Mycotoxin Fumonisin B1 Alters the Cytokine Profile and Decreases the Vaccinal Antibody Titer in Pigs
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《毒物学科学杂志》
Pharmacology and Toxicology Laboratory, UR 66, National Institute of Agronomic Research INRA, Toulouse, France
IBNA, Institute of Biology and Animal Nutrition, Balotesti, Romania
Pasteur Institute, Bucarest, Romania
Department of Mycotoxicology, Veterinary School of Toulouse, Toulouse, France
Ottawa-Carleton Institute of Chemistry, Carleton University, Ottawa, Canada
ABSTRACT
Fumonisin B1 (FB1), a mycotoxin produced by Fusarium verticillioides, may contaminate feed and food. In the present study, we investigated the effect of FB1 on the modulation of the cytokine profile and on the establishment of a vaccinal antibody response. In vitro investigations on pig peripheral blood mononuclear cells (PBMC) indicate that FB1 decreased interleukin-4 (IL-4) and increased interferon-gamma (IFN-) synthesis at both the protein and mRNA levels. A short in vivo exposure (7 days) of weanling piglets to 1.5 mg/kg body weight of purified FB1 altered the cytokine balance in mesenteric lymph nodes and spleen similarly to the in vitro PBMC results. We also investigated the effect of FB1 on the antibody response during a vaccination process. A prolonged in vivo exposure (28 days) of weanling piglets to feed contaminated with 8 mg FB1/kg significantly decreased the expression of IL-4 mRNA by porcine whole blood cells and diminished the specific antibody titer after vaccination against Mycoplasma agalactiae. By contrast, ingestion of the contaminated feed had no effect on the serum concentration of the immunoglobulin subset (IgG, IgA, and IgM). Taken together, our data suggest that FB1 alters the cytokine profile and decreases the specific antibody response built during a vaccination protocol. These results may have implications for humans or animals eating contaminated food or feed.
Key Words: fumonisin B1; PBMC; swine; cytokine; Th1/Th2; specific antibody; immunoglobulin.
INTRODUCTION
Mycotoxins are secondary metabolites of fungi, which may contaminate animal and human feeds. Because of their toxicological effects, their global occurrence is considered an important risk factor for human and animal health (Mannon and Johnson, 1985). Fumonisins are a family of cytotoxic and carcinogenic mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that commonly contaminate maize. Recent surveys on fumonisins in food and feed throughout the world, including the United States and most European countries, have raised concerns about the extent of FB1 contamination of maize and its implications for food safety (IPCS, 2000; Murphy et al., 1993). The mechanism(s) of FB1 toxicity is complex and may involve several molecular sites (Riley et al., 1998). The primary biochemical effect of fumonisins is the inhibition of the ceramide synthase leading to the accumulation of sphingoid bases and sphingoid base metabolites, and the depletion of more complex sphingolipids (Merrill et al., 2001; Riley et al., 1998).
At high concentrations, FB1 causes a variety of species-specific acute toxicological effects in domestic and laboratory animals. It induces leukoencephalomalacia in horses, pulmonary edema in pigs, and nephrotoxicity in rats, rabbits, and lambs. It also causes hepatotoxicity in all species thus far examined (Bolger et al., 2001; Haschek et al., 2001). This toxin has also been reported to be a carcinogen in rodents, and there is evidence that it is a contributing factor in human esophageal cancers (IPCS, 2000). The effects of ingestion of low doses of FB1 are less completely documented, but recent studies have shown that it does not have a major effect on clinical signs in mice or in swine (Bondy et al., 2000; Rotter et al., 1996; Zomborszky-Kovacs et al., 2002). However, ingestion of low doses of FB1 revealed pathological alterations of the lungs and an increase of intestinal colonization by opportunistic pathogenic bacteria in piglets (Halloy et al., submitted; Oswald et al. 2003).
Cellular immune responses generated after antigen stimulation of lymphocyte populations can be characterized by the distinct cytokines that are produced (Abbas et al., 1996). CD4+ T cell clones are broadly divided into two subsets, based on the cytokines they secrete: the Th1-producing forms (interleukin-2 [IL-2], interferon-gamma [IFN-], and tumor necrosis factor [TNF]) or Th2-producing forms (IL-4, IL-5, IL-6, IL-10, and IL-13) (Abbas et al., 1996; Asnagli and Murphy, 2001). The development of one or the other T-cell subset is particularly relevant in response to the development of protective immunity to many pathogens and to the development of vaccinal immunity (Abbas et al., 1996; Sher and Coffman, 1992).
Several dietary factors are able to alter the balance between Th1 and Th2 cytokines. For example, the n-3 fatty acids present in fish oil, along with a food-restricted diet, modulate the Th1/Th2 shift toward maintenance of a Th1 response and lead to a decrease of lupus erythematosus manifestation in autoimmune-prone (NZB/NZW)F1 mice (Jolly et al., 2001). Vitamin D and vitamin D analogs facilitate the development of a Th2 response in humans (Jirapongsananuruk et al., 2000; Matheu et al., 2003). By contrast, the effect of food/feed contaminants such as mycotoxins on the Th1/Th2 cytokine balance has been poorly investigated (Choi et al., 2000). In vitro exposure of primary cells or cell lines to low doses of FB1 (1–100 μM) affect their function (Bouhet et al., 2004; Dombrink-Kurtzman et al., 2000; Liu et al., 2002), as well as their ability to produce cytokines and other metabolites (Dresden Osborne et al., 2002; Theumer et al., 2002). In vivo, subcutaneous injection of the toxin, via a slow delivery route, has been shown by Sharma and colleagues to alter inflammatory cytokine production in mice (Bhandari and Sharma, 2002; Sharma et al., 2003).
In many countries, swine are potentially exposed to high levels of fumonisin in maize-based diets. As monogastric animals, swine are also considered a good model for extrapolation to humans (Almond et al., 1996; Miller and Ullrey, 1987). The aims of the present study, in which a piglet model was used, were to analyze the effect of FB1 on the cytokine balance and to investigate the consequences of ingestion of low dose of toxin on the development of a vaccinal immune response.
MATERIALS AND METHODS
Animals and Toxins.
Thirty-two crossbred weanling piglets housed in floored indoor pens were used in this study. They were acclimatized for 1 week prior to being used in experimental protocols and were given ad libitum access to water and feed. They were cared for in accordance with the National Institutes of Health Guide and the French Ministry of Agriculture standards for the care and use of laboratory animals.
Fumonisin B1 (>98% pure by nuclear magnetic resonance [NMR], and high performance liquid chromatography [HPLC]) used for in vitro studies was obtained from PROMEC/MRC (Tygerberg, South Africa) and diluted in sterile water. FB1 (>98% pure by NMR, MS, and HPLC) used in the short-term toxin exposure trial was prepared as previously described (Miller et al., 1994) and obtained from Dr. J. D. Miller (Department of Chemistry, Carleton University, Ottawa, Ontario, Canada). Fumonisin extract obtained after in vitro culture of the Fusarium verticilloides strain NRRL 34281 (Oswald et al., 2003) was incorporated into a pig basal diet to provide a feed diet containing 8 mg FB1/kg, which was used in the long-term toxin exposure trial.
Experimental Design for In Vivo Studies.
A long- and short-term toxin trial was performed. In the long-term trial, piglets of initial average body weight of 12.3 ± 0.8 kg were studied for 28 days. They were fed on a corn-soybean meal basal diet (Marin et al., 2002) supplemented or not with fumonisin extract. At day 8 and 22 of the experiment, animals were immunized subcutaneously with 1 ml of Agavac, a vaccine made with a combination of formol inactivated Mycoplasma agalactiae strains (Pasteur Institute, Bucharest, Romania). Blood samples were obtained throughout the experiment to measure total and specific antibody levels as well as cytokine mRNA expression upon mitogenic stimulation. In the short-term trial, 12 piglets with an initial average body weight of 7.3 ± 0.4 kg were studied for 7 days. Treated pigs were given by gavage 1.5 mg/kg body weight/day of purified toxin diluted in water. Control animals
Experimental Design for In Vitro Studies.
Blood, collected from the jugular vein of piglets, was used to isolate porcine peripheral blood mononuclear cells (PBMC) as described elsewhere (Dozois et al., 1997). The PBMC were resuspended in RPMI-1640 (Eurobio, Les Ulis, France) supplemented with glutamine (2 mM), penicillin (100 U/ml), streptomycin (50 μg/ml), amphotericin B (2.5 μg/ml), and 10 % fetal calf serum (FCS: Hyclone, Perbio, Brebières, France). Cell viability was evaluated by trypan blue exclusion (Eurobio) before in vitro culture.
Determination of IL-4 and IFN- Protein Concentration by ELISA.
Porcine PBMC, cultured at a density of 5 x106 cells/well and stimulated with 10 μg/ml concanavalin A (ConA) (type IV, Sigma, St. Quentin Fallavier, France), were incubated with various concentrations of FB1 for 96 h. We previously verified that this time point was optimal for IFN- secretion and corresponded to a plateau for IL-4 synthesis. Culture supernatants were then collected and analyzed for cytokine content with the CytoSet ELISA kit (Biosource Europe, CliniSciences, Montrouge, France) according to the manufacturer's instructions. Briefly, a purified fraction of anti-swine IL-4 and IFN- (clone A155B 16F2 and clone A151D 5B8, respectively) were used as capture antibodies in conjunction with the biotinylated anti-swine IL-4 and IFN- monoclonal antibodies (clone A155B 15C6 and clone A151D 13C5, respectively). Streptavidin-horseradish peroxidase (HRP) (Biosource) and tetramethylbenzidene [TMB] (Fermentas, Hanover, MD) were used for detection. Absorbance was read at 450 nm with an ELISA plate reader (Spectra thermo, Tecan, NC). Dilution of recombinant swine IL-4 and IFN- were used as standards, and results were expressed as picograms of cytokine per milliliter.
Determination of the Expression of mRNA Encoding for IL-4 and IFN- by Semiquantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR).
The mRNA expression of IL-4 and IFN- was analyzed in tissues and blood samples from control animals and piglets exposed in vivo to FB1 as well as in PBMC treated in vitro or not with FB1. Tissues samples (spleen and mesenteric lymph nodes) collected after euthanasia were maintained in Extract-all (Eurobio) at –80°C before they were homogenized for mRNA extraction (Fournout et al., 2000). Blood samples, aseptically collected before euthanasia, were diluted 1/10 in complete RPMI, and stimulated for 24 h with 10 μg/ml ConA or 10 μg/ml phytohemagglutinin (PHA, Sigma). Isolated PBMC were stimulated with 10 μg/ml ConA and cultured in vitro for 24 h, in the presence or not of 100 μM FB1. Whole blood and PBMC cell pellets were then resuspended in Extract-all and stored at –80°C before RNA analysis.
Total RNA (from tissues or cells) was extracted following the manufacturer's recommendations. RNA, resuspended in diethylpyrocarbonate water, was quantified by spectrophotometry. Semi-quantitative determination of IL-4, IFN-, and cyclophilin, chosen as a housekeeping gene, was carried out using RT-PCR performed as previously described (Fournout et al., 2000). Briefly, 1 and 1.5 μg of mRNA for blood and tissues samples, respectively, were reverse transcribed with M-MLV Reverse Transcriptase, RNase H- (Promega, Charbonnières, France) and amplified with 1 U Taq DNA Polymerase (Gibco, Life Technologies, Cergy Pontoise, France) using the already published primer sequences (Dozois et al., 1997). Amplified DNA was analyzed by electrophoresis and quantified densitometrically using the Quantity One program (Bio-Rad, Hercules, CA). To compare the relative cytokine mRNA expression levels among samples, the values were presented as the ratio of the band intensity of the cytokine-specific RT-PCR product over that of the corresponding constitutively expressed housekeeping gene, cyclophilin.
Measurement of Total Immunoglobulin Subsets (IgG, IgA, IgM).
Total concentration of the immunoglobulin subsets was measured by ELISA (Bethyl, Interchim, Montlucon, France). The sera were diluted 1/100,000, 1/2000, and 1/8000 in Tris–buffered saline to detect IgG, IgA, and IgM, respectively, and processed according to the manufacturer's instructions.
Measurement of Specific Antibody to Mycoplasma agalactiae.
Antibody titers against M. agalactiae were measured by ELISA as described elsewhere (Marin et al., 2002). Briefly, ELISA plates were coated with supernatant from ultrasonicated M. agalactiae culture. Serum samples diluted 1/100, were then added to the plates, and the anti-mycobacterial antibodies were detected with peroxidase-labeled anti-pig IgG. The absorbance at 405 nm was recorded with an ELISA plate reader, and values were expressed as optical density (OD).
Statistical Analysis.
Student's t-test was used to analyze cytokine and antibody production; p values < 0.05 were considered significant.
RESULTS
In Vitro Effect of FB1 on the Cytokine Production
We first examined the ability of FB1 to modulate in vitro the cytokine profile at the protein and mRNA levels. The IFN- and IL-4 concentrations were measured in supernatants of PBMC stimulated with mitogen in the presence of increasing concentrations of FB1. As expected, ConA stimulated the production of both IFN- and IL-4 by porcine PBMC (Fig. 1). The cytokine profile was greatly affected by the presence of FB1 in the culture media. Indeed, the toxin stimulated in a dose-dependent manner the synthesis of IFN- by porcine PBMC. An increase of 220 ± 11% in IFN- concentration was observed in PBMC treated for 96 h with 100 μM FB1. By contrast, the synthesis of IL-4 was significantly reduced in the presence of FB1 at concentrations higher than 10 μM, and a 84.5 ± 0.5% decrease was noted in culture stimulated with 100 μM of toxin. The alteration of the cytokine profile toward the production of IFN- was also observed at the mRNA level (Fig. 2). For instance, the treatment of porcine PBMC with 100 μM FB1 increased the expression of mRNA encoding for IFN- by 40.8 ± 3.3% (p < 0.05) and decreased the expression of mRNA encoding for IL-4 by 59.9 ± 8.0% (p < 0.05). The alteration of cytokine profile was not associated with an increase in apoptosis. The percentage of PBMC in the sub-G1 phase of the cell cycle was measured by flow cytometry after propidium staining. This percentage increased only by 3% when comparing PBMC stimulated with ConA and PBMC treated with ConA and 100 μM FB1.
In Vivo Effect of FB1 on Cytokine Production
Blood samples obtained from piglets exposed or not to the toxin, were stimulated with mitogen and analyzed for their IL-4 and IFN- mRNA expression levels (Table 1). After in vitro stimulation with phytohemagglutinin (PHA), porcine blood cells expressed mRNA encoding for IFN- and IL-4, but the cytokine expression pattern was altered in samples from animals that ingested the toxin. As already observed in vitro, exposure of the piglets to FB1 for 4 weeks significantly decreased IL-4 mRNA expression in whole blood cells after mitogenic stimulation. By contrast, the ingestion of the toxin did not have a significant effect on the expression of IFN- mRNA in whole blood cells (Table 1).
A similar decrease in IL-4 was observed in blood samples obtained from piglets exposed for a shorter period (7 days) to purified FB1 (Table 1). In these animals, a decreased expression of IL-4 mRNA and an increased expression of IFN- mRNA was also observed in the spleen and the mesenteric lymph nodes (Fig. 3).
In Vivo Influence of FB1 on Antibody Production
Piglets, fed for 4 weeks with a FB1-contaminated or control diet, were vaccinated with M. agalactiae vaccine, and their total and specific antibody responses were analyzed. Table 2 shows that the concentration of the different immunoglobulin subsets (IgA, IgG, and IgM) increased with the age of the animals. However, FB1 treatment did not have any influence on the concentration of these immunoglobulin subsets. By contrast, FB1 treatment induced a small but significant decrease of the specific immune response toward a vaccinal antigen (Fig. 4). Animals immunized at day 8 and 22 with M. agalactiae vaccine developed a specific antibody response. The ingestion of FB1 decreased the specific antibody response observed after the second immunization. Indeed, at day 28 of the experiment, the mean OD for the specific antibody level was 1.012 ± 0.118 in the serum of vaccinated control animals receiving a normal diet versus 0.781 ± 0.06 (p < 0.05) in the serum of vaccinated piglets receiving a diet contaminated with 8 mg FB1/kg.
DISCUSSION
Our results showed that FB1 alters the balance between Th1 and Th2 cytokines. A decrease in IL-4 and an increase in IFN- level were observed upon FB1 treatment both in vivo (Fig. 3) and in vitro (Fig. 1 and Fig. 2). The in vivo effect of FB1 on the production of IL-10, another Th2 cytokine has also been reported by Theumer (2002). These authors demonstrated that splenocytes from rats exposed for 12 weeks to 100 μg/g FB1-contaminated feed and further stimulated with ConA produced more IL-10 than splenocytes from control animals. By contrast, in this study, in vitro treatment of lymphocytes with 10 μM FB1 induced no change in IL-10 or IL-4 synthesis. Our results may suggest that swine lymphocytes are more susceptible to the effect of FB1 than rat lymphocytes. Indeed, in vitro treatment of swine lymphocytes with 10 μM of FB1 significantly decreased IL-4 production (Fig. 1).
We demonstrated that FB1 increases IFN- production by swine PBMC (Fig. 1 and Fig. 2). This effect was observed both at the protein and the mRNA levels. Bhandari et al. (2002a) also observed an increased IFN- expression in liver of mice treated subcutaneously or per os with a single dose of FB1. In a further study, the same group demonstrated a reduction of the pathological effect of FB1 in IFN- knockout mice (Sharma et al., 2003). The pro-inflammatory effect of FB1 has been demonstrated in several studies. Stimulation of nitric oxide (NO) production by peritoneal macrophages from mice fed with 150 mg/g of FB1 and challenged with Trypanosoma cruzi was observed by Dresden Osborne et al. (2002). Additionally, FB1 stimulated not only the basal production of NO by rat macrophages (Dombrink-Kurtzman et al., 2000) but also the lipopolysaccharide (LPS)-induced synthesis of TNF by mice macrophages (Dugyala et al., 1998). In mice, subcutaneous injection of FB1 caused an increased expression of TNF- and IL-12 p40, but this increase was localized in the liver and not seen in other organs such as kidney or spleen (Bhandari et al., 2002b). In our porcine model, the ingestion of mycotoxin increased IFN- level in both mesenteric lymph nodes and spleen (Fig. 3), suggesting either species differences depending on the animal species or a generalized induction of IFN- when the animals are fed with the mycotoxin.
By its structural analogy with sphingosine and sphinganine, FB1 interferes with the sphingolipid metabolism causing the accumulation of free sphingoid bases (Merrill et al., 1997). Free sphingoid bases inhibit the growth of several cell types including lymphocytes (Nakamura et al., 1996; Merrill, 2002), and Th2 lymphocytes are more sensitive to the action of exogenous sphingosine than Th1 lymphocytes (Tokura et al., 1996). Thus, it is possible that FB1 selectively acts on Th2 cells to decrease the synthesis of the Th2 cytokine (Fig. 1 and Fig. 2). FB1 also modulates glycolipid synthesis (Merrill, 2002). It was first anticipated that FB1 decreases the synthesis of all complex glycosphigolipids. However, more recent data indicate that the picture is more complicated and that FB1 has a different action on gangliosides and globosides (Meivar-Levy and Futerman, 1999). Gangliosides are known to modulate immunoglobulin and cytokine production (Kanda and Tamaki, 1999; Kanda and Watanabe, 2000, 2001). For example GD1b and GT1b enhance Th1 cytokine production, suppress Th2 cytokine production and inhibit immunoglobulin synthesis (Kanda and Tamaki, 1999; Kanda and Watanabe, 2001). By contrast, GQ1b, GM2, and GD1a increase in vitro the synthesis of immunoglobulin (Kanda and Watanabe, 2000). Further studies are needed to determine the role of the modulation of glycolipid synthesis by FB1 on lymphocyte functions.
Th2 cytokines are implicated in the development of the humoral immune response and antibody production (Abbas et al., 1996). We therefore have postulated that the decrease of IL-4 induced by the FB1 treatment may diminish the specific antibody response mounted during vaccination. Indeed, a prolonged exposure (28 days) to feed contaminated with 8 mg FB1/kg induced a small but significant decrease in the specific antibody response against M. agalactiae (Fig. 4). It would have been interesting to study the antibody response on a longer period to analyze if the difference obtained at 28 days increases over time. An experimental challenge would have permitted to determine if the small but significant difference observed in the specific antibody production would influence the protective immunity of the piglets.
A decrease of the specific antibody production has been also observed in turkey vaccinated against Newcastle virus (Li et al., 2000) and in rodents immunized with sheep red blood cells (Martinova and Merrill 1995; Tryphonas et al., 1997). By contrast, exposure of piglets for up to 4 months to feed contaminated with 1, 5, and 10 mg FB1/kg had no significant effect on their antibody titers against Aujeszky's disease (Tornyos et al., 2003).
The observed effect of FB1 on the Th1/Th2 balance may also explain the effect of this mycotoxin on animal susceptibility to infectious diseases. Indeed, resistance to Escherichia coli infection is antibody mediated, and we have recently shown that ingestion of FB1 increases the intestinal colonization by these bacteria (Oswald et al., 2003). By contrast, resistance to intracellular parasites such as Trypanosoma cruzi require the development of cellular immunity, and recent data indicate that mice fed with FB1 are more resistant to this parasite than mice fed with a control mycotoxin-free diet (Dresden Osborne et al., 2002).
In conclusion, we found that the ingestion of FB1 alters the cytokine production and decreases the vaccinal antibody response. This could have important consequences in human and animal health as the breakdown in vaccinal immunity and may lead to the occurrence of disease even in properly vaccinated animals and/or humans. Considering the high levels of FB1 that may be present in animal feeds and human food preparations (IPCS, 2000; Murphy et al., 1993), further studies are needed to determine the minimal concentration that modulates the immune response. Epidemiological studies are also needed to assess the extent to which fumonisins are involved in the failure of vaccinal immunity in humans and animals.
ACKNOWLEDGMENTS
This work was supported by funds from the region Midi-Pyrénées, France (DAER-Rech/99008345), and the Transversalité INRA (mycotoxines-P00263). Dr. I. Taranu was the recipient of an INRA post-doctoral fellowship. D. Marin was supported by the "Réseau Formation-Recherche," a bilateral project between France and Romania granted by the "Ministère de l'éducation Nationale" and the "Ministère de la Recherche" (Paris, France). S. Boulet was supported by a fellowship from the "Ministére de L'éducation Nationalé." Thanks are also due to Dr. Neil Ledger for his help with the English text.
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IBNA, Institute of Biology and Animal Nutrition, Balotesti, Romania
Pasteur Institute, Bucarest, Romania
Department of Mycotoxicology, Veterinary School of Toulouse, Toulouse, France
Ottawa-Carleton Institute of Chemistry, Carleton University, Ottawa, Canada
ABSTRACT
Fumonisin B1 (FB1), a mycotoxin produced by Fusarium verticillioides, may contaminate feed and food. In the present study, we investigated the effect of FB1 on the modulation of the cytokine profile and on the establishment of a vaccinal antibody response. In vitro investigations on pig peripheral blood mononuclear cells (PBMC) indicate that FB1 decreased interleukin-4 (IL-4) and increased interferon-gamma (IFN-) synthesis at both the protein and mRNA levels. A short in vivo exposure (7 days) of weanling piglets to 1.5 mg/kg body weight of purified FB1 altered the cytokine balance in mesenteric lymph nodes and spleen similarly to the in vitro PBMC results. We also investigated the effect of FB1 on the antibody response during a vaccination process. A prolonged in vivo exposure (28 days) of weanling piglets to feed contaminated with 8 mg FB1/kg significantly decreased the expression of IL-4 mRNA by porcine whole blood cells and diminished the specific antibody titer after vaccination against Mycoplasma agalactiae. By contrast, ingestion of the contaminated feed had no effect on the serum concentration of the immunoglobulin subset (IgG, IgA, and IgM). Taken together, our data suggest that FB1 alters the cytokine profile and decreases the specific antibody response built during a vaccination protocol. These results may have implications for humans or animals eating contaminated food or feed.
Key Words: fumonisin B1; PBMC; swine; cytokine; Th1/Th2; specific antibody; immunoglobulin.
INTRODUCTION
Mycotoxins are secondary metabolites of fungi, which may contaminate animal and human feeds. Because of their toxicological effects, their global occurrence is considered an important risk factor for human and animal health (Mannon and Johnson, 1985). Fumonisins are a family of cytotoxic and carcinogenic mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that commonly contaminate maize. Recent surveys on fumonisins in food and feed throughout the world, including the United States and most European countries, have raised concerns about the extent of FB1 contamination of maize and its implications for food safety (IPCS, 2000; Murphy et al., 1993). The mechanism(s) of FB1 toxicity is complex and may involve several molecular sites (Riley et al., 1998). The primary biochemical effect of fumonisins is the inhibition of the ceramide synthase leading to the accumulation of sphingoid bases and sphingoid base metabolites, and the depletion of more complex sphingolipids (Merrill et al., 2001; Riley et al., 1998).
At high concentrations, FB1 causes a variety of species-specific acute toxicological effects in domestic and laboratory animals. It induces leukoencephalomalacia in horses, pulmonary edema in pigs, and nephrotoxicity in rats, rabbits, and lambs. It also causes hepatotoxicity in all species thus far examined (Bolger et al., 2001; Haschek et al., 2001). This toxin has also been reported to be a carcinogen in rodents, and there is evidence that it is a contributing factor in human esophageal cancers (IPCS, 2000). The effects of ingestion of low doses of FB1 are less completely documented, but recent studies have shown that it does not have a major effect on clinical signs in mice or in swine (Bondy et al., 2000; Rotter et al., 1996; Zomborszky-Kovacs et al., 2002). However, ingestion of low doses of FB1 revealed pathological alterations of the lungs and an increase of intestinal colonization by opportunistic pathogenic bacteria in piglets (Halloy et al., submitted; Oswald et al. 2003).
Cellular immune responses generated after antigen stimulation of lymphocyte populations can be characterized by the distinct cytokines that are produced (Abbas et al., 1996). CD4+ T cell clones are broadly divided into two subsets, based on the cytokines they secrete: the Th1-producing forms (interleukin-2 [IL-2], interferon-gamma [IFN-], and tumor necrosis factor [TNF]) or Th2-producing forms (IL-4, IL-5, IL-6, IL-10, and IL-13) (Abbas et al., 1996; Asnagli and Murphy, 2001). The development of one or the other T-cell subset is particularly relevant in response to the development of protective immunity to many pathogens and to the development of vaccinal immunity (Abbas et al., 1996; Sher and Coffman, 1992).
Several dietary factors are able to alter the balance between Th1 and Th2 cytokines. For example, the n-3 fatty acids present in fish oil, along with a food-restricted diet, modulate the Th1/Th2 shift toward maintenance of a Th1 response and lead to a decrease of lupus erythematosus manifestation in autoimmune-prone (NZB/NZW)F1 mice (Jolly et al., 2001). Vitamin D and vitamin D analogs facilitate the development of a Th2 response in humans (Jirapongsananuruk et al., 2000; Matheu et al., 2003). By contrast, the effect of food/feed contaminants such as mycotoxins on the Th1/Th2 cytokine balance has been poorly investigated (Choi et al., 2000). In vitro exposure of primary cells or cell lines to low doses of FB1 (1–100 μM) affect their function (Bouhet et al., 2004; Dombrink-Kurtzman et al., 2000; Liu et al., 2002), as well as their ability to produce cytokines and other metabolites (Dresden Osborne et al., 2002; Theumer et al., 2002). In vivo, subcutaneous injection of the toxin, via a slow delivery route, has been shown by Sharma and colleagues to alter inflammatory cytokine production in mice (Bhandari and Sharma, 2002; Sharma et al., 2003).
In many countries, swine are potentially exposed to high levels of fumonisin in maize-based diets. As monogastric animals, swine are also considered a good model for extrapolation to humans (Almond et al., 1996; Miller and Ullrey, 1987). The aims of the present study, in which a piglet model was used, were to analyze the effect of FB1 on the cytokine balance and to investigate the consequences of ingestion of low dose of toxin on the development of a vaccinal immune response.
MATERIALS AND METHODS
Animals and Toxins.
Thirty-two crossbred weanling piglets housed in floored indoor pens were used in this study. They were acclimatized for 1 week prior to being used in experimental protocols and were given ad libitum access to water and feed. They were cared for in accordance with the National Institutes of Health Guide and the French Ministry of Agriculture standards for the care and use of laboratory animals.
Fumonisin B1 (>98% pure by nuclear magnetic resonance [NMR], and high performance liquid chromatography [HPLC]) used for in vitro studies was obtained from PROMEC/MRC (Tygerberg, South Africa) and diluted in sterile water. FB1 (>98% pure by NMR, MS, and HPLC) used in the short-term toxin exposure trial was prepared as previously described (Miller et al., 1994) and obtained from Dr. J. D. Miller (Department of Chemistry, Carleton University, Ottawa, Ontario, Canada). Fumonisin extract obtained after in vitro culture of the Fusarium verticilloides strain NRRL 34281 (Oswald et al., 2003) was incorporated into a pig basal diet to provide a feed diet containing 8 mg FB1/kg, which was used in the long-term toxin exposure trial.
Experimental Design for In Vivo Studies.
A long- and short-term toxin trial was performed. In the long-term trial, piglets of initial average body weight of 12.3 ± 0.8 kg were studied for 28 days. They were fed on a corn-soybean meal basal diet (Marin et al., 2002) supplemented or not with fumonisin extract. At day 8 and 22 of the experiment, animals were immunized subcutaneously with 1 ml of Agavac, a vaccine made with a combination of formol inactivated Mycoplasma agalactiae strains (Pasteur Institute, Bucharest, Romania). Blood samples were obtained throughout the experiment to measure total and specific antibody levels as well as cytokine mRNA expression upon mitogenic stimulation. In the short-term trial, 12 piglets with an initial average body weight of 7.3 ± 0.4 kg were studied for 7 days. Treated pigs were given by gavage 1.5 mg/kg body weight/day of purified toxin diluted in water. Control animals
Experimental Design for In Vitro Studies.
Blood, collected from the jugular vein of piglets, was used to isolate porcine peripheral blood mononuclear cells (PBMC) as described elsewhere (Dozois et al., 1997). The PBMC were resuspended in RPMI-1640 (Eurobio, Les Ulis, France) supplemented with glutamine (2 mM), penicillin (100 U/ml), streptomycin (50 μg/ml), amphotericin B (2.5 μg/ml), and 10 % fetal calf serum (FCS: Hyclone, Perbio, Brebières, France). Cell viability was evaluated by trypan blue exclusion (Eurobio) before in vitro culture.
Determination of IL-4 and IFN- Protein Concentration by ELISA.
Porcine PBMC, cultured at a density of 5 x106 cells/well and stimulated with 10 μg/ml concanavalin A (ConA) (type IV, Sigma, St. Quentin Fallavier, France), were incubated with various concentrations of FB1 for 96 h. We previously verified that this time point was optimal for IFN- secretion and corresponded to a plateau for IL-4 synthesis. Culture supernatants were then collected and analyzed for cytokine content with the CytoSet ELISA kit (Biosource Europe, CliniSciences, Montrouge, France) according to the manufacturer's instructions. Briefly, a purified fraction of anti-swine IL-4 and IFN- (clone A155B 16F2 and clone A151D 5B8, respectively) were used as capture antibodies in conjunction with the biotinylated anti-swine IL-4 and IFN- monoclonal antibodies (clone A155B 15C6 and clone A151D 13C5, respectively). Streptavidin-horseradish peroxidase (HRP) (Biosource) and tetramethylbenzidene [TMB] (Fermentas, Hanover, MD) were used for detection. Absorbance was read at 450 nm with an ELISA plate reader (Spectra thermo, Tecan, NC). Dilution of recombinant swine IL-4 and IFN- were used as standards, and results were expressed as picograms of cytokine per milliliter.
Determination of the Expression of mRNA Encoding for IL-4 and IFN- by Semiquantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR).
The mRNA expression of IL-4 and IFN- was analyzed in tissues and blood samples from control animals and piglets exposed in vivo to FB1 as well as in PBMC treated in vitro or not with FB1. Tissues samples (spleen and mesenteric lymph nodes) collected after euthanasia were maintained in Extract-all (Eurobio) at –80°C before they were homogenized for mRNA extraction (Fournout et al., 2000). Blood samples, aseptically collected before euthanasia, were diluted 1/10 in complete RPMI, and stimulated for 24 h with 10 μg/ml ConA or 10 μg/ml phytohemagglutinin (PHA, Sigma). Isolated PBMC were stimulated with 10 μg/ml ConA and cultured in vitro for 24 h, in the presence or not of 100 μM FB1. Whole blood and PBMC cell pellets were then resuspended in Extract-all and stored at –80°C before RNA analysis.
Total RNA (from tissues or cells) was extracted following the manufacturer's recommendations. RNA, resuspended in diethylpyrocarbonate water, was quantified by spectrophotometry. Semi-quantitative determination of IL-4, IFN-, and cyclophilin, chosen as a housekeeping gene, was carried out using RT-PCR performed as previously described (Fournout et al., 2000). Briefly, 1 and 1.5 μg of mRNA for blood and tissues samples, respectively, were reverse transcribed with M-MLV Reverse Transcriptase, RNase H- (Promega, Charbonnières, France) and amplified with 1 U Taq DNA Polymerase (Gibco, Life Technologies, Cergy Pontoise, France) using the already published primer sequences (Dozois et al., 1997). Amplified DNA was analyzed by electrophoresis and quantified densitometrically using the Quantity One program (Bio-Rad, Hercules, CA). To compare the relative cytokine mRNA expression levels among samples, the values were presented as the ratio of the band intensity of the cytokine-specific RT-PCR product over that of the corresponding constitutively expressed housekeeping gene, cyclophilin.
Measurement of Total Immunoglobulin Subsets (IgG, IgA, IgM).
Total concentration of the immunoglobulin subsets was measured by ELISA (Bethyl, Interchim, Montlucon, France). The sera were diluted 1/100,000, 1/2000, and 1/8000 in Tris–buffered saline to detect IgG, IgA, and IgM, respectively, and processed according to the manufacturer's instructions.
Measurement of Specific Antibody to Mycoplasma agalactiae.
Antibody titers against M. agalactiae were measured by ELISA as described elsewhere (Marin et al., 2002). Briefly, ELISA plates were coated with supernatant from ultrasonicated M. agalactiae culture. Serum samples diluted 1/100, were then added to the plates, and the anti-mycobacterial antibodies were detected with peroxidase-labeled anti-pig IgG. The absorbance at 405 nm was recorded with an ELISA plate reader, and values were expressed as optical density (OD).
Statistical Analysis.
Student's t-test was used to analyze cytokine and antibody production; p values < 0.05 were considered significant.
RESULTS
In Vitro Effect of FB1 on the Cytokine Production
We first examined the ability of FB1 to modulate in vitro the cytokine profile at the protein and mRNA levels. The IFN- and IL-4 concentrations were measured in supernatants of PBMC stimulated with mitogen in the presence of increasing concentrations of FB1. As expected, ConA stimulated the production of both IFN- and IL-4 by porcine PBMC (Fig. 1). The cytokine profile was greatly affected by the presence of FB1 in the culture media. Indeed, the toxin stimulated in a dose-dependent manner the synthesis of IFN- by porcine PBMC. An increase of 220 ± 11% in IFN- concentration was observed in PBMC treated for 96 h with 100 μM FB1. By contrast, the synthesis of IL-4 was significantly reduced in the presence of FB1 at concentrations higher than 10 μM, and a 84.5 ± 0.5% decrease was noted in culture stimulated with 100 μM of toxin. The alteration of the cytokine profile toward the production of IFN- was also observed at the mRNA level (Fig. 2). For instance, the treatment of porcine PBMC with 100 μM FB1 increased the expression of mRNA encoding for IFN- by 40.8 ± 3.3% (p < 0.05) and decreased the expression of mRNA encoding for IL-4 by 59.9 ± 8.0% (p < 0.05). The alteration of cytokine profile was not associated with an increase in apoptosis. The percentage of PBMC in the sub-G1 phase of the cell cycle was measured by flow cytometry after propidium staining. This percentage increased only by 3% when comparing PBMC stimulated with ConA and PBMC treated with ConA and 100 μM FB1.
In Vivo Effect of FB1 on Cytokine Production
Blood samples obtained from piglets exposed or not to the toxin, were stimulated with mitogen and analyzed for their IL-4 and IFN- mRNA expression levels (Table 1). After in vitro stimulation with phytohemagglutinin (PHA), porcine blood cells expressed mRNA encoding for IFN- and IL-4, but the cytokine expression pattern was altered in samples from animals that ingested the toxin. As already observed in vitro, exposure of the piglets to FB1 for 4 weeks significantly decreased IL-4 mRNA expression in whole blood cells after mitogenic stimulation. By contrast, the ingestion of the toxin did not have a significant effect on the expression of IFN- mRNA in whole blood cells (Table 1).
A similar decrease in IL-4 was observed in blood samples obtained from piglets exposed for a shorter period (7 days) to purified FB1 (Table 1). In these animals, a decreased expression of IL-4 mRNA and an increased expression of IFN- mRNA was also observed in the spleen and the mesenteric lymph nodes (Fig. 3).
In Vivo Influence of FB1 on Antibody Production
Piglets, fed for 4 weeks with a FB1-contaminated or control diet, were vaccinated with M. agalactiae vaccine, and their total and specific antibody responses were analyzed. Table 2 shows that the concentration of the different immunoglobulin subsets (IgA, IgG, and IgM) increased with the age of the animals. However, FB1 treatment did not have any influence on the concentration of these immunoglobulin subsets. By contrast, FB1 treatment induced a small but significant decrease of the specific immune response toward a vaccinal antigen (Fig. 4). Animals immunized at day 8 and 22 with M. agalactiae vaccine developed a specific antibody response. The ingestion of FB1 decreased the specific antibody response observed after the second immunization. Indeed, at day 28 of the experiment, the mean OD for the specific antibody level was 1.012 ± 0.118 in the serum of vaccinated control animals receiving a normal diet versus 0.781 ± 0.06 (p < 0.05) in the serum of vaccinated piglets receiving a diet contaminated with 8 mg FB1/kg.
DISCUSSION
Our results showed that FB1 alters the balance between Th1 and Th2 cytokines. A decrease in IL-4 and an increase in IFN- level were observed upon FB1 treatment both in vivo (Fig. 3) and in vitro (Fig. 1 and Fig. 2). The in vivo effect of FB1 on the production of IL-10, another Th2 cytokine has also been reported by Theumer (2002). These authors demonstrated that splenocytes from rats exposed for 12 weeks to 100 μg/g FB1-contaminated feed and further stimulated with ConA produced more IL-10 than splenocytes from control animals. By contrast, in this study, in vitro treatment of lymphocytes with 10 μM FB1 induced no change in IL-10 or IL-4 synthesis. Our results may suggest that swine lymphocytes are more susceptible to the effect of FB1 than rat lymphocytes. Indeed, in vitro treatment of swine lymphocytes with 10 μM of FB1 significantly decreased IL-4 production (Fig. 1).
We demonstrated that FB1 increases IFN- production by swine PBMC (Fig. 1 and Fig. 2). This effect was observed both at the protein and the mRNA levels. Bhandari et al. (2002a) also observed an increased IFN- expression in liver of mice treated subcutaneously or per os with a single dose of FB1. In a further study, the same group demonstrated a reduction of the pathological effect of FB1 in IFN- knockout mice (Sharma et al., 2003). The pro-inflammatory effect of FB1 has been demonstrated in several studies. Stimulation of nitric oxide (NO) production by peritoneal macrophages from mice fed with 150 mg/g of FB1 and challenged with Trypanosoma cruzi was observed by Dresden Osborne et al. (2002). Additionally, FB1 stimulated not only the basal production of NO by rat macrophages (Dombrink-Kurtzman et al., 2000) but also the lipopolysaccharide (LPS)-induced synthesis of TNF by mice macrophages (Dugyala et al., 1998). In mice, subcutaneous injection of FB1 caused an increased expression of TNF- and IL-12 p40, but this increase was localized in the liver and not seen in other organs such as kidney or spleen (Bhandari et al., 2002b). In our porcine model, the ingestion of mycotoxin increased IFN- level in both mesenteric lymph nodes and spleen (Fig. 3), suggesting either species differences depending on the animal species or a generalized induction of IFN- when the animals are fed with the mycotoxin.
By its structural analogy with sphingosine and sphinganine, FB1 interferes with the sphingolipid metabolism causing the accumulation of free sphingoid bases (Merrill et al., 1997). Free sphingoid bases inhibit the growth of several cell types including lymphocytes (Nakamura et al., 1996; Merrill, 2002), and Th2 lymphocytes are more sensitive to the action of exogenous sphingosine than Th1 lymphocytes (Tokura et al., 1996). Thus, it is possible that FB1 selectively acts on Th2 cells to decrease the synthesis of the Th2 cytokine (Fig. 1 and Fig. 2). FB1 also modulates glycolipid synthesis (Merrill, 2002). It was first anticipated that FB1 decreases the synthesis of all complex glycosphigolipids. However, more recent data indicate that the picture is more complicated and that FB1 has a different action on gangliosides and globosides (Meivar-Levy and Futerman, 1999). Gangliosides are known to modulate immunoglobulin and cytokine production (Kanda and Tamaki, 1999; Kanda and Watanabe, 2000, 2001). For example GD1b and GT1b enhance Th1 cytokine production, suppress Th2 cytokine production and inhibit immunoglobulin synthesis (Kanda and Tamaki, 1999; Kanda and Watanabe, 2001). By contrast, GQ1b, GM2, and GD1a increase in vitro the synthesis of immunoglobulin (Kanda and Watanabe, 2000). Further studies are needed to determine the role of the modulation of glycolipid synthesis by FB1 on lymphocyte functions.
Th2 cytokines are implicated in the development of the humoral immune response and antibody production (Abbas et al., 1996). We therefore have postulated that the decrease of IL-4 induced by the FB1 treatment may diminish the specific antibody response mounted during vaccination. Indeed, a prolonged exposure (28 days) to feed contaminated with 8 mg FB1/kg induced a small but significant decrease in the specific antibody response against M. agalactiae (Fig. 4). It would have been interesting to study the antibody response on a longer period to analyze if the difference obtained at 28 days increases over time. An experimental challenge would have permitted to determine if the small but significant difference observed in the specific antibody production would influence the protective immunity of the piglets.
A decrease of the specific antibody production has been also observed in turkey vaccinated against Newcastle virus (Li et al., 2000) and in rodents immunized with sheep red blood cells (Martinova and Merrill 1995; Tryphonas et al., 1997). By contrast, exposure of piglets for up to 4 months to feed contaminated with 1, 5, and 10 mg FB1/kg had no significant effect on their antibody titers against Aujeszky's disease (Tornyos et al., 2003).
The observed effect of FB1 on the Th1/Th2 balance may also explain the effect of this mycotoxin on animal susceptibility to infectious diseases. Indeed, resistance to Escherichia coli infection is antibody mediated, and we have recently shown that ingestion of FB1 increases the intestinal colonization by these bacteria (Oswald et al., 2003). By contrast, resistance to intracellular parasites such as Trypanosoma cruzi require the development of cellular immunity, and recent data indicate that mice fed with FB1 are more resistant to this parasite than mice fed with a control mycotoxin-free diet (Dresden Osborne et al., 2002).
In conclusion, we found that the ingestion of FB1 alters the cytokine production and decreases the vaccinal antibody response. This could have important consequences in human and animal health as the breakdown in vaccinal immunity and may lead to the occurrence of disease even in properly vaccinated animals and/or humans. Considering the high levels of FB1 that may be present in animal feeds and human food preparations (IPCS, 2000; Murphy et al., 1993), further studies are needed to determine the minimal concentration that modulates the immune response. Epidemiological studies are also needed to assess the extent to which fumonisins are involved in the failure of vaccinal immunity in humans and animals.
ACKNOWLEDGMENTS
This work was supported by funds from the region Midi-Pyrénées, France (DAER-Rech/99008345), and the Transversalité INRA (mycotoxines-P00263). Dr. I. Taranu was the recipient of an INRA post-doctoral fellowship. D. Marin was supported by the "Réseau Formation-Recherche," a bilateral project between France and Romania granted by the "Ministère de l'éducation Nationale" and the "Ministère de la Recherche" (Paris, France). S. Boulet was supported by a fellowship from the "Ministére de L'éducation Nationalé." Thanks are also due to Dr. Neil Ledger for his help with the English text.
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