(A)). In addition, RT-PCR analysis revealed that t10c12-CLA significantly suppressed (P < 0·001) TNF-α mRNA expression in LPS-stimulated PBMC compared with vehicle plus LPS-treated controls. This suppression of TNF-α mRNA expression by t10c12-CLA was reversed (P < 0·05) by GW9662; however, TNF-α mRNA expression was restored with a level lower than that of vehicle plus LPS-treated controls (Fig. 6(B)).
The results of the present study showed that t10c12-CLA up-regulated TNF-α expression in porcine PBMC. A similar increase in TNF-α mRNA levels in response to t10c12-CLA treatment has also been reported in isolated adipocytes(Reference Tsuboyama-Kasaoka, Takahashi and Tanemura21). t10c12-CLA also enhanced TNF-α and IL-6 gene expression in white adipose tissue(Reference Poirier, Shapiro and Kim22), and increased TNF-α secretion in mouse splenocytes(Reference Kelley, Warren and Simon23). In porcine PBMC, the t10c12-CLA-induced enhancement of TNF-α production up-regulated the phagocytic capacity of porcine polymorphonuclear cells(Reference Kang, Lee and Jeung24). In addition, t10c12-CLA has been found to enhance the chemotaxis of porcine polymorphonuclear cells through IL-8 produced by CLA-treated PBMC(Reference Son, Kang and Lee25). These findings suggest that t10c12-CLA has an immunostimulating effect mediated by the production of pro-inflammatory cytokines, including TNF-α and IL-8.
The t10c12-CLA-mediated activation of NF-κB p65 DNA binding in porcine PBMC found in the present study has also been reported in human umbilical vein endothelial cells(Reference Nakamura and Omaye26). The activation of NF-κB is followed by the transcription of various pro-inflammatory genes including TNF-α(Reference Wright, Singh and Hasday18). t10c12-CLA increased TNF-α production through the activation of NF-κB in human adipocytes(Reference Chung, Brown and Provo27). Moreover, t10c12-CLA directly induced IL-6 secretion in 3T3-L1 adipocytes by an NF-κB-dependent mechanism(Reference Poirier, Shapiro and Kim22). These observations indicate that t10c12-CLA may participate in the expression of inflammatory mediators through the activation of NF-κB.
In contrast with its effect on LPS-naive PBMC, t10c12-CLA down-regulated TNF-α expression in LPS-stimulated PBMC. Similarly, t10c12-CLA attenuated the production and gene expression of TNF-α in weaned pigs challenged with LPS(Reference Changhua, Jindong and Defa13). t10c12-CLA also decreased pro-inflammatory cytokines including TNF-α in interferon-γ-stimulated macrophages(Reference Yu, Correll and Vanden Heuvel10), and CLA has been reported to increase IL-10 production and decrease IL-12 production in LPS-stimulated murine dendritic cells(Reference Loscher, Draper and Leavy28). IL-10 is known to be an anti-inflammatory cytokine capable of inhibiting the synthesis of pro-inflammatory cytokines and blocking NF-κB activity(Reference Eskdale, Kube and Tesch29, Reference Moore, de Waal Malefyt and Coffman30). The decrease in TNF-α expression in LPS-stimulated PBMC treated with t10c12-CLA found in the present study could therefore be related to an increase in IL-10 production. In the present study, t10c12-CLA suppressed NF-κB p65 DNA-binding activity in LPS-stimulated porcine PBMC. c9t11-CLA, which is also known to have an anti-inflammatory effect, decreased phorbol ester-induced NF-κB activation in HR-1 mouse skin cells by down-regulating inhibitory-κB degradation(Reference Hwang, Kundu and Shin31). t10c12-CLA prevented TNF-α gene expression by inhibiting NF-κB-binding activity in PBMC from weaned pigs challenged with LPS(Reference Zhao, Yin and Li32). Also, treatment with a CLA mixture (isomers 9, 11 and 10, 12) down-regulated inducible NO synthase and cyclo-oxygenase 2 expression, as well as the subsequent production of NO and PGE2 in LPS-stimulated RAW 264.7 macrophages through the inhibition of NF-κB DNA-binding activity(Reference Cheng, Lii and Chen33). These findings suggest that t10c12-CLA may suppress TNF-α expression in LPS-stimulated porcine PBMC by inhibiting NF-κB activation.
In the present study, PPARγ activity and mRNA expression in porcine PBMC were enhanced by t10c12-CLA treatment regardless of LPS stimulation. These findings are in agreement with several studies which have shown that CLA stimulated PPARγ gene expression in different tissues and cells, including skeletal muscle(Reference Meadus, MacInnis and Dugan34), adipocytes(Reference Evans, Park and Pariza35) and macrophages(Reference Yu, Correll and Vanden Heuvel10). Moreover, treatment with a CLA mixture enhanced PPARγ DNA-binding activity in cardiomyocytes(Reference Alibin, Kopilas and Anderson36). The activity of t10c12-CLA in PBMC could therefore be associated with both increased levels of PPARγ protein and activation of PPARγ.
PPAR activators have been reported to increase TNF-α production in mouse hepatocytes(Reference Nakatani, Tsuboyama-Kasaoka and Takahashi37), and t10c12-CLA, which activates PPARγ, enhanced TNF-α and PPARγ expression in RAW macrophages(Reference Song, Kang and Lee38). Furthermore, t10c12-CLA and c9t11-CLA increased both PPARγ and NF-κB activity in human umbilical vein endothelial cells(Reference Nakamura and Omaye26). In contrast, PPARγ activation inhibited the production of inflammatory cytokines, including TNF-α, in LPS-stimulated dendritic cells(Reference Szatmari, Rajnavolgyi and Nagy39). The PPARγ agonists troglitazone and PUFA attenuated the activation of NF-κB and the production of IL-6, IL-8 and inducible NO synthase in IL-1β-stimulated intestinal-like Caco-2 cells and in LPS-stimulated human dendritic cells(Reference Marion-Letellier, Butler and Déchelotte40). t10c12-CLA suppressed the production of TNF-α, IL-1β and IL-6, and enhanced PPARγ activation and gene expression in LPS-stimulated PBMC(Reference Changhua, Jindong and Defa13). t10c12-CLA and c9t11-CLA also inhibited platelet-derived growth factor-induced NF-κB activation in human vascular smooth muscle cells by a PPARγ-dependent mechanism(Reference Ringseis, Gahler and Eder41). These findings support the idea that the activation of PPARγ by t10c12-CLA can modulate TNF-α expression through the up- and down-regulation of NF-κB activity in LPS-naive and LPS-stimulated PBMC, respectively.
The present results showed that t10c12-CLA increased TNF-α expression and NF-κB activation in porcine PBMC. In contrast, in LPS-stimulated porcine PBMC, t10c12-CLA decreased TNF-α expression and NF-κB activation. t10c12-CLA also up-regulated PPARγ activity and mRNA expression in both LPS-naive and LPS-stimulated porcine PBMC. To elucidate the role of PPARγ on NF-κB activity and TNF-α expression in CLA-treated porcine PBMC, we used a PPARγ antagonist, GW9662. GW9662 negated the effects of CLA on TNF-α expression and NF-κB activation in both LPS-naive and LPS-stimulated porcine PBMC. These results strongly suggested that the effects of t10c12-CLA on NF-κB activation and TNF-α expression may be dependent on the PPARγ pathway. In the present study, however, t10c12-CLA, LPS and GW9662 were added simultaneously, which might preclude mechanistic insights due to the potential for interference or interactions between the various drugs. An experimental method employing sequential additions may be necessary to clarify more precisely the mechanism involved.
Recent nutritional immunology studies have revealed an important role for dietary CLA in the attenuation of inflammation-associated diseases. The preventive administration of a CLA mixture before the onset of porcine bacterial-induced colitis attenuated inflammatory lesion development and growth failure(Reference Hontecillas, Wannemeulher and Zimmerman42). Dietary CLA supplementation (mixture of isomers 9, 11 and 10, 12) up-regulated colonic PPARγ expression and contributed to delaying the onset of experimental inflammatory bowel disease in a pig model(Reference Bassaganya-Riera and Hontecillas43). It has also been demonstrated that CLA ameliorates dextran sodium sulphate-induced colitis(Reference Bassaganya-Riera, Reynolds and Martino-Catt44) and prevents colorectal tumour formation, partly through a PPARγ-dependent mechanism in PPARγ-null mice(Reference Evans, Misyak and Schmelz45). c9t11-CLA reduced allergic sensitisation and airway inflammation in mice, most probably via a PPARγ-related mechanism and by reducing eicosanoid precursors(Reference Jaudszus, Krokowski and Möckel46). Recently, t10c12-CLA and c9t11-CLA have been found to suppress muscle wasting, which is mediated by TNF-α, in a model of human muscle cell inflammation(Reference Larsen, Cameron-Smith and Crowe47). In the present study, we have shown that t10c12-CLA treatment increases TNF-α expression in naive PBMC but suppresses LPS-induced TNF-α expression. This apparently conflicting result following LPS stimulation may be related directly to NF-κB p65 activity and may be mediated at least partly through a PPARγ-dependent mechanism. Although it is currently difficult to determine why t10c12-CLA yields such a conflicting effect, the conflict could explain discrepancies in previous reports over whether or not CLA has a beneficial effect on health. Besides the effects on IL-10 and PGE2 production mentioned above, another factor that might explain TNF-α expression signalling modulated by t10c12-CLA could be the ubiquitin-editing enzyme A20, also known as TNF-α-induced protein 3(Reference Vereecke, Beyaert and van Loo48). Ubiquitin-editing enzyme A20 is a key player in the negative feedback regulation of NF-κB signalling(Reference Coornaert, Carpentier and Beyaert49), but there is yet no evidence to directly explain the effects of CLA. Recently, it has been reported that the ubiquitin-editing enzyme A20 is required for the termination of NF-κB signalling in response to LPS and TNF-induced NF-κB responses(Reference Hitotsumatsu, Ahmad and Tavares50). To clarify the effects of t10c12-CLA, further research into the overall TNF signalling pathway, including the ubiquitin-editing enzyme A20, will be necessary.
In conclusion, t10c12-CLA has an immunostimulating effect on porcine PBMC, which is mediated by the enhancement of NF-κB activity and TNF-α expression. By contrast, t10c12-CLA has an anti-inflammatory effect through the suppression of NF-κB activity and TNF-α expression in LPS-stimulated PBMC. These immunostimulating and anti-inflammatory effects of t10c12-CLA on porcine PBMC are mediated by a PPARγ-dependent pathway.
The present study was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD; KRF-2007-313-E00540). D.-I. K. performed the study, analysed the data and wrote the manuscript; K.-H. K. performed the study and analysed the data; J.-H. K. designed the study, analysed the data and wrote the manuscript; E.-M. J. and S.-S. K. performed the study; E.-B. J. analysed the data; M.-P. Y. designed the study, analysed the data, wrote the present manuscript. All authors approved the final manuscript. All authors declare that they had no conflict of interest.