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Nutritional Immunology and Molecular Nutrition Laboratory, Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061;
* Loders Croklaan BV, Lipid Nutrition, Channahon, IL 60410; and
Iowa State University, Ames, IA 50010
3To whom correspondence should be addressed. E-mail: jbassaga{at}vt.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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production in CD4+ T cells. At the molecular level, these cellular immunoregulatory properties were associated with differential patterns of peroxisome proliferator-activated receptor (
and ) mRNA expression between diets in virally infected pigs.
KEY WORDS: • lipid nutrition • lymphoid depletion • growth suppression • conjugated linoleic acid
Nutritional immune modulation has often been examined by identifying changes of the immune system linked to specific dietary regimens. We utilized this experimental approach earlier to investigate the immunomodulatory properties of conjugated linoleic acid (CLA).3 Our initial research led to the discovery that CLA increases the numbers of CD8+ T cells in peripheral blood and thymus of pigs (1,2). By feeding the CLA mixture or purified isomers, Yamasaki and colleagues (3) confirmed our initial findings and further demonstrated that cis-9, trans-11 CLA is responsible for expanding CD8+ T cells in the spleen of mice.
The interactions between CLA and adaptive immunity can be examined further in the context of immune responses to specific viral or bacterial antigens (2,4). By immunizing pigs against pseudorabies virus (Prv), a relative of the human herpes simplex virus, we found that the magnitude of Prv-specific recall responses of CD8+ T cells was greater in immunized pigs fed CLA (4). Even though this finding was consistent with the concept that CD8+ T lymphocytes are crucial effectors of antiviral immunity (5), its implications for viral disease prevention remain unknown.
On the basis of the greater proliferation and granzyme activities of Prv-specific CD8+ T cells and increased numbers of CD8+ T cells in CLA-fed pigs (4), we hypothesized that the nutritional immune modulation of this T-cell subset by CLA would enhance protection from viral disease. To test this hypothesis, we infected pigs with type 2 porcine circovirus (PCV2), a small, negative-sense, single-stranded DNA virus of the family Circoviridae, which causes a disease characterized by clinical signs such as wasting, dyspnea and pallor, and pathological lesions including interstitial pneumonia and lymphoid depletion. Because of the high incidence and rapid spread of PCV2 worldwide, the public health risk associated with xenogeneic transmissions to humans has received some attention (6–8).
Here, we used the PCV2 challenge of pigs as a model of lymphoid depletion and pulmonary pathology to understand the cellular and molecular regulation of viral disease pathogenesis by CLA. Lymphoid depletion is a hallmark immunological lesion associated with immunosuppressive viral infections such as HIV, type-1 human T-cell leukemia virus or PCV2 (9–11). Thus, nutritional interventions targeting these viral diseases must enhance immune protection and attenuate the immunologic and inflammatory damage caused by these viruses. The prevention and/or amelioration of lymphoid depletion is particularly important in the elderly in whom lymphoid regeneration can be impaired by immune senescence (12).
We investigated the kinetics of PCV2-specific immune responses, gene-nutrient interactions and lesion development after a live PCV2 challenge in pigs fed either control or CLA-supplemented diets. We proposed that peroxisome proliferator-activated receptors (PPAR) were likely molecular targets of CLA actions in the immune system (13). Although the modulation of T cell responses by synthetic agonists of PPAR- and - was investigated previously (14–16), this is the first report that examines the differential modulation of PPAR by a natural PPAR- agonist.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Serum samples were collected from 110 pregnant sows of a breeding herd with no history of PCV2-associated disease and low PCV2 antibody titers 21 d before the estimated delivery date. Sera were tested for the presence of antibodies to the open reading frame-2 (ORF2) of PCV2-specific (a recombinant major capsid protein) gene product by indirect ELISA. An indirect fluorescent antibody (IFA) assay was used to detect overall anti-PCV2 antibodies. The offspring from sows with the lowest PCV2 antibody concentrations were purchased and transported to large animal isolation facilities. The original breeding herd was free of other pathogens such as porcine respiratory and reproductive virus, swine influenza virus, Mycoplasma hyopneumoniae, Prv or porcine parvovirus. Pigs (n = 32) with an initial body weight of 4.3–5.1 kg were weaned at 14 d, penned individually, fed the experimental diets with free access to water and handled according to the practices of animal care established by the Institutional Animal Care Committee. Blocks of pigs were designed on the basis of litter of origin, maternal concentration of anti-PCV2 antibodies, initial body weight, age and gender.
Pigs (n = 32, 16 blocks of 2 pigs each) were distributed into two dietary treatments: 1) control (n = 16) or 2) CLA-supplemented diet (n = 16). Pigs were fed the experimental diets for 42 d before the live PCV2 challenge. Before 8 blocks of pigs were challenged, the experimental design was a randomized complete block. After intrainguinal lymph node challenge with PCV2 of 8 blocks of pigs, the design became a 2 x 2 factorial arrangement within a split-plot design. Pigs within block were the experimental units for dietary treatment, and blocks of two pigs each were the experimental units for the infection treatment (i.e., PCV2-infected or uninfected).
Dietary treatments.
Either a 1.33 g CLA/100 g diet or an isoenergetic and isonitrogenous soybean oil–supplemented control diet (Table 1) was randomly allotted to pens within blocks as previously described (1). Pigs had free access to feed for 63 d in four phases (I, wk 1–2; II, wk 3–4; III, wk 5–6; and IV, wk 7–9). Phase feeding is consistent with the NRC nutrient recommendations for pigs (17). Between treatments and within phases, diets were formulated to be isoenergetic and isonitrogenous to avoid energy- and/or protein-derived immunological changes (18,19). Thus, in control diets, 2.21 g CLA source/100 g diet was replaced by 2.21 g soybean oil/100 g diet to keep both the CLA-supplemented and the control diets isoenergetic within phases. The CLA source was alkali-isomerized sunflower oil (Loders Croklaan, Lipid Nutrition, Channahon, IL). The fatty acid composition of similar diets was published previously (20). Diets were formulated to maintain or exceed current recommended nutritional requirements of the NRC (17) for pigs. Pigs, feeders and feed were weighed on a weekly basis before and after challenge to evaluate modifications in growth and feed intake.
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A PK-15 cell suspension was prepared in MEM growth medium at a concentration of 4 x 108 cells/L and inoculated with PCV2 designated ISUVDL 98–15237 (21) at a concentration of 108 50% tissue culture infective doses (TCID50)/L at a ratio of 1 mL of virus per 50 mL of cell suspension. Inoculated cells were seeded into a 165-cm2 tissue culture flask and incubated for 24 h. The resulting cell monolayers were treated with 300 mmol/L D-glucosamine for 20 min and incubated for another 48 h. PCV2-infected PK-15 cells were then frozen/thawed twice, harvested and pelleted by centrifugation at 3000 x g for 10 min. Supernatant containing the propagated virus was titrated and utilized in the viral challenge. The supernatant from uninfected PK-15 cells served as a sham control. The absence of PCV1 in the virus preparation was confirmed by IFA and PCR before the challenge. The virus used in this study represented 12 in vitro passages in cell culture.
Viral challenge.
Challenge inoculum consisted of one dose of lysate of PCV2-infected PK-15 cell supernatant inoculated in the external inguinal lymph nodes (i.e., 1 mL each) at 109.2 TCID50/L. Pigs were distributed into two separate rooms (n = 16 pigs/room). On d 42 of the experiment, pigs in the "challenged room" were inoculated with PCV2 and pigs in the "nonchallenged room" were inoculated with lysate of uninfected PK-15 cells. Biosecurity measures were implemented to prevent contamination of the uninfected room with PCV2.
Production of recombinant capsid protein ORF2 of PCV2.
Antigen for ex vivo restimulation assays [e.g., lymphocyte blastogenesis test, intracellular interferon (IFN)- detection, and PKH67 proliferation assay] and the initial ELISA-based screening of the herd of origin was partially purified protein from Hi-Five insect cells (Invitrogen, Carlsbad, CA) infected with either wild-type baculovirus (AcMNP.wt) or recombinant baculovirus carrying the ORF2 gene of PCV2 (AcMNP.ORF2) (22). At 72 h postinfection, recombinant baculovirus–infected Hi-Five cells were transferred into a 50-mL tube and centrifuged at 800 x g for 10 min. The cell pellet was washed three times with cold PBS and subjected to three rounds of freezing and thawing (-70°C/37°C) to release viral proteins before clarification by centrifugation 800 x g for 10 min. The supernatant was diluted in PBS, laid over 6 mL of 400 g/L sucrose in PBS and centrifuged at 270,000 x g for 6 h using a Ti 41 SW rotor (Beckman, Palo Alto, CA). The viral antigen was clarified by centrifugation at 10,000 x g at 4°C for 10 min. Protein concentrations were measured using the Bio-Rad Bradford-basal protein assay (Hercules, CA).
IFA assay for PCV2.
Serum samples were collected on d 0, 28, 42, 49, 56 and 63 of the experiment. The IFA assay was performed as described previously (21).
Recombinant capsid protein (ORF2)-based ELISA.
Serum samples were collected from the pregnant sows. The indirect recombinant open reading frame 2 protein (rORF2) ELISA was performed using a modification of a previously described procedure (22). rORF2 and wild-type (WT) antigens were diluted 1:800 in the carbonate-bicarbonate coating buffer, pH 9.5. The Immulon 2HB polystyrene microtiter plates were coated with 100 µL of diluted ORF2 or WT antigen. The coated plates were incubated at 4°C for 36–40 h and stored at -20°C. The plates were then thawed and washed with PBST washing buffer (0.1 mol/L PBS and 0.1% Tween 20). Sera were diluted 1:40 in PBS. Diluted sera (100 µL) were incubated with positive and negative antigen at 37°C for 30 min. Excess antibodies were removed by washing five times with PBST buffer. Plates were blotted dry between each wash and gently tapped after the last wash. Peroxidase-labeled anti-pig IgG (H+L) (100 µL) in 50 g/L milk diluent was added to each well. After incubation at 37°C for 30 min, plates were washed five times. The 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonate) substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) (100 µL) was added and incubated at 37°C for 15 min. The ELISA reaction was stopped by adding equal amounts of 1% SDS to each well. Optical densities were measured at 405 nm in a microtiter plate reader (Tecan, Research Triangle Park, NC).
Viral isolation.
The virus was isolated using PCV-free PK-15 cells from external inguinal lymph nodes (EILN) collected on d 63 of the experiment as previously described (21,23–25).
PCR for detecting PCV2.
A multiplex PCR assay was used to detect and differentiate between nucleic acid of PCV1 and PCV2 in serum and tissues as previously described (21).
Necropsy procedures.
On d 63 of the experiment pigs were killed by injection of a sodium pentobarbital solution (Fort Dodge Animal Health, Fort Dodge, IA) into the vena cava. Peripheral blood (40 mL) was collected from the subclavian vein into 50-mL conical tubes containing 5 mL of PBS with 1000 U of heparin (Elkins-Sinn, Cherry Hill, NJ). Lesions were macroscopically evaluated without knowledge of treatment. Sections of lungs, tonsils, EILN, spleen, liver, ileum, thymus and kidney were obtained, fixed in 10% buffered neutral formalin, later embedded in paraffin, and then sectioned and stained with hematoxylin and eosin (H&E) for histological examination. Samples of EILN were frozen in RNA Later (Ambion, Austin, TX) for isolation of total RNA and analysis of PPAR-, PPAR-, interleukin (IL)-12, IL-18, B cell leukemia/lymphoma-xl (Bcl-xl) and Bcl-2 homologous antagonist/killer (Bak) mRNA expression. For immunohistochemistry, samples of EILN were placed in tissue freezing medium (Triangle Biomedical Sciences, Durham, NC) and snap-frozen in dry ice. Samples for RNA expression analysis and immunohistochemistry were stored at –70°C in RNAlater (Ambion) and tissue freezing medium (Fisher Scientific, Fairlawn, NJ), respectively. Thymic and EILN samples were also collected in RPMI-1640 (Mediatech, Herndon, VA) and stored at -4°C for immunophenotyping and proliferation assays.
Proliferation assays.
Complete medium was prepared as described previously (1). A lymphocyte blastogenesis assay (LBT) was performed using a modification of previously described protocols (1,4). Flat-bottomed, 96-well microtiter plates (Falcon 3072; Becton Dickinson, Lincoln Park, NJ) were seeded with 100 µL of whole blood and 100 µL of medium alone (nonstimulated wells), medium containing the rORF2 of PCV2 (20 mg/L) or medium containing concanavalin A (Con-A) (5 mg/L) (Sigma Chemical, St. Louis, MO) as an internal proliferation control. Preliminary validation assays using Con-A at 2.5, 5, 10 and 20 mg/L were conducted to assess the optimal lymphocyte stimulation level to both Con A and rORF2 within our experimental conditions and genetic background of the pigs. The supernatant of Hi-Five insect cells infected with WT baculovirus lacking the rORF2 (20 mg/L) was used in preliminary validation LBT and PKH assays to examine background proliferation on d 49, 56 and 63. Proliferation of lymphocytes in wells stimulated with WT baculovirus did not differ from proliferation of lymphocytes in wells containing medium alone. Overall lymphocyte proliferation was expressed as stimulation indices, which were calculated by dividing the counts per minute of antigen-stimulated wells by the counts per minute of nonstimulated wells (4).
The PKH assay was performed using a modification of a protocol published previously (2,4). Cells (200,000) were added to 96-well flat-bottomed microtiter plates containing 100 µL of complete medium (nonstimulated), medium plus 20 mg/L of rORF2 of PCV2 or medium plus 5 mg/L of Con-A. Cells were incubated under the same conditions as those for LBT. As cells divide, PKH67-GL membrane staining diminishes, resulting in a decreased mean fluorescence intensity (26). After 5 d, cultured cells were prepared for immunophenotyping. The subset-specific proliferation was presented as relative proliferation indices as previously described (4). The percentages of proliferating (i.e., PKH67dim) cells (IgM+, CD4+, CD4+CD8+ or CD8+) in antigen-stimulated wells were divided by their counterparts in nonstimulated wells.
Flow cytometry.
Mononuclear cells derived from peripheral blood, thymus or EILN (i.e., 2 x 109 viable cells/L) and PKH67-stained, cultured lymphocytes were labeled with anti-pig primary antibodies as previously described (2). The primary antibodies were phycoerythrin (PE)-labeled anti-pig-CD4 (clone 74–12-4), biotinylated IgG2a mouse anti-pig-CD8 (clone 76–2-11) (27), IgG2a mouse anti-pig-CD8ß (PG164A), IgG1 mouse anti-pig-CD45 (K252.1E4, pan-leukocyte marker) (VMRD, Pullman, WA), IgG1 mouse anti-pig-CD3
(8E6), IgG2b mouse anti-pig-SWC3 (74–22-15, VMRD) and IgM mouse anti-pig-IgM (PG145A, VMRD). Flow cytometric data acquisition and analysis were performed as previously described (2,4).
Intracellular IFN- staining.
Peripheral blood mononuclear cells (PBMC) were incubated in 96-well, flat-bottomed microtiter plates at a concentration of 109 cells/L with medium alone or with the rORF2 antigen (20 mg/L), in a final volume of 200 µL. On d 5, cultures were stimulated with 50 µg/L of phorbol myristate acetate (PMA) (Sigma) and 100 µg/L ionomycin (Sigma). Golgi transport was blocked by the addition of 4 µL of Golgi Stop (Pharmingen, San Diego, CA) for every 6 mL of cell culture. After CD4/CD8ß cell surface staining, cells were fixed and permeablized with the Cytofix-cytoperm kit (Pharmingen) following the manufacturer’s instructions. PE-labeled mouse IgG1 anti-porcine IFN- (P2G10, Pharmingen) was used for intracellular cytokine detection. Equally stained non-PMA and ionomycin-stimulated cultures were used as negative controls, and mouse IgG1-PE (Sigma) was used as an isotype control in the flow cytometric examination. Results are presented as relative IFN- production ratios, which were calculated by dividing the percentages of IFN-+ cells bearing the marker of interest (i.e., CD4 and/or CD8 molecules) of antigen-stimulated wells by the percentages of their counterparts from nonstimulated wells.
Histopathological evaluation of tissue samples and quantification of lesions.
H&E-stained EILN, thymus, spleen, tonsil, liver, kidney and ileal sections were evaluated histologically for lymphoid depletion, i.e., 0-no depletion, 1-mild depletion, 2-moderate depletion and 3-severe depletion). Lung slides were evaluated for interstitial pneumonia/bronchiolitis/peribronchiolar lymphoid hyperplasia (0–3). Liver and kidney were examined for the presence of lymphoplasmacytic infiltrates (0–3), i.e., periportal hepatitis and interstitial nephritis, respectively. H&E slides were labeled with accession numbers lacking any reference to dietary or infective treatments and were evaluated without knowledge of treatment.
Immunohistochemical examination of external inguinal lymph nodes.
Frozen EILN sections embedded in tissue-freezing medium were cut to a thickness of 6 µm on a cryostat at -18°C. Sections were placed on poly-L-lysine coated slides, fixed in 95% methanol for 2 min and soaked in cryopreservative (0.5 mol/L sucrose, 0.006 mol/L MgCl2, 50% glycerol) for 10 min. Slides were stored at -20°C until stained. Immunohistochemical staining for T [CD4+, 
T-cell receptors (TCR), CD3+ and CD8+] and B cells (IgM+) was performed as previously described (20).
RNA isolation and RT-PCR for the detection of Bcl-xl, Bak, IL-12, IL-2, IL-18, PPAR-, PPAR- and ß-actin mRNA.
Total RNA was isolated from the EILN using the total RNA isolation MiniKit (Qiagen, Valencia, CA) according to the manufacturer’s instructions, and quantified using a GeneQuant II spectrophotometer (Pharmacia, Peapack, NJ). Expression of Bcl-xl, Bak, IL-12, IL-2, IL-18, PPAR-, PPAR- and ß-actin in external inguinal lymph nodes was examined by RT-PCR as previously described (20). The following primer pairs were used: sense 5'-GGCTGGGACACTTTTGTGG-3'; antisense 5'-TTCCGACTGAAGAGCGAACC-3' for Bcl-xl (140 bp); sense 5'-TGACATCAACCGGCGATACG-3'; antisense 5'-GCTGGAGGCGATCTTGGTG-3' for Bak (103 bp); sense 5'-CTCTTCCAGCCCTCCTTCCT-3'; antisense 5'-GCCAGACAGCACCGTGTT-3' for ß-actin (123 bp); sense 5'-AAGACGGGGTCCTCATCTCC-3'; antisense 5'-CGCCAGGTCGCTGTCATCT-3' for PPAR- (149 bp); sense 5'-CCCTCGTCCGTCACCTAC-3'; antisense 5'-AGGCTTTGTCCCCACAGATT-3' for PPAR- (97 bp); sense 5'- ACCAAATCTCAGCCAAGGTT-3', antisense 5'- GACACAGATGCCCATTCAC-3' for IL-12p40 (103 bp); sense 5'-AACCTCAACTCCTGCCAC-3', antisense 5'-TCCTTGATATTTGCTGAGTCA-3' for IL-2 (550 bp); sense 5'- CTGGAATCGGATTACTTTGG-3', antisense 5'- CGGTCTGAGGTGCATTATCT-3' for IL-18 (148 bp). PCR products were separated electrophoretically on a 2% agarose gel and stained with 0.5 g/L of ethidium bromide. The density of the bands was quantitated using AlphaImager (Proteome Systems, Boston, MA) 2000 v3.3b software. The specific gene expression level was determined semiquantitatively by calculating the ratio of densitometric value of each PCR product in relation to the density of the internal standard represented by ß-actin.
Statistical analysis.
Before the viral challenge, data were analyzed as a randomized complete block design. Two-way ANOVA was used to determine the main effects of the dietary treatment or the infective status and the interaction between dietary treatment and infective status. ANOVA was performed using the general linear model procedure of SAS (28) as previously described (20). Differences with P < 0.05 were considered significant. Postchallenge data were analyzed as a 2 x 2 factorial arrangement of treatments within a split-plot design. In the model, pig within block was the experimental unit for the dietary treatment (subplot), and the blocks of pigs within infective status were the experimental units for infection treatment (whole plot). The whole plot error (i.e., error A) was block within infective status and the subplot error (i.e., 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. The statistical model utilized was Yijk = µ + Infectioni + error Aik + Dietj + (Infection x Diet)ij + error Bijk, where µ was the general mean, Infectioni was the main effect of the ith level of the challenge effect, Dietj was the main effect of the jth level of the dietary effect, (Infection x Diet)ij was the interaction effect between infection and diet, and errors A and B represented the random errors for the whole plot and the subplot, respectively.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and antibody production after the viral challenge.
Seroconversion associated with the PCV2 challenge, as measured by IFA, was observed on d 56 of the experiment (i.e., 14 d postinfection) and the maximum antibody titers were detected on d 63 (21 d postinfection) (Fig. 1). PCV2-specific antibody concentrations did not differ between dietary treatments. Cellular immune responses against PCV2 (i.e., proliferation of lymphocytes after ex vivo stimulation with rORF2) were detectable on d 49, peaked on d 56 and declined on d 63 of the experiment (Fig 1). Lymphocytes recovered from infected pigs fed CLA tended to have greater (P < 0.08) PCV2-specific proliferative responses than those recovered from pigs fed the control diet (Fig 1). Further examination using PKH67 proliferation assays revealed that these numerical differences in overall proliferation were attributable primarily to greater (P < 0.05) PCV2-specific proliferation of CD8+ T cells on d 56 and 63 (Fig. 2). CD4+ and CD8+ T cells recovered from virally infected pigs produced IFN- upon ex vivo restimulation with PCV2 antigens. CLA suppressed IFN- production in CD4+ but not in CD8+ T cells (Fig. 3). Proliferation of B cells in response to ex vivo stimulation with PCV2 was very limited and not influenced by dietary CLA-supplementation (data not shown).
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The percentages of CD8+ PBMC and CD4+CD8+ thymocytes in CLA-fed pigs on d 63 of dietary supplementation were greater than those in pigs fed the control diet, regardless of the infective status. The percentages of CD4+CD8+ thymocytes were greater in PCV2-infected than uninfected pigs. In peripheral blood, CLA-fed pigs had greater percentages of CD8+CD45RC+ and CD8+CD29low cells than those fed the control diet. However, the predominance of a naïve CD8+ T cell phenotype in CLA-fed pigs was attenuated after infection with PCV2. Specifically, dietary CLA interacted with the viral infection to increase the surface expression of IL-2 receptor (CD25) in CD8+ T cells in CLA-fed pigs (Table 2).
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Flow cytometric examination of the lymphocyte subsets in peripheral blood indicated that the PCV2 infection significantly depleted B cells on d 49, 56 and 63 of the experiment. The magnitude of the B-cell depletion in blood was lower in PCV2-infected pigs fed CLA than in infected pigs fed the control diet (Fig. 4). Further phenotypic examination of peripheral blood B cells (i.e., two-color flow cytometry) indicated that the most depleted B-cell subset (IgM+) expressed an immature phenotype characterized by expression of a myeloid marker, i.e., SWC3.
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The viral infection promoted a lymphoid depletion with histiocytic and/or macrophagic replacement in the lymphoid follicles (i.e., B cell–rich areas) located in the cortex of EILN (Fig. 5A). Flow cytometric and immunohistochemical analysis of the EILN confirmed that similar to what was observed in peripheral blood, the lymphoid depletion was comprised primarily of B cells. Topologically, the B-cell depletion was more accentuated in the lymphoid follicles than in the interfollicular areas. The magnitude of the B-cell depletion was lower (P < 0.04) in the EILN of CLA-fed pigs than in those of pigs fed the control diet (Fig. 5B). These cortical lesions were sometimes accompanied by multinucleated giant cells in the medulla of the lymph nodes (data not shown). Immunohistochemical staining using rabbit hyperimmune serum prepared against purified PCV2 indicated that PCV2 also accumulated in the lymphoid follicles (Fig. 5C). Intracytoplasmatic PCV2 inclusions within macrophages and dendritic cells were also observed in the EILN of PCV2-infected pigs.
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and PPAR- mRNA expression in EILN.
Bcl-xl protects cells, including B lymphocytes, against apoptosis, whereas Bak promotes cell death. Bcl-xl mRNA expression was suppressed and Bak mRNA expression was enhanced in PCV2-infected pigs compared with uninfected pigs (P < 0.03). Dietary CLA attenuated the suppressive effects of the viral infection on Bcl-xl expression in infected pigs fed CLA (Fig. 6A).Additionally, infection with PCV2 suppressed PPAR- and enhanced PPAR- expression. PPAR- mRNA expression in PCV2-infected pigs fed CLA resembled that of uninfected pigs. Also, the magnitude of the PPAR- increase in infected pigs fed CLA was lower than that in infected pigs fed the control diet. (Fig. 6B). IL-12 and IL-18 are two cytokines produced by macrophages and dendritic cells, which contribute to regulating the induction of IFN-. The PCV2 infection completely abrogated IL-18 mRNA expression, regardless of the dietary treatment and upregulated IL-12 mRNA expression in pigs fed the control diet. CLA downmodulated IL-12 expression in infected pigs and IL-18 in uninfected pigs. IL-2 is produced by T cells (CD4+ and CD8+) and induces proliferation in T and B cells. Infection with PCV2 suppressed IL-2 mRNA expression in EILN of pigs fed the control diet and enhanced IL-2 mRNA expression in those fed CLA (Fig. 6C).
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The gross pathological examination of tissues conducted during necropsy revealed that infection with PCV2 induced an enlargement of the lymph nodes (i.e., external inguinal and mediastinal), which were threefold larger in PCV2-infected than uninfected pigs (P < 0.0001). However, the dietary treatments did not affect the development of macroscopic lesions. Microscopic pathology demonstrated that the lymphoid depletion of PCV2-infected pigs was detectable in lymph nodes, spleen, tonsils and ileum but not in the thymus (Table 3). In the lymph nodes, the depletion affected primarily B cell–rich areas of the lymphoid follicles. Lymphoid depletion scores in the spleen and lymph nodes tended to be lower (P = 0.15 and 0.23, respectively) in CLA-fed pigs than in those fed the control diet. Also, the liver and kidneys of PCV2-infected pigs contained a mild lymphoplasmacytic infiltrate with a pattern of mild-to-moderate interstitial nephritis and hepatitis, respectively. Interstitial pneumonia and peribronchiolar lymphoid hyperplasia in the lungs tended to be lower (P < 0.14) in infected pigs fed CLA compared with those fed the control diet.
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Before infection with PCV2, average daily gain (ADG) and feed intake (ADFI) did not differ between the diet groups (data not shown). After the challenge with PCV2, ADG was decreased in control-fed pigs from d 49 to 63 (Table 4). ADG in infected pigs fed CLA did not differ from that of uninfected pigs. From d 56 to 63, dietary treatment and infective status tended to interact (P < 0.11) for ADFI. Specifically, in uninfected pigs, ADFI was greater in control than CLA-fed pigs. Conversely, infected pigs fed CLA had greater feed intakes than infected pigs fed the control diet (Table 4). Clinical signs of disease were consistent with mild-to-moderate cases of respiratory disease (i.e., coughing and heavy breathing) in PCV2-infected pigs. The outbreak of respiratory disease peaked between 7 and 12 d postinfection (d 49 to 55 of the experiment) both in the number of pigs affected and the severity of the disease (data not shown). Even though PCV2 DNA was detected in the EILN of all PCV2-infected pigs by PCR, the virus was recovered from 25% of the infected pigs fed CLA and 50% of the infected pigs fed the control diet.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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At the molecular level, the PCV2 infection suppressed the expression of the antiapoptotic molecule Bcl-xl and stimulated the expression of Bak, which promotes cell death, in the EILN. This predominantly proapoptotic expression profile in EILN of PCV2-infected pigs was consistent with the B-cell depletion in blood and lymph nodes. The B-cell depletion and the suppression of Bcl-xl were attenuated in infected pigs fed CLA-supplemented diets. This increased Bcl-xl expression and suppressed B-cell depletion were linked to the greater expression of IL-2 mRNA in EILN. Addition of IL-2 in cultures of resting B cells augmented Bcl-xl expression and diminished apoptosis (29). The involvement of PPAR-–dependent mechanisms in preventing B cell depletion is unlikely because synthetic PPAR- agonists accentuate apoptosis of B cells (30). However, dietary CLA-supplementation suppressed the in vivo synthesis of arachidonic acid (AA) and its metabolites (31,32). The plasma concentrations of AA in pigs fed the control and CLA-supplemented diets were 1.86 and 0.85g/100 g total fatty acids, respectively. Both AA and eicosanoids have profound negative effects on B-cell survival and hematopoiesis (33,34). Hence, the diminished B-cell depletion could be linked to increased IL-2 expression and suppressed synthesis of AA in CLA-fed pigs.
The humoral immune responses against PCV2 were induced more slowly than the cellular responses and were not influenced by dietary treatment. The concentrations of PCV2-specific antibodies in sera of infected pigs did not differ between dietary treatments, but protection from disease was greater in CLA-fed pigs. The IgM+SWC3+ B-cell subset, which was the most depleted, has been characterized as an immature phenotype highly susceptible to apoptosis (35). This finding led to the conclusion that PCV2 targeted immature B cells rather than plasma cells secreting PCV2-specific antibodies. Hence, the B-cell depletion did not suppress antibody production. A better understanding of the main components of the immune system, which were damaged by the viral infection and/or involved in immune protection, will be of importance for the rational design of effective vaccines against PCV2-associated disease. Currently, these vaccines are not available.
For cellular immunoregulation, the proliferation kinetics of individual lymphocyte subsets indicated that CLA significantly enhanced antigen-specific recall responses of CD8+ T cells after the live viral challenge. The kinetics of CD8+ T-cell proliferation resembled that of IFN- production by CD8+ T cells. Consistent with the greater proliferative abilities of CD8+ T cells, a greater percentage of CD8+ T cells recovered from infected pigs fed CLA expressed the IL-2 receptor (CD25) than those recovered from either infected pigs fed the control diet or uninfected pigs. Previously, it was reported by others that CLA enhanced IL-2 expression in mitogen-stimulated lymphocyte cultures (36). Because the expression of IL-2 receptor was not enhanced in uninfected pigs fed CLA, this compound acted in combination with the PCV2 infection, i.e., TCR engagement, to stimulate the expression of CD25. The ability of CLA to enhance CD8+ T cell proliferation and CD25 expression plays a critical role in the protective immune responses against virally infected and neoplastic cells (37,38) and maintenance of immunological memory (39–41). In uninfected pigs, CLA favored a CD8+ T cell phenotype with a greater predominance of naïve cells (CD8+CD29low and CD8+CD45RC+). This naïve CD8+ T cell phenotype in the peripheral blood of uninfected pigs fed CLA originated from an enhanced thymic output of CD8+ T cells. After the viral challenge, it was overshadowed by the expansion of antigen-specific CD8+ T cells and increased CD25 expression.
The pulmonary inflammatory pathology tended to be more severe in infected pigs fed the control diet than in those fed CLA. These pathological findings were suggestive of ameliorated pulmonary disease and consistent with the faster growth (d 50–63) and greater feed intake (d 57–63) in infected pigs fed CLA. Synthetic PPAR- activators are important therapeutic targets in human respiratory diseases (42). Although the inflammatory lesions characteristic of PCV2-associated disease may be immune mediated (43), the specific component(s) of the immune system responsible for the lung immunoinflammatory pathogenesis have not been identified.
Previously, we demonstrated that CLA ameliorated CD4+ T cell–mediated lesions in the intestine (20). Others have shown that IFN-–producing CD4+ T cells were linked to the development of a lung immunopathology after adoptive transfer of naïve, TCR transgenic CD4+ T cells (44). Like synthetic PPAR- agonists (45), CLA suppressed the production of the effector cytokine IFN- by CD4+ T cells. In addition to the TCR engagement, the induction of IFN- in T cells is controlled by IL-12 and IL-18 produced by macrophages and dendritic cells (46). The infection of pigs with PCV2 downmodulated IL-18, regardless of the dietary treatment, and upregulated IL-12 transcript levels in pigs fed the control diet. During a viral infection, downmodulated IFN- production may result in immune deviation of CD8+ cytolytic T cell function (47). However, CD8+ T cell effector functions were not impaired in CLA-fed pigs because they maintained their ability to produce virus-specific IFN-. Furthermore, we demonstrated previously (4) that CLA enhanced the granzyme activities of peripheral blood lymphocytes after stimulation with viral antigens.
In examining the mechanism of T-cell immunoregulation, dietary CLA differentially modulated PPAR- and PPAR- expression in PCV2-infected pigs. Both PPAR isoforms are expressed in most immune cell types, including T cells (14,16). The greater expression of PPAR- in the lymph nodes of pigs fed the control diets was associated with attenuated CD8+ T-cell responses. Conversely, the enhanced PPAR- expression in lymph nodes of pigs fed CLA was linked to greater CD8+ responses. In vitro experiments demonstrated that PPAR- and - expression in T cells is linked to the suppression and activation of the T-cell function, respectively (14,15). However, the inhibitory actions of PPAR- agonists on CD4+ T cells are not consistent with a PPAR- involvement in T-cell activation (45,48). The suppressed IFN- production by CD4+ T cells induced by CLA is consistent with the inhibitory actions of PPAR- agonists on CD4+ T cells. However, the modulation of CD8+ T cells by PPAR- has not been investigated. We propose that CD8+ T cells are the primary targets of PPAR- within the T-cell subset. Additional experimentation is required to examine the modulatory actions of PPAR on CD8+ T cells and to elucidate the mechanism(s) through which PPAR- agonists, including CLA, regulate CD8+ T-cell function.
Results of studies using individual isomers indicate that cis-9, trans-11 and trans-10, cis-12 CLA have important immunomodulatory properties. The effects of CLA on CD8+ T-cell numbers were due mainly to cis-9, trans-11, whereas the trans-10, cis-12 isomer differentially modulated B-cell effector functions in mice (3). In addition, in in vitro studies in macrophages, cis-9, trans-11 CLA inhibited tumor necrosis factor- production (49). Furthermore, recent studies in humans indicated that the effects of a 50:50 isomeric mixture on B cells were greater than those of a more purified preparation consisting of 80% cis-9, trans-11 CLA (50). Hence, both CLA isomers are active and differentially modulate specific components of the immune response. Future studies to examine the nutritional and immunological interactions between these two CLA isomers are warranted.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by grants from the National Pork Board and Loders Croklaan awarded to J.B.-R.
4 Abbreviations used: AA, arachidonic acid; ADG, average daily gain; ADFI, average daily feed intake; Bak, Bcl-2 homologous antagonist/killer; Bcl-xl, B-cell leukemia/lymphoma-xl; CLA, conjugated linoleic acid; Con-A, concanavalin A; EILN, external inguinal lymph nodes; IFA, indirect fluorescent antibody; IFN-, interferon-; IL, interleukin; LBT, lymphocyte blastogenesis assay; ORF2, open reading frame-2; PBMC, peripheral blood mononuclear cells; PCV2, type-2 porcine circovirus; PE, phycoerythrin; PMA, phorbol myristate acetate; PPAR, peroxisome proliferator-activated receptor; Prv, pseudorabies virus; rORF2, recombinant ORF2; TCR, T-cell receptor; WT, wild-type.
Manuscript received 7 May 2003. Initial review completed 16 June 2003. Revision accepted 8 July 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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