![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
*
Veterinary Medical Research Institute, Nutritional Immunology, College of Veterinary Medicine, Ames, IA 50011;
Department of Animal Science, Iowa State University, Ames, IA 50010; and
U.S. Department of Agriculture–Animal and Plant Health Inspection Service, Ames, IA 50010
3To whom correspondence should be addressed. E-mail: bassy{at}iastate.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
(PPAR-) mRNA expression were assayed in colonic lymph nodes and colon of pigs. Colonic mucosal lesions and lymphocyte subset distribution were evaluated by histology and immunohistochemistry. Supplementation of CLA in the diet before the induction of colitis decreased mucosal damage; maintained cytokine profiles (i.e., interferon- and interleukin-10) and lymphocyte subset distributions (i.e., CD4+ and CD8+), resembling those of noninfected pigs; enhanced colonic expression of PPAR-; and attenuated growth failure. Therefore, CLA fed preventively before the onset of enteric disease attenuated inflammatory lesion development and growth failure.
KEY WORDS: • lipid nutrition • colitis • conjugated linoleic acid • interferon- • PPAR- • growth suppression
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
,2
) to applying this fundamental knowledge in the development of nutritionally based therapies for both human and animal diseases (3
,4
). Human epidemiological studies and animal feeding trials have uncovered a role for nutrients and nutraceuticals (5
) in preventing or ameliorating chronic recurring diseases (i.e., neoplasic, inflammatory or autoimmune). Inflammatory bowel disease (IBD4
) is a chronic recurring illness that can be nutritionally controlled (6
,7
). Histopathological features consistent with IBD include mucosal inflammation and ulceration with crypt abscesses, chronic mucosal damage and branching of the crypts (8
). IBD is characterized by two diseases of unknown etiology, Crohn’s disease (CD) and ulcerative colitis (UC) (9
). Although UC is typically a diffuse change without segmentation that is confined to the colon, CD is characterized by segmental distribution with sharply demarcated boundaries and may occur in any section of the gastrointestinal tract. Inflammatory lesions of the porcine colonic mucosa triggered by the bacterial enteric pathogen Brachyspira hyodysenteriae resemble those of human CD. Similarly to what occurs in 65 to 75% of patients with CD (10
,11
), B. hyodysenteriae–induced colitis results in weight loss. Furthermore, in both human IBD and B. hyodysenteriae–induced colitis, an immunoinflammatory etiology contributes to the mucosal damage associated with the onset of enteric disease (12
,13
).
Predisposition to IBD is controlled in part by genetic factors (14
–16
). However, environmental influences including nutrition may contribute to either preventing or promoting the onset of disease. Nutritional interventions that ameliorate IBD, both dietary 1,25-dihydroxycholecalciferol (6
) and (n-3) polyunsaturated fatty acids (PUFA) (7
), have been shown to attenuate the symptoms of IBD. Vitamin D status modulates the enteric immune and inflammatory dysfunction by targeting vitamin D receptors (VDR) (17
), whereas the targets for PUFA and their bioactive derivatives (18
) are peroxisome proliferator-activated receptors (PPAR) (19
). PPAR are novel members of the nuclear receptor superfamily with several isoforms (
, ß and ), of which PPAR- is the predominant isoform in immune cells and enterocytes. Functionally, PPAR- agonists modulate immune function (20
) and decrease mucosal inflammation (21
,22
).
Two factors determine the role of lipid nutrition in health and disease: 1) the composition and 2) the total amount of fat in the diet (23
). The present study was designed to examine the cellular and molecular mechanisms by which the dietary fatty acid composition modulates colonic health. More specifically, we examined the direct influence of conjugated linoleic acid (CLA) on mucosal inflammation. CLA is a mixture of positional (9,11; 10,12; 11,13; etc.) and geometric (cis or trans) isomers of octadecadienoic acid with conjugated double bonds. We previously demonstrated that dietary CLA supplementation enhances the numbers of peripheral blood CD8+ T-cells (24
). Depletion of this T-cell subset exacerbated inflammatory lesions in a murine model of respiratory disease (25
).
On the basis of the preventive role of dietary CLA on diseases with an inflammatory pathogenesis (26
–28
), the CLA-induced immunomodulation of CD8+ cells (29
) and because PPAR- agonists prevented murine experimental IBD (21
,22
), we hypothesized that colonic inflammation can be ameliorated by dietary CLA supplementation. To test this hypothesis, pigs were challenged with an enteric bacterial pathogen (i.e., B. hyodysenteriae). Through the bacterial challenge of pigs fed either a control or CLA-supplemented diet, we were able to investigate the role of CLA on the progression of enteric inflammatory diseases.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Two experiments were performed using a total of 64 pigs (ISU swine nutrition farm, Ames, IA) with an initial body weight of 4.8–5.5 kg. Pigs were weaned at 14 d, penned individually, fed experimental diets with free access to water and handled according to the practices of animal care established by the Committee of Animal Care at Iowa State University. In both experiments, to decrease variation, blocks of pigs were designed based on litter of origin, initial body weight, age and gender. On d 0, antibody titers of the 64 pigs to B. hyodysenteriae were tested by using an ELISA as previously described (30
) and found to be negative.
Experiment 1. A total of 16 pigs (i.e., eight blocks of two pigs each) were distributed into two dietary treatments: soybean oil–supplemented diet (n = 8) or conjugated linoleic acid–supplemented diet (n = 8) and fed the experimental diets for 72 d before the induction of colitis. Before challenging four of the blocks of pigs, the experimental design was a randomized complete block. After oral challenge with B. hyodysenteriae of four of the blocks, it became a 2 x 2 factorial arrangement within a split-plot design.
Experiment 2. A total of 48 pigs (i.e., 16 blocks of three pigs each) were distributed into three immunomodulatory (i.e., diet or immunization) treatments: 1) soybean oil–supplemented diet (n = 16), 2) conjugated linoleic acid–supplemented diet (n = 16) or 3) soybean oil–supplemented diet and immunized with a proteinase-digested B. hyodysenteriae bacterin. Pigs in expt. 2 were fed the experimental diets for 49 d before challenge.
Before challenging eight of the blocks of pigs, the experimental design was a randomized complete block. After oral challenge with B. hyodysenteriae of eight of the blocks, it became a 2 x 3 factorial arrangement within a split-plot design. Pigs within the block were the experimental units for dietary treatment, and blocks of three pigs each were the experimental units for challenge status (i.e., infected or noninfected).
Dietary and immunization treatments.
Either a 1.33 g CLA/100 g of diet or an isocaloric and isonitrogenous soybean oil–supplemented control diet (Table 1
) was randomly allotted to pens within blocks as previously described (24
). Before challenge, pigs were given free access to feed for 72 d in four phases (I, 1 to 2 wk; II, 3 to 4 wk; III, 5 to 8 wk; and IV, 9 to 11 wk) and 49 d in three phases (I, 1 to 2 wk; II, 3 to 4 wk; and III, 5 to 7 wk) in the first and second studies, respectively. Between treatments, diets were formulated to be isocaloric and isonitrogenous to avoid energy- and/or protein-derived immunological changes (31
,32
). Thus, in control diets, 2.21 g CLA source/100 g of diet was replaced by 2.21 g soybean oil/100 g of diet to maintain both the CLA-supplemented and the control diets isocaloric within phases. Pigs were fed either a CLA-supplemented or a control diet for the entire experimental period. The CLA source was alkali-isomerized sunflower oil (ConLinco, Detroit Lakes, MN). Diets were formulated to maintain or exceed current recommended nutritional requirements of the National Research Council (33
) for pigs. In the second experiment, on d7, d21 and d35, the immunization treatments (i.e., squalene control, or proteinase-digested B. hyodysenteriae bacterin) were randomly assigned to half of the blocks. Pigs were intramuscularly (i.m.) inoculated with 2 mL of a proteinase-digested B. hyodysenteriae bacterin strain B204 in squalene as previously described (30
). Pigs, feeders and feed were weighed on a weekly basis before and after challenge to evaluate modifications in growth [i.e., average daily gain (ADG)] and appetite [i.e., average daily feed intake (ADFI)].
|
Brachyspira hyodysenteriae strain B204 was grown under anaerobic conditions in trypticase soy broth (Becton Dickinson, Cockeysville, MD) supplemented with 5% horse serum (Hyclone; Logan, Utah), 0.5% yeast extract (Difco, Detroit, MI) and VPI salts. All cultures used for infection studies were > 90% motile and had been passed in vitro 23 to 25 times. Challenge inoculum consisted of two doses of 1010 B. hyodysenteriae organisms given orally on 2 consecutive days (i.e., d72 and d73 or d49 and d50 in expt. 1 and expt. 2, respectively) that was administered to half of the blocks of pigs. Challenge inoculum was given 14 d after the third vaccination in vaccinated pigs of expt. 2. B. hyodysenteriae is the causative agent of the porcine dysentery.
Determination of fat content and fatty acid analyses.
Fat content in the diet was determined by weighing 2 g of sample into a 50-mL test tube with 20 mL solvent (chloroform:methanol = 2:1, v/v), and homogenized with a Brinkman polytron (Type PT 10/35) for 10 s at high speed. Butylated hydroxyanisole (BHA, 10%) dissolved in 98% ethanol (25 µg) was added to the sample before homogenization. The homogenate was filtered through a Whatman no. 1 filter paper (Whatman, Clifton, NJ) into a 100-mL graduated cylinder to which was added 5 mL of a solution of 8.8 g NaCl/L. After the cylinder was capped with a glass stopper, the filtrate was mixed well. The inside of the cylinder was washed twice with 10 mL of Folch 2 (CHCl3:CH3OH:H2O = 3:47:48), and the contents were stored until the aqueous and organic layers clearly separated. The upper layer was siphoned off and 400 mL of the lower layer (chloroform layer) was transferred to a 20-mL test tube and dried at 50°C under nitrogen flow. Fatty acid analyses (triplicate sample readings from each diet) of diets were conducted using a Hewlett–Packard (HP) 6890 gas chromatograph (Hewlett–Packard, Wilmington, DE) equipped with an autosample injector and flame-ionization detector (FID). A combined column [HP-225 column (7.5 m, 0.25 mm i.d., 0.25 µm nominal), an HP wax column (15 m, 0.25 mm i.d., 0.25 µm nominal) and an HP wax column (30 m, 0.25 mm i.d., 0.25 µm nominal) that were connected using zero dead-volume column connectors (J & W Scientific, Folsom, CA)] was used to improve separation. A split inlet (19:1) was used to inject samples (1 µL) into the capillary column. Ramped oven-temperature conditions (180°C for 1 min, increased to 230°C at 2.5°C/min, then held at 230°C for 14 min) were used. Temperatures of both inlet and detector were 280°C. Helium was used as a carrier gas, and a constant column flow of 1.1 mL/min was used. Detector (FID) air, H2, and make-up gas (He) flows were 350, 35 and 43 mL/min, respectively. The composition of fatty acids was calculated as percentage composition of total peak area (pA·s). Column performance and detector response were verified using commercially available (Nu-Chek-Prep, Elysian, MN) fatty acid standards {e.g., myristic (14:0), palmitic (16:0), palmitoleic [16:1(n-9)], heptadecanoic [17:1(n-9)], stearic (18:0), oleic [18:1(n-9)], linoleic [18:2(n-6)], linolenic [18:3(n-3)], arachidic (20:0), arachidonic [20:4(n-6)], eicosapentanoic [20:5(n-3)], docosapentanoic [22:5(n-3)], docosahexanoic [22:6(n-3)], c9,t11 CLA, t10,c12 CLA, t9,t11 CLA and c10,c12 CLA}.
Necropsy procedures.
Pigs were anesthetized by administration of Rompun (Bayer, Shawnee, KS)/Telazol (Fort Dodge Laboratories, Fort Dodge, IA) i.m. and euthanized via electrocution. 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). At necropsy, swabs of cecal and spiral colon contents were streaked onto modified BJ blood agar plates containing antibiotics for isolation of B. hyodysenteriae (35
). Sections of spiral colon and cecum were obtained, fixed in 10% buffered formalin, later embedded in paraffin and then sectioned for histological examination. Samples of colon and mesenteric lymph nodes were embedded in RNAlater (Ambion, Austin, TX) for posterior isolation of total RNA and analysis of cytokine expression (colonic samples). For immunohistochemistry, samples of colonic tissue were placed in tissue-freezing medium (Triangle Biomedical Sciences, Durham, NC) and snap-frozen in an ethanol/dry ice bath. Both samples for mRNA expression analysis and for immunohistochemistry were stored at -70°C.
Histopathological and immunohistochemical evaluation of colonic samples.
Hematoxilin-eosin (H&E)–stained colonic sections were histologically evaluated on the basis of mucosal thickness (i.e., expressed as crypt depth as a function of crypt width) and epithelial erosions [i.e., 1) no erosion, 2) mild erosion and 3) severe erosion]. H&E slides were labeled with accession numbers lacking any reference to either the immunomodulatory or infective treatment and were evaluated by a board-certified pathologist.
For the evaluation of colonic lymphocyte subset distributions, frozen colonic tissue sections embedded in tissue-freezing medium were cut on a cryostat at -18°C at thicknesses from 5 to 10 µm. 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. Before staining tissues with monoclonal antibodies, slides were warmed to room temperature and rehydrated in 0.5 mol/L Tris solution. Endogenous peroxidase activity was blocked by adding 0.3% hydrogen peroxide for 10 min. Nonspecific binding was blocked with the addition of the immunohistochemistry buffer containing 5% normal goat serum/3% bovine serum albumin/Tris buffer (NGS/BSA/Tris) solution at room temperature for 2 h. Slides were incubated with the primary antibody solution overnight at 4°C. Primary antibodies were diluted in NGS/BSA/Tris. Mouse anti-pig CD4 primary antibody was 10x supernatant from the mouse cell line HB147 used at 1:25 dilution. Mouse anti-pig CD8 antibody was supernatant from the mouse cell line HB143 used at 1:100 dilution. Both antibodies were grown in our laboratory. The mouse anti-pig TCR
(Po-Tcr1-N4) antibody was purchased from VMRD (Pullman, WA) and used at 1:100 dilution. The mouse anti-pig CD3 was concentrated supernatant from the mouse cell line 8E6 and used at 1:10,000 dilution. Before the incubation with the secondary antibodies, slides were rinsed with Tris solution to wash unbound primary antibody. Peroxidase-conjugated goat anti-mouse IgG (H + L) (Jackson ImmunoResearch, West Grove, PA) was added to slides stained with mouse anti-pig CD4, mouse anti-pig CD3 and mouse anti-pig TCR. The goat anti-mouse IgG (H + L) was diluted 1:300 with NGS/BSA/Tris and incubated for 2 h at room temperature. For the mouse anti-pig CD8 primary antibody, the secondary used was biotin-conjugated goat F(ab')2 anti-mouse IgG2a (Southern Biotechnologies Associates, Birmingham, AL) diluted 1:250 (in NGS/BSA/Tris), and incubated for 2 h at room temperature. After the second incubation, slides were treated with peroxidase-conjugated strepavidin, diluted in Tris solution (1:500) and incubated for 1 h at room temperature. The chromagen used was diaminobenzediene (Biomedia Corporation, Foster City, CA). Slides were counterstained with Instant Hematoxylin (Shandon, Pittsburg, PA), coverslipped with Immu-mount (Shandon) and numbers of CD4+, CD8+, CD3+ and TCR+ cells enumerated. Stained colonic sections were observed at x400 magnification. Five randomly chosen sections (i.e., area 0.375 mm2) were enumerated for each pig and antibody treatment. Data were presented as number of cells per square millimeter.
Isolation of total RNA.
Colonic lymph nodes and colonic tissue were recovered during the necropsy procedure and kept in RNAlater (Ambion) at -70°C. Total RNA was isolated using the total RNA isolation MiniKit (Qiagen, Valencia, CA), treated with DNA-free (Ambion) and kept in 0.02% diethyl pyrocarbonate (DEPC)–treated water at -20°C according to the manufacturer’s instructions. RNA in samples were quantified, and the purity was determined using a spectrophotometer at an optical density (OD)260 and OD260/OD280 ratios, respectively. All samples had OD260/OD280 ratios above 1.80, corresponding to 90–100% pure nucleic acid.
Reverse-transcriptase–polymerase chain reaction (RT-PCR).
Expression of interleukin-10 (IL-10), interferon- (IFN-), PPAR- and ß2-microglobulin (i.e., housekeeping gene) in colonic lymph nodes was determined using the RT-PCR procedure. Briefly, after isolation of total RNA, 1 µg of each RNA isolate from each pig was added to a 5-µL DNA digestion reaction containing 4 µL of M-MLV RT reaction buffer (Promega, Madison, WI), 0.4 µL of RNase-free water, 0.5 µL of SUPERase In (Ambion) and 0.1 µL of Dnase I (Sigma Chemical, St. Louis, MO). Cycle parameters for DNA digestion were 1 cycle of 37°C, 15 min; 1 cycle of 94°C, 10 min; 1 cycle of 4°C, 5 min. For the melting of the secondary structure, 1 µL of Promega random hexamers were added to the digested RNA. Cycle parameters for the melting reaction were 1 cycle of 94°C, 5 min. After the melting reaction, the reaction mixture was placed onto ice for 1 min. RNA was then reverse transcribed in a 10-µL reaction containing 6 µL of the previously described reactions plus 1 µL of M-MLV RT reaction buffer (Promega), 1.25 µL Sigma dNTP mix, 0.75 µL of RNase-free water and 1 µL of Promega M-MLV RT (200 U reverse transcriptase/µL). Cycle parameters for the reverse-transcription procedure were 1 cycle of 37°C, 60 min; 1 cycle of 94°C, 5 min; and 1 cycle of 4°C, 5 min. The entire 10-µL reaction was then subjected to PCR amplification in a PCR reaction with a total volume of 50 µL containing 3 µL of Promega 25 mM MgCl2, 4 µL of Gibco PCR buffer without MgCl2, 35.5 µL of PCR water, 1 µL of forward primer, 1 µL of bacward primer, and 0.5 µL of Taq polymerase (Life Technologies, Rockville, MD). Cycle parameters for PCR amplification were 1 cycle of 94°C, 2.5 min; 32 cycles of (94°C, 1 min; 55°C, 1 min; 72°C, 1 min); 1 cycle of 72°C, 10 min; and 1 cycle of 4°C, 5 min.
To amplify IL-10, IFN-, PPAR- and ß2-microglobulin cDNA fragments, the sequences of PCR primers were as follows: upstream, 5'-GCTCTATTGCCTCATCTTCC-3'; downstream, 5'-GCACTCTTCACCTCCTCCAC-3' for the IL-10; upstream, 5'-TGTACCTAATGGTGGACCTC-3'; downstream, 5'-TCTCTGGCCTTGGAACATAG-3' for IFN-, upstream; 5'-TTCAAACACATCACCCCCCTGC-3'; downstream, 5'-GCTTCACATTCAGCAAACCTGGGC-3' for the PPAR- (36
); and upstream, 5'-CTGCTCTCACTGTCTGG-3'; downstream, 5'-ATCGAGAGTCACGTGCT-3' for ß2-microglobulin. PCR-amplified products were electrophoretically separated on a 1.5% agarose gel. After electrophoresis, gels were stained in ethidium bromide and photographed. A 100-kbp ladder (100 Kbplus; Life Technologies) was used as size standard.
Statistical analysis.
Postchallenge data were analyzed as a 2 x 2 (i.e., expt. 1) or 2 x 3 (i.e., expt. 2) factorial arrangement of treatments within the split-plot design. In the model, pig within block was the experimental unit for immunomodulatory treatment (subplot), and blocks of pigs within infective status were the experimental units for infection treatment (whole plot). Analysis of variance (ANOVA) was used to determine the main effects of the immunomodulatory treatment (control diet, CLA-supplemented diet, or vaccine), the infective status (i.e., infected or noninfected) and the interaction between immunomodulatory treatment and infective status. ANOVA was performed using the general linear model (GLM) procedure of the SAS software using the TEST statement to define the whole plot and subplot within the model in the program (37
). Differences with P < 0.05 were considered significant. In expt. 2, the whole plot error (i.e., error A) is the block within infective status [i.e., 14 degrees of freedom (df)] and the subplot error (i.e., error B) is the residual degrees of freedom after accounting for the immunomodulatory treatment (i.e., diet or immunization) variance and the variance for the interaction between immunomodulatory treatment and infective status (i.e., 28 df). The statistical model used in expt. 2 was Yijk = µ + Infectioni + error Aik + Immunomodulationj + (Infection x Immunomodulation)ij + error Bijk, where µ is the general mean, Infectioni is the main effect of the ith level of the challenge effect, Immunomodulationj is the main effect of the jth level of the immunomodulatory effect, (Infection x Immunomodulation)ij is the interaction effect between infection and immunomodulation, and errors A and B represent the random errors for the whole plot and the subplot, respectively. Data for expt. 1 were analyzed similarly using the following model: Yijk = µ + Infectioni + error Aik + Dietj + (Infection x Diet)ij + error Bijk. In expt. 1, the whole plot error (i.e., error A) is the block within infective status (i.e., 6 df) and the subplot error (i.e., error B) is the residual degrees of freedom after accounting for the dietary treatment variance and the variance for the interaction between dietary treatment and infective status (i.e., 6 df).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
The major difference between dietary treatments was the presence of CLA isomers in the CLA-supplemented diet, replacing primarily linoleic acid from the control diet (Table 2
). The concentration of palmitic acid (16:0) was slightly lower in CLA-supplemented diets than that in soybean oil–supplemented diets because of lower palmitic acid concentrations in the source of CLA. In phase IV the concentration of linolenic acid was greater than that in the other phases because of a greater percentage of corn in the diet. Based on the total fat content of the diets, control and CLA-supplemented diets provided the same amount of energy within phases.
|
Before infection with B. hyodysenteriae, diet groups did not differ in average daily gain or feed intake (data not shown). However, when immunization with the proteinase-digested B. hyodysenteriae bacterin was included as a positive control (expt. 2), the growth rate of vaccinated pigs after the third immunization was less than that of nonimmunized groups (data not shown). After challenge with B. hyodysenteriae, colitis-induced growth suppression in challenged pigs fed CLA-supplemented diets was attenuated compared with that of infected pigs fed the isocaloric control diet in expt. 1 (data not shown) and expt. 2 (Table 3
).
|
mRNA expression.
In expt. 1, after infection of pigs with B. hyodysenteriae, colonic lymph nodes recovered from pigs fed CLA-supplemented diets expressed cytokine profiles (e.g., INF- and IL-10) more similar to those of noninfected pigs than to those of infected pigs fed the control diet (Fig. 1
). In addition, expression of PPAR- in colonic tissue samples recovered from infected pigs fed CLA-supplemented diets was greater than that in colonic samples recovered from infected pigs fed control diets (Fig. 2
). Moreover, expression of PPAR- mRNA was not detected in colonic tissue from noninfected pigs.
|
|
Both immunization with a proteinase-digested B. hyodysenteriae bacterin and dietary CLA supplementation decreased the epithelial erosion associated with B. hyodysenteriae–induced colitis (Table 4
). However, only dietary CLA supplementation prevented the enlargement of the colonic mucosa (Table 4
, Fig. 3A
). Furthermore, the cellular infiltrate of healthy colon was primarily lymphoplasmacytic (Fig. 3
B). Infected pigs either immunized with the proteinase-digested B. hyodysenteriae bacterin or fed CLA-supplemented diets maintained a lymphoplasmacytic infiltrate with numbers of CD4+ and CD8+ cells not different from those of noninfected pigs (Table 5
). However, infected pigs fed the control diet showed a mixed cellular infiltrate including neutrophils (Fig. 3
B), with numbers of CD4+ and CD8+ T-cells substantially decreased (Table 5)
.
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
) and (n-3) PUFA (7
), ameliorated or prevented colonic inflammation. The model of colitis used involved a noninvasive bacterial agent (i.e., B. hyodysenteriae). Thus, mucosal lesions were not directly caused by B. hyodysenteriae but induced by the pig’s inflammatory response. CLA ameliorated the inflammation-associated mucosal damage, decreased growth suppression and delayed the onset of clinical disease associated with bacterial-induced colitis. The expression of IFN- and IL-10 within colonic lymph nodes in infected pigs fed CLA-supplemented diets resembled that of noninfected pigs. Conversely, expression of IFN- and IL-10 in colonic lymph nodes of infected pigs fed control diets was up-regulated. These findings suggest that supplementation of diets with CLA before the onset of clinical disease may help prevent mucosal damage. This is a first-time observation demonstrating that dietary CLA supplementation prevents or ameliorates the onset of experimental colitis in pigs.
The increased expression of IFN- in the pigs with more severe lesions (i.e., fed the control diet) is consistent with the predominantly T helper (Th) 1 nature of lesions found in patients with IBD as well as for the model of experimental colitis used in these studies. In support of this, adoptive transfer of T-cells, which preferentially express Th1 cytokines, induced Crohn’s-like lesions to mice that were otherwise healthy (38
). In a murine model of dextran sulfate sodium (DSS)–induced chronic colitis, neutralizing antibodies against IFN- facilitated lesion restitution (39
). IFN- exacerbates lesion development by initiating leukocyte recruitment from the vascular space into the tissues by modulating surface expression of endothelial adhesion molecules. These microcirculatory changes may contribute to the enhanced inflammatory cell infiltrate found in the lamina propria of pigs fed the control diet. Although enhanced mRNA expression of IL-10 correlates with increased severity of colonic injury (40
), the up-regulation of IL-10 may be a part of a homeostatic mechanism to balance Th1 polarization. At least three functional outputs of terminally differentiated Th cells have been characterized based on cytokine production and homing capacity: Th1, Th2, and nonpolarized cells (41
). The development of immune-mediated mucosal damage can be triggered by both Th1 and Th2 effector CD4+ cells (42
) and prevented by CD8+ T-cells (25
). Dietary CLA may have prevented this immunopathology by enhancing numbers of CD8+ cells and favoring differentiation toward a nonpolarized Th cell subset. Consistent with a mechanism of nutritional immunoregulation by T-cells, we previously demonstrated that CD4+ Th responses to bacterial antigens were attenuated by dietary CLA (24
,43
). In addition, two ligands for PPAR- (i.e., 15d-PGJ2 and ciglitazone) inhibited proliferative responses of murine Th cell clones and freshly isolated splenocytes (20
).
At the molecular level, the concentration of cytokines in tissues is controlled in part by mechanism(s) of transcriptional regulation. Nuclear factor-kappa B (NF-
B) is included among the transcription factors involved in up-regulating the expression of IFN- (44
). Interestingly, PPAR- activation was previously demonstrated to antagonize the activities of several transcription factors including NF-B (45
). As a result of this interference with the NF-B signaling pathway, the expression of proinflammatory cytokines (i.e., TNF-, IL-6 and IL-1ß) is suppressed (22
) and macrophage apoptosis induced (46
), both effects with likely consequences in inflammation. Here, we have shown that PPAR- expression is up-regulated after colonic inflammation and the concentration of PPAR- is greater in infected pigs fed CLA-supplemented diets than that in infected pigs fed the control diets.
PPAR-–independent mechanism(s) have also been shown to significantly contribute to the anti-inflammatory actions of compounds, such as CLA, that are PPAR- agonistic ligands (47
). In the case of CLA, PPAR-–independent mechanisms of action would include CLA-induced regulation of lipid mediator synthesis. Consistent with previous observations in liver of mice (48
), the analysis of the fatty acid composition of plasma (data not shown) revealed that dietary CLA supplementation decreased the concentration of linoleic and arachidonic acids. The latter is a precursor for the generation of first-phase eicosanoids (i.e., two series prostaglandins and four series leukotrienes) involved in early microinflammatory events (i.e., polymorphonuclear neutrophilic leukocyte chemotaxis and release of superoxide anions) (49
). Enhanced intestinal eicosanoid concentrations closely correlate with severe histological signs of colonic inflammation (50
). Therefore, the enteric health benefits of dietary CLA may derive in part from the generation of an array of lipid mediators (i.e., hydroxy-containing fatty acids, prostaglandins, lipoxins and leukotrienes) that are either anti-inflammatory or not proinflammatory (18
).
Dietary CLA supplementation was more effective than immunization in preventing growth suppression and lesion development in infected pigs. B. hyodysenteriae were recovered from the colon of all infected pigs, regardless of dietary treatment. All infected pigs were positive for B. hyodysenteriae, suggesting that CLA modulates the host’s immune effector mechanisms instead of directly targeting the bacterial agent. Histological evaluation of colonic tissues demonstrated that both CLA and systemic immunization decreased epithelial erosion compared to that of the control diet. However, only CLA prevented the enlargement of the colonic mucosa. Comparable to immunization with a B. hyodysenteriae bacterin (13
), CLA increased the numbers of TCRCD8 cells in peripheral blood (29
) and maintained numbers of CD4+ and CD8+ cells in the colonic mucosa.
This is the first report of efficacy of CLA in ameliorating disease associated with colitis. Future studies will be designed to distinguish the PPAR-–independent and the PPAR-–dependent mechanisms by structurally elucidating T-lymphocyte–derived lipid mediators and defining the phenotype of tissue-targeted (i.e., enterocytes, CD8+ T-lymphocytes or CD4+ T-lymphocytes) PPAR-–deficient mice, respectively. These experimental approaches may yield novel nutritional therapies for both inflammatory and immune pathologies.
|
FOOTNOTES |
|---|
2 Supported by the National Pork Board (grant 01134, awarded to J.B.-R.).
4 Abbreviations used: ADFI, average daily feed intake; ADG, average daily gain; CD, Crohn’s disease; CLA, conjugated linoleic acid; DSS, dextran sulfate sodium; 15d-PGJ2, 15-deoxy-
12,14-prostaglandin J2; FACS, fluorescence-activated cell sorting; HBSS, Hanks’ balanced salt solution; IBD, inflammatory bowel disease; IEL, intraepithelial lymphocyte; IFN-, interferon-; IL-10, interleukin-10; PBMC, peripheral blood mononuclear cell; PPAR-, peroxisome proliferator-activated receptor-; PUFA, polyunsaturated fatty acid; SP, single-positive; TCR, T-cell receptor; UC, ulcerative colitis.
Manuscript received 11 February 2002. Initial review completed 18 March 2002. Revision accepted 10 April 2002.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Cantorna, M. T., Nashold, F. E. & Hayes, C. E. (1994) In vitamin A deficiency multiple mechanisms establish a regulatory T helper cell imbalance with excess Th1 and insufficient Th2 function. J. Immunol. 152:1515-1522.[Abstract]
2. Cantorna, M. T., Nashold, F. E., Chun, T. Y. & Hayes, C. E. (1996) Vitamin A down-regulation of IFN-gamma synthesis in cloned mouse Th1 lymphocytes depends on the CD28 costimulatory pathway. J. Immunol. 156:2674-2679.[Abstract]
3. Cantorna, M. T., Hayes, C. E. & DeLuca, H. F. (1996) 1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis. Proc. Natl. Acad. Sci. USA 93:7861-7864.
4. Cantorna, M. T. & Hayes, C. E. (1996) Vitamin A deficiency exacerbates murine Lyme arthritis. J. Infect. Dis. 174:747-751.[Medline]
5. Bassaganya-Riera, J., Hontecillas, R. & Wannemuehler, M.J. (2002) Nutritional impact of conjugated linoleic acid: a model functional food ingredient. In Vitro Cell. Dev. Biol. 38:241-246.
6. Cantorna, M. T., Munsick, C., Bemiss, C. & Mahon, B. D. (2000) 1,25-Dihydroxycholecalciferol prevents and ameliorates symptoms of experimental murine inflammatory bowel disease. J. Nutr. 130:2648-2652.
7. Geerling, B. J., Badart-Smook, A., van Deursen, C., van Houwelingen, A. C., Russel, M. G., Stockbrugger, R. W. & Brummer, R. J. (2000) Nutritional supplementation with N-3 fatty acids and antioxidants in patients with Crohn’s disease in remission: effects on antioxidant status and fatty acid profile. Inflamm. Bowel Dis. 6:77-84.[Medline]
8. Crawford, J. M. (1994) The gastrointestinal tract. Cotran, R. S. Kumar, V. Robbins, S. L. eds. Pathologic Basis of Disease 1994 WB Saunders Philadelphia, PA. .
9. Kirsner, J. B. (1991) Inflammatory bowel disease. Part I: Nature and pathogenesis. Dis. Mon. 37:607-666.
10. Fleming, C. R. (1995) Nutrition in patients with Crohn’s disease: another piece of the puzzle. J. Parenter. Enteral Nutr. 19:93-94.[Medline]
11. Geerling, B. J., Badart-Smook, A., Stockbrugger, R. W. & Brummer, R. J. (1998) Comprehensive nutritional status in patients with long-standing Crohn disease currently in remission. Am. J. Clin. Nutr. 67:919-926.[Abstract]
12. Nagura, H., Ohtani, H., Sasano, H. & Matsumoto, T. (2001) The immuno-inflammatory mechanism for tissue injury in inflammatory bowel disease and Helicobacter pylori-infected chronic active gastritis. Roles of the mucosal immune system. Digestion 63:12-21.
13. Waters, W. R., Sacco, R. E., Dorn, A. D., Hontecillas, R., Zuckermann, F. A. & Wannemuehler, M. J. (1999) Systemic and mucosal immune response of pigs to parenteral immunization with a pepsin-digested Serpulina hyodysenteriae bacterin. Vet. Immunol. Immunopathol. 69:75-87.[Medline]
14. Podolsky, D. K. (1991) Inflammatory bowel disease (1). N. Engl. J. Med. 325:928-937.[Medline]
15. Ogura, Y., Bonen, D. K., Inohara, N., Nicolae, D. L., Chen, F. F., Ramos, R., Britton, H., Moran, T., Karaliuskas, R., Duerr, R. H., Achkar, J. P., Brant, S. R., Bayless, T. M., Kirschner, B. S., Hanauer, S. B., Nunez, G. & Cho, J. H. (2001) A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 411:603-606.[Medline]
16. Hugot, J. P., Chamaillard, M., Zouali, H., Lesage, S., Cezard, J. P., Belaiche, J., Almer, S., Tysk, C., O’Morain, C. A., Gassull, M., Binder, V., Finkel, Y., Cortot, A., Modigliani, R., Laurent-Puig, P., Gower-Rousseau, C., Macry, J., Colombel, J. F., Sahbatou, M. & Thomas, G. (2001) Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 411:599-603.[Medline]
17. Veldman, C. M., Cantorna, M. T. & DeLuca, H. F. (2000) Expression of 1,25-dihydroxyvitamin D(3) receptor in the immune system. Arch. Biochem. Biophys. 374:334-338.[Medline]
18. Serhan, C. N., Clish, C. B., Brannon, J., Colgan, S. P., Chiang, N. & Gronert, K. (2000) Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing. J. Exp. Med. 192:1197-1204.
19. Jump, D. B. & Clarke, S. D. (1999) Regulation of gene expression by dietary fat. Annu. Rev. Nutr. 19:63-90.[Medline]
20. Clark, R. B., Bishop-Bailey, D., Estrada-Hernandez, T., Hla, T., Puddington, L. & Padula, S. J. (2000) The nuclear receptor PPAR and immunoregulation: PPAR mediates inhibition of helper T cell responses. J. Immunol. 164:1364-1371.
21. Su, C. G., Wen, X., Bailey, S. T., Jiang, W., Rangwala, S. M., Keilbaugh, S. A., Flanigan, A., Murthy, S., Lazar, M. A. & Wu, G. D. (1999) A novel therapy for colitis utilizing PPAR- ligands to inhibit the epithelial inflammatory response. J. Clin. Invest. 104:383-389.[Medline]
22. Desreumaux, P., Dubuquoy, L., Nutten, S., Peuchmaur, M., Englaro, W., Schoonjans, K., Derijard, B., Desvergne, B., Wahli, W., Chambon, P., Leibowitz, M. D., Colombel, J. F. & Auwerx, J. (2001) Attenuation of colon inflammation through activators of the retinoid X receptor (RXR)/peroxisome proliferator-activated receptor (PPAR) heterodimer. A basis for new therapeutic strategies. J. Exp. Med. 193:827-838.
23. Calder, P. C. (1998) Dietary fatty acids and the immune system. Nutr. Rev. 56:S70-S83.[Medline]
24. Bassaganya-Riera, J., Hontecillas-Magarzo, R., Bregendahl, K., Wannemuehler, M. J. & Zimmerman, D. R. (2001) Effects of dietary conjugated linoleic acid in nursery pigs of dirty and clean environments on growth, empty body composition, and immune competence. J. Anim. Sci. 79:714-721.
25. Jones, H. P., Tabor, L., Sun, X., Woolard, M. D. & Simecka, J. W. (2002) Depletion of CD8(+) T cells exacerbates CD4(+) Th cell-associated inflammatory lesions during murine mycoplasma respiratory disease. J. Immunol. 168:3493-3501.
26. Ha, Y. L., Storkson, J. & Pariza, M. W. (1990) Inhibition of benzo(a)pyrene-induced mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic acid. Cancer Res. 50:1097-1101.
27. Houseknecht, K. L., Vanden Heuvel, J. P., Moya-Camarena, S. Y., Portocarrero, C. P., Peck, L. W., Nickel, K. P. & Belury, M. A. (1998) Dietary conjugated linoleic acid normalizes impaired glucose tolerance in the Zucker diabetic fatty fa/fa rat [published erratum appears in Biochem. Biophys. Res. Commun. 1998 Jun 29;247(3):911[. Biophys. Res. Commun. 244:678-682.[Medline]
28. Lee, K. N., Kritchevsky, D. & Pariza, M. W. (1994) Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 108:19-25.[Medline]
29. Bassaganya-Riera, J., Hontecillas, R., Zimmerman, D. R. & Wannemuehler, M. J. (2001) Dietary conjugated linoleic acid modulates phenotype and effector functions of porcine CD8(+) lymphocytes. J. Nutr. 131:2370-2377.
30. Waters, W. R., Pesch, B. A., Hontecillas, R., Sacco, R. E., Zuckermann, F. A. & Wannemuehler, M. J. (1999) Cellular immune responses of pigs induced by vaccination with either whole cell sonicate or pepsin-digested Brachyspira (Serpulina) hyodysenteriae bacterin. Vaccine 18:711-719.[Medline]
31. Lim, B. O., Jolly, C. A., Zaman, K. & Fernandes, G. (2000) Dietary (n-6) and (n-3) fatty acids and energy restriction modulate mesenteric lymph node lymphocyte function in autoimmune-prone (NZB x NZW)F1 mice. J. Nutr. 130:1657-1664.
32. Sato, M., Iwakabe, K., Kimura, S. & Nishimura, T. (1999) The influence of dietary protein antigen on Th1/Th2 balance and cellular immunity. Immunol. Lett. 70:29-35.[Medline]
33. National Research Council (1998) Nutrient requirements of swine 1998 National Academy Press Washington, DC. .
34. AOAC (1990) Official Methods of Analysis 1990 Association of Official Analytical Chemists Arlington, VA. .
35. Kunkle, R. A. & Kinyon, J. M. (1988) Improved selective medium for the isolation of Treponema hyodysenteriae. J. Clin. Microbiol. 26:2357-2360.
36. Houseknecht, K. L., Bidwell, C. A., Portocarrero, C. P. & Spurlock, M. E. (1998) Expression and cDNA cloning of porcine peroxisome proliferator-activated receptor (PPAR). Gene 225:89-96.[Medline]
37. SAS Institute Inc (1988) SAS/STAT User’s Guide, Version 6.03 1988 SAS Institute Cary, NC. .
38. Aranda, R., Sydora, B. C., McAllister, P. L., Binder, S. W., Yang, H. Y., Targan, S. R. & Kronenberg, M. (1997) Analysis of intestinal lymphocytes in mouse colitis mediated by transfer of CD4+, CD45RBhigh T cells to SCID recipients. J. Immunol. 158:3464-3473.[Abstract]
39. Obermeier, F., Kojouharoff, G., Hans, W., Scholmerich, J., Gross, V. & Falk, W. (1999) Interferon- (IFN-)- and tumour necrosis factor (TNF)-induced nitric oxide as toxic effector molecule in chronic dextran sulphate sodium (DSS)-induced colitis in mice. Clin. Exp. Immunol. 116:238-245.[Medline]
40. Egger, B., Bajaj-Elliott, M., MacDonald, T. T., Inglin, R., Eysselein, V. E. & Buchler, M. W. (2000) Characterisation of acute murine dextran sodium sulphate colitis: cytokine profile and dose dependency. Digestion 62:240-248.[Medline]
41. Langenkamp, A., Messi, M., Lanzavecchia, A. & Sallusto, F. (2000) Kinetics of dendritic cell activation: impact on priming of TH1, TH2 and nonpolarized T cells. Nat. Immunol. 1:311-316.[Medline]
42. Iqbal, N., Oliver, J. R., Wagner, F. H., Lazenby, A. S., Elson, C. O. & Weaver, C. T. (2002) T helper 1 and T helper 2 cells are pathogenic in an antigen-specific model of colitis. J. Exp. Med. 195:71-84.
43. Bassaganya-Riera, J., Hontecillas, R., Zimmerman, D. R. & Wannemuehler, M. J. (2002) Long-term influence of lipid nutrition on the induction of CD8+ responses to viral or bacterial antigens. Vaccine 20:1435-1444.[Medline]
44. Schindler, H., Lutz, M. B., Rollinghoff, M. & Bogdan, C. (2001) The production of IFN- by IL-12/IL-18-activated macrophages requires STAT4 signaling and is inhibited by IL-4. J. Immunol. 166:3075-3082.
45. Ricote, M., Li, A. C., Willson, T. M., Kelly, C. J. & Glass, C. K. (1998) The peroxisome proliferator-activated receptor- is a negative regulator of macrophage activation. Nature 391:79-82.[Medline]
46. Chinetti, G., Griglio, S., Antonucci, M., Torra, I. P., Delerive, P., Majd, Z., Fruchart, J. C., Chapman, J., Najib, J. & Staels, B. (1998) Activation of proliferator-activated receptors and induces apoptosis of human monocyte-derived macrophages. J. Biol. Chem. 273:25573-25580.
47. Chawla, A., Barak, Y., Nagy, L., Liao, D., Tontonoz, P. & Evans, R. M. (2001) PPAR- dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation. Nat. Med. 7:48-52.[Medline]
48. Belury, M. A. & Kempa-Steczko, A. (1997) Conjugated linoleic acid modulates hepatic lipid composition in mice. Lipids 32:199-204.[Medline]
49. Levy, B. D., Clish, C. B., Schmidt, B., Gronert, K. & Serhan, C. N. (2001) Lipid mediator class switching during acute inflammation: signals in resolution. Nat. Immunol. 2:612-619.[Medline]
50. Hommes, D. W., Meenan, J., de Haas, M., ten Kate, F. J., von dem Borne, A. E., Tytgat, G. N. & van Deventer, S. J. (1996) Soluble Fc receptor III (CD 16) and eicosanoid concentrations in gut lavage fluid from patients with inflammatory bowel disease: reflection of mucosal inflammation. Gut 38:564-567.
This article has been cited by other articles:
|
|
 |
|
  D. E. Butz, G. Li, S. M. Huebner, and M. E. Cook A mechanistic approach to understanding conjugated linoleic acid's role in inflammation using murine models of rheumatoid arthritis Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2007; 293(2): R669 - R676. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. D Whigham, A. C Watras, and D. A Schoeller Efficacy of conjugated linoleic acid for reducing fat mass: a meta-analysis in humans Am. J. Clinical Nutrition, May 1, 2007; 85(5): 1203 - 1211. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kohno, R. Suzuki, Y. Yasui, S. Miyamoto, K. Wakabayashi, and T. Tanaka Ursodeoxycholic Acid versus Sulfasalazine in Colitis-Related Colon Carcinogenesis in Mice Clin. Cancer Res., April 15, 2007; 13(8): 2519 - 2525. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Bassaganya-Riera, A. J Guri, A. M Noble, K. A Reynolds, J. King, C. M Wood, M. Ashby, D. Rai, and R. Hontecillas Arachidonic acid and docosahexaenoic acid enriched formulas modulate antigen-specific T cell responses to influenza virus in neonatal piglets Am. J. Clinical Nutrition, March 1, 2007; 85(3): 824 - 836. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L Dubuquoy, C Rousseaux, X Thuru, L Peyrin-Biroulet, O Romano, P Chavatte, M Chamaillard, and P Desreumaux PPAR{gamma} as a new therapeutic target in inflammatory bowel diseases. Gut, September 1, 2006; 55(9): 1341 - 1349. [Full Text] [PDF]
|
|
|
|
 |
|
 R. Jonasson, M. Andersson, T. Rasback, A. Johannisson, and M. Jensen-Waern Immunological alterations during the clinical and recovery phases of experimental swine dysentery. J. Med. Microbiol., July 1, 2006; 55(Pt 7): 845 - 855. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-M. Gaullier, J. Halse, K. Hoye, K. Kristiansen, H. Fagertun, H. Vik, and O. Gudmundsen Supplementation with Conjugated Linoleic Acid for 24 Months Is Well Tolerated by and Reduces Body Fat Mass in Healthy, Overweight Humans J. Nutr., April 1, 2005; 135(4): 778 - 784. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Changhua, Y. Jindong, L. Defa, Z. Lidan, Q. Shiyan, and X. Jianjun Conjugated Linoleic Acid Attenuates the Production and Gene Expression of Proinflammatory Cytokines in Weaned Pigs Challenged with Lipopolysaccharide J. Nutr., February 1, 2005; 135(2): 239 - 244. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-M. Gaullier, J. Halse, K. Hoye, K. Kristiansen, H. Fagertun, H. Vik, and O. Gudmundsen Conjugated linoleic acid supplementation for 1 y reduces body fat mass in healthy overweight humans Am. J. Clinical Nutrition, June 1, 2004; 79(6): 1118 - 1125. [Abstract] [Full Text] [PDF]
|
|
|
|
|
