Nutritional Immunology

Nutritional Immunology Conjugated Linoleic Acid Ameliorates Viral Infectivity in a Pig Model of Virally Induced Immunosuppression1,2 Josep Bassaganya-Riera,3 Roman M. Pogranichniy,† Scott C. Jobgen, Pat G. Halbur,† Kyoung-Jin Yoon,† Marianne O’Shea,* Inge Mohede* and Raquel Hontecillas 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 ABSTRACT We investigated the cellular and molecular immunoregulatory actions of conjugated linoleic acid (CLA) of relevance to viral disease pathogenesis and antiviral responses. To test the hypothesis that CLA ameliorates viral disease, we developed a viral challenge model by infecting pigs with type-2 porcine circovirus (PCV2). After 42 d of dietary supplementation with either soybean oil (n 16) or CLA (n 16), half of the pigs in each group were challenged with PCV2. We examined the effect of CLA on the development of lesions (i.e., lymphoid depletion and pneumonia) and observed the kinetics of the immune responses against PCV2. The viral Downloaded from jn.nutrition.org by on October 19, 2010 infection depleted immature B cells (IgM SWC3 ) and favored proapoptotic mRNA expression profiles [i.e., suppressed B-cell leukemia/lymphoma-xl (Bcl-xl) and stimulated Bcl-2 homologous antagonist/killer (Bak)] in the external inguinal lymph nodes. B-cell depletion was more accentuated in pigs fed the control diet, whereas interleukin (IL)-2 mRNA expression was downregulated. Histopathological examination of the lungs revealed that the interstitial pneumonia tended to be more severe in infected pigs fed the control diet, which were also affected by growth retardation. CD8 T cells were the primary cellular targets of CLA action in peripheral blood (CD8 CD29low and CD8 CD45RC ) and thymus (CD8 and CD4 CD8 ). CLA interacted with PCV2 to increase the proliferation of CD8 T cells and to suppress PCV2-specific interferon (IFN)- 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. J. Nutr. 133: 3204 –3214, 2003. KEY WORDS: ● lipid nutrition ● lymphoid depletion ● growth suppression ● conjugated linoleic acid Nutritional immune modulation has often been examined trans-11 CLA is responsible for expanding CD8 T cells in the by identifying changes of the immune system linked to specific spleen of mice. dietary regimens. We utilized this experimental approach ear- The interactions between CLA and adaptive immunity can lier to investigate the immunomodulatory properties of con- be examined further in the context of immune responses to jugated linoleic acid (CLA).4 Our initial research led to the specific viral or bacterial antigens (2,4). By immunizing pigs discovery that CLA increases the numbers of CD8 T cells in against pseudorabies virus (Prv), a relative of the human peripheral blood and thymus of pigs (1,2). By feeding the CLA herpes simplex virus, we found that the magnitude of Prv- mixture or purified isomers, Yamasaki and colleagues (3) con- specific recall responses of CD8 T cells was greater in immu- firmed our initial findings and further demonstrated that cis-9, nized 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 1 Presented in part at Experimental Biology 03, April 11–15, 2003, San Diego, disease prevention remain unknown. CA [Bassaganya-Riera, J., Hontecillas, R., Halbur, P. G., Yoon, K. J., Wanne- muehler, M. J. & Pogranichniy, R. M. Modulation of anti-viral activity and viral- On the basis of the greater proliferation and granzyme induced lymphoid depletion by conjugated linoleic acid FASEB J. 17: A2 (abs.)]. activities of Prv-specific CD8 T cells and increased numbers 2 Supported by grants from the National Pork Board and Loders Croklaan of CD8 T cells in CLA-fed pigs (4), we hypothesized that the awarded to J.B.-R. 3 To whom correspondence should be addressed. E-mail: jbassaga@vt.edu. nutritional immune modulation of this T-cell subset by CLA 4 Abbreviations used: AA, arachidonic acid; ADG, average daily gain; ADFI, would enhance protection from viral disease. To test this average daily feed intake; Bak, Bcl-2 homologous antagonist/killer; Bcl-xl, B-cell hypothesis, we infected pigs with type 2 porcine circovirus leukemia/lymphoma-xl; CLA, conjugated linoleic acid; Con-A, concanavalin A; EILN, external inguinal lymph nodes; IFA, indirect fluorescent antibody; IFN- , (PCV2), a small, negative-sense, single-stranded DNA virus of interferon- ; IL, interleukin; LBT, lymphocyte blastogenesis assay; ORF2, open the family Circoviridae, which causes a disease characterized by reading frame-2; PBMC, peripheral blood mononuclear cells; PCV2, type-2 por- clinical signs such as wasting, dyspnea and pallor, and patho- cine circovirus; PE, phycoerythrin; PMA, phorbol myristate acetate; PPAR, peroxisome proliferator-activated receptor; Prv, pseudorabies virus; rORF2, recom- logical lesions including interstitial pneumonia and lymphoid binant ORF2; TCR, T-cell receptor; WT, wild-type. depletion. Because of the high incidence and rapid spread of 0022-3166/03 $3.00 © 2003 American Society for Nutritional Sciences. Manuscript received 7 May 2003. Initial review completed 16 June 2003. Revision accepted 8 July 2003. 3204

LIPID NUTRITION AND VIRAL DISEASE 3205 PCV2 worldwide, the public health risk associated with xeno- TABLE 1 geneic transmissions to humans has received some attention (6 – 8). Dietary composition (as-fed basis) Here, we used the PCV2 challenge of pigs as a model of Control diets1 lymphoid depletion and pulmonary pathology to understand the cellular and molecular regulation of viral disease patho- Phase I Phase II Phase III Phase IV genesis by CLA. Lymphoid depletion is a hallmark immunological lesion associated with immunosuppressive viral infec- % tions such as HIV, type-1 human T-cell leukemia virus or PCV2 (9 –11). Thus, nutritional interventions targeting these Ingredient viral diseases must enhance immune protection and attenuate Corn 32.69 49.59 63.08 72.36 Soybean meal2 12.00 21.20 31.00 22.00 the immunologic and inflammatory damage caused by these Dried whey 22.00 17.00 — — viruses. The prevention and/or amelioration of lymphoid de- Spray-dried plasma 7.50 3.00 — — pletion is particularly important in the elderly in whom lym- Dried blood cells — 2.00 0.50 — phoid regeneration can be impaired by immune senescence Dry skim milk 21.00 1.50 — — (12). CLA mixture3 — — — — We investigated the kinetics of PCV2-specific immune Soybean oil 2.21 2.21 2.21 2.21 DL-Methionine 0.18 0.23 0.07 0.12 responses, gene-nutrient interactions and lesion development L-Lysine — 0.19 0.20 0.39 after a live PCV2 challenge in pigs fed either control or Sodium chloride 0.25 0.25 0.25 0.25 CLA-supplemented diets. We proposed that peroxisome pro- Dicalcium phosphate 1.09 1.72 1.60 1.50 liferator-activated receptors (PPAR) were likely molecular Calcium carbonate 0.78 0.81 0.79 0.90 targets of CLA actions in the immune system (13). Although Vitamin premix4 0.20 0.20 0.20 0.20 the modulation of T cell responses by synthetic agonists of Trace mineral premix5 0.05 0.05 0.05 0.05 Downloaded from jn.nutrition.org by on October 19, 2010 Selenium premix6 0.05 0.05 0.05 0.05 PPAR- and - was investigated previously (14 –16), this is Calculated composition the first report that examines the differential modulation of Crude protein 24.36 21.17 20.30 16.43 PPAR by a natural PPAR- agonist. Lysine 1.76 1.50 1.30 1.15 Methionine cystine 0.97 0.86 0.71 0.65 Calcium 1.05 0.93 0.75 0.70 MATERIALS AND METHODS Phosphorus available 0.70 0.55 0.35 0.30 Experimental design. Serum samples were collected from 110 Metabolizable energy, MJ/kg 14.43 14.15 14.25 14.27 pregnant sows of a breeding herd with no history of PCV2-associated 1 Phase I, wk 1–2; II, wk 3– 4; III, wk 5– 6; and IV, wk 7–9. disease and low PCV2 antibody titers 21 d before the estimated 2 48 g/100 g crude protein. delivery date. Sera were tested for the presence of antibodies to the 3 In conjugated linoleic acid (CLA)-supplemented diets, 2.21% of open reading frame-2 (ORF2) of PCV2-specific (a recombinant major CLA was replaced by 2.21% of soybean oil to keep the diets isoener- capsid protein) gene product by indirect ELISA. An indirect fluores- getic within phases. cent antibody (IFA) assay was used to detect overall anti-PCV2 4 Supplied per kilogram of diet: retinyl acetate, 1516 g; cholecal- antibodies. The offspring from sows with the lowest PCV2 antibody ciferol, 26 g; dl- -tocopheryl acetate, 22 mg; riboflavin, 6.6 mg; pan- concentrations were purchased and transported to large animal iso- tothenic acid, 17.6 mg; niacin, 33 mg; and vitamin B-12, 22 g. lation facilities. The original breeding herd was free of other patho- 5 Supplied per kilogram of diet: Zn, 165 mg (ZnO); Fe, 193 mg gens such as porcine respiratory and reproductive virus, swine influ- (FeSO4 H2O); Mn, 66 mg (MnO); Cu, 19.29 mg (CuSO4 5H2O); and I, enza virus, Mycoplasma hyopneumoniae, Prv or porcine parvovirus. Pigs 0.2 mg (ethylene diamine dihydroiodide). (n 32) with an initial body weight of 4.3–5.1 kg were weaned at 6 Supplied per kilogram of diet: Se, 0.1 mg (Na2SeO3). 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 acid composition of similar diets was published previously (20). Diets pigs were designed on the basis of litter of origin, maternal concen- were formulated to maintain or exceed current recommended nutri- tration of anti-PCV2 antibodies, initial body weight, age and gender. tional requirements of the NRC (17) for pigs. Pigs, feeders and feed Pigs (n 32, 16 blocks of 2 pigs each) were distributed into two were weighed on a weekly basis before and after challenge to evaluate dietary treatments: 1) control (n 16) or 2) CLA-supplemented diet modifications in growth and feed intake. (n 16). Pigs were fed the experimental diets for 42 d before the live Viral propagation. A PK-15 cell suspension was prepared in PCV2 challenge. Before 8 blocks of pigs were challenged, the exper- MEM growth medium at a concentration of 4 108 cells/L and imental design was a randomized complete block. After intrainguinal inoculated with PCV2 designated ISUVDL 98 –15237 (21) at a lymph node challenge with PCV2 of 8 blocks of pigs, the design concentration of 108 50% tissue culture infective doses (TCID50)/L became a 2 2 factorial arrangement within a split-plot design. Pigs at a ratio of 1 mL of virus per 50 mL of cell suspension. Inoculated within block were the experimental units for dietary treatment, and cells were seeded into a 165-cm2 tissue culture flask and incubated for blocks of two pigs each were the experimental units for the infection 24 h. The resulting cell monolayers were treated with 300 mmol/L treatment (i.e., PCV2-infected or uninfected). D-glucosamine for 20 min and incubated for another 48 h. PCV2- Dietary treatments. Either a 1.33 g CLA/100 g diet or an infected PK-15 cells were then frozen/thawed twice, harvested and isoenergetic and isonitrogenous soybean oil–supplemented control pelleted by centrifugation at 3000 g for 10 min. Supernatant diet (Table 1) was randomly allotted to pens within blocks as previ- containing the propagated virus was titrated and utilized in the viral ously described (1). Pigs had free access to feed for 63 d in four phases challenge. The supernatant from uninfected PK-15 cells served as a (I, wk 1–2; II, wk 3– 4; III, wk 5– 6; and IV, wk 7–9). Phase feeding sham control. The absence of PCV1 in the virus preparation was is consistent with the NRC nutrient recommendations for pigs (17). confirmed by IFA and PCR before the challenge. The virus used in Between treatments and within phases, diets were formulated to be this study represented 12 in vitro passages in cell culture. isoenergetic and isonitrogenous to avoid energy- and/or protein- Viral challenge. Challenge inoculum consisted of one dose of derived immunological changes (18,19). Thus, in control diets, 2.21 g lysate of PCV2-infected PK-15 cell supernatant inoculated in the CLA source/100 g diet was replaced by 2.21 g soybean oil/100 g diet external inguinal lymph nodes (i.e., 1 mL each) at 109.2 TCID50/L. to keep both the CLA-supplemented and the control diets isoener- Pigs were distributed into two separate rooms (n 16 pigs/room). On getic within phases. The CLA source was alkali-isomerized sunflower d 42 of the experiment, pigs in the “challenged room” were inocu- oil (Loders Croklaan, Lipid Nutrition, Channahon, IL). The fatty lated with PCV2 and pigs in the “nonchallenged room” were inoc-

3206 BASSAGANYA-RIERA ET AL. ulated with lysate of uninfected PK-15 cells. Biosecurity measures diatech, Herndon, VA) and stored at 4°C for immunophenotyping were implemented to prevent contamination of the uninfected room and proliferation assays. with PCV2. Proliferation assays. Complete medium was prepared as de- Production of recombinant capsid protein ORF2 of PCV2. An- scribed previously (1). A lymphocyte blastogenesis assay (LBT) was tigen for ex vivo restimulation assays [e.g., lymphocyte blastogenesis performed using a modification of previously described protocols test, intracellular interferon (IFN)- detection, and PKH67 prolifer- (1,4). Flat-bottomed, 96-well microtiter plates (Falcon 3072; Becton ation assay] and the initial ELISA-based screening of the herd of Dickinson, Lincoln Park, NJ) were seeded with 100 L of whole origin was partially purified protein from Hi-Five insect cells (Invitro- blood and 100 L of medium alone (nonstimulated wells), medium gen, Carlsbad, CA) infected with either wild-type baculovirus (Ac- containing the rORF2 of PCV2 (20 mg/L) or medium containing MNP.wt) or recombinant baculovirus carrying the ORF2 gene of concanavalin A (Con-A) (5 mg/L) (Sigma Chemical, St. Louis, MO) PCV2 (AcMNP.ORF2) (22). At 72 h postinfection, recombinant as an internal proliferation control. Preliminary validation assays baculovirus–infected Hi-Five cells were transferred into a 50-mL tube using Con-A at 2.5, 5, 10 and 20 mg/L were conducted to assess the and centrifuged at 800 g for 10 min. The cell pellet was washed optimal lymphocyte stimulation level to both Con A and rORF2 three times with cold PBS and subjected to three rounds of freezing within our experimental conditions and genetic background of the and thawing ( 70°C/37°C) to release viral proteins before clarifica- pigs. The supernatant of Hi-Five insect cells infected with WT tion by centrifugation 800 g for 10 min. The supernatant was baculovirus lacking the rORF2 (20 mg/L) was used in preliminary diluted in PBS, laid over 6 mL of 400 g/L sucrose in PBS and validation LBT and PKH assays to examine background proliferation centrifuged at 270,000 g for 6 h using a Ti 41 SW rotor (Beckman, on d 49, 56 and 63. Proliferation of lymphocytes in wells stimulated Palo Alto, CA). The viral antigen was clarified by centrifugation at with WT baculovirus did not differ from proliferation of lymphocytes 10,000 g at 4°C for 10 min. Protein concentrations were measured in wells containing medium alone. Overall lymphocyte proliferation using the Bio-Rad Bradford-basal protein assay (Hercules, CA). was expressed as stimulation indices, which were calculated by divid- IFA assay for PCV2. Serum samples were collected on d 0, 28, ing the counts per minute of antigen-stimulated wells by the counts 42, 49, 56 and 63 of the experiment. The IFA assay was performed as per minute of nonstimulated wells (4). described previously (21). The PKH assay was performed using a modification of a protocol Recombinant capsid protein (ORF2)-based ELISA. Serum published previously (2,4). Cells (200,000) were added to 96-well Downloaded from jn.nutrition.org by on October 19, 2010 samples were collected from the pregnant sows. The indirect recom- flat-bottomed microtiter plates containing 100 L of complete me- binant open reading frame 2 protein (rORF2) ELISA was performed dium (nonstimulated), medium plus 20 mg/L of rORF2 of PCV2 or using a modification of a previously described procedure (22). rORF2 medium plus 5 mg/L of Con-A. Cells were incubated under the same and wild-type (WT) antigens were diluted 1:800 in the carbonate- conditions as those for LBT. As cells divide, PKH67-GL membrane bicarbonate coating buffer, pH 9.5. The Immulon 2HB polystyrene staining diminishes, resulting in a decreased mean fluorescence in- microtiter plates were coated with 100 L of diluted ORF2 or WT tensity (26). After 5 d, cultured cells were prepared for immunophe- antigen. The coated plates were incubated at 4°C for 36 – 40 h and notyping. The subset-specific proliferation was presented as relative stored at 20°C. The plates were then thawed and washed with proliferation indices as previously described (4). The percentages of PBST washing buffer (0.1 mol/L PBS and 0.1% Tween 20). Sera were proliferating (i.e., PKH67dim) cells (IgM , CD4 , CD4 CD8 or diluted 1:40 in PBS. Diluted sera (100 L) were incubated with CD8 ) in antigen-stimulated wells were divided by their counterparts positive and negative antigen at 37°C for 30 min. Excess antibodies in nonstimulated wells. were removed by washing five times with PBST buffer. Plates were Flow cytometry. Mononuclear cells derived from peripheral blotted dry between each wash and gently tapped after the last wash. blood, thymus or EILN (i.e., 2 109 viable cells/L) and PKH67- Peroxidase-labeled anti-pig IgG (H L) (100 L) in 50 g/L milk stained, cultured lymphocytes were labeled with anti-pig primary diluent was added to each well. After incubation at 37°C for 30 min, antibodies as previously described (2). The primary antibodies were plates were washed five times. The 2,2 -azino-di-(3-ethylbenzthiazo- phycoerythrin (PE)-labeled anti-pig-CD4 (clone 74 –12-4), biotinyl- line-6-sulfonate) substrate (Kirkegaard & Perry Laboratories, Gaith- ated IgG2a mouse anti-pig-CD8 (clone 76 –2-11) (27), IgG2a ersburg, MD) (100 L) was added and incubated at 37°C for 15 min. mouse anti-pig-CD8 (PG164A), IgG1 mouse anti-pig-CD45 The ELISA reaction was stopped by adding equal amounts of 1% SDS (K252.1E4, pan-leukocyte marker) (VMRD, Pullman, WA), IgG1 to each well. Optical densities were measured at 405 nm in a micro- mouse anti-pig-CD3 (8E6), IgG2b mouse anti-pig-SWC3 (74 –22- titer plate reader (Tecan, Research Triangle Park, NC). 15, VMRD) and IgM mouse anti-pig-IgM (PG145A, VMRD). Flow Viral isolation. The virus was isolated using PCV-free PK-15 cytometric data acquisition and analysis were performed as previously cells from external inguinal lymph nodes (EILN) collected on d 63 of described (2,4). the experiment as previously described (21,23–25). Intracellular IFN- staining. Peripheral blood mononuclear PCR for detecting PCV2. A multiplex PCR assay was used to cells (PBMC) were incubated in 96-well, flat-bottomed microtiter detect and differentiate between nucleic acid of PCV1 and PCV2 in plates at a concentration of 109 cells/L with medium alone or with serum and tissues as previously described (21). the rORF2 antigen (20 mg/L), in a final volume of 200 L. On d 5, Necropsy procedures. On d 63 of the experiment pigs were killed cultures were stimulated with 50 g/L of phorbol myristate acetate by injection of a sodium pentobarbital solution (Fort Dodge Animal (PMA) (Sigma) and 100 g/L ionomycin (Sigma). Golgi transport Health, Fort Dodge, IA) into the vena cava. Peripheral blood (40 was blocked by the addition of 4 L of Golgi Stop (Pharmingen, San mL) was collected from the subclavian vein into 50-mL conical tubes Diego, CA) for every 6 mL of cell culture. After CD4/CD8 cell containing 5 mL of PBS with 1000 U of heparin (Elkins-Sinn, Cherry surface staining, cells were fixed and permeablized with the Cytofix- Hill, NJ). Lesions were macroscopically evaluated without knowledge cytoperm kit (Pharmingen) following the manufacturer’s instructions. of treatment. Sections of lungs, tonsils, EILN, spleen, liver, ileum, PE-labeled mouse IgG1 anti-porcine IFN- (P2G10, Pharmingen) thymus and kidney were obtained, fixed in 10% buffered neutral was used for intracellular cytokine detection. Equally stained non- formalin, later embedded in paraffin, and then sectioned and stained PMA and ionomycin-stimulated cultures were used as negative con- with hematoxylin and eosin (H&E) for histological examination. trols, and mouse IgG1-PE (Sigma) was used as an isotype control in Samples of EILN were frozen in RNA Later (Ambion, Austin, TX) the flow cytometric examination. Results are presented as relative for isolation of total RNA and analysis of PPAR- , PPAR- , inter- IFN- production ratios, which were calculated by dividing the leukin (IL)-12, IL-18, B cell leukemia/lymphoma-xl (Bcl-xl) and percentages of IFN- cells bearing the marker of interest (i.e., CD4 Bcl-2 homologous antagonist/killer (Bak) mRNA expression. For and/or CD8 molecules) of antigen-stimulated wells by the percent- immunohistochemistry, samples of EILN were placed in tissue freez- ages of their counterparts from nonstimulated wells. ing medium (Triangle Biomedical Sciences, Durham, NC) and snap- Histopathological evaluation of tissue samples and quantification frozen in dry ice. Samples for RNA expression analysis and immuno- of lesions. H&E-stained EILN, thymus, spleen, tonsil, liver, kidney histochemistry were stored at –70°C in RNAlater (Ambion) and and ileal sections were evaluated histologically for lymphoid deple- tissue freezing medium (Fisher Scientific, Fairlawn, NJ), respectively. tion, i.e., 0-no depletion, 1-mild depletion, 2-moderate depletion and Thymic and EILN samples were also collected in RPMI-1640 (Me- 3-severe depletion). Lung slides were evaluated for interstitial pneu-

LIPID NUTRITION AND VIRAL DISEASE 3207 monia/bronchiolitis/peribronchiolar lymphoid hyperplasia (0 –3). infection and diet, and errors A and B represented the random errors Liver and kidney were examined for the presence of lymphoplasma- for the whole plot and the subplot, respectively. cytic infiltrates (0 –3), i.e., periportal hepatitis and interstitial nephritis, respectively. H&E slides were labeled with accession numbers RESULTS lacking any reference to dietary or infective treatments and were evaluated without knowledge of treatment. PCV2-specific lymphocyte proliferation, intracellular Immunohistochemical examination of external inguinal lymph IFN- and antibody production after the viral challenge. nodes. Frozen EILN sections embedded in tissue-freezing medium Seroconversion associated with the PCV2 challenge, as mea- were cut to a thickness of 6 m on a cryostat at 18°C. Sections were sured by IFA, was observed on d 56 of the experiment (i.e., placed on poly-L-lysine coated slides, fixed in 95% methanol for 2 min 14 d postinfection) and the maximum antibody titers were and soaked in cryopreservative (0.5 mol/L sucrose, 0.006 mol/L detected on d 63 (21 d postinfection) (Fig. 1). PCV2-specific MgCl2, 50% glycerol) for 10 min. Slides were stored at 20°C until antibody concentrations did not differ between dietary treat- stained. Immunohistochemical staining for T [CD4 , T-cell receptors (TCR), CD3 and CD8 ] and B cells (IgM ) was performed ments. Cellular immune responses against PCV2 (i.e., prolif- as previously described (20). eration of lymphocytes after ex vivo stimulation with rORF2) RNA isolation and RT-PCR for the detection of Bcl-xl, Bak, IL-12, IL-2, IL-18, PPAR- , PPAR- and -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 -actin in external inguinal lymph nodes was examined by RT-PCR as previously described (20). The fol- Downloaded from jn.nutrition.org by on October 19, 2010 lowing primer pairs were used: sense 5 -GGCTGGGA- CACTTTTGTGG-3 ; antisense 5 -TTCCGACTGAAGA- GCGAACC-3 for Bcl-xl (140 bp); sense 5 -TGACATCAAC- CGGCGATACG-3 ; antisense 5 -GCTGGAGGCGATCTT- GGTG-3 for Bak (103 bp); sense 5 -CTCTTCCAGCCCTC- CTTCCT-3 ; antisense 5 -GCCAGACAGCACCGTGTT-3 for -actin (123 bp); sense 5 -AAGACGGGGTCCTCAT- CTCC-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 semiquantita- tively by calculating the ratio of densitometric value of each PCR product in relation to the density of the internal standard represented by -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). Differ- ences with P 0.05 were considered significant. Postchallenge data were analyzed as a 2 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 FIGURE 1 Humoral (right axis) and cellular (left axis) immune within infective status and the subplot error (i.e., error B) was the responses against porcine circovirus type 2 (PCV2) in uninfected pigs residual df after accounting for the dietary treatment variance and the (panel A) and PCV2-infected (panel B) pigs fed control or conjugated variance for the interaction between dietary treatment and infective linoleic acid (CLA)-supplemented diets. Pigs were challenged with status. The statistical model utilized was Yijk Infectioni error PCV2 on d 42 of the experiment. Values are means SEM (n 8). Aik Dietj (Infection Diet)ij error Bijk, where was the Differences (P 0.05) between treatments attributed to the main ef- general mean, Infectioni was the main effect of the ith level of the fects of infection (†), diet (*) and the interaction between infection and challenge effect, Dietj was the main effect of the jth level of the diet ([yen]) are indicated. If an interaction was found, the significance of dietary effect, (Infection Diet)ij was the interaction effect between the main effects was not reported. IFA, indirect fluorescent antibody.

3208 BASSAGANYA-RIERA ET AL. (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). Immunophenotyping of CD8 T cells in peripheral blood and thymus. 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 con- FIGURE 2 Antigen-specific proliferation of CD8 T cells (i.e., Downloaded from jn.nutrition.org by on October 19, 2010 TCR CD8 ) against porcine circovirus type 2 (PCV2) in cultures of peripheral blood mononuclear cells recovered from uninfected (panel A) and PCV2-infected (panel B) pigs fed control or conjugated linoleic acid (CLA)-supplemented diets. Pigs were challenged with PCV2 on d 42 of the experiment. Values are means SEM (n 8). Differences (P 0.05) between treatments attributed to the main effects of infection (†), diet (*) and the interaction between infection and diet ([yen]) are indicated. If an interaction was found, the significance of the main effects was not reported. FIGURE 3 Antigen-specific interferon- (IFN- ) production by were detectable on d 49, peaked on d 56 and declined on d 63 CD8 (panel A) or CD4 (panel B) T cells recovered from porcine circovirus type 2 (PCV2)-infected pigs fed control or conjugated linoleic of the experiment (Fig 1). Lymphocytes recovered from in- acid (CLA)-supplemented diets. Pigs were challenged with PCV2 on d fected pigs fed CLA tended to have greater (P 0.08) PCV2- 42 of the experiment. Values are means SEM (n 8). Differences (P specific proliferative responses than those recovered from pigs 0.05) between treatments attributed to the main effects of infection fed the control diet (Fig 1). Further examination using PKH67 (†), diet (*) and the interaction between infection and diet ([yen]) are proliferation assays revealed that these numerical differences indicated. If an interaction was found, the significance of the main in overall proliferation were attributable primarily to greater effects was not reported.

LIPID NUTRITION AND VIRAL DISEASE 3209 trol diet, regardless of the infective status. The percentages of cells, including B lymphocytes, against apoptosis, whereas Bak CD4 CD8 thymocytes were greater in PCV2-infected than promotes cell death. Bcl-xl mRNA expression was suppressed uninfected pigs. In peripheral blood, CLA-fed pigs had greater and Bak mRNA expression was enhanced in PCV2-infected percentages of CD8 CD45RC and CD8 CD29low cells than pigs compared with uninfected pigs (P 0.03). Dietary CLA those fed the control diet. However, the predominance of a attenuated the suppressive effects of the viral infection on naıve CD8 T cell phenotype in CLA-fed pigs was attenuated ¨ Bcl-xl expression in infected pigs fed CLA (Fig. 6A). Addi- after infection with PCV2. Specifically, dietary CLA inter- tionally, infection with PCV2 suppressed PPAR- and en- acted with the viral infection to increase the surface expres- hanced PPAR- expression. PPAR- mRNA expression in sion of IL-2 receptor (CD25) in CD8 T cells in CLA-fed pigs PCV2-infected pigs fed CLA resembled that of uninfected (Table 2). pigs. Also, the magnitude of the PPAR- increase in infected B-cell depletion in peripheral blood. Flow cytometric ex- pigs fed CLA was lower than that in infected pigs fed the amination of the lymphocyte subsets in peripheral blood in- control diet. (Fig. 6B). IL-12 and IL-18 are two cytokines dicated that the PCV2 infection significantly depleted B cells produced by macrophages and dendritic cells, which contrib- on d 49, 56 and 63 of the experiment. The magnitude of the ute to regulating the induction of IFN- . The PCV2 infection B-cell depletion in blood was lower in PCV2-infected pigs fed completely abrogated IL-18 mRNA expression, regardless of CLA than in infected pigs fed the control diet (Fig. 4). the dietary treatment and upregulated IL-12 mRNA expres- Further phenotypic examination of peripheral blood B cells sion in pigs fed the control diet. CLA downmodulated IL-12 (i.e., two-color flow cytometry) indicated that the most de- expression in infected pigs and IL-18 in uninfected pigs. IL-2 pleted B-cell subset (IgM ) expressed an immature phenotype is produced by T cells (CD4 and CD8 ) and induces prolif- characterized by expression of a myeloid marker, i.e., SWC3. eration in T and B cells. Infection with PCV2 suppressed IL-2 Lymphoid depletion in EILN, and detection of PCV2 mRNA expression in EILN of pigs fed the control diet and protein. The viral infection promoted a lymphoid depletion enhanced IL-2 mRNA expression in those fed CLA (Fig. 6C). with histiocytic and/or macrophagic replacement in the lym- Effects of the dietary treatment and the infective status on Downloaded from jn.nutrition.org by on October 19, 2010 phoid follicles (i.e., B cell–rich areas) located in the cortex of lesion development. The gross pathological examination of EILN (Fig. 5A). Flow cytometric and immunohistochemical tissues conducted during necropsy revealed that infection with analysis of the EILN confirmed that similar to what was PCV2 induced an enlargement of the lymph nodes (i.e., ex- observed in peripheral blood, the lymphoid depletion was ternal inguinal and mediastinal), which were threefold larger comprised primarily of B cells. Topologically, the B-cell de- in PCV2-infected than uninfected pigs (P 0.0001). How- pletion was more accentuated in the lymphoid follicles than in ever, the dietary treatments did not affect the development of the interfollicular areas. The magnitude of the B-cell depletion macroscopic lesions. Microscopic pathology demonstrated that was lower (P 0.04) in the EILN of CLA-fed pigs than in the lymphoid depletion of PCV2-infected pigs was detectable those of pigs fed the control diet (Fig. 5B). These cortical in lymph nodes, spleen, tonsils and ileum but not in the lesions were sometimes accompanied by multinucleated giant thymus (Table 3). In the lymph nodes, the depletion affected cells in the medulla of the lymph nodes (data not shown). primarily B cell–rich areas of the lymphoid follicles. Lymphoid Immunohistochemical staining using rabbit hyperimmune se- depletion scores in the spleen and lymph nodes tended to be rum prepared against purified PCV2 indicated that PCV2 also lower (P 0.15 and 0.23, respectively) in CLA-fed pigs than accumulated in the lymphoid follicles (Fig. 5C). Intracytoplas- in those fed the control diet. Also, the liver and kidneys of matic PCV2 inclusions within macrophages and dendritic cells PCV2-infected pigs contained a mild lymphoplasmacytic in- were also observed in the EILN of PCV2-infected pigs. filtrate with a pattern of mild-to-moderate interstitial nephritis Regulation of Bcl-xl, Bak, IL-12, IL-2, IL-18, PPAR- and hepatitis, respectively. Interstitial pneumonia and peri- and PPAR- mRNA expression in EILN. Bcl-xl protects bronchiolar lymphoid hyperplasia in the lungs tended to be TABLE 2 The phenotype of peripheral blood mononuclear cells (PBMC) and thymocytes in pigs fed control or conjugated linoleic acid (CLA)-supplemented diets and infected and uninfected with type-2 porcine circovirus (PCV2)1,2 Uninfected pigs PCV2-Infected pigs3 P-value4 Control diet CLA diet Control diet CLA diet SEM D IN D IN % Thymocytes CD4 9.34 7.23 5.64 4.45 1.34 0.34 0.15 0.89 CD8 CD4 54.13c 60.40b 65.57b 72.72a 5.17 0.02 0.12 0.04 CD8 8.88b 13.14a 9.55b 14.17a 2.10 0.05 0.98 0.23 PBMC CD8 CD29low 7.12b 10.91a 6.27b 7.18b 1.14 0.03 0.43 0.54 CD8 CD45RC 9.40b 15.89a 9.17b 10.39a 2.16 0.05 0.68 0.24 CD8 CD25 2.29b 2.17b 2.73b 4.02a 0.81 0.18 0.40 0.04 1 PBMC were isolated from peripheral blood and thymocytes were isolated from the thymus of pigs on d 63 of the experiment. 2 Least-square mean values (n 8) in a row for a particular marker with different superscripts differ, P 0.05. 3 On d 42, eight blocks of 2 pigs each were infected. 4 The P-values represent the main effect of the diet (D), the main effect of infection (IN) and the PCV2 interaction between the infection and the dietary treatments (D IN).

3210 BASSAGANYA-RIERA ET AL. 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. DISCUSSION Nutritional immunology studies have revealed a critical role of dietary CLA in the modulation of the numbers (1,3) and effector functions (2,4) of CD8 T cells. In the absence of antigenic stimulation, CLA increased the numbers of CD8 T cells in peripheral blood and thymus. After immunization with viral antigens, CLA enhanced effector functions of CD8 T cells that are critical for the clearance of viruses, i.e., antigen- Downloaded from jn.nutrition.org by on October 19, 2010 specific proliferation and granzyme activities (4). Here, we examined whether the nutritional immune modulation of CD8 T cells by CLA enhanced prevention of PCV2-associated disease (i.e., lymphoid depletion and pulmonary pathology). We found that PCV2 infection led to inflammatory FIGURE 4 Flow cytometric analysis of B lymphocytes in peripheral blood mononuclear cells recovered from uninfected (panel A) and porcine circovirus type 2 (PCV2)-infected (panel B) pigs fed control or conjugated linoleic acid (CLA)-supplemented diets. Pigs were challenged with PCV2 on d 42 of the experiment. A depletion of a B-cell subset was detected during the first 15 d postchallenge. Values are means SEM (n 8). Differences (P 0.05) between treatments attributed to the main effects of infection (†), diet (*) and the interaction FIGURE 5 Photomicrographs of hematoxylin and eosin (H&E) between infection and diet ([yen]) are indicated. If an interaction was staining and immunostaining by anti-porcine circovirus type 2 (PCV2) found, the significance of the main effects was not reported. antibodies on paraffin-embedded external inguinal lymph node (EILN) and lung sections of pigs fed control or conjugated linoleic acid (CLA)- supplemented diets. (A) Histopathological appearance of representa- lower (P 0.14) in infected pigs fed CLA compared with tive external inguinal lymph nodes recovered from uninfected pigs (left), those fed the control diet. PCV2-infected pigs fed the control diet (center), and PCV2-infected PCV2-associated disease: clinical disease, growth, PCR pigs fed the CLA-supplemented diet (right). In PCV2-infected pigs the and viral isolation. Before infection with PCV2, average majority of the lymphoid depletion was situated within the lymphoid follicle. Cytoplasmic PCV2 staining was observed within the lymphoid daily gain (ADG) and feed intake (ADFI) did not differ follicles (see inset). (B) Histopathological appearance of representative between the diet groups (data not shown). After the challenge lung sections recovered from uninfected pigs (left), PCV2-infected pigs with PCV2, ADG was decreased in control-fed pigs from d 49 fed the control diet (center), and PCV2-infected pigs fed the CLA- to 63 (Table 4). ADG in infected pigs fed CLA did not differ supplemented diet (right). Interstitial pneumonia was more severe in from that of uninfected pigs. From d 56 to 63, dietary treat- PCV2-infected pigs fed the control diet than those fed CLA-supple- ment and infective status tended to interact (P 0.11) for mented diets.

LIPID NUTRITION AND VIRAL DISEASE 3211 lesions in the lungs, liver and kidney, and induced lymphoid depletion in the lymph nodes, peripheral blood, spleen, tonsils and ileum but not in the thymus. The absence of lesions in the thymus suggests that PCV2 does not target T cells. In addition, the depletion of the lymphoid follicles, the presence of PCV2 antigens in the B cell-rich areas of the lymph nodes and the B cell depletion in blood suggest that PCV2 targets B cells in addition to macrophages. 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). How- ever, dietary CLA-supplementation suppressed the in vivo Downloaded from jn.nutrition.org by on October 19, 2010 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 abili- vated receptor (PPAR)- and PPAR- (panel B), and interleukin (IL)-12, IL-2, and IL-18 (panel C) mRNA expression in external inguinal lymph nodes of uninfected (panel A) and porcine circovirus type 2 (PCV2)- infected (panel B) pigs fed control or conjugated linoleic acid (CLA)- supplemented diets. Values of ratios of each gene to -actin were calculated for each individual pig. Values are means SEM (n 8). Differences (P 0.05) between treatments attributed to the main effects of infection (†), diet (*) and the interaction between infection and FIGURE 6 B cell leukemia/lymphoma-xl (Bcl-xl) and Bcl-2 homol- diet ([yen]) are indicated. If an interaction was found, the significance of ogous antagonist/killer (Bak) (panel A), peroxisome proliferator-acti- the main effects was not reported.

3212 BASSAGANYA-RIERA ET AL. TABLE 3 The development of microscopic lesions in pigs fed control or conjugated linoleic acid (CLA)-supplemented diets and infected and uninfected with type-2 porcine circovirus (PCV2)1,2 Uninfected pigs PCV2-Infected pigs3 P-value4 Control diet CLA diet Control diet CLA diet SEM D IN D IN Tissue Lymph nodes 0.00b 0.00b 1.00a 0.87a 0.18 0.53 0.04 0.53 Thymus 0.00 0.00 0.00 0.00 — — — Spleen 0.00 0.00 0.37 0.00 0.16 0.62 0.20 0.63 Tonsils 0.00b 0.00b 0.37a 0.37a 0.15 0.74 0.006 0.74 Ileum 0.00 0.00 0.25 0.37 0.13 0.76 0.08 0.81 Liver 0.00b 0.00b 0.75a 0.62a 0.15 0.86 0.004 0.93 Kidney 0.00 0.00 0.25 0.12 0.10 0.75 0.14 0.86 Lungs 0.00c 0.00c 1.37a 0.37ab 0.28 0.14 0.008 0.35 1 Pigs were killed on d 63, external inguinal lymph nodes, spleen, tonsils, ileum, liver, kidney, and lungs were collected for histopathological examination of lesions. 2 Least-square mean values (n 8) in a row for a particular marker with different superscripts differ, P 0.05. 3 On d 42, eight blocks of 2 pigs each were infected. 4 The P-values represent the main effect of the diet (D), the main effect of infection (IN) and the PCV2 interaction between the infection and the dietary treatments (D IN). Downloaded from jn.nutrition.org by on October 19, 2010 ties of CD8 T cells, a greater percentage of CD8 T cells cells. After the viral challenge, it was overshadowed by the recovered from infected pigs fed CLA expressed the IL-2 expansion of antigen-specific CD8 T cells and increased receptor (CD25) than those recovered from either infected CD25 expression. pigs fed the control diet or uninfected pigs. Previously, it was The pulmonary inflammatory pathology tended to be more reported by others that CLA enhanced IL-2 expression in severe in infected pigs fed the control diet than in those fed mitogen-stimulated lymphocyte cultures (36). Because the ex- CLA. These pathological findings were suggestive of amelio- pression of IL-2 receptor was not enhanced in uninfected pigs rated pulmonary disease and consistent with the faster growth fed CLA, this compound acted in combination with the PCV2 (d 50 – 63) and greater feed intake (d 57– 63) in infected pigs infection, i.e., TCR engagement, to stimulate the expression fed CLA. Synthetic PPAR- activators are important thera- of CD25. The ability of CLA to enhance CD8 T cell pro- peutic targets in human respiratory diseases (42). Although liferation and CD25 expression plays a critical role in the the inflammatory lesions characteristic of PCV2-associated protective immune responses against virally infected and neo- disease may be immune mediated (43), the specific compo- plastic cells (37,38) and maintenance of immunological mem- nent(s) of the immune system responsible for the lung immu- ory (39 – 41). In uninfected pigs, CLA favored a CD8 T cell noinflammatory pathogenesis have not been identified. phenotype with a greater predominance of naıve cells ¨ Previously, we demonstrated that CLA ameliorated CD4 (CD8 CD29low and CD8 CD45RC ). This naıve CD8 T ¨ T cell–mediated lesions in the intestine (20). Others have cell phenotype in the peripheral blood of uninfected pigs fed shown that IFN- –producing CD4 T cells were linked to the CLA originated from an enhanced thymic output of CD8 T development of a lung immunopathology after adoptive trans- TABLE 4 Growth and feed intake of pigs fed control or conjugated linoleic acid (CLA)-supplemented diets and infected and uninfected with type-2 porcine circovirus (PCV2)1,2 Uninfected pigs PCV2-Infected pigs3 P-value4 Control diet CLA diet Control diet CLA diet SEM D IN D IN Growth g/d d 42–49 863 917 1034 936 96 0.54 0.16 0.08 d 50–56 892a 815a 601b 848a 84 0.36 0.76 0.03 d 57–63 943a 952a 752b 914a 90 0.18 0.004 0.05 Feed intake d 42–49 1514 1459 1496 1600 51 0.50 0.12 0.31 d 50–56 1624b 1556b 1712a 1886a 73 0.64 0.001 0.26 d 57–63 2081a 1855ab 1571c 1824b 110 0.98 0.005 0.11 1 Pigs, feeders and waste feed were weighed on a weekly basis, and mean daily gain and mean daily feed intake were calculated. 2 Least-square mean values (n 8) in a row for a particular marker with different superscripts differ, P 0.05. 3 On d 42, eight blocks of 2 pigs each were infected. 4 The P-values represent the main effect of the diet (D), the main effect of infection (IN) and the PCV2 interaction between the infection and the dietary treatments (D IN).

LIPID NUTRITION AND VIRAL DISEASE 3213 fer of naıve, TCR transgenic CD4 T cells (44). Like syn- ¨ nursery pigs of dirty and clean environments on growth, empty body composition, and immune competence. J. Anim. Sci. 79: 714 –721. thetic PPAR- agonists (45), CLA suppressed the production 2. Bassaganya-Riera, J., Hontecillas, R., Zimmerman, D. R. & Wanne- of the effector cytokine IFN- by CD4 T cells. In addition to muehler, M. J. (2001) Dietary conjugated linoleic acid modulates phenotype the TCR engagement, the induction of IFN- in T cells is and effector functions of porcine CD8( ) lymphocytes. J. Nutr. 131: 2370 –2377. 3. Yamasaki, M., Chujo, H., Hirao, A., Koyanagi, N., Okamoto, T., Tojo, N., controlled by IL-12 and IL-18 produced by macrophages and Oishi, A., Iwata, T., Yamauchi-Sato, Y., Yamamoto, T., Tsutsumi, K., Tachibana, dendritic cells (46). The infection of pigs with PCV2 down- H. & Yamada, K. (2003) Immunoglobulin and cytokine production from spleen modulated IL-18, regardless of the dietary treatment, and lymphocytes is modulated in C57BL/6J Mice by dietary cis-9, trans-11 and upregulated IL-12 transcript levels in pigs fed the control diet. trans-10, cis-12 conjugated linoleic acid. J. Nutr. 133: 784 –788. 4. Bassaganya-Riera, J., Hontecillas, R., Zimmerman, D. R. & Wanne- During a viral infection, downmodulated IFN- production muehler, M. J. (2002) Long-term influence of lipid nutrition on the induction of may result in immune deviation of CD8 cytolytic T cell CD8( ) responses to viral or bacterial antigens. Vaccine 20: 1435–1444. function (47). However, CD8 T cell effector functions were 5. Doherty, P. C., Allan, J. E., Lynch, F. & Ceredig, R. (1990) Dissection of an inflammatory process induced by CD8 T cells. Immunol. Today 11: 55–59. not impaired in CLA-fed pigs because they maintained their 6. Yoo, D. & Giulivi, A. (2000) Xenotransplantation and the potential risk ability to produce virus-specific IFN- . Furthermore, we dem- of xenogeneic transmission of porcine viruses. Can. J. Vet. Res. 64: 193–203. onstrated previously (4) that CLA enhanced the granzyme 7. Fenaux, M., Halbur, P. G., Gill, M., Toth, T. E. & Meng, X. J. (2000) Genetic characterization of type 2 porcine circovirus (PCV-2) from pigs with activities of peripheral blood lymphocytes after stimulation postweaning multisystemic wasting syndrome in different geographic regions of with viral antigens. North America and development of a differential PCR-restriction fragment length In examining the mechanism of T-cell immunoregulation, polymorphism assay to detect and differentiate between infections with PCV-1 dietary CLA differentially modulated PPAR- and PPAR- and PCV-2. J. Clin. Microbiol. 38: 2494 –2503. 8. Morozov, I., Sirinarumitr, T., Sorden, S. D., Halbur, P. G., Morgan, M. K., expression in PCV2-infected pigs. 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S., Nielsen, J., Stadejek, T., Storgaard, T., Kra- that PPAR- and - expression in T cells is linked to the kowka, S., Ellis, J., McNeilly, F., Allan, G. & Botner, A. (2002) Reproduction of suppression and activation of the T-cell function, respectively postweaning multisystemic wasting syndrome (PMWS) in immunostimulated and non-immunostimulated 3-week-old piglets experimentally infected with porcine (14,15). However, the inhibitory actions of PPAR- agonists circovirus type 2 (PCV2). Vet. Microbiol. 89: 97–114. on CD4 T cells are not consistent with a PPAR- involve- 12. Small, T. N., Avigan, D., Dupont, B., Smith, K., Black, P., Heller, G., ment in T-cell activation (45,48). The suppressed IFN- pro- Polyak, T. & O’Reilly, R. J. (1997) Immune reconstitution following T-cell duction by CD4 T cells induced by CLA is consistent with depleted bone marrow transplantation: effect of age and posttransplant graft rejection prophylaxis. Biol. Blood Marrow Transplant. 3: 65–75. the inhibitory actions of PPAR- agonists on CD4 T cells. 13. Bassaganya-Riera, J., Hontecillas, R. & Beitz, D. C. (2002) Colonic However, the modulation of CD8 T cells by PPAR- has not anti-inflammatory mechanisms of conjugated linoleic acid. Clin. Nutr. 21: 451– been investigated. We propose that CD8 T cells are the 459. 14. Jones, D. C., Ding, X. & Daynes, R. A. (2002) Nuclear receptor per- primary targets of PPAR- within the T-cell subset. Addi- oxisome proliferator-activated receptor alpha (PPARalpha) is expressed in resting tional experimentation is required to examine the modulatory murine lymphocytes. The PPARalpha in T and B lymphocytes is both transacti- actions of PPAR on CD8 T cells and to elucidate the vation and transrepression competent. J. Biol. Chem. 277: 6838 – 6845. 15. Cunard, R., DiCampli, D., Archer, D. C., Stevenson, J. L., Ricote, M., mechanism(s) through which PPAR- agonists, including Glass, C. K. & Kelly, C. J. (2002) WY14, 643, a PPAR alpha ligand, has CLA, regulate CD8 T-cell function. profound effects on immune responses in vivo. J. Immunol. 169: 6806 – 6812. Results of studies using individual isomers indicate that 16. Wang, Y. L., Frauwirth, K. A., Rangwala, S. M., Lazar, M. A. & Thompson, cis-9, trans-11 and trans-10, cis-12 CLA have important im- C. B. (2002) Thiazolidinedione activation of peroxisome proliferator-activated receptor gamma can enhance mitochondrial potential and promote cell survival. munomodulatory properties. The effects of CLA on CD8 J. Biol. Chem. 277: 31781–31788. T-cell numbers were due mainly to cis-9, trans-11, whereas the 17. National Research Council (1998) Nutrient Requirements of Swine. trans-10, cis-12 isomer differentially modulated B-cell effector National Academy Press, Washington, D.C. 18. Lim, B. O., Jolly, C. A., Zaman, K. & Fernandes, G. (2000) Dietary (n-6) functions in mice (3). In addition, in in vitro studies in and (n-3) fatty acids and energy restriction modulate mesenteric lymph node macrophages, cis-9, trans-11 CLA inhibited tumor necrosis lymphocyte function in autoimmune-prone (NZB NZW)F1 mice. J. Nutr. 130: factor- production (49). Furthermore, recent studies in hu- 1657–1664. 19. Sato, M., Iwakabe, K., Kimura, S. & Nishimura, T. (1999) The influence mans indicated that the effects of a 50:50 isomeric mixture on of dietary protein antigen on Th1/Th2 balance and cellular immunity. Immunol. B cells were greater than those of a more purified preparation Lett. 70: 29 –35. consisting of 80% cis-9, trans-11 CLA (50). Hence, both CLA 20. Hontecillas, R., Wannemeulher, M. J., Zimmerman, D. R., Hutto, D. L., isomers are active and differentially modulate specific compo- Wilson, J. H., Ahn, D. U. & Bassaganya-Riera, J. (2002) Nutritional regulation of porcine bacterial-induced colitis by conjugated linoleic acid. J. Nutr. 132: nents of the immune response. Future studies to examine the 2019 –2027. nutritional and immunological interactions between these two 21. Pogranichnyy, R. M., Yoon, K. J., Harms, P. A., Swenson, S. L., Zimmer- CLA isomers are warranted. man, J. J. & Sorden, S. D. (2000) Characterization of immune response of young pigs to porcine circovirus type 2 infection. Viral Immunol. 13: 143–153. 22. Nawagitgul, P., Harms, P. A., Morozov, I., Thacker, B. J., Sorden, S. D., Lekcharoensuk, C. & Paul, P. S. (2002) Modified indirect porcine circovirus ACKNOWLEDGMENTS (PCV) type 2-based and recombinant capsid protein (ORF2)-based enzyme- linked immunosorbent assays for detection of antibodies to PCV. Clin. Diagn. We thank T. Opriessnig, E. Thacker and S. Yu for technical Lab. Immunol. 9: 33– 40. assistance and P. S. Paul and P. Nawagitgul for kindly providing the 23. Ellis, J., Hassard, L., Clark, E., Harding, J., Allan, G., Willson, P., Stro- recombinant baculovirus carrying the ORF2 gene of PCV2 and kappe, J., Martin, K., McNeilly, F., Meehan, B., Todd, D. & Haines, D. (1998) Isolation of circovirus from lesions of pigs with postweaning multisystemic wast- antibodies anti-PCV2. ing syndrome. Can. Vet. J. 39: 44 –51. 24. Allan, G. M., McNeilly, F., Kennedy, S., Daft, B., Clarke, E. G., Ellis, J. A., Haines, D. M., Meehan, B. M. & Adair, B. M. (1998) Isolation of porcine LITERATURE CITED circovirus-like viruses from pigs with a wasting disease in the USA and Europe. J. Vet .Diagn. Investig. 10: 3–10. 1. Bassaganya-Riera, J., Hontecillas, R., Bregendahl, K., Wannemuehler, 25. Pogranichniy, R. M., Yoon, K. J., Harms, P. A., Sorden, S. D. & Daniels, M. J. & Zimmerman, D. R. (2001) Effects of dietary conjugated linoleic acid in M. (2002) Case-control study on the association of porcine circovirus type 2

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Nutritional Immunology

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