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Infection and Immunity, June 2007, p. 2886-2893, Vol. 75, No. 6
0019-9567/07/$08.00+0     doi:10.1128/IAI.01431-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Fatty Acids Isolated from Toxoplasma gondii Reduce Glycosylphosphatidylinositol-Induced Tumor Necrosis Factor Alpha Production through Inhibition of the NF-B Signaling Pathway

Françoise Debierre-Grockiego,^1* Khamran Rabi,¹ Jörg Schmidt,¹ Hildegard Geyer,² Rudolf Geyer,² and Ralph T. Schwarz^1,3

Institut für Virologie, AG Parasitologie, Hans-Meerwein-Str. 2, D-35043 Marburg, Germany,¹ Institut für Biochemie, Friedrichstrasse 24, D-35392 Giessen, Germany,² Unité de Glycobiologie Structurale et Fonctionnelle UMR CNRS/USTL 8576 - IFR 118, F-59655 Villeneuve D'Ascq, France³

Received 6 September 2006/ Returned for modification 24 October 2006/ Accepted 9 March 2007

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Glycosylphosphatidylinositols (GPIs) are involved in the pathogenicity of protozoan parasites and are known to induce inflammatory cytokines. However, we have previously shown that the family of six GPIs of Toxoplasma gondii extracted together from tachyzoites could not induce tumor necrosis factor alpha (TNF-) secretion by macrophages, whereas GPIs individually separated from this extract by thin-layer chromatography (TLC) were able to stimulate the cells. In the present study we show that the TLC step makes it possible to eliminate inhibitors extracted together with the T. gondii GPIs. Among the non-GPI molecules we have isolated fatty acids able to inhibit the secretion of TNF- induced by the T. gondii GPIs. Myristic and palmitic acids reduce the production of TNF- through the inhibition of tyrosine phosphorylation of cytoplasmic proteins and the inhibition of NF-B activation in a peroxisome proliferator-activated receptor-independent pathway and after a rapid entry into the cytoplasm of macrophages. GPIs are considered toxins inducing irreversible damage in the host, and fatty acids produced in parallel by the parasite could reduce the immune response, thus favoring the persistence of parasite infection.

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Glycosylphosphatidylinositols (GPIs) constitute a class of glycolipids that have various functions, the most fundamental being to link proteins to eukaryotic cell membranes. GPIs are involved in the pathogenicity of protozoan parasites and are known to induce tumor necrosis factor alpha (TNF-) production that is reversed by antibodies raised against GPIs (34, 35, 41). We have shown that GPIs purified from Toxoplasma gondii tachyzoites induce TNF- production in macrophages (10). To be sure that no contaminating molecules such as bacterial compounds were responsible for this stimulation, the absence of endotoxin was checked. The specific effect of GPIs was confirmed further by using a chemically synthesized GPI of T. gondii (10). A chloroform-methanol-water mixture that extracts polar lipids was used to extract GPIs. We have previously shown that the chloroform-methanol-water extract of T. gondii glycolipids was unable to induce TNF- production by macrophages (10). Thus, a phase partition between water and water-saturated n-butyl alcohol was performed to further purify GPIs. The family of the six free GPIs of T. gondii was recovered in the n-butyl alcohol phase, which again could not induce TNF- secretion by macrophages. However, after separation of the n-butyl alcohol phase by thin-layer chromatography (TLC), each individual GPI was able to stimulate the cells (10). We have shown in a recent study that phospholipids and other hydrophobic components were recovered by the solvents used to extract GPIs (2). These contaminants had high R_f values (0.63 to 0.99) that were different from the T. gondii GPIs with lower R_f values (0.2 to 0.6) that were separated after TLC. The low solubility of GPIs in water-saturated n-butyl alcohol permitted complete removal of the contaminants (2). The n-butyl alcohol phase was dried under a stream of nitrogen until a white precipitate was formed and could be separated from the supernatant by centrifugation. The white precipitate contained all of the GPIs devoid of contaminants, whereas the supernatant contained cholesterol, triglyceride, sphingomyelin, phospholipids, and other components. The discovery of contaminating molecules extracted with T. gondii GPIs raised the hypothesis that the TNF- production could have been inhibited by one or more molecules present in the chloroform-methanol-water extract (9). We have previously shown that fatty acids isolated from Plasmodium falciparum are able to inhibit TNF- production induced by the malarial toxin, GPI Pf, in macrophages (11). Here we show that fatty acids present in T. gondii tachyzoites are also able to reduce the production of TNF- induced by T. gondii GPIs. In addition, we demonstrate that these fatty acids exert their inhibitory action at an intracellular level through inhibition of the signal pathway leading to NF-B transcription factor activation in a peroxisome proliferator-activated receptor (PPAR)-independent manner.

(A part of this study is presented as a fulfillment of the doctoral thesis in medicine of K. Rabi.)

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Materials. [³H]Glucosamine hydrochloride (25 Ci/mmol) was purchased from Hartmann Analytic GmbH (Braunschweig, Germany). Myristic, palmitic, stearic, and oleic acids were obtained from Sigma (Deisenhofen, Germany). All solvents used were of analytical or high-performance liquid chromatography grade and were obtained from Riedel-de Haen (Seelze, Germany).

Extraction and purification of GPIs. Cultures of T. gondii tachyzoites (strain RH) grown in Vero cells (free of Mycoplasma) were washed twice with glucose-free Dulbecco modified Eagle medium containing 20 mM sodium pyruvate. GPI labeling was performed using the same medium supplemented with 0.5 mCi [³H]glucosamine for 4 h at 37°C in 5% CO₂ atmosphere. Tachyzoites were released from host cells with the help of glass beads and the Mixer Mill homogenizer (Retsch, Haan, Germany) and purified by glass wool filtration (14). The purity of the tachyzoite suspension was monitored microscopically. GPIs from labeled and unlabeled cultures were extracted according to the method of Menon et al. (27) as described previously. Briefly, glycolipids were extracted with chloroform-methanol-water (10:10:3, by volume) and partitioned between water and water-saturated n-butyl alcohol. GPIs and non-GPI contaminants present in n-butyl alcohol were separated by precipitation of GPIs under a stream of nitrogen as described by Azzouz et al. (2). The six different T. gondii GPIs (GPI I to GPI VI [39]) were then separated by TLC, with [³H]glucosamine metabolically labeled T. gondii GPIs used as tracers. Chromatograms were scanned for radioactivity, and areas corresponding to individual T. gondii GPIs were scraped off, re-extracted with chloroform-methanol-water (10:10:3, by volume) by sonication (Branson 3200, 47 MHz; Branson Ultrasonics Corp., Danbury, CT), and recovered in the n-butyl alcohol phase after water-saturated n-butyl alcohol-water partition.

Purification of lipid classes. The lipid classes of T. gondii tachyzoites were extracted as previously described (11) by using aminopropyl-bonded silica gel (LC-NH₂), weak cation-exchanger (LC-WCX) cartridges, and different solvents (6): fraction 1 (cholesterol, cholesteryl esters, triglycerides, diglycerides, fatty alcohols, fatty acid methyl esters) eluted with hexane-ethyl acetate (17:3, by volume), fraction 2 (cholesterol, monoglycerides, free ceramides, N-methyl derivatives of sphingoid bases) eluted with chloroform-methanol (23:1, by volume), fraction 3 (normal and -hydroxy free fatty acids) eluted with di-isopropyl ether-acetic acid (98:5, by volume), fraction 4 (ceramide mono-, di-, tri-hexosides, globoside) eluted with acetone-methanol (9:1.35, by volume) and chloroform-methanol (9:3, by volume), fraction 5 (sphingosine, dihydrosphingosine, phytosphingosine) eluted with 1 N acetic acid in methanol, and fraction 6 (sphingomyelin, phosphatidylcholine, lysolecithin, lysophosphatidylcholine, lecithin, phosphatidylethanolamine) eluted with chloroform-methanol (2:1, by volume). All solvents were dried under a stream of nitrogen, and molecules were stored in chloroform-methanol-water (40:30:4).

TLC. The third fraction containing fatty acids was separated by TLC using the solvent system chloroform-methanol (50:2, by volume). Unsaturated fatty acids that were visualized by staining with iodine vapor and silica were scraped off in eight parts according to the positions of the yellow spots. Lipids of each scraping were extracted with chloroform-methanol-water (10:10:3, by volume) by sonication, dried, and individually tested on macrophages in the presence of GPIs. The fraction presenting a biological effect was submitted to a second TLC using the solvent system chloroform-methanol- 2 M aqueous ammonia (65:25:4, by volume). Unsaturated fatty acids were detected with iodine, and silica were scraped off in eight parts. Lipids were extracted from the silica with chloroform-methanol-water (10:10:3, by volume) by sonication, and each part was individually tested on macrophages.

Fatty acid analysis. Fatty acids were released as methyl ester derivatives by treatment with 500 µl of 1 M HCl and 10 M H₂O in methanol for 16 h at 100°C according to the method of Gaver and Sweeley (13). Free fatty acids were converted into methyl esters with diazomethane (5). Fatty acid methyl esters were recovered by a threefold phase partition using n-hexane and analyzed by gas chromatography-mass spectrometry using a VF 5ms capillary column (60 m, 0.25-mm inner diameter, 0.1-µm film thickness; Varian, Darmstadt, Germany) and 2.5 ml of helium/min as the carrier gas. The temperature was maintained at 50°C for 2 min, raised to 130°C with 40°C/min and to 300°C with 6°C/min, and was finally maintained at 300°C for 5 min. Fatty acid methyl esters were registered by mass spectrometry in the positive ion mode after chemical ionization with methanol using a PolarisQ instrument (Thermo Finnigan, Egelsbach, Germany).

TNF- production by macrophages. RAW 264.7 macrophages were diluted to a concentration of 10⁶ cells/ml with the serum free medium Panserin 401 (Pan Biotech GmbH, Aidenbach, Germany), and 0.2-ml aliquots were dispensed into the wells of a 96-well plate. The cells were allowed to adhere for 3 h, washed, and incubated at 37°C in 5% CO₂ atmosphere for 24 h with medium containing the molecules to test. Absence of endotoxin was checked in all molecules with the Limulus amebocyte lysate kit QCL-100 (Bio-Whittaker, Walkersville, MD). The amount of GPIs and sphingolipid classes needed for one experiment was dried under a stream of nitrogen to remove the solvent. The culture medium was added, and molecules were resuspended in this medium by sonication. The molecules tested for their potential inhibitory effect were added 30 min before GPIs. For the PPAR inhibition assay, GW9662 (Calbiochem, Darmstadt, Germany) was added at 2 µM 30 min or 12 h prior to T. gondii fatty acids (fraction 3), which had been added 30 min prior to T. gondii GPIs (from 10⁸ tachyzoites). Macrophages were incubated with medium alone for negative control and with lipopolysaccharide (LPS) at 200 ng/ml (from Escherichia coli serotype O55:B5; Sigma) for positive control. Measurement of TNF- levels in the macrophage culture supernatants was performed by using a specific sandwich enzyme-linked immunosorbent assay (BD Biosciences, San Jose, CA).

Immunofluorescence. The fluorescent hexadecanoic acid BODIPY FL C₁₆ (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid, Molecular Probes, Inc., Eugene, OR) was added to macrophages at 50 µM in culture medium with or without T. gondii GPIs (from 10⁸ tachyzoites) during 15 min and 1 h at 37°C. Cells were washed two times with phosphate-buffered saline (PBS), fixed during 30 min with 4% paraformaldehyde in PBS, and washed three times with PBS. Fluorescence was observed with a Zeiss AxioPhot microscope (Carl Zeiss MicroImaging GmbH, Göttingen, Germany).

NF-B p65 transcription factor assay. RAW 264.7 macrophages were incubated for 30 min with medium alone, with T. gondii fatty acids (fraction 3 isolated from 4 x 10⁸ tachyzoites), or with 20 µM myristic and palmitic acids together. Cells were further incubated for 30 min with medium alone or T. gondii GPIs (isolated from 2 x 10⁸ tachyzoites). Nuclear proteins (50 µg) were analyzed for NF-B activation, using the Trans-AM NF-B p65 transcription factor assay kit, according to the manufacturer's protocol (Active Motif, Rixensart, Belgium). Recombinant NF-B protein was used for the standard curve (Active Motif).

Expression of TNF- mRNA. RAW 264.7 macrophages (5 x 10⁵) were incubated for 30 min with medium alone or with 10 µM myristic acid and palmitic acid together. Cells were further incubated for 20 h with medium alone or T. gondii GPIs isolated from 2 x 10⁸ tachyzoites. Isolation of total macrophage RNA was performed with an RNeasy minikit (QIAGEN, Hilden, Germany), and the material was treated with RNase-free DNase (New England Biolabs, Ipswich, MA). Conversion of RNA to cDNA was performed with an Omniscript reverse transcription system (QIAGEN). Synthesis of cDNA was done at 37°C for 60 min in a final volume of 20 µl containing reverse transcription buffer, 0.5 mM of the deoxynucleoside triphosphate mix, 10 U of RNase inhibitor (Invitrogen, Carlsbad, Germany), 4 U of Omniscript reverse transcriptase, 1 µM oligo(dT) primer, and 1.5 µg of total RNA. Real-time PCR was performed by using the LightCycler 2.0 (Roche Diagnostics, Mannheim, Germany) in 20-µl microcapillary tubes containing 12.4 µl of water, 1.6 µl (3 mM) of MgCl₂, 2 µl (0.5 µM) of forward (CCA-CGT-CGT-AGC-AAA-CCA-C) and reverse (TGG-GTG-AGG-AGC-ACG-TAG-T) TNF- primers (Tib Molbiol Syntheselabor, Berlin, Germany), 2 µl of LightCycler DNA master SYBR green I mix (Roche), and 2 µl of cDNA product. The real-time PCR protocol was composed by a denaturation program (10 min at 95°C), an amplification program of 45 cycles (10 s at 95°C, 10 s at 60°C, and 7 s at 72°C), a melting curve program (60 to 95°C with a heating rate of 0.1°C/s), and a cooling program down to 40°C. The threshold cycle (C_T) value, i.e., the cycle number when the fluorescence exceeds baseline, was used to calculate the amount of PCR product and the fragment size (189 bp) was checked on a 2% agarose gel stained with ethidium bromide (DNA molecular weight from Fermentas, St. Leon-Rot, Germany). The optical density of the bands was measured by using TINA 2.09 software (Raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany).

Determination of protein tyrosine phosphorylation by Western blot. RAW 264.7 macrophages (10⁶) were preincubated during 30 min with medium alone, with T. gondii fatty acids (fraction 3 isolated from 4 x 10⁸ tachyzoites), or with 20 µM myristic acid and palmitic acid together and then further incubated for 1 h with medium alone or with GPIs purified from 2 x 10⁸ tachyzoites. Cells were then centrifuged at 1,000 x g for 5 min. The cells were lysed in 20 mM Tris-HCl (pH 8.0), 1% Triton X-100, 1 mM sodium o-vanadate, 10% glycerol, 137 mM NaCl, 1 mM EDTA, and protease inhibitors cocktail {Sigma, final concentrations: 0.5 mM EDTA, 1 mM AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride], 65 µM bestatin, 0.7 µM E-64, 0.5 µM leupeptin, 0.15 µM aprotinin}. The cells were placed on ice for 20 min and centrifuged (10,600 x g for 15 min at 4°C). Cell lysates were mixed (vol/vol) with sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2.3% sodium dodecyl sulfate, 100 mM dithiothreitol, and 0.005% bromophenol blue and boiled for 5 min. The samples were then subjected to sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis, electrophoretically transferred to nitrocellulose membrane (GE Healthcare, Piscataway, NJ), and incubated with 2 µg of horseradish peroxidase-conjugated mouse monoclonal anti-phosphotyrosine 4G10 antibody (Upstate, Lake Placid, NY)/ml. Enhanced chemiluminescence reagents (GE Healthcare) were used for the detection of peroxidase activity. The optical density of the proteins was measured by using TINA 2.09 software (Raytest Isotopenmessgeräte GmbH).

Statistics. An unpaired Student t test was used for statistical evaluation, and a P value of <0.05 was considered significant.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Inhibition of GPI-induced TNF- by T. gondii fatty acids. To determine whether inhibitory fatty acids exist in Toxoplasma, we extracted lipid classes from the parasites by using aminopropyl-bonded silica gel (LC-NH₂), as well as weak cation-exchanger (LC-WCX) cartridges and different solvents. Six fractions containing neutral lipids, free ceramides, fatty acids, neutral glycosphingolipids, free sphingoid bases, and sphingomyelin, respectively, were obtained and individually tested. None of the six fractions separated from T. gondii induced TNF- production by macrophages (Fig. 1A). The six fractions were then tested in the presence of T. gondii GPIs. When the six fractions were tested in the presence of the GPIs, only the fraction 3 containing fatty acids inhibited the TNF- secretion (Fig. 1B, fraction T3). The inhibitory effect of the fraction T3 was obtained for each type of GPI tested among the six different GPIs isolated from T. gondii tachyzoites (GPI I to GPI VI) (39). The inhibitory effect of fraction T3 was not due to cell death as the viability of macrophages was checked microscopically in the presence of trypan blue and found unaltered (not shown).

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FIG. 1. The fraction T3 containing fatty acids inhibits the T. gondii GPI-induced TNF- production. (A) Macrophages were incubated for 24 h with medium alone (M) or with the six different fractions T1 to T6 isolated from 107 and 108 T. gondii tachyzoites by using aminopropyl-bonded silica gel (LC-NH2) and weak cation exchanger (LC-WCX) cartridges. LPS at 200 ng/ml (L) was used as a positive control. (B) Macrophages were incubated for 24 h with medium alone (M), GPI alone (from 108 T. gondii tachyzoites) or with the six fractions T1 to T6 (from 2 x 108 T. gondii tachyzoites) added 30 min before GPI was added. Values represent the means ± the standard deviations for triplicate samples of a representative experiment.

Identification of inhibitory fatty acids. To know which fatty acid in the panel was responsible for the inhibitory effect, TLC performed with the solvent system chloroform-methanol (50:2, by volume) was used to subfractionate the fraction T3. Iodine vapors were used to stain the unsaturated fatty acids. The whole surface was scraped off in eight parts, lipids were extracted from the silica, and a part of each sample was tested on macrophages in the presence of GPIs. The second area inhibited GPI-induced TNF- production by macrophages (Fig. 2A). This contained unsaturated fatty acids detected in a large yellow spot with iodine, suggesting a mixture of several molecules. A second TLC was done with the rest of this inhibitory fraction to further separate the fatty acids. The solvent system chloroform-methanol- 2 M aqueous ammonia (65:25:4, by volume) was used, and the unsaturated fatty acids were again stained by iodine vapors. Once again, eight areas were scraped off and tested on macrophages after extraction of the lipid compounds. The last fraction had the inhibitory effect (Fig. 2B). Its analysis by GC-MS revealed 4 major fatty acids, which were assigned as myristic acid (C_14:0), palmitic acid (C_16:0), oleic acid (C_18:1), and stearic acid (C_18:0) according to standard fatty acids (Fig. 2C). To finally discriminate between these four molecules, commercially available fatty acids were tested on macrophages stimulated by T. gondii GPIs. Among the molecules tested, only myristic acid and palmitic acid were able to reduce the production of TNF- in response to T. gondii GPIs in macrophages (Fig. 3). When both fatty acids were added together, the TNF- inhibition was stronger (Fig. 3E).

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FIG. 2. Isolation of the inhibitory fatty acids by TLC. (A) Macrophages were incubated for 24 h with medium alone (M []), with GPI alone (from 108 T. gondii tachyzoites []), or with the eight different fractions T3/1 to T3/8 obtained after separation of the fraction T3 by TLC (from 2 x 108 T. gondii tachyzoites) and added 30 min before GPI was added (). (B) Macrophages were incubated for 24 h with medium alone (M []), with GPI alone (from 108 T. gondii tachyzoites []), or with the eight fractions T3/2/1 to T3/2/8 obtained after separation of the fraction T3/2 by TLC (from 2 x 108 T. gondii tachyzoites) and added 30 min before GPI was added (). Values represent the means ± the standard deviations of triplicate samples of a representative experiment. (C) Analysis of fatty acids derived from T. gondii. Fatty acid methyl esters were separated by capillary gas chromatography and registered by mass spectrometry after chemical ionization. The upper profile shows fatty acids found in the fraction T3/2/8; the lower profile shows standard fatty acid methyl esters. Numbers: 1, C14:0; 2, C15:1; 3, C15:0; 4, C16:1; 5, C16:0; 6, C17:1; 7, C17:0; 8, C18:1; 9, C18:1 (trans isomer); 10, C18:0; 11, C20:1; 12, C20:0; 13, C21:0; 14, C22:1; 15, C22:0; 16, C23:0; 17, C24:1; 18, C24:0.

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FIG. 3. Myristic acid and palmitic acid inhibit GPI-induced TNF- production. Macrophages were incubated for 24 h with medium alone, with GPI alone (from 108 T. gondii tachyzoites), or with myristic acid (A), palmitic acid (B), oleic acid (C), or stearic acid (D) at 1 or 10 µM, added 30 min before the GPI was added. (E) Myristic acid and palmitic acid (10 µM) were added together 30 min before the GPI was added. Values represent the means ± the standard deviations of triplicate samples of a representative experiment. *, P < 0.01; **, P < 0.02.

Palmitic acid crosses the macrophage plasma membrane. In all experiments, inhibitory molecules were added 30 min prior to the stimulatory GPIs. Therefore, it was interesting to explore the time-dependent cell response. Due to the limited availability of purified material, the whole fraction T3 was used in all of the following experiments. As shown in Fig. 1B, fatty acids added 30 min prior to the GPIs reduced the TNF- production (Fig. 4A). In contrast, fatty acids added simultaneously with the GPIs were unable to inhibit it (Fig. 4A). Protozoan GPIs have been shown to activate cells through the Toll-like receptors TLR2 and TLR4 (7, 20). The fact that fatty acids must be added 30 min prior to GPIs suggested a competition for a surface receptor such as TLR. In order to visualize the binding of fatty acids at the cell surface, a fluorescent palmitic acid (BODIPY FL C₁₆) was used. In contrast to our hypothesis, this fatty acid did not localize at the surface but in the cytoplasm of macrophages (Fig. 4B), indicating its entrance through the plasma membrane. Thus, fatty acids were not in competition with GPIs for a surface receptor, but the presence of GPIs could perturb fatty acid penetration. To evaluate this possibility, T. gondii GPIs were added to macrophages simultaneously with the fluorescent palmitic acid. As predicted, the intracellular fluorescence was strongly reduced at both 15 min and 1 h of incubation (Fig. 4B). This suggests that GPIs block the access of fatty acids to the plasma membrane or form complexes with them.

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FIG. 4. Fatty acids require to be added before GPI to inhibit the TNF- production. (A) Macrophages were incubated for 24 h with medium alone (open bar), with GPI alone (from 108 T. gondii parasites, black bar), or with fraction T3 (from 2 x 108 tachyzoites) added simultaneously (0', dark gray bar) or 30 min (30', clear gray bar, P = 0.0062) before the addition of GPI. (B) Macrophages were incubated for 15 min (15') or 1 h (1 h) with fluorescent palmitic acid alone (FL C16) or with fluorescent palmitic acid and GPI (from 109 T. gondii parasites) simultaneously (GPI + FL C16).

T. gondii fatty acids do not inhibit GPI activity through PPAR. A remaining issue concerned the mechanism by which the T. gondii fatty acids triggered the intracellular signaling in the host cells. A hypothesis was that fatty acids signal via PPARs, which control a variety of genes in several pathways of lipid metabolism. Among the large amount of endogenous and exogenous PPAR ligands are fatty acids, such as palmitic acid, oleic acid, linoleic acid, and arachidonic acid (3, 18). Saturated short-chain fatty acids (shorter than C₁₀) were poorer activators of PPAR than longer-chain fatty acids (C₁₀ to C₁₆) (12). A better association of fatty acids with PPAR is observed in the presence of unsaturated acyl chains (26). Polyunsaturated and monounsaturated fatty acids have a preference for PPAR, followed by PPAR and PPARβ (19). PPAR ligation leads to inhibition of gene expression in part by antagonizing the activities of the transcription factors AP-1, STAT, and NF-B (31). A polyunsaturated fatty acid, docosahexaenoic acid, inhibits TLR4 agonist (LPS)-induced upregulation of CD40, CD80, and CD86 expression and inflammatory cytokine production in dendritic cells (43). To study the involvement of PPAR in T. gondii fatty acids signaling, we have used a cell-permeable, selective and irreversible PPAR antagonist, GW9962 (25). Since the 50% inhibitory concentration of GW9962 is 3.3 nM, 32 nM, or 2 µM for PPAR, PPAR, or PPAR, respectively, the concentration chosen here was 2 µM. GW9962 was added to macrophages 30 min prior to the T. gondii fatty acids (fraction T3), followed by T. gondii GPIs after 30 min. As shown in Fig. 5A, the PPAR antagonist did not block the inhibitory effect of fatty acids on GPI-induced TNF- production. To be sure that the incubation time was not a limiting factor, GW9962 was given to the cells during 12 h before fatty acids. However, this long period of preincubation did not change the result, and fatty acids were still able to inhibit the TNF- production in response to GPIs (Fig. 5B). GW9962 did not induce TNF- in macrophages and had no effect on GPI-induced TNF- secretion (Fig. 5).

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FIG. 5. T. gondii fatty acids do not inhibit GPI activity through PPAR activation. (A) Macrophages were incubated with the PPAR specific inhibitor GW9662 for 30 min prior to fatty acids (fraction T3 from 2 x 108 T. gondii tachyzoites) added 30 min prior to T. gondii GPI (from 108 T. gondii tachyzoites). (B) Macrophages were incubated with GW9662 for 12 h prior to fatty acids (fraction T3 from 2 x 108 T. gondii tachyzoites) added 30 min prior to GPI (from 108 T. gondii tachyzoites).

T. gondii fatty acids reduce GPI-induced NF-B activation and protein phosphorylation. We have previously shown that T. gondii GPIs activate NF-B and its nuclear translocation in macrophages, suggesting the involvement of NF-B in TNF- gene expression (10). PPAR ligands inhibit NF-B by PPAR-dependent and PPAR-independent mechanisms. For example, fatty acids reduce interleukin-12 (IL-12) production induced by IFN- and LPS in macrophages in a PPAR-independent pathway (44). To determine whether NF-B is involved in the inhibition of TNF- production in response to the fatty acids of T. gondii, NF-B activation was measured in macrophages in response to T. gondii GPIs alone or with fatty acids. Cells were stimulated 30 min with or without T. gondii fatty acids (fraction T3 or commercial fatty acids) prior to further 30 min of incubation with T. gondii GPIs. The proteins of macrophages were extracted and tested in a NF-B assay. As previously shown (10), very low basal activity of NF-B was observed in absence of GPI stimulation, whereas activation of NF-B was observed in nuclear extract of macrophages stimulated during 30 min with T. gondii GPIs (Fig. 6A). Preincubation of macrophages with T. gondii fatty acids (P = 0.003), as well as with a mixture of myristic acid and palmitic acid (P = 0.03), reduced the GPI-induced NF-B activation (Fig. 6A). To evaluate the impact of the reduction of NF-B activation on the TNF- production, the amounts of TNF- mRNA were estimated by real-time PCR. Macrophages were stimulated 30 min with or without myristic acid plus palmitic acid and incubated for 20 h with T. gondii GPIs. Total mRNA was isolated and subjected to real-time PCR with primers specific for TNF- after a reverse transcription step as described in Materials and Methods. The PCR product size of 189 bp was checked on agarose gel. In cells incubated with medium alone TNF- was slightly transcripted (Fig. 6B) (LightCycler C_T value of 17.3). Stimulation of macrophages with T. gondii GPIs induced a strong transcription of TNF- (LightCycler C_T value of 14.0; Fig. 6B, optical density in lane 2/optical density in lane 1 = 7.5), whereas preincubation with myristic acid and palmitic acid together reduced this upregulation (LightCycler C_T value of 15.4; Fig. 6B, optical density in lane 3/optical density in lane 2 = 0.65). This confirms that the reduction of NF-B activation correlates with the reduction of TNF- expression. In order to answer the question of whether the inhibition of NF-B by fatty acids resulted in an upstream inhibition in the signal pathway, tyrosine phosphorylation of proteins was studied in macrophages. Cells were stimulated for 30 min with or without fatty acids (fraction T3 isolated from T. gondii or commercial fatty acids) prior to incubation with T. gondii GPIs during 1 h. The cytoplasmic proteins of macrophages were extracted, and tyrosylphosphorylation was detected by Western blotting. GPI-treated macrophages (Fig. 6C, lane 2) showed an increase in the level of constitutively phosphorylated substrates (Fig. 6C, lane 1) with apparent molecular masses of 72, 82, 110, and 120 kDa, with fold increases of 1.05, 1.03, 1.15, and 1.03, respectively. Pretreatment of macrophages with myristic acid and palmitic acid together (Fig. 6C, lane 3) abolished this elevation in protein tyrosine phosphorylation (optical density in lane 3/optical density in lane 2 = 0.90, 0.88, 0.61, and 0.80 for 72, 82, 110, and 120 kDa, respectively). A similar inhibition was observed with the fraction T3 containing T. gondii fatty acids (not show). This result shows that fatty acids exert their inhibitory effect through the inhibition of the signal pathway leading to NF-B activation.

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FIG. 6. Fatty acids inhibit GPI-induced NF-B activation, TNF- gene transcription, and protein tyrosine phosphorylation. (A) Macrophages were incubated for 1 h with medium alone (), for 30 min with medium followed by 30 min with GPI (from 2 x 108 T. gondii tachyzoites []), for 30 min with T. gondii fatty acids (fraction T3 from 4 x 108 tachyzoites) followed by 30 min with GPI (), or with myristic acid and palmitic acid together (M-P, 20 µM) followed by 30 min with GPI (). Nuclear proteins were extracted and tested using an NF-B p65 activation kit. (B) Macrophages were incubated for 20 h with medium alone, for 30 min with medium followed by 20 h with GPI (from 2 x 108 T. gondii tachyzoites), or for 30 min with myristic acid and palmitic acid (M-P) together (20 µM) followed by 20 h with GPI. TNF- transcription was determined by real-time PCR by using the LightCycler 2.0 and the LightCycler DNA master SYBR green I kit. PCR products (189 bp) were visualized on agarose gel. (C) Macrophages were incubated for 90 min with medium alone, for 30 min with medium followed by 1 h with GPI (from 2 x 108 T. gondii tachyzoites), or for 30 min with myristic acid and palmitic acid (M-P) together (20 µM) followed by 1 h with GPI. Cytoplasmic proteins were extracted, and protein tyrosine phosphorylation was tested by Western blotting with the anti-phosphotyrosine 4G10 antibody. The immunoreactive bands were visualized by enhanced chemiluminescence (right panel). Ponceau red staining shows that equivalent amounts of proteins were loaded (left panel).

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In this study, we show that fatty acids from T. gondii are able to inhibit the TNF- production induced by T. gondii GPIs in macrophages. After identification of myristic acid, palmitic acid, oleic acid, and stearic acid in the inhibitory fraction, use of standards has shown that myristic acid and palmitic acid present an inhibitory activity. Concerning the mechanism used by the fatty acids to exert their inhibitory effect on macrophages, we first hypothesized a competition for a T. gondii GPI receptor such as TLR. This assumption was supported by several studies performed by Lee et al. demonstrating that saturated fatty acids activate TLR2 and TLR4 (21-24). However, the use of fluorescent palmitic acid revealed that the fatty acids did not localize at the macrophage cell surface, arguing against its binding to a surface receptor or coreceptor. Fluorescence was observed in the cytoplasm of macrophages as soon as after 1 min (not shown), indicating a rapid entrance through the plasma membrane. Different mechanisms can be involved in fatty acid penetration, and the most simple would be a passive transmembrane diffusion. Flip-flop of free fatty acids from the outer to the inner leaflet of the phospholipid bilayer is very fast (16). Although membrane proteins are not required for transmembrane transport of free fatty acids, fatty acid binding plasma protein (FABPpm), fatty acid transport protein (FATP), or fatty acid transporter (FAT/CD36) are potentially responsible for lipid acquisition (15, 33, 38).

After having shown that fatty acids localize in target cell cytoplasm, we thought that the inhibitory mechanism might involve receptors of the PPAR family. Indeed, activation of PPAR by a synthetic ligand (troglitazone) reduced RANTES, macrophage inflammatory protein 1, IL-10, and IL-12 secretion induced by LPS in dendritic cells (1). These inhibitory effects on TLR4-induced dendritic cell activation were mediated via inhibition of the NF-B and mitogen-activated protein kinase pathways while not affecting the phosphatidylinositol 3-kinase/Akt signaling. The natural ligand of PPAR, 15d-PGJ₂, inhibited the phosphorylation of ERK1, as well as p38 (1). Docosahexaenoic acid inhibits TLR4 agonist (LPS)-induced upregulation of the costimulatory molecules, major histocompatibility complex class II, and cytokine production (43). Furthermore, the anti-inflammatory effects of fish oil may result from the inhibitory effects of oxidized omega-3 fatty acids on NF-B activation via a PPAR-dependent pathway (28). To demonstrate that PPAR family might be responsible for the inhibitory effect of T. gondii fatty acids on the GPI-induced signaling, we used a specific PPAR antagonist. In contrast to our expectation, the specific PPAR inhibitor GW9962 did not suppress the inhibitory activity of fatty acids. Reduced phosphorylation of pp42 in 15d-PGJ₂-treated dendritic cells was apparent 15 min after incubation with the TLR ligand (1). Thus, the incubation time of 30 min could be not sufficient for GW9962 to antagonize PPAR. We decided to prolong the incubation time to 12 h, but the result was unchanged. This indicated that PPAR are not involved in the inhibition of GPI signaling by T. gondii fatty acids.

Other studies indicate that some PPAR ligands induce anti-inflammatory response in a PPAR-independent pathway (17, 29). Although the natural PPAR ligand 15d-PGJ₂ is a potent inhibitor of TNF- and IL-6 secretion in LPS-stimulated macrophages, this activity is not diminished in PPAR-deficient macrophages, indicating that alternative signaling pathways are involved (9). These observations are consistent with the findings that 15d-PGJ₂ significantly inhibited LPS-induced degradation of IB and NF-B DNA binding without inhibiting nuclear entry of NF-B and that anti-inflammatory cyclopentenone prostaglandins directly inhibit and modify the IKKβ subunit of IKK (32, 37). In agreement with these data, we found that T. gondii fatty acids were able to inhibit the NF-B activation due to GPIs in macrophages, resulting in a reduction of the TNF- gene transcription. Our experiment with fluorescent palmitic acid showed that it did not enter the nucleus even after 1 h of incubation, indicating that the reduction of TNF- expression is not due to the inhibition of the binding of NF-B onto its promoter consensus sequence. Our study on tyrosine phosphorylation of cytoplasmic proteins gives the first evidence that T. gondii GPIs are able to activate tyrosine kinases in macrophages. The core glycan of P. falciparum GPI induces activation of the protein tyrosine kinase p59^hck in macrophages, whereas GPI-associated diacylglycerols activate the protein kinase C (40). Both signals collaborate in regulating the downstream NF-B-dependent gene expression of IL-1, TNF-, and inducible NO synthase (40). Although macrophages express surface phospholipases capable of cleaving T. gondii GPIs (20), further experiments are needed to establish whether the core glycans and the diacylglycerols of T. gondii GPIs induce two independent intracellular signals. Our study shows that fatty acids abolished the GPI-induced tyrosine phosphorylation of macrophage proteins, explaining the downstream reduction of NF-B activation and TNF- expression.

A remaining issue concerns the mechanisms by which T. gondii fatty acids come into contact with macrophages. The parasite diverts host cell precursors to contribute to the synthesis of major phospholipids, as well as some unknown lipids (4). However, some of the Toxoplasma-specific glycerolipids may require functional apicoplast fatty acid synthesis (4). Both de novo production and recycling of host cell components are likely to coexist. This lipid acquisition would be facilitated by the intimate and specific apposition of the membrane of the parasitophorous vacuole with the host cell lipid biosynthesis apparatus, i.e., the endoplasmic reticulum and the mitochondria (36). T. gondii sequesters host cell-derived lipids in nonsecretory lipid bodies, as well as within endomembranes (8). The concentrated lipids are gradually mobilized from the lipid bodies to other organelles (endoplasmic reticulum and Golgi) and are probably used for the construction of complex lipids, providing a source for membrane biogenesis (8). Interestingly, the presence of lipid inclusions in the Toxoplasma-containing vacuole was revealed by labeling with Nile Red upon incubation with excess free fatty acids (30). These lipids are probably liberated in the extracellular compartment after host cell disruption. This could explain how the toxoplasmal fatty acids come into contact with cells of the host immune system. Fatty acids from host cells could also directly participate in the modulation of inflammatory cytokine production in response to T. gondii infection. Indeed, macrophages challenged with tachyzoites resulted in a high mobilization of arachidonic acid in the culture medium (42). Both parasite and host cell phospholipase A₂ are involved in arachidonic acid release from phospholipids. Other fatty acids with an inhibitory activity could be released from the host cell membrane in the same way.

As shown for P. falciparum (11), a balance between GPIs and fatty acids seems to exist in T. gondii and could be a feature conserved among apicomplexa parasites. GPIs are considered as toxins inducing irreversible damage in the host, and fatty acids could reduce the immune response favoring the persistence of parasite infection.

 
This study was supported by a grant from the Deutsche Forschungsgemeinschaft to R.T.S. and to R.G. (SFB 535, project Z1), the Fonds der Chemischen Industrie, Stiftung P. E. Kempkes, and Hessisches Ministerium für Wissenschaft und Kunst.

 
* Corresponding author. Mailing address: Institut für Virologie, AG Parasitologie, Hans-Meerwein-Str. 2, D-35043 Marburg, Germany. Phone: (49)-6421-28-65493. Fax: (49)-6421-28-68976. E-mail: debierre{at}staff.uni-marburg.de

Published ahead of print on 26 March 2007.

Editor: J. F. Urban, Jr.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Infection and Immunity, June 2007, p. 2886-2893, Vol. 75, No. 6
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