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College of Animal Science and Technology, China Agricultural University, Beijing, P.R. China
2To whom correspondence should be addressed. E-mail: defali{at}public2.bta.net.cn.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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] induced by the LPS challenge. CLA enhanced the expression of interleukin-10 (IL-10) and peroxisome proliferator-activated receptor-
(PPAR) in spleen and thymus. To further elucidate the inhibitory effects and the mechanism of action of CLA on cytokine profiles (i.e., IL-1ß, IL-6, and TNF-), PBMCs were isolated from weaned pigs and cultured in media containing cis-9, trans-11 (9c,11t) CLA and trans-10, cis-12 (10t,12c) CLA. Each CLA isomer suppressed the production and expression of IL-1ß, IL-6, and TNF-, and enhanced PPAR activation and gene expression in cultured PBMCs. At the molecular level, the inhibitory actions of CLA on IL-1ß, IL-6, and TNF- are attributable mainly to 10t,12c-CLA and the anti-inflammatory properties of CLA are mediated, at least in part, through a PPAR-dependent mechanism.
KEY WORDS: • proinflammatory cytokine • conjugated linoleic acid • PPAR • pigs • growth suppression
Cytokines play an important role in immunoregulation. Interleukin (IL)3 -1, IL-6, and tumor necrosis factor (TNF)- are among the most important cytokines produced by monocytes and macrophages. These cytokines can mediate the systemic effects of inflammation such as fever, loss of appetite, mobilization of protein and fat, and acute phase protein synthesis. The production of appropriate amounts of IL-1ß, IL-6, and TNF- is clearly beneficial in response to infection, but inappropriate amounts or overproduction can be dangerous; these cytokines, especially TNF-, are implicated in causing some of the pathological responses that occur in inflammatory conditions or some inflammation-associated diseases (i.e., cancer, atheromatosis, and arthritis) (1). Therefore, to alleviate inflammation, it is important to inhibit the production of proinflammatory cytokines. Nutrition strategies may be desirable to manipulate the secretion of proinflammatory cytokines.
One strategy to attenuate the negative effects of proinflammatory cytokines is to supplement the diet with conjugated linoleic acid (CLA). CLA comprises an isomeric mixture of linoleic acid that have conjugated double bonds. CLA has been shown to have anti-inflammatory activities in guinea pigs (2) and rats (3) and to alleviate inflammation-associated diseases; these include anti-carcinogenic activities in mice (4), rats (5), and humans (6), anti-atherogenic activities in rabbits (7), and attenuation of inflammatory lesion development induced by bacterial infection in pigs (8).
Research in several species, including poultry (9), rats (10), and mice (11), showed that dietary CLA can reduce the release of proinflammatory cytokines. However, there is little information on the relation between proinflammatory cytokine profiles (i.e., IL-1ß, IL-6, and TNF-) and CLA supplementation in swine. Furthermore, the mechanism(s) by which CLA regulates the synthesis of proinflammatory cytokines must be more clearly elucidated. A recent study (12) showed that dietary CLA is associated with an elevated expression of a number of genes that are responsive in part to peroxisome proliferator-activated receptors (PPAR), which are involved in modulating the activities of several transcription factors that regulate cytokine expression. Further understanding of the mechanisms underlying the anti-inflammatory activity of CLA may yield novel nutritional approaches to inflammation.
Therefore, to investigate the anti-inflammatory role and mechanism(s) of action of CLA in weaned pigs, we created a model of acute inflammation using the lipopolysaccharide (LPS) challenge in pigs and determined the effects of dietary CLA and individual CLA isomers on the production and mRNA expression of proinflammatory cytokine profiles (i.e., IL-1ß, IL-6, and TNF-), as well as the subsequent activation and expression of nuclear transcription factors PPAR in an in vivo animal feeding trial and in vitro peripheral blood mononuclear cell (PBMC) culture experiments.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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On d 14 and 21, blood samples from each pig were obtained by puncturing the vena cava; blood was collected in 5-mL heparinized vacutainer tubes (Becton Dickinson Vacutainer Systems) at 3.0 h postinjection. The blood samples were centrifuged (3500 x g for 5 min) to collect plasma. Plasma was stored at –20°C until analysis for IL-1ß, IL-6, TNF-, IL-10, and prostaglandin E2 (PGE2). The pigs’ response to LPS after injection was observed and recorded. At the end of the 4-wk feeding trial, pigs were injected with either LPS or sterile saline as described above; 3 h postinjection, they were killed by bleeding after i.m. injection of a sodium pentobarbital solution. Spleen and thymus were removed and immediately frozen in liquid nitrogen, then stored at –80°C for isolation of total RNA and analysis of IL-1ß, IL-6, TNF-, IL-10, and PPAR mRNA expression profiles.
Isolation and preparation of peripheral blood mononuclear cells.
Two crossbred (Large White x Landrace x Pietran) barrows weighing 
9.8–10.0 kg were fed control diets for 7 d. Blood samples were collected from the vena cava as described above.
The PBMCs were isolated from heparinized whole blood by Ficoll-Paque density gradient centrifugation. Briefly, blood samples were centrifuged (2500 x g, 30 min), and the mononuclear cell layer was recovered and washed twice with RPMI-1640 (Gibco, Invitrogen) medium. Subsequently, the cells were resuspended in complete RPMI-1640 medium supplemented with 10% (v:v) heat-inactivated fetal calf serum, 105 U/L of penicillin, 100 mg/L of streptomycin, and 25 mmol/L HEPES buffer (Sigma). The pH of the media was adjusted to 7.4 with the addition of a sterile solution of 56 g/L sodium bicarbonate. Then cells were counted on a hemocytometer using a light microscope; viability was determined by trypan blue exclusion method.
Cell culture.
Cell suspensions (4 x 109 cells/L culture medium) were incubated with culture media containing 9c,11t-CLA, 10t,12c-CLA or 1:1 CLA mixture (9c,11t-CLA:10t,12c-CLA = 1:1) at a final concentration of 100 µmol/L in a total volume of 1 mL in a 24-well plate. Cells were incubated at 37°C in 5% CO2 humidified atmosphere for 24 h. LPS was added to each well at a final concentration of 10 mg/L and incubated for another 3 h. Supernatants were collected after centrifugation (800 x g at 4°C for 10 min) and stored at –80°C for analysis of IL-1ß, IL-6, TNF-, PGE2, and 15-deoxy-
12,14-prostaglandin J2 (15d-PGJ2). The cells were harvested and stored at –80°C to extract nuclear protein and isolate total RNA. Bovine serum albumin (BSA) was the vehicle control. Cultures were set up in groups of 6. The fatty acids used to incubate cells were prepared as described by Lin et al. (14).
Nuclear protein extraction and PPAR activation test.
At the end of the incubation of the cells (3 h after adding LPS), cells were harvested after centrifugation (800 x g at 4°C for 10 min) to extract nuclear protein by Nuclear Extract Kit (Active Motif). The protein yield of this kit for nuclear extract is 0.15–0.25 mg at 3–5 g/L from 8.8 x 106 cells. PPAR activation was tested using TransAMTM PPAR Transcription Factor Assay Kits (Active Motif). TransAM provides quantitative results from 0.75 to 7.5 µg of nuclear extract/well. Extraction and assay were performed according to the manufacturer’s instructions.
Total RNA isolation and reverse transcription (RT). Total RNA was isolated from the spleen, thymus, and PBMCs using the TRIzol Reagent (Invitrogen, Life Technologies) according to the manufacturer’s instructions. RNA integrity was verified electrophoretically by ethidium bromide staining, and RNA in samples was quantified. The RNA purity was determined using UV-clear Microplates (TECAN) at OD260 and an OD260/OD280 ratio. The OD260/OD280 ratio of all samples was >1.80. Total RNA was reverse transcribed as describing in the following. Briefly, 2 µg of each RNA isolated from each pig or each cell sample was added to a 40-µL reaction system containing 1.0 µL of Oligo-dT18 (Promega), 1.0 µL of dNTPs (Sigma), 1.0 µL of RNasin inhibitor (Promega), 2.0 µL of M-MLV transcriptase (Promega), 8.0 µL of M-MLV RT reaction buffer (Promega), and 25 µL of RNase-free water. Cycle parameters for the RT procedure were 1 cycle of 37°C, 2 h; 1 cycle of 75°C, 5 min; and 1 cycle of 4°C, 5 min. The RT products (cDNA) were stored at –20°C for relative quantification by PCR.
Real-time PCR for quantification of cytokines and PPAR mRNA.
Quantitative analysis of PCR was carried out in the DNA Engine Opticon 2 fluorescence detection system (MJ Research) according to optimized PCR protocols and DyNAmo SYBR Green qPCR kit (Finnzymes), in which SYBR Green I was a double-stranded DNA-specific fluorescent dye. The PCR reaction system (20 µL) contained 10 µL DyNAmo SYBR Green qPCR mix, 5 µL primer (0.3 µmol/L forward and 0.3 µmol/L reverse), and 5 µL cDNA template (<10 µg/L). For the PCR reaction, the following experimental run protocol was used: enzyme incubation (50°C for 2 min), denaturation program (95°C for 10 min), amplification and quantification program repeated 38 times (94°C for 20 s, different annealing temperature for different target genes for 20 s, 72°C for 20 s with a single fluorescence measurement), melting curve program (65–95°C with a heating rate of 0.1°C/s and a continuous fluorescence measurement) and finally 72°C for 10 min. The annealing temperature for IL-1ß, IL-6, TNF-, IL-10, PPAR, and ß-actin was 57.5, 62.0, 60.0, 59.5, 58.5, and 57.5°C, respectively. All samples were measured in triplicate.
To amplify IL-1ß, IL-6, TNF-, IL-10, PPAR, and ß-actin cDNA fragments, the following sequences of PCR primers pairs were used: forward 5'-CAACGTGCAGTCTATGG-AGT-3', reverse 5'-GAGGTGCTGATGTACCAGTT-3' for IL-1ß (372 bp); forward 5'-GGCTGCTTCTGGTGATGGCTA-3', reverse 5'-TTGCCTCAGGGTCTGGATCAGT-3' for IL-6 (419 bp); forward 5'-CCACGTTGTAGCCAATGTCA-3', reverse 5'-CAGCAAAGTCCAGATAGTCG-3' for TNF- (375 bp); forward 5'-GAAGGACCAGATGGGCGACTT-3', reverse 5'-CACCTCCTCCACGGCCCTTG-3' for IL-10 (256 bp); forward 5'-CTCTGTGGACCTGTCGGTGAT-3', reverse 5'-GGAGTTGGAAGGCTCTTCGTG-3' for PPAR (262 bp); forward 5'-TTCCTGGGTATGGAATCCTG-3', reverse 5'-CACCTTCACCGTTCCAGTTT-3' for ß-actin (485 bp).
The relative standard curve methods were used for quantification of gene expression. Briefly, copy numbers were determined from 2 independent cDNA preparations of any sample. Copy numbers were calculated relative to a dilution series of the respective reference plasmids, comprising 103–107 copies. The reference plasmids contained the cloned RT-PCR products obtained with these primers. The housekeeping gene, ß-actin, was used as an internal standard for the PCR reaction. The Ct-value (number of cycles halfway through the exponential phase) was determined and was used to calculate the relative expression level compared with ß-actin.
Determination of cytokines, PGE2, and 15d-PGJ2 levels.
IL-1ß was measured using a commercially available porcine ELISA kit (BioSource International). The minimum detectability was 15 ng/L with 4.1 and 8.9% intra- and interassay CV, respectively. The mean recovery of porcine IL-1ß in porcine serum was 94%. IL-6, TNF-, and IL-10 were also measured using commercially available porcine ELISA kits (R&D Systems). The minimum detectable dose was 10 ng/L for IL-6, 3.7 ng/L for TNF-, and 3.5 ng/L for IL-10. The intra- and interassay CV were 2.9 and 8.5% for IL-6, 4.9 and 8.9% for TNF-, and 3.1 and 9.5% for IL-10, respectively. The mean recovery of porcine IL-6, TNF-, and IL-10 in porcine serum was 95, 105, and 94%, respectively. PGE2 was measured using a commercially available 125I RIA kit (College of Medical Science of Suzhou University, Jiangsu, China). Minimum detectability of porcine plasma prostaglandin E2 was 6.25 ng/L with an intra-assay CV < 10%. There were cross-reactivities of 4.5, 2.4, and < 0.1% in the kit with PGE1, PGE3, and arachidonic acid or its metabolites, respectively. 15d-PGJ2 was tested using Correlate-EIA kit (Assay Designs). The sensitivity of this kit was 36.8 ng/L with 5.7 and 13.0% intra- and interassay CV, respectively. The recovery of 15d-PGJ2 in porcine plasma was 105%. Assays were performed in duplicate and analyzed according to the manufacturer’s instructions.
Statistical analysis. After the LPS challenge, data were analyzed using the GLM procedures of SAS (15) with a 2 x 2 factorial arrangement of treatments in a randomized block design. In the model, pig within block was the experimental unit for the dietary treatment (subplot), and the blocks of pigs within infective status were the experimental units for infection treatment (whole plot). The statistical model utilized was Yijk = µ + Injectioni + Dietj + (Injection x Diet)ij + error Aik + error Bijk, where µ was the general mean, Injectioni was the main effect of the ith level of the challenge effect, Dietj was the main effect of jth level of the dietary effect, (Injection x Diet)ij was the interaction effect between injection and diet, and errors A and B represented the random errors for the whole plot and the subplot, respectively. Data in the in vitro experiment were analyzed using one-way ANOVA. Fisher’s Protected Least Significant Difference test was used to determine which means differed. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Dietary CLA and cytokines in plasma and tissues.
The plasma concentrations of IL-1ß in LPS-challenged pigs were higher than those in pigs not challenged by LPS, whereas on d 14 and 21, pigs fed the CLA diets had lower IL-1ß concentrations in plasma than those fed the control diets (Table 2). The plasma IL-6 and TNF- concentrations in LPS-challenged pigs fed the CLA diets were lower than those in LPS-challenged pigs fed the control diets after 2 injections of LPS (Table 2). However, on d 14 and 21, plasma IL-10 in LPS-challenged pigs fed the CLA diets was higher than that in LPS-challenged pigs fed the control diets (Table 2). Pigs challenged with LPS had greater IL-1ß mRNA expression in spleen and thymus than unchallenged pigs (Table 3). However, the increased IL-6 and TNF- mRNA expression in spleen and thymus in LPS-injected pigs were attenuated by dietary CLA supplementation (Table 3). Compared with soybean oil, dietary CLA enhanced IL-10 mRNA expression in spleen and thymus in LPS-challenged pigs (Table 3).
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production (Table 4) and mRNA expression in PBMCs (Table 5), compared with PBMCs cultured in the vehicle. Furthermore, the inhibitory actions of 10t,12c-CLA on cytokine profiles (i.e., IL-1ß, IL-6, and TNF-) were greater than those of 9c,11t-CLA.
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PPAR activation and mRNA expression.
The mRNA expressions of PPAR in spleen and thymus tissue samples recovered from pigs fed the CLA-supplemented diet were greater than those in pigs fed the soybean oil–supplemented diet, regardless of challenge status (Table 3). LPS-challenged pigs had higher PPAR expression in spleen and thymus compared with unchallenged pigs fed the CLA and control diets. The 9c,11t-CLA, 10t,12c-CLA, and 1:1mixture all enhanced PPAR activation (Table 4) and expression in cultured PBMCs (Table 5) compared with the vehicle control. Moreover, the enhancing effects of 10t,12c-CLA on PPAR activation and expression in cultured cells were greater than those of 9c,11t-CLA.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and depressed growth.
However, synthesis of the proinflammatory cytokines, IL-6 and TNF-, was reduced at both the protein and mRNA levels, and the production and expression of the anti-inflammatory cytokine IL-10 were enhanced in injected pigs by dietary CLA supplementation. These findings suggested that the supplementation of CLA may help attenuate inflammation induced by LPS challenge. The in vitro study further demonstrated that the inhibitory actions of CLA on the production and mRNA expression of IL-1ß, IL-6, and TNF- in PBMCs are due mainly to 10t,12c-CLA. This also indicated that, at the molecular level, the synthesis of these cytokines is controlled in part by the mechanism of transcriptional regulation. This is a first time it was demonstrated that the supplementation of CLA ameliorates the production and expression of proinflammatory cytokine profiles (i.e., IL-6 and TNF-) in LPS-injected pigs. Consistent with the results of this study, dietary CLA reduced the release of proinflammatory cytokines in chickens (9), rats (10), and mice (11). CLA also ameliorated the inflammation-associated mucosal damage, decreased growth suppression, and delayed the onset of clinical disease associated with bacterial-induced colitis (8). Furthermore, compared with 10t,12c-CLA, 9c,11t-CLA significantly stimulated TNF- production in spleen lymphocytes of mice in vivo (20). Thus, the prevention of depressed growth in LPS-challenged pigs by dietary CLA may be associated with CLA inhibiting the production of these cytokines.
Dietary CLA increased IL-10 production in plasma and mRNA expression in spleen and thymus in injected pigs, compared with uninjected pigs fed the CLA or the control diets. IL-10 is an anti-inflammatory cytokine that is produced by Th2. Although the enhanced production and expression of IL-10 are simultaneous with an acute inflammation response induced by LPS challenge, the upregulation of IL-10 may be part of a homeostatic mechanism to balance the action of Th1 (8). At least 3 functional outputs of terminally differentiated Th cells were characterized on the basis of cytokine production and homing capacity, i.e., Th1, Th2, and nonpolarized cells (21).
Dietary CLA decreased the plasma level of PGE2 in LPS-challenged pigs, and CLA isomers inhibited the production of PGE2 in cultured PBMCs, thus indicating that CLA has an anti-inflammatory effect. CLA might modulate the accumulation of arachidonate in phospholipids, resulting in a reduced arachidonate pool and decreased production of downstream eicosanoid products such as PGE2 (22), which are involved in early inflammatory events. A previous study (23) also reported that CLA decreased PGE2 production by interfering with the synthesis of arachidonic acid from linoleic acid. However, the anti-inflammatory effect of CLA via suppressing the synthesis of proinflammatory cytokines (i.e., IL-1ß, IL-6, and TNF-) seems not to be related to the decrease in PGE2 that suppresses the production of IL-1ß, IL-6, TNF-, IL-2, and interferon- (24). This suggests that CLA regulates the production of IL-1ß, IL-6, and TNF- through other modulatory pathways.
In the present study, we observed that PPAR expression was upregulated and the synthesis of IL-6 and TNF- were decreased simultaneously in CLA-fed pigs after an inflammatory challenge. Furthermore, PPAR expression in mononuclear cells cultured in media containing 10t,12c-CLA was also greater than that cultured in media containing 9c,11t-CLA in vitro. Therefore, we speculated that the modulatory effect of CLA on the synthesis of proinflammatory cytokines is a PPAR-dependent mechanism. Study showed that activation of PPAR in the colon inhibits mucosal production of IL-1ß and TNF- by downregulation of the nuclear factor-
B and mitogen-activated protein kinase signal pathways (25). In addition, the anti-inflammatory effects of the PPAR agonists, rosiglitazone or ciglitazone, were abolished by the PPAR antagonists, bisphenol A diglycidyl ether (26) and GW9660 (27).
The increased production of 15d-PGJ2 in cultured mononuclear cells further demonstrated that the modulatory effect of CLA on proinflammatory cytokines is related in part to a PPAR-dependent mechanism. 15d-PGJ2 is the key end metabolite of PGD2 and is a natural high-affinity ligand for PPAR. Recent studies suggested that 15d-PGJ2 has activity as an agonist of PPAR (28). Furthermore, CLA also is a potent activator of PPARs (23), and CLA has structural and physiologic characteristics similar to the PPAR ligand peroxisome proliferators (29). CLA was shown to activate PPAR and decrease production of proinflammatory products in mice macrophages (30). Here, we showed that after an increase in 15d-PGJ2 levels in cultured PBMC supernatant from 9c,11t-CLA, 10t,12c-CLA, or the 1:1 CLA mixture culture media, PPAR activation and mRNA expression were enhanced, whereas the synthesis of cytokines levels was reduced. These results suggest that the activation and expression of PPAR are associated in part with 15d-PGJ2 production or CLA supplementation. However, the link between the action of CLA and 15d-PGJ2 synthesis has not been reported and the exact mechanisms of CLA regulating 15d-PGJ2 production are not known. Future investigations should be conducted to evaluate the exact mode of action of CLA on PPAR, including using a PPAR antagonist or knocking out or down the expression of mRNA for PPAR in the experimental design.
The responses in pigs’ performance and the change in inflammation response to the first LPS challenge were more obvious than they were for the second challenge. The different responses may be related to a tolerance phenomenon (31). However, the production of cytokines on d 21 was lower than on d 14, indicating that the inhibitory effect of CLA on the modulation of the inflammatory response has a greater effect over time. Furthermore, the concentrations of IL-1ß, IL-6, TNF-, and PPAR in spleen were higher than those in thymus, indicating that their expression is tissue dependent in the immune system. There is little information concerning CLA and isomer specificity in the regulation of proinflammatory cytokine production induced by LPS challenge. Further study should also be designed to investigate the tissue-targeted effect of CLA and individual CLA isomers.
In conclusion, the supplementation of CLA suppresses the inflammation response in LPS-challenged pigs, and especially inhibits the synthesis of proinflammatory cytokines at both the protein and mRNA levels. The modulatory mechanism by which CLA reduces the production of proinflammatory cytokines is in part via activation of the PPAR pathway.
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FOOTNOTES |
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3 Abbreviations used: BSA, bovine serum albumin; CLA, conjugated linoleic acid; 9c,11t-CLA, cis-9, trans-11 CLA; 10t,12c-CLA, trans-10, cis-12 CLA; 15d-PGJ2, 15-deoxy-12,14-prostaglandin J2; IL, interleukin; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cell; PGE2, prostaglandin E2; PPAR-, peroxisome proliferator-activated receptor-; TNF, tumor necrosis factor.
Manuscript received 16 August 2004. Initial review completed 1 September 2004. Revision accepted 7 October 2004.
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