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Arachidonic acid–and docosahexaenoic acid–enriched formulas modulate antigen-specific T cell responses to influenza virus in neonatal piglets^1,2,3

¹ From the Nutritional Immunology & Molecular Nutrition Laboratory, Department of Human Nutrition Foods and Exercise (JB-R, AJG, AMN, KAR, JK, and RH), and the Department of Animal and Poultry Sciences (CMW and MA), Virginia Tech University, Blacksburg, VA, and Mead Johnson Nutritionals/Bristol Myers Squibb, Evansville, IN (DR)

² Supported by a grant from Mead Johnson Nutritionals/Bristol Myers Squibb (to JBR) and by the Nutritional Immunology & Molecular Nutrition Laboratory.

³ Reprints not available. Address correspondence to J Bassaganya-Riera, Nutritional Immunology & Molecular Nutrition Laboratory, 319 Wallace Hall, Virginia Tech University, Blacksburg, VA 24060. E-mail: jbassaga{at}vt.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: Whereas the immunomodulatory effects of feeding either arachidonic acid (AA) or docosahexaenoic acid (DHA) separately have been previously investigated, little is known about the immunomodulatory efficacy of AA or DHA when they are fed in combination as infant formula ingredients.

Objective: The objective of this study was to investigate the ability of AA- and DHA(AA/DHA)-enriched infant formula to modulate immune responses in the neonate in response to an inactivated influenza virus vaccine.

Design: Neonatal piglets (n = 48) were weaned on day 2 of age and distributed into 16 blocks of 3 littermate piglets each. Within each block, piglets were randomly assigned to a control formula, AA/DHA-enriched formula (0.63% AA and 0.34% DHA), or sow milk for 30 d. On day 9, 8 blocks of piglets were immunized with an inactivated influenza virus vaccine. On days 0, 9, 16, 23, and 30 after weaning, we measured influenza virus–specific T cell proliferation and phenotype of T subsets in peripheral blood. A delayed-type hypersensitivity reaction test was administered on day 28. Cytokine messenger RNA expression was determined by quantitative real time reverse transcriptase–polymerase chain reaction on day 30.

Results: The influenza virus–specific CD4⁺ and CD8⁺ T cell ex vivo lymphoproliferative responses were significantly lower on day 23 after immunization in piglets receiving dietary AA/DHA supplementation and sow milk than in those receiving the unsupplemented control formula. The immunomodulatory effects of AA/DHA-enriched formulas were consistent with up-regulation of interleukin 10 in peripheral blood mononuclear cells.

Conclusion: Overall, it appears that the AA/DHA-enriched formula modulated antigen-specific T cell responses in part through an interleukin 10–dependent mechanism.

Key Words: Arachidonic acid • docosahexaenoic acid • immunity • growth • piglets

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The nutritional requirements of essential fatty acids (ie, linoleic and -linolenic acids) for growth and development are well established. Breast milk is the best source of nutrition for young infants; however, when it is not available, infant formulas are the only source of nutrition for many infants during the first 4–6 mo of life. Therefore, the minimum concentrations of 29 nutrients, including linoleic acid, are tightly regulated in the United States under the provisions of the Infant Formula Act of 1980, its 1986 amendments, and the Code of Federal Regulations (21 CFR). In addition, new ingredients added to infant formulas are regulated under the Food, Drug and Cosmetic Act of 1938. Matching the ingredient composition of infant formulas with the nutritional composition of human milk or breastfeeding performance is a widely used strategy for improving infant formulas.

Human breast milk contains, in addition to essential fatty acids, other polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA), docosahexaenoic acid (DHA), and conjugated linoleic acid (CLA). The presence of these PUFAs in human milk suggests that they may be required for optimal health of infants and children. Whereas AA and DHA can be synthesized by term and preterm infants born at 33 wk gestation (1), the limited rates of desaturation and elongation from linoleic and linolenic acids to AA and DHA, respectively, in young infants may not meet the needs for optimal infant growth and development (2-5). Hence, whether long-chain PUFAs are essential or conditionally essential nutrients required for optimal infant nutrition remains controversial (1, 6).

Supplementation of infant formulas with AA and DHA is believed to favorably modulate the development of the nervous system (7), the retina (8), the auditory system (9), and the digestive system (10). Some preliminary data also suggest a benefit of AA and DHA on immune system development and function in the early neonatal period. For instance, Field et al (11) showed that, in preterm infants receiving AA and DHA supplementation, the lymphocyte populations, the phospholipid composition of lymphocytes, and markers of immune cell maturity were similar to those in breastfed infants. Moreover, the concentrations of interleukin 10 (IL-10) in infants fed AA- and DHA- (AA/DHA) enriched formula resembled those in breastfed infants, whereas IL-10 production was suppressed in infants fed the control formula (11). Even though the effects of dietary AA and DHA supplementation on immune function were investigated previously in adults and found to elicit opposing immunomodulatory actions (12, 13), little is known about their immunomodulatory efficacy when fed in combination in the early neonatal period. The primary goal of the current study was to determine whether AA/DHA-enriched formulas would modulate adaptive T cell responses in the neonate. To achieve this goal, we used a neonatal piglet model, because piglets are comparable in size, physiology, and immunity to human infants.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Experimental design
Ten pregnant (primiparous or first parity) Yorkshire x Landrace sows were purchased from a herd with low or null influenza virus antibody titers and transported to the infant formula testing facilities at Virginia Tech University. The original breeding herd was also free of other pig pathogens, such as porcine respiratory and reproductive virus, Mycoplasma hyopneumoniae, pseudorabies virus, Salmonella typhimurium, or porcine parvovirus. The sows were housed in a temperature- and humidity-controlled facility. Airflow was adjusted on the basis of the sows’ comfort level and the age of the piglets. Before parturition, sows were fed 2.00–2.20 kg of sow ration each day at 0900. After parturition, sows were twice daily (ie, 0900 and 1700) fed increasing amounts of sow ration starting at 2.5 kg/feeding on day 1 and ending at 3.84–4.20 kg/feeding on day 30.

The temperature in the infant formula testing facility was set between 70 and 72 °F throughout the course of the study, and local heat was provided by heat lamps. In the adjustable lactation crates, zone heating was provided by heat lamps, which were turned on while sows were farrowing. In addition, the umbilical cords were tied off and disinfected with iodine immediately after birth. Between 6 and 24 h after parturition, neonatal piglets were given 0.5 cc penicillin intramuscularly, ear tagged, and weighed. Naxcel (0.5 cc at 5 mg/kg) was administered intramuscularly every other day starting on day 1. On day 2, 48 neonatal piglets with an initial body weight of 1.5–1.8 kg were selected from a total of 92 neonatal piglets born in the infant formula testing facility. All the piglets were given 1 cc iron dextran (100 mg; intramuscularly) to prevent anemia, and, in line with the policy of reducing the number of animals used for research, the piglets as well as the sows not included in this study were returned to a production facility. Sixteen blocks of 3 pigs each were designed on the basis of litter of origin, maternal concentration of antiinfluenza virus titers, initial body weight, age, and sex.

Piglets within the blocks were randomly assigned to the 3 treatment groups—unsupplemented formula, AA/DHA-enriched formula, or suckling from the sow as a model for breastfeeding. Forty-eight piglets were weaned from the sows at age 2 d; 32 piglets were penned individually in stainless steel cages with appropriate bedding and heat lamps and fed the experimental formulas through a semiautomatic feeding system, consisting on a modification of the method first proposed by McClead et al (14). Sixteen piglets (littermates from the formula-fed piglets) were maintained with 2 sows in 2 separate lactation crates (8 piglets/sow). The individual cages and the lactation crates were cleaned and disinfected daily. Piglets were fed experimental liquid formulas ad libitum from sterilized enteral containers connected through vinyl tubing to a cleft palate nipple. The containers were refilled 3–5 times/d. The survival rate for the piglets selected for this study was 100%. During the entire experiment, piglets, feeders, and waste formula were weighed daily. The sow milk intake was not measured because the only available method (eg, the weigh-suckle-weigh method) could have altered the immunologic variables under investigation.

All of the animals were handled according to the practices of animal care established by the Virginia Tech and Bristol-Myers Squibb animal care and use committees. The animal care and use protocols met or exceeded the guidelines of the National Institutes of Health Office of Laboratory Animal Welfare and the policy of the Public Health Service.

Dietary treatments
The dry formula was prepared by mixing 150 g of a concentrated, fat-free basal mix with 50 g fat blend by using a dry formula mixer. For the AA/DHA-enriched formula, the AA and DHA were mixed with the fat blend under a nitrogen blanket before the fat blend was mixed with the rest of the ingredients. The dry infant formula (200 g; Table 1) was reconstituted with 1 L deionized sterile water in a heavy-duty homogenizer for 2 min. The reconstituted formula provided 4074 kJ or 970 kcal/kg. It is estimated that human breast milk provides a mean value of 670 kcal/kg from 1–24 mo of lactation (15, 16). This formula met or exceeded the requirements for growing piglets as set by the National Research Council (17), and it can support the growth of piglets from day 1 to day 30 of age. The piglet formula was prepared 3–5 times/d and stored at 4 °C, and the semiautomatic feeders were refilled every 4 h. The ratio of AA to DHA in the enriched formula was maintained at 1.9:1. AA was provided in the form of a triacylglycerol oil source rich in AA (Martek Biosciences Corp, Columbia, MD) generated by the fungus Morteriella alpina, which contains AA at 40% of fatty acids. Other fatty acids found in the source of AA include palmitic acid (5–15%), stearic acid (10–20%), oleic acid (12–39%), linoleic acid (5–15%), and linolenic acid (2.5–5%). DHA was provided in the form of a triacylglycerol oil source rich in DHA that is produced commercially (Martek Biosciences) from Crypthecodinium cohni, a marine dinoflagelate, which contains DHA as 40% by weight of fatty acids, with the balance being myristic acid (10–20%), palmitic acid (15–20%), oleic acid (10–30%), and linoleic acid (1–5%). In addition to the fungal or algal oils, both products contain high-oleic sunflower oil, tocopherols, and ascorbyl palmitate as ingredients. Both sources of AA and DHA used in this study are Generally Recognized as Safe (GRAS) by the Food and Drug Administration (GRAS Notice no. GRN 000041). GRAS status means that a food ingredient is not anticipated to cause harm at the approved concentrations.

Harvesting of peripheral blood mononuclear cells
On days 0, 9, 16, 23, and 30 of the study, whole blood was obtained by vena cava puncture with sterile, heparinized, 10-mL evacuated tubes. Peripheral blood mononuclear cells (PBMCs) were isolated by using a previously described gradient centrifugation procedure (21). Briefly, PBMCs were isolated by overlaying lymphoprep (Mediatech, Herndon, VA) with whole blood diluted 1:4 in phosphate-buffered saline (PBS). Mononuclear cells located in the interface between the diluted plasma and the lymphoprep were recovered by using a sterile Pasteur pipette. PBMCs were washed twice with PBS and resuspended in complete medium. Complete medium was prepared by supplementing RPMI-1640 with 25 mmol HEPES buffer/L (Sigma, St. Louis, MI), 100 units penicillin/mL (Sigma), 0.1 mg streptomycin/mL (Sigma), 5 x 10^–5 mol 2-mercaptoethanol/L (Sigma), 1 mmol essential amino acids/L (Mediatech), 1 mmol nonessential amino acids/L (Sigma), 2 mmol L-glutamine/L (Sigma), 1 mmol sodium pyruvate/L (Sigma) and 10% fetal bovine serum. Media pH was measured with a pH meter (Orion Research Inc, Beverly, MA) and adjusted to 7.4 with addition of a solution of 7.5% sodium bicarbonate (Fisher Scientific, Pittsburgh, PA). The remaining peripheral blood was used to determine the total white blood cell counts by using a single-particle counter (Coulter Z1; Beckman Coulter Corp, Miami, FL). Differential counts were performed by using flow cytometry based on forward and side scatter after the lysis of red blood cells.

Proliferation assays
The proliferation assays were performed on PBMCs isolated from all of the piglets on days 0, 9 (before the vaccination), 16, 23, and 30 after weaning. To measure overall lymphocyte proliferation, we used a lymphocyte blastogenesis test based on the incorporation of titrated thymidine. Briefly, flat-bottomed, 96-well microtiter plates (Falcon 3072; Becton Dickinson, Lincoln Park, NJ) were seeded with 100 µL PBMCs at 2 x 10⁶ cells/mL and 100 µL of media alone (nonstimulated wells), media containing the influenza virus antigens (20 µg/mL), or media containing concanavalin A (ConA; 5 µg/mL) (Sigma) as an internal proliferation control. Preliminary validation assays using ConA or influenza antigen at 2.5, 5, 10, and 20 µg/mL were conducted to assess the optimal lymphocyte stimulation under the current experimental conditions and within the genetic background and age of the pigs. Plates were incubated for 5 d at 37 °C in a 5% CO₂ humidified atmosphere. After 5 d of culture, 0.5 µCi methyl-[³H] thymidine (specific radioactivity: 6.7 Ci mmol^–1; Amersham Life Science, Arlington Heights, IL) in 10 µL medium was added to each well, and the plates were incubated for an additional 20 h. Twenty hours after thymidine addition, cells were harvested onto glass fiber filters with a Combicell harvester (Skatron Instruments, Sterling, VA), and incorporated radioactivity was measured by liquid scintillation counting (LS 6500; Beckman Instruments, Palo Alto, CA). Overall lymphocyte proliferation results were expressed as stimulation indexes, which were calculated by dividing the counts per minute of antigen-stimulated wells by the counts per minute of nonstimulated wells.

To determine subset-specific proliferation, PBMCs were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE; Molecular Probes, Eugene, OR), a fluorescent dye used for tracking cell division. CFSE is an ester that diffuses into cells, where it reacts with amine groups, becoming fluorescent. The label is stably retained by proteins and, after cell division, it is equally distributed between daughter cell populations. Briefly, 25 x 10⁷ cells were incubated for 15 min at room temperature in 1 mL complete RPMI containing 1 mmol CFSE/L. After 2 extra washes, cells were resuspended in complete RPMI and enumerated, and the cell concentration was normalized to 25 x 10⁶/mL. Cells were cultured with media alone or stimulated with influenza virus antigens or ConA. Cells were harvested on day 5, seeded in 96 round-bottom microtiter plates, and stained with anti-pig CD4 (clone 74–12-4), anti-pig CD8 (clone 76–2-11), anti-pig-CD8ß (clone PG164A), anti-pig-IgM (clone PG145A), and appropriate secondary antibody combinations. Data acquisition was performed in a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).

Immunophenotyping of peripheral blood mononuclear cells
PBMCs were labeled with anti-pig primary antibodies as previously described (21). The primary antibodies were phycoerythrin-labeled anti-pig-CD4 (clone 74–12-4), biotinylated immunoglobulin G (IgG)2a mouse anti-pig-CD8 (clone 76–2-11) (22), IgG2a mouse anti-pig-CD8ß (clone PG164A), IgG1 mouse anti-pig-CD45RA (clone PG96A), IgG2b mouse anti-pig-SWC3 (clone 74–22-15), and IgM mouse anti-pig-IgM (clone PG145A) (all: VMRD Inc, Pullman, WA), IgM mouse anti-pig-CD45RC (clone MIL15) (Serotec, Oxford, United Kingdom), and appropriate secondary antibody combinations (Southern Biotechnology Associates, Birmingham, AL). Flow cytometric data acquisition and analyses were performed as previously described (21, 23).

Necropsy procedures and tissue collection and storage
During the course of the experiment, we monitored piglets for signs of hypothermia, lethargy, and enteric disease according to a disease activity index. On day 30 of the study, piglets were euthanized by an intravenous injection of Sleepaway (Fort Dodge Laboratories, Fort Dodge, IA), a sodium pentobarbital solution. Then, the mediastinal lymph nodes were collected in sterile conditions. All organs (including the heart, lungs, thymus, brain, liver, stomach, pancreas, spleen, kidneys, duodenum, jejunum/ileum, and colon) were excised and weighed, and samples were snap-frozen in liquid nitrogen for subsequent storage at –80 °C. Ileal samples were also collected in tissue freezing medium in cryomolds and frozen at –80 °C for immunohistochemical evaluation of mucosal secretory IgA and in RNAlater solution (Ambion, Austin, TX) for RNA isolation and gene expression analyses. Samples of spleen, thymus, ileum, and colon were also fixed in a phosphate-buffered neutral 10% formalin solution for subsequent histologic analyses.

Delayed-type hypersensitivity reaction for influenza virus antigens
DTH represents a widely accepted in vivo measurement of cell-mediated immunity. The current study examined the effects of infant formula and influenza virus vaccination on the induction of cell-mediated, influenza virus–specific responses in vivo. Briefly, on day 28 of the study, the ear was disinfected with a 70% ethanol solution. All pigs received an intradermal injection of influenza virus antigen (100 µg in 150 µL) or PBS (negative control for nonspecific inflammation due to physical irritation or injection) at different locations on the dorsal aspect of the right ear. Injection sites were examined at 0 and 24 h for erythema, edema, and induration. In addition, induration was measured by using calipers and recorded in millimeters. The percentage increase in skin thickness was calculated as follows: [(vaccine antigen 24 h –vaccine antigen 0 h)/vaccine antigen 0 h] –[(PBS 24 h –PBS 0 h)/PBS 0 h]. DTH to influenza virus antigens has previously been used to investigate the immunomodulatory actions of nutrients in vivo (24).

Real-time, quantitative reverse transcriptase–polymerase chain reaction
For RNA isolation, PBMCs and spleen samples were stored in RNAlater (Ambion), and total RNA was isolated by using the Mini RNA isolation kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Total RNA (1 µg) from each sample was used to generate complementary DNA (cDNA) template by using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). The total reaction volume was 20 µL. The reaction was incubated as follows in an iCycler iQ thermal cycler (Bio-Rad): 5 min at 25 °C, 30 min at 42 °C, 5 min at 85 °C, and hold at 4 °C. Controls were also performed with no RNA template (no template) and by omitting the reverse transcriptase enzyme (no RT). The PCR primer pairs were designed, on the basis of sequences previously published by GeneBank, with the use of the OLIGO 6 primer design software (version 5; Molecular Biology Insights, Cascade, CO), and predicted amplicon sequences were checked online by using a BLAST search. The PCR primer pair sequences, annealing temperatures, accession numbers, and PCR product lengths are outlined in Table 2.

Statistical analysis
Analysis of variance (ANOVA) was used to determine the main effects of the dietary treatment or the immunization status and the interaction between dietary treatment and immunization status. ANOVA was performed by using the general linear model procedure in SAS software (version 9.1.3) (26) as previously described (19, 25). P < 0.05 was considered to be significant. Before the influenza virus immunizations, data were analyzed as a repeated-measures randomized complete block design. For data collected at the end of the study as a single timepoint measurement, postimmunization data were analyzed as a 2 x 3 factorial arrangement of treatments within a split-plot design. In the model, 3 pigs within 1 block was the experimental unit for the dietary treatment (subplot), and the blocks of pigs within each immunization status were the experimental units for immunization treatment (whole plot). The whole-plot error (ie, error A) was block within immunization status (ie, 7 df), and the subplot error (ie, error B) was the residual df after accounting for the dietary treatment variance and the variance for the interaction between dietary treatment and infective status (ie, 35 df). The statistical model utilized was given in the following equation:

(1)

where µ was the general mean, Immunization_i was the main effect of the i_th level of the vaccine effect, Diet_j was the main effect of the j_th level of the dietary effect, (Immunization x Diet)_ij was the interaction effect between immunization and diet, and errors A and B represented the random errors for the whole plot and the subplot, respectively.

For analyzing the measurements on the kinetics of the immune response and body weight data over time, we used a 3-factor repeated-measures ANOVA. For this analysis, in addition to the main effects of diet, vaccine, and the 2-factor interaction between diet and immunization (as shown above), the model included the main effect of time, the diet x time and immunization x time interactions, and the 3-factor interaction (diet x immunization x time). The ANOVA was followed by Sheffe’s post hoc multiple comparison analyses.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Effect of an arachidonic acid– and docosahexaenoic acid–enriched formula on body weight and organ weight
Body weight and formula intake were measured daily to determine the effect of AA/DHA-enriched formula on growth and appetite. All piglets within a block were very similar (ie, sex, litter, and initial body weight) on day 0 of the study. No significant differences were found in body weight between treatment groups during the first 2 d of the study. However, the sow-reared piglets grew significantly faster than did piglets fed the control or AA/DHA-enriched formulas between days 3 and 15 of the study (Figure 1). The influenza virus vaccination had no effect on piglet body weight throughout the study (Figure 1B and C).

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Least-squares mean (±SEM) body weights before (days 0–9; A) and after (days 10–30) immunization treatment with an influenza virus vaccine in nonimmunized (B) and immunized (C) piglets fed control formula (), arachidonic acid–and docosahexaenoic acid–(AA/DHA; ) enriched formula, or sow milk (). Piglets were immunized with an inactivated influenza virus vaccine on day 9 of the experiment. n = 16 from day 0 to day 9; n = 8 from day 10 to day 30. The error bars at younger ages are smaller than the symbols and cannot be seen in the figure. Before the immunization treatment, data were analyzed as a repeated-measures randomized complete-block design. After the immunization treatment, data were analyzed as a repeated-measures factorial arrangement within a split-plot design. Immunization status represents the whole plot, and dietary treatments represent the subplot. The experimental unit for the whole plot was a block of 3 littermate piglets, and that for the subplot was 3 piglets within 1 block. The 3-factor interaction (diet x immunization x time) was not significant. However, the 2-factor diet x time interaction was significant (P < 0.0001). Scheffe’s multiple-comparisons test was used after ANOVA. *Significant differences among treatments attributed to the effects of diet over time, P < 0.05.

Table 3

Table 4

Table 5

View this table: [in this window] [in a new window]   TABLE 4 Effect of dietary treatments on total numbers of CD4, CD4CD8, and CD8 peripheral blood lymphocytes in neonatal piglets fed control, arachidonic acid– (AA) and docosahexaenoic acid– (DHA) enriched formula, or sow milk before and after immunization with an influenza virus vaccine1

View this table: [in this window] [in a new window]   TABLE 5 Effect of dietary treatments on total numbers of CD8ßCD45RChiCD45RAlo, CD8ßCD45RChiCD45RAhi, CD8ßCD45RCloCD45RAlo, and CD8ßCD45RCloCD45RAhi peripheral blood lymphocytes in neonatal piglets fed control, arachidonic acid– (AA) and docosahexaenoic acid– (DHA) enriched formula, or sow milk before and after immunization with an influenza virus vaccine1

Table 6

Figures 2

3

4

View larger version (13K): [in this window] [in a new window]   FIGURE 2. Least-squares mean (±SEM) antigen-specific proliferation of CD4+ T cells against influenza virus in cultures of peripheral blood mononuclear cells recovered from nonimmunized (A) and influenza virus–immunized (B) piglets fed control formula (), arachidonic acid–and docosahexaenoic acid–(AA/DHA) enriched formula (), or sow milk (). Piglets were immunized with an inactivated influenza virus vaccine on day 9 of the experiment. n = 16 from day 0 to day 9; n = 8 from day 10 to day 30. The error bars in nonimmunized piglets or at early timepoints are smaller than the symbols and cannot be seen in the figure. Before the immunization treatment, data were analyzed as a repeated-measures randomized complete-block design. After the immunization treatment, data were analyzed as a repeated-measures factorial arrangement within a split-plot design. Immunization status represents the whole plot, and dietary treatments represent the subplot. The experimental unit for the whole plot was a block of 3 littermate piglets, and that for the subplot was 3 piglets within 1 block. The 3-factor interaction (diet x immunization x time) was not significant. However, the 2-factor immunization x time (P < 0.001) and immunization x diet (P < 0.04) interactions were significant. Scheffe’s multiple-comparisons test was used after ANOVA. *Significant differences among treatments attributed to the interaction between immunization and diet, P < 0.05.

View larger version (12K): [in this window] [in a new window]   FIGURE 3. Least-squares mean (±SEM) antigen-specific proliferation of CD8+ T cells against influenza virus in cultures of peripheral blood mononuclear cells recovered from nonimmunized (A) and influenza virus–immunized (B) piglets fed control formula (), arachidonic acid–and docosahexaenoic acid–(AA/DHA) enriched formula (), or sow milk (). Piglets were immunized with an inactivated influenza virus vaccine on day 9 of the experiment. n = 16 from day 0 to day 9; n = 8 from day 10 to day 30. The error bars in nonimmunized piglets or at early timepoints are smaller than the symbols and cannot be seen in the figure. Before the immunization treatment, data were analyzed as a repeated-measures randomized complete-block design. After immunization, data were analyzed as a repeated-measures factorial arrangement within a split-plot design. Immunization status represents the whole plot, and dietary treatments represent the subplot. The experimental unit for the whole plot was a block of 3 littermate piglets, and that for the subplot was 3 piglets within 1 block. The 3-factor interaction (diet x immunization x time) was not significant. However, the 2-factor diet x time (P < 0.01) and immunization x time (P < 0.0001) interactions were significant. Scheffe’s multiple-comparisons test was used after ANOVA. *Significant differences among treatments attributed to the effects of diet and immunization over time, P < 0.05.

View larger version (12K): [in this window] [in a new window]   FIGURE 4. Least-squares mean (±SEM) antigen-specific proliferation of CD8ß+ T cells (ie, TCRßCD8ß) against influenza virus in cultures of peripheral blood mononuclear cells recovered from nonimmunized (A) and influenza virus–immunized (B) piglets fed control formula (), arachidonic acid–and docosahexaenoic acid–(AA/DHA) enriched formula (), or sow milk (). Piglets were immunized with an inactivated influenza virus vaccine on day 9 of the experiment. n = 16 from day 0 to day 9; n = 8 from day 10 to day 30. The error bars in nonimmunized piglets or at early timepoints are smaller than the symbols and cannot be seen in the figure. Before the immunization treatment, data were analyzed as a repeated-measures randomized complete-block design. After the immunization treatment, data were analyzed as a repeated-measures factorial arrangement within a split-plot design. Immunization status represents the whole plot, and dietary treatments represent the subplot. The experimental unit for the whole plot was a block of 3 littermate piglets, and that for the subplot was 3 piglets within 1 block. The 3-factor interaction (diet x immunization x time) (P < 0.002) and the 2-factor immunization x diet (P < 0.01), diet x time (P < 0.0001), and immunization x time (P < 0.0005) interactions were significant. Scheffe’s multiple-comparisons test was used after ANOVA. *Significant differences among treatment groups, P < 0.05.

Figure 5

View larger version (22K): [in this window] [in a new window]   FIGURE 5. Least-squares mean (±SEM) delayed-type hypersensitivity (DTH) responses in the ear of nonimmunized () and influenza virus–immunized () piglets fed control formula, arachidonic acid–and docosahexaenoic acid–(AA/DHA) enriched formula, or sow milk. n = 8. Piglets were immunized with an inactivated influenza virus vaccine on day 9 of the experiment; the DTH antigens were administered on day 28 and measured on day 29 of the experiment. PBS, phosphate-buffered saline. DTH values (n = 8) are calculated as % increase = [(vaccine antigen 24 h –vaccine antigen 0 h)/vaccine antigen 0 h]–[(PBS 24 h –PBS 0 h)/PBS 0 h]. Data were analyzed as a 2 x 3 factorial arrangement (ie, 2 immunization status and 3 dietary treatments) within a split-plot design. Immunization status represents the whole plot, and dietary treatments represent the subplot. The experimental unit for the whole plot was a block of 3 littermate piglets, and that for the subplot was 3 piglets within 1 block. The 2-factor immunization x diet interaction and the main effect of the diet were not significant, but the main effect of immunization was significant, P < 0.0001. Scheffe’s multiple- comparisons test was used after ANOVA.

Figure 6

View larger version (21K): [in this window] [in a new window]   FIGURE 6. Least-squares mean (±SEM) interleukin 10 mRNA expression in lymphocytes of piglets fed control formula, arachidonic acid–and docosahexaenoic acid–(AA/DHA) enriched formula, or sow milk on day 30 of the study. n = 8. Data were analyzed as a 2 x 3 factorial arrangement (ie, 2 immunization status and 3 dietary treatments) within a split-plot design. Immunization status (, nonimmunized; , immunized) represents the whole plot, and dietary treatments represent the subplot. The experimental unit for the whole plot was a block of 3 littermate piglets, and that for the subplot was 3 piglets within 1 block. The 2-factor immunization x diet was not significant. However, the main effect of the diet (P < 0.04) and the main effect of immunization (P < 0.02) were significant. Post hoc analyses comparing the 3 dietary treatment groups indicated that interleukin 10 expression in piglets fed AA/DHA-enriched formula and sow-suckled piglets was significantly (P < 0.05) greater than that in control formula-fed piglets. Scheffe’s multiple-comparisons test was used after ANOVA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We found that the body weight and formula intake did not differ significantly between the piglets fed the control formula and those fed the AA/DHA-enriched formula. Thus, immunologic differences between these 2 groups could not be explained on the basis of growth- or energy intake–related differences. These results with respect to body weight are consistent with previous reports indicating that the detrimental effects of DHA in growth can be overcome by feeding it in combination with AA (30, 31). The inclusion of a third group of sow-reared piglets was intended to provide a means of comparing the 2 experimental formulas with a reference group resembling breastfeeding. In this regard, infant formula is defined by law as a food for use in infants that simulates human milk or that is suitable as a complete or partial substitute of human milk. In line with the greater body weight and length associated with breastfeeding, we found that sow-reared piglets grew at a faster rate from days 3 to 15 of the study, which can be explained by the fact that the energy content of sow milk during the initial days of lactation is greater than that of the experimental formulas used in the current study (1363 and 970 kcal/kg, respectively) (32).

The kinetics of cell-mediated immune responses to influenza virus immunization, as measured by antigen-specific lymphoproliferation, followed the typical pattern of a primary antigen-specific response to immunization, challenge, or both (19, 21). Specifically, the response was detectable on day 16 (ie, day 7 after immunization), peaked on day 23 (ie, day 14 after immunization), and returned to nearly baseline on day 30 (ie, day 21 after immunization). On day 23, the proliferative abilities of CD4⁺, CD8⁺, and CD8ß⁺ T cells to influenza virus stimulation were significantly greater in immunized piglets fed the control formula than in immunized piglets fed AA/DHA-enriched formula or sow-reared vaccinated piglets. Thus, it appears that the AA/DHA-enriched formula and sow milk may have suppressed influenza virus–specific T cell responses on day 23 after feeding.

Porcine CD8⁺ lymphocytes can be subdivided phenotypically into 5 subpopulations on the basis of T cell receptor (TCR) and coreceptor expression: 1) CD8⁺ TCR⁺CD4^–; 2) CD8⁺TCRß⁺CD4⁺; 3) CD8⁺CD3^–CD16⁺ (a subset of natural killer cells); 4) CD8⁺TCRß⁺CD4^– (potentially immunoregulatory); and 5) CD8ß⁺TCRß⁺CD4^– (cytotoxic) (21). Conversely, the porcine CD4⁺ T cell population is more homogeneous and can be subdivided into 2 populations: 1) CD8⁺TCRß⁺ and 2) CD8^–TCRß⁺ (33). CD4⁺CD8^–TCRß⁺ (Figure 2) and CD8ß⁺TCRß⁺CD4^– (Figure 4) correspond to human CD4⁺ and CD8⁺ T cells, respectively. The modulatory actions of AA/DHA observed ex vivo were consistent with important numerical differences between groups in the DTH reaction test, an in vivo assessment of cell mediated immunity, although the numerical differences in the DTH response were not statistically significant. It is interesting that the DTH response observed in the AA/DHA-enriched group was similar to that observed in the sow milk–fed group.

AA and DHA are known to elicit opposing immunomodulatory actions (12, 13). Even though the ratio of AA to DHA in the formula, which is consistent with worldwide ranges of AA and DHA in breast milk, was favorable to AA (1.9:1), the final immunologic outcome (ie, suppressed immune responsiveness) was more consistent with the previously reported immunosuppressive effects of DHA (13), which suggested that DHA may have neutralized the ability of AA to enhance influenza virus–specific immune responses. AA/DHA-enriched formulas decreased antigen-specific responses against influenza virus on day 23, but it is important to note that AA/DHA-enriched formulas have shown no adverse effects on the immune response in infants (11, 34).

In a recent study from our laboratory that examined the nutritional interaction between CLA and n–3 PUFAs in experimental inflammatory bowel disease, we found that, when CLA was fed in combination with n–3 PUFAs, n–3 PUFAs abrogated the beneficial effects of CLA in clinical activity by blocking CLA-induced activation of peroxisome proliferator–activated receptor (PPAR-; 35). In this regard, DHA has been found to suppress transactivation of PPAR- and expression of the PPAR-–responsive gene, CD36, by the synthetic PPAR- agonist ciglitazone in a colon tumor cell line (36). Thus, DHA may have blocked the ability of AA to modulate immune function through a similar molecular mechanism.

It is intriguing that the levels of lymphoproliferation in piglets fed AA/DHA-enriched formula did not differ significantly from those in sow-reared piglets. In this regard, it is known that the prevalences of atopies, eczema, and food allergies in infants and children have risen in parallel with the decrease in the practice of breastfeeding. We propose that AA and DHA may prevent or ameliorate autoimmune and allergic reactions in infants by down-modulating T cell responses. In support of this hypothesis, we found that IL-10 mRNA was upregulated on day 30 in piglets fed AA/DHA-enriched formula and sow-reared piglets. IL-10 is an antiinflammatory cytokine produced by induced regulatory T cells, which has major suppressive effects on immune and inflammatory responses, and its absence or suppression in an area of the body constantly exposed to antigens (ie, intestine) leads to chronic inflammation (37). The upregulated IL-10 expression observed in piglets fed AA/DHA-enriched formula is consistent with the findings of a clinical study indicating that IL-10 production was lower in preterm infants fed control formula than in those fed an AA/DHA-enriched formula or human breast milk (11). However, because IL-10 expression was measured systemically in the current study, when the antigenic load is limited or null, the benefits of this effect are not clear, whereas potential disadvantages associated with suppressed systemic adaptive immune responses may include decreased resistance against bacterial or viral infection (or both). These findings suggest that future studies should aim at assessing the role of AA/DHA-enriched formula in preventing or ameliorating allergies, autoimmunity, and intestinal inflammation. Essentially, these data show that both the supplementation of the diet with AA and DHA and the feeding of sow milk during the neonatal period modulated antigen-specific T cell responses to an inactivated influenza virus and up-regulated IL-10 expression.

	ACKNOWLEDGMENTS

 
JB-R, RH, and DR designed the experiments. JB-R and RH wrote the manuscript, conducted the isolation of peripheral blood mononuclear cells and lymphoproliferation assays, managed the logistics of the animal research, and performed the statistical analyses. AJG, AMN, and JK contributed to the animal research, laboratory assays, and data entry. MA and CMW contributed to the animal research and necropsy procedures. KAR contributed to the laboratory analyses. None of the authors had any personal or financial conflict of interest.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Uauy R, Mena P, Wegher B, Nieto S, Salem N Jr. Long chain polyunsaturated fatty acid formation in neonates: effect of gestational age and intrauterine growth. Pediatr Res 2000;47:127–35.[Medline]
2. Cunnane SC. The Canadian Society for Nutritional Sciences 1995 Young Scientist Award Lecture. Recent studies on the synthesis, beta-oxidation, and deficiency of linoleate and alpha-linolenate: are essential fatty acids more aptly named indispensable or conditionally dispensable fatty acids? Can J Physiol Pharmacol 1996;74:629–39.[Medline]
3. Mathews SA, Oliver WT, Phillips OT, Odle J, Diersen-Schade DA, Harrell RJ. Comparison of triglycerides and phospholipids as supplemental sources of dietary long-chain polyunsaturated fatty acids in piglets. J Nutr 2002;132:3081–9.[Abstract/Free Full Text]
4. Blanaru JL, Kohut JR, Fitzpatrick-Wong SC, Weiler HA. Dose response of bone mass to dietary arachidonic acid in piglets fed cow milk-based formula. Am J Clin Nutr 2004;79:139–47.[Abstract/Free Full Text]
5. Fleith M, Clandinin MT. Dietary PUFA for preterm and term infants: review of clinical studies. Crit Rev Food Sci Nutr 2005;45:205–29.[Medline]
6. Cunnane SC, Francescutti V, Brenna JT, Crawford MA. Breast-fed infants achieve a higher rate of brain and whole body docosahexaenoate accumulation than formula-fed infants not consuming dietary docosahexaenoate. Lipids 2000;35:105–11.[Medline]
7. Saste MD, Carver JD, Stockard JE, Benford VJ, Chen LT, Phelps CP. Maternal diet fatty acid composition affects neurodevelopment in rat pups. J Nutr 1998;128:740–3.[Abstract/Free Full Text]
8. Suh M, Wierzbicki AA, Lien EL, Clandinin MT. Dietary 20:4n–6 and 22:6n–3 modulates the profile of long- and very-long-chain fatty acids, rhodopsin content, and kinetics in developing photoreceptor cells. Pediatr Res 2000;48:524–30.[Medline]
9. Haubner LY, Stockard JE, Saste MD, et al. Maternal dietary docosahexanoic acid content affects the rat pup auditory system. Brain Res Bull 2002;58:1–5.[Medline]
10. Jarocka-Cyrta E, Perin N, Keelan M, et al. Early dietary experience influences ontogeny of intestine in response to dietary lipid changes in later life. Am J Physiol 1998;275:G250–8.[Medline]
11. Field CJ, Thomson CA, Van Aerde JE, et al. Lower proportion of CD45R0+ cells and deficient interleukin-10 production by formula-fed infants, compared with human-fed, is corrected with supplementation of long-chain polyunsaturated fatty acids. J Pediatr Gastroenterol Nutr 2000;31:291–9.[Medline]
12. Kelley DS, Taylor PC, Nelson GJ, Schmidt PC, Mackey BE, Kyle D. Effects of dietary arachidonic acid on human immune response. Lipids 1997;32:449–56.[Medline]
13. Kelley DS, Taylor PC, Nelson GJ, et al. Docosahexaenoic acid ingestion inhibits natural killer cell activity and production of inflammatory mediators in young healthy men. Lipids 1999;34:317–24.[Medline]
14. McClead RE Jr, Lentz ME, Vieth R. A simple technique to feed newborn piglets. J Pediatr Gastroenterol Nutr 1990;10:107–10.[Medline]
15. Garza C, Butte NF. Energy concentration of human milk estimated from 24-h pools and various abbreviated sampling schemes. J Pediatr Gastroenterol Nutr 1986;5:943–8.[Medline]
16. Prentice AM, Lucas A, Vasquez-Velasquez L, Davies PS, Whitehead RG. Are current dietary guidelines for young children a prescription for overfeeding? Lancet 1988;2:1066–9.[Medline]
17. National Research Council. Nutrient requirements of swine. Washington, DC: National Academy Press, 1998.
18. Wurzer WJ, Planz O, Ehrhardt C, Giner M, Silberzahn T, Pleschka S, Ludwig S. Caspase 3 activation is essential for efficient influenza virus propagation. Embo J 2003;22:2717–28.[Medline]
19. Bassaganya-Riera J, Pogranichniy RM, Jobgen SC, et al. Conjugated linoleic acid ameliorates viral infectivity in a pig model of virally induced immunosuppression. J Nutr 2003;133:3204–14.[Abstract/Free Full Text]
20. Pleschka S, Wolff T, Ehrhardt C, et al. Influenza virus propagation is impaired by inhibition of the Raf/MEK/ERK signalling cascade. Nat Cell Biol 2001;3:301–5.[Medline]
21. Bassaganya-Riera J, Hontecillas R, Zimmerman DR, Wannemuehler MJ. Dietary conjugated linoleic acid modulates phenotype and effector functions of porcine cd8(+) lymphocytes. J Nutr 2001;131:2370–7.[Abstract/Free Full Text]
22. Pescovitz MD, Lunney JK, Sachs DH. Murine anti-swine T4 and T8 monoclonal antibodies: distribution and effects on proliferative and cytotoxic T cells. J Immunol 1985;134:37–44.[Abstract]
23. Bassaganya-Riera J, Hontecillas R, Zimmerman DR, Wannemuehler MJ. Long-term influence of lipid nutrition on the induction of CD8(+) responses to viral or bacterial antigens. Vaccine 2002;20:1435–44.[Medline]
24. Vos AP, Haarman M, Buco A, et al. A specific prebiotic oligosaccharide mixture stimulates delayed-type hypersensitivity in a murine influenza vaccination model. Int Immunopharmacol 2006;6:1277–86.[Medline]
25. Hontecillas R, Wannemeulher MJ, Zimmerman DR, et al. Nutritional regulation of porcine bacterial-induced colitis by conjugated linoleic acid. J Nutr 2002;132:2019–27.[Abstract/Free Full Text]
26. SAS. SAS/STAT User’s guide (Release 6.0.3). Cary, NC: SAS Institute, Inc, 1988.
27. Bailey M, Plunkett F, Clarke A, Sturgess D, Haverson K, Stokes C. Activation of T cells from the intestinal lamina propria of the pig. Scand J Immunol 1998;48:177–82.[Medline]
28. Bassaganya-Riera J, Hontecillas R, Bregendahl K, Wannemuehler MJ, Zimmerman DR. Effects of dietary conjugated linoleic acid in nursery pigs of dirty and clean environments on growth, empty body composition, and immune competence. J Anim Sci 2001;79:714–21.[Abstract/Free Full Text]
29. Yamasaki M, Chujo H, Hirao A, et al. Immunoglobulin and cytokine production from spleen lymphocytes is modulated in C57BL/6J mice by dietary cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid. J Nutr 2003;133:784–8.[Abstract/Free Full Text]
30. Vanderhoof J, Gross S, Hegyi T, et al. Evaluation of a long-chain polyunsaturated fatty acid supplemented formula on growth, tolerance, and plasma lipids in preterm infants up to 48 weeks postconceptional age. J Pediatr Gastroenterol Nutr 1999;29:318–26.[Medline]
31. Vanderhoof J, Gross S, Hegyi T. A multicenter long-term safety and efficacy trial of preterm formula supplemented with long-chain polyunsaturated fatty acids. J Pediatr Gastroenterol Nutr 2000;31:121–7.[Medline]
32. Noblet J, Etienne M. Estimation of sow milk nutrient output. J Anim Sci 1989;67:3352–9.[Abstract/Free Full Text]
33. Hontecillas R, Bassaganya-Riera J, Wilson J, Hutto DL, Wannemuehler MJ. CD4+ T-cell responses and distribution at the colonic mucosa during Brachyspira hyodysenteriae-induced colitis in pigs. Immunology 2005;115:127–35.[Medline]
34. Lucas A, Stafford M, Morley R, et al. Efficacy and safety of long-chain polyunsaturated fatty acid supplementation of infant-formula milk: a randomised trial. Lancet 1999;354:1948–54.[Medline]
35. Bassaganya-Riera J, Hontecillas R. CLA and n–3 PUFA differentially modulate clinical activity and colonic PPAR-responsive gene expression in a pig model of experimental IBD. Clin Nutr 2006;25:454–65.[Medline]
36. Lee JY, Hwang DH. Docosahexaenoic acid suppresses the activity of peroxisome proliferator-activated receptors in a colon tumor cell line. Biochem Biophys Res Commun 2002;298:667–74.[Medline]
37. Strober W, Fuss IJ, Blumberg RS. The immunology of mucosal models of inflammation. Annu Rev Immunol 2002;20:495–549.[Medline]

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